4TH EDITION

Pocket Guide to Clinical Microbiology

Christopher D. Doern 4TH EDITION

POCKET GUIDE TO Clinical Microbiology

4TH EDITION

POCKET GUIDE TO Clinical Microbiology

Christopher D. Doern, PhD, D(ABMM) Assistant Professor, Pathology Director of Clinical Microbiology Virginia Commonwealth University Health System Medical College of Virginia Campus

Washington, DC Copyright © 2018 Ameri­­can Society for Microbiology. All rights re­served. No part of this publi­ ­ca­tion may be repro­ ­duced or trans­mit­ted in whole or in part or re­used in any form or by any means, elec­tronic or me­chan­i­cal, in­clud­ing pho­to­copy­ing and re­cord­ing, or by any in­for­ma­tion stor­age and re­trieval sys­tem, with­out­ per­mis­sion in writ­ing from the pub­lish­er.

Disclaimer: To the best of the pub­lish­er’s knowl­edge, this pub­li­ca­tion pro­ vi­des in­for­ma­tion con­cern­ing the sub­ject mat­ter cov­ered that is ac­cu­rate as of the date of pub­li­ca­tion. The pub­lisher is not pro­vid­ing le­gal, med­ical,­ or other pro­fes­sional ser­vices. Any ref­er­ence herein to any spe­cific com­mer­cial prod­ucts, pro­ce­dures, or ser­vices by trade name, trade­mark, man­u­fac­turer, or oth­er­wise does not con­sti­tute or im­ply en­dorse­ment, rec­om­men­da­tion, or fa­vored sta­tus by the Ameri­­can Society for Microbiology (ASM). The views and opin­ions of the au­thor(s) ex­pressed in this pub­li­ca­tion do not nec­es­sar­ily state or re­flect those ofASM, and they shall not be used to ad­ver­tise or en­ dorse any prod­uct.

Library of Congress Cataloging-in-Publication Data

Names: Doern, Chris­to­pher D., au­thor. Title: Pocket guide to clin­i­cal mi­cro­bi­ol­ogy / Chris­to­pher D. Doern. Description: Fourth edi­tion. | Wash­ing­ton, DC : ASM Press, 2018. Identifiers: LCCN 2018008388 | ISBN 9781683670063 (pbk. : alk. pa­per) Subjects: LCSH: Medical mi­cro­bi­ol­o­gy—Handbooks, man­u­als, etc. Classification: LCC QR46 .M92 2018 | DDC 616.9/041—dc23 LC re­cord avail­­able at https://​lccn.​loc.​gov/​2018008388 doi:10.1128/9781683670070

All Rights Reserved Printed in the United States of Amer­i­ca

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Send or­ders to ASM Press, P.O. Box 605, Hern­don, VA 20172, USA Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 E-mail: books@asmusa.​org Online: http://​www.​asmscience.​org To Drs. Carey-Ann Burnham, Mike Dunne, and Betz Forbes: invaluable mentors and dear friends.

Contents

Preface xiii About the Author xv

SECTION 1 Taxonomic Classification of Medically Important Microorganisms 1 Gee ­n ­ral Comments 2 Taxonomic Classification of Bacteria 2 Taxonomic Classification of Human Viruses 9 Taxonomic Classification of Fungi 13 Taxonomic Classification of Parasites 13

SECTION 2 Indigenous and Pathogenic Microbes of Humans 19 Ge­ne­ral Comments 20 Table 2.1 Human Indigenous Flora 22 Microbes Responsible for Human Disease 34 Summary of Notifiable Infectious Diseases: United States, 2015 54 Table 2.2 Arthropod Vectors of Medically Important Diseases 58 Table 2.3 Fungal Pathogens and Geographic Distribution 61 Table 2.4 Parasitic Pathogens and Geographic Distribution 71

SECTION 3 Specimen Collection and Transport 81 Ge­ne­ral Comments 82 Table 3.1 Bacteriology: Collection and Transport Guidelines 84 viii Contents Table 3.2 Specimen Collection and Transport Guidelines for Infrequently Encountered Bacteria 105 Table 3.3 Guidelines for Collection of Specimens for Anaerobic Culture 107 Virology: Ge­ne­ral Specimen Guidelines 107 Virology: Specific Specimen Guidelines 108 Table 3.4 Recommended Blood Volumes to Collect for Blood Cultures 111 Table 3.5 Mycology: Collection and Transport Guidelines 112 Table 3.6 Parasitology: Specimen Guidelines 117 Table 3.7 Guidelines for Processing Stool Specimens for Parasites 124

SECTION 4 127 Bacterial Diagnosis 127 Ge­ne­ral Comments 128 Table 4.1 Detection Methods for Bacteria 129 Table 4.2 Recommendations for Gram Stain and Plating Media 133 Table 4.3 Screening Specimens for Routine Bacterial Culture 137 Table 4.4 Processing Specimens for Mycobacterial Identification 138 Microscopy 139 Primary Plating Media: Bacteria 141 Primary Plating Media: Mycobacteria 154 Specific Diagnostic Tests 157 Aerobic Gram-Positive Cocci 157 Aerobic Gram-Positive Rods 158 Acid-Fast and Partially Acid-Fast Gram-Positive Rods 160 Aerobic Gram-Negative Cocci 161 Aerobic Gram-Negative Rods 161 Anaerobic Bacteria 168 Curved and Spiral-Shaped Bacteria 170 Mycoplasma spp. and Obligate Intracellular Bacteria 172 Identification Tables 175 Contents ix SECTION 5 Viral Diagnosis 219 Ge­ne­ral Comments 220 Table 5.1 Detection Methods for Viruses 221 Table 5.2 Cells Used for Viral Isolation 224 RNA Viruses 225 DNA Viruses 232 Transmissible Spongiform Encephalopathies 239

SECTION 6 Fungal Diagnosis 241 Mycology Specimen Collection and Transport Guidelines 242 Table 6.1 Methods for the Identification of Fungi 245 Microscopy 246 Table 6.2 Characteristic Fungal Elements Seen by Direct Examination of Clinical Specimens 248 Primary Plating Media 254 Table 6.3 Mycology Plating Guide 256 Specific DiagnosticTests 258 Aspergillus Species 258 Blastomyces dermatitidis 258 Candida Species 259 Coccidioides Species 259 Cryptococcus Species 260 Histoplasma capsulatum 260 Malassezia Species 261 Paracoccidioides brasiliensis 261 Talaromyces (Penicillium) marneffei 262 Pneu­mo­cys­tis jiroveci 262 Sporothrix schenckii 262 Zygomycetes 262 Identification Tables 264 x Contents SECTION 7 Parasitic Diagnosis 285 Ge­ne­ral Comments 286 Table 7.1 Detection Methods for Parasites 287 Microscopy 290 Specific Diagnostic Tests 291 Free-Living Amebae 291 Intestinal and Urogenital Protozoa 292 Blood and Tissue Protozoa 294 Microsporidia 297 Helminths: Nematodes 297 Helminths: Trematodes 299 Helminths: Cestodes 300 Identification Tables and Figures 302 Table 7.2 Trophozoites of Common Intestinal Amebae 302 Table 7.3 Cysts of Common Intestinal Amebae 305 Figure 7.1 Intestinal Amebae of Humans 308 Table 7.4 Trophozoites of Flagellates 309 Table 7.5 Cysts of Flagellates 311 Figure 7.2 Intestinal and Urogenital Flagellates of Humans 312 Table 7.6 Morphological Characteristics of Ciliates, Coccidia, Microsporidia, and Tissue Protozoa 313 Table 7.7 Morphological Characteristics of Protozoa Found in Blood 315 Table 7.8 Morphological Characteristics of Blood and Tissue Nematodes 317 Table 7.9 Morphological Characteristics of Helminths 318 Figure 7.3 Relative Sizes of Helminth Eggs 320

SECTION 8 Vaccines, Susceptibility Testing, and Methods of Organism Identification 323 Ge­ne­ral Comments 324 Table 8.1 Recommended pe­di­at­ric im­mu­ni­za­tion sched­ule 325 Contents xi Table 8.2 Recommended adult im­mu­ni­za­tion sched­ule 328 Table 8.3 Clinical and Laboratory Standards Institute (CLSI) doc­u­ments re­lated to an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing from hu­mans 330 Table 8.4 Summary of CLSI an­ti­mi­cro­bial sus­cep­ti­bil­ity test meth­ods for se­lect bac­te­ria, my­co­bac­te­ria, and fun­gi 332 Table 8.5 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­mi­cro­bial agents 337 Table 8.6 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­fun­gal agents 342 Table 8.7 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­par­a­sitic agents 343 Table 8.8 Antibacterial agents for specific­ bacte­ ria­ 345 Table 8.9 Intrinsic re­sis­tance of se­lected Gram-negative bacte­ ­ria 354 Table 8.10 Intrinsic resis­ tance­ of selected­ Gram pos­i­tive bac­te­ria 356 Table 8.11 Important mech­a­nisms of mul­ti­drug re­sis­tance in bac­te­ria. 358 Table 8.12 Organisms included­ in CLSI and EUCAST breakpoint tables­ 361 Table 8.13 Guide to in­ter­pre­tive cri­te­ria for se­lect or­gan­isms for com­monly tested an­ti­mi­cro­bi­als 364 Table 8.14 Summary of MALDI-TOF MS iden­ti­fi­ca­tion of bac­te­ria, my­co­bac­te­ria, and fun­gi 374 Table 8.15 Gene se­quenc­ing tar­gets for or­gan­ism iden­ti­fi­ca­tion 380

Index 383

Preface

The Pocket Guide to Clinical Microbiology is unique among ref­er­ ence texts in that its pri­mary pur­pose is to pres­ent us­able in­for­ma­ tion in a con­cise and ac­ces­si­ble man­ner. Now in its fourth edi­tion, this iconic text has re­mained pop­u­lar over the years be­cause it has suc­cess­fully con­densed most of the in­for­ma­tion needed by the prac­ tic­ing clin­i­cal mi­cro­bi­ol­o­gist into a pock­et-sized guide. As Dr. Pat­ rick Mur­ray ar­tic­u­lated in the pref­ace of the third edi­tion, with each it­er­a­tion of this text the for­mat has been honed to en­hance its us­abil­ity. Although the over­all for­mat of the fourth edi­tion re­ mains true to the tem­plate of pre­vi­ous edi­tions, this edi­tion re­flects the tre­men­dous evo­lu­tion that has oc­curred in clin­i­cal mi­cro­bi­ol­ogy over the past de­cade. In par­tic­u­lar, three key ad­vances have re­shaped the prac­tice of clin­i­cal mi­cro­bi­ol­ogy, and have there­fore, re­shaped this edi­tion. First, the wide­spread adop­tion of ma­trix-assisted la­ser de­sorp­tion/ ionization time-of-flight mass spec­trom­e­try (MALDI-TOF MS) for or­gan­ism iden­ti­fi­ca­tion has al­tered the way many lab­o­ra­to­ries prac­tice clin­i­cal mi­cro­bi­ol­ogy. Despite the fact that those us­ing MALDI-TOF MS are less de­pen­dent on bio­chem­i­cal re­ac­tions to iden­tify or­gan­isms, un­der­stand­ing bio­chem­i­cal pro­files is still re­ quired for the trou­ble­shoot­ing of failed or am­big­u­ous MALDI-TOF MS iden­ti­fi­ca­tions. As such, this text has up­dated, but main­tained, much of the bio­chem­i­cal re­ac­tions re­quired to iden­tify or­gan­isms the “old school” way. It is my be­lief that un­der­stand­ing and us­ing these re­ac­tions to con­firm or­gan­ism iden­ti­fi­ca­tions, re­mains one of the foun­da­tions of good clin­i­cal mi­cro­bi­ol­ogy. This pocket guide should pro­vide quick ref­er­ence to ev­ery­thing the mi­cro­bi­ol­o­gist needs to con­fi­dently iden­tify most or­gan­isms en­coun­tered in the clin­i­cal lab­o­ra­tory. In ad­di­tion, new in­for­ma­tion has been added in Section 8 that out­lines the spe­cif­ics of MALDI-TOF MS per­for­ mance for in­di­vid­ual bac­te­ria, my­co­bac­te­ria, and fun­gi. Second, the de­vel­op­ment of mo­lec­u­lar tech­niques has rev­o­lu­ tion­ized the di­ag­no­sis of in­fec­tious dis­eases. Most no­ta­bly, nu­cleic acid am­pli­fi­ca­tion test­ing (NAATs), also re­ferred to as po­ly­mer­ase chain re­ac­tion (PCR), has dras­ti­cally changed the prac­tice of clin­i­cal xiii xiv Preface vi ­rol­ogy to the ex­tent that many lab­o­ra­to­ries no lon­ger per­form vi­ral cul­ture. Information re­gard­ing vi­ral cul­ture has been re­tained in this guide for labs still per­form­ing those tech­niques, but sig­nif­ i­cant ad­di­tions have been made through­out­ this edi­tion to re­flect the use of mo­lec­u­lar di­ag­nos­tics. Also, be­cause gene se­quenc­ing is now a com­mon tech­nique used to iden­tify bac­te­ria and fungi, Sec­ tion 8 con­tains new in­for­ma­tion to help in­ter­pret these re­sults. Third, the con­tin­ued emer­gence of an­ti­mi­cro­bial re­sis­tance poses sig­nif­ cant chal­lenges to clin­i­cal mi­cro­bi­ol­o­gists as we strug­gle to pro­vide treat­ment op­tions for in­creas­ingly dif­fi­cult-to-treat or­gan­ isms. To re­flect this re­al­ity, sig­nif­i­cant changes have been made to this pocket guide to help the mi­cro­bi­ol­o­gist bet­ter per­form and in­ ter­pret an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing in the era of mul­ti­drug re­sis­tance. Acknowledging the in­ter­na­tional ap­peal of this text, I added new ta­bles out­lin­ing guid­ance pro­vided by both the Clini­ cal and Laboratory Standards Institute (CLSI) and the Eu­rop­ ean Committee on Antimicrobial Susceptibility Testing (EUCAST). The goal of these ta­bles is to help the reader eas­ily un­der­stand what meth­ods can be used, and what in­ter­pre­tive cri­te­ria ex­ist, for most or­gan­ism/antibiotic com­bi­na­tions. Also in­cluded are ta­bles with im­ por­tant in­trin­sic re­sis­tance pro­files for com­monly en­coun­tered or­ gan­isms, as well as key mech­a­nisms of re­sis­tance. Sir Isaac Newton once said, “If I have seen fur­ther than oth­ers, it is by stand­ing on the shoul­ders of gi­ants.” Although I have no il­lu­sions that I have “seen fur­ther” than any­one else, the hu­mil­ity ex­pressed in this quote res­o­nates with me as I have un­der­taken the task of up­dat­ing a text that was conc­ eived and au­thored by Dr. Pat­ rick Mur­ray, one of the true gi­ants of clin­i­cal mi­cro­bi­ol­ogy. So first and fore­most, I thank him for his work mak­ing this pocket guide the re­spected re­source that it is. I hope that the fourth edi­tion will do jus­tice to the tra­di­tion of this text. In ad­di­tion, I want to thank the tal­ented, and pa­tient, pro­fes­sion­als at ASM Press. Specifically, Chris­tine Charlip and Larry Klein, along with what I’m sure are count­less oth­ers at ASM, war­rant spe­cial thanks for all ­their work. And last, I thank my wife Kelli, who pro­vided sup­port and un­der­ stand­ing through the many late nights and week­ends it took to com­plete this pro­ject. I could not have fin­ished this pro­ject were it not for her sup­port. The prac­tice of clin­i­cal mi­cro­bi­ol­ogy is a won­der­ful dis­ci­pline, re­quir­ing judge­ment, in­ves­ti­ga­tion, and crit­i­cal de­ci­sion-making to pro­duce qual­ity re­sults. It is my hope that you will find this pocket guide to be a us­er-friendly ref­er­ence that en­hances your abil­ity to do all­ of these things, and ul­ti­mately pro­vide the best pa­tient care pos­si­ble. Chris­to­pher D. Doern About the Author

Christopher Doern, PhD, D(ABMM), is an Assistant Professor of Pathology and the Director of Clinical Microbiology at the Vi­ rginia Commonwealth University Health System, Richmond, ­Virginia. He earned his undergraduate and doctoral degrees from Wake Forest University in Winston-­Salem, North Carolina. Doern went on to a fellowship in Medical and Public Health Microbiology at the Washington University School of Medicine, St. Louis, Missouri, and is certified by the American Board of Medical Microbiology (ABMM).

Doern is an active member of the clinical microbiology community and serves on the ABMM, ASM Clinical Laboratory Practices Com­ mittee, Clinical Chemistry Trainee Council, and several Clinical and Laboratory Standards Institute document development and revision committees. He is an editor for the Clinical Microbiology Newsletter and serves on the Journal of Clinical Microbiology and Pediatric Infectious Diseases Journal editorial boards.

Doern is involved in educational programs that reach an interna­ tional audience. Among ­these is the Medical Microbiology Question of the Day (www​.­pathquestions​.­com), for which he has been an edi­ tor since 2011. This ser­vice provides freely accessible educational material to participants in more than 60 countries

xv

SECTION 1 Taxonomic Classification of Medically Important Microorganisms

Ge­ne­ral Comments 2 Taxonomic Classification of Bacteria 2 Taxonomic Classification of Human Viruses 9 Taxonomic Classification of Fungi 13 Taxonomic Classification of Parasites 13

doi:10.1128/9781683670070.ch1 2 SECTION 1 In or­der to re­main true to the tra­di­tion set forth by the first three edi­tions of this pocket guide, the first sec­tion will be de­voted to de­ scrib­ing the tax­on­omy of com­mon (and some un­com­mon) or­gan­ isms which are as­so­ci­ated with hu­mans and may be iso­lated by the Clinical Microbiology Laboratory. Unfortunately, in the 13 or so years that have passed since the third edi­tion of the Pocket Guide was pub­lished, the rate of tax­o­nomic changes has con­tin­ued to in­ crease such that pub­lish­ing a tax­o­nomic list of or­gan­isms would be out­ of date be­fore this book goes to pub­li­ca­tion. This is a prod­uct of con­tin­ued pro­lif­er­a­tion of new spe­cies of or­gan­isms which are be­ing iden­ti­fied by in­creas­ingly so­phis­ti­cated ge­no­mic an­a­ly­ses. As such, the re­vi­sed goal of this sec­tion will be to out­­line some high- level tax­o­nomic group­ings and pro­vide the re­sources and ref­er­

Taxonomic Classification Taxonomic ences one would need to iden­tify the most up-to-date tax­o­nomic clas­si­fi­ca­tions. It should be ap­pre­ci­ated that de­spite ap­pear­ances, changes in no­ men­cla­ture are reg­u­lated by a sys­tem of rules with over­sight gov­ erned by the International Code of Biological Nomenclature (www​ .​biosis.​org.​uk/​zrdocs/​codes/​codes.​htm). The International Code of Nomenclature of Bacteria gov­erns bac­te­rial tax­on­omy, and all ­bac­ te­ria named af­ter 1980 must be val­idly pub­lished in the Interna- tional Journal of Systematic and Evolutionary Microbiology. A cur­rent list­ing of bac­te­ria can be found at http://​www.​bacterio.​net, http://​www.​bacterio.​cict.​fr/​, and https://​www.​dsmz.​de/​.​ The Inter­ national Committee on Taxonomy of Viruses (ICTV) governs viral taxonomy, and all currently recognized viruses can be found at https://​talk.​ictvonline.​org//​.​ The International Code of Botanical Nomenclature gov­erns fun­gal clas­si­fi­ca­tion, and ad­di­tional in­for­ma­ tion can be found at http://​www.​iapt-​taxon.​org/​nomen/​main.​php/. Taxonomic Classification of Bacteria Classification and tax­on­omy of pro­kary­otes (bac­te­ria) is com­pli­ cated and is gov­erned by the International Code of Nomenclature of Bacteria (last re­vi­sed in 1990). By def­i­ni­tion, each pro­kary­otic spe­cies must in­clude a ge­nus-level name that is in­cluded within a hi­er­ar­chy or ranks, which in­cludes (from high­est to low­est rank) sub­tribe, tribe, sub­fam­ily, fam­ily, sub­or­der, or­der, sub­class, class, di­vi­sion (or phy­lum), and do­main (or em­pire). To fur­ther com­pli­cate mat­ters, the tribe and sub­tribe do not ac­tu­ally in­clude names and are there­fore not used. Most im­por­tantly, there is no such thing as an of­fi­cial clas­si­fi­ ca­tion of pro­kary­otes. This is be­cause in con­trast to eu­kary­otes, the pro­kary­otic des­ig­na­tions are a mat­ter of sci­en­tific judg­ment. There­ fore, the clos­est things that we have to “of­fi­cial” tax­o­nomic des­ig­ na­tions are those names that are gen­er­ally ac­cepted by the mi­cro­bi­ol­ogy com­mu­nity. Despite this fact, mi­cro­bi­ol­o­gists have Taxonomic Classification of Important Microorganisms 3 achieved some amount of con­sen­sus by re­ly­ing on re­sources such as the International Journal of Systematic and Evolutionary Micro­ biology and Bergey’s Manual of Systematic Bacteriology. As of the writ­ing of this fourth edi­tion Pocket Guide, the pro­ kary­otes were di­vided into 2 do­mains, 35 phyla, 80 clas­ses, 1 sub­ class, 178 or­ders, 20 sub­or­ders, 402 fam­i­lies, and 2,552 gen­era. The fol­low­ing is a con­sol­i­dated tax­o­nomic out­­line, which will fo­cus on the tax­o­nomic or­ga­ni­za­tion or those or­gan­isms that are most likely to be en­coun­tered in the clin­i­cal mi­cro­bi­ol­ogy lab­o­ra­tory. This is not meant to be an ex­haus­tive list of all­ bac­te­ria. Rather, it is in­tended to pro­vide some con­text to the re­la­tion­ships be­tween some of the most com­monly en­coun­tered or­gan­isms in hu­man clin­i­cal spec­i­mens. The tax­on­omy of bac­te­rial clas­si­fi­ca­tion is ar­ranged in the fol­ low­ing way. . . Classification Taxonomic

Domain Phyla Class Subclass Order Suborder Family Genera

Domain: Bacteria Class. Actinobacteria Family. Actinomycetaceae Genus. Actinobaculum Genus. Actinomyces Genus. Arcanobacterium Genus. Mobiluncus Genus. Trueperella Family. Corynebacteriaceae Genus. Corynebacterium Genus. Turicella Family. Dietziaceae Genus. Dietzia Family. Mycobacteriaceae Genus. Mycobacterium Family. Nocardiaceae Genus. Gordonia Genus. Nocardia Genus. Rhodococcus Family. Tsukamurellaceae Genus. Tsukamurella Family. Propionibacteriaceae Genus. Propionibacterium 4 SECTION 1 Family. Streptocmycetaceae Genus. Streptomyces Family. Nocardiopsaceae Genus. Nocardiopsis Family. Bifidobacteriaceae Genus. Alloscardovia Genus. Bifidobacterium Genus. Gardnerella Family. Brevibacteriaceae Genus. Brevibacterium Family. Cellulomonadaceae Genus. Cellulomonas Genus. Oerskovia

Taxonomic Classification Taxonomic Genus. Tropheryma Family. Dermabacteraceae Genus. Dermabacter Genus. Helcobacillus Family. Dermacoccaceae Genus. Dermacocccus Genus. Kytococcus Family. Microbacteriaceae Genus. Leifsonia Genus. Microbacterium Family. Micrococcaceae Genus. Arthrobacter Genus. Kocuria Genus. Micrococcus Genus. Rothia Genus. Stomatococcus Class. Coriobacteriia Family. Atopobiaceae Genus. Atopobium Family. Eggerthellaceae Genus. Eggerthella Genus. Slackia Class. Bacteroidia Family. Bacteroidaceae Genus. Bacteroides Family. Porphyromonadaceae Genus. Dysgonomonas Genus. Microbacter Genus. Parabacteroides Genus. Porphyromonas Genus. Tannerella Family. Prevotellaceae Genus. Prevotella Taxonomic Classification of Important Microorganisms 5 Class. Flavobacteriia Family. Flavobacteriaceae Genus. Bergeyella Genus. Capnocytophaga Genus. Chryseobacterium Genus. Elizabethkingia Genus. Empedobacter Genus. Flavobacterium Genus. Weeksella Class. Sphingobacteriia Family. Sphingobacteriaceae Genus. Sphingobacterium Class. Chlamydiae

Family. Chlamydiaceae Classification Taxonomic Genus. Chlamydia Genus. Chlamydophila Class. Bacilli or Fibribacteria Family. Bacillaceae Genus. Bacillus Family. Listeriaceae Genus. Listeria Family. Paenibacillaceae Genus. Paenibacillus Family. Staphylococcaceae Genus. Staphylococcus Family. Unassigned Genus. Gemella Family. Aerococcaceae Genus. Abiotrophia Genus. Aerococcus Genus. Dolosicoccus Genus. Facklamia Genus. Globicatella Family. Carnobactericeae Genus. Alloiococcus Genus. Dolosigranulum Genus. Granulicatella Family. Enterococcaceae Genus. Enterococcus Genus. Vagococcus Family. Lactobacillaceae Genus. Lactobacillus Genus. Pediococcus Family. Leuconostocaceae Genus. Leuconostoc Genus. Weissella 6 SECTION 1 Family. Streptococcaceae Genus. Lactococcus Genus. Streptococcus Class. Clostridia Family. Clostridiaceae Genus. Clostridium Genus. Sarcinia Family. Peptococcaceae Genus. Peptococcus Family. Peptostreptococcaceae Genus. Peptostreptococcus Family. Unassigned Genus. Anaerococcus

Taxonomic Classification Taxonomic Genus. Finegoldia Genus. Helcococcus Genus. Peptoniphilus Class. Erysipelotrichia Family. Erysipelotrichaceae Genus. Erysipelothrix Class. Negativicutes Family. Veillonellaceae Genus. Veillonella Class. Fusobacteriia Family. Fusobacteriaceae Genus. Fusobacterium Family. Leptotrichiaceae Genus. Leptotrichia Genus. Sneathia Genus. Streptobacillus Class. Alphaproteobcteria Family. Caulobacteraceae Genus. Brevundimonas Family. Bartonellaceae Genus. Bartonella Family. Brucellaceae Genus. Brucella Genus. Ochrabactrum Family. Rhizobiaceae Genus. Agrobacterium Genus. Rhizobium Family. Rhodobacteraceae Genus. Paracoccus Family. Acetobacteraceae Genus. Roseomonas Family. Rhodospirllaceae Genus. Inquilinus Family. Anaplasmataceae Taxonomic Classification of Important Microorganisms 7 Genus. Anaplasma Genus. Ehrlichia Genus. Wolbachia Family. Rickettsiaceae Genus. Orientia Genus. Rickettsia Family. Sphingomonadaceae Genus. Sphingomonas Class. Betaproteobacteria Family. Alcaligenaceae Genus. Achoromobacter Genus. Alcaligenes Genus. Bordetella

Genus. Oligella Classification Taxonomic Family. Burkholderiaceae Genus. Burkholderia Genus. Cupriavidis Genus. Pandoraea Genus. Ralstonia Family. Comamonadaceae Genus. Acidovorax Genus. Comamonas Genus. Defltia Family. Oxalobacteraceae Genus. Herbaspirillum Family. Neisseriaceae Genus. Eikenella Genus. Kingella Genus. Neisseria Family. Sprillaceae Genus. Genera Genus. Sprillium Class. Epsilonproteobacteria Family. Campylobacteraceae Genus. Arcobacter Genus. Campylobacter Family. Helicobacteraceae Genus. Helicobacter Class. Gammaproteobacteria Family. Enterobacteriaceae Genus. Cedecea Genus. Citrobacter Genus. Cronobacter Genus. Edwardsiella Genus. Enterobacter Genus. Erwinia Genus. Escherichia 8 SECTION 1 Genus. Hafnia Genus. Klebsiella Genus. Kluybera Genus. Leclercia Genus. Morganella Genus. Pantoea Genus. Plesiomonas Genus. Pro­teus Genus. Providencia Genus. Raoltella Genus. Salmonella Genus. Serratia Genus. Shigella

Taxonomic Classification Taxonomic Genus. Yersinia Genus. Yokenella Family. Vibrionaceae Genus. Vibrio Family. Aeromonadaceae Genus. Aeromonas Family. Shewanellaceae Genus. Shewanella Family. Cardiobacteriaceae Genus. Cardiobacterium Genus. Suttonella Family. Coxiellaceae Genus. Coxiella Family. Legionellaceae Genus. Legionella Family. Pasteurellaceae Genus. Actinobacillus Genus. Aggregatibacter Genus. Haemophilus Genus. Pasteurella Family. Moraxelaceae Genus. Acinetobacter Genus. Branhamella Genus. Moraxella Family. Pseudomonadaceae Genus. Chryseomonas Genus. Flavimonas Genus. Pseudomonas Family. Francisellaceae Genus. Francisella Class. Spirochaetes Family. Brachyspiraceae Genus. Brachyspira Taxonomic Classification of Important Microorganisms 9 Family. Leptospiraceae Genus. Leptospira Family. Borreliaceae Genus. Borrelia Family. Spirochaetaceae Genus. Treponema Class. Mollicutes Family. Mycoplasmataceae Genus. Mycoplasma Genus. Ureaplasma Taxonomic Classification of Human Viruses

The tax­on­omy of vi­ral clas­si­fi­ca­tion is ar­ranged in the fol­low­ing Classification Taxonomic way. . . Order Family Subfamily Genus Species Practically speak­ing, most clin­i­cal mi­cro­bi­ol­o­gists or­ga­nize vi­ ruses in terms of ge­nome struc­ture, fam­ily, and ge­nus, and only rarely are the sub­fam­ily or spe­cies des­ig­na­tions uti­lized. As such, the fol­low­ing tax­o­nomic struc­ture is pre­sented in terms of what would be most use­ful to the prac­tic­ing clin­i­cal mi­cro­bi­ol­o­gist.

Single-stranded, nonenveloped DNA vi­rus­es Family. Parvoviridae Genus. Erythrovirus Species. Human par­vo­vi­rus B19 vi­rus

Double-stranded, nonenveloped DNA vi­rus­es Family. Polyomaviridae Genus. Polyomavirus Species. JC poly­oma­vi­rus, BK poly­oma­vi­rus Family. Papillomaviridae Genus. Papillomavirus Species. Human pap­il­lo­ma­vi­rus Family. Adenoviridae Genus. Mastadenovirus Species. Human ad­e­no­vi­ruses (spe­cies A to G)

Double-stranded, en­vel­oped DNA vi­rus­es Family. Poxviridae Genus. Orthopoxvirus 10 SECTION 1 Species. Vaccinia vi­rus, va­ri­ola vi­rus small­pox vi­rus, cow­pox vi­rus, mon­key­pox vi­rus Genus. Molluscipoxvirus Species. Molluscum contagiosum vi­rus Genus. Parapoxvirus Species. Orf vi­rus Family. Hepadnaviridae Genus. Orthohepadnavirus Species. Hepatitis B vi­rus Family. Herpesviridae Genus. Simplexvirus Species. Human her­pes­vi­rus 1 (her­pes sim­plex vi­rus type 1; HHV-1), hu­man her­pes­vi­rus 2 (her­pes

Taxonomic Classification Taxonomic sim­plex vi­rus type 2; HHV-2) Genus. Varicellovirus Species. Human her­pes­vi­rus 3 (var­i­cel­la-zoster vi­rus [VZV]; HHV-3) Genus. Lymphocryptovirus Species. Human her­pes­vi­rus 4 (Ep­stein-Barr vi­rus [EBV]; HHV-4) Genus. Cytomegalovirus Species. Human her­pes­vi­rus 5 (CMV; HHV-5) Genus. Roseolovirus Species. Human her­pes­vi­rus 6 (ro­se­ola vi­rus; HHV-6), hu­man her­pes­vi­rus 7 (HHV-7) Genus. Rhadinovirus Species. Human her­pes­vi­rus 8 (HHV-8)

Single-stranded, pos­i­tive-sense, nonenveloped RNA vi­rus­es Family. Picornaviridae Genus. Enterovirus Species. Enterovirus A (hu­man coxsackievirus A2, hu­man en­tero­vi­rus 71) Enterovirus B (hu­man coxsackievirus B1, hu­man echo­vi­rus), Enterovi- rus C (hu­man po­lio­vi­rus 1 to 3, hu­man coxsacki­ evirus A1), Enterovirus D (hu­man en­tero­vi­rus 68, 70, and 94), Rhinovirus A, B, and C. Genus. Aphthovirus Species. Foot-and-mouth dis­ease vi­rus Genus. Hepatovirus Species. Human hep­a­ti­tis A vi­rus (HHAV) Family. Caliciviridae Genus. Norovirus Species. Nor­walk vi­rus Genus. Sapovirus Species. Sap­poro vi­rus Family. Astroviridae Taxonomic Classification of Important Microorganisms 11 Genus. Astrovirus Species. Human astrovirus

Single-stranded, pos­i­tive-sense, en­vel­oped RNA vi­rus­es Family. Coronaviridae Genus. Coronavirus Species. Human co­ro­na­vi­rus, Severe acute re­spi­ra­tory syn­drome (SARS) vi­rus, Middle east­ern re­spi­ra­tory syn­drome (MERS) vi­rus Genus. Torovirus Species. Human torovirus Family. Togaviridae Genus. Alphavirus

Species. Sindbis vi­rus, Eastern equine en­ceph­a­li­tis Classification Taxonomic (EEE) vi­rus, Western equine en­ceph­a­li­tis (WEE) vi­rus, Ven­e­zu­e­lan equine en­ceph­a­li­tis (VEE) vi­rus, Chickungunya vi­rus, many other vi­rus­es Genus. Rubivirus Species. Rubella vi­rus Family. Flaviviridae Genus. Flavivirus Species. Yellow fe­ver vi­rus, West Nile vi­rus, St. Louis en­ceph­a­li­tis (SLE) vi­rus, Jap­a­nese en­ceph­a­li­tis (JE) vi­rus, Dengue vi­rus (types 1 through 4), Zika vi­rus, many other vi­rus­es Genus. Hepacivirus Species. Hepatitis C vi­rus (HCV)

Single-stranded, neg­a­tive-sense, en­vel­oped RNA vi­rus­es Family. Rhabdoviridae Genus. Lyssavirus Species. Rabies vi­rus Family. Filoviridae Genus. “Mar­burg-like vi­rus­es” Species. Mar­burg vi­rus Genus. “Ebo­la-like vi­rus­es” Species. Ebo­la vi­rus Family. Orthomyxoviridae Genus. Influenzavirus A Species. Influenza A vi­rus Genus. Influenzavirus B Species. Influenza B vi­rus Genus. Influenzavirus C Species. Influenza C vi­rus Family. Paramyxoviridae Genus. Respirovirus 12 SECTION 1 Species. Sen­dai vi­rus, Human para­in­flu­enza vi­rus (types 1 and 3) Genus. Rubulavirus Species. Mumps vi­rus, Human para­in­flu­enza vi­rus (types 2 and 4) Genus. Morbillivirus Species. Measles vi­rus Genus. Henipavirus Species. Hendra vi­rus, Nipah vi­rus Genus. Pneumovirus Species. Human re­spi­ra­tory syn­cy­tial vi­rus (RSV) Genus. Metapneumovirus Species. hu­man metapneumovirus

Taxonomic Classification Taxonomic Family. Bunyaviridae Genus. Orthobunyavirus Species. Bunyamwera vi­rus, Cal­i­for­nia en­ceph­a­li­tis vi­rus, La Crosse vi­rus, many other vi­rus­es Genus. Hantavirus Species. Hantaan vi­rus, Sin Nombre vi­rus, other vi­rus­es Genus. Nairovirus Species. Cri­me­an-Congo hem­or­rhagic fe­ver vi­rus (CCFV), other vi­rus­es Genus. Phlebovirus Species. Rift Valley fe­ver vi­rus, other vi­rus­es Family. Arenaviridae Genus. Arenavirus Species. Lymphocytic cho­rio­men­in­gi­tis (LCM) vi­rus, Lassa vi­rus, Junin vi­rus, Machupo vi­rus, Sabia vi­rus, other vi­rus­es

Double-stranded, en­vel­oped RNA vi­rus­es Family. Retroviridae Genus. Deltaretrovirus Species. Human T-lymphotropic vi­rus type 1 (HTLV-1), hu­man T-lymphotropic vi­rus type 2 (HTLV-2) Genus. Lentivirus Species. Human im­mu­no­de­fi­ciency vi­rus type 1 (HIV-1), hu­man im­mu­no­de­fi­ciency vi­rus type 2 (HIV-2) Family. Reoviridae Genus. Rotavirus Species. Rotavirus (types A, B, and C) Genus. Coltivirus Species. Col­o­rado tick fe­ver vi­rus Taxonomic Classification of Important Microorganisms 13 Taxonomic Classification of Fungi The tax­o­nomic clas­si­fi­ca­tion of fun­gal or­gan­isms is com­plex be­cause fungi can be clas­si­fied by dif­fer­ent meth­ods. The phylogentic tax­ on­omy for fungi is rep­re­sented in this chap­ter and is sub­ject to the International Code of Nomenclature (ICN) for al­gae, fungi, and plants (http://​www.​iapt-​taxon.​org). This or­ga­ni­za­tion was for­mally known as the International Code of Botanical Nomenclature (ICBN). Fungi are di­vided into four di­vi­sions (phy­lum or sub­phy­lum): Mucormycotina, Entomophthoromycotina, Ascomycota, and Basid­ iomycota. The Protozoa and Chromista king­doms in­clude some mem­bers that pos­sess a fun­gus-like ap­pear­ance and are clin­i­cally rel­e­vant, such as Rhinosporidium and Pythium.

The tax­on­omy of fun­gal clas­si­fi­ca­tion is ar­ranged in the fol­low­ Classification Taxonomic ing way. . .

Phylum Subphylum Class Order Family Genus Species

Since the last writ­ing of this Pocket Guide, a sig­nif­i­cant change has oc­curred in the world of fun­gal tax­on­omy. As of Jan­u­ary 1, 2013, only one name will be used to iden­tify fungi, and the “cor­ rect” name will be that which was first iden­ti­fied. Why has this oc­ curred? Most sci­en­tists agree that the con­ven­tion of hav­ing mul­ti­ple names for fungi to rep­re­sent the dif­fer­ing states of a fun­ gus is no lon­ger nec­es­sary with the use of DNA se­quence an­a­ly­ses. The mul­ti-name con­ven­tion is con­fus­ing, and is es­pe­cially so for the clin­i­cal mi­cro­bi­ol­o­gist who is try­ing to com­mu­ni­cate un­der­ stand­able and ac­tion­able in­for­ma­tion to those car­ing for pa­tients. Because fun­gal tax­o­nomic nam­ing con­ven­tions are chang­ing more quickly than for bac­te­ria, par­a­sites, and vi­ruses, pub­lish­ing an ex­ten­sive list of fun­gal tax­on­omy would be ren­dered in­ac­cu­rate as soon as this Pocket Guide was pub­lished. In fact, many of the names that would be in­cluded as of the writ­ing of this book will likely cease to ex­ist. Taxonomic Classification of Parasitesa The term “par­a­site” re­fers to a group of eu­kary­otic or­gan­isms, about 200 of which are med­i­cally rel­e­vant hel­minths, and 80 of which are med­i­cally rel­e­vant pro­to­zoan spe­cies. Within this sub­set of nearly 14 SECTION 1 300 par­a­sites, about 100 spe­cies are com­monly found in hu­mans, and an even smaller num­ber within that cause a dis­pro­por­tion­ate num­ber of im­por­tant dis­eases. Presented in the fol­low­ing ta­ble are the tax­o­nomic clas­si­fi­ca­tions of some of the most im­por­tant hu­man par­a­sites. Since the last edi­tion of this Pocket Guide was pub­lished, some sig­nif­ica­ nt tax­o­nomic changes have oc­curred. The two most clin­i­ cally rel­e­vant changes re­late to the Microsporidia and Blastocystis hominis. Due to re­cent ge­nome-wide an­a­ly­ses, Microsporidia now be­long to the king­dom Fungi. The tax­on­omy of Blastocystis hom- inis has been con­tro­ver­sial, and it has pre­vi­ously been con­sid­ered a fun­gus and also as pro­to­zoa. Recent ge­nome an­a­ly­ses sug­gest it is most closely re­lated to Proteromonas, though this or­gan­ism is

Taxonomic Classification Taxonomic a flag­el­late and B. hominis does pos­sess a fla­gel­lum and is not mo­tile. It is now part of the king­dom Chromista rather than the Protozoa.

Kingdom. Protozoa Phylum. Metamonada (flag­el­lates) Class. Trepomonadea (in­tes­ti­nal flag­el­lates) Order. Diplomonadida Genus. Giardia duodenalis Class. Retortamonadea (in­tes­ti­nal flag­el­lates) Order. Retortamonadida Genus. Chilomastix mesnili, Genus. Retortamonas intestinalis Class. Trichomonadea (in­tes­ti­nal and re­lated flag­el­lates) Order. Trichomonadida Genus. Dientamoeba fragilis Genus. Trichomonas va­gi­na/is Genus. Trichomonas tenax Genus. Pentatrichomonas hominis Phylum. Percolozoa Class. Heterolobosea (flag­el­lated amoe­bae) Order. Schizopyrenida Genus. Naegleria fowleri Phylum. Euglenozoa Class. Kinetoplastea (blood and tis­sue flag­el­lates) Order. Trypanosomatida Genus. Leishmania Don­o­van Genus. Leishmania in­fan­tum (= L. chagasi) Genus. Leishmania ma­jor Genus. Leishmania tropica Genus. Leishmania braziliensis Genus. Leishmania mexicana Genus. Trypanosoma cruzi Genus. Trypanosoma brucei gambiense Taxonomic Classification of Important Microorganisms 15 Genus. Trypanosoma brucei rhodesiense Genus. Trypanosoma rangeli Phylum. Amoebozoa Class. Amoebaea Order. Acanthopodida Genus. Acanthamoeba spp. Genus. Balamuthia mandrillaris Class. Archamoebea (in­tes­ti­nal amoe­bae) Order. Euamoebida Genus. Entamoeba histolytica Genus. Entamoeba coli Genus. Entamoeba dispar Genus. Entamoeba hartmanni

Genus. Entamoeba gingivalis Classification Taxonomic Genus. Entamoeba polecki Genus. Endolimax nana Genus. Iodamoeba buetschlii Phylum. Sporozoa (spo­ro­zo­ans) Class. Coccidea Order. Eimeriida Genus. Cryptosporidium parvum Genus. Toxoplasma gondii Genus. Cyclospora cayetanensis Genus. Cystoisospora (Isospora) bel­li Genus. Sarcocystis hominis Order. Piroplasmida Genus. Babesia microti Genus. Babesia divergens Genus. Babesia gibsoni Order. Haemosporida Genus. Plasmodium fal­cip­a­rum Genus. Plasmodium malariae Genus. Plasmodium ova­le Genus. Plasmodium vi­vax Genus. Plasmodium knowlesi Phylum. Ciliophora (cil­i­ates) Class. Litostomatea Order. Trichostomatia Genus. Balantidium coli

Kingdom. Chromista Phylum. Bigyra Class. Blastocystea Genus. Blastocystis hominis

Kingdom. Animalia Phylum. Nemathelminthes (Nematodes, Roundworms) 16 SECTION 1 Class. Adenophorea (Asphasmidea) Family. Trichinellidae (Trichuridae) Genus. Trichinella spiralis Genus. Trichuris trichiura Genus. Capillaria spp. Class. Secernentea (Phasmidea) Family. Ancylostomatidae Genus. Ancylostoma duodenale Genus. Necator americanus Family. Angiostrongylidae Genus. Parastrongylus (Angiostrongylus) cantonensis Genus. Parastrongylus (Angiostrongylus)

Taxonomic Classification Taxonomic costaricensis Family. Ascarididae Genus. Ascaris lumbrioides Genus. Toxocara canis Genus. Toxocara cati Genus. Bayliascaris procyonis Family. Dracunculidae Genus. Dracunculus medinensis Family. Onchocercidae Genus. Brugia malayi Genus. Loa loa Genus. Wuchereria bancrofii Genus. Onchocerca vol­vu­lus Genus. Brugia timori Genus. Dirofilaria immitis Genus. Mansonella ozzardi Genus. Mansonella perstans Family. Oxyuridae Genus. Enterobius vermicularis Family. Strongyloididae Genus. Strongyloides stercoralis Genus. Strongyloides fuelleborni Family. Gnathostomatidae Genus. Gnathostoma spinigerum

Phylum. Platyhelminthes Class. Trematoda (flukes) Order. Diplostomida Family. Schistosomatidae Genus. Schistosoma haematobium Genus. Schistosoma japonicum Genus. Schistosoma mansoni Genus. Schistosoma mekongi Genus. Schistosoma intercalatum Taxonomic Classification of Important Microorganisms 17 Order. Plagiorchiida Family. Fasciolidae Genus. Fasciola he­pat­i­ca Genus. Fasciola gigantica Genus. Fasciolopsis buski Family. Heterophyidae Genus. Heterophyes heterophyes Family. Opisthorchidae Genus. Clonorchis sinensis Genus. Opisthorchis felineus Genus. Opisthorchis viverrini Family. Paragonimidae Genus. Paragonimus westermani

Genus. Paragonimus kellicotti Classification Taxonomic Family. Paragonimidae Genus. Dicrocoelium dentriticum Class. Cestoda (tape­worms) Order. Pseudophyllidea Family. Diphyllobothriidae Genus. Diphyllobothrium latum Order. Cyclophyllidea Family. Dipylidiidae Genus. Dipylidium caninum Family. Hymenolepididae Genus. Hymenolepis nana Genus. Hymenolepis diminuta Family. Taeniidae Genus. Taenia saginata Genus. Taenia solium Genus. Echinococcus granulosus Genus. Echinococcus multilocularis

aAdapted from J. H. Jorgensen, M. A. Pfaller, K. C. Car­roll, G. Funke, M. L. Landry, S. S. Rich­ter, D. W. Warnock (ed.), Manual of Clinical Microbiology, 11th ed., ASM Press, Wash­ing­ton, D.C., 2015.

SECTION 2 Indigenous and Pathogenic Microbes of Humans

Ge­ne­ral Comments 20 Table 2.1 Human Indigenous Flora 22 Microbes Responsible for Human Disease 34 Summary of Notifiable Infectious Diseases: United States, 2015 54 Table 2.2 Arthropod Vectors of Medically Important Diseases 58 Table 2.3 Fungal Pathogens and Geographic Distribution 61 Table 2.4 Parasitic Pathogens and Geographic Distribution 71

doi:10.1128/9781683670070.ch2 20 SECTION 2 Thea re­l ­tion­ship be­tween hu­mans and mi­crobes can be de­fined in one of three ways: 1) tran­sient col­o­ni­za­tion, 2) per­sis­tent col­o­ni­za­ tion, 3) or path­o­genic in­fec­tion. The ma­jor­ity of or­gan­isms are un­ able to es­tab­lish per­ma­nent col­o­ni­za­tion/infection on the skin or mu­co­sal sur­faces and are con­sid­ered an in­sig­nif­ cant find­ing when re­cov­ered in clin­i­cal spec­i­mens. Examples in­clude the molds and many of the nonfermentative Gram-negative ba­cilli that can be iso­ lated in soil, veg­e­ta­tion, wa­ter, and food prod­ucts. These or­gan­ isms are un­able to com­pete with the nor­mal mi­cro­bial pop­u­la­tion of the body or can­not sur­vive on the skin sur­face. Other or­gan­isms are a­ ble to es­tab­lish long-term res­i­dency on or in the hu­man body. The suc­cesses of these in­ter­ac­tions are influ­ enced by com­plex mi­cro­bial and host fac­tors (e.g., fa­vor­able en­vi­ ron­ment [pH, at­mo­sphere, mois­ture, avail­­able nu­tri­ents], abil­ity to ad­here to sur­faces, re­sis­tance to bac­te­rio­cins, an­ti­bi­ot­ics, and phago­cytic cells). These mi­crobes gen­er­ally ex­ist in a sym­bi­otic re­ la­tion­ship with their hu­man host and pro­duce dis­ease only when Microbes of Humans Microbes they in­vade nor­mally ster­ile body sites such as tis­sues and body flu­ ids. Table 2.1 is a list­ing of the or­gan­isms most com­monly re­cov­ ered from the body sur­faces of healthy in­di­vid­u­als. This ta­ble is in­tended to serve as an in­ter­pre­tive guide­line for cul­tured spec­i­ mens. It should be re­mem­bered that many or­gan­isms can­not be de­tected when pres­ent in a mixed pop­u­la­tion (typ­i­cal of many body sites). With the emer­gence of next gen­er­a­tion se­quenc­ing microbi­ ome ex­per­i­men­ta­tion, our un­der­stand­ing of the hu­man microbiome has ex­panded sig­nif­i­cantly and dem­on­strated a greater di­ver­sity of com­men­sal or­gan­isms than had been pre­vi­ously ap­pre­ci­at­ed. Additionally, re­cent ad­vances in di­ag­nos­tic tech­nol­ogy have changed the prac­tice of clin­i­cal mi­cro­bi­ol­ogy for­ever. In par­tic­u­lar, the use of ma­trix-assisted la­ser de­sorp­tion/ionization time-of-flight mass spec­trom­e­try (MALDI-TOF MS) has al­lowed mi­cro­bi­ol­o­gists to iden­tify or­gan­isms rap­idly and with more ac­cu­racy than ever be­ fore. Consequently, the mi­cro­bi­ol­o­gist must un­der­stand a greater depth of or­gan­isms than ever be­fore, and it re­mains crit­i­cal that only those or­gan­isms which are clin­i­cally sig­nif­ cant be re­port­ed. MALDI-TOF MS is a pow­er­ful tool that has greatly im­proved our abil­ity to di­ag­nose in­fec­tious dis­eases. However, if used in­cor­rectly, MALDI-TOF MS can lead to mis­di­ag­noses, pro­vider con­fu­sion, and un­nec­es­sary an­ti­mi­cro­bial ther­a­py. The quan­ti­ta­tive and qual­i­ta­tive pres­ence of spe­cific mi­crobes will also vary with the in­di­vid­ual host, inc­ lud­ing dra­matic changes in the in­dig­e­nous flora in hos­pi­tal­ized pa­tients. Thus, only qual­i­ta­ tive data (pres­ence or ab­sence of the or­gan­isms) are pre­sented. Data for vi­ruses are not listed be­cause rep­li­ca­tion of vi­ruses gen­er­ally is as­so­ci­ated with host tis­sue de­struc­tion or an im­mu­no­logic re­sponse (al­though this can range from a clin­i­cally asymp­tom­atic in­fec­tion to host death). Indigenous and Pathogenic Microbes of Humans 21 Most dis­eases in hu­mans are caused by in­fec­tions with en­dog­e­ nous bac­te­ria and yeasts or ex­po­sure to op­por­tu­nis­tic molds, par­a­ sites, and vi­ruses. However, some in­ter­ac­tions be­tween mi­crobes and hu­mans com­monly lead to dis­ease. The most com­mon mi­crobes re­spon­si­ble for hu­man dis­ease are sum­ma­rized in this sec­tion. Selected path­o­gens are mon­i­tored rou­tinely, with all­ clin­i­cal lab­ o­ra­to­ries re­quired to re­port spe­cific or­gan­isms or dis­eases to their state pub­lic health de­part­ment. This group of or­gan­isms and the dis­ eases as­so­ci­ated with them are re­ported weekly in Morbidity and Mortality Weekly Report. Data for 2015 are sum­ma­rized in this sec­ tion. In ad­di­tion, the Department of Health and Human Services (HHS) and the U.S. Department of Agriculture (USDA) have pub­ lished a list of se­lect agents and tox­ins. This list is pre­sented in this sec­tion and can be found at this web­site (https://​www.​selectagents​ .​gov/​selectagentsandtoxinslist.​html). Arthropods, par­a­sites in their own right, can also serve as vec­ tors for hu­man dis­ease. A list­ing of the most com­mon ar­thro­pod vec­tors and their as­so­ci­ated dis­eases is in­cluded in Table 2.2. of Humans Microbes Tables 2.3 and 2.4 are list­ings of fungi and par­a­sites iso­lated from hu­mans and their geo­graphic dis­tri­bu­tion. For ad­di­tional in­for­ma­ tion about in­dig­e­nous and path­o­genic mi­crobes, please con­sult the ref­er­ence texts listed in the Bibliography. 22 SECTION 2 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + GI tract 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + Resp tract

a a ­ r ous flo ­ n ­ e ig

­ d .

spp . ­ tis uman in H spp . spp. spp.

spp. spp Organism defectiva Abiotrophia laidlawii Acholeplasma Actinomyces actinomycetemcomitans Aggregatibacter christensenii Aerococcus Aerococcus viridans Aerococcus urinae Alloiococcus oti Anaerococcus hydrogenalis Anaerococcus lactolyticus forcosus Anaerorhabdus Arcanobacterium caccae Bacteroides Alistipes Aeromonas Acidaminococcus fermentans Acinetobacter baumannii Acinetobacter junii Acinetobacter lwoffii Acinetobacter radioresistens Anaerococcus prevotii Atopobium Bacillus Table 2.1 Indigenous and Pathogenic Microbes of Humans 23 (continued) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + Microbes of Humans Microbes 0 0 0 0 + + + + + + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + ­ p com lex Bacteroides distasonis Bacteroides Bacteroides fragilis Bacteroides ovatus splanchnicus Bacteroides thetaiotaomicron Bacteroides adolescentis Bifidobacterium bifidum Bifidobacterium catenulatum Bifidobacterium dentium Bifidobacterium Bilophila wadsworthia Blastocystis hominis capitatus Blastoschizomyces cepacia Burkholderia Butyrivibrio fibrisolvens concisus Campylobacter Brevibacterium casei Brevibacterium epidermidis Bacteroides eggerthii Bacteroides merdae Bacteroides vulgatus Bifidobacterium breve Bifidobacterium longum Campylobacter curvus Campylobacter gracilis Campylobacter jejuni 24 SECTION 2 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + + GI tract 0 0 0 0 + + + + + + + + + + + + + + + + + + + Resp tract (continued) a a ­ r ous flo ­ n ­ e ig ­ d us ­ t . uman in H spp

Campylobacter rec Organism showae Campylobacter Candida albicans Candida glabrata Candida guilliermondii Candida kefyr Candida krusei Candida lusitaniae Candida parapsilosis Candida tropicalis Capnocytophaga gingivalis Capnocytophage granulosum haemolytica Capnocytophaga Capnocytophaga ochracea hominis Cardiobacterium Cantipeda periodontii Chilomastix mesnili Corynebacterium accolens Corynebacterium afermentans Citrobacter freundii Citrobacter koseri Clostridium Capnocytophaga sputigena Table 2.1 Indigenous and Pathogenic Microbes of Humans 25 (continued) 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + Microbes of Humans Microbes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + ­ tum ­ a spp. Corynebacterium amycolatum Corynebacterium auris Corynebacterium duram Corynebacterium Corynebacterium glucuronolyticum jeikeium Corynebacterium Corynebacterium kroppenstedtii macginleyi Corynebacterium matruchotii Corynebacterium Corynebacterium minutissimum propinquum Corynebacterium Corynebacterium pseudodiphtheriticum riegelii Corynebacterium simulans Corynebacterium Corynebacterium stri Corynebacterium urealyticum Cryptococcus albidus Dermacoccus nishinomiyaensis pigra Desulfomonas Dysgonomonas corrodens Eikenella nanaEndolimax Corynebacterium diphtheriae Corynebacterium ulcerans Dermabacter hominis Eggerthella lenta 26 SECTION 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 + + + + + + + + + + + + + + + + + GI tract 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + Resp tract (continued) a a ­ r ous flo ­ n ­ e ig ­ d uman in spp. spp. H

Ewingella americana Entamoeba coli Organism Entamoeba gingivalis hartmanni Entamoeba Enterobacter Epidermophyton floccosum Eubacterium magnus Finegoldia gonidiaformans Fusobacterium periodonticum Fusobacterium Escherichia coli Escherichia fergusonii Escherichia hermanii Escherichia vulneris Fusobacterium russii Fusobacterium mortiferum Fusobacterium naviforme Fusobacterium necrophorum Fusobacterium nucleatum Fusobacterium alocis Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Table 2.1 Indigenous and Pathogenic Microbes of Humans 27 (continued) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + Microbes of Humans Microbes 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + ­ lus ­ i m i ­ u i ­ c ­ r ­ doph ­ i . spp. spp. spp. spp. spp. spp spp. spp. spp. Kingella Fusobacterium va Fusobacterium sul Gardnerella vaginalis Gemella haemolysans Gemella morbillum Granulicatella Haemophilus Hafnia alvei kunziiHelcococcus Helicobacter sedantariusKytococcus Lactobacillus ac Lactobacillus breve Lactobacillus casei Lactobacillus cellobiosus fermentum Lactobacillus Lactobacillus salivarius Lactococcus adeocarboxylata Leclercia Geotrichum Lactobacillus reuteri Leminorella Klebsiella Kocuria 28 SECTION 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + GI tract 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + Resp tract (continued) a a ­ r ous flo ­ n ­ e ig ­ d . ­ cros uman in spp. spp H spp.

Organism Leptotrichia bucalis Leuconostoc luteus Micrococcus lylae Micrococcus Micromonas mi Microsporum multiacidus Mitsuokella Mobiluncus curtisii Mobiluncus mulieris wisconsensisMoellerella catarrhalis Moraxella Mycoplasma buccale Mycoplasma faucium Mycoplasma genitalium Mycoplasma hominis Mycoplasma lipophilum Mycoplasma orale penetrans Mycoplasma Morganella morganii Mycoplasma fermentans Listeria monocytogenes Malassezia Megasphaera elsdenii Table 2.1 Indigenous and Pathogenic Microbes of Humans 29 (continued) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + Microbes of Humans Microbes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + + a ­ s o ­ c spp. Mycoplasma pneumoniae Mycoplasma primatum Mycoplasma salivarium Mycoplasma spermatophilum ureolyticaOligella urethralisOligella Pasteurella bettyae multocidaPasteurella distasonis Parabacteroides hominis Pentatrichomonas niger Peptococcus asaccharolyticusPeptoniphilus lacrimalisPeptoniphilus anaerobiusPeptostreptococcus productus Peptostreptococcus vaginalis Peptostreptococcus Neisseria cinerea Neisseria flavescens Neisseria lactamica Neisseria meningitidis Neisseria mu Neisseria polysaccharea Neisseria sicca Neisseria subflava Pantoea 30 SECTION 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + GI tract 0 0 0 + + + + + + + + + + + + + + + + + + + + Resp tract (continued) a a ­ r ous flo ­ n ­ e ig ­ d uman in H

Organism asaccharolytica Porphyromonas catoniae Porphyromonas endodontalis Porphyromonas Porphyromonas gingivalis Prevotella bivia Prevotella buccae Prevotella buccalis Prevotella corporis Prevotella dentalis denticola Prevotella Prevotella disiens Prevotella enoeca heparinolytica Prevotella Prevotella intennedia Prevotella loescheii Prevote/la melaninogenica Prevotella nigrescens Prevotella oralis Prevotella oris Prevotella oulorum Prevotella zoogleoformans Prevotella tannerae Prevotella veroralis Table 2.1 Indigenous and Pathogenic Microbes of Humans 31 (continued) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + Microbes of Humans Microbes 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + es ­ n ­ us is spp. ­ l is i spp ­ r ­ b ­ ga ­ ra eus penneri eus vul eus mi ­ t ­ t ­ t Pro Providencia rettgeri Pro Propionibacterium ac Propionibacterium propionicum innocuum Propionferax stuartii Providencia aeruginosa Pseudomonas intestinalis Retortamonas dentocariosa Rothia Rothia mucilaginosa productus Ruminococcus Saccharomyces Selenomonas liquefaciens Serratia Staphylococcus au­ re Staphylococcus auricularis Staphylococcus capitis Staphylcoccus caprae epidermidis Staphylococcus Staphylococcus haemolyticus Pro Serratia marcescens Serratia odorifera Propionibacterium avidum Propionibacterium granulosum 32 SECTION 2 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + Skin, and ear, eye : b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + iage in ­ r GU tract Microbes of Humans Microbes Prevalence of car 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + GI tract 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + Resp tract (continued) a a ­ r ous flo ­ n ­ e ig ­ d ­ ta uman in H

Organism hominis Staphylococcus Staphylococcus lugdunensis Staphylococcus pasteuri Staphylococcus saccharolyticus Staphylococcus saprophyticus Staphylococcus simulans Staphylococcus xylosus warneri Staphylococcus Streptobacillus moniliformis Streptococcus agalactiae Streptococcus anginosus Streptococcus bovis constellatus Streptococcus Streptococcus cricetus Streptococcus cris dysdalactiae Streptococcus Streptococcus equisimilis Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguis Table 2.1 Indigenous and Pathogenic Microbes of Humans 33 - ated ­ l 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + ally iso ­ c ­ i nt; 0, not typ ­ e only pres ­ m 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + + , com + ary tract; ­ n ri Microbes of Humans Microbes ­ u o ­ t ­ i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + . 2012. Jun. 2012. 13; 486:207–14. al tract; GU, gen ­ n i ­ t es ­ t n ­ i ro ­ t 0 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + L. Collier, A. Balows, and M. Sussman (ed.), Topley & Wilsons Microbiology and Microbial In nx; GI, gas ­ y har ­ p nx and oro ­ y har ­ p o ­ s ng na ­ i on, 1998, and Human Microbiome Project Consortium, Nature ­ d lud ay, Humanay, microbiota, 295–306, p. in ­ c ­ r ­ nis old, Lon ­ gui ls. ­ n ­ a ­ u ory tract in id ­ t spp. spp. ­ v i ­ r ­ d spp. ­ s 9th ed., Ar Adapted R. Mur from P. Resp, a re pi a b in healthy in fections, Streptococcus pneumoniae Streptococcus pyogenes Streptococcus Streptococcus salivarius Streptococcus san Streptococcus sobrinus vestibularis Streptococcus dextrinosolvens Succinivibrio denticola Treponema maltophilum Treponema minutum Treponema phagedenis Treponema Treponema refringens socranskii Treponema vincentii Treponema tenax Trichomonas Trichophyton Trichosporon otitidisTuricella parvum Ureaplasma urealyticum Ureaplasma Weeksella virosa Veillonella Tissierella praeacuta 34 SECTION 2 Microbes Responsible for Human Disease

BONE AND JOINT INFECTIONS Arthritis Bacteria Neisseria gonorrhoeae Staphylococcus au­re­us Borrelia burgdorferi Brucella spp. Pseudomonas aeruginosa Pasteurella multocida Eikenella corrodens Streptobacillus moniliformis Mycoplasma hominis Ureaplasma urealyticum Mycobacterium marinum (and other

Microbes of Humans Microbes Mycobacterium spp.) Kingella kingae (chil­dren) Viruses Rubella vi­rus Hepatitis B vi­rus Mumps vi­rus Lymphocytic cho­rio­men­in­gi­tis vi­rus Parvovirus B19 Human im­mu­no­de­fi­ciency vi­rus Chickungunya Dengue Fungi Sporothrix schenckii Candida spp. Coccidioides immitis Osteomyelitis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Streptococcus, be­ta-hemolytic groups Streptococcus pneumoniae Escherichia coli Salmonella spp. (and other Enterobacteriaceae) Pseudomonas aeruginosa Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Fungi Candida spp. Aspergillus spp. Indigenous and Pathogenic Microbes of Humans 35 Cryptococcus neoformans Blastomyces dermatitidis Coccidioides immitis CARDIOVASCULAR INFECTIONS Endocarditis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Streptococcus, viridans group (pri­mar­ily S. mitis, S. oralis, S. sanguis, and S. mutans) Streptococcus bovis group (es­pe­cially S. gallolyticus subsp. gallolyticus) Streptococcus pneumoniae Abiotrophia defectiva Granulicatella adiacens Rothia mucilaginosa of Humans Microbes Enterococcus spp. (pri­mar­ily E. faecalis and E. faecium) HACEK group or­gan­isms Haemophilus parainfluenzae Aggregatibacter actinomycetemcomitans Aggregatibacter aphrophilus (for­merly Haemophilus aphrophilus) Aggregatibacter paraphrolius Cardiobacterium hominis Eikenella corrodens Kingella kingae Salmonella spp. Serratia spp. (and other en­teric Gram-negative rods) Pseudomonas aeruginosa Brucella spp. Bartonella spp. (pri­mar­ily B. henselae) Corynebacterium spp. (pri­mar­ily in dam­aged or pros­thetic valves) Erysipelothrix rhusiopathiae Coxiella burnetii Chlamydophila psittaci Fungi Candida spp. (C. parapsilosis, C. albicans, C. tropicalis, and oth­ers) Aspergillus spp. Histoplasma capsulatum 36 SECTION 2 Myocarditis Bacteria Corynebacterium diphtheriae Clostridium perfringens Streptococcus pyogenes Borrelia burgdorferi Neisseria meningitidis Staphylococcus au­re­us Salmonella spp. Mycoplasma pneumoniae Chlamydophila spp. (C. pneumoniae and C. psittaci) Rickettsia rickettsii Orientia tsu­tsu­ga­mu­shi Viruses Coxsackievirus groups A and B Echoviruses Microbes of Humans Microbes Poliovirus Mumps vi­rus Rubeola vi­rus Influenza A and B vi­rus­es Herpesvirus group Adenoviruses Flaviviruses Arenaviruses Fungi Aspergillus spp. Candida spp. Cryptococcus neoformans Parasites Trypanosoma spp. Trichinella spiralis Toxoplasma gondii Pericarditis Bacteria Streptococcus pneumoniae Staphylococcus au­re­us Neisseria spp. (pri­mar­ily N. meningitidis and N. gonorrhoeae) Mycoplasma pneumoniae Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Viruses Coxsackievirus groups A and B Echovirus Adenovirus Mumps vi­rus Indigenous and Pathogenic Microbes of Humans 37 Influenza A and B vi­rus­es Herpesvirus group Fungi Histoplasma capsulatum Coccidioides immitis Blastomyces dermatitidis Cryptococcus neoformans Candida spp. Aspergillus spp. Parasites Toxoplasma gondii Entamoeba histolytica Schistosoma spp. Sepsis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Enterobacteriaceae (com­mon­ly: Escherichia coli, of Humans Microbes Klebsiella spp., Enterobacter spp., Pro­teus mi­ra­bi­lis, Serratia spp., Citrobacter spp., Salmonella spp.) Enterococcus spp. (pri­mar­ily E. faecalis and E. faecium) Streptococcus pneumoniae Pseudomonas aeruginosa Streptococcus, be­ta-hemolytic (pri­mar­ily groups A, B, C, and F) Streptococcus, viridans group Acinetobacter spp. Mycobacterium avium com­plex Mycobacterium tu­ber­cu­lo­sis Fungi Candida albicans Candida glabrata Candida parapsilosis Candida tropicalis Candida krusei Cryptococcus neoformans Trichosporon spp. Malassezia spp. Histoplasma capsulatum Fusarium spp. Transfusion-associated sep­sis Bacteria Yersinia enterocolitica Staphylococcus, co­ag­u­lase-negative spp. Pseudomonas fluorescens/putida Salmonella spp. Serratia marcescens (and other Enterobacteriaceae) 38 SECTION 2 Campylobacter jejuni Treponema pallidum Bacillus ce­re­us Borrelia spp. Viruses Hepatitis vi­ruses (pri­mar­ily types A, B, C, and D) Cytomegalovirus Ep­stein-Barr vi­rus Human im­mu­no­de­fi­ciency vi­rus Human T-cell leu­ke­mia vi­rus Parvovirus B19 Col­o­rado tick fe­ver vi­rus Parasites Plasmodium spp. Babesia microti Toxoplasma gondii Trypanosoma cruzi Microbes of Humans Microbes Leishmania spp. Suppurative throm­bo­phle­bi­tis Bacteria Staphylococcus au­re­us Klebsiella (and other Enterobacteriaceae) Pseudomonas aeruginosa Enterococcus spp. (pri­mar­ily E. faecalis and E.faecium) Bacteroides fragilis group Campylobacter fe­tus Fungi Candida spp. Malassezia spp. CENTRAL NERVOUS SYSTEM INFECTIONS Acuten men­i ­gi­tis Bacteria Escherichia coli (Neonates) Streptococcus agalactiae (group B) (Neonates and el­der­ly) Streptococcus pneumoniae Neisseria meningitidis Listeria monocytogenes (Neonates and el­der­ly) Haemophilus influenzae (rare in post-vaccine era) Other Gram-negative rods (e.g., Klebsiella and Pseudomonas spp.) Staphylococcus au­re­us (and other Staphylococcus spp.) (shunts, neu­ro­sur­gi­cal pro­ce­dures) Indigenous and Pathogenic Microbes of Humans 39 Propionibacterium ac­nes (shunts, neu­ro­sur­gi­cal pro­ce­dures) Nocardia spp. Treponema pallidum Borrelia burgdorferi Leptospira spp. Mycobacterium tu­ber­cu­lo­sis Mycobacterium avium com­plex (and other Mycobacterium spp.) Rickettsia spp. Orientia tsu­tsu­ga­mu­shi Ehrlichia spp. Viruses Enteroviruses (echo­vi­rus and coxsackievirus groups A and B) Orbivirus (Col­o­rado tick fe­ver vi­rus) Mumps vi­rus Measles vi­rus of Humans Microbes Adenovirus Herpes sim­plex vi­rus Human im­mu­no­de­fi­ciency vi­rus Fungi Cryptococcus neoformans (HIV in­fect­ed) Cryptococcus gattii Histoplasma capsulatum Coccidioides immitis Candida spp. Parasites Naegleria fowleri Bayliasciaris spp. Acanthamoeba spp. Angiostrongylus cantonensis Chronic men­in­gi­tis Bacteria Brucella spp. Borrelia burgdorferi Treponema pallidum Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Nocardia spp. Fungi Coccidioides immitis Histoplasma capsulatum Cryptococcus neoformans Sporothrix schenckii 40 SECTION 2 Parasites Acanthamoeba spp. Angiostrongylus cantonensis Encephalitis Bacteria Listeria monocytogenes Treponema pallidum Leptospira spp. Actinomyces spp. Nocardia spp. Borrelia spp. (as­so­ci­ated with Lyme dis­ease and re­laps­ing fe­ver) Rickettsia rickettsii Coxiella burnetii Mycoplasma pneumoniae Mycobacterium tu­ber­cu­lo­sis Viruses Microbes of Humans Microbes Enteroviruses (po­lio­vi­rus, coxsackievirus, echo­vi­rus, and hep­a­ti­tis A vi­rus) Herpesvirus group Alphaviruses (Eastern, Western, and Ven­e­zu­e­lan equine en­ceph­a­li­tis vi­rus­es) Flaviviruses (St. Louis en­ceph­a­li­tis vi­rus, West Nile vi­rus, Jap­a­nese en­ceph­a­li­tis vi­rus, and den­gue vi­rus) Bunyaviruses (La Crosse vi­rus and Rift Valley vi­rus) Arenaviruses (lym­pho­cytic cho­rio­men­in­gi­tis vi­rus, Machupo vi­rus, Lassa vi­rus, and Ju­nin vi­rus) Filoviruses (Ebola vi­rus and Mar­burg vi­rus) Rabies vi­rus Human im­mu­no­de­fi­ciency vi­rus Mumps vi­rus Measles vi­rus Rubella vi­rus Adenovirus Fungi Cryptococcus neoformans Histoplasma capsulatum Parasites Naegleria fowleri Acanthamoeba spp. Toxoplasma gondii Plasmodium fal­cip­a­rum Trypanosoma spp. Indigenous and Pathogenic Microbes of Humans 41 Brain ab­scess Bacteria Staphylococcus au­re­us Enterobacteriaceae (Pro­teus, Escherichia, Klebsiella, and other spp.) Pseudomonas aeruginosa Streptococcus, viridans group (S. anginosus group) Bacteroides spp. (and other an­aer­o­bic Gram-negative rods) Peptostreptococcus spp. (and other an­aer­o­bic Gram-positive coc­ci) Actinomyces spp. Clostridium spp. Listeria monocytogenes Nocardia spp. Rhodococcus equi Mycobacterium tu­ber­cu­lo­sis Nocardia spp. of Humans Microbes Fungi Cryptococcus neoformans Candida spp. Coccidioides immitis Aspergillus spp. Mucorales Cladiophialophora spp. Scedosporium spp. Exophiala spp. Parasites Acanthamoeba spp. Toxoplasma gondii EAR INFECTIONS Otitis externa Bacteria Pseudomonas aeruginosa Staphylococcus au­re­us Streptococcus pyogenes Fungi Aspergillus spp. (pri­mar­ily A. fumigatus and A. niger) Candida albicans Pseudallescheria boydii Malassezia spp. Otitis me­dia Bacteria Streptococcus pneumoniae Haemophilus influenzae 42 SECTION 2 Moraxella catarrhalis Staphylococcus au­re­us Streptococcus pyogenes Turicella otitidis (con­tro­ver­sial—look for pure/predominant growth) Mixed an­aer­obes Viruses Respiratory syn­cy­tial vi­rus Influenza vi­rus Enterovirus Rhinovirus EYE INFECTIONS Conjunctivitis Bacteria Streptococcus pneumoniae Streptococcus agalactiae Microbes of Humans Microbes Streptococcus, viridans group Staphylococcus au­re­us Moraxella catarrhalis Haemophilus aegyptius Neisseria gonorrhoeae Pseudomonas aeruginosa Corynebacterium diphtheriae Corynebacterium macginleyi Francisella tularensis Borrelia burgdorferi Bartonella henselae Chlamydia trachomatis Viruses Adenovirus Herpesvirus group Papillomavirus Rubella vi­rus Influenza vi­rus Measles vi­rus Fungi Candida spp. Sporothrix schenckii Parasites Onchocera vol­vu­lus Loa loa Wuchereria bancrofti Leishmania donovani Microsporidia (most com­monly Encephalitozoon spp.) Toxocara canis Indigenous and Pathogenic Microbes of Humans 43 Endophthalmitis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Pseudomonas aeruginosa Propionibacterium spp. Corynebacterium spp. Bacillus ce­re­us (and other Bacillus spp.) Rapidly grow­ing my­co­bac­te­ria (pri­mar­ily M. chelonae and M. abscessus) Viruses Herpesvirus group Rubella vi­rus Measles vi­rus Fungi Candida albicans (and other Candida spp.) Aspergillus spp. Histoplasma capsulatum of Humans Microbes Opportunistic fun­gi Parasites Toxoplasma gondii Toxocara spp. Cysticercus cellulosae Keratitis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Streptococcus pneumoniae Streptococcus pyogenes Enterococcus faecalis Pseudomonas aeruginosa Pro­teus mi­ra­bi­lis (and other en­teric Gram-negative rods) Bacillus spp. (pri­mar­ily B. ce­re­us) Clostridium per:fringens Neisseria gonorrhoeae Viruses Herpesvirus group Adenovirus Measles vi­rus Fungi Fusarium spp. Aspergillus spp. Candida spp. Parasites Onchocerca vol­vu­lus Acanthamoeba spp. 44 SECTION 2 Leishmania braziliensis Trypanosoma spp. Microsporidia (pri­mar­ily Nosema and Encephalitozoon spp.) GASTROINTESTINAL INFECTIONS Esophagitis Viruses Cytomegalovirus Herpes sim­plex vi­rus Human im­mu­no­de­fi­ciency vi­rus Fungi Candida albicans (and other Candida spp.) Noninflammatory di­ar­rhea Bacteria Escherichia coli Microbes of Humans Microbes Staphylococcus au­re­us Bacillus ce­re­us Clostridium perfringens Vibrio spp. (pri­mar­ily V. cholerae and V. parahaemolyticus) Viruses Rotaviruses Caliciviruses (Norovirus) Adenoviruses Astroviruses Coronaviruses Inflammatory di­ar­rhea Bacteria Escherichia coli Salmonella spp. Shigella spp. Campylobacter spp. Clostridium dif­fi­cile Yersinia enterocolitica Vibrio parahaemolyticus Plesiomonas shigelloides Edwardsiella tarda Aeromonas spp. Viruses Adenoviruses Cytomegalovirus Fungi Mucorales Parasites Giardia duodenalis Indigenous and Pathogenic Microbes of Humans 45 Entamoeba histolytica Neobalantidium (Balantidium) coli Cryptosporidium parvum Cryptoisospora (Isospora) bel­li Microsporidia Cyclospora cayetanensis Diphyllobothrium tatum Trichinella spiralis Strongyloides stercoralis Schistosoma spp. (pri­mar­ily S. mansoni and S. japonicum) GENITAL INFECTIONS Cervicitis Bacteria Neisseria gonorrhoeae Neisseria meningitidis of Humans Microbes Chlamydia trachomatis Actinomyces spp. Viruses Herpes sim­plex vi­rus Cytomegalovirus Adenovirus Measles vi­rus Papillomavirus Genital ul­cers and skin nod­ules Bacteria Treponema pallidum Haemophilus ducreyi Chlamydia trachomatis Klebsiella granulomatis Mycobacterium ulcerans Mycobacterium tu­ber­cu­lo­sis Viruses Herpes sim­plex vi­rus Molluscipoxviruses Fungi Histoplasma capsulatum Urethritis Bacteria Neisseria gonorrhoeae Chlamydia trachomatis Ureaplasma urealyticum Mycoplasma genitalium Parasites Trichomonas vaginalis 46 SECTION 2 Vaginitis Bacteria Mobiluncus spp. Gardnerella vaginalis Mycoplasma hominis Absence of Lactobacillus spp. Fungi Candida spp. Parasites Trichomonas vaginalis GRANULOMATOUS INFECTIONS Bacteria Brucella spp. Francisella tularensis Listeria monocytogenes Burkholderia pseudomallei Microbes of Humans Microbes Actinomyces spp. Bartonella henselae Tropheryma whippelii Mycobacterium spp. Chlamydia trachomatis Coxiella burnetii Treponema pallidum Nocardia spp. Corynebacterium kroppenstedtii (gran­u­lo­ma­tous breast ab­scess­es) Viruses Cytomegalovirus Measles vi­rus Mumps vi­rus Ep­stein-Barr vi­rus Fungi Cryptococcus neoformans Candida spp. Sporothrix schenckii Histoplasma capsulatum Paracoccidioides brasiliensis Coccidioides immitis Blastomyces dermatitidis Aspergillus spp. Phialophora spp. Exophiala spp. Fonsecaea spp. Taloromyces marneffei (for­merly Penicillium) Pseudallescheria boydii Indigenous and Pathogenic Microbes of Humans 47 Parasites Leishmania spp. Toxoplasma gondii Schistosoma spp. Toxocara spp. INTRA-ABDOMINAL INFECTIONS Peritonitis Bacteria Escherichia coli Klebsiella pneumoniae (and other en­teric Gram-negative rods) Pseudomonas aeruginosa Streptococcus pneumoniae Streptococcus anginosus Staphylococcus au­re­us Enterococcus spp. Bacteroides fragilis group (and other of Humans Microbes Bacteroides spp.) Fusobacterium spp. Clostridium spp. Peptostreptococcus spp. (and other an­aer­o­bic Gram-positive coc­ci) Neisseria gonorrhoeae Chlamydia trachomatis Mycobacterium tu­ber­cu­lo­sis Fungi Candida albicans Parasites Strongyloides stercoralis Dialysis-associated peri­to­ni­tis Bacteria Staphylococcus au­re­us (and other Staphylococcus spp.) Streptococcus spp. Corynebacterium spp. Propionibacterium spp. Escherichia coli (and other Enterobacteriaceae) Pseudomonas aeruginosa Acinetobacter spp. Fungi Candida albicans Candida parapsilosis (and other Candida spp.) Aspergillus spp. Fusarium spp. Exophiala spp. 48 SECTION 2 Visceral ab­scess­es Bacteria Escherichia coli (and other Enterobacteriaceae) Enterococcus spp. Staphylococcus au­re­us Bacteroides fragilis group Fusobacterium spp. Actinomyces spp. Mixed aer­obes and an­aer­obes Yersinia enterocolitica Streptococcus anginosus Mycobacterium tu­ber­cu­lo­sis Mycobacterium avium com­plex (and other Mycobacterium spp.) Fungi Candida albicans (and other Candida spp.) Parasites Microbes of Humans Microbes Entamoeba histolytica (pri­mar­ily he­patic ab­scess­es) Echinococcus (he­patic ab­scess­es) RESPIRATORY TRACT INFECTIONS Pharyngitis Bacteria Streptococcus pyogenes Streptococcus dysgalactiae (groups C and G) Fusobacterium necrophorum Arcanobacterium haemolyticum Chlamydophila pneumoniae Neisseria gonorrhoeae Corynebacterium diphtheriae Corynebacterium ulcerans Mycoplasma pneumoniae Yersinia enterocolitica Treponema pallidum Viruses Respiratory syn­cy­tial vi­rus Rhinovirus Coronavirus Adenovirus Herpes sim­plex vi­rus Parainfluenza vi­rus Influenza vi­rus Coxsackievirus A Ep­stein-Barr vi­rus Indigenous and Pathogenic Microbes of Humans 49 Cytomegalovirus Human im­mu­no­de­fi­ciency vi­rus Laryngitis Bacteria Mycoplasma pneumoniae Chlamydophila pneumoniae Streptococcus pyogenes Viruses Rhinovirus Influenza vi­rus Parainfluenza vi­rus Adenovirus Coronavirus Laryngotracheobronchitis (croup) Bacteria Mycoplasma pneumoniae Viruses Parainfluenza vi­rus of Humans Microbes Influenza A and B vi­rus­es Respiratory syn­cy­tial vi­rus Adenovirus Rhinovirus Enterovirus Sinusitis Bacteria Streptococcus pneumoniae Haemophilus influenzae Moraxella catarrhalis Mixed an­aer­obes Staphylococcus au­re­us Streptococcus pyogenes Chlamydophila pneumoniae Pseudomonas aeruginosa (and other Gram-negative rods) Viruses Rhinovirus Influenza vi­rus Parainfluenza vi­rus Adenovirus Fungi Aspergillus spp. (al­ler­gic si­nus­i­tis) Hyphomycetes (al­ler­gic si­nus­i­tis) Zygomycetes (in­va­sive dis­ease) Bronchitis Bacteria Mycoplasma pneumoniae 50 SECTION 2 Chlamydophila pneumoniae Bordetella per­tus­sis Moraxella catarrhalis Haemophilus influenzae Viruses Rhinovirus Coronavirus Parainfluenza vi­rus Influenza vi­rus Respiratory syn­cy­tial vi­rus Adenovirus Empyema Bacteria Staphylococcus au­re­us Streptococcus pneumoniae Streptococcus pyogenes Bacteroides fragilis Microbes of Humans Microbes Klebsiella pneumoniae (and other Gram-negative rods) Actinomyces spp. Nocardia spp. Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Fungi Aspergillus spp. Community-acquired pneu­mo­nia Bacteria Streptococcus pneumoniae Staphylococcus au­re­us Klebsiella pneumoniae Haemophilus influenzae Moraxella catarrhalis Neisseria meningitidis Mycoplasma pneumoniae Chlamydia trachomatis Chlamydophila spp. (pri­mar­ily C. pneumoniae and C. psittaci) Pseudomonas aeruginosa Legionella spp. Bacteroides fragilis (and other an­aer­obes in mixed in­fec­tions) Nocardia spp. Rhodococcus equi Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Coxiella burnetii Many other bac­te­ria Indigenous and Pathogenic Microbes of Humans 51 Viruses Respiratory syn­cy­tial vi­rus Parainfluenza vi­rus Influenza vi­rus Adenovirus Rhinovirus Enteroviruses Herpesviruses Measles vi­rus Fungi Pneu­mo­cys­tis jiroveci (ca­ri­nii) Cryptococcus neoformans Cryptococcus gattii Histoplasma capsulatum Blastomyces dermatitidis Coccidioides immitis Paracoccidioides brasiliensis Zygomycetes (pri­mar­ily Rhizopus and Mucor spp.) of Humans Microbes Parasites Ascaris lumbricoides Strongyloides stercoralis Toxoplasma gondii Paragonimus spp. Hospital-acquired pneu­mo­nia Bacteria Streptococcus pneumoniae Staphylococcus au­re­us Haemophilus influenzae Klebsiella pneumoniae Enterobacter spp. Escherichia coli Serratia marcescens Stenotrophomonas maltophilia Acinetobacter spp. Moraxella catarrhalis Pro­teus mi­ra­bi­lis Citrobacter spp. Enterococcus spp. Viruses Cytomegalovirus Respiratory syn­cy­tial vi­rus Fungi Aspergillus fomigatus Mucorales (pri­mar­ily Rhizopus and Mucor spp.) Parasites Toxoplasma gondii 52 SECTION 2 SKIN AND SOFT TISSUE INFECTIONS Primary pyo­der­mas Bacteria Staphylococcus au­re­us Streptococcus pyogenes Pseudomonas aeruginosa Bacillus anthracis Treponema pallidum Haemophilus ducreyi Francisella tularensis Corynebacterium diphtheriae Mycobacterium spp. (pri­mar­ily M. ulcerans and M. marinum) Fungi Candida spp. Sporothrix schenckii

Microbes of Humans Microbes Gangrenous cel­lu­li­tis Bacteria Streptococcus pyogenes Pseudomonas aeruginosa Clostridium spp. (pri­mar­ily C. perfringens, C. septicum, C. sordellii and C. novyi) Vibrio vulnificus Aeromonas hydrophila Erysipelothris rhusiopathiae Mixed aer­obes and an­aer­obes (e.g., E. coli, Bacteroides spp., and Peptostreptococcus spp.) Fungi Aspergillus spp. Zygomycetes (pri­mar­ily Rhizopus, Absidia, and Mucor spp.) Nodular le­sions Bacteria Staphylococcus au­re­us Nocardia spp. Mycobacterium marinum Bartonella spp. Fungi Candida spp. Sporothrix schenckii Parasites Leishmania spp. Secondary skin in­fec­tions Bacteria Staphylococcus au­re­us Streptococcus pyogenes Indigenous and Pathogenic Microbes of Humans 53 Pseudomonas aeruginosa Enterobacter spp. (and other Enterobacteriaceae) Anaerobic Gram-positive coc­ci Pasteurella spp. (pri­mar­ily P. multocida and P. canis) Fungi Candida spp. Aspergillus spp. Disseminated in­fec­tions with cu­ta­ne­ous man­i­fes­ta­tions Bacteria Staphylococcus au­re­us Streptococcus pyogenes Neisseria spp. (pri­mar­ily N. meningitidis and N. gonorrhoeae) Pseudomonas aeruginosa Salmonella enterica serovar Typhi Listeria monocytogenes Leptospira interrogans Streptobacillus moniliformis of Humans Microbes Burkholderia spp. (pri­mar­ily B. pseudomallei and B. mal­lei) Bartonella spp. Mycobacterium tu­ber­cu­lo­sis (and other Mycobacterium spp.) Nocardia spp. Fungi Candida spp. Blastomyces dermatitidis Aspergillus spp. Coccidioides immitis Fusarium spp. URINARY TRACT INFECTIONS Cystitis and py­elo­ne­phri­tis Bacteria Escherichia coli Enterococcus spp. (pri­mar­ily E. faecalis and E. faecium) Pro­teus mi­ra­bi­lis Klebsiella spp. Pseudomonas aeruginosa Corynebacterium urealyticum Enterobacter spp. Staphylococcus au­re­us Staphylococcus saprophyticus (and other Staphylococcus spp.) Streptococcus agalactiae (group B) 54 SECTION 2 Aerococcus urinae Aerococcus sanguinicola Mycobacterium tu­ber­cu­lo­sis Viruses Adenovirus Cytomegalovirus BK vi­rus Fungi Candida glabrata Candida albicans (and other Candida spp.) Parasites Schistosoma haematobium Renal cal­cu­li Bacteria Pro­teus spp. Morganella morganii Klebsiella pneumoniae Microbes of Humans Microbes Corynebacterium urealyticum Staphylococcus saprophyticus Ureaplasma urealyticum Prostatitis Bacteria Escherichia coli Klebsiella spp. Pro­teus mi­ra­bi­lis Enterobacter spp. Enterococcus spp. Neisseria gonorrhoeae Mycobacterium spp. Fungi Candida spp. Cryptococcus neoformans Summary of Notifiable Infectious Diseases: United States, 2015a Bacterial (2015 to­tals in Brucellosis (Brucella spp.) pa­ren­the­ses) (126) Anthrax (Bacillus Campylobacteriosis anthracis) (2) (54,556) Botulism, food-borne Chancroid (Haemophilus (Clostridium ducreyi) (11) bot­u­li­num) (37) Chlamydia (Chlamydia Botulism, in­fant (138) trachomatis) (1,526,658) Botulism, Cholera (Vibrio other (20) cholerae) (5) Indigenous and Pathogenic Microbes of Humans 55 Diphtheria Salmonellosis (Corynebacterium (Salmonella spp.) diphtheriae) (0) (55,108) Ehrlichiosis and Shiga tox­in-producing Anaplasmosis Escherichia coli (STEC) Anaplasma (7,059) phagocytophilum Shigellosis (Shigella spp.) (3,656) (23,590) Ehrlichia chaffeensis Spotted fe­ver rickettsiosis (1,288) (Rickettsia rickettsii) Ehrlichia ewingii (14) (4,198) Gonorrhea (Neisseria Streptococcal toxic shock gonorrhoeae) (395,216) (S. pyogenes) (335) Haemophilus influenzae, Syphilis, all­ stages in­va­sive (4,138) (Treponema pallidum) Serotype B (29) (74,702) Hemolytic ure­mic syn­ Syphilis, con­gen­i­tal drome, post di­ar­rhe­al (487) of Humans Microbes Hansen dis­ease, lep­rosy Tetanus (Clostridium (Mycobacterium tetani) (29) leprae) (89) Toxic shock syn­drome Invasive pneu­mo­coc­cal (other than dis­ease (Streptococcus strep­to­coc­cal) (64) pneumoniae) (16,163) Tuberculosis Legionellosis (Legionella (Mycobacterium spp.) (6,079) tu­ber­cu­lo­sis) (9,557) Leptospirosis (40) Tularemia (Francisella Listeriosis (Listeria tularensis) (314) monocytogenes) (768) Typhoid fe­ver (Salmonella Lyme dis­ease (Borrelia enterica serovar burgdorferi) (38,069) Typhi) (367) Meningococcal dis­ease Vancomycin-intermediate (Neisseria meningitidis) Staphylococcus (372) au­re­us (VISA) Serogroup B (111) in­fec­tion (183) Pertussis (Bordetella Vancomycin-resistant per­tus­sis) Staphylococcus (20,762) au­re­us (VRSA) Plague (Yersinia pestis) (16) in­fec­tion (3) Psittacosis (Chlamydophila Vibriosis (any spe­cies psittaci) (4) of the fam­ily Q fe­ver (Coxiella burnetii), Vibrionaceae, other acute (122) than toxi­genic Q fe­ver (Coxiella burnetii), Vibrio cholerae 01 chronic (34) or 0139) (1,323)

(continued) 56 SECTION 2 Summary of Notifiable Infectious Diseases: United States, 2015a (continued) Viral James­town Canyon Chickungunya vi­rus vi­rus dis­ease Neuroinvasive (6) Neuroinvasive (4) Nonneuroinvasive (5) Nonneuroinvasive (892) La Crosse vi­rus dis­ease Dengue vi­rus in­fec­tion Neuroinvasive (51) Dengue (929) Nonneuroinvasive (4) Dengue-like Measles ill­ness (16) Indigenous (162) Severe den­gue (6) Imported (26) Encephalitis, Mumps (1,329) Cal­i­for­nia (0) Novel in­flu­enza A vi­rus Encephalitis, Eastern in­fec­tions (7)

Microbes of Humans Microbes equine (6) Poliomyelitis, par­a­lytic (0) Encephalitis, Powassan Poliovirus in­fec­tion, Neuroinvasive (6) nonparalytic (0) Nonneuroinvasive (1) Rabies, an­i­mal (5,491) Encephalitis, St. Lou­is Rabies, hu­man (2) Neuroinvasive (19) Rubella (5) Nonneuroinvasive (4) Rubella (con­gen­i­tal) (1) Encephalitis, West Nile Severe acute re­spi­ra­tory Neuroinvasive (1455) syn­drome as­so­ci­ated Nonneuroinvasive (720) co­ro­na­vi­rus dis­ease Encephalitis, Western (SARS-CoV) (0) equine (0) Smallpox (0) Hantavirus in­fec­tion, Varicella (Morbidity) non-Hantavirus (9,789) pul­mo­nary Varicella (Morality) (6) syn­drome (3) Viral hem­or­rhagic fe­ver Hantavirus pul­mo­nary Cri­me­an-Congo syn­drome (21) hem­or­rhagic fe­ver Hepatitis A, acute (1,390) vi­rus (0) Hepatitis B, acute (3,370) Ebola vi­rus (0) Hepatitis B, chronic Lassa vi­rus (1) (14,147) Lujo vi­rus (0) Hepatitis B, peri­na­tal Mar­burg vi­rus (0) in­fec­tion (37) New World Hepatitis C, acute (2,447) ar­e­na­vi­rus Hepatitis C, past or pres­ent Guanarito vi­rus (0) (179,584) Ju­nin vi­rus (0) HIV di­ag­noses (33,817) Machupo vi­rus (0) Influenza-associated Sabia vi­rus (0) pe­di­at­ric mor­tal­ity (130) Yellow fe­ver (1) Indigenous and Pathogenic Microbes of Humans 57 Fungal Cyclosporiasis Coccidioidomycosis (Cyclospora (Coccidioides immitis) cayetanensis) (11,072) (645) Giardiasis (Giardia Parasitic Iamblia) (14,485) Babesiosis (2,100) Malaria (Plasmodium Cryptosporidiosis spp.) (1,390) (Cryptosporidium Trichinosis (Trichinella parvum) (9,735) spiralis) (14)

aData from Morb. Mortal. Wkly. Rep. Summary of Notificable Infectious Diseases and Conditions—United States, 2015(64):1-143, 2017. Num­bers in pa­ren­the­ses rep­ re­sent the num­ber of cases re­ported in 2015. Microbes of Humans Microbes 58 SECTION 2 er ­ v ase ­ e ase ­ e ­ ing fe hus aps ­ p ­ l ­ ease orm dis er ­ phus orm ­ v ­ w ­ w Epidemic ty Trench fe Epidemic re Gnathostomiasis Disease Filariasis Chagas Chagas dis Diphyllobothriasis Guinea worm dis Paragonimiasis Plague Murine ty Rat tape Plague Dog tape Microbes of Humans Microbes spp. spp. a s ­ e as ­ e uchereria, Brugia Borrelia recurrentis Bartonella quintana Bartonella Rickettsia prowazekii Gnathostoma spinigerum Etiologic agent W Trypanosoma cruzi Trypanosoma Dracunculus medinensis Paragonimus Diphyllobothrium Hymenolepis diminuta Hymenolepis Yersinia pestis Rickettsia typhi Yersinia pestis caninumDipylidium ant dis ­ t or ­ p ally im ­ c ­ i

ors of med ­ t eans) ­ c ­ ta ­ fish) rthropod vec (i.e., louse) ng bug) i ­ A

ulex, Oropsylla Xenopsylla, Nosophyllus P Pediculus Panstrongylus, Rhodnius, Triatoma Rhodnius, Panstrongylus, Aedes (i.e., kiss(i.e., Ctenocephalides opepods (i.e., crus iphonaptera flea) (i.e., S Anopleura Decapods cray (i.e., Diptera (i.e., flies) C Hemiptera

Insecta Crustacea Arthropod

 Table 2.2 Indigenous and Pathogenic Microbes of Humans 59 (continued) ess ­ n er, zikaer, ­ v er ­ v ng sick ­ i ow fe ow r ­ l ­ ve ­ ease ­ can sleep i ­ r Encephalitis Filariasis Encephalitis Af Loiasis Tularemia Malaria Filariasis Encephalitis Filariasis Onchocerciasis Filariasis Dengue, yel Lyme dis Anaplasmosis Boutonneuse fe Babesiosis Leishmaniasis Bartonellosis Sandfly fe Microbes of Humans Microbes us ­ l u ­ es ­ v us ­ r spp. i spp. spp. ­ v o ­ b spp. Other ar Arboviruses Francisella tularensis Plasmodium Wuchereria Mansonella Trypanosoma brucei Loa loa Onchocerca vol Wuchereria, Brugia Arboviruses Mansonella ozzardi Mansonella Flaviviruses Leishmania Borrelia burgdorferi Babesia Anaplasma phagocytophilum Anaplasma Rickettsia conorii Bartonella bacilliformis Phlebovirus Anopheles Culex Culicoides Glossina Chrysops Simulium Phlebotomus, Lutzomyia Ixodes cari (ticks) A

Arachnida 60 SECTION 2 er ­ v is er er er ­ s ­ v ­ v ­ v ­ o ­ i hagic hagic fe ­ r ich er ed fe ed fe ed fe ­ l r ­ t ­ t ­ t ­ v ­ o er er gic gic fe ­ v ­ v ytic ehr ­ c er ­ rha ­ v r ongo ongo hem ­ o hus C - ­ p n ­ a ado tick fe ­ r ­ o ­ me Relapsing fe Boutonneuse Boutonneuse fe Rocky Mountain spot Omsk hem Tularemia Human mono Rocky Mountain spot Cri Disease Rocky Mountain spot Tularemia Col Rickettsialpox Scrub ty Microbes of Humans Microbes i ­ sh u ­ m er SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th a ­ t ­ g su ­ t (continued) a s spp. ­ e as ­ e rientia rientia tsu Borrelia Rickettsia rickettsii Rickettsia conorii Flavivirus Francisella tularensis Ehrlichia chaffeensis Rickettsia rickettsii Nairovirus Francisella tularensis Coltivirus Etiologic agent Rickettsia rickettsii O Rickettsia akari Rickettsia ant dis ­ t or ­ p ally im oll Funke KC, G, Landry ML, Rich ­ r ­ c ­ i ors of med ­ t rthropod vec on, D.C., 2015. on, D.C., ­ t A ng ­ i

Ornithodoros Rhipicephalus Amblyomma Hyalomma Dermacentor Leptotrombidium Acari (mites) Adapted from Jorgensen JH, Pfaller MA, Car a Liponyssoides

Press, Wash

Arthropod Table 2.2 Indigenous and Pathogenic Microbes of Humans 61 ers ­ m on­ ­ r i ­ v oirs (continued) ­ v eon and ­ er ed trees ­ i ­ g ers ­ t ings and soil ted in en ­ p ary tracts of ­ u ­ t en only pi ­ trib ­ m ­ m ated with these ­ i ­ n ls ­ i ngs cts ­ ma ­ pi ­ u am ­ t Gum and other var Most com drop Widely dis ment, soil, beach sand, dairy prod other bird drop con Plants, al Climates with hot, dry sum and mild, win wet Environmental Environmental res mam only ­ m ion ­ t ­ bu ri Microbes of Humans Microbes ­ t ope ­ r Worldwide Worldwide Worldwide Worldwide Worldwide Southern Eu Geographic dis but most com Worldwide, ­ tem; ated us ­ c ­ o li e ­ p ­ n only only ion ous sys ­ t a ly ly

ng ng any part ­ t ­ i ­ m ­ m ­ v ­ ­ i d u ­ bu ­ e ar ­ c ut ions ions ­ t ­ t om om o ­ o ri olv ­ m ­ c ­ c ­ t ­ c ect ­ v ec ec ­ f ­ f ­ f ral ner ­ t en rarely im en un en in en pri en un ­ g ­ g ­ g ­ g ­ g ase with ­ o ­ o ­ o ­ o ­ o al of mu ­ e ­ s ­ temic in ­ temic in nt raphic dis ­ te en ­ g e ­ m ­ p ion. ion. Disease in ­ t a ­ com ­ n o anes ­ i ng ng lungs and cen ated in sys ated in sys ­ i ­ n ­ ease ­ c ­ c m ­ br u li li olv ­ se ­ m ­ p ­ p ­ v ens and geo im Opportunistic path in dis dis Opportunistic path Primarily lung dis im Opportunistic path in other body sites can be in im Human body sites mem Opportunistic path Opportunistic path of of body, com ­ g ­ o , other spp. ungal path F spp.

ndida eotrichum ryptococcus ryptococcus gattii C spp. C Ca Cryptococcus neoformans Blastoschizomyces capitatus G  

Yeasts Fungi  Table 2.3 62 SECTION 2 als ­ m ned) ­ fi ­ es ains, oirs ­ t ­ v ush ­ er ­ br looded mam b - als ­ m ­ i ub ub grout, tooth ­ t Probably soil (not de well Skin of warm Moist skin, cur shower bath Environmental Environmental res Plants Soil, an Mammalian lung a ­ c i ­ r ouri and ion ­ s ­ t ins; ­ bu ­ s ions of Af ­ t ippi River ri ­ s Microbes of Humans Microbes ­ t is da, South ­ s ­ a as River ba rn Can ca, and por ­ s ­ i ­ e an eys, aseys, as well Mis ­ l ­ k val Ar south Amer Worldwide Ohio and Mis Worldwide Geographic dis Worldwide Worldwide Worldwide is, ­ t i­ al, i ­ t ­ c ­ t ­ i a (continued) o ary ro­ ­ s ­ n ­ m ­ p ral o ­ t only ion ion ions), ­ m ­ t ly ly om ­ s ng ng cap ng ng skin ­ i ­ m ­ c ­ i ­ i o ­ bu ase (as ar ions ­ n ­ e ­ t om ri olv olv ­ m us le ­ c ­ t ­ v ­ v ec ­ o ion ion to skin ­ f ­ mu ly ly a pul ­ t e ­ i a ­ n ­ n ar ­ i ­ ta en un en in en pri en in y) u ­ g ­ g ­ g ­ g ­ m ­ p ­ c ­ temic dis ­ a ­ o ­ o ­ o ­ o em ory tract ­ s ­ temic in ­ t al hairs (white piedra); raphic dis ions in im ­ r ­ ra ­ g ­ t pi ec ­ s ­ tem ­ f ary tract, bone, and cen ients ­ n ­ t us and sub is) and sys ­ t ri ­ o i ng ng re ­ l ated in sys e ­ i ­ u ion ion with dis ­ c ­ u ­ t ­ n o ary, or cru ous sys ­ t ace (tinea versicolor, atopic der ­ l li ­ i ic ­ v l ­ f ­ ta ­ temic in olv ec ­ l ­ p ­ i ­ f ­ v ens and geo ner (cu gen Opportunistic path ax sys Opportunistic path im Opportunistic path Opportunistic path in ated with lipid ther in Human body sites Blastomycosis is pri sur mised pa Opportunistic cause fungemia of fol ­ g ­ o

spp. ­ gi spp. is ungal path ­ t spp. F spp. ­ cys

) jiroveci o ii ­ m ­ n i ­ r alassezia hodotorula neu richosporon T ( ca M B dermatitidis R P

Fungi Pseudozyma Dimorphic fun  lastomyces  Table 2.3 Indigenous and Pathogenic Microbes of Humans 63 ions al ­ g on­ ­ c ­ r i ­ v (continued) rid re ­ a al tracts of ­ n i ­ t ated with fe es ­ t ­ n ­ i ated from en ­ l am ­ t ors hot, semi ­ v als er from birds and bats ­ t ­ m ­ i Soil con mat Soil, fa an Rarely iso ment, soil, in Unknown ­ a ut as ­ c ­ o ­ e ­ i a rn ­ c ­ e ­ i ions of ast sippi ­ sippi River ­ t ­ e ween 20°N nces of arly Ohio, ­ t ­ sis ­ i ico, ico, a ­ l al, and ­ c ­ x ­ c ­ u ­ i Microbes of Humans Microbes as in Central ­ i ­ e ions through rn por ic and, Laos, ­ t ­ l ­ e ­ g ica (be rn Me ­ r ­ e al re ­ c ar, and south ­ i est ina, and other dry ar rn Thai ­ t ­ m ­ e ­ w da, and ar rop ­ a ­ souri, and Mis en eys, eys, south ­ t ­ l ­ g and South Amer Can Temperate, trop sub the world, par Mis val Southwestern United States, north Ar Centralof and South Amer Central Af and 20°S) Central and South Amer Mountainous prov north Myan China a­ ­ t an u ­ c uent ­ c al ­ i ary ary ion ion to ­ q o ­ n ­ n ­ t ral ­ n ­ c e ary and only to ­ t a o ­ r ous o ­ n al tract, ­ n ­ v ­ m ­ i ­ m ­ m ­ n ri ary em ­ u ­ ti ­ t ng ng bone, ­ n ly a ly ­ i em o ­ i o ­ t ues es ­ s ­ i ­ t ­ s ar ­ m only to lymph n ral ner olv ­ i ­ t ­ m ­ v ly ly a pul ­ m ion ion to cen ion ion com ly ly a pul ­ i ­ t ­ t ­ i ous sys ­ tro a a ar ­ v ar ­ n ­ n al glands, mu ­ i ­ i ues is; pul ­ n ­ m ­ s ­ m ­ s ion ion in on with more fre e ­ t o ­ r em em ­ m ­ s ­ s ion ion with dis ­ m ec ­ t ­ f ral ner ­ t las ec ­ f al tracts, cen ­ p ­ n is) is pri ­ tem, ad ­ to ­ s ­ ti aces, and other tis o al al glands ­ f ent of skinent of and bones ­ c es ­ n ­ t y ary in e ­ m n ­ r ­ n ­ i ­ m ion ion with dis lession com ion ion with dis an his o ­ t ­ t ­ t ous sys ­ c ­ to em, and other tis i us sur ­ tro ­ v ­ m ­ t olve ec ec ec ­ r ­ o ­ f ­ f ­ f ­ v ner ne Af Histoplasmosis is pri in glands, and cen in in skin, bone, joints, lymph nodes, ad nose and mouth, less com nodes, spleen, liver, gas Coccidioidomycosis is pri pul Paracoccidioidomycosis (South Amer and ad Disseminated in skin, lung, lymph nodes, gen blas sys in gas , var.

laromyces . capsulatum. istoplasma Penicillium ) H duboisii H capsulatum Coccidioides Coccidioides posadasii Coccidioides immitis Coccidioides Paracoccidioides brasiliensis Ta ( marneffei      64 SECTION 2 nic ­ ga oirs ­ v ­ er ng ng or ents ­ i ­ d ay ­ c er ­ t Humans Humans Cats and dogs Humans Soil and small ro Environmental Environmental res Plants, soil, de mat l ­ a i ­ r rn rn e ­ e ly inly rn ­ t ion ­ e ­ i ­ t ­ e ar ly inly soil ­ i ­ bu ­ m i; rarely in ri ar ­ t Microbes of Humans Microbes ­ t ­ m ia), east ca or west ­ r ­ i e ng ng plant ma ­ g ­ i ay ­ c ope, and Hai ope ope ica (Ni ica, East Asia, east ­ r ­ r ­ r ­ r ­ r Eu Af North Amer Worldwide but priWorldwide Worldwide Eu Af Worldwide Eu Worldwide Geographic dis Worldwide, priWorldwide, and de ects ­ f ion ion (continued) ­ t ion of of ion a arly of al ­ t ­ l ­ n ­ n s ­ i ­ u ec ion ion ­ e ­ f ­ t er ic g ­ t ­ t em ­ a ­ s ­ bu ri ­ t rous skin ren; rarely in ren, in ­ b ­ d ­ d cs to lymph nodes ­ i ng skinng and ­ i hat ­ p olv ues with dis ­ v ­ s raphic dis only, to otheronly, in rous skin in all ­ g ­ m ­ b us tis ­ o e ­ n a only via lym ­ t u ns ­ m ­ c ­ ga ens and geo adults the groin and feet Infection scalp of in chil Infection scalp of in chil beard and gla Infection scalp of Sporotrichosis in sub com Human body sites Infection nails of and skin, par Infection scalp of and gla and, less com or ­ g ­ o ­ gi ungal path F

crosporum crosporum canis crosporum crosporum Mi audouinii Mi Mi ferrugineum Sporothrix schenckii Sporothrix Epidermophyton floccosum Mi gypseum   Fungi Cutaneous fun   Table 2.3 Indigenous and Pathogenic Microbes of Humans 65 ls er ­ t ­ ma (continued) ng ng plant mat ­ i nts ay ­ c ­ de oil Humans Humans Humans and small mam small and Humans Water, Water, de Small ro S Humans Humans a ­ c i l, ­ r an ­ ga ­ c i u ­ t ­ r ands, ­ l ica, ­ r ica, and ­ r sia and Af i; rare in ­ a arly Por rn United Microbes of Humans Microbes inia), Af ­ d ­ l ­ e ­ d ­ u a un ca, Af ­ c ic ast ­ r ­ i ­ i ions Af of as Central of and ­ t ­ e ­ e ­ g ­ ca ­ i ope (par ion of Bu of ion ­ r ­ t Worldwide Primarily in Eur Worldwide Tropical ar South Amer Asia; south States Worldwide Tropical re Asia, and Central and South Amer Southwestern Pacific is Southeast Asia, Central and South Amer Western Hemisphere Western Spain, and Sar Eu na lly on ­ a ­ m ng ng ­ i ion ­ s ic hairsic a lud ­ b ­ c ­ c aces in ­ f arly) feetarly) ­ l rous skin, and feet ary, or pu ­ l ­ u us) and oc ­ b l hyte) ­ v ­ i ic ­ p ­ t ra) palms of hands of o ­ g ­ t ody sur a rous skin rous skin, scalp, and beard ­ b ­ b b ­ ­ m lly, the dorsally, the of feet ­ a ion ­ s a ­ c only beard, ax ­ genic der ­ m ­ o nails and skin Infection scalp of (fa path Infection (black piedra) scalp of hair; less com Infection all of Infection skin of and nails (most com nails, hair, and (par and, oc Infection (tinea ni Infection scalp, of gla Infection gla of Infection gla of

) Exophiala richophyton richophyton richophyton richophyton werneckii ( Phaeoannellomyces T schoenleinii Mi persicolor Piedraia hortae mentagrophytes rubrumTrichophyton T concentricum T megninii T



 crosporum    66 SECTION 2 ent. nic ­ m ­ ga oirs on ­ r ­ v i ­ v ­ er ng ng or ­ i s ay ­ e ­ c ­ al i ­ r e ­ t mans Hu Humans Cattle and hors Humans Ubiquitous in the en ma Environmental Environmental res Soil, plants, de a ion ­ c ­ t i ca, and ­ i ­ r ion arly in the ­ l ­ t ­ bu u ­ u ri ­ b Microbes of Humans Microbes ic ­ t ­ t ri ­ t ica, Middle East, ­ r ­ ca o ­ i ­ c ope, South Amer ­ xi ­ r United States and Latin Amer Worldwide dis North Af Worldwide Geographic dis Central Af and West Worldwide, par Eu Me Worldwide Worldwide ions ­ t (continued) ec ­ f rous skin, ion ion ­ t ­ b eys on ­ n ­ bu ­ m ri ­ t atic mucormycosis es, and kid ocks ­ t ­ m ­ g ions, as as well in ­ t in raphic dis ­ n ec ­ g ­ f es feet of ­ l s ­ e ac ­ f en), as as well skin and nails ­ g ­ o ens and geo Infection scalp, of beard, nails, and other skin sur path theof skin, me Pulmonary in Human body sites Infection scalp of and hair Infection scalp of (most com Infection scalp, of as as well gla nails, and so Rare cause trau of Subcutaneous mucormycosis limbs, of chest, back, or but ­ g ­ o ) Absidia ungal path ( F

asidiobolus asidiobolus ichtheimia richophyton richophyton richophyton richophyton T verrucosum corymbifera T soudanense T tonsurans T violaceum Apophysomyces elegans L B ranarum  Fungi    Mucormycosis    Table 2.3 Indigenous and Pathogenic Microbes of Humans 67 (continued) Environmental, soil, plants Plants ; ) as la, ­ e ­ e u ean or ­ z ­ n ­ e al ar a ­ r ­ c ­ i ly ly in ­ i er ) ­ t ­ i ar rop Microbes of Humans Microbes ­ t ­ m ia, Ven as C. geniculata ­ e ­ n a and, United States, ­ l . lunata ­ m C al ar ­ c ­ i l ( al and sub ­ ga il ­ c rop ­ i ­ e ­ z ica, Ro ­ t ia, Thai ­ r ­ d Worldwide Primarily in Med sub Worldwide Worldwide, Worldwide, pri trop In Af Worldwide Worldwide Bra United States ( Sen ues ated us ­ s ­ n nd ­ o al ­ i ent of ent of ­ o e ­ s ­ n ues ­ m a em ­ s ­ t ­ s ent tis ated ­ c olve ­ n a ­ i ­ j ­ v us tis ­ o in dogs) em e ­ s ­ n ive mucormycosis,ive R. oryzae most a ­ s ary or dis ­ t ated, or cu a ­ n u ­ n ­ v o ­ i ng ng spread from ­ c ­ i ­ m em olv ­ s ­ v A. falciforme is the sec C. genicu/ata on cause in the United States) nx to brain. ­ m ­ y arly in ­ l on. ­ u har ­ p ­ m osa, with spread into ad ic o ­ t ­ c ­ s com na par Primary cause in of mucormycosis Uncommon cause of dis Pulmonary, dis mucormycosis Occasional cause rhinocerebral of mucormycosis, as as well in mu mucormycosis Mycetoma ( Subcutaneous mucormycosis na of Rare cause of pul most com bone, skin, and sub Mycetoma ( ) . ) C ­ ma ­ to , A. spp. ­ ce C. lunata spp. ( , spp. , A. recifei spp. rvularia ucor hizopus M R kiliense ( A. falciforme C coronatus Cunninghamella bertholletiae Rhizomucor pusillus vasiformis Saksenaea A geniculata Cu

 onidiobolus Eumycotic my  cremonium   68 SECTION 2 er ­ t oirs ng ng ­ v ­ i ial (i.e., ial (i.e., ­ r er, er, food, air ­ er e ay ­ t ­ t ­ c ng ng plant mat ­ i ay ­ c ion, soil, wa ­ t a ­ t ­ e Water, Water, de Environmental Environmental res Soil, hard plant ma thorns) veg Environmental, de sh a, a, ­ i uay, uay, ­ g ­ na ia, M. ­ tin ­ d ­ ti en en ­ g ion ia, South il, Brit ­ g ­ t ­ z ­ d a, a, a, a, Uru ­ dan, ico, ico, ­ bu M. on, Guinea, ope, In ­ x ­ tin ­ tin ­ r ­ o ri la, la, Ar l, In r ia, Zaire ( Microbes of Humans Microbes ­ t ­ e ia ( ia en en ­ e ­ d ­ l ­ l u ­ ga ­ g ­ g and, and, Ar ia a a ­ z ­ l ­ e ) ­ dia, Su ­ n ­ e ­ m ­ m a ­ m la, la, Ar la, la, Ar ia, Cam ­ e ­ e ia, In l, So l, So ­ l ­ ca ia, Sen ); ); Ven u u ­ n ­ i uay, uay, Chile, Bra ­ l ­ z ­ z ­ ga ­ ga a a ­ g aya, aya, Thai ­ e ­ e ­ tra ­ e ­ e ­ a ­ l ­ m ­ m orldwide ­ dia, Ro Ma United States, Eu Ven mycetomatis Ven Geographic dis United States, Me In So Par Indies,West In grisea Sen Aus Amer Sen Ro W ­ es, or (continued) ­ s o ­ c ­ i ion ion ­ t le le of ­ b a ­ bu ­ p ri ­ t ase, tox ­ e s s phaeohyphomy on cause in United , and A. niger are the ­ ou ­ m ens; ca e ­ g ­ n ­ o ive ive dis a ­ s ­ t a u raphic dis ­ v ­ c ­ g ­ tis i , A. flavus ­ n on path ­ to ­ m ion, in ­ t a ­ z i ­ gy ­ n ­ o ­ ler ens and geo Mycetoma; Mycetoma; sub cosis; peri Mycetoma Human body sites Mycetoma (most com States) Mycetoma Mycetoma A. fumigatus most com al col ­ g ­ o i

­ g ungal path ) spp. spp F

otestudina rosatii spergillus niliaceous fun Madurella ( Exophiala jeanselmei Exophiala Trematosphaeria Pseudallescheria boydii Pyrenochaeta romeroi Ne A  Fungi 

Mo

 Table 2.3 Indigenous and Pathogenic Microbes of Humans 69 l ­ a i ated ­ r cts ­ n e ent, ­ i ­ t ­ u (continued) ­ m ­ tam on ­ r i ­ v ng ng plant ma ­ i ng ng food prod er ­ i ­ t ay ects at ­ c ­ s ­ r ­ o i ­ r e ­ t Ubiquitous in en Environmental, con de grain, wa Soil, in Soil, de Plants, soil Microbes of Humans Microbes Worldwide Worldwide Worldwide Worldwide Worldwide us ­ n - is, is, ens; ­ t only, only, ants) ­ i ­ g ­ n ates are

is and, ­ m ­ i ­ l ­ o ­ t us i on ­ n ­ t ails and (less ­ a ­ m am ­ t ­ n ion, and skin is, is, si ­ t uent lab are the most ­ t is, skin and nail on path i ­ t ­ q ­ i ec ­ d F. monili, and F. ­ f ­ m a , most iso ary tract (this ge us ar ­ m ­ n ­ n ­ c ion o ­ t ­ t o ion ion of toe ails ­ t e ­ d ­ n ary in ec ­ c ­ f ec ­ n ens; cause ker ion and,ion less com ­ f er ion of Talormyces of ion o ­ t ion, si ­ t ­ g ­ g ated as lab con ­ t ­ l is the most com ­ o ­ m ec ep ­ f ec ue in ­ c ­ f ­ s only, only, en P. lilacinus and P. ants ant; in , F. oxysporum ­ m uses, and uri ­ n ­ n ­ i ­ i ­ n is, pul en, as as well a fre ­ t on path only) only) fin are the most com ion, and my ­ g ­ t ­ m ­ m ­ o am uently iso hri ­ tam ­ t ­ temic in ­ q ec ­ p ­ f Penicillium ) marneffei sys cause in eye S. brevicaulis in con P. variotii P. com Phaeohyphomycosis bone, of skin, ears, si eyes, F. solani forme less com and other dematiaceous fungi are fre path com con ne With the ex and soft tis ( ­ gi spp. spp. spp. spp. aecilomyces enicillium Fu P Scopulariopsis A P sarium spp. Dematiaceous fun lternaria

70 SECTION 2 oirs ­ v ­ er Soil, plants Plants Environmental Environmental res Soil, plants as ­ e on in ion ­ m al ar ­ t ­ c ­ i ­ bu ri rop Microbes of Humans Microbes ­ t ­ t al and sub ­ c ­ i er SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th ­ t Worldwide Worldwide Geographic dis Worldwide, most com Worldwide, trop (continued)

) ty ­ i ion ion ­ t al ­ t ­ bu ri ­ t em. Opporunistic. ­ t uses, bone, eyes, C. carrionii ­ n ant ant ­ n ­ n ous sys ­ i ­ i ase, high mor ­ v raphic dis ­ e oll Funke KC, G, Landry ML, Rich ­ r ­ g ­ tam ­ tam ) ral ner ­ t ens and geo C. bantiana C. ( Chromoblastomycosis ( Common con skin, cen Phaeohyphomycosis, si Human body sites Common con Neurotropic dis ­ g ­ o spp. spp. ungal path on, D.C., 2015. on, D.C., ­ t F and ) ng

­ i spp. Adapted from Jorgensen JH, Pfaller MA, Car a C. bantiana C. Bipolaris Fungi Cladosporium Cladophialophora ( C. carrionii Table 2.3 Press, Wash Indigenous and Pathogenic Microbes of Humans 71 (continued) ion ­ t ­ bu ri ­ t Worldwide Worldwide Worldwide Unknown Worldwide Worldwide Worldwide Worldwide Worldwide Worldwide Worldwide Geographic dis Worldwide Worldwide Microbes of Humans Microbes ly thely same as other um um; extraintestinal sites um um um um ­ b ­ c ­ c ­ c ­ c ­ c ­ c ­ a ion ­ tine ­ t es ­ t ­ bu ri ­ t on and ce on and ce on and ce on and ce on and ce on and ce ­ l ­ l ­ l ­ l ­ l ­ l spp. a ­ s raphic dis lude liver, lung, brain,lude liver, skin ­ g ­ c Brain, skin, lung eye, Entamoeba Brain, CSF Lumen co of de Newly Lumen co of in cribed, prob Lumen co of Mouth Lumen co of Lumen co of Primarily large in Colon Human body sites Lumen co of Brain, CSF ens and geo ­ g ­ o spp. ­ lates ­ bae ­ el arasitic path P

aegleria fowleri ntamoeba coli Acanthamoeba Balamuthia mandrillaris nanaEndolimax Entamoeba bangladeshi Entamoeba histolytical/dispar hartmanni Entamoeba Entamoeba gingivalis Entamoeba polecki Iodamoeba butschlii Chilomastix mesnili Dientamoeba fragilis E N

Parasites Protozoa: amoe Protozoa: flag

Table 2.4 72 SECTION 2 a; ­ c ­ i ope, ­ r rn United ara Desert ica, ara Desert ­ e ­ r ­ h ­ h ean, Eu ­ n a ia, Middle East, a ­ c ­ d ­ r ­ i a ­ n er ion i ­ t ­ t ­ t ­ i ­ wide in North Af en ­ bu ­ g ica south Sa of ri ica south Sa of ­ r ­ t ­ tum ­ r an ­ f in China, In ies world ­ c L. in ­ can Republic ­ i in ­ ca; i ­ m ­ r Worldwide Do Worldwide Worldwide East Central Af Western Hemisphere from south States south to Ar Many spe L. donovani Geographic dis Worldwide L. chagasi in Central and South Amer Af West CentralWest Af Central and South Amer Southwest Asia, Med ow ow us Microbes of Humans Microbes ­ r ­ o e r ­ n ­ e a ­ t iac and smooth is ­ d ­ m ­ y tes id ­ cy ­ d ­ i (continued) is: amastigotes in cu is ­ s ­ s ­ tate, ep ­ a ­ a is: amastigotes in bone mar ion ion ­ i ­ i ­ t ­ s ­ a ­ i an ­ bu ­ m ­ man an ri ­ t e ­ m hra, pros ­ t ­ tin es ­ t ates from spleen, lymph nodes, or liv ­ r i le, glialle, cells, and phago ­ p ­ c raphic dis ions ­ g ­ s Cecum Cutaneous Cutaneous leish Mouth Vagina, ure As b. gambiense with T. Trypomastigotes in blood; amastigotes and epimastigotes in pseudocysts in car mus Cutaneous leish le Small Small in Visceral leish Human body sites Trypomastigotes in brain, blood, CSF, lymph nodes, and spleen or as , ) ens and geo or ­ g ­ o , other ­ jor , L. donovani Trichomonas , ( , L. ma arasitic path spp. ) duodenalis P (aka intestinalis G.

iardia eishmania chagasi rypanosoma brucei gambiense hominis Leishmania L. braziliensis G. lamblia L. infantium G Leishmania tropica Leishmania waltoni Pentatrichomonas tenex Trichomonas vaginalis Trichomonas Trypanosoma brucei rhodesiense cruzi Trypanosoma T L Parasites 





 Table 2.4 Indigenous and Pathogenic Microbes of Humans 73

­ ) with ope; (continued) ­ r

a; orted in all ­ c ­ i ­ p , B. equi cs and ­ i ia ica and Asia; ca and the ­ l ­ i cs and ­ r ­ i ca and Eu ­ tra ­ i te and warm ­ a er arly Af ion ion in trop ­ l ­ p ­ t ­ u u ope, Aus ­ b ic ted in trop ­ r ­ t ica, Southeast Asia, Eastern ­ u ­ r esistant strains re ­ tri r - rib t ­ in North Amer ept Central Amer n cs, par ­ c ­ a uine ­ i ­ ics ide dis e ean, Af ­ q es ­ b ­ w ­ b ­ t o a ­ r rop rop ib ib ica and Asia ­ t ­ t as ex ­ r ­ r ­ r ­ m ­ e r Af and Western Eu North, Central, and South Amer Ca Worldwide world B. microti otherspecies B. divergens (e.g., cli Widespread in tem sub Worldwide disWidely sub chlo a Ca Microbes of Humans Microbes - o­ ­ r tes of ally um ­ c ryth ­ cy ­ n ­ i ­ e o u ­ j ­ r ial cells; ­ l ­ tem he ­ t ­ i ­ tent exo is ry sys nts not typ ect eryth ­ s ­ a al ep ­ f um and je ­ o ­ i ­ n ­ n e um um enterocytes ­ ti ­ d ­ n es ­ o u ­ t tes in ­ j ly samely as other Crypto ­ b ­ cy ­ a o ­ t e ­ cytes ­ m tes and schiz ite of in ite of du ­ i ­ s ­ site of je ­ s o o ­ r ­ a ­ a ­ a ­ z ral blood; no per ory tract and bil ­ t ­ e a ho ­ r ­ p iph spp. pi ­ r ­ s ­ s s; tro ­ e ­ ag sporidium Intracellular par de Newly Intracellular par cribed, prob Intracellular par Ring forms and ga all Parasite Parasite of eryth Colon also in re cytic stage seen in pe ) coli ­ li ) bel ­ rum ­ a ( Balantidium ( Isospora ­ i ates spp. ystoisospora C Neobalantidium B Cryptosporidium parvum Cryptosporidium viatorum Cyclospora cayetanensis Plasmodium fal ­ cip

Protozoa: cil Protozoa: apicomplexans abesia

74 SECTION 2 al te ia, ­ c ­ a ­ s ­ i ar, er ia ay orted ­ p ­ m ­ s ­ l ­ p ay ­ l ines; also arly in ­ p ­ l p ­ u ­ i ic cs (e.g., trop cs (e.g., ies in tem ­ t ­ i cs such as West cs such as West ­ c ­ i on in Ma rop ­ m ­ t on than other ­ m ica (par ion ar, Sri Lanka, Ma ant spe ­ r ­ t esistant strains re ­ n r ­ m ­ i - ­ bu ua New Guinea, Myan only in trop ri om al Af ­ t ­ p ­ d uine ­ c cs and sub ­ m ­ i ­ i ­ q o ­ r ia, Myan a ia, Pa ­ d and ­ s ica), Guinea, New Phil ­ l ia ­ an e ­ r ia) butia) less com ­ d ­ n ­ s o e a; chlo ­ d ­ n ­ c ­ mo i ica, In o orted in Southeast Asia as: less com ­ r ­ r orldwide ­ d ­ e ­ p re West Af West W Present in trop Worldwide; pre ar Af Present in trop plas in In and Guy Worldwide Af In Geographic dis Southeast Asia, most com and Thai ze ze Microbes of Humans Microbes ­ i ­ i tic it it ­ s ­ s ­ cy ­ a ­ a o ­ r tes; m; cysts ­ u al cells ­ cy i ist for yearsist for atic ­ l o ryth ­ s ­ m ­ l ­ p tes par tes par tes in ­ e ­ u he ­ t hy ­ cy ­ cy ­ cy ­ i ic ­ c ­ t o o o ­ t ­ t ­ t with band ium, brain; ate ring forms, in dots; late en e e e ­ t ­ c ­ d ­ r ­ i a al ep ­ m ­ m ­ m ar fluid, bronchoal­ ist in he ar ­ tent exo ­ l ­ n ­ s ­ m ­ c ­ u is ­ ti le ­ s ­ c es ­ t nce for re atic pa (continued) ­ e ­ p r ­ e ear as del P. malariae P. le, le, myo ble ­ ble chro ­ p ­ c ion ion nts, and ga nts, and ga nts, and ga le le ­ t iac mus ­ o ­ o ­ o ­ b ite of in ­ d tes; no per ­ s ­ bu ist in he ­ id ­ a ­ s tes ap ri evel parasitemiaevel can per ­ cy ­ i ­ sem ­ t al mus l o ­ t - o ­ r ­ e ­ z al cells tes; hypnozoites per lly with dou tes, with pref age flu tes tes re ­ m ­ i ­ p ­ a ­ v al and car ­ cy o ­ t ­ cy ion hy ­ z ­ e o o ­ t ­ l ­ c ion ­ r a ­ s ­ u ho ure eryth raphic dis a en ­ t ­ m ­ p ic ­ r ­ c ­ g ­ t hypnozoites per pa Intracellular par veolar la eryth ma stage, but low re Early tro oc ho Trophozoites, schiz Trophozoites, schiz Trophozoites, schiz Cysts in skel tachyzoites in oc blood, CSF, in skel Human body sites tro for ens and geo ­ g ­ o ­ le ­ vax arasitic path spp. P

arcocystis S Plasmodium knowlesi Plasmodium malariaePlasmodium Plasmodium ova Plasmodium vi gondii Toxoplasma

Parasites

Table 2.4 Indigenous and Pathogenic Microbes of Humans 75 rn aii; ­ e ­ w umed ca, ­ s ­ i (continued) an, Egypt, ia, Ha ­ w ­ s ea, south e ­ r ica, China, ­ n ­ r o rn lndonesian ­ e ­ d a ­ c rn Af ­ i ­ e ines, Ko an, In ­ p ooked fish is con p ­ c ­ w ­ i arly in warm, moist ­ l ­ u and, Japan, Tai ­ l ic ­ t iti, Tai ope, north ­ h ­ r ­ bia ­ ana only in Cuba, Central Amer i ­ dia ­ go ­ s om ­ m ­ i ­ l ­ a ines, Thai el ­ p and, Ta ­ p p ­ l ­ i ions) hi ­ dia, Japan ­ c ­ g less com Central and South Amer Southern Eu In Lesser Sunda lslands east of ar Worldwide China, In Thai and Lou re Phil Worldwide where unWorldwide Worldwide (parWorldwide Iran, Co Microbes of Humans Microbes lly lly ­ a ­ a ion ine, liver, ion ­ s ­ t ­ s a a ional es ­ c ­ c ­ t ­ g ae and eggs a: blood Southeast Asia, Phil a: blood ­ v ­ i ­ i ar ar ae: oc ­ l ­ l on, re ­ l ­ v es ­ fi ­ fi ­ tine; oc ­ c es; lar ro ro ­ i es ­ t ­ c ­ c er ae: small in ­ t es; lar ­ v um, co ­ c ­ c ch or in ­ tem; mi ­ tem; mi eric ar ­ a ­ t us, ce ine; eggs: fe ine; lar n t t ­ ­ ue ­ e ­ e ­ s es es ­ t ­ t es al il ­ c g g tis ­ n hatic sys hatic sys ­ tine; eggs: fe i es ­ p ­ p ­ in ­ c ­ m ­ er ­ t ound ­ r dults: liv Adults: small in Adults: small in Adults: lym A Adults: in es lymph nodes, mes Larvae: wall of stom Larvae and young adults in CSF Adults: ter in sur found in fe Adults: lym in extraintestinal sites extraintestinal in lungs; eggs: fe ) ) ­ ca ­ i ( Angiostrongylus ( Angiostrongylus at ­ p spp. pillaria pillaria he nisakis rugia timori Ca Ancylostoma duodenale Parastrongylus Parastrongylus lumbricoides Ascaris Brugia malayi Capillaria philippinensis A cantonensis costaricensis B

Nematodes

  76 SECTION 2 umed ­ s ia, ­ s ica on) ­ r e ara Desert, ­ o ama) and r ­ n ­ h ­ n ands ­ e ­ l ­ a al, and warm ­ c ­ i ca, West Indiesca, West ­ i ean is ico, Pa ico, rop ­ b ­ t ia, Mel ­ x rn coastal and ia, Cam and, Japan ­ d ib ­ r ­ l ­ e ­ r e ion ­ g ooked fish is con ­ t ­ c ica south Sa of al, sub sts Central of and West ­ r ara Desert ­ bu ­ c ­ e ­ h ­ i ri ­ t ines, Thai ca (e.g., Me ca (e.g., rn Asia, In ­ i ions; south ­ p ­ e ­ g ca, some Ca p ­ i ­ i sts Central of Af and West ern ­ ern United States ­ e te re ia ­ a ­ s ast rn part South of Amer e ­ e ­ e er ­ n ­ p ica, south ica south Sa of ­ r ­ r south Worldwide Worldwide Worldwide in trop tem Worldwide Central Amer north Geographic dis Worldwide where unWorldwide (rare) (e.g., (e.g., Zaire, Ghana, Ni West and CentralWest Af Rain for China, Phil South Amer Western Hemisphere, Central and South Af Poly Equatorial rain for Af Microbes of Humans Microbes eal ­ n ­ to a: skin ­ i a: blood a: blood ­ i ­ i ar ­ l ar ar um; eggs: ­ l ­ l es, es, peri ­ t ­ fi ­ i ­ fi ­ fi ro er ro ro es ­ c ­ t ­ c ­ c ­ c ­ tines ­ en on, rec es ­ l ­ t les ­ u ue; mi ues; mi ue; mi (continued) a ­ s ­ s ­ s ­ e ty, ty, mes ty, ty, in ions ix, co ­ i ­ i ­ s ­ d ion ion ­ t ine; eggs: fe en us tis us tis us tis ­ t a: blood ­ p ­ i ­ o ­ o ­ o ary nod us le ­ bu e e e al cav al cav es ­ n ­ o ­ t ar ­ n ­ n ­ n ­ n ­ n ri o ­ l e ­ i ­ i ­ t a a a ­ n ­ fi ­ t ­ t ­ t ­ m a u u u ­ t ro ues om om ­ c ­ c ­ c um, ap ­ s ­ c ­ d ­ d ­ c ted in perianal ar ­ i raphic dis os ues; mi ­ s ­ p ­ g Larvae in pul Adults in cu Adults: sub Adults: sub de Human body sites Adults: ab Adults: ce Adults: ab snips Larvae: Larvae: tis tis Adults: small in Adults: sub ens and geo ­ g ­ o spp. spp. arasitic path P

cator americanus ansonella perstans nathostoma nterobius vermicularis oa loa oa M Dirofilaria immitis Dirofilaria Dracunculus medinensis Eustrongyloides ozzardi Mansonella streptocerca Mansonella E G Ne L

Parasites

Table 2.4 Indigenous and Pathogenic Microbes of Humans 77 ia, rn ­ d ­ e ia, il) ­ s ara ous ­ z (continued) ands, ­ h ­ r ­ l ay cs (In ­ o ca ­ l ­ i al ­ i ­ c iv ­ i il, West il, West ­ b rop ­ z ca (south ­ t ­ i or, or, Bra ope and North ­ d ia, Ma ­ r ines, Sri Lanka, a ­ s ­ p ­ u e p ­ n ­ i o ­ d ica south Sa of only in trop ­ r ia, Ec arly in warm, moist arly in warm, moist arly in warm, moist ly inly Eu cs and sub ted with her ­ l ­ l ­ l ­ i ­ i ­ b ­ m ­ a ­ u ­ u ­ u i ar ­ c ala), South Amer ic ic ic om am, South Pacific is ­ t ­ t ­ t o ­ l ­ m ­ m ­ n ­ s e ­ t en, Central Amer ­ m la, Co esh, China, In ­ e a; less com ­ d u ­ c ­ z ­ i ries) and, Viet la als) ­ t ­ e ­ l ico, ico, Gua ­ g ua Guinea, New Phil ica, Egypt, Costa Rica, Bra ­ x ions) ­ m ions) ions) ­ r ­ p ­ i ­ g ­ g ­ g Pa Thai Af Indies) Ban Widespread in trop re coun (parWorldwide Worldwide Worldwide (as an Amer Worldwide (priWorldwide West and CentralWest Af Desert; Ye Me re re (parWorldwide (Ven Worldwide (parWorldwide Microbes of Humans Microbes ue ­ s us ­ o ous ­ i ­ v a: skin ­ i le le tis ­ c ar ­ l ix; eggs: a: blood ­ d ­ fi ­ i ral ner ­ t ro en ar es ­ l ­ c ­ p ­ c es ­ fi ­ c st in mus ro ­ c ­ cy ae found in var ae: ae: fe les; mi ­ v um, ap ­ v ­ u ­ c ae: en ­ tem; mi ­ v e; e; lar ine; eggs: fe us nod ­ t ­ tin rans; lar ­ tine, ce ­ o ­ g e es es lly blood or urine es ­ t ­ t ­ n ­ t ­ a a ng liver, eye, and eye, cen liver, ng ­ t ­ i hatic sys ­ tines; lar u ion ­ p ­ c ­ s lud a ­ t ­ c ­ c ues in ­ tem es ­ s ­ c Adults: lym Adults: small in Adults: in es Adults: large in fe Adults: sub snips; oc tis Visceral larva mi sys Adults: Adults: small in us ­ l spp. u ­ v spp. uchereria bancrofti trongyloides stercoralis oxocara W O spiralis Trichinella Trichostrongylus trichiura Trichuris T S

nchocerca vol

78 SECTION 2 am ia, : ­ n ­ d ica, am ­ r an states aising ­ n ia, In ­ k r ­ s - ar, Viet e ­ n ­ m rn Af o ­ e ­ d er USSR ea, Viet ­ r ­ m a ­ c ­ i ion ­ t er USSR ia, Myan and, In ­ l arly in sheep ­ d ­ m ­ l o ­ bu and, Laos; O. felineus ­ u ­ l ­ b ri ic ­ t ­ t er USSR, north an, Japan, Ko an, Thai ope, for : Thai ­ m ­ r ­ w ­ w esh, Cam ­ d a rn Asia, Far East, Western Hemisphere ries) ­ e la ­ d ­ t ­ a ­ g ope, for ia, Southeast Asia, for ­ r ­ d north In China, Tai Eu Southeast Asia China, Tai Ban East andNile River Southeast delta, Turkey, Asia China, Japan, Southeast Asia, Bal Can Northwest North Amer Southeast Asia Geographic dis Worldwide Worldwide (par coun O. viverrini Eastern Eu Microbes of Humans Microbes es es es es es es ­ c ­ c ­ c ­ c ­ c ­ c es ­ c es es es es es ­ c ­ c ­ c ­ c ­ c (continued) ion ion ­ t ine; eggs: fe ine; eggs: fe ine; eggs: fe ine; eggs: fe ine; eggs: fe ine; eggs: fe t t t t t t ­ ­ ­ ­ ­ ­ on; eggs: fe ­ l ­ bu es es es es es es ­ t ­ t ­ t ­ t ­ t ­ t ri ­ t ­ c raphic dis ­ g Adults: bile ducts; eggs: fe Adults: bile ducts; eggs: fe Adults: small in Adults: small in Adults: ce Adults: small in Adults: small inum, co Adults: bile ducts; eggs: fe Adults: small in Adults: small in Human body sites Adults: bile ducts; eggs: fe Adults: bile ducts; eggs: fe ens and geo ­ g ­ o a ­ c ­ i at spp. arasitic path ­ p P

pisthorchis asciola he Clonorchis sinensis Clonorchis Dicrocoelium dendriticum hortense Echinostoma buskiFasciolopsis hominis Gastrodiscoides Heterophyes heterophyes yokogawai Metagonimus conjunctusMetorchis Nanophyetus salmineola Neodiplostomum seoulense F O

Parasites Trematodes

Table 2.4 Indigenous and Pathogenic Microbes of Humans 79 a) ­ l ­ e u ­ z ca, ­ i rn ­ e ­ e (continued) and ­ l ia ­ d ies in Latin a ­ c ­ c i ­ r ame, Ven ­ n sia, ­ sia, Thai i ng ng Puerto Rico, ian Peninsula, Iraq, ian Peninsula, e ­ r ­ i ­ b ­ b ­ n o and orm) ­ l lud ­ d ­ c ­ w il, Su ries, North Amer ­ t ­ z on, Turkey, In on, Turkey, ea; other spe ar, Ara ar, Ara ies found in Alaska, Peru, ­ r ­ n ­ c ­ c ­ c ­ a s s ia, Thai ­ ga ­ ga pines, ­ pines, In ands, in ­ d ­ l ic ic coun ca (Bra ­ a ­ a p ­ t ­ i ­ i ­ bo orm in cold lakes north of ­ w ean is ca, Southeast Asia, Af ­ i ­ b ib ope, Bal ica, Mad ica, Mad ­ r ­ r ­ r ­ r f hina, Phil aos, Cam Af Iran, Syria, Leb C A Ca South Amer L Eu (dog tapeWorldwide Southeast Asia Southeast Asia Japan; other spe and Japan Amer Southeast Asia China, Japan, Ko Microbes of Humans Microbes es Fish tape es m; ­ c ­ c um ­ t ­ u ­ sy ­ e ns; p um; ­ o es ­ t ­ ga ­ c ids: fe ids: fe ­ t ­ t ine; eggs: ine; eggs: al bi ­ t ­ t ­ t lot lot es or spu ­ g ­ g ­ c lly in es es ­ t ­ t ­ a um, fe ­ t es es es er and rec ion ­ c ­ c ­ c ues, and or ­ d es, rec ­ s ­ s ­ c on il and lower a ­ l ­ c ive ive tis a; oc ­ t ­ m ec er wall or rec ses blad of ses small of in ses co of ses small of in ­ n ­ d ­ u ­ u ­ u ­ u hy ine; eggs: fe ine; eggs: fe ine; eggs and pro ine; eggs and pro ine; eggs: fe t t t t ­ ­ ­ ­ ­ t ­ c ­ tem; eggs: fe ues; brain; eggs: fe ­ sy ­ sy es es es es es ­ s en ­ t ­ t ­ t ­ t ­ t p p ­ r ­ o ­ o al sys us tis ­ t ­ n ­ n ­ n ­ n al bi al bi ­ o ­ t ­ t psy blad of e al wall, con ­ o ­ n ­ n ­ i a ­ t u om atic por ­ c es, rec es, rec ­ d ­ p ­ c ­ c eggs: bi Adults: lung pa Adults: small in Adults: small in Adults: ve Adults: ve fe ous plex Adults: ve he ous plex Adults: ve ous plex fe Adults: small in ous plex Adults: small in ab Adults: small in sub ­ ma haneropsolus bonnei Paragonimus westermani Paragonimus Prosthodendrium molenkampi Pygidiopsis sum Schistosoma haematobium Schistosoma japonicum Schistosoma mansoni Schistosoma mekongi latumDiphyllobothrium caninumDipylidium P

Cestodes

80 SECTION 2 ca, ­ i arly in ia, New ­ l ­ l ia, North ­ u ­ d ra ­ t ic rn rn South ­ t ico, ico, Latin ­ e ­ e ­ x am ­ n orm) da, north orm), par ries, Me orm) ­ w ope, North Amer ­ t ion ­ a ica, south ­ r ­ w ­ t ­ w orm) ­ r ries ries (e.g., Aus ries (e.g., ­ w ­ t ­ t ­ bu ea, Viet ­ r ri ­ t rn Af ­ e ean coun ope, Japan, China, In ia, ia, China ­ r ­ p ­ ca ­ d ­ i o ­ r aising coun aising coun a); parts Eu of r r ca (Alaska, Can ca, In ­ c - - ­ i ­ i ­ i le le Eu and, south ­ d ­ l Sheep Latin Amer Worldwide (beefWorldwide tape Sheep Northern Eu midwestern United States) (ratWorldwide tape (dwarfWorldwide tape China, Japan, Ko States United Amer Amer Geographic dis Worldwide Worldwide (pork tape Zea Amer and the Orient mid Microbes of Humans Microbes es es; le, le, ­ c ­ c ­ c ids: fe ids: fe le ­ t ­ t ­ c ues, mus lot lot ­ s ­ g ­ g ues ­ s ae form cysts in es es ­ v us tis ae form cysts in any ­ c ­ c ­ o ­ v e ) form cysts in us tis ­ n em ­ o ­ t a e r ­ t ­ n ­ e u ase; lar a (continued) ­ c ­ t ­ e ase; lar u ­ e ng ng brain and mus ­ c ­ i ion ion ous sys ­ t ­ v ine; eggs: fe ine; eggs: fe ine; eggs and pro ine; eggs and pro arly liv t t t ­ ­ ­ ­ t id disid lud ­ l ­ t ­ c ­ bu ­ u id disid a es es es es ­ t ­ t ­ t ­ t ­ t ­ d ri ic a ­ t ­ t ­ d ng liver, lung, and liver, ng brain ral ner ­ i ­ t ues in ­ s ­ g ­ g lud ­ c Cysticercus cellulosae ue, par ­ s us tis e ( ­ o raphic dis ue in ­ i ­ va ­ s ­ g Unilocular hy tis Multilocular hy any tis Adults: small in Adults: small in Larvae mi Larvae mi Larvae form cysts in andeye, cen sub rate to brain Adults: small in rate in sub Human body sites Adults: small in lar var ens and geo ­ g ­ o d. ­ i al flu ­ n arasitic path pi ­ s P ­ b

­ r aenia solium aenia Echinococcus granulosus Echinococcus multilocularis Echinococcus Echinococcus vogeli diminuta Hymenolepis Hymenolepis nana Spirometra mansoni mansonoidesSpirometra multiceps Taenia saginata Taenia T CSF, CSF, e ce ro a

Parasites

Table 2.4 SECTION 3 Specimen Collection and Transport

Ge­ne­ral Comments 82 Table 3.1 Bacteriology: Collection and Transport Guidelines 84 Table 3.2 Specimen Collection and Transport Guidelines for Infrequently Encountered Bacteria 105 Table 3.3 Guidelines for Collection of Specimens for Anaerobic Culture 107 Virology: Ge­ne­ral Specimen Guidelines 107 Virology: Specific Specimen Guidelines 108 Table 3.4 Recommended Blood Volumes to Collect for Blood Cultures 111 Table 3.5 Mycology: Collection and Transport Guidelines 112 Table 3.6 Parasitology: Specimen Guidelines 117 Table 3.7 Guidelines for Processing Stool Specimens for Parasites 124

doi:10.1128/9781683670070.ch3 82 SECTION 3 The qual­ity of any lab­o­ra­tory di­ag­nos­tic test is di­rectly de­pen­dent on the qual­ity of the spec­i­men that is sub­mit­ted for anal­y­sis. This is par­tic­u­larly true for the di­ag­no­sis of in­fec­tious dis­eases, where spec­i­ men in­teg­rity is of par­a­mount im­por­tance. Not only is it crit­i­cal to col­lect an ap­pro­pri­ate spec­i­men that would con­tain the path­o­gen of in­ter­est, but it is also im­por­tant that the spec­i­men be trans­ported to the lab­o­ra­tory in a timely man­ner and un­der con­di­tions that pre­serve or­ gan­ism vi­a­bil­ity and en­sure the re­li­abil­ity of the di­ag­nos­tic pro­ce­dure (e.g., cul­ture, mi­cros­copy, and an­ti­gen or an­ti­body tests). The fol­low­ ing guide­lines can be used for the most com­monly sub­mit­ted spec­i­ mens. For fur­ther in­for­ma­tion, please con­sult the ASM Manual of Clinical Microbiology, 11th ed. As a gen­eral guide­line for all­ spec­i­ mens, the fol­low­ing con­sid­er­ations should be kept in mind.

1. Appropriate safety pre­cau­tions must be used for the col­lec­tion and trans­port of all ­spec­i­mens. Specimens should al­ways be con­sid­ ered in­fec­tious. Therefore, gloves should al­ways be worn when han­ dling spec­i­mens, and all­ pro­ce­dures should be per­formed be­hind bar­rier pro­tec­tion, pref­er­a­bly in a bio­safety cab­i­net. 2. Many in­fec­tions are caused by mem­bers of the pa­tient’s in­dig­ e­nous mi­cro­bial pop­u­la­tion. For this rea­son, it is im­por­tant to avoid

Specimen Collection con­tam­i­na­tion of the spec­i­men with these or­gan­isms. In many cases, the pres­ence of abun­dant ep­i­the­lial (i.e., squa­mous or re­spi­ ra­tory) in­di­cate a sub­op­ti­mal spec­i­men that may con­tain com­ men­sal flor which may ob­scure cul­ture re­sults. 3. Specimens should be col­lected from the ar­eas where or­gan­ isms are pres­ent and rep­li­cat­ing. Although it seems ob­vi­ous, this prin­ci­ple is of­ten ig­nored. For ex­am­ple, pus typ­i­cally con­tains rel­a­ tively few vi­a­ble or­gan­isms. A more ap­pro­pri­ate spec­i­men would be scrapi­ngs or a bi­opsy spec­i­men from the wall of an ab­scess. Likewise, the ma­te­rial col­lected from the sur­face of a wound is of­ten not rep­re­sen­ta­tive of the or­gan­isms pres­ent deep in the wound. Finally, the di­ag­no­sis of a lower re­spi­ra­tory tract in­fec­tion re­quires col­lec­tion of ma­te­rial from that site (e.g., spu­tum) and not from the mouth (e.g., sa­li­va). 4. The quan­tity of spec­i­men col­lected must be suf­fi­cient to en­ sure that all­ re­quested tests (cul­tures, mi­cros­copy, an­ti­gen tests, nu­cleic acid probes, and am­pli­fi­ca­tion tests) can be per­formed prop­erly. If only a lim­ited amount of spec­i­men can be col­lected, tests should be per­formed se­lec­tively. If too many tests are at­ tempted, no test will be per­formed ad­e­quate­ly. 5. Traditionally, cot­ton-wrapped swabs have been dis­cour­aged for spec­i­men col­lec­tion. However, with the advent of the flocked swab with liq­uid Amies trans­port me­dia, this is now an ac­cept­able and in some cases a pre­ferred spec­i­men col­lec­tion sys­tem. Flocked swab sys­tems are par­tic­u­larly use­ful for those labs uti­liz­ing au­to­ mated spec­i­men pro­ces­sors. Specimen Collection and Transport 83 6. Transport of spec­i­mens should main­tain the vi­a­bil­ity of the eti­o­logic agent (if cul­ture is per­formed) and pre­vent over­growth with con­tam­i­nat­ing or­gan­isms. 7. Specimens should al­ways be trans­ported in a leak­proof con­ tainer in­serted in a leak­proof plas­tic bag with a sep­a­rate com­part­ ment for the req­ui­si­tion. Use of plas­tic bags al­lows the spec­i­men to be ex­am­ined be­fore the bag is opened. Every ef­fort should be made to col­lect a sec­ond spec­i­men if the orig­i­nal spec­i­men is re­ceived in a leak­ing con­tainer. However, if an ad­di­tional spec­i­men can­not be col­lected, the lab­o­ra­tory should at­tempt to pro­cess the spec­i­men if it can be done safe­ly. 8. For off-site spec­i­men trans­port guide­lines, re­fer to the Interna­ tional Air Transport Association (lATA) Dangerous Goods Regula­ tions (http://​www.​iata.​org/​publications/​dgr/​Pages/​index.​aspx), the U.S. Department of Transportation (https://​www.​phmsa.​dot.​gov​ /​hazmat), and the International Civil Aviation Organization (ICAO) reg­u­la­tions. When pre­par­ing a spec­i­men for trans­port, al­ways check the spec­i­men trans­port guide­lines of the re­ceiv­ing lab­o­ra­to­ry. Specimen Collection 84 SECTION 3 ng ­ i ion ion ­ s iz ­ n ­ o ace ­ f uce col ­ d ro olved inolved the ­ t ess. ­ v ­ c ive. cess wall are most ­ t ­ s ia not in ial will in ­ r ­ tious pro ­ r uc e e ­ t ­ d ­ t ec ­ f Comments pro Samples the of base the of le and ab Contamination with sur ma bac in ng ng ­ i ays ays ia is ­ w ect two, ­ d ­ l on swabs ­ t le flockedle ­ g id meid ng. Preserveng. ial by plac ure and one for ­ i ­ u ­ t ­ r te for both e ­ t ­ a en. If cot rt’s or Amiesrt’s ri ­ a ­ p ior to ior a swab ­ m ­ r ­ i ium. A sin ure and Gram stain. ro e ­ d ­ t ­ p ­ p 2 h, RT 2 h, RT Tissue or fluid is al Transport time and temp su spec must be used, col one cul for Gram stain swab ma inswab liq ap cul ≤ ≤ in Stu me Specimen Collection a,b ­ tem ort ­ p ice and/or ines ­ l ­ v ort sys ­ p m vol ­ mu ort guide ­ i em, 1 ml ­ t ­ p naerobic trans Transport de min sys Swab Swab trans A ion ion ­ s ­ ­ rial ate ic ic e ­ t ­ b ­ d le le ol inge; er” ­ i ­ o ­ u ­ r ­ h ­ d ines le orle pass o ­ l er all er ­ b ­ f ­ c ­ a i ­ s le thele ­ ce ess ess ma ­ p i ace ex ­ v ­ f ­ sc le andle sy ­ d ng withng ster ally trans ­ i ort de ­ c i ­ p ­ rial into an ­ t e ­ t ine or 70% al ­ l sion’s ­ sion’s “fresh bor sa Collection guide by wip by trans Remove Remove sur Aspirate if pos deepa swab into the le asep ma to firmly sam with nee le Aspirate ab acteriology: Collection and trans B

al ­ r ­ ne e G Open Closed Specimen type Specimen Abscess

Table 3.1 Specimen Collection and Transport 85 ls le. le. ­ a ro­ i ­ b ­ c i ­ b i ­ s ­ m ro i ­ c ­ t (continued) i ithin 24 h ­ m i ­ w ­ t ls). ls). ain two or ls if pos ithin min 10 ­ a ­ a ­ t i ures are i ­ a ­ t i ode, an ­ b ­ b ­ w ­ s ­ b ­ i ro ­ cro ­ cro ase, an i i ­ c ­ e i ­ m ­ m y: y: two sets from y: y: two or three sets i i ate sites, within h, 1–2 ate sites 1 h apart, ­ m ­ l ­ l ate sites all ­ t ­ t i ­ r ­ r ­ r rile ep ­ t te te ­ a ­ a ­ a ­ b als no closer than 3 h ­ a ­ a ­ v i i ­ d ­ d ive at 24 ive h, ob ate sites, all er e e ­ t ­ r ­ t a ­ ­ a ­ fore an ­ fore an ore an ls to be started or changed ­ m ­ m ­ f ­ a (be bi im sep Acute fe Nonacute dis at in will not be started or changed im from sep (be be Endocarditis, acute: three sets from sep from sep neg three more sets. within 24 h. If cul red) ion ion ­ e ­ t al bite ov ­ m ec ­ i ­ c ­ f ure an ­ t 12 h old (agents h old 12 nt. lly not re ­ e ­ a ess signs of in ­ l 2 h, RT Do not cul un wounds ≤ are usu are pres ≤ Specimen Collection evel me me, l uires - ­ u ­ u les for les for ­ q ­ t me most ren (even ren (even ­ u ure bot ­ d ­ t m safe vol l l blood vol ive) mia that re ­ t es) have low ia; adult, 20 ml/set ­ e ­ t ­ r ­ mu ­ ma a uc ­ i ­ i e er ­ n ­ t ­ t ­ d ­ o ne Blood cul bac (higher vol pro Pediatric, draw the max many chil bac max le; ­ t yl yl ­ p ic ic to ­ l o ro ­ n ­ p ure bot ­ t ers and wait ­ p ol ol or phe er stop ­ h ly ly 70% iso ­ b o ­ p ­ c See Abscess See Disinfect cul 1 min ap al rub Bite wound Blood 86 SECTION 3 us le ure ­ t ­ o ­ t ­ u in ­ t l bot ween ­ t ­ ga ial cul ­ c ent con y; y; rarely ­ l ­ m te ­ u ate that an ive thanive the ic ic or fun ­ a ­ c ­ t i ­ b i le. Pediatric: ­ d ­ t ­ o ­ d uc e ­ d ­ m ic bot mia with hours be ­ b ary to doc ­ e ­ o ­ s ional aer s er ­ t ems (e.g., Isolator, Bactecems (e.g., i er ­ t ­ e ­ t ­ tures. ­ a ­ d Some data in ad is more pro an Collect im nec bac Comments cul Mycobacteria: spe Use sys Bactec 13A, Myco/F Lytic). ain ­ t in: ­ g ­ i iod. If ­ r e nown or h p - ­ k ive at 24–48ive h, ob ate sites 1 h apart ­ t ­ r ng a 24ng ­ a ­ i ­ a dur neg two or three more sets two or three sets from sep Transport time and temp Fever of un Specimen Collection (continued) a,b ice and/or ines ­ l ­ v ng on weight of of onng weight ­ i m vol end ­ p ­ mu ort guide ­ i ient ­ p ­ t Infant and child, ml/ 1–20 set de pa Transport de min le le ­ i unc­ er, er, unc­ ­ t ­ ster ­ p ­ p i ally, ally, i ure, ut ines ­ n ­ c ol. ­ t ­ n ­ l ­ o ­ h ri ine to dry. ore ­ t o ­ f ­ d ­ c ate vein at unc ine ine from the en ­ p ­ p ­ d ­ d ­ c i ion. ­ n ­ t a ion ion of ve ­ r ­ t ol. ng atng the cen ­ i ­ a ­ h ove io ove ec o ­ f ­ m fter ve o not pal ­ c leanse site with70% wab wab con n ­ i with an io prep start  S re skin with al  A  D this point with glove.  C al 3. Allow3. the io 4. 5. Collect5. blood. 6. Collection guide 2. ture site: 1. Disinfection of ve Palpate vein be ture site dis acteriology: Collection and trans B

Specimen type Specimen Table 3.1 Specimen Collection and Transport 87 ial ion­ ­ r t ­ ow ow ered. e ­ r ­ t ­ d (continued) tion, ­ tion, psy a ure only. ure only. ­ rial, ­ o ­ t ­ t e ectly onto n ­ t ­ r ers for ers for ow is rarelyow ure (Maki ­ t ­ r ­ t m when e ­ ­ me ­ i ic culic l al ar ure may or may ­ vance. ­ mu ures are or ­ a ­ b ­ t i ­ t ­ e ­ er ­ o ­ t er ated di ral, CVP Hickman, ­ l ive ive cul les bums of may be ­ p ures are ques of ­ t ­ t ­ t ­ u ble. Culturesble. of ia. Routine bac ­ p m punch bi a ­ riph c ­ a mes bone of mar ­ t ­ d al rel i ­ o m ­ u ­ t ­ c - ive ive cul l, l, hy ng. ­ i ­ t anz. ­ i en is op a ­ ca ­ t G d): cen uan ­ i i - ­ m ­ o ­ t ­ q ­ i to 4 il ead ace sam ure me ure bone of mar - ­ l ­ ful. ­ f ­ b ­ t ­ t may be in cul cul use Small vol spec mis A 3 quan Process aer for Quantitative cul Catheter cul able clin semi not be valu sur cath i.v. Acceptable meth Broviac, pe um Swan ure ­ t le orle tube ­ t 24 h, RT 24 h, RT, if in24 cul h, RT, 15 min,15 RT ≤ ≤ bot ≤ Specimen Collection ­ r; en ­ ga ­ e u ainer ­ m ­ f ­ t ­ i em ain ry ate; y ­ t ­ t ure ri ­ t ­ d ­ t ­ t ­ i ged due ­ to ­ u ­ a a ures ­ r ­ t le le con ­ ibil ­ o ­ i ap tube or our ort sys c ap con is cen uc ­ c - c ­ p ­ s ­ d - y ­ l e ­ pro ­ at i ed to lab ort in ster ­ d ­ er ­ p ate or swab ex le le screw le le or a ly ­ r ­ i ­ t i iv ­ me ­ l ­ p Inoculate blood cul bot tion tube; plated spec as trans to to low re de Tissue is placed into a ster should be dis or swab transor swab Quantitative cul im Sterile screw cup ory ­ t tely tely to a ­ r ­ a i ­ o ove ove ­ d e ion ng. ­ m ­ i ­ s > site with i ­ m ectly into a ride the burn y lab ­ c ure site as ­ r ­ t ­ b er(’ ­ og ­ t l ­ e ­ o al in i ent dry er and clip 5 cm of ­ c ­ b ­ v i ­ t ol. le le tube. ­ e ­ h ­ g ­ i al tip di ro t ­ o ­ c septically re ­ c leanse the skin around ransport ransport im  A cath dis ster to pre  T mi the cath C  al Prepare punc for sur Clean and de 1. 1. 2. 3. 3. ­ r ate ­ r i i.v. ­ p Bone mar as ow Burn Catheter

88 SECTION 3 l­ ­ o i ­ b ured ­ t ro ens in ­ c ­ g tubeit two ure. ­ o ­ t ­ m ected, it ­ l ure also. If only ens cul ­ t ­ m ed to mi ­ i ­ t ise sub ial path it ­ t ­ w ­ m r gy. gy. Aspirate of brain en ­ e ble ble for cul ­ o ­ t l ­ a ­ o i ­ b ept ty of spec ­ c ­ i ro ­ c or ­ n Comments Not ac Yield of po mi to mi one tube CSF is of col should be sub ogy first; oth Obtain blood cul for te; ­ a r ­ e rig ­ f 15 min,15 RT Transport time and temp ≤ Bacteria: never re 15 min,15 RT Specimen Collection (continued) a,b inge ia, 21 ml; ice and/or ­ r ines ­ r ap tubesap ­ l ­ v e c ­ t - ­ ed m vol end ­ m ort not ­ p ­ mu ort guide ­ i uired: bac ­ om ­ p ­ q Transport de min Sterile tube (sy trans rec Sterile screw Minimum amount re AFB, 25 ml le le ­ i ion ion 5, 5, ary. ­ t ng ng er ­ s ng ng L ­ i a ­ t ­ i s - ­ e le andle ­ m a ­ d el intoel ­ r ines ion. ­ p ion ion with pace. ­ l ine or ap tube. ­ t ­ t am ­ s ­ l c a le withle A, L4 ­ fl - ­ r er I ­ ga ine into ­ d ­ t i ­ a ­ l - ­ tal flo ol. ­ r le sale ­ h ­ i m in ure, since growth o 1 in ­ t only the cen ­ c S - ­ m ­ mu ­ i le screwle ­ i et at L3 ents dis inge; ir ine prep inge, and ex ­ l ine may be nec ­ s ­ r ­ r ­ l spirate the area of ­ d spirate sa isinfect site with leanse wip site by e nsert a nee ­ r  max  A I sty or L5 a small amount ster of sa rather than the lead edge) with a nee sy (com ster  A sy C  with ster 70% al  D io Collection guide 2. Do not cul rep 1. 2. 1. 1. 3. 3. acteriology: Collection and trans B

ey ­ l ate from ­ r i Fo ­ p Specimen type Specimen Cellulitis, as area of CSF Table 3.1 Specimen Collection and Transport 89 zed ic ies ies ­ i ­ b ­ r ­ o psy en may ial ­ to ­ o (continued) ion andion er ­ c ­ t a ­ m ­ a ­ r ­ i ized ate is the es no ­ o ­ r ec ­ n ­ d ­ t i en a of i g ue bi ­ p ­ v ­ s ­ o ­ m ­ i ect ect an ­ t ide spe ion, it should not ­ t ites. ­ v le asle a ions should be ­ s psy spec ­ s ­ d ­ a ­ o er pro ­ m ­ c or ed. A tis ­ f ­ t ion ion of rec ary to de ­ t it ­ s ­ a us ul en of choice. ­ t ­ m ­ es ia or par al in ­ gens. iques the for de er le orle nee ­ m ­ r ­ bi ­ c ­ i essed essed only by lab ­ o ­ n ­ p e ­ i ­ m u ­ c ­ t cess or a bi ­ c ­ s ab be nec bac Since spec a swab de spec clin be sub sam Periodontal le pro equipped to pro enu path tech 2 h, RT 2 h, RT ≤ ≤ Specimen Collection ic) or ic) ­ b ort ­ o ­ p ­ tem (for ic ic sys ­ b ­ o er ue) ­ tem ­ a ­ s Sterile tube (aer an tis sys Anaerobic trans en er. ­ c iva, iva, ove ove ­ m ­ l ­ i ial er it ­ m ­ r ­ f al ­ t ­ te on et and ove ove sa ate ­ si ­ l in and psy is not ic ic don ­ r ­ tem. ­ o i ­ g ace with ­ b ­ m ­ o ully re ng ng the ­ f ­ p ­ f ­ i ­ o oid oid space, ine. ­ tory ma er ­ l le bi le ­ n a ­ a ­ p ­ m ort sys al mar ial and trans ble, ble, as roof tubes. ­ v ­ p ­ r ­­ a rach i le le sa ­ p ace to re e ove the styove ­ i ect 1–2 m1 of fluid of m1 ect 1–2 ris, and plaque. ­ a am ­ g ­ t ­ f ­ l ­ b ­ fl ­ m sing a peri leanse sur arefully cleanse f a sam  I avail in from the base the of ul  U scaler, care subgingival le ma to an an trans sub col  U re into each three of leak  C ster de supragingival tooth sur  C gin 3. 3. pon reach A swab is notA swab the spec of choice 1. 2. 1. 1. 2. al, ­ t s ­ ti al, al, ­ ti ­ v ent’s a ­ odon ­ c ­ gi ­ ture: ­ m ­ cer Decubitus ul peri periapical, Vin gin sto Dental cul 90 SECTION 3 le le yn­ ­ b ­ r i ­ s ha ­ p o pon ose. ­ s ­ s ­ p ures are not ­ t ia and should not be ­ d ed for that pur ive ive of agents re is me ­ t ­ t ­ t it ic ­ m ­ d Comments Results throat of or na geal cul swab pre otifor sub 2 h, RT ≤ Transport time and temp Specimen Collection (continued) a,b ice and/or ines ­ l ­ v ium, or ­ tem ­ d m vol ic ic sys ­ b ort me ­ o ­ p ­ mu ort guide ­ i er ­ p ­ a Sterile tube, swab trans Transport de min an is ect ­ t le le ­ l en ent, ­ b ­ r ­ i ­ m ­ i rum, ur ­ served ­ c ­ d ique ique ines um. ­ l ­ l ­ n rum, ­ tent oti ­ u ­ d al with is inge ­ n ­ s ­ r ion ion and col ­ t ated, re ured ear u ­ c ­ t ­ l ion ion tech act ear li ­ t ­ t ng ng with spec ­ p ory spec ­ i a on. ­ t ­ i ­ r ect fluid onflex ected in the same ­ l ­ l ­ di ­ pi ia repare smear for or rup or in ­ d col shaft via swab an au  F col  P stain fash fluid via sy as (tympanocentesis).  F clean ear ca soap so 2. Collection guide 1. or chronic per me 3. 3. Tympanocentesis re for com acteriology: Collection and trans B

Inner Specimen type Specimen Ear Table 3.1 Specimen Collection and Transport 91 al a­ ace ­ t ­ c ­ f ngs ngs ­ i re oc ­ p ­ thetic ected ibit ibit ­ c ia. are the s ­ f ­ s ­ h o er ­ p ­ e ­ t ­ t us (continued) ­ o ­ the r en is s ­ o ­ e ­ m ­ i ia. ore an ine the ora. The ts this ­ f ­ d ­ i ist in in ­ fl ­ m le le both ­ s ib er an ­ p uired since sur ro ­ t er ­ h ­ q ­ t ­ c roach, on the rely l me ­ p al spec ia. Corneal scrap ­ v ­ ga ­ r ure. i ated from the in ­ t t ­ e ­ l ­ t le, le, sam iva, even if only even iva, one is ion, which may in ­ t ous mi ­ b is externa, vig ­ t is. ained af i ion apion ­ t unc ­ t ing is re ing may miss strep ­ n ­ t a ­ t ­ j ­ s i ected can eye serve as a ­ e ­ b ­ b ­ c ­ l ­ f unc rol with which to com ec ected, do so be li ­ j ­ t n ig ­ l ­ l ­ lu ected, to de ­ i ­ p ­ f ­ d con in Gram stain to as If con col in con tion of cul some bac Include fun un agents iso If costeye. pro col ap are ob If pos swab swab cel For oti 2 h, RT 15 min, 15 RT; 2 h; RT 15 min,15 RT ≤ Plates: ≤ swabs: ≤ ≤ Specimen Collection ion: ­ t a­ a ­ l ­ l ion: ­ u ­ u ­ t c c a ­ l ­ o ­ o ­ u c ­ o ort ort ­ p ure in ure in ­ p ­ t ­ t ory in ory mold agar ­ t ­ t ­ i a ­ r ib ­ o ­ h tions: BHI with 10% sheep blood, CHOC, and in Direct cul Swab trans Direct cul swab trans BAP and CHOC; lab al. ng ng ated ­ i ­ n a. ­ l ng ng m. ­ i ­ v ared ions, ­ u la, i ­ u ­ s c ­ p ­ t ­ u ion. ­ o ion; roll ­ di ected ­ t m area ­ t ­ l ris or ­ gist. al. c le by by le ­ b ­ o ­ n unc - ec ec ­ j ­ p ­ l ­ l ol ng theng swab ­ i ned to swab le le spat ate scrap ­ m ned with ­ i ers or le ­ l ­ e at ine) by roll ­ e ­ c ­ t ­ l ­ u c hal ­ t ­ o oist ate swabs ­ r le le sa ­ m ove any de ove ­ i ­ a dium may be in ectly ectly onto me ­ r ­ m btain a sam sing ster se moist mear may be pre pecimen is col ample eachwith eye di  U and in firmly ro scrape ul  O in the outer ca at time col of  Me at time col of S  of slide. swab over 1–2 over swab S  by oph  U re crust from the ear ca (pre ster over each con  S sep . 2. 1. 1. 1 2. 1. 2. 3. ngs ­ i Corneal scrap Outer Conjunctiva

Eye 92 SECTION 3 a­ ­ t ogic ­ l ul ­ s ­ o ens are ients. ­ g ­ con ­ t ­ o ept for ut ­ c should be ine stool ­ o ­ t 3 days and the ia. Anesthetics is was not ­ d ­ s o ian. Tests for for ian. Tests ients whose length ­ c ­ n ine path ­ t i ­ t g ory to some eti l me ­ s ­ t is, with ­ a ­ i ended ex ­ t orm rou ­ i ­ ga ­ f ib ­ m er ­ h al stay is > ­ t m ­ t red for these pa i n ­ o ing di ­ e ­ e ­ p ­ t id it ures pa for ­ s ­ tro ­ t ants (see Rectal swabs). ­ m ­ f Include fun Comments agents. may be in Do not per cul hosof gas ad tion with phy Clostridium djfficile con not rec in Swabs for rou for Swabs 24 h, ≤ 1 h, RT 1 h, RT m: ­ u i ­ d 15 min,15 RT ≤ Transport time and temp Holding me Unpreserved: ≤ Unpreserved: RT Specimen Collection ia ­ d (continued) ng ng a,b ­ i ainer or ­ t ice and/or ines ap tube or ion ion of small ­ l ­ v ­ t c - a roof; ­ l ­ p lair hold 2 g) ­ u B c > - m vol ­ o outh con m ­ mu - ium ( ort guide ­ i ­ d ­ p ect in ­ r Transport de min Sterile screw amount fluid of onto me di wide Clean, leak me use Cary r; ­ e gy ­ o l ain m le le ­ t ­ o c i ­ d er to - ­ b ­ f ines ectly ­ l ­ r ial from ng d. ro ­ i ­ i ­ r ­ c e ­ t en di ­ m ­ i la onto 1–2 lair hold ing ma ory within 1 h of ion of flu ion ion or trans ­ u ­ t ort to mi ­ t ­ t ­ b B a ­ um. ­ p a - i ­ r ­ r repare two smears by ec i ­ o ­ d ­ l  P rub spat area of slide. ­ p Collection guide 3. 3. Pass spec into a clean, dry con trans me Prepare nee for eye lab col Cary as acteriology: Collection and trans B

ates ­ r i ­ ture ­ p Routine Routine cul Vitreous fluid as Specimen type Specimen Feces

Table 3.1 Specimen Collection and Transport 93 l l h - ­ to i ected ­ b ­ l (continued) ieve thisieve y. ­ l ng threeng to al cramps ­ t e. ­ i ­ i ults in rapid ­ n ­ u ­ s ­ i et among HEC iv ended. ­ s ­ t ­ E ­ m om ure for 0157:H7 0157:H7 ure for er er than sor ­ d ­ t ­ t m al val st yield. Shiga id stoolsid col ­ c ­ o ­ i ­ e ­ u 20°C re oxin ac ­ t o ­ t ay for all for ay id orid soft stools per 24 ­ s le le clin ­ t ­ u ypes is bet ­ t iod. Testing of formed of iod. Testing or hard y. ients with ab ­ l ­ t ­ r ­ ro e p loss of cy stool is not rec Freezing at − Patients should be pass liq five se within on 6 days of pa thehave high toxin as MacConkey cul on Bloody or liq Controversial: many be is of lit

24 h, nite,

≤ f­ e ­ tem: ­ d r l h, RT l h, RT 1 h, RT ­ e ort sys ­ p 24 or h, 4°C RT 1 h, RT; 1–24 h, 1–24 4°C; 1 h, RT; ≤ —20°C or cold Swab Swab trans Unpreserved: ≤ Unpreserved: ≤ Unpreserved: ≤ Unpreserved: Formalin/PVA: in RT Specimen Collection

ainer, or ainer or ng ­ tainer, ­ t ­ t ­ i roof, roof, roof, ­ p ­ p ­ p in and/or PVA; ­ l 2 g) a > ­ m outh con outh con outh con lair hold B m m m - - - - ium ( ­ d 5 ml 2 mi Sterile, leak wide > wide Sterile, leak Cary me Sterile, leak wide > 10% for r ­ e r ng ng gy ­ e ort ­ i ­ o ­ p l ain ­ t in or ­ o ain er to i ­ l um ­ t r; ­ f ­ s a ­ b ­ e ­ m ro ­ c ain ­ t ite trans ectly into a ­ r ­ s ­ a id orid soft stool orid bloody stool r; soft stool is es di ­ u ­ u ­ e ory within 1 h of ­ c ion, or trans ­ t ort to mi ­ t a ­ p ain ­ r ned as stool as ec ­ t ­ tem (10% for ­ o ­ l ectly into a clean, dry ­ fi ­ r Pass liq di con into a clean, dry con de Pass liq the shape its of con lab col trans sys ova ova and par Pass fe clean, dry con PVA) - ­ - ypes ­ c ­ tion ­ t ­ tious hea) ducing ducing ­ fi ­ r r ­ om ients who ­ ro ec . coli ­ tec ­ t ­ f ­ a oxin E 0157:H7 and other Shiga t pro se ­ ture Leukocyte Leukocyte de (not rec for mended use with pa have acute in di

C. dif cul ile

94 SECTION 3 - ds ­ i us ­ r le le ng of a of ng ­ t ion. ion. One ­ i ­ t lex vi lex Neisseria occi, or for a ­ p , Campylo ­ r ­ c ­ a o ng ng ­ t ­ i ds are best ­ i ure bot ­ t orted in an ect es sim ­ t ­ p ­ p em and need not be and other ­ t iage group of B , Shigella ­ r ble to ble pass a ­ a ic sys ic en. ­ b uged prior to Gram ated at bed site may be , and her ned Gram by stain emolytic strep ­ f ng. Otherng. flu ­ l ic ic blood cul ­ o ­ i rmed. ­ m h ­ i ­ i ­ u ­ b - er m ­ fo c ients un ­ o a ­ a ­ t ­ t ­ a ­ o Amniotic and culdocentesis flu centri ex should be trans an stain Comments cytocentrifuged prep in per aer Reserved for de gonorrhoeae spec and anal car Streptococcus be pa bacter 2 h, RT 15 min,15 RT ≤ Transport time and temp ≤ Specimen Collection ap (continued) c - a,b tely to ure ­ t ­ a ort i ia; ­ p ­ d ice and/or ines ­ r ­ l e ­ v e ­ t le le screw ­ m ort ­ i ­ p m vol ­ ry o ­ t ort im ­ p ­ mu ­ ra ort guide ­ i le le for bac ­ t ­ tem, ster ­ o ­ p lab Anaerobic trans sys tube, or blood cul bot trans Bacteria, ml >1 Transport de min Swab trans le le ion. ­ b ­ t ­ i le a ery. ­ r ­ d heal ng ng skin ­ r ­ g ­ a ­ i ines r ­ l en via y ­ a le; never ­ l it asit much ert a swab ­ b ­ m i ­ s ­ i ond the anal ­ m ­ s us nee ­ y ate the swab ­ o ­ t e ine prep le thele anal ­ n ­ d ion ion or sur ­ p ­ ter. ­ t ion ion of di ­ ta ­ gens. it a swab dipped a swab it in ­ t a u ­ o ­ r ­ m i ­ c d. ec ­ i ­ t ­ p ently ro btain spec lways sub lways arefully in eces should be vis  O per as  crypts. G to sam  flu A fluid as pos sub  F path on the for swab de with io  D ca. 1 in. be  C sphinc 2. 3. 3. Collection guide 1. isinfect over 1. 1. 2. 3. See Abscess See acteriology: Collection and trans B

al, al, ­ n ­ d ­ i ial, ­ ne al, ­ otic, ­ v o es, bile, i ­ r ectal swab ­ ca ­ t ­ t om o i ­ n R ­ d ­ n ­ c Specimen type Specimen paracentesis, perir peri pleu sy ial, thoracentesis

am as Fluids: ab Fistula joint, Table 3.1 Specimen Collection and Transport 95 al n­ ­ n

ia ­ i ­ r ­ me e ace or essed psy or ­ t ngs. ngs. ­ f ­ c ­ i ­ o ac ormed. (continued) ble ble ­ f ­ b ng ng for ­ a ­ i o ­ c pt ial for ­ t ion ion of vag ium ­ t ­ ce ­ d en a ric wash ­ ing. ­ t ­ r ­ t ling sur of i ac ­ p ­ p a. ue. ue. Tissue bi ­ r ause my ­ s not ­ c en must be pro ial ial test tion ­ tion with the com te when hold ­ m ­ b ­ i al flo ­ a ­ na ial tis ­ n ro dly in gas ­ i ­ i ­ i ion ion should be per ­ c ­ c on ­ t ions is ­ fi ­ t ­ b a am ­ r ­ mi ­ t ause the of po er i re ar ­ c ­ ti ­ p ­ c ­ p ­ c Discourage sam su as promptly be die rap Neutralize with so bi The spec >1 h. Culture may be needed for an se Swabbing or as be con sal vag ze ­ i ral ion ­ t ­ t ec ­ l 2 h, RT 15 min, RT, or min, neu 15 RT, ≤ within col 1 h of <1 h, RT <1 ≤ Specimen Collection ort ­ p ort ­ p roof ­ p 1 ml r ≥ ­ e ­ um i ­ tain ­ tem, ­ d con Sterile, leak Sterile tube with trans me Anaerobic trans sys l­ ­ ­ o ­ te er ­ tric ng ng y ­ t er. ch. ­ i s ­ t ­ p n ­ a o ­ e ­ cen ­ ga ­ c roof, ng ng r. o io ­ i ­ p ­ s ­ e ­ tro le andle y os ­ n ­ p ­ d age with ­ er ­ v ­ tain iv ­ tilled wa ­ l ect dur ients eat and ng ng en ­ l ­ t ­ i le le dis le con an de ­ i ­ i ­ e ecover sam erform la ntroduce a na ore pa 25–50 ml chilled, of ster  P ist dur place in a leak  R ster  I tube into the stom ­ f ­ sar ­ g See Abscess See Collect in early morn while they are still in bed. 1. be 3. 3. 2. Aspirate Aspirate via am sis, or col ce Collected by gas o ale e­ ­ t i ­ m ­ r ac o ­ l ­ b d ­ co age for ­ i ­ v ria Wash Wash or la my Amniotic flu Biopsy Biopsy for H. py ­ su Gangrenous tise Gastric

Genital, Genital, fe

96 SECTION 3 ion andion ­ t ec ­ l and Neisseria ort need Chlamydia for ­ p Comments See the col text for trans trachomatis gonorrhoeae. 2 h, RT 2 h, RT 2 h, RT 2 h, RT Transport time and temp ≤ ≤ ≤ ≤ Specimen Collection (continued) a,b ort ort ort ­ p ­ p ­ p ice and/or ines ­ l ­ v ort ­ p 1 ml 1 ml 1 ml m vol ≥ > ≥ ­ mu ort guide ­ i ­ tem, ­ tem, ­ tem, ­ p Transport de min Swab trans Anaerobic trans sys sys Anaerobic trans sys le ­ p l l d Anaerobic trans ­ na ­ i ix er. ­ t ­ v ­ e le swab. le ines ion. ­ i ­ l ­ t ­ port um ant.

­ l a us and ­ c ­ r ­ u ­ c ­ a ri ire amount to ­ t ate or flu ­ b ­ r ng ng cath u i ard the swab. ­ i ­ l ­ p ­ c bic ­ bic trans al or with swab, ions from the ut ­ t ­ o ­ c ate via a cop i ­ o ­ r ­ s er re i ­ v ine prep ­ tem. ng ng a spec ­ a ­ c ­ i ­ p ­ d isinfect skin with isualize the cer emove mu emove ollect transcervical ransfer en irmly gently sam yet se  R cer and dis sys  T an  with ster a new F the endocervical ca with  D io V   C as tele us Collection guide 2. Aspirate fluid from ducts. 1. 1. 1. 2. 3. Submit as 1. 2. acteriology: Collection and trans B ac ac s

- ions ions ions e ­ t ­ t ­ t d - ue and d re re re ­ i ­ s ­ c ­ c ­ c Bartholin gland se Cervical se flu Endometrial tis se Cul Specimen type Specimen

Table 3.1 Specimen Collection and Transport 97 ire ­ t ­ mit is. ­ s o ure of ­ n ­ t ained, ­ i ­ t ng ng (continued) ­ i ainer and ion, sub ­ t ­ t a ead ­ l ­ z ate it, and i ial vag ­ t ended the for ices, place en ­ r ­ n ­ v ion. e ­ m le le con ­ o ­ t ­ t ­ i m ­ o orp ne de hra, ro ess lochia, cul ­ s ­ i ­ t al col ­ c ­ c ion of group of ion B harge can be ob ­ t ter ­ c ­ u oc is of bac ec ­ s ­ c ­ t ra ate ab o ­ t al swab. o it at RT. Gram stain, at RT. it not ­ t e into a ster ­ t ­ t ­ i ­ n ­ c ure, is rec er. Inserter. a small 2–4 swab cm ­ m i g il ­ t ults. ­ t ­ v ­ a ­ s ­ c re de cul which mis may give sub Do not pro strep di For de For in into the ure in it placeleave at least for 2 s to fa a rec If no dis wash the periurethral area with Betadine soap and rinse with wa 2 h, RT 2 h, RT 2 h, RT ≤ ≤ ≤ Specimen Collection ic ic ­ b ­ o er ­ a ­ tem ort ort ­ p ­ p ort sys ­ p trans Swab trans Sterile tube or an Swab trans e. ial ce. ic ic ­ g ­ r an ­ b ­ fi ng e ette. ­ e ­ i ­ i ­ t er ­ o rane ­ p ­ t ales, tely ar ­ b is ­ char ag er ate ­ a ­ s a. ­ s ­ s ­ m i ­ a d. ­ d ­ n ­ d le le ­ e i hra against ­ u hy ­ i ­ t e ­ g ion of of ion ions from ­ p hral or at ­ tem. ­ t ­ t ­ t ­ m al wall with ­ n harge ma re ­ n al mem ­ c ­ i nd swab. ­ c ­ s ­ o r. o ­ e hra; fe for ­ c ­ t ort sys le swab or pi swab le ry, im er to an an ions ions and dis bic ­ bic sym ained by ce ­ i ­ t ­ p ­ f ­ e ­ t age the ure ain ­ s ­ t ue in a ster re ­ liv ­ s ipe old away ­ c btain se emove emove old ex ollect dis ubmit a por f ob f a smear is also needed, ient has uri de  I trans trans a ster of theof vag the mu  O  I use a sec con  S tis  R from the ure  W se ­ t  C the ure on a swab by mas by on a swab mas the pu through the va 2. 2. Collect at least 1 h af pa 1. 1. 1. 3. 1. 1. 2. ion ­ t ions ions ­ t ­ t ­ cep re re ­ c ­ c on Products of c Urethral se Vaginal se

98 SECTION 3 er

ect

­ b ­ t er ive ive ­ t ­ t ions a ­ t ­ t i re ured ­ t ­ t ­ c slip, and - ed cham ion ion to de ­ t ­ fi ­ i a ore and af ­ n id ­ i ­ f tely to the ­ tatic se ­ a ot be cul ­ m m i ia. ­ a ­ n ­ d ed by quan ­ d e ­ fi i ­ t ­ m can ate, add cover eld eld ex ­ d ial me fi u - ­ c ­ s ory in a hu ­ fi ort im ­ t i age. Ejaculateage. may also be ­ t a ­ p ­ r ­ s ri dish with moist gauze). ure urine of be ­ t ­ t ­ tured. ­ o Comments , touch pallidum a glass slide toT. the tran trans lab (pe pallidum T. For dark on ar may be iden Pathogens in pros cul mas cul 2 h, RT 2 h, RT Transport time and temp ≤ ≤ Specimen Collection le le (continued) ­ i a,b ice and/or ines ­ l ­ v 1 ml of ort ort or ster > ­ p ­ p m vol en. ­ m ­ mu ­ i ium for ort guide ­ i ­ d ­ p Transport de min Swab trans spec me Swab transSwab us ­ t ine, ­ a ­ l le ­ i er. ­ t ressed le swab swab le le sale ines ­ i ­ p ­ i ­ l ion’s ion’s ate to . ­ s ­ tate ­ d ng theng base hral me ­ id ­ i u ­ t ­ tum. ­ s ate on a ­ t ion, firmly rub ate. ­ s ove le ­ l u ect flu ­ m ­ l el blade. ­ m le le swab. ace with a ster ­ p ­ i u ­ f hile press ­ c assage pros llow llow tran ollectex fluid leanse ure lean with ster ac through rec  A  M of theof le  W to col base with a ster  C from pros ster with soap and wa  C scal sur  C and re . 3. 3. Collection guide 1 1. 1. 2. 2. 3. 3. acteriology: Collection and trans B

ion ­ s ale or Prostate ­ m Specimen type Specimen male Genital, Genital, fe male le Table 3.1 Specimen Collection and Transport 99 ­ ­ u ect ­ l × is of ec ­ s ­ s ­ y ble ble to en, a (continued) ­ a ive ive r). r). ­ m 10 ­ t ­ i a ≤ ­ la ­ t i field (10 field ­ u ­ t it anit early × ive ive anal ion. Theion. best ­ t ­ t ­ m a oc ist should col ­ t ine. ients un i × ­ l en on 3 con ­ p ­ t ­ t ­ a um spec ­ m ­ t ­ i ic ic pa ­ r t en via suc ­ a i ory ther en should have ­ m ­ t ous cells/100 ­ d is brushings, of place brush ­ i ive ive and 10 ng ng spec a ­ s ­ t ­ i ­ m al 40–80 of ml fluid of is ­ r uce a spu ­ m ­ i ­ t ­ y ­ d ec pi ­ j ­ s A to needed for quan BAL fluid. For quan mlinto sa of 1.0 anal For pe pro re a spec spec squa ob Mycobacteria: sub morn tive days. 2 h, RT 2 h, RT 2 h, RT ≤ ≤ ≤ Specimen Collection 1 ml 1 ml > > ort ­ p ­ tainer, ­ tainer, 1 ml > ia, ­ r e ­ t Sterile con Swab trans Minimum amount: bac Sterile con a. ­ r er ion. ­ d ­ t le le ­ i ion ion of en (not ­ s i ian. um trap. orp ­ m ­ t ­ c er to ­ v ­ i ­ s i en un ­ t ­ s uce a lower er hral ng ng or ­ m d). ­ t ­ d ­ i ­ i ­ p ­ i ess oral flo ient to cough ­ t ­ c ate ab ient rinse or ­ t ­ t ­ i ate and swab, al flu ory spec ­ t ­ t il ect su ­ s ­ r a ­ c a ate in a spu ­ r le withle wa ainer with 1 ml of ­ n ­ r ove ove ex ­ t ­ g i ine. pi ­ l ­ m ­ p ­ s ave pa ave ollect spec ollect wash en, ro lace brush in ster nstruct pa  P con sa re H gar   I re deeply to pro post as  C  C the di a nurse or phy ­ m . nsert a small 2–4 swab leave it in it placeleave at least for 2 s to fa cm into the ure lu Abscess See I 1. 2. 1 3. 2. ­ er ­ heal ­ ed ­ c at ­ r ate ­ tra o ­ r o ­ t i age, age, brush ­ d olar ­ p ­ v ec Urethra Bronchoal ve la or wash, en as Pilonidal cyst ­ p Respiratory, low Sputum, ex 100 SECTION 3 ial ­ r ates are e ­ r ­ t i ­ p um, ­ t psy ­ o le le as ling of ­ d ­ p ue for bac ­ s . ens choice. of ­ ed ­ m at ­ i ial tis ­ r ion. ion. Tissue bi ens or nee ­ c o ­ t ­ t ­ fi ­ a ­ m ­ i ­ u ec er ­ p ­ p su the spec Discourage sam Comments eval spec Same spu as for above ex 2 h, RT 2 h, RT ≤ Transport time and temp ≤ Specimen Collection le le (continued) ­ i a,b 1 ml > ice and/or ines ­ l ­ v ort or ster ­ p ­ tainer, r m vol ­ e ­ mu ort guide ­ i ­ tain ­ p Swab transSwab con Transport de min Sterile con er ­ t ions ients ­ t ely ely 25 ­ t le le ­ t ­ i ard this ion ion le le le le a re ­ c ­ s ­ i ­ i ines er af ­ c ­ l ­ t ­ m ­ i rox ­ p ient rinse r. r. ­ t ris from the ­ e ­ e ­ b ng gumsng and izer, have pa ­ i ­ l ain ain ace the of le ­ u ­ t ­ t ale ap ­ f ine. ith the aid a of ­ l ­ h ave pa ave ollect in a ster ollect in a ster emove emove oral se in sa  W neb ml 3–10% of ster con  C con  C sur with a swab; dis and de swab.  R brush tongue.  H mouth with wa Collection guide 1. 1. 2. 4. 4. 1. 3. acteriology: Collection and trans B

­ per Oral ­ duced Specimen type Specimen Respiratory, up Sputum, in Table 3.1 Specimen Collection and Transport 101

(continued)

­ tients rs or ­ e i ng ng ­ r ­ i ures are ­ t ures are ­ t ect ­ t ed in pa al car ions. ­ c ­ cat ­ s i oc ­ d ­ c n o i al le ­ l - ­ s ­ y ra ­ t erved for de ­ s re Anterior nose cul staph for for na Throat cul swab con with epiglottitis. for Swabs 2 h, RT 2 h, RT ≤ Plates: min, swabs: 15 RT; 2 h, RT ≤ Specimen Collection ion ion ­ t ion ion a ­ t ­ l a ­ u ­ n c ­ i ort ­ o m ­ p ­ a ort ort ium in ­ p ­ p ­ d ide or ex ­ s le, le, swab trans ­ b Swab trans ta Swab trans Direct me at bed

ior ior ine ­ r

oist­ ­ l e al ­ t ng ng ­ m le le ­ i ­ m a. ­ p ium ­ s or. le sale ­ i ­ c ­ s o nd swab, orb ­ c ­ o ­ s es. ely ely 1–2 cm res ­ t ­ r nx via the ert a small ­ p a ­ s ­ y as nor of ­ m on, avoid al mu ­ i usly sam ­ e ­ s ions. ­ si har ­ o ­ t ate or flocked r ­ p ­ n ue. rox ­ o re o i ­ s ­ s ­ p ­ c ­ g ently in epress tongue with a otate the against swab otate swab slowly otate slowly swab nsert pre a swab,  U vig the le any ar  R the na tis  R 5 s to abfor se  I ened with ster (or use flockedswab), ap into the na  D tongue de  G intoswab) the pos al swab (e.g., cal (e.g., swab na nose. 1. 1. 2. sing a sec 2. 1. 1. 1. 1. 2. ­ nx ­ y Nasal Throat or phar Nasophar

ynx 102 SECTION 3

ue as ­ s ntaining ue is co ­ s ion of of ion - ­ t ace a of ­ f al 70°C in case − ­ co ort at RT. ess tis ­ p ­ c ium and plated h 12 it asit much tis es are needed. Never ­ d ue at ­ i ion. ion. JEMBEC, ­ m ect more than 30 ml ­ t ­ s ­ l ec ­ l le. le. If ex al tis ort me ble, save a por save ble, it a swab that a swab it has been ­ b ­ c ­ p ­ a i i her stud r r col er for transer for ­ s ­ m ue. ­ t ­ g ­ t ­ s ­ te Comments Neisseria gonorrhoeae should in char placed be trans sub Always pos avail sur for NAAT Do not col fur sub rubbed the over sur af tis Biobags, andBiobags, the GonoPak are bet 2 h, RT; 24 h, 4°C 15 min,15 RT Transport time and temp ≤ ≤ Unpreserved: ≤ Specimen Collection (continued) ort le le ­ i r ­ p a,b r; add ­ e ­ e ort ur ­ t ­ p ed by ice and/or ines ain le, le, ­ l ­ t ­ v ­ fi ­ i ac ­ i ­ f ­ u m vol ap con c ue moist. - ­ s ­ mu ium spec ral drops ster of ort guide ­ i ­ e ­ tem or ster ­ d ­ p ine to keep small pieces ­ l Transport de min sev sa Sterile tube or trans of tisof me NAAT man Anaerobic trans sys screw ery ­ g ior ior ­ r ines ­ te ­ l psy ils, and ­ s ­ o as with a ng ng sur ­ e ­ i us bi ­ o nx, ton e ­ y le le swab. ­ n ure ­ i amed ar a ­ d ­ t ­ fl e ample the pos ­ c ster in  S phar Collection guide 2. or cu pro Collected dur acteriology: Collection and trans B and

- ) NAAT ale, first ­ m Specimen type Specimen void (for Chlamydia gonor N. rhoeae Urine Male and fe Tissue Table 3.1 Specimen Collection and Transport 103 uce ­ d (continued) ro ­ t hral flora into ­ t rease the risk ion. ­ t ­ c ec ­ f er and in ­ d ­ genic in ers the of ure o ­ r ­ b of iat mem Catheterization may in the blad 2 h, RT; 2 h, RT; 2 h, RT 2 h, RT; 24 h, RT 24 h, RT ≤ ≤ ­ served: ­ served: Unpreserved: ≤ Unpreserved: pre ≤ Unpreserved: ≤ Unpreserved: pre Specimen Collection ic ic ic ic ­ r ­ r ­ r outh outh ive ive ive m m roof ­ t ­ t ­ t - - a a a ­ p 1 ml, or urine 1 ml, or urine ­ v ­ v ­ v ≥ ≥ ­ ser ­ ser ­ ser ort tube with bo ort tube with bo ort tube with bo ­ p ­ p ­ p ainer, ainer, ainer or urine ­ t ­ t ­ t trans trans trans Sterile, wide con acid pre Sterile, wide con acid pre Sterile, leak acid pre con in ia ­ g ers ers ­ b ­ t ­ t ion ion ion i i ­ t ­ t ­ l ­ l i i ng. ­ l ­ l ial ­ i ­ r ect a ect a ion ion ion e ­ l ­ l ­ t ­ t ure. ­ t ng ng with ­ t ing the ing the ­ i er. ­ p ­ p ­ t ng theng ng theng la racted, be ­ i ­ i ­ t ral mil ral mil ream por ream por ­ e ­ e ­ gin void ­ st ­ st ­ stop ­ stop ut ut ream por ream por ng. kin re ­ o ­ o ­ i ­ st ­ st hral open ­ s ­ t ­ ture. hile hold hile hold fter sev fter sev inse area with wet he mid he mid horoughly cleanse the flow of flow urine. of flow urine.  R gauze pads. mid mid with with has passed, col has passed, col  A  A is used bac for is used cul for  T cul  T W fore T soap and wa   ure void  W apart, be 3. 3. 3. 1. 1. 1. 2. 1. 1. 2. 2. tream tream ­ s ­ s er ­ t id ­ e Male, mid Female, m

Straight cath

104 SECTION 3 al ­ n pi ers ­ t ­ s ­ e ro ­ b e ­ r ess they ­ l ng ng cath ect urine , 11th ed., ASM , 11th ­ i ­ l ia in their ­ r e well ­ t c. ­ d ­ i ients un t ­ t ­ a ateagar; ce CSF, om ­ l ­ t ­ o ers. Do not col ­ d ays have bac have ays ­ w Comments Patients Patients with in al blad from these pa are symp ion;CHOC, choc ­ s u ­ f 2 h, RT; 2 h, RT; ure. ­ t 24 h, RT ­ a ≤ er ­ p ­ served: Transport time and temp Unpreserved: ≤ Unpreserved: pre Specimen Collection er SS, Warnock DW. (ed.),er SS, Manual Warnock DW. of Clinical Microbiology ­ t ive; RT, room tem ­ t (continued) ­ a ­ tainer a,b ol ol fix ­ h ice and/or ines o ort tube ­ l ­ c ­ v ­ p roof con ­ p yl yl al age; BAP, blood agarage;BAP, plate;BHI, brain heart in ­ n ive ­ v i ­ t ic acidic m vol ­ r ­ v a y ­ v ­ l ­ mu ort guide ­ i ­ ser ­ p Transport de min with bo pre Sterile leak or urine trans oll Funke KC, G, Landry ML, Rich er. ­ r ng ng ous; PVA, po er ­ i ­ t ­ d ­ n ­ e e er le le inge ­ v ­ t ­ i ­ r ow ow ect ­ e le tubele ­ l ra ­ l ­ i ­ t ines ert ­ l ­ s i;BAL, bronchoalveolarla ­ l ng ng ect in tub ­ i ely ely 15 ml to ­ l il ­ t ­ c ­ er. a ally col ect urine to be le andle sy ­ l r. ow ­ c ­ d ­ l i ­ m ed in a ster ­ e ure; i.v., in ­ t ain ­ i ­ t ion portion with 70% ­ s ­ t er er into the blad ­ t it ol. ol. Clamp cath ­ t astba f ain ­ e ­ h - ec rox ­ t ­ m ­ l ow portow and al o d ­ p ­ l ­ i fter al se nee septically, in ­ c isinfect the cath ransfer to a ster to asep ml5– urine. of 10  U  A ap con pass, col sub  A cath  T or con col  D be urine to col al 10–20for min. ous pres ­ n Collection guide 3. 3. 1. 4. 4. 2. 3. See Abscess See acteriology: Collection and trans on, D.C., 2015. on, D.C., ral ve ­ t ­ t B ng ­ i

er ­ t ­ e Indwelling cath d; CVP, cen ­ i Adapted from Jorgensen J.H, Pfaller MA, Car Abbreviations: AFB,ac a b Specimen type Specimen

Wound flu Press, Wash Table 3.1 Specimen Collection and Transport 105 (continued) ate ic ic health ­ l ­ v ue PCR ­ s ies) ­ gy ­ r ­ o ­ to ble ble at pub ol a ­­ a ­ r ­ r ­ o iemsa stain blood of smear, ies and some pri ­ r G o - ­ t gy (avail nce nce lab a ­ o ­ e ­ r ­ o ol ­ er ­ r a Culture and se ref se lab Diagnostic of Test Choice Serology Serology Wright Culture ia ­ r e ­ t le en ­ b ­ z i ­ s ered bac le ­ t ure; ure le; hand ­ t ­ t ­ b ­ i i ­ a ­ a ­ s er er ­ p ­ p ­ coun itely at—70°Citely Serology and/or tis ory if pos le; at hold n ­ t ­ i i­ a f­ ­ r e ­ o ­ d ort on ice or fro ure sent on ice; keep entrifugation tube is ­ t ory if pos ­ p c ­ t Specimen Collection - a ­ sues ­ r is uently uently en il tested for or at—7°C ­ o ­ s ­ t ­ q ue moist and ster re ­ s ­ f ic ic ly ­ r ent; trans t ­ a ­ ful ­ m i ue moist and ster ­ d ­ s aterial cul for Transport at room tem pe help 4–20°C un ship 1 week at 4°C; in Hand carry to lab Transport Transport is tis M PCRfor test Keep tis carry to lab ines for in ­ l ngs Transport at room tem ­ i

psy, psy, ­ o in or ant), ow ion ion ort guide ­ l ­ r ­ r ­ s ­ u ­ a ­ p ace scrap g ­ f ­ a o ­ s ­ c ue, lymph node ­ s ­ ti ­ rum psy at le ry, ry, blood, CSF ­ o ­ e se ate b ­ r i iph ­ r ­ p ion ion and trans ­ t Blood, bone mar Blood, tis as Blood smear (blood) Specimen choice of Tissue, sub ur Skin bi CSF, pe Blood smear, skin bi blood hep (with EDTA an ec ­ l

s) er) ­ si ­ v ­ o e; e; ­ i ­ l is) ­ s ­ na an ­ lo ­ lich ­ ing fe ui ­ m el pecimen col ­ g ­ c S aps ­ l (hu (cat scratch (cat

ytic ehr (bru (re ­ c oma oma in ­ l o ­ l ­ u ­ u er) ­ ease) aplasma ­ v artonella orrelia burgdorferi (Lyme  Brucella  B fe (gran Organism Organism (dis­ ease)  An Klebsiella granulomatis donovanosis)  B dis Borrelia gran Table 3.2 106 SECTION 3 , 11th ed., ASM , 11th ­ gy ­ o ol ­ r ­ gy ­ o ol ­ r (continued) a Culture and se Blood PCR Culture Serology Diagnostic of Test Choice Phase I (chronic) and Phase II (acute) se ia ­ r e ­ t ent; ium ­ m ­ d ute 1:10 ute 1:10 ered bac ­ l ­ t in and store ory or ­ t en at—70°C en at—70°C ­ m erred a ­ z u ­ f ­ r en for PCRen for test ­ b ­ coun ­ o ­ z ze with so le; at hold 4–20°C ­ i ­ i 1 h or di < ral le le pre ­ t ­ t um al ure sent on ice; keep ­ r ­ t Specimen Collection ue are fro ­ sues ­ s er SS, Warnock DW, (ed.),er SS, Warnock Manual DW, of Clinical Microbiology ort to lab ­ t uently uently en ­ p ­ q ine se ­ v ­ ate re 1 h; urine, ­ f olume bot < on ort on ice or fro v ­ b - ­ p r ue moist and ster il il shipped il tested ship for or at—70°C ­ t ­ t ­ s ­ ca Rapid trans freeze; ship on dry ice Blood, trans bi un in 1% boin 1% at 4–20°C or neu High Material cul for tis Transport Transport is un ines for in d ­ l ­ i er first ants psy, psy, ­ t - ­ l ­ o psy, psy, ­ u ­ o g ate ue Blood and tis ate, in or ant), CSF, ­ a ­ r ­ r ­ s ­ l ort guide ­ r i o ­ u ­ a ­ p ­ p ­ c g ­ ti ion ion bi ­ a ­ s o oll Funke KC, G, Landry ML, Rich ates joint of flu ­ r ­ r um ­ c i ­ t i ­ t ­ p ngs, le ­ i ­ rum ontaining an ion ion and trans ­ t Lymph Lymph node as scrap blood, spu Serum, blood (cit c should not be used), CSF (firstweek), urine(af week) Blood, as se Serum, blood, tis Specimen choice of Blood smear, skin bi blood hep (with EDTA an ec ­ l is) ­ s ers; d. s) a) ­ i o ­ v ­ r ­ si er) ­ mi ­ o pi ­ v ­ i ­ s ­ re al flu ed fe ­ n a ­ t ­ to er), (rat bite ­ l pecimen col ­ lich ­ v pi on, DC. 2015. ­ s ­ t S ­ hill fe (tu (lep ng er ­ b ­ i

(spot (ehr ­ v ­ r (Q fe hus) er; er; Ha ­ p ­ v CSF, CSF, e ce ro oxiella Adapted from Jorgensen JH, Pfaller MA, Car a b Leptospira Francisella ty  fe Rickettsia Ehrlichia Organism Organism (dis ­ ease)  C Streptobacillus Press, Wash Table 3.2 Specimen Collection and Transport 107 Table 3.3 Guidelines for col­lec­tion of spec­i­mens for an­aer­o­bic cul­turea Acceptable ma­te­ri­al Unacceptable ma­te­ri­al Aspirate (by nee­dle and sy­ringe) Bronchoalveolar la­vage wash­ing Bartholin’s gland in­flam­ma­tion or Cervical se­cre­tions se­cre­tions Endotracheal se­cre­tions (as­pi­rate) Blood (ve­ni­punc­ture) Lochia se­cre­tions Bone mar­row (as­pi­rate) Nasopharyngeal swab Bronchoscopic se­cre­tions Perineal swab (pro­tected spec­i­men brush) Prostatic or sem­i­nal flu­id Culdocentesis fluid (as­pi­rate) Sputum (ex­pec­to­rated or in­duced) Fallopian tube fluid or tis­sue Stool or rec­tal swab sam­ples (as­pi­rate/biopsy) Tracheostomy se­cre­tions Intrauterine de­vice, for Actinomyces spp. Urethral se­cre­tions Nasal si­nus (as­pi­rate) Urine (voided or from cath­e­ter) Placenta tis­sue (via ce­sar­ean Vaginal or vul­var se­cre­tions (swab) de­liv­ery) Stool, for Clostridium dif­fi­cile Surgery (as­pi­rate, tis­sue) Specimen Collection Transtracheal as­pi­rate Urine (suprapubic as­pi­rate) aAdapted from Mur­ray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (ed.), Manual of Clinical Microbiology, 8th ed., ASM Press, Wash­ing­ton, D.C., 2003.

Virology: Ge­ne­ral Specimen Guidelines

1. The tim­ing of spec­i­men col­lec­tion is crit­i­cal be­cause the du­ra­ tion of vi­ral shed­ding is influ­enced by the type of vi­rus, the or­gan or tis­sue in­volved, and the im­mu­no­com­pe­tence of the pa­tient. For op­ti­ mal re­cov­ery of most vi­ruses, spec­i­mens should be col­lected within 3 to 7 days af­ter on­set of symp­toms. 2. The method of col­lec­tion can have a pro­found ef­fect on de­tec­ tion of vi­ruses. If vi­ral cul­ture is at­tempted, the vi­a­bil­ity of the vi­rus must be main­tained in ap­prop­ ri­ate trans­port me­dium. If nu­cleic acid am­pli­fi­ca­tion is at­tempted, the swab com­po­si­tion and an­ti­co­ag­u­ lants can af­fect the as­say. 3. If only a lim­ited amount of ma­te­rial can be col­lected, the num­ber of tests re­quested should also be lim­it­ed. 4. Because vi­ruses are ob­li­gate in­tra­cel­lu­lar path­o­gens, wound and skin spec­i­mens (e.g., ves­i­cles) should con­tain cel­lu­lar ma­te­ri­al. 5. Specimens should be trans­ported to the lab­o­ra­tory as quickly as pos­si­ble, par­tic­u­larly for spec­i­mens sub­mit­ted for vi­ral cul­ture. Via­ bility is not re­quired for an­ti­gen or nu­cleic acid am­pli­fi­ca­tion tests. 108 SECTION 3 6. Viral trans­port me­dium (VTM) should be used to pro­tect spec­i­ mens from dry­ing. VTM is not re­quired for ce­re­bro­spi­nal flu­ids, blood, urine, bronchoalveolar la­vage spec­i­mens, am­ni­otic fluid, and fe­ces. 7. Specimens other than blood should be main­tained at 4°C if held for more than 1 h af­ter col­lec­tion. Freezing should be avoided un­less a de­lay of more than 24 h is an­tic­i­pated. Recovery of some en­vel­oped vi­ruses (e.g., re­spi­ra­tory syn­cy­tial vi­rus, her­pes sim­plex vi­rus, cy­to­ meg­a­lo­vi­rus, and var­i­cel­la-zoster vi­rus) is com­pro­mised by freez­ing. 8. A va­ri­ety of com­mer­cial VTM are avail­able. Most con­tain pro­tein to sta­bi­lize the vi­rus, an­ti­bi­ot­ics to pre­vent bac­te­rial and fun­gal growth, and a buffer to con­trol pH. Culturette swabs with Stu­art’s me­dium can also be used.

Virology: Specific Specimen Guidelines Blood 1. An 8 to 10ml vol­ume of blood is col­lected us­ing ap­prop­ ri­ate asep­tic tech­niques. 2. Anticoagulant tubes (EDTA [pur­ple top], hep­a­rin [green top], and ac­id-citrate-dextrose [yel­low top]) are used for de­tec­tion

Specimen Collection of vi­ruses in plasma or leu­ko­cytes. Heparin in­hib­its PCR and the in­fec­tiv­ity of some vi­rus­es. 3. Plasma can be ob­tained by centri­fug­ing blood col­lected in tubes with an­ti­co­ag­u­lants. Plasma par­ti­tion­ing tubes can fa­cil­i­ tate sep­a­ra­tion of plasma from cel­lu­lar ma­te­ri­al. 4. Plasma for nuc­ leic acid am­pli­fi­ca­tion of RNA vi­ruses should be sep­a­rated within 4 to 6 h of col­lec­tion and re­frig­er­ated for up to 72 h or fro­zen at −70°C for lon­ger pe­ri­ods. 5. For se­ro­logic test­ing, acute-phase se­rum should be col­lected within the first few days of clin­i­cal on­set, and con­va­les­cent-phase se­rum should be col­lected 2 to 4 weeks lat­er.

Bone mar­row 1. Bone mar­row as­pi­rates are col­lected from the pos­te­rior il­iac crest, the an­te­rior il­iac crest (in­fants and chil­dren), or the ster­num or the tibia (in­fants youn­ger than 18 months). 2. Leukocytes from bone mar­row spec­i­mens can be cul­tured for cy­to­meg­a­lo­vi­rus (CMV). Varicella-zoster vi­rus (VZV) and hu­man her­pes­vi­rus 6 can also be cul­tured from bone mar­row. PCR is used to di­ag­nose par­vo­vi­rus B19 in­fec­tions.

CSF NOTE: Viral cul­ture of CSF is only rarely per­formed be­cause higher sen­si­tiv­ity al­ter­na­tives ex­ist for most of the vi­ruses that could be cul­tured from the CSF. Specimen Collection and Transport 109 1. Enteroviruses and herpes simplex virus (HSV) are the most common viruses grown from cerebral spinal fluid (CSF). Arbo­ viruses are important causes of sporadic encephalitis but are dif­ ficult to culture. These in­fec­tions are most com­monly di­ag­nosed by nu­cleic ac­id–based tests. Likewise, CMV, VZV, Ep­stein-Barr vi­rus (EBV), and JC vi­rus in­fec­tions are most com­monly di­ag­nosed by nu­cleic ac­id–based tests. 2. Viral ti­ters are gen­er­ally low in CSF; there­fore, the spec­i­men should not be di­luted and tests should be se­lec­tively per­formed. 3. CSF should be col­lected in a ster­ile tube with­out­ trans­port me­di­um. 4. CSF for vi­ral cul­ture does not re­quire spe­cial pro­cess­ing.

Respiratory spec­i­mens (throat, na­sopha­ ­ryn­geal swab, na­sopha­ ­ryn­geal as­pi­rate, na­sal wash­ings, and bronchoalveolar la­vage [BAL]) spec­i­mens) 1. Influenza vi­ruses, para­in­flu­enza vi­ruses, RSV, ad­e­no­vi­ ruses, and rhi­no­vi­ruses are most fre­quently iden­ti­fied in re­spi­ra­ tory spec­i­mens. Metapneumoviruses and co­ro­na­vi­ruses are also im­por­tant re­spi­ra­tory path­o­gens. 2. With the adop­tion of the flocked swab, it is now thought that Specimen Collection na­so­pha­ryn­geal as­pi­rates are equiv­a­lent to na­so­pha­ryn­geal swabs for spec­i­men col­lec­tion. 3. Nasal washes do not con­tain a large num­ber of vi­rus- infected cells; how­ever, they are of­ten used when na­sal as­pi­ra­ tion is con­tra­in­di­cat­ed. 4. Nasopharyngeal as­pi­rates are typ­i­cally trans­ported in ap­ pro­pri­ate vi­ral trans­port me­dium (VTM). The use of VTM is op­ tional for na­sal washes and BAL spec­i­mens. 5. All spec­i­mens can be used for vi­ral cul­ture. However, na­ so­pha­ryn­geal as­pi­rates and washes are pre­ferred for an­ti­gen de­tec­tion. 6. Mucus in spec­i­mens can af­fect an­ti­gen de­tec­tion by fluo­res­ cent-antibody (FA) as­say and en­zyme im­mu­no­as­says (EIA). Mucus can in­hibit the fix­a­tion of cells to slides (FA as­say) and can cause non­spe­cific fluo­res­cence. It can also in­ter­fere with pen­e­tra­tion of the spec­i­men into EIA mem­brane de­vices. Therefore, spec­i­mens should be bro­ken up by glass beads or as­pi­ra­tion through a small-bore pi­ pette be­fore pro­cess­ing.

Urine 1. Urine is an im­por­tant spec­i­men for de­tec­tion of CMV, ent­ero­ vi­ruses, ad­e­no­vi­ruses, and BK vi­rus. Mumps and ru­bella vi­ruses are rarely iso­lated in urine be­cause of the use of vac­cines. 2. Urine should be cul­tured for CMV within 7 days of birth to de­tect con­gen­i­tal in­fec­tion. 110 SECTION 3 3. Midstream urine should be col­lected in a ster­ile con­tainer. VTM is not re­quired. 4. Before urine is cul­tured, it should be neu­tral­ized with 7.5% so­dium bi­car­bon­ate so­lu­tion and fil­tered through a 0.2-μm-pore- size fil­ter to re­move con­tam­i­nat­ing bac­te­ria.

Feces 1. Many vi­ruses re­spon­si­ble for gas­tro­en­ter­i­tis (e.g., en­teric ­ad­e­no­vi­rus, calicivirus, astrovirus, and ro­ta­vi­rus) can­not be cul­ti­vat­ed. 2. Enveloped vi­ruses are not nor­mally re­cov­ered in fe­ces, with the ex­cep­tion of CMV in im­mu­no­com­pro­mised pa­tients. 3. Fecal spec­i­mens (2 to 4 g) are pre­ferred to fe­cal swabs be­ cause an in­ad­e­quate amount of ma­te­rial is col­lected on swabs.

Eyes 1. HSV and ad­e­no­vi­rus are the most com­monly iso­lated vi­ rus­es; en­tero­vi­rus 70 and coxsackievirus A24 may be de­tected by PCR. 2. Conjunctival swabs are col­lected from the lower con­junc­

Specimen Collection tiva with a flex­i­ble, fi­ne-shaft swab moist­ened with ster­ile sa­line and placed in VTM. 3. Scrapings of cor­nea or con­junc­tiva should also be placed in VTM. 4. Aqueous and vit­re­ous flu­ids can in­hibit PCR; there­fore, the spec­i­men must be di­luted or ex­tracted to re­move in­hib­i­tors.

Tissue 1. Many vi­ruses can be iso­lated from tis­sues. 2. Tissues should be trans­ported in VTM. 3. As much tis­sue as pos­si­ble should be col­lected and sub­ mit­ted to the clin­i­cal mi­cro­bi­ol­ogy and sur­gi­cal pa­thol­ogy lab­o­ra­to­ries. 4. Upon re­ceipt in the lab­o­ra­tory, tis­sues should be ground and centri­fuged and the su­per­na­tant should be used for pro­cess­ing. 5. Tissue for nu­cleic acid de­tec­tion should be minced, treated with pro­teo­lytic en­zymes, and ex­tracted with chaotropic salts or or­ganic sol­vents.

Genital spec­i­mens 1. HSV-2 and HSV-1 are the most com­monly iso­lated vi­ruses from ex­ter­nal gen­i­tal le­sions. HSV-1, HSV-2, and CMV are fre­quently iso­ lated from the cer­vix, va­gina, and ure­thra. These vi­ruses can be read­ ily cul­tured from these sites. Human pap­il­lo­ma­vi­rus, an im­por­tant cause of cer­vi­cal canc­ er, can be de­tected by mo­lec­u­lar tests. Specimen Collection and Transport 111 2. Genital le­sions should be swabbed vig­or­ously to col­lect cel­ lu­lar ma­te­rial, and the spec­i­men should be trans­ported to the lab­ o­ra­tory in VTM. 3. Cervical spec­i­mens are col­lected by in­sert­ing a clean swab 1 cm into the cer­vi­cal ca­nal and ro­tat­ing it for 5 s. The swab is trans­ ported in VTM. 4. To col­lect ure­thral spec­i­mens, ex­u­dates should be ex­pressed and dis­carded. The pa­tient should not have uri­nated for at least 1 h prior to spec­i­men col­lec­tion. A flex­i­ble, fi­ne-shafted swab is in­ serted 4 cm into the ure­thra, ro­tated two or three times, re­moved, and placed in VTM.

Skin 1. Rubella vi­rus, mea­sles vi­rus, ad­e­no­vi­ruses, and en­tero­vi­ruses can cause der­mal rashes and be iso­lated in cul­ture. Parvovirus B19 can cause a rash but is re­cov­ered from other sources. HSV, VZV, and en­tero­vi­ruses can be re­cov­ered from ve­sic­u­lar le­sions. 2. Fresh der­mal le­sions (not crusted, heal­ing le­sions) should be used for re­cov­ery of vi­rus­es. 3. Vesicular fluid and cells from the base of the le­sion should be col­lected and trans­ported to the lab­o­ra­tory in VTM. Specimen Collection

Table 3.4 Recommended blood vol­umes to col­lect for blood cul­turesa Volume of Recommended blood equal to blood vol­ume Total blood 1% of pa­tient’s Patient per cul­ture vol­ume for two to­tal blood weight (lb) (ml) cul­tures (ml) vol­ume (ml) <18 1 2 2 18–30 3 6 6–10 30–60 5 10 10–20 60–90 10 20 20–30 90–120 15 30 30–40 >120 20 40 >40 aJorgensen J H, Pfaller MA, Car­roll KC, Funke G, Landry ML, Rich­ter SS, Warnock DW (ed.), Manual of Clinical Microbiology, 11th ed., ASM Press, Wash­ing­ton, D.C., 2015. 112 SECTION 3 ion ­ t a ­ tems es. ­ n ­ s ­ i o ­ c am ry of ­ t ­ e ov ­ c ially those emic my ­ c ositive rates.ositive e ­ d p ­ p - entrifugation sys c les and note color if - ng ng en ­ u ­ i ­ ent. Comments pres Examine grains for or gran Lysis are good re for caus They give high con and false molds, es ­ er ­ a ­ g er ort ess in ­ p ­ p ­ c 2 h, RT. 2 h, RT; if lon 2 h, RT; 16 h. Time and tem ture trans for If ≤ RT. As above As above; pro ≤ If ≤ um Specimen Collection ­ t reat ­ t entrifugation ens. c ­ dure - ­ m arly to spu e ­ i ­ l ­ c ­ i a,b Processing pro If thick, pre spec sim Automated (BacT/ Alert, BACTEC, VersaTrek) Lysis Manual ines ­ l ine; ­ d ort guide ­ p ol. Collectol. ­ h o ures; ­ c ­ t ure io of ­ t inge. If open, use ate ­ r ial cul ­ r ral edge with a ­ r i ­ e e ines ­ p ­ t ­ l iph ­ r m amount blood of ­ ed le andle sy ace with 70% al ­ d ­ f ­ mu ­ tem or as ive pe ive ­ i end ­ t ­ m ect skin with tinc ­ f le neele ­ i n ­ om ­ i Collection guide Clean sur from ac ster swab swab sys Collect as bac for dis use max rec ycology: Collection andycology: trans M

­ d ­ u Specimen type Specimen (drain,Abscess ex wound) ate, pus, Blood Table 3.5 Specimen Collection and Transport 113

ristle esce b ­ r (continued) - ated with ­ r ve oilve or a ­ i ant agar of ­ u ­ r is versicolor ­ s ), ol ­ a i ­ r ush. ­ y ­ br er a Wood’s light. er a Wood’s ected with a soft er disk sat ­ l ve oilve should be placed on ­ d ­ p ­ i M. furfurM. the first quad Select hairs that fluo un Hair and skin can be col ol plate. tooth For pit ( pa Pediatric Isolator tubes are best. er, er, ­ g er er ­ g ­ g hytes hytes are te, as ­ p ­ a r ­ to ive ive to cold. ­ e a ­ t le), Never 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; 72 hr, RT (very (very hr, RT 72 ­ si ­ m ­ b rig ­ f 15 min, RT, If min,15 lon RT, If ≤ RT. If ≤ sta der sen ≤ RT. re If ≤ As above RT. le le ­ i ow ow ­ r le. ers ­ b Specimen Collection ­ t ­ a i ng edgeng ­ i les ­ v ­ t ble. ­ a ens should pt ­ m ­ i ushes ushes should entrifugation ion shouldion be ­ ce en non ­ s c ­ t led, as cen - ­ br ­ ac ­ p Only the lead a le of sam are of All spec be pressed gently into the agar with a ster swab; do not streak agar plates. If used, tooth be pressed gently into agar as well. Clotted bone mar is is un Biphasic Biphasic bot Lysis el el ­ p al ol ­ n ­ h el. el. o ainer or zed ­ t ­ p ope. ­ i ­ c ­ l ant, e in ­ t ristle ­ v ­ r b ­ a or - ­ p conle ­ i usly brushusly in a it 10 to 12 hairs to 12 10 it ­ o ope r ­ l ­ m e ­ o ­ v ainer or en entrifugation tube ­ t c - ally in a hep ypes with 70% al ­ c er en is i er nail should also be ­ s ­ t ion withion a soft ­ t ­ p ­ d ­ t ate in swab outer ear ca ­ t ush. le dryle con ­ i ng isng best; sub ar mo ­ i ­ br ­ l u inge or ly ­ c ­ r tooth Nails: clip or scrape with a scal Material un scraped. Submit in a ster clean dry pa cir Firmly ro or glass slide or vig pluck Skin: scrape with dull edge a scal of in ster Hair: hair root is most im Disinfect all Collect asep sy As above al ow ­ r ­ n er ­ t ­ gi Dimorphic/ filamentous fun Ear, Ear, ex Cutaneous (hair, skin, nails) Bone mar

114 SECTION 3 lly ion ­ a ­ t ia ­ r ec e ally. ally. ­ t ­ t ng ­ c ia with en. en. ­ i ­ i ­ d ac ­ m um is an ­ b ­ i ­ t ow es. ­ l o cop ­ s ­ gens. ial is usu ays beays ­ s ­ c pu ­ r o ­ o ­ w e ­ c ro ­ t ion ion are not ­ t ent for de h s ­ c ­ l - a ide. ­ n el ival time for ble ble spec ­ i ­ m ­ c ­ v ­ a ­ i le le ma ­ t ble. hic path pt am ex ­ a ­ p emic my ned ned mi ­ t ble. ble. Avoid me ­ i ­ h ­ d ­­ a age has a high yield. This ­ ce pt or ­ s on m ­ ac ­ c ­ clo ­ ce ­ a ­ m Very Very lit de avail cy Saliva or 24 un Methods for my ac Comments Short sur di Fluid should al ex The first urinefol mas fluid is ex of en ­ er er, er, er er ­ a ­ g ­ g ­ g ­ g er ort ­ p ­ p 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; If ≤ RT. Time and tem ture trans for If ≤ 4˚C. If ≤ RT. If ≤ RT. ens ia ia ­ d ort in ­ m ­ d ­ i ­ p Specimen Collection ate ­ l outh ia ia reated ­ u ­ t ­ d c m - ­ o haped ion; use uged to ­ t s ­ f - r. ­ ga rate their ory spec ­ e ­ t ­ t u ent for me (continued) ­ dure ­ f a ­ r e en le le wide ain ri ­ m ion ­ c ­ i a,b ­ c ­ tents. ­ t ­ t ­ i ­ t pi ectly or trans ­ r ­ s Corneal: in in X or C noninhibitory me mo Viscous lower re Processing pro Concentrate by cen con should be pre and centri con sed di and smears Inoculate me ster con ines ­ l d ng ng y ­ l ­ i ­ ­ i ians ­ c i ort guide ect ­ s ­ r ­ p ected ­ l ide. ial agents ­ b ­ m rs. Inoculate ­ i ro er, ander, then ­ e ­ c ­ d ex i ated di ­ l ­ h ain um col ­ m ­ u ­ t ­ t i ally. Placeally. all lo c ­ t ­ c ­ c ­ o i ines ­ g ­ l ­ cy le le con ng spung ng ng an ut ­ i ng: taken phy by ion ­ i ­ i ate gland to yield flu ­ i ­ t ­ o ­ t a ­ r ng teeth.ng Collect brush ain i ­ i ­ t ­ p ient empty blad ia/slides in ­ t ­ d les in ster age pros ia con ­ p ­ s ­ d er brush ­ t am af Collection guide Corneal scrap and me Use firstUse morn with and with Needle as s me and BAL fluid sur Have pa Have mas ycology: Collection andycology: trans M d ­ i

ngs ­ i d ­ i ate, um) ­ r ­ t i Corneal scrap Vitreous flu ­ p Specimen type Specimen Respiratory, (BAL,lower wash, brush, as spu Eye

Prostatic flu Table 3.5 Specimen Collection and Transport 115 ion ion ­ t ry ectly a ­ e ­ r enic ended ­ m ov ­ g ­ m ­ c 2 ml, (continued) o or ­ f < m ent should ­ m ue for ­ o ned ­ s ­ m ase. Examine ­ i ­ i ­ e mes m ­ ble. ­ u ­ a i ally. ally. With ­ s us tis ­ c ­ es. ­ i psy rec ­ o ­ o e ged ive ive dis ess ­ n ­ s ­ a en vol cop a a ­ s ­ sc les and see in ­ t ­ m ­ v u ­ u ­ i ia are best re for our ro ­ c ­ d ­ c ng asng much fluid on each ­ c ­ i ­ ways be ex Selective and chro me of Candida Sterile fluid sed al mi spec plate as pos fluid should platedbe di us Specimen should be dis Tissue bi for for in sub gran for ab te. er, er, er, er, ­ a ­ g ­ g r ­ e rig ­ f 2 h, RT; if 2 h, RT; 2 h, RT; if 2 h, RT; er, 4˚C. er, RT. 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; ­ g ­ g Oral: if ≤ lon Sinus: If ≤ lon RT. RT. Never re If ≤ RT. If ≤ le ­ i le le le ­ i us ­ i ­ n ort le le Specimen Collection r; add ure ­ i ­ p ­ t ­ e al ent blood ain ­ t ­ v ­ ge ds in ster r. ­ i yn ­ e ­ r ry of yeast. entrifugation in or in a ­ e c les. Place si ­ r ha - ing. Except for ain ents in ster ­ p le scanle be used for em for oralem for and ­ t ­ t ­ t ­ a ­ p ­ t ­ t ov ine to keep moist is ­ l ­ c ­ s se trans swab U sys oro con con sam clot tube to pre bot re ly blood cul CSF, body flu Except CSF, put sterExcept CSF, vacutainer tubes with hep sa a few dropsa few ster of Sterile con ent for ity ents ­ m ­ t ­ t ­ i y ­ g ­ o l ng tongue.ng Use ­­ o ­ i gy. gy. Concentrate i us con ­ r ­ o ­ n l e ­ t ­­ o i le swab for for swab le ­ r ­ b e ­ i ­ t ion, large quan ­ t ion, and use sed ions, avoid ­ t ec ­ s ­ l ­ ga u nx. Collect si ­ f ion. ion. Clots should be ground ­ ly ­ y ­ t ri ­ t a al ­ l ­ c har i ­ u ­ p c uired than for bac ­ g ­ q ­ o a thin wire or flex Swab oralSwab le oro sur Collect as for bac by by cen in Surgical col re us ­ n psy ial ar­ ­ o ­ v al, ­ c yn­ o en ­ r ­ n es) ­ ne ­ m o ­ i ­ pl ­ t ­ pha ds (CSF ds) ­ i ­ i ­ p Respiratory, Respiratory, up oro er (oral, geal, and si sam Sterile body flu and peri dial, peri and sy flu Stool Tissue bi spec 116 SECTION 3 ion. ­ t ent for ent for a ­ n ­ m ia best for ­ i ia or ­ i ­ d ­ d m , 11th ed., ASM , 11th Candida. ­ a ry of Use sed Use ­ e enic agars are best ­ g copic copic ex ov o ­ s ­ c ­ m Microbiology ro

l ­ c Comments Chromogenic me Candida. mi Antibacterial me chro for for re ­ er, er, er, er, ­ a ng ng to ­ g ­ g ort ­ i er ­ p ort ­ p ­ p ord ­ tions. ­ c a ­ d urer n ­ t er ac c ­ g ­ me ­ fa m ­ u ems can stay at RT 2 h, RT; if lon 2 h, RT; 2 h, RT; if lon 2 h, RT; ­ t ­ o Time and tem ture trans for If ≤ Urine4˚C. trans sys for lon man rec If ≤ RT. ure; h, hours. ­ t ­ tem ­ a er ent. Specimen Collection ­ p outh er SS, Warnock (ed.), DW Manual of Clinica ­ m ­ t ainer for ­ i ­ t m ­ tem. ort sys - ion ion and ­ p ­ t ­ ga ens by le conle u ­ i ate sed ort sys (continued) ­ dure ngs ­ f ­ l ­ m ­ i ­ p e ­ i ­ u ainer or urine ri ­ c a,b ­ t ­ t c ­ o d; RT, roomd; tem RT, ­ i Processing pro Concentrate spec Sterile wide trans con cen in Swab Swab trans or ster wash ines ­ l al flu ­ n pi ­ s ro orning ­ b zed e ort guide ­ i m ­ r - ­ p er y ­ l ­ t ­ e oll Funke KC, G, Landry ML, Rich ­ r ens are ­ m ­ i age; CSF, ceage; CSF, ures; ear ures ­ t ­ t ­ v ines ­ l spec h - erred, or cath ­ f ial cul ial cul ­ r ­ r e e ­ t ­ t ged. ­ a en pre ens; 24 ur ­ m ­ m ­ i ­ i ­ co spec Collection guide As for bac spec dis As for bac ycology: Collection andycology: trans on, D.C., 2015. on, D.C., ­ t M ng

­ i Adapted from Jorgensen JH, Pfaller MA, Car Abbreviations: BAL, bronchoalveolar la a b Specimen type Specimen Urine Vaginal Table 3.5 Press, Wash Specimen Collection and Transport 117 ine ­ d ion. uires i ­ t ­ r ient ­ q a crip­ ­ c ­ s ­ c ­ fi (continued) ­ fi ling in i e l: re ­ t ­ p ­ d ­ e o ains ac ­ n ­ t ­ mu ion toion some the of ies lev ­ t i ­ c ype iden ­ t ­ d spp., much more spp., o ­ n rit tube con omes, and microfilariae.It ­ c ­ s ­ o ble on theble blood films. ­ a at usly, in ad usly, ng microfilariaeng or ­ i ies or ge rs may not match the de ano ­ o ion stain)ion works, stip ­ m i b ­ c ­ t ect ­ o ­ p ­ t a ­ v sms to the spe ­ i ­ n i n ites try ­ b ­ s aria and some microfilariae. ­ ga ­ a ased stain (sheathed microfilariae). ­ l ites (he b ­ s sms; col - ­ i ­ a ion of mov of ion n in ­ t ral blood is used from im ­ l ­ e ­ y aria or ­ ga ec ­ l ­ t ant par ox iemsa com iph ­ v en used spe for ­ t ­ r ­ t ­ e a G - sms should be de ­ i aria, Babesia, ­ m ­ l n ify ma ­ t ­ ga ct is of ­ u spp. (pe ay testay kits ma for le, andle, the or ­ s ites); he ­ b s ­ s ­ i ­ a ­ a o ng. le le to iden ­ n ­ i ­ b i ­ s ­ mu ng methodng blood for par Specimen Collection ­ i os ­ p Leishmania ­ blood par ­ ly). ls train of ­ e ­ somes lly im ­ a ive ive for no ­ t i aria may not be vis msa (all ­ s nge), has been used ma for ients ients on ­ l ­ pa ­ e ­ t ­ a Sequencing PCR of prod or is usu Sensitivity is not higher than thick for filmsfor Plasmodium sen Commercial im high lev Buffy coat, fresh blood filmsfor de try pa QBC, a screen tions. However, with othertions. stains However, (those listed pre ma “quick” blood stains), the or Gi Although stain Wright’s (Wright Recommended stain(s) and rel les ­ p a erred) ­ f lood for b yzed ­ l b ol fixedol or - o ly ly with ­ lines ­ n ­ m ­ b ­ a i ­ s ­ dures ds: e ­ o ­ c en blood sam ­ z ens (he ct in some tests) ­ g ­ a ion: EDTA lood, eth i ­ t ­ t b er - ­ t um or plasma for ec lood (fill EDTA tube ­ r ­ t b - lood ng ng an ­ i b ated blood, pos - yzed or fro ­ l at ­ l letely with a blood and then ­ l ­ u o ­ p xed thin and thick blood films, u aria, se g ­ l ­ c ­ fi ­ a ­ m he blood can in PCR: EDTA co un ma cir Antigen de Concentration meth EDTA or EDTA com Specimen Specimen and pro Microscopically: thin and thick blood films. Freshblood (pre mix). Parasitology: Specimen guide

Body site Blood Table 3.6 118 SECTION 3 ue

­ s ure ­ t msa, ­ e y. ine agar/ ­ t ­ t ­ i ile amoeba ­ t il ­ t ble), EITB ogic ue cul ­ l ­ a ­ s o ­ t ures. Culture for ­ t Taenia solium Taenia ge mo ge ­ a ine his our ­ t ially avail ­ c ured in tis ­ todes: ­ t ­ c ine (microsporidia) (tis er opy, look for mo look for opy, ­ m ­ c ­ m uired cul for a msa, trichrome, or Calcofluor al ces ­ q b ­ n ­ e ­ v ros ­ c he ­ t ites ­ s en re ); Gi ­ a spp. do spp. not grow in the rou ­ m gy (lar ­ i ­ o eld mield er me ites), and IFA. spp. can spp. be cul fi ­ s ­ v - ­ tol ­ a ant par ­ v ­ e Balamuthia ine his ­ t ) ion: ­ t ended microsporidia for in rou spp.). Bright Toxoplasma gondii Toxoplasma ites). Sterile spec ­ m ­ s ­ cep ed trichrome, sil m ­ a ble ble for some par ­ fi ­ o ites ­­ a —PAM, Acanthamoeba, Balamuthia— GAE]); Gi ­ i ­ s ­ a omes, ­ s ae (ex ­ b Specimen Collection ay method).ay Toxoplasma ­ l lood par ano ­ p only used are EIA (many test kits com spp. (or T. spp. cruzi b ­ Echinococcus ially avail ­ m Naegleria ­ c ions); H&E, PAS, rou ­ t e e [ er ained prep). slide (wet Slides warmed en to 35˚C a iving ame erci, ast, PAS, mod ­ r l ­ m ­ st ­ ba f ­ c ­ a - ia. - msa (all msa (try ­ ti ­ d d ­ e ­ e ­ i Recommended stain(s) and rel Most com (com (ame Gram stains also rec ac prep Gi in un bacterial over Leishmania Gi cys Free me PCR blood for par um (continued) ­ r psy a ine) ­ o ­ l b yzed blood ­ lines ­ l ion: se ates al sa ­ t ­ r o ­ c ial in EDTA i ­ i ­ r ­ dures ec ­ m ­ p ate or bi g e ­ t e ­ r ­ t ­ c i ­ lo ­ p ­ o al fluid and CSF ected in EDTA ­ i ­ l ­ n ems in some tests) ­ um ­ l i le male ion, stained smears), ody de ­ i ­ t ­ d le as les or as ­ i ­ b a ­ p ­ t ­ n ­ i ate col ­ r psy (touch or squash i m ions, stained) ­ o ­ t ­ a ­ p ated blood (he a ­ l ial (in phys ­ ture me ­ r ­ r ­ u ­ a e g ­ t ­ a Specimen Specimen and pro Specifici an can cause prob or plasma, anticoagulated or co Cultures: ster Biopsy Biopsy sam Microscopy: thin and thick films with as or cul Culture: ster ma brain bi prep (wet (wet ex Microscopy: spi Parasitology: Specimen guide ous ­ v ow

­ r ­ tem Body site Bone mar Central ner sys Table 3.6 Specimen Collection and Transport 119 tes ­ i o ast ­ z (continued) f - tes, cysts); ho d ­ i ­ i ­ p o ype ype ­ t ­ t ­ z o o ­ n ­ n ho ­ p ion Loa Loa, ­ t ic troic za msa for tro ­ b ­ i ­ e erci, er ies and ge ies and ge ­ c ­ t erci, Loa loa, Toxoplasma ­ c ­ c c ­ c ­ ti i ­ a t ­ ine stain, PAS, ac ]); Gi ­ m a msa (ame ­ n spp. spp. ­ e gy (cys ast, microsporidia) EM (for he ­ o gy (cys ­ t f ype char ­ o - ­ t ­ tol d o ol ­ i y); Giy); t ­ ­ l ­ n er me ­ v Acanthamoeba ine his ­ t microsporidial spe microsporidial spe ine his ­ t ae [ ­ b ies and ge ­ c ae and Toxoplasma ae and Toxoplasma ­ b ­ b erred) or sil ­ f Toxoplasma, Toxoplasma, inths: spe ng ameng ng ameng ­ i ­ i ae, ae, ­ m ­ b ­ b ae); H&E rou for iv iv Specimen Collection l l ­ b - - spp.) er methamine stain, ac PAS, Acanthamoeba cysts spp. on ­ v ion. ­ t ­ tion a ­ c ­ ca ed trichrome (pre ); sil iving ame iving ame ­ fi ­ fi l l ­ fi i i - - ­ i ­ t ­ t oxoplasma Protozoa and hel Calcofluor ( Calcofluorfor cysts only(ame (micropsoridial spores; H&E, rou Cultures: free Free ­ T Cultures: free mod iden and cysts (ame gondii Free iden ­ ­ ­ i ­ i en en ­ z ­ z a­ og og ol ­ r ­ l ­ l ial, ­ n ­ a ­ o ­ o ­ r ­ a ­ i ­ i e ­ t ions) ­ t ial ­ r psy e ­ o ­ t ol, or fro ol, or fro ­ n ­ n ­ tion act lens, ial (see above) ial (see above) ­ t ­ a ­ a u ­ r ­ r psy ma le le with le le to avoid ­ l le le to avoid e e ­ o ­ b ­ b ­ t ­ t ial in phys ial in phys i ­ b ogic sec i ate bi i ­ l ­ r ­ r ­ s ­ r ­ s ­ s i e e ented with psy ma ented with ­ t ­ t ­ to ­ p ­ o ­ m ­ m le ngs, con le ive ma ive ive ma ive ­ i ­ p en, or fixed in eth ­ t ­ t ­ p ­ z ­ r ive ive ma ive ma cs if pos ­ t ­ t cs if pos cs, if pos ion, his ­ p ­ i ­ i ­ i ­ t t t t a ent of lens so ial ial growth ial growth ­ o ial (smears, touch or squash ­ o ­ o ­ r ­ r ­ r i i i ­ r ­ a ­ m e e ­ b ­ b ­ b e ­ i ­ t ­ t i i i ive, froive, ­ t ­ t ­ t ­ t ­ t PCR:i as Microscopy: as ma ate or bi Culture: na bac PCR: na bac PCR: na in sup PBS na prep an cal NaCl oreth PBS, cal NaCl oreth PBS, Culture: na tion), scrap sed an Microscopy: bi an in sup PBS (smears, touch or squash prep Cutaneous ul ­ cers Eye 120 SECTION 3 ion ion spp. spp., ­ t or - and a ­ t ae of orm, or in spp., ­ l ­ v en ­ w a ion of most of ion ­ t ­ m ­ m ­ i a ­ c ed trichrome ­ fi i ­ fi ial is also used); ­ t ­ i ­ r ion (larion E. histolytica, ­ t e ­ t ents). Proglottids ra ­ t ined sed spp., hook spp., ­ m ats ( ion ion of for ­ b ­ t en oan cysts may also be spp., Cyclospora ­ m a ­ c ­ z ies iden ­ t o ­ c oa); mod ­ t spp., Cyclospora en ­ z o ­ m ­ t ode seg ­ i ­ t spp., Diphyllobothrium ) b ridge for ­ t ion ion or com ­ t us or spe al pro Taenia solium ouse tests Taenia for a ites ­ t ­ n ­ n h ­ s ate sed Taenia - ­ t ­ ti ­ a ­ e lude Schistosoma uired) es ­ c ­ t ­ q ue stains. oa); flo inth eggs and pro ­ s ­ z Cryptosporidium thyl thyl ac spp.) In spp.) o ­ m ant par in (in us level ( us level ­ t ngs inngs e E. vermicularis ­ l ion of unpreserved of ion fresh ma ­ i ­ v ­ n - ­ t ­ y ­ e ished. ast ( a ­ l f in ng tisng ­ n ­ l - ­ i ox ­ i ­ t s a d ­ i a m tes; hel ­ u ­ m ­ i ­ a on find ­ m ble. Primersble. ge for o ut E. histolytica, Cryptosporidium ­ a ­ z ­ m ­ o ays (e.g., EIA, FA), car EIA, (e.g., FA), ays inth agar ova); or Baermann con ­ s ed ac s ho ­ fi ed to the ge ­ m les (most pro ­ a ­ i ­ p oa are pub ect ex gy ( ds: for ­ fi ­ p ­ r o ­ z i ­ o ­ o ­ t ­ n o ­ t ­ tol ­ mu ose tape, no stain ( ds (hel Specimen Collection ­ l spp.) oan tro spp.) with spp.) ­ o spp.; unpreserved stool re unpreserved stool spp.; u ­ z ial test avail ­ l o ­ c ­ t ine his ­ t er spp. lly be iden spp., and spp., Cryptosporidium ­ m ­ a d) xed stool sam ­ e fi ion methion inths and pro ile pro - ­ t ­ t a ­ m ­ t ­ tect Carmine stains (rarely used adult for worms or ces can usu Hymenolpesis Cystoisospora Adhesive cel Commercial im Taenia saginata. Taenia comNo Concentration meth Giardia SAF hel Trichuris microsporidia); less com Recommended stain(s) and rel (microsporidia); mod H&E, rou Direct smear wet (di de flo Strongyloides (mo Stain: trichrome or iron he l en ­ a ents ­ i ­ z (continued) ­ r opy opy ­ m a e ­ c gy or ­ t s ion ion ive or ive ­ o b ­ t ­ o ­ t ol ra ­ t ty of ­ t ­ lines ions for ions for ­ i oid ­ t ents (all al ma ­ t a en orm seg ­ m ­ n ­ r ent ­ dures bil ­ c ­ a ­ ti ­ d ­ w e ­ a ­ c es ial, fresh, fro en ­ t ­ r ­ p al con e ­ t ­ n l ion: fresh na l; suit e ­ a ­ t ­ a ion ion smear i ­ d i erved 4), (see Table ­ r ­ s ­ o ­ r ec ­ s e ­ t fixed e ­ t ­ t - res ive ive ma ids) ­ t ­ p ol ­ t ­ n g ial, du ­ a ­ ods ­ in ­ r ion ion is test de ­ glot en ma e ­ t ­ t ­ z ect wet smear, con ­ a ­ r Antigen de Microscopy: fixedfor his touch or squash prep ma fro PCR: na Biopsy ma stain fix Microscopy: stool, sig di Adult worms or tape Specimen Specimen and pro Stool and other in fresh or pre Anal im (pro or eth meth Parasitology: Specimen guide

Body site tract Intestinal Table 3.6 Specimen Collection and Transport 121

er ­ v ble ble for ­­ a gy ), eggs (continued) ty test ­ i ­ o il ae); sil ol ­ b ­ b t ­ red in ­ a ­ e ov ­ c y) ed trichrome, EM ­ g ine his ­ fi ­ t ­ o ­ i ial for vi ol spp., ame spp., ­ t ­ r e ­ t tes), tes), protoscolices of msa stain ( Leishmania ­ i spp.) ­ e o ine blue stains avail ­ z ­ d ine his ­ i Gi ­ t . u ho ­ l ­ p spp.) can spp.) be re ­ ca cyst ma ­ i at (tro spp.); H&E, spp.); rou ­ p ine, to spp., Dirofilaria ­ m Echinococcus a ue Gram stains, mod ­ s ­ n he ­ t Echinococcus ast, tis f ion ion (e.g., E. multilocularis - ­ t ets ( er me ens. a d ­ l n) y ­ v ­ i ­ c ­ p ­ m ­ fi ­ i ­ a i ­ mo ­ t ion ion of her ­ t ­ t a ­ l o msa, sil ­ u Ascaris lumbricoides, Strongyloides stercoralis ae Strongyloides lumbricoides, ( Ascaris ­ e ­ m c ­ v ory spec ­ o oa and microsporidia); H&I (rou ­ t ype iden ­ z a (not com (not ­ t ast stains (Cryptosporidium ­ r o Specimen Collection f o ­ t spp.), or spp.), hook Capillaria or spp. Capillaria eggs of he - pi ­ n d ­ s is jiroveci. ­ i ­ t inth lar erm che ine stain, ac PAS, t - ys ­ m ­ m ­ c a o ­ n ­ m ained re he ­ t er er long ­ st ­ t Paragonimus Pneu Some hel ( un (microsporidia); Gi (Strongyloides stercoralis, Paragonimus stercoralis, (Strongyloides me Examination smear wet of E. for histolytica For Leishmania Modified ac spp., otherspp., pro Intraperitoneal in Echinococcus af Species or ge en ive ive ­ z ­ t ate, ­ r i le le for ­ p ­ i al and l l ­ s ­ a ­ a ial, i ial in i ial, fro en or ­ r ­ r ­ r ­ r ­ r le le le le ­ tion ­ z e e ; ster e ­ i ­ i ­ t ­ t ­ te ­ te ion ion of na a ­ t ates ­ t ­ r ­ r ­ sy ate, ate, brush a i ­ a um, na ­ r p ­ r ­ t ­ p i ­ o ­ a age fluid and ­ p ­ v ial, fro ­ r ive ive ma xed ma xed ma tive ­ tive ma tive ­ tive ma ­ t e ion: ster ion: ster ol ­ fi ­ fi ­ t ­ t ­ t ­ n a a ung bi ­ a ­ l ­ l le le prep l hial as les or as ­ i ­ u ­ u - ­ c ­ p c c al NaCl; fixed for ­ d ­ c ­ o ­ o xed na harge, bronchoalveolar ­ i on ive ive ma ­ c ­ fi ­ t tion ­ tion of na tion ­ tion of na ­ br og ­ gy ­ al a a ­ l i ­ r ­ r ­ o ol ol fixed ­ o ­ r ried smears. ­ i ­ a ­ a ­ n heo e ol d psy, psy, open ­ a ­ t ­ t age fluid, transbronchial as us dis ­ c - ­ o ­ v ­ n Microscopy: un PCR: na la tra si Microscopy: un ma Animal in Biopsy Biopsy sam Culture: ster eth Sputum, in bi uced spu bronchoalveolar la air treated for smear prep PCR: un or fixed in eth prep his Animal in phys prep Liver andLiver spleen tract Respiratory 122 SECTION 3 ng. ­ i i); us ­ c ­ o uenc e ­ q ion ion with er us, ­ n ­ t ­ l ­ c ods a a u ­ p ­ t ­ ti ­ c ­ v ­ fi i ­ t hro uent se ­ t ­ q ng ng cu ­ i e ­ s spp., cys ies iden ­ c ae caus ­ v uires sub Onchocerca vol orms), and ar ­ q ­ w b Trichinella leishmaniae, Acanthamoeba gy ( ained (spe ­ o ­ st ue Gram stains, EM (rare ites ol gy ( ­ s ­ s t ­ ­ o ­ a ol ­ t ies level re ed un ­ c ­ fi spp., hook spp., i ast, tis ine his ­ t f ­ t - d ant par ­ i ine his ­ v ­ t ­ e ble. ­­ a microsporidia), other lar ion ion to the spe ­ t Strongyloides a ite avail ­ c ­ s ­ fi spp. can be iden ­ a i otic ­ t ­ n er (above). ­ c ine stain, ac PAS, Specimen Collection ­ m a ions (microfilariae) ­ n ­ t a ae PCR). by H&E, rou ­ r ained smears or H&E, rou he rans (zoo ­ v ­ t ­ a st ­ g - a ­ s ies and other mites) ies le larle ­ b m er me ­ g ­ e ­ v sil microsporidia). Microsporidial iden (sca Dipetalonema Dirofilaria streptocerca, repens, larva mi Primers for most par spp., Entamoeba histolytica, sin Wet prep Gi Recommended stain(s) and rel Larvae Trichinella of See Cutaneous ul gy ­ o (continued) ol a ­ t en or b ­ z ngs, ion, ­ i ­ t en or fixed a ­ lines ­ z ­ n ­ i ­ dures m e ive, froive, ­ a ­ c ­ t ive, froive, xed, touch and ­ t ­ fi ions or fixedfor l ­ t ­ a a i ­ r ­ r ­ a e ­ t xed or na xed na ­ fi ­ fi ol ­ n gy and EM ­ a ­ o ol ol fixed y ­ s ­ n ­ a ­ tol ­ op stained smear (or fixedfor his or EM) PCR: un squash prep Aspirates, skin snip, scrap Specimen Specimen and pro Biopsy ma Microscopy: un his PCR: un bi Microscopy: wet ex in eth eth Parasitology: Specimen guide

Skin Body site Muscle Table 3.6 Specimen Collection and Transport 123 ­ ­ a ally ine eph ­ t ­ c ­ c ­ i ue ­ s cop ope ic en ­ l ­ s ­ b e 11th ed., ASM 11th ­ v ro

­ c ast, tis f - ­ tic en ous ame d ­ t ­ i a ); Delafield’s ­ m ected mi o ­ l ­ t ­ u uences is the method of ial plas ­ q ­ c er ­ m (com tes can be de ive gene se ive ­ i ­ t o ­ i ody; GAE, gran ­ z ­ b et i ­ t ­ p ho microfilariae);PAS, ac Trichomonas vaginalis Trichomonas ­ p ed trichrome (microsporidia); H&E, rou ent an ­ fi d–Schiff stain. ­ c ­ i ­ i T. vaginalisT. ng ng tro es ­ i ­ r dic ac ­ o ion ion of i gents (FA) ( ­ t ­ r ­ a ion of highly of ion re ­ ga ­ t ­ a y; FA, fluo ble); mov ec ­ p ­­ a ­ t ay ay re o ­ s ­ c s ­ a ained smears). is; PAS, pe o ­ t ­ cros Specimen Collection i ­ n st ­ l - er SS, Warnock (ed.), DW Manual of Clinical Microbiology, ­ a ­ t a ­ mu ­ s ­ tems avail Schistosoma haematobium, in (microfilariae); mod eph ­ l m ron mi ­ c ­ t ­ e ­ y y ( ox ­ og ­ t ic en a ­ b ure sys ol msa, im ­ t ­ t ­ e ­ m cul Gram stains, EM or PCR (microsporidia). Identification and prop (or in Gi he Gi his PCR based on the de choice. ay; ay; EM, elec ­ s ary ame s ­ a ­ m o ure ­ n ) en, ­ t u en or ­ m ­ m ­ z ­ i ure, ­ t hral hral ­ t ions ­ t ­ t ions; ive ive ­ t en, a ­ t oal), cul in; PAM, pri ­ r yme im ial, fro ­ s ­ c ­ m re ­ r ­ z ­ a ­ i ­ c ine swab, en. e ope cul oll Funke KC, G, Landry ML, Rich ­ l ­ t ­ l Toxoplasma ­ r ­ m e ­ i ­ v al or ure ure): na ­ n ion ion ( ­ t ­ i ­ t in and eo ­ tatic se ­ l ive ive ma a ­ t d; EIA, en ­ l ­ y ol ­ tic en ­ i ­ u harge, sa ­ n ent, stained smears ox c ­ c ­ a ­ t le le unpreserved spec ­ o a ­ m ­ g ­ i al flu ­ m ­ n ­ al ort (no char swab pi i morning spec unpreserved spec ­ s ­ p - ­ r ried ure smear FA; for ium, plas harge, pros harge or swab prep e d h ro ­ ly ­ d ­ t ­ c ­ c - - ­ b e ­ r urine, sin ma dis 24 air ear Molecular: na PCR (and/or cul Animal in Vaginal dis me Microscopy: smears, wet smears of urine sed Cultivation: vag dis trans fixed in eth al; H&E, he ­ n i ­ t es ­ t on, D.C., 2015. on, D.C., n ­ t ­ i ro ng ­ t ­ i Adapted from Jorgensen JH, Pfaller MA, Car Abbreviations: CSF, ce is; Gl, gas a b ­ t Amniotic Fluid Urogenital ­ tem sys li Press, Wash 124 SECTION 3 ­ ive ive ­ t ­ i ion, le le ­ t ies of ies of ­ b i ient. ­ r ec ­ t ­ s ­ l en is ­ m em is ­ i ­ l ay timeay ends on the d ­ p ion ion (rare), but - ­ t ec en. In a se oa are shed ­ f ­ z ­ m ­ i o ­ t ient for the pa en the case that not all ist, proper col ­ t ­ n ended 10 ­ g itic in e ­ s ­ o ­ m l ­ v ­ a ­ o ive and/orive may be pos i m ­ t on ­ i ­ b ­ o ­ c le stoolle exam de ro ­ g ­ c ial par sms. ­ i ens, is of it ­ m nd (or third) stool spec and microsporidia are pos n o ­ o ­ m ­ c ­ i ­ ga ­ o ies of stools;ies of pro ­ r ite load in the spec ens are pos ­ s ays theays chance that the prob ­ a ­ m ­ w ­ i nt nt or tions. ­ e ­ a ally. ally. May be in r nce of the mi ­ c er ­ e ­ i ­ f ­ e i d ­ r id ­ o ected within the rec ­ s e i ­ l ated to a nos ­ r ­ p ­ l hree spec Cons There is al re Cryptosporidium con Diagnosis from a sin and the par three stool spec ex t for for dif Assumes the sec col frame a se for pe itic ­ s tic oms - ­ a ­ a ­ t ens may Specimen Collection al Ent om ­ m ­ t ­ n omes ­ i ion ion of the ­ c ­ t ­ ti a,b ions, the ­ t ec a es ion ion and ients may ­ l ­ t ­ t ­ t ­ n ites ­ i a osed in the first lly to other ­ s reases ( ­ p 22.7%; Giardia ­ n ­ a i m ­ c ­ a ient be r ­ t g ­ a ted not to par ­ st ­ e ­ a er col uent spec ­ u ome symp er been they have ­ t ­ q ­ t ­ c e ions, pa oa in rib ­ t ­ s ­ t ­ z ary. ­ s o ec Dientamoeba and Dientamoeba 11.3%; tic af ­ t s ­ f ional ex ­ a ­ e hea af ­ t i ­ r lly at r ­ d 31.1%). ites are di ens ens for par om ­ a ­ a ­ s ­ t ate with con ions but gen ­ a s. ­ m itic in le orle if the pa ients for a few days;ients symp a few for ­ t hea. ­ n ­ i ­ e ­ s ­ t ­ p ­ r ­ a a ec er ­ f ­ p ­ ar ­ t Patients may be with di in Pros are usu in caus If par sam asymp first stool, sub not be nec par However, However, with some in al di yield of proyield of amoeba histolytica, duodenalis, fragilis, With ad ient ­ t ng ng stool spec ng ng en ­ i ­ i ive. ­ m ­ t ens is ­ i i ess ain ­ s ­ m ­ c ional ­ i ­ t ­ m i ­ d ients who have ients have who ­ t a ­ p ive andive the pa ient re ­ t ­ t est that 40–50% of ­ a uire ad ­ g nd stool spec ­ q le stoolle (O&P 3 days. ­ o ­ g c. ­ i t nt willnt be found only by a idelines idelines for pro ­ a m; any pa ­ e ­ a al > for ­ t om Gu i ­ t ­ p tic would re

ion). Data sug ­ a ­ t er the first is neg ble, but threeble, are more sen a ­ t ­ a sms pres ­ n om ­ i ­ i ­ t n ng. Examinationng. two spec of le le stool ex ­ i m ept ­ g ­ a ­ c ­ ga Rejection stools of from in been in hos Option test Examination a sin of symp ex or sin ac Examination a sec of only af is still symp Table 3.7 Specimen Collection and Transport 125 ion ion ­ t nt innt oa ­ e ion ion ­ z ens for ion ion ­ t ec o ay. ay. If ­ t ­ l ­ t u ­ m ­ s ry stool). ­ l (continued) ­ i s ent stain ions with ­ e ­ a ­ t ­ n ion of three of ion o a ­ t , and D. ion ion and ec ­ n ens, this ­ t ­ f ­ m ec ­ l ­ m ra ­ i ­ t ­ mu ae) might be ­ v ate the col en itic in ­ n ­ c i ­ s ble timeble frame. ive, ive, other pro ­ d ­ a ­ a tic, then if the even ­ t r ormed on ev , E. dispar ­ a ­ f ­ a ar ­ o on oms (off and on), so it ate the col ­ s ­ p ­ t en and im om ­ n ­ t i ­ m ver, ver, with a per ­ d ly bely the best next op ng of the of ng three spec ­ i ble timeble frame. ended. Organisms pres ­ e r ­ i ­ b ­ a roach (con ­ o ion (eggs,ion lar ­ a ­ m ­ t ­ p ult to co ay ay is neg on ­ c ­ s m ­ s ec s ­ fi ­ o ­ f ibit sympibit ­ a E. histolytica o ­ h ens in a rea ains symp ­ n ard ap ion; how ­ m ­ t ult to co ult to rule out ­ d ­ m ­ i ­ c ­ c ers may be missed due to the di ra ­ mu inth in ­ fi le le stool spec ­ t ­ fi ­ b ­ g ­ m en im ent stained smear per ­ c ). ens in a rea ient re ­ n ­ t a ­ m ­ i ormed on each the of three spec or. or. May be dif roach would prob ­ f ­ m ­ t er the stan ­ p ­ t Patients may ex may be dif only a sin the pa Giardia fac may be missed ( Procedure not rec small num fragilis of threeof spec Light hel missed due to the pool the con per ap af May beMay dif per spec ly ly ­ b ­ a ize Specimen Collection ­ m sms are as the of ­ i ­ i ­ e n ected 7 over ected 7 over ­ l ­ l ide, prob uired. ­ s ­ ga ­ q ive andive the ­ t ­ a oa in ar ­ z o ­ t oms sub ng isng re ­ t ­ i ens are col ens are col ­ m ­ m on. ­ i ­ i ­ m ms are neg ­ a her test ry pro of ­ t ry where these or ­ e ­ t ense. ient’s ient’s symp ov ­ t ­ p ­ c If the ex pa no fur Three spec Three spec to 10 days and timeto may save 10 and ex coun to 10 days; thisto 10 would max most com re ion; ion; ­ t ­ t a a ent ­ n ­ n ­ n ­ i ­ i a m m ­ m ­ a ­ a ent stain are ­ n a ridge) ­ m ­ t ble; one O&P exam ens for ex ens for ex ory pools the ormed. ­ a ­ t ­ f le stoolle and an ­ m ­ m a ­ i ­ i ­ g ­ r ept ­ o ­ c roach is a mix: one roach. ­ p ­ p rate and three per ­ t en rate and one per ­ c ay ay (EIA, FA car ay is ac ­ t ­ s ­ s s s en ); this ap ­ a ­ a ­ c o o ens. ­ n ­ n le le con ­ m ­ g ­ i ormed. The lab ­ f ­ mu ­ mu Giardia Examination a sin of im ( im is not the best ap per spec one con stained smears are per a sin Pooling of three spec Pooling of three spec 126 SECTION 3 ures ient ­ d ow ow ­ t ­ l e ive. ­ c ­ t tic), ­ a ­ a ients from reak ­ t ive, and ive, the ­ b ng ng the ive andive the ate pa ­ t ­ i ­ t ­ c om a ­ t ay ay pro li ec rd ­ v ­ s ­ f ­ p s ar risk group er ­ a ­ ga ­ l ­ s , 11th ed., ASM Press,, 11th o ­ u ­ n ren from day care en and fix ult in poorly ic ­ s ­ d ­ t ies, and pa ­ m ess an out ­ i ­ l ­ c ­ mu ion thation would al ially re ­ t ly rely ­ c a ien e ­ b ­ c ­ m ­ a ­ p ­ fi ow unow ­ l or ­ f en is not cost ef ­ m ng of spec of ng ne de ient in a par ­ i ­ i 5 yr old, chil ­ t io of stool of io to pre ens, es ­ t ­ mu ­ m eive in eive ­ i olved. ­ c ­ v ren < ended. This would com ­ d ­ m m ended ra ­ o ion andion very prob ­ m ry stool spec ­ t ion ion is in ­ e ive rateive will be be ­ t m reaks. Performance of im erved spec ers (who may orers may not be (who symp ­ t ec ­ a ­ i ­ t ­ s ­ o ­­ b ­ l ients with im ­ u ­ t Cons col Not Not rec pre rec lack proper of mix out such as chil on ev sit cen pa pos them to place a pa Labs rarely re a­ ures ­ m ite. ­ d ­ s e ens. ­ a or ­ c ­ f ­ m Specimen Collection ­ i (continued) ive thanive ients is a,b ­ t ­ t l. ay ay pro ver, ver, in er SS, Warnock (ed.), DW Manual of Clinical Microbiology ec ­ a ­ t ­ s ­ f ­ e ens would ites s ­ s ­ a uired for ­ m ers. ­ a ­ i o ­ q le vile ­ d ­ n ­ g ­ mu ens; how ion ion re eived eived with spec ­ t ­ c ­ m a ­ i ody; O&P, ova and par ­ c te test or ­ b ­ u ng ng im i ­ a ­ i ­ t uired to group pa ens ens for par ri ­ q ­ p ­ spec ­ m ­ i orm ro ­ f uire only a sin ent an en not re ­ p ­ t ­ q ­ c Pooling the of spec Pros re Would be moreWould cost ef per on all tion re of Client ed ap es ­ r oll Funke KC, G, Landry ML, Rich ­ r ies, ients ng ng stool spec ­ t ­ c ­ i ay; ay; FA, fluo ­ s s ien ­ a ess ­ c o le vialle ­ c ­ n ­ fi reaks) for ­ g les stool of u e ­­ b ­ p ­ d ected pa ­ m ­ l o ­ n ren from day care ­ mu ­ d yme im heal out ­ r ­ z r ions are placed in a ­ t ays ays on se ­ a ­ s s ec ­ l ­ a oa. idelines idelines for pro o ­ z ient isa sin given ­ n ­ t ­ to Gu ­ mu ients with im 5 years, chil ­ t

< hree col ­ t on, D.C., 2015. al pro ients from di ­ t ­ t ­ n ren ren i ng ­ t ­ d ­ i ers, pa le vialle (pa ­ t ­ g es ­ ly). ­ t Adapted from Jorgensen JH, Pfaller M. Car Abbreviations: EIA, en a b Collection three of stools; sam from all on Option sin cen Perform im and pa (chil in Table 3.7 Wash SECTION 4 Bacterial Diagnosis

Ge­ne­ral Comments 128 Table 4.1 Detection Methods for Bacteria 129 Table 4.2 Recommendations for Gram Stain and Plating Media 133 Table 4.3 Screening Specimens for Routine Bacterial Culture 137 Table 4.4 Processing Specimens for Mycobacterial Identification 138 Microscopy 139 Primary Plating Media: Bacteria 141 Primary Plating Media: Mycobacteria 154 Specific Diagnostic Tests 157 Aerobic Gram-Positive Cocci 157 Aerobic Gram-Positive Rods 158 Acid-Fast and Partially Acid-Fast Gram-Positive Rods 160 Aerobic Gram-Negative Cocci 161 Aerobic Gram-Negative Rods 161 Anaerobic Bacteria 168 Curved and Spiral-Shaped Bacteria 170 Mycoplasma spp. and Obligate Intracellular Bacteria 172 Identification Tables 175

doi:10.1128/9781683670070.ch4 128 SECTION 4 This sec­tion pro­vi­des guide­lines for the se­lec­tion and pro­cess­ing of spec­i­mens for the de­tec­tion of spe­cific bac­te­ria. Testing can be sub­di­vided into mi­cros­copy, cul­ture, an­ti­gen tests (in­clud­ing im­mu­ no­as­says and mo­lec­u­lar di­ag­nos­tic tests), and an­ti­body tests. Al- though it is im­pos­si­ble to pro­vide guide­lines for all­ pos­si­ble in­fec­tions, the most com­mon bac­te­ria as­so­ci­ated with hu­man dis­ease are in­cluded. This sec­tion has been ex­panded to in­clude sum­mary ta­bles of iden­ti­fi­ca­tion tests as well as a more de­tailed dis­cus­sion of the im­mu­no­log­i­cal de­tec­tion of or­gan­isms where ap­pro­pri­ate. Bacterial Diagnosis Bacterial Diagnosis 129 cs ­ ti s ­ no g (continued) ­ a B B C C C C C A D D A D A D D Molecular di

n ­ tio B D D D D D D D D D D D D D D ­ tec de Antibody

n ­ tio a a ­ tec ­ ri Antigen ­ te de nt nt bac ­ va BD AAAC ABACAD AD AD AD AD AD AD AD AD AD ­ e Culture lly rel ­ ca ­ i B B B A A A A A A A A A A A A Bacterial Diagnosis Microscopy ns for clin ­ me ­ i l spec ­ ca ­ i s from clin ex s complex ­ od ­ pl -fast Gram-positive rods ­ si ­ ci ­ id ­ lo ­ cu other spp. ­ ber ally ac ­ ti group A group B spp. spp. tection meth spp. De spp. spp.

Staphylococcus ­ us au ­ re Streptococcus, Streptococcus, pneumoniae Streptococcus Bacillus anthracis Corynebacterium, Gardnerella vaginalis Enterococcus Listeria Erysipelothrix Mycobacterium tu Corynebacterium diphtheriae Mycobacterium avium com Nocardia Rhodococcus Aerobic Gram-positiveAerobic coc Organism Acid-fast and par Aerobic Gram-positiveAerobic rods Table 4.1 130 SECTION 4 cs ­ ti s ­ no g ­ a C B C B B B C B D D D D A D D D D D D Molecular di n ­ tio C B B D D D D D D D D D D D D D D D D ­ tec de Antibody n (continued) ­ tio a a ­ tec ­ ri Antigen ­ te de nt nt bac ­ va AD AD AD AD AD AD ADAD AD AC AD AD AB AD ADADADAD AD ­ e Culture lly rel ­ ca ­ i B A A A A A A A A A A A A A A A A A A Bacterial Diagnosis Microscopy ns for clin ­ me ­ i l spec ­ ca ­ i s from clin ­ od ­ ci serovar Typhi serovar spp. spp. spp. spp. tection meth other serovars spp. De spp. spp. spp.

Kingella Eikenella Escherichia coli Neisseria gonorrhoeae catarrhalis Moraxella Actinobacillus Capnocytophaga Cardiobacterium Streptobacillus influenzae Haemophilus ducreyi Haemophilus enterica Salmonella Salmonella, Neisseria meningitidis Pasteurella Shigella Yersinia pestis Yersinia enterocolitica Other Enterobacteriaceae Organism coc Gram-negative Aerobic rods Gram-negative Aerobic Table 4.1 Bacterial Diagnosis 131 (continued) B C B C C C C B B D D D D D D A D D D D A D D C C B D D D D D D D D D D D D D A D A A A D A D A BA BA B BD BD BD AC AD A ADAD ADAD ADAD AD AD AD AD AD AD AC AA B B B B B B B C A A A A A A A A A A A A A A A Bacterial Diagnosis ­ plex com group s spp. ­ num ­ ri ­ si ­ li ­ cile s ­ lo ­ u spp. ­ fi ­ ria ­ tu ­ te spp. spp. spp. spp. spp. spp. other spp. Clostridium perfringens Clostridium Helicobacter py Aeromonas aeruginosa Pseudomonas pseudomallei Burkholderia cepacia Burkholderia Stenotrophomonas Campylobacter Clostridium bot tetani Clostridium Clostridium dif Bacteroides fragilis Acinetobacter spp. Bordetella per Vibrio cholerae Vibrio, Actinomyces spp. Fusobacterium spp. Francisella Brucella Legionella Bartonella Mobiluncus Anaerobic bac 132 SECTION 4 ­ tic cs s ­ ti s ­ no ­ ag ­ no g ­ a al di B B B C B A A A A D D D ­ er l for genl for fu ­ Molecular di m use ­ do n ­ tio B B C A A A A A A A A A ­ tec de Antibody on; C, test sel ­ ti c n ­ fe (continued) ­ tio a a ­ tec ­ ri Antigen ­ te de fic formsfic of in nt nt bac ­ ci ­ va CCBDCD CD BA CD CD DDDD DD DD DD ­ e Culture s of spes of ­ si lly rel ­ no ­ ag ­ ca ­ i l. B B B B B C D A D D D A Bacterial Diagnosis ­ fu Microscopy ns for clin ly usely ­ al ances or the for di ­ me ­ er ­ i ­ st a m ­ ri ­ cu ­ te l spec ­ ca in cir ­ i r bac ­ ta ­ la ­ lu r cer l ­ de ­ ce a ce labs; test D, not gen l un ­ tr ­ ria ­ en s from clin fu ­ ­ te ­ er ­ od te in ­ ga ­ li le in ref ­­ ab l; test B, use ­ fu ral-shaped ­ ral-shaped bac tection meth ly usely spp. ­ al spp. and ob De spp. other spp. ­ er spp.

ses but may be avail ­ po Leptospira spp. pallidum Treponema Mycoplasma pneumoniae trachomatis Chlamydia Chlamydophila psittaci pneumoniae Chlamydophila Rickettsia rickettsii Ehrlichia Anaplasma Coxiella Borrelia burgdorferi Borrelia, A, test gen a ur p Organism Curved and spi Mycoplasma Table 4.1 Bacterial Diagnosis 133 s y ns ­ it ­ er es r

­ ­ me ­ tu ­ i ­ um c (continued) ­ fa –10%, dy cav 2 ­ u ­ bo es should be ­ tl l tract spec te large vol ions in 5% O at 42°C for all for at 42°C ­ t 2 ­ na ­ ba re bot i c ns all for ­ t ­ da s ­ cu ­ tu ­ te ­ me ­ i ­ men ­ in s. Following man –85% N ro 2 ­ t ­ om ­ id gas used to in Comments Blood cul CO of specof flu rec C. jejuni/coli a ­ di BA BA BBA BBA BBA LKV BBE BBA B B g ­ in l- ­ to a Anaerobic me ­ bi ­ di a,b ); sor a ­ al ­ di on ­ ti Bacterial Diagnosis g me Aerobic Aerobic me B Mac HE Ca EB B C B C Th B C B C B C Mac CNA B C Th B C B C B B C Mac B C B C B Mac HE Ca EB (op MAC/chromogenic agar/Shiga toxin test ­ in Gram stain Gram x x x x x x x x x x x ­ ism /swab ­ id ­ id s ­ gan commendations Gram for stain and plat ­ id l l flu l flu ne) Re ­ row ­ na ­ ti ­ na r

­ ity flu ­ ter ­ te (rou heter tip heter PD F (shunt) ne mar r r ex r in ces ricardial leural eritoneal ynovial CSF CS Pe P P CA S Bo Cat Ea Ea Fe Rectal swab Specimen or or Body cav Eye Gastrointestinal tract

Table 4.2 134 SECTION 4 re is ­ tu c s of BV ­ si ­ no ­ ag Gram stain and NOT cul the method choice the of for di Comments a ­ di ired c ­ qu ope ­ bi ­ o ­ sc r g re ­ ae ­ in ho re BBA LKV re BBA ­ c ­ tu BBA LKV BBE Protected bron brush cul BBA LKV BBE for for an a Anaerobic me ­ di (continued) a,b re to B a ­ tu ­ di l ­ cu Bacterial Diagnosis g me Aerobic Aerobic me B TM TM B C Mac TM Selective broth, sub B C Mac (Cystic Fibrosis); BCSA Salt Mannitol B C Mac B C Mac CNA B C Mac CNA B C Mac Th CNA ­ in Gram stain Gram X X X x x x x x ­ ing ­ id l screen ing, ­ ing, wash ­ ca ­ vage flu c ­ ism ­ co e y y tract ­ to ­ gan commendations Gram for stain and plat ­ rat ­ tor ­ pi Re ­ ra

pi ­ s heal heal as oup B strep utum ther rethra/penis U O Gr Sp Trac Bronchoalveolar la Bronchoscopy brush Specimen or or Vaginal/cervix Lower Lower re Tissue Table 4.2 tract Genital Bacterial Diagnosis 135 (continued) rted in ­ po ive ­ t ­ si re only if em ­ t ­ tu te agar for ­ la e sys ­ o ­ at i te swab trans ve ­ pr ­ ti ­ ra o ­ si ­ a ­ pr epiglottitis ap Add choc Anaerobic cul NAAT more sen Shiga toxin EIA more or NAAT sen sep BBA LKV BBE BBA LKV BBE m or ­ ru c CNA le ­­ ab Bacterial Diagnosis B Mac or Chromo- genic agar B C Mac CNA Regan Lowe B C or Cysteine-tellurite se Loeffler’s CNA if not above avail B C B or SSA B B C Ma CCFA Sorbitol-Mac Chromogenic agar C or BCYE x x and B. parapertussis ­ sis ­ cile ­ fi ­ tus y y tract ms ­ is ­ tor ­ scess ­ ra ­ gan spp. ­ spi oli O157:H7 (EHEC) se rynebacterium diphtheriae spirate asopharynx hroat rucella A B Co N No T Swab Clostridium dif E. c Francisella tularensis Bordetella per Urine Selected or

Upper Upper re ab or Wound 136 SECTION 4 ­ est for . ­ qu ­ um ­ di us act; TCBS, thio ing “school ­ eo ­ tr ­ bl m gas ­ cin; BBE, bacteroides ­ se ­ my c ­ co n doesn not re have al yeast ex ­ co ­ me here here at 35–37°C ­ i ­ sp ­ mo ed char e spece ­ cin and van of fish” Comments Gram stain re Campylobacter at 30°C wk up to for 13 ­ er ­ at i ­ my ­ pr o a ­ pr ­ di anif ap ­ ci ­ si ely. Call ely. phy ­ at i

­ pr um or n o a Anaerobic me ­ di ­ ci ­ pr r SS, Yolken RH SS,r Yolken (ed.), Manualof Clinical Microbiology, ed., ASM Press, 11th ­ di (continued) ve agar,ve TM, Thayer-Martin; BCYE, buff ­ te ­ my ­ ti ­ late blood agar, Mac, MacConkey agar; Th, thioglycolate broth; Ca, Campylobacter a,b c ­ o a ­ co ­ le rtedap ­ di ­ cella blood agar; laked LKV, blood with kana se ­ po g/ml) r BHI Bacterial Diagnosis g me Aerobic Aerobic me C + van (3 μ B o Fletcher’s me Fletcher’s EMJH TM BCYE TCBS CIN BCYE e-cefoxitin-fructose agar; EMJH, Ellinghausen-McCullough-Johnson-Harris me ­ in ­ in r ­ se o Streptococcus ­ cl ctedand trans ­ le ­ sis; blood B, agar; C, choc ­ y Gram stain Gram x n isn col ­ al ; CCFA, cy llFunke KC, G, Landry ML, Rich ­ me ­ id ­ i ­ ro ­ neal di ­ to est,if spec ­ ment broth; SSA, group A n-nalidixic ac ­ tory peri ­ qu ­ ti ­ la s ­ rich ­ li ­ bu co ­ bic reupon re ­ o ­ crose; CIN, cefsulodin-Irgasan-novobiocin; bru BBA, r ­ ri ­ ic; EB, en ­ ism ­ tu ­ ae ­ lo ­ ter cul ­ gan commendations Gram for stain and plat ­ bic spp. re. Re ­ o ­ tu

­ aer n, D.C., 2015. spp. cul ­ rate bile salt su ­ to inia enterocolitica g sseria gonorrhoeae licobacter py ­ bic ­ in ­ o ocardia ibrio egionella eptospira spp. r Haemophilus ducreyi Haemophilus He L L Nei N V Yers ­ fate cit CAPD, fluid from chronic am Setup an Adapted fromJorgensen JH, Pfaller MA, Car ­ ae a b c Specimen or or

agar; HE, Hektoen en Table 4.2 sul bile esculin; CNA, an Wash an Bacterial Diagnosis 137 ­ ­ i m al es on ­ ta ­ ti

­ gi la into n ­ o ­ tu ­ te an l con ol ­ ci ­ ca tes biofilm on ­ ph ­ si te fis ­ ca le) ­ ca ­ di t are SEC ­ na l mor ­ di r or fe o ­ en ­ ti ­ te ­ ca on: phy ly ly in ­ e ­ ti ­ al p cted(100x) in 20 fields ­ ce ­ te g cath good ra ns usu ­ in b a de r from GI tract. ­ ge ­ ri ­ des ell ­ o ­ de on. Mixed fe ­ te ­ vi ­ ti ­ dw 2 + SEC and no PMNs 3 days (ex 10 SEC/average10 field 10x cells of 1% pres 10 SEC/average 10 field and 10x no Unacceptable > > > Gram stain may in > bac > Growth three of or more po path in na blad pro a ­ ri ­ te sults of screen ­ e test le. Onele. ­ re is an Re ­ as 3 ­ ab er ­ li ­ t on, but no ­ ti e es t c ­ en ­ fe ­ cyt t are SECs ­ ko e in ­ en ­ bl 10 PMNs/mm > ­ si ck ck leu g r SS, Yolken RHr SS, Yolken (ed.), Manual of Clinical Microbiology, ed., ASM Press, 11th ­ in ­ sti ­ te r or pos um per oilcor field power ­ to a ­ ri cted in at(100x) least 20 1 of fields lt lt or see ­ ca ­ te re ­ te onds toonds 10,000 CFU/ml in the urine. ­ di ­ su 2 + SEC, PMNs pres 3 days 10 SEC/average10 field 10x SEC/average10 field and 10x bac cells of 1% pres Bacterial Diagnosis ­ tu Acceptable < < < < in method has proved truly yet re bac ≤ de sp re Positive Positive dip al cul ­ ri on of on of on of on of ­ te ­ ti ­ ti ­ ti ­ ti ­ na ­ na ­ na ­ na ­ i ­ i ­ i ­ i ­ od l ­ am ­ am ­ am ­ am ne bac ­ ta ­ ti nt al cells. ll Funke KC, G, Landry ML, Rich ­ pi ­ li e ­ ro ­ me ­ i ­ th ­ i us ep ns for rou icroscopic ex ­ mo Microscopic ex Screening meth Gram-stained smear Microscopic ex Gram-stained smear M Gram-stained smear Microscopic ex Gram-stained smear Days Days in hos Urinalysis, Gram stain or urine sed ­ me ­ i e; SEC, squa ­ vage ­ as r ­ rate eening spec ­ te al ­ pi ­ ri Scr ­ te ­ cyte es

n, D.C., 2015. ­ ko ns ­ to g ­ ge ­ o ­ in ­ id LE, leu Adapted from Jorgensen JH, Pfaller MA, Car a b Sputum Specimen Endotracheal as Bronchoalveolar la flu Superficialwound Stool for bac path Urine Wash Table 4.3 138 SECTION 4 Table 4.4 Processing spec­i­mens for my­co­bac­te­rial iden­ti­fi­ca­tion Solid and liq­uid me­dia at: Specimen typea Smearb 35°C 30°C Abscess RX Blood/bone mar­row NX Biopsy spec­i­men Lung RX No t lung, lymph node, skin, OX or synovium Sk in, synovium, and lymph OX X nodec Superficial skin, wound, or RX X tis­suec Eye OX X Fluids Not joint or sy­no­vial flu­id OX Joint and sy­no­vial flu­id O X X Gastric wash­ing RX Respiratory (not mouth) RX Stool OX Urine OX aSources not gen­er­ally rec­om­mended for my­co­bac­te­rial cul­ture in­clude gen­i­tal sites, ears, cath­e­ter, mouth, and rec­tal swabs. Consult lab­o­ra­tory prior to mak­ing a re­quest. bR, stain­ing should be per­formed rou­tine­ly; 0, stain­ing is op­tional and should be per­formed if re­quest­ed; N, stain­ing should not be per­formed un­less the re­quest is dis­cussed with the phy­si­cian. Bacterial Diagnosis cSuspect rap­idly grow­ing my­co­bac­te­ria, M. haemophilum, or M. marinum. Bacterial Diagnosis 139 Microscopy Acid-Fast Stain Acid-fast stain­ing is use­ful for se­lect group of bac­te­ria that in­cludes the Mycobacteria spp. (both rap­idly and slowly grow­ing), Nocardia, Rhodococcus, Tsukamurella, Gordonia, and Legionella micdadei. What dif­fer­en­ti­ates these bac­te­ria is that they have long-chain fatty ac­ids, or mycolic ac­ids, that make up their cell walls and make them re­sis­tant to de­col­or­iza­tion. Thus, the prin­ci­ple of the ac­id-fast stain is the use of a ro­bust de­col­or­iz­ing agent (3% ac­id-alcohol [Kinyoun and Ziehl-Neelsen], 0.5–1% sul­fu­ric acid [mod­i­fied ac­id- fast]). The pri­mary dif­fer­ence be­tween the Ziehl-Neelsen (ZN) stain and the Kinyoun mod­i­fi­ca­tion is that the ZN pro­ce­dure re­ quires a lengthy heat­ing pro­ce­dure and a slow cool­ing pro­cess. In the Kinyoun pro­ce­dure, there is no heat­ing. In all­ ac­id-fast stains, the pri­mary stain­ing re­agent is carbol fuch­sin and the counter stain is meth­y­lene blue.

Acridine Orange Stain Acridine or­ange is a fluo­res­cent dye that in­ter­ca­lates into nu­cleic acid (na­tive and the de­na­tured). At neu­tral pH, bac­te­ria, fungi, and cel­lu­lar ma­te­rial (e.g., leu­ko­cytes and squa­mous ep­i­the­lial cells) stain red-orange. At acidic pH (pH 4.0), bac­te­ria re­main red-orange but the back­ground ma­te­rial stains green yel­low. Optimal de­tec­tion of fluo­res­cence re­quires the use of a 420- to 490-nm ex­ci­ta­tion fil­ Bacterial Diagnosis ter and a 520-nm bar­rier fil­ter. This stain can be use­ful in many sit­u­a­tions. It can be use­ful to as­sess whether am­big­u­ous ob­jects seen on Gram stain are real, and it can be used to screen low or­gan­ism den­sity spec­i­mens for or­gan­isms at a lower mag­ni­fi­ca­tion (i.e., ster­ ile body flu­ids, or pos­i­tive blood cul­ture bot­tles with no or­gan­isms vi­su­al­ized on Gram stain).

Auramine-Rhodamine Stain Auramine and rho­da­mine are non­spe­cific fluo­ro­chromes that bind to mycolic ac­ids and are re­sis­tant to de­col­or­iza­tion with ac­id-alcohol (ac­id-fast stain). Acid-fast or­gan­isms ap­pear or­ange-yellow. If the sec­ond­ary stain is not used, or­gan­isms will fluo­resce a yel­low-green color. Potassium per­man­ga­nate is used as a coun­ter­stain. It is a strong ox­i­diz­ing agent that in­ac­ti­vates the un­bound fluo­ro­chrome dyes, pro­ duc­ing a black back­ground for the stained spec­i­mens. Fluorochrome- stained smears can be restained by the Kinyoun or Ziehl-Neelsen meth­ods. Optimal de­tec­tion of fluo­res­cence re­quires use of a 420- to 490-nm ex­ci­ta­tion fil­ter and a 520-nm bar­rier fil­ter. 140 SECTION 4 Direct Fluorescent-Antibody Stain A va­ri­ety of or­gan­isms (e.g., Legionella spp., and Chlamydia tracho­ matis) can be di­rectly de­tected in clin­i­cal spec­i­mens by us­ing spe­cific fluo­res­ce­in-labeled an­ti­bod­ies. The la­beled an­ti­bod­ies bind to the or­ gan­isms and fluo­resce green un­der UV light. The sen­si­tiv­ity and spec­i­fic­ity of the stain are de­ter­mined by the qual­ity of the an­ti­bod­ies used in the re­agents. Optimal de­tec­tion of fluo­res­cence re­quires the use of ei­ther a 420- to 490-nm (wide-band) or 470- to 490-nm (nar­ row-band) ex­ci­ta­tion fil­ter and a 510- to 530-nm bar­rier fil­ter.

Gram Stain Gram stain is the most com­monly used stain in clin­i­cal mi­cro­bi­ol­ ogy lab­o­ra­to­ries. It is used to sep­a­rate bac­te­ria into Gram-positive (blue) and Gram-negative (red) groups. Variations in the per­for­ mance of this stain are com­mon­place; how­ever, the stain­ing prin­ ci­ple is con­stant. After the spec­i­men is fixed to a glass slide (by ei­ther heat­ing or treat­ment with 95% meth­a­nol), it is ex­posed to the ba­sic dye crys­tal vi­o­let. Iodine is added and forms a com­plex with the pri­mary dye. During the de­col­or­iza­tion step, this com­plex is re­ tained in Gram-positive or­gan­isms but lost in Gram-negative or­gan­ isms. The Gram-negative or­gan­isms are de­tected with a coun­ter­stain (e.g., saf­ra­nin). The de­gree to which an or­gan­ism re­tains the stain is a func­tion of the spe­cies, cul­ture con­di­tions, and stain­ing skills of the mi­cro­bi­ol­o­gist. Older cul­tures tend to de­col­or­ize read­ily. Some no­ ta­ble Gram stain re­ac­tions to be aware of in­clude the fol­low­ing: Bacterial Diagnosis • Treponema, Mycoplasma, Chlamydia, and Rickettsia ei­ther lack a cell wall or are too small and can­non be vi­su­al­ized by Gram stain. • Bacillus spp. and Clostridium spp. fre­quently de­col­or­ize and ap­pear Gram neg­a­tive. • Faintly stain­ing Gram-negative rods, such as Campylobacter, Francisella, Brucella, Legionella, Helicobacter, and oth­ers, can be count­er-stained with carbol fuch­sin rather than saf­ra­nin for bet­ter vi­su­al­i­za­tion. • Organisms that have been treated with an­ti­bi­ot­ics (par­tic­u­ larly with cell-wall act­ing an­ti­bi­ot­ics, i.e. be­ta-lactams) can have dis­torted ap­pear­ance, which may com­pli­cate their iden­ti­fi­ca­tion. • Bipolar stain­ing is a fea­ture most com­monly at­trib­uted to Yer­ sinia; how­ever, a num­ber of other mem­bers of the Enterobacteria­ ceae can dis­play this fea­ture as well.

Spore Stain The Wirtz-Conklin spore stain is a dif­fer­en­tial stain for de­tec­tion of spores. This is very use­ful for the iden­ti­fi­ca­tion of Bacillus and Bacterial Diagnosis 141 Clostridium spe­cies. Using this pro­ce­dure, spores stain green while the rest of the cell stains pink. Non-spore-forming bac­te­ria are pink. In this pro­ce­dure, the slide is flooded with 5 to 10% aque­ous mal­a­chite green. The stain is left on the slide for 45 min. Alternatively, the slide can be heated gently to steam­ing for 3 to 6 min­utes. Heating to steam­ing en­hances the up­take of the stain into the spores. The slide is then rinsed with wa­ter. Aqueous saf­ ra­nin (0.5%) is used as a coun­ter­stain for 30 s. The slide is then washed, blot­ted dry, and ex­am­ined by light mi­cros­copy at x1,000 mag­ni­fi­ca­tion. Primary Plating Media: Bacteria Ashdown Medium Ashdown me­dium is used for the se­lec­tive iso­la­tion and char­ac­ter­ iza­tion of Burkholderia pseudomallei from clin­i­cal spec­i­mens such as spu­tum. The me­dium con­tains crys­tal vi­o­let and gen­ta­mi­cin as se­lec­tive fac­tors. It is en­riched with glyc­erol and con­tains neu­tral red. B. pseudomallei pro­duces flat, wrin­kled, pur­ple col­o­nies on this me­dium. Both Ashdown agar and Ashdown broth can be mod­ i­fied by the ad­di­tion of an­ti­mi­cro­bial for the se­lec­tive cul­ture of B. pseudomallei. Modified Ashdown broth re­mains the stan­dard for iso­la­tion of B. pseudomallei from throat swabs in pa­tients with sus­pected melioidosis.

Bacteroides Bile-Esculin (BBE) Agar Bacterial Diagnosis Bacteroides bile-esculin agar is a se­lec­tive, dif­fer­en­tial agar me­ dium used for the re­cov­ery of the Bacteroides fragilis group and Bilophila wadsworthia. The me­dium con­tains oxgall (bile), esculin, fer­ric am­mo­nium cit­rate, he­min, vi­ta­min, and gen­ta­mi­cin in a ca­ sein and soy­bean agar base. Growth of non-B. fragilis group or­gan­ isms is in­hib­ited by the bile and the gen­ta­mi­cin. Supplementation of the agar with he­min and vi­ta­min K1 stim­u­lates the growth of Bacteroides spp. Esculin hy­dro­ly­sis is de­tected when esculin is con­ verted to esculin and re­acts with fer­ric am­mo­nium cit­rate to pro­ duce black col­o­nies.

Bile-Esculin (Enterococcal Selective) Agar Bile-esculin agar can be made se­lec­tive for the re­cov­ery of van­co­ my­cin-resistant en­tero­cocci by add­ing 6 μg of van­co­my­cin per mil­li­li­ter to it. Enterococci are ab­ le to grow in the pres­ence of bile and hy­dro­lyze esculin. Vancomycin-resistant strains pro­duce black col­o­nies on this agar, but sus­cep­ti­ble strains fail to grow. In ad­di­tion, van­co­my­cin re­sis­tant lac­to­ba­cilli can break through and grow on this me­dia. 142 SECTION 4 Bismuth Sulfite Agar Bismuth sul­fite agar is a dif­fer­en­tial, se­lec­tive me­dium used for the iso­la­tion and iden­ti­fi­ca­tion of Salmonella enterica server Typhi and other en­teric rods. The me­dium con­tains di­gests of ca­sein and an­i­ mal tis­sue, beef ex­tract, glu­cose, fer­ric sul­fate, and bis­muth sul­fite. Most com­men­sal or­gan­isms are in­hib­ited by the bis­muth sul­fite. S. enterica server Typhi col­o­nies ap­pear black with a me­tal­lic sheen. This me­dium may be in­hib­i­tory for some spe­cies of Shigella.

Blood Agar Many types of blood agar me­dia are used in clin­i­cal lab­o­ra­to­ries. The me­dium is used for the iso­la­tion and de­tec­tion of he­mo­lytic or­ gan­isms. Alpha he­mo­lytic or­gan­isms will pro­duce a green­ish color in the me­dia sur­round­ing the col­ony, whereas be­ta-hemolytic or­gan­ isms pro­duce a clear zone around the col­ony. The na­ture of be­ta- hemolysis can be char­ac­ter­is­tic of cer­tain or­gan­isms. The two ba­sic com­po­nents are the basal me­dium (e.g., brain heart in­fu­sion, bru­ cella, Co­lum­bia, Shaedler’s, tryp­tic soy) and blood (e.g., sheep, horse, rab­bit). Additional sup­ple­ments are com­monly used to en­hance the growth of spe­cific or­gan­isms or to sup­press the growth of un­wanted or­gan­isms.

Bor ­det-Gengou Agar Recovery of Bordetella per­tus­sis and Bordetella parapertussis is Bacterial Diagnosis in­hib­ited by fac­tors such as fatty ac­ids, metal ions, sul­fi­des, and per­ ox­ides that are com­monly pres­ent in me­dia. Starch, char­coal, se­rum al­bu­min, blood, or sim­i­lar com­po­nents are added to the me­dium to neu­tral­ize these in­hib­i­tors. Bor­det-Gengou agar is a po­tato in­fu­sion– glyc­er­ol-based agar me­dium sup­ple­mented with 20 to 30% sheep, horse, or rab­bit blood. Potato in­fu­sion is re­quired for the growth of Bordetella spp., and glyc­erol is added to con­serve mois­ture in the me­dium. Antibiotics such as meth­i­cil­lin or ceph­a­lexin are com­ monly added to sup­press the growth of bac­te­ria such as staph­y­lo­ cocci, which in­hibit the growth of Bordetella spp. Because this me­dium must be made fresh (it has a shelf life of less than 1 week), it has largely been re­placed by Regan-Lowe agar.

Brain Heart Infusion Agar and Broth Brain heart in­fu­sion agar is a gen­er­al-purpose me­dium used for the iso­la­tion of a wide va­ri­ety of path­o­gens. The ba­sic for­mula in­cludes in­fu­sion from brains and beef heart, as well as meat pep­tones, yeast ex­tract, and dex­trose. Vitamin K and he­min can be added for the en­riched growth of an­aer­obes. The an­aer­o­bic for­mu­la­tion is in­fe­ Bacterial Diagnosis 143 rior for the iso­la­tion of an­aer­o­bic gram neg­a­tive or­gan­isms. Broth for­mu­la­tions sup­ple­mented with 6.5% so­dium chlo­ride are used for the iso­la­tion of salt-tolerant strep­to­cocci and en­tero­cocci. The me­ dium is par­tic­u­larly use­ful for cul­tur­ing strep­to­cocci, pneu­mo­cocci, and me­nin­go­coc­ci.

Brilliant Green Agar Brilliant green agar is a se­lec­tive, dif­fer­en­tial me­dium used for the iso­la­tion of Salmonella serovars, ex­cept S. enterica server Typhi. The nu­tri­tive base con­tains meat and ca­sein pep­tones. Brilliant green dye at a high conce­ n­tra­tion in­hib­its most Gram-positive and Gram-negative bac­te­ria, in­clud­ing Shigella spp. and S. enterica server Typhi. Phenol red is the pH in­di­ca­tor. Yeast ex­tract pro­vi­des ad­di­tional nu­tri­ents. Acid pro­duc­tion from the fer­men­ta­tion of su­ crose or lac­tose pro­duces yel­low-green col­o­nies with a yel­low-green zone around the col­ony. Nonfermenters (i.e., Salmonella) may range in color from white to red­dish pink with a red zone.

Brucella Agar and Broth Brucella agar is a me­dium de­signed orig­i­nally for the iso­la­tion of Bru­ cella spp. Brucella agar sup­ple­mented with 5% horse blood can be used as a gen­er­al-purpose me­dium for the iso­la­tion of both aer­o­bic and an­aer­o­bic or­gan­isms. The nu­tri­tive base in­cludes meat pep­tones, dex­trose, and yeast ex­tract. The agar for­mu­la­tion can be sup­ple­ mented with he­min and vi­ta­min K for re­cov­ery of fas­tid­i­ous an­aer­ Bacterial Diagnosis obes or with cefoxitin and cy­clo­ser­ine for the se­lec­tive re­cov­ery of Clostridium dif­fi­cile. The broth con­tains so­dium bi­sul­fite as a re­duc­ ing agent and has been used for cul­ti­va­tion of Campylobacter spp.

Buffered Charcoal-Yeast Extract (BCYE) Agar Buffered char­coal-yeast ex­tract agar is se­lec­tive for the re­cov­ery of Legionella, Nocardia, and Francisella spp. It con­tains agar, yeast ex­ tract, char­coal, and salts and is sup­ple­mented with L-cysteine, fer­ric py­ro­phos­phate, ACES [Ar-(2-acetamido)-2- aminoethanesulfonic acid] buffer, and a-ketoglutarate. The char­coal de­tox­i­fies the me­ di­um; the yeast ex­tracts are rich in nu­tri­ents; and the L-cysteine, fer­ ric py­ro­phos­phate, and al­pha-Ketoglutarate stim­u­late the growth of Legionella spp. The ad­di­tion of ACES is re­quired to buffer the me­ dium be­cause Legionella spp. have a nar­row pH tol­er­ance (growth is op­ti­mal at pH 6.9). Various an­ti­bi­ot­ics such as po­ly­myxin B, aniso- mycin, cefamandole, van­co­my­cin, and cy­clo­hex­i­mide are added to in­hibit the growth of other bac­te­ria when nonsterile clin­i­cal and en­ vi­ron­men­tal spec­i­mens are cul­tured. 144 SECTION 4 Burkholderia cepacia Selective Agar (BCSA) Burkholderia cepacia se­lec­tive agar is an en­riched, se­lec­tive me­ dium used for the iso­la­tion of B. cepacia. Trypticase pep­tone, yeast ex­tract, so­dium chlo­ride, su­crose, and lac­tose form the nu­tri­tive base; and po­ly­myxin B, gen­ta­mi­cin, van­co­my­cin, and crys­tal vi­o­ let are added as se­lec­tive agents. This agar is the most sen­si­tive and se­lec­tive me­dium for the re­cov­ery of B. cepacia.

Campylobacter Selective Medium A large num­ber of me­dia have been de­vel­oped for the se­lec­tive iso­ la­tion of Campylobacter spp. from stool spec­i­mens. Most con­tain a bru­cella basal me­dium, which pref­er­en­tially sup­ports the growth of Campylobacter spp. Blood is added, as are var­i­ous com­bi­na­tions of an­ti­bi­ot­ics (e.g., ceph­a­lo­thin, van­co­my­cin, tri­meth­o­prim, am­pho­ ter­i­cin, and po­ly­myxin in the Blaser-Wang for­mu­la­tion; cy­clo­hex­ i­mide, cefazolin, no­vo­bi­o­cin, bac­i­tra­cin, and co­lis­tin in the Butzler for­mu­la­tion; cy­clo­hex­i­mide, cefoperazone, and van­co­my­cin in the Karmali for­mu­la­tion; and cy­clo­hex­i­mide, ri­fam­pin, tri­meth­o­prim, and po­ly­myxin in the Pres­ton for­mu­la­tion).

Cefsulodin-lrgasan-Novobiocin (CIN) Agar Cefsulodin-Irgasan-novobiocin agar is a se­lec­tive, dif­fer­en­tial agar me­dium used for the iso­la­tion of Yersinia enterocolitica, most com­ monly from stool spec­i­mens. The me­dium con­sists of di­gests of an­i­ Bacterial Diagnosis mal tis­sue and gel­a­tin, beef and yeast ex­tracts, so­dium py­ru­vate, so­dium deoxycholate, neu­tral red, crys­tal vi­o­let, cefsulodin, Irgasan, and no­vo­bi­o­cin. The an­ti­bi­ot­ics and so­dium deoxycholate in­hibit the growth of most or­gan­isms in stool spec­i­mens. However, Yer­ sinia spp. are re­sis­tant and can fer­ment man­ni­tol in the me­dium. This fer­men­ta­tion pro­duces col­o­nies with a bull’s-eye ap­pear­ance (i.e., deep red cen­ters with trans­par­ent edges some­times re­ferred to as a “bull’s eye”).

Chromogenic Media A large num­ber of chro­mo­genic me­dia ex­ist for the se­lec­tive and dif­fer­en­tial iso­la­tion of bac­te­ria and yeast. These me­dia can in­ clude an­ti­bi­ot­ics and can be used to iso­late drug-resistant bac­te­ria such as van­co­my­cin re­sis­tant en­tero­cocci, meth­i­cil­lin re­sis­tant Staphylococcus au­re­us, and re­sis­tant Gram-negative or­gan­isms. In many cases, the com­po­nents of the me­dia are pro­pri­e­tary and can­ not be dis­cussed here. The list of chro­mo­genic me­dia in­cludes, but is not lim­ited to, the iso­la­tion and iden­ti­fi­ca­tion of ESBL pro­duc­ ing Enterobacteriaceae, carbapenemase re­sis­tant Enterobacteria­ ceae, MRSA, VRE, P. aeruginosa, Salmonella, Streptococcus Bacterial Diagnosis 145 agalactiae, Vibrio, C. dif­fi­cile, Enterobacteriaceae, Yersinia en­ terocolitica, Acinetobacter, and oth­ers.

Chocolate Agar Chocolate agar is an en­riched me­dium that de­rives its name from its color. Blood or he­mo­glo­bin is added im­me­di­ately af­ter the me­ dium is heated, and the heat causes the added com­po­nent to lyse and turn brown. This me­dium sup­ports the growth of most bac­te­ ria and is re­quired for the re­cov­ery of many Haemophilus spp. and some path­o­genic Neisseria strains. The so-called nu­tri­tion­ally de­ fi­cient strep­to­cocci (Abiotrophia and Granulicatella) will also grow on choc­o­late, but not on blood agar. A va­ri­ety of for­mu­la­tions of this me­dium have been used, but the most com­mon con­sists of a pep­tone base en­riched with 2% he­mo­glo­bin or IsoVitaleX. Catalase-negative bac­te­ria (e.g., Streptococcus pneumoniae) grow less well on this me­dium than on blood agar be­cause cat­a­lase from rup­tured eryth­ro­ cytes in blood agar is not avail­­able to pro­tect the bac­te­ria from per­ ox­ides that ac­cu­mu­late in the me­di­um.

Chopped-Meat Broth Chopped-meat broth is an en­riched broth used for the re­cov­ery of a va­ri­ety of bac­te­ria, par­tic­u­larly an­aer­obes, from clin­i­cal spec­i­ mens. Extracts as well as solid par­ti­cles of beef or horse meat are sus­pended in broth with pep­tones, yeast ex­tract, sug­ars, starch, and Bacterial Diagnosis L-cysteine. The L-cysteine helps main­tain a low Eh (ox­i­da­tion- reduction po­ten­tial), which sup­ports the growth of an­aer­obes.

Colistin-Nalidixic Acid (CNA) Agar Colistin-nalidixic acid agar is a se­lec­tive me­dium used for the re­ cov­ery of aer­o­bic and an­aer­o­bic Gram-pos­i­tive bac­te­ria. The me­ dium con­sists of Co­lum­bia agar base sup­ple­mented with na­li­dixic acid, co­lis­tin, and blood. Nalidixic acid in­hib­its most aer­o­bic Gram- negative rods, as does co­lis­tin. The B. fragilis group is usu­ally re­ sis­tant to these an­ti­bi­ot­ics, but other an­aer­o­bic Gram-negative rods can be in­hib­ited by co­lis­tin.

Co­lum­bia Agar and Broth Co­lum­bia agar with 5% sheep blood is a gen­er­al-purpose me­dium used for the iso­la­tion of com­mon bac­te­ria. The me­dium con­tains meat and ca­sein pep­tones, beef ex­tract, yeast ex­tract, and corn­ starch as the nu­tri­tive base. Sheep blood al­lows the de­ter­mi­na­tion of he­mo­lytic re­ac­tions and pro­vi­des X fac­tor. However, the sub­stan­ tial car­bo­hy­drate con­tent may make beta he­mo­lytic strep­to­cocci 146 SECTION 4 ap­pear to be al­pha-hemolytic or take on a green­ish hue. Use of horse or rabbi­ t blood im­proves the he­mo­ly­sis. NADase in sheep blood de­ stroys the V fac­tor (NAD); there­fore, or­gan­isms that re­quire this fac­tor do not grow. Salt and Tris buff­ers are added to the broth for­ mu­la­tion to en­hance the growth of or­gan­isms and in­crease the buff­ er­ing ca­pac­ity, re­spec­tive­ly.

Cycloserine-Cefoxitin-Egg Yolk-Fructose Agar (CCFA) Cycloserine-cefoxitin-egg yolk-fructose agar is a se­lec­tive, dif­fer­ en­tial me­dium used for the re­cov­ery of Clostridium dif­fi­cile. The me­dium con­sists of an­i­mal tis­sue di­gest, fruc­tose, cy­clo­ser­ine, cefoxitin, and neu­tral red. Cycloserine and cefoxitin in­hibit most in­tes­ti­nal bac­te­ria. C. dif­fi­cile can fer­ment fruc­tose, pro­duc­ing a more acidic pH, which is de­tected by the in­di­ca­tor dye neu­tral red (shift from red to yel­low me­dium sur­round­ing the col­o­nies). Vari- ous mod­i­fi­ca­tions of this me­dium are used, in­clud­ing sup­ple­men­ ta­tion with egg yolk to stim­u­late the growth of Clostridia.

Cystine Tellurite Blood Agar Cystine tellurite blood agar me­dium is a se­lec­tive, dif­fer­en­tial me­dium used for the re­cov­ery of Corynebacterium diphtheriae. The me­dium con­sists of heart in­fu­sion agar, po­tas­sium tellurite, L-cystine, and rabbi­ t blood. Potassium tellurite in­hib­its the growth of most com­men­sal or­gan­isms and al­lows C. diphtheriae to grow. Bacterial Diagnosis The or­gan­ism pro­duces hy­dro­gen sul­fide from cys­tine, and the re­ac­ tion of tellurite with hy­dro­gen sul­fide re­sults in brown ha­los sur­ round­ing the col­o­nies of C. diphtheriae.

Egg Yolk Agar Egg yolk agar (mod­i­fied McClung-Toabe agar) is a se­lec­tive, dif­ fer­en­tial me­dium used for the iso­la­tion and dif­fer­en­ti­a­tion of Clos­ tridium spp. Degradation of lec­i­thin re­sults in an opaque pre­cip­i­tate around the bac­te­rial col­ony, and li­pase de­stroys fats in the egg yolk, re­sult­ing in an ir­i­des­cent sheen on the col­ony sur­face. Proteolysis can also be de­ter­mined on the ba­sis of a trans­lu­cent clear­ing of the me­dium around the col­ony. Addition of neo­my­cin makes the egg yolk agar mod­er­ately se­lec­tive by in­hib­it­ing some fac­ul­ta­tive an­ aer­o­bic gram-neg­a­tive rods.

Ellinghausen-McCullough-Johnson-Harris Medium The mod­i­fied bo­vine al­bu­min Tween 80 me­dium is se­lec­tive for the growth of Leptospira spp. The basal me­dium, con­sist­ing of glyc­ erol, so­dium py­ru­vate, and thi­a­mine, is sup­ple­mented with bo­vine Bacterial Diagnosis 147 al­bu­min, Tween 80, vi­ta­min Bi2, and salts of iron, cal­cium, mag­ne­ sium, zinc, and cop­per.

Enterococcosel agar This me­dium is used for the rapid, se­lec­tive iso­la­tion of en­tero­cocci. It is also used for the cul­ti­va­tion of staph­y­lo­cocci and L. monocy­ togenes. The me­dium con­tains di­gest of ca­sein, iron, Esculin, and yeast ex­tract. The ad­di­tion of van­co­my­cin can al­low for the se­lec­ tive de­tec­tion of van­co­my­cin re­sis­tant en­tero­coc­ci.

Eosin-Methylene Blue (EMB) Agar Eosin-methylene blue agar is a dif­fer­en­tial, se­lec­tive me­dium used for the iso­la­tion and dif­fer­en­ti­a­tion of lac­tose-fermenting and nonfer- menting Gram-negative rods. The agar me­dium con­sists of ca­sein di­gests, lac­tose, su­crose, eo­sin Y, and meth­y­lene blue. The Le­vine for­mu­la­tion does not in­clude su­crose. Growth of Gram-positive bac­ te­ria is sup­pressed by the meth­y­lene blue, which, to­gether with eo­ sin Y, also serves as an in­di­ca­tor for car­bo­hy­drate fer­men­ta­tion (dyes pre­cip­i­tate at an acidic pH). Bacteria that fer­ment lac­tose (e.g., Escherichia, Klebsiella, and Enterobacter spp.) form col­o­nies that have a green me­tal­lic sheen or that are blue-black to brown. Nonfer- mentative bac­te­ria (e.g., Pro­teus, Salmonella, and Shigella spp.) have col­or­less or light pur­ple col­o­nies. Bacterial Diagnosis Fletcher Medium Fletcher me­dium is a semi­solid me­dium used for the re­cov­ery of Leptospira spp. The me­dium con­sists of 0.15% agar, salt, pep­tones, beef ex­tract, and rab­bit se­rum. Leptospira spp. usu­ally grow within 1 to 2 weeks in this me­di­um.

Gram-Negative (G N; Hajna) Broth This me­dium is used for the se­lec­tive cul­ti­va­tion of Salmonella and Shigella spp. The me­dium con­tains di­gest of ca­sein and an­i­mal tis­ sue, cit­rate, man­ni­tol, gluco­ se, and deoxycholate. Commensal or­ gan­isms will over­grow the en­teric path­o­gens if the broth is in­cu­bated for more than 4 to 6 h.

Haemophilus Test Medium (HTM) Agar and Broth Haemophilus test me­dium is an en­riched me­dium used for sus­cep­ ti­bil­ity test­ing of Haemophilus spp. The me­dium con­tains beef and ca­sein ex­tracts. Yeast ex­tract, he­min, and NAD pro­vide the nec­es­ sary growth fac­tors and en­rich­ments. Antagonists to sul­fon­amides 148 SECTION 4 and tri­meth­o­prim are re­moved by thy­mi­dine phos­phor­y­lase. The ad­van­tage of the agar me­dium is that it is a clear agar base so that sharp growth end-point in­ter­pre­ta­tions can be made. The cal­cium and mag­ne­sium con­cen­tra­tions are ad­justed to the con­cen­tra­tions rec­om­mended by the CLSI.

Hektoen Enteric Agar Hektoen en­teric agar is a se­lec­tive me­dium used for the iso­la­tion of Salmonella and Shigella spp. and dif­fer­en­ti­a­tion of these or­gan­ isms from other Gram-negative rods that may be re­cov­ered on this me­dium. It con­sists of a pep­tone base agar sup­ple­mented with bile salts, lac­tose, su­crose, sal­i­cin, fer­ric am­mo­nium cit­rate, and the pH in­di­ca­tors brom­thy­mol blue and acid fuch­sin. The bile in­hib­its all­ Gram-positive bac­te­ria and many Gram-negative rods. Acids pro­ duced by fer­men­ta­tion of lac­tose, su­crose, or sal­i­cin re­act with brom­thy­mol blue to pro­duce a yel­low color and with acid fuch­sin

to pro­duce a red color. Hydrogen sul­fide (H2S) pro­duced by the me­ tab­o­lism of so­dium thio­sul­fate is de­tected when a black pre­cip­i­tate forms af­ter the ad­di­tion of fer­ric am­mo­nium cit­rate. Lactose-fer- menting bac­te­ria (e.g., E. coli) are slightly in­hib­ited on this agar and ap­pear as or­ange or salmon pink col­o­nies. Salmonella col­o­nies typ­i­cally ap­pear blue-green with black cen­ters. Shigella col­o­nies ap­pear green with no black cen­ter. Pro­teus spp. are in­hib­it­ed; their col­o­nies are col­or­less. Bacterial Diagnosis Kanamycin-Vancomycin Laked Blood (LKV) Agar Kanamycin-vancomycin laked blood agar is a se­lec­tive, dif­fer­en­tial me­dium used for the re­cov­ery of an­aer­o­bic Gram-negative rods, es­ pe­cially Bacteroides and Prevotella spp. The me­dium con­sists of ca­sein and soy­bean meal agar sup­ple­mented with kana­my­cin, van­ co­my­cin, vi­ta­min K, and lysed (laked) sheep blood. Kanamycin in­ hib­its most fac­ul­ta­tive, Gram-negative rods; and van­co­my­cin in­hib­its most Gram-positive or­gan­isms and Porphyromonas spp.; Vitamin K stim­u­lates the growth of some Prevotella strains, which also de­velop a black pig­ment in the pres­ence of lysed blood.

LIM Broth LIM broth is a se­lec­tive en­rich­ment broth used for the re­cov­ery of group B strep­to­cocci. The me­dium con­sists of Todd-Hewitt broth sup­ple­mented with yeast ex­tract, co­lis­tin, and na­li­dixic acid. Most aer­o­bic and an­aer­o­bic Gram-negative rods are in­hib­ ited by the an­ti­bi­ot­ics, whereas group B strep­to­cocci grow well in this broth. Bacterial Diagnosis 149 L o f f­e r M e d i u m Loff­ler me­dium is an en­riched me­dium used for the re­cov­ery of Co­ rynebacterium diphtheriae. The me­dium con­sists of an­i­mal di­gests, heart mus­cle in­fu­sion, beef se­rum, egg, and glu­cose. C. diphtheriae grows rapid­ ly on this me­dium, and Gram stains of col­o­nies dem­on­ strate char­ac­ter­is­tic meta­chro­matic gran­ules.

MacConkey (MAC) Agar MacConkey agar is a se­lec­tive agar me­dium used for the iso­la­tion and dif­fer­en­ti­a­tion of lac­tose-fermenting and nonfermenting Gram- negative rods. The me­dium con­sists of di­gests of pep­tones, bile salts, lac­tose, neu­tral red, and crys­tal vi­o­let. Bile salts and crys­tal vi­o­let in­hibit the growth of Gram-positive bac­te­ria and some fas­tid­i­ ous Gram-negative bac­te­ria. Colonies that fer­ment lac­tose (e.g., Escherichia, Klebsiella, and Enterobacter spp.) pro­duce acid, which causes a red color shift in the neu­tral red pH in­di­ca­tor and pre­cip­i­ tates the bile salts. Colonies ap­pear red to pink, while nonfermenting col­o­nies (e.g., Pro­teus, Salmonella, and Shigella spp.) ap­pear yel­low, col­or­less, or trans­lu­cent. MacConkey agar will typ­i­cally in­hibit Pro­ teus from swarm­ing over the plate.

MacConkey Agar with Sorbitol—see Sorbitol- MacConkey Agar

Mannitol Salt Agar Bacterial Diagnosis Mannitol salt agar is a se­lec­tive me­dium used for the iso­la­tion of staph­y­lo­cocci. The me­dium con­sists of di­gests of ca­sein and an­i­mal tis­sue, beef ex­tract, man­ni­tol, salt, and phe­nol red in­di­ca­tor. If the or­gan­ism can grow in the pres­ence of 7.5% salt and fer­ment man­ni­ tol, the acid turns the in­di­ca­tor yel­low. Most strains of Staphylococ­ cus au­re­us pro­duce yel­low col­o­nies, whereas co­ag­u­lase-negative staph­y­lo­cocci do not fer­ment the man­ni­tol and thus re­main red. Most other or­gan­isms are in­hib­ited by the high salt con­cen­tra­tion.

Martin-Lewis Agar Martin-Lewis agar, a for­mu­la­tion of the mod­i­fied Thayer-Martin (MTM) agar, is an en­riched se­lec­tive me­dium for the iso­la­tion of Neisseria gonorrhoeae. The nu­tri­tive base is choc­o­late agar. The spe­cific dif­fer­ences from MTM agar are a higher con­cen­tra­tion of van­co­my­cin (4 ver­sus 3 µg/ml) and re­place­ment of nys­ta­tin with anisomycin. Trimethoprim and co­lis­tin are also in­cor­po­rated. Some path­o­genic Neisseria strains have been re­ported to be in­hib­ited by van­co­my­cin and tri­meth­o­prim. 150 SECTION 4 McBride Listeria agar This me­dium is used for the se­lec­tive iso­la­tion of L. monocytogenes from clin­i­cal spec­i­mens con­tain­ing mixed bi­o­tas. The me­dium con­ tains gly­cine, di­gest of ca­sein and an­i­mal tis­sue, beef ex­tract, phenylethyl al­co­hol, and lith­ium chlo­ride.

Mueller-Hinton Agar and Broth Mueller-Hinton agar and broth are rec­om­mended by CLSI for the rou­tine sus­cep­ti­bil­ity test­ing of non-fastidious or­gan­isms. Supple- mentation of this agar with 5% sheep blood is used for sus­cep­ti­bil­ity test­ing of fas­tid­i­ous or­gan­isms such as Streptococcus pneumoniae. Beef and ca­sein ex­tracts and sol­u­ble starch form the nu­tri­tive base. Calcium and mag­ne­sium con­cen­tra­tions are con­trolled.

New York City Agar New York City agar is a se­lec­tive me­dium used for the iso­la­tion of path­o­genic Neisseria spp. The me­dium con­sists of pep­tones, corn­ starch, yeast di­al­y­sate, glu­cose, he­mo­glo­bin, horse plasma, and a mix­ture of an­ti­bi­ot­ics (van­co­my­cin, co­lis­tin, am­pho­ter­i­cin B, and tri­meth­o­prim). It can be used in­stead of Thayer-Martin agar.

Oxidative-Fermentative Polymyxin

Bacterial Diagnosis B-Bacitracin-Lactose (OFPBL) Agar Oxidative-fermentative po­ly­myxin B-bacitracin-lactose agar is a se­ lec­tive, dif­fer­en­tial me­dium used for the iso­la­tion of Burkholderia cepacia. The nu­tri­tive base is an ox­i­da­tive-fermentative me­dium with pep­tones. When acid is pro­duced from the uti­li­za­tion of lac­ tose, as with B. cepacia, the brom­thy­mol blue in­di­ca­tor changes the col­ony from green to yel­low. Polymyxin B and bac­i­tra­cin are se­ lec­tive agents that in­hibit some Gram-negative and Gram-positive or­gan­isms, re­spec­tively. Other or­gan­isms may grow on this me­dium and are dif­fer­en­ti­ated from B. cepacia by the in­abil­ity to pro­duce acid from lac­tose.

Phenylethyl Alcohol (PEA) Blood Agar Phenylethyl al­co­hol blood agar is a se­lec­tive me­dium that con­sists of ca­sein and soy­bean agar sup­ple­mented with phenylethyl al­co­hol and blood. Facultative Gram-negative rods are in­hib­ited by the phenylethyl al­co­hol (e.g., the growth of swarm­ing Pro­teus spp. is sup­pressed). Most Gram-positive and Gram-negative an­aer­obes, as well as aer­o­bic Gram-positive bac­te­ria, will grow on this me­dium. Pseudomonas spp. are not in­hib­it­ed. Bacterial Diagnosis 151 Regan-Lowe Agar Medium Regan-Lowe agar me­dium, for the se­lec­tive iso­la­tion of Bordetella spp., con­tains beef ex­tract, gel­a­tin di­gest, starch, char­coal, ni­a­cin, 10% horse blood, and ceph­a­lexin (40 µg/ml). The char­coal and horse blood are re­quired to neu­tral­ize fatty ac­ids and other in­hib­i­tory fac­ tors pres­ent in the me­dium. Sheep but not hu­man blood can re­place horse blood. Cephalexin can de­lay the de­tec­tion of Bordetella spp. on this me­dium, but the use of an ad­di­tional non­se­lec­tive me­dium is not con­sid­ered nec­es­sary. The shelf life of this me­dium is 6 to 8 weeks.

Salmonella-Shigella (SS) Agar Salmonella-shigella agar is a highly se­lec­tive me­dium for the re­ cov­ery of Salmonella spp. The me­dium is not rec­om­mended for the pri­mary iso­la­tion of Shigella spp. The me­dium con­sists of beef ex­ tract and pep­tone di­gests, lac­tose as a car­bo­hy­drate source, bile salts, so­dium cit­rate, so­dium thio­sul­fate, neu­tral red, bril­liant green, and fer­ric cit­rate. Bile salts, so­dium cit­rate, and bril­liant green are in­hib­i­tory for all­ Gram-positive and se­lected Gram-negative bac­ te­ria. Bacteria that grow on the me­dium and pro­duce hy­dro­gen sul­ fide from the me­tab­o­lism of so­dium thio­sul­fate are de­tected by the black pre­cip­i­tate formed with fer­ric cit­rate. Acid pro­duced from lac­ tose fer­men­ta­tion is de­tected with the pH in­di­ca­tor neu­tral red. All lac­tose-fermenting bac­te­ria form pink or red col­o­nies, while non- fermenting bac­te­ria form ei­ther col­or­less (e.g., Shigella spp.) or black (e.g., Salmonella spp.) col­o­nies. Bacterial Diagnosis

Schaedler’s Agar Schaedler’s agar is a gen­er­al-purpose me­dium used for the iso­la­ tion of an­aer­o­bic bac­te­ria. The nu­tri­tive base in­cludes veg­e­ta­ble and meat pep­tones, dex­trose, and yeast ex­tract. Sheep blood, vi­ta­min Ku, and he­min pro­vide other ad­di­tives that stim­u­late the growth of fas­tid­i­ous an­aer­obes. Because of the high car­bo­hy­drate con­tent, col­ o­nies with be­ta-hemolytic re­ac­tions may have a green­ish hue. Acid pro­duc­tion may also lead to rapid cell death.

Selenite Broth Selenite broth is a se­lec­tive en­rich­ment broth used for the iso­la­tion of Salmonella spp. from stools and other con­tam­i­nated spec­i­mens. It con­sists of pep­tones, so­dium phos­phate, lac­tose, and so­dium sel­ e­nite. E. coli and other Gram-negative rods are in­hib­ited by so­dium sel­e­nite. The broth should be sub­cul­tured within 8 to 12 h af­ter in­ oc­u­la­tion with the spec­i­men, or else the en­teric path­o­gens will be over­grown with com­men­sal or­gan­isms. 152 SECTION 4 Skirrow Brucella Medium Skirrow me­dium is an en­riched se­lec­tive blood agar me­dium used for the iso­la­tion of Campylobacter spp. The nu­tri­tive agar base is bru­cella agar. Hematin is pro­vided by sheep blood. The se­lec­tive agents are tri­meth­o­prim, van­co­my­cin, and po­ly­myxin B, which in­ hibit the nor­mal flora found in fe­cal spec­i­mens.

Sorbitol-MacConkey Agar Sorbitol-MacConkey agar is a se­lec­tive dif­fer­en­tial agar used for the iso­la­tion of E. coli 0157. Lactose is re­placed with sor­bi­tol. Most E. coli strains fer­ment sor­bi­tol; how­ever, E. coli 0157 does not, and there­fore its col­o­nies are col­or­less on this agar.

Streptococcus se­lec­tive me­di­um This me­dium is used for the se­lec­tive iso­la­tion of strep­to­cocci from clin­i­cal spec­i­mens. The me­dium con­tains pep­tone, starch, and blood. It also con­tains Colistin sul­fate and oxolinic acid as se­lec­tive fac­tors. It is a com­monly used me­dium for throat cul­tures.

StrepB Carrot Broth This is a propr­ i­e­tary broth that is used for de­tect­ing the pres­ence of GBS in­fec­tions in preg­nant women. The me­dium con­tains pep­ tone, starch, morpholinepropanesfulfonic acid, glu­cose, py­ru­vate, Bacterial Diagnosis growth fac­tors, and se­lec­tive fac­tors. It is a mod­i­fi­ca­tion of Gra­nada me­dium con­sist­ing of a one-step method for screen­ing preg­nant women for the pres­ence of GBS. Tubes show an or­ange-to-red color change, typ­i­cal of GBS. The pro­duc­tion of or­ange, red, or brick red pig­ment is a unique char­ac­ter­is­tic of he­mo­lytic GBS due to re­ac­ tions with sub­strates such as starch, pro­te­ose pep­tone, se­rum, and fo­late path­way in­hib­i­tors.

Tetrathionate Broth, Hajna Tetrathionate broth is a se­lec­tive en­rich­ment broth used for the ­re­cov­ery of Salmonella spp. (ex­cept Salmonella Typhi and S. enterica subsp. arizonae) from stool and urine spec­i­mens. It con­ sists of a pep­tone base sup­ple­mented with yeast ex­tract, man­ni­tol, glu­cose, so­dium deoxycholate, so­dium thio­sul­fate, cal­cium car­bon­ ate, and bril­liant green. The so­dium deoxycholate, so­dium thio­sul­ fate, and bril­liant green in­hibit Gram-positive and Gram-negative bac­te­ria. The broth should be sub­cul­tured for 12 to 24 h af­ter in­oc­u­ la­tion to pre­vent over­growth of Salmonella spp. with com­men­sal or­gan­isms. Bacterial Diagnosis 153 Thayer-Martin (Modified) Agar Many mod­i­fi­ca­tions of Thayer-Martin me­dium have been de­vel­ oped for the iso­la­tion of path­o­genic Neisseria spp. The blood agar base me­dium is en­riched with he­mo­glo­bin and sup­ple­ments. The growth of un­wanted bac­te­ria can be sup­pressed by the ad­di­tion of an­ti­bi­ot­ics such as co­lis­tin (which in­hib­its most Gram-negative bac­ te­ria ex­cept Pro­teus spp.), tri­meth­o­prim (which in­hib­its Pro­teus spp.), van­co­my­cin (which in­hib­its most Gram-positive bac­te­ria), and nys­ta­tin (which in­hib­its yeasts). Some strains of N. gonor­ rhoeae are in­hib­ited by van­co­my­cin, and so non­se­lec­tive me­dia (e.g., choc­o­late agar) should be used for pri­mary iso­la­tion.

Thioglycolate Broth Thioglycolate broth is an en­rich­ment broth used for the re­cov­ery of aer­o­bic and an­aer­o­bic bac­te­ria. Various for­mu­la­tions are used, but most in­clude ca­sein di­gest, glu­cose, yeast ex­tract, cys­te­ine, and so­dium thioglycolate. Supplementation with he­min and vi­ta­min K will en­hance the re­cov­ery of an­aer­o­bic bac­te­ria.

Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar Thiosulfate cit­rate bile salts su­crose agar is a se­lec­tive, dif­fer­en­tial me­dium used for the re­cov­ery of Vibrio spp. The me­dium con­sists of di­gests of ca­sein and an­i­mal tis­sue, yeast ex­tract, so­dium cit­rate, so­dium cho­late, oxgall (bile), sucr­ ose, fer­ric cit­rate, thy­mol blue, Bacterial Diagnosis and brom­thy­mol blue. Sodium cit­rate, so­dium cho­late, and bile in­ hibit com­men­sal or­gan­isms. Vibrio cholerae col­o­nies are yel­low on this me­dium due to fer­men­ta­tion of su­crose with the acid, re­sult­ing in a yel­low color shift of the in­di­ca­tor, brom­thy­mol blue. Vibrio parahaemolyticus fails to fer­ment su­crose, and the col­o­nies are there­fore blue-green. Some en­teric rods and en­tero­cocci may grow, but the col­o­nies are usu­ally small and trans­lu­cent. Sucrose-ferment- ing Pro­teus strains pro­duce yel­low col­o­nies that are sim­i­lar to Vib­ rio col­o­nies.

Tinsdale Agar Tinsdale agar is a se­lec­tive dif­fer­en­tial me­dium used for the iso­la­tion of Corynebacterium diphtheriae from up­per re­spi­ra­tory spec­i­mens. The me­dium con­sists of pep­tones, salt, yeast ex­tract, L-cysteine, po­tas­ sium tellurite, and se­rum. The po­tas­sium tellurite in­hib­its the growth of most com­men­sal or­gan­isms in the up­per re­spi­ra­tory tract and al­lows the growth of C. diphtheriae and re­lated Corynebacterium spe­cies. C. diphtheriae col­o­nies can be dis­tin­guished by the brown halo that de­vel­ops around the black col­o­nies. These ha­los re­sult from the 154 SECTION 4 re­ac­tion of tellurite with hy­dro­gen sul­fide, which C. diphtheriae pro­ duces from the cys­te­ine in the me­di­um.

Tryptic or Trypticase Soy Agar (TSA) and Broth (TSB) Tryptic(ase) soy agar with 5% sheep blood is a gen­er­al-purpose me­ dium used for the iso­la­tion of a wide va­ri­ety of or­gan­isms. The me­dium con­tains soy­bean and ca­sein pep­tones as the nu­tri­tive base. The ad­di­tion of sheep blood en­riches the me­dium and al­lows the growth of more fas­tid­i­ous or­gan­isms by pro­vid­ing he­min (X fac­ tor). V fac­tor (NAD) is in­ac­ti­vated by en­zymes in the sheep blood. Sheep blood is used for the in­ter­pre­ta­tion of he­mo­lytic re­ac­tions. The broth for­mu­la­tion is rec­om­mended for prep­a­ra­tion of in­oc­ula for sus­cep­ti­bil­ity test­ing. Addition of 6.5% so­dium chlo­ride to the broth for­mu­la­tion can be used for iso­la­tion of salt-tolerant or­gan­ isms, and Fildes en­rich­ment can be added to the broth for re­cov­ery of fas­tid­i­ous or­gan­isms such as Haemophilus spp.

Xylose-Lysine-Deoxycholate (XLD) Agar Xylose-lysine-deoxycholate agar is a mod­er­ately se­lec­tive me­ dium used for the iso­la­tion and dif­fer­en­ti­a­tion of en­teric path­o­ gens. The me­dium con­sists of yeast ex­tract with xy­lose, ly­sine, lac­tose, su­crose, so­dium deoxycholate, so­dium thio­sul­fate, fer­ric am­mo­nium cit­rate, and phe­nol red. The ma­jor­ity of the non­patho­ genic en­teric rods fer­ment lac­tose, su­crose, or xy­lose, pro­duc­ing Bacterial Diagnosis yel­low col­o­nies (the phe­nol red in­di­ca­tor is yel­low at acidic pH). Because Shigella spp. do not fer­ment these car­bo­hy­drates, the col­o­nies are red. Salmonella and Edwardsiella spp. fer­ment xy­ lose, but they also de­car­box­yl­ate ly­sine to an al­ka­line di­amine, ca­dav­er­ine. This di­amine neu­tral­izes the acid prod­ucts of fer­men­ ta­tion by de­car­box­yl­ation of ly­sine and pro­duces red col­o­nies. If the or­gan­ism pro­duces hy­dro­gen sul­fide (e.g., Salmonella and Edwardsiella spp.), the cen­ter of the col­o­nies will blacken. Sodium deoxycholate in­hib­its the growth of many non­patho­genic or­gan­isms (in the pres­ence of acid, it pre­cip­i­tates, pro­duc­ing yel­low, opaque col­o­nies).

Primary Plating Media: Mycobacteria Amer ­i­can Tru­deau Society Medium Amer­i­can Tru­deau Society me­dium con­tains co­ag­u­lated egg yolks, po­tato flour, glyc­erol, and mal­a­chite green. The con­cen­tra­tion of mal­ a­chite green is lower than in Lowenstein-Jensen me­dium, al­low­ing ear­lier de­tec­tion of my­co­bac­te­rial col­o­nies (other than M. leprae), but the me­dium is also more eas­ily over­grown by con­tam­i­nants. Bacterial Diagnosis 155 Dubos Broth (Dubos Tween al­bu­min broth) Dubos broth, a non­se­lec­tive broth, con­tains ca­sein di­gests, salt so­ lu­tions, L-asparagine, fer­ric am­mo­nium cit­rate, al­bu­min or se­rum, and Tween 80. The growth of most spe­cies of my­co­bac­te­ria is rapid in this me­dium, al­though the ad­di­tion of an­ti­bi­ot­ics is re­quired when spec­i­mens from con­tam­i­nated sites are pro­cessed. Tween 80 is a sur­fac­tant that fa­cil­i­tates the dis­persal of clumps of my­co­bac­ te­ria and re­sults in more rapid, ho­mo­ge­neous growth.

Lowenstein-Jensen (LJ) Medium Lowenstein-Jensen me­dium con­sists of glyc­erol, po­tato flour, de­ fined salts, and co­ag­u­lated whole eggs (to so­lid­ify the me­dium). Malachite green is added to in­hibit con­tam­i­nat­ing bac­te­ria, par­tic­ u­larly Gram-positive bac­te­ria. LJ me­dium has a long shelf life (sev­ eral months) and sup­ports the growth of most my­co­bac­te­ria, in part be­cause lec­i­thin in the eggs neu­tral­izes many toxic fac­tors pres­ent in clin­i­cal spec­i­mens. A prob­lem with LJ me­dium is that the con­tam­i­nants (com­monly Pseudomonas aeruginosa from Cys- tic Fibrosis sputa) that grow on this me­dium can com­pletely hy­ dro­lyze it.

Lowenstein-Gruft Modification The Gruft mod­i­fi­ca­tion of LJ me­dium con­tains RNA, pen­i­cil­lin, and na­li­dixic acid, which fur­ther sup­press the growth of con­tam­i­ nat­ing or­gan­isms. Because the growth of my­co­bac­te­ria can be de­ Bacterial Diagnosis layed with this se­lec­tive me­dium, it should al­ways be used with a tube of non­se­lec­tive me­dium. Mycobacterium tu­ber­cu­lo­sis ap­pears as gran­u­lar, rough, dry col­o­nies. M. kansasii ap­pears as smooth to rough, photochromogenic col­o­nies. M. gordonae ap­pears as smooth, yel­low-orange col­o­nies. M. avium ap­pears as smooth, col­or­less col­ o­nies. M. smegmatis ap­pears as wrin­kled, creamy white col­o­nies.

Lowenstein-Jensen Medium, Mycobactosel Modification The Mycobactosel mod­i­fi­ca­tion of LJ me­dium con­tains cy­clo­hex­i­ mide, lin­co­my­cin, and na­li­dixic acid to sup­press the growth of con­tam­i­nants.

Middlebrook 7H9 Broth The 7H9 for­mu­la­tion of Middlebrook broth is the same as Middle­ brook 7H10 agar, ex­cept that the agar and mal­a­chite green are ab­sent. The growth of most my­co­bac­te­ria is rapid in this me­dium, al­though an­ti­bi­ot­ics must be added to sup­press the growth of 156 SECTION 4 con­tam­i­nants. It is also used for per­form­ing my­co­bac­te­rial sus­cep­ti­ bil­ity test­ing.

Middlebrook 7H10 Agar Middlebrook 7H10 agar is a non­se­lec­tive me­dium that con­tains de­ fined salts, vi­ta­mins, co­fac­tors, oleic acid, al­bu­min, cat­a­lase, glyc­ erol, glu­cose, and mal­a­chite green. The ad­di­tion of glyc­erol en­hances the growth of Mycobacterium avium/intracellulare. Pyruvic acid can be added if M. bovis is sus­pected, and 0.25% L-asparagine or 0.1% po­tas­sium as­par­tate must be added for max­i­mal pro­duc­tion of ni­a­cin. The me­dium has a rel­a­tively short shelf life (ap­prox­i­ma­tely 1 month), and ex­po­sure to heat or light may re­sult in its de­te­ri­o­ra­tion and in the re­lease of form­al­de­hyde. Growth of my­co­bac­te­ria can be de­tected ear­lier on this me­dium than on egg-based me­dia.

Middlebrook 7H11 Agar Middlebrook 7H11 agar is pre­ferred over 7H10 agar be­cause the ad­ di­tion of ca­sein hy­dro­ly­sates im­proves the re­cov­ery of iso­ni­a­zid- resistant strains of M. tu­ber­cu­lo­sis, which have be­come prev­a­lent in some com­mu­ni­ties. It is also par­tic­u­larly use­ful for the cul­ti­va­tion of fas­tid­i­ous strains of tu­ber­cle ba­cilli that oc­cur fol­low­ing treat­ment of tu­ber­cu­lo­sis with sec­ond­ary an­ti­tu­ber­cu­lar drugs.

Bacterial Diagnosis Middlebrook 7H11 Agar, Mitchison’s Modification Mitchison’s mod­i­fi­ca­tion of 7H11 me­dium con­tains carbenicillin, po­ly­myxin B, tri­meth­o­prim, and am­pho­ter­i­cin B. The carbenicil- lin is par­tic­u­larly use­ful for sup­press­ing the growth of Pseudomo­ nas spp.

Middlebrook 7H13 Broth Middlebrook 7H13 broth is based on the 7H9 broth for­mu­la­tion sup­ple­mented with ca­sein hy­dro­ly­sate, po­ly­sor­bate 80, so­dium poly- anetholesulfonate, cat­a­lase, and [14C] pal­mitic acid. This broth is used in the BACTEC sys­tem.

Petragnani Medium Petragnani me­dium is a non­se­lec­tive my­co­bac­te­rial me­dium that con­tains co­ag­u­lated whole eggs, egg yolks, whole milk, po­tato, po­ tato flour, glyc­erol, and mal­a­chite green. This me­dium is more in­ hib­i­tory than LJ me­dium be­cause it con­tains a higher con­cen­tra­tion of mal­a­chite green. It should be re­stricted to use with heavily con­ tam­i­nated spec­i­mens. It is par­tic­u­larly use­ful for the cul­ti­va­tion and main­te­nance of M. smegmatis. Bacterial Diagnosis 157 Specific Diagnostic Tests for Pathogen Detection Aerobic Gram-Positive Cocci Enterococcus spp. Microscopy and cul­ture are the most com­monly used de­tec­tion meth­ods. Selective me­dia can be used to re­cover the bac­te­ria from spec­i­mens con­tam­i­nated with Gram-negative bac­te­ ria, and se­lec­tive me­dia sup­ple­mented with van­co­my­cin can be used to re­cover van­co­my­cin-resistant en­tero­coc­cal (VRE) strains. Direct de­tec­tion from spec­i­mens us­ing mo­lec­u­lar meth­ods may be used for in­fec­tion con­trol screen­ing for VRE. In ad­di­tion, com­mer­cially avail­­able mo­lec­u­lar meth­ods ex­ist for the iden­ti­fi­ca­tion of en­tero­ cocci and van­co­my­cin re­sis­tance from pos­i­tive blood cul­ture broth.

Staphylococcus au­re­us. Microscopy and cul­ture are the most com­monly used de­tec­tion meth­ods. Selective me­dia (e.g., man­ni­ tol salt agar, co­lis­tin-nalidixic acid agar, and phenylethyl al­co­hol agar) can be used for re­cov­ery from heavily con­tam­i­nated spec­i­ mens (most com­monly used for cul­ture of Cystic Fibrosis sputa). Molecular assays are avail­­able for de­tect­ing meth­i­cil­lin-resistant S. au­re­us both for in­fec­tion con­trol screen­ing pur­poses as well as from pos­i­tive blood cul­tures.

Coagulase Negative Staphylococci (CONS). Microscopy and cul­ture are the most com­monly used de­tec­tion meth­ods. CONS will read­ily grow on rou­tinely used me­dia (blood agar, choc­o­late agar, CNA agar). However, S. saccharolyticus is a strict an­aer­obe and Bacterial Diagnosis re­quires in­cu­ba­tion in an­aer­o­bic con­di­tions. Assess spec­i­men Gram stain re­sults for the pres­ence of ep­i­the­lial cells, which may in­di­cate a poorly col­lected spec­i­men and in­sig­ni­fi­cance of CONS. Virtually any CONS can be path­o­genic in the set­ting of a hard­ware-associated in­fec­tion.

Streptococcus, Group A. Group A Streptococcus grows read­ily in cul­ture. Streptococcus se­lec­tive me­dia may be used to en­hance iso­ la­tion of GAS from clin­i­cal spec­i­mens. Microscopy is not use­ful for di­ag­no­sis of phar­yn­gi­tis but is use­ful for cu­ta­ne­ous in­fec­tions. Nu- merous di­rect an­ti­gen tests are avail­­able for strep­to­coc­cal phar­yn­gi­ tis. Although the tests are highly spe­cific (for S. pyogenes but not other spe­cies of group A Streptococcus), the sen­si­tiv­ity is <80% and the Centers for Disease Control (CDC) rec­om­mends that neg­a­tive re­ac­tions must be con­firmed by cul­ture for pe­di­at­ric, but not adult, pa­tients. Antibody tests are used to con­firm an­te­ced­ent group A strep­to­coc­cal phar­yn­gi­tis or pyo­derma in pa­tients with sus­pected rheu­matic fe­ver or ne­phri­tis. The most pop­u­lar tests are the an­ti- streptolysin O (ASO) and an­ti-DNase B tests. Both tests have a sen­ si­tiv­ity of 85%, and they should be per­formed to­gether. The ASO 158 SECTION 4 test is non­re­ac­tive in pa­tients with ne­phri­tis fol­low­ing strep­to­coc­cal pyo­derma. False-positive ASO ti­ters can oc­cur in pa­tients with liver dis­ease and in­fec­tions with strep­to­coc­cal groups C or G. Anti-DN- ase B is spe­cific for group A strep­to­coc­ci; there is no re­ac­tiv­ity with other strep­to­coc­cal groups. Peak ASO and an­ti-DNase B ti­ters oc­ cur 2 to 3 weeks af­ter the pri­mary in­fec­tion and per­sist for 6 months or more. Positive ti­ters are two or more di­lu­tions above the up­per limit of nor­mal. Other tests (e.g., Streptozyme) are less sen­si­tive and re­pro­duc­ible.

Streptococcus, Group B. Group B Streptococcus grows read­ily in cul­ture, al­though 10% of strains are non­he­mo­lytic and may not be de­tected in mixed cul­tures. Enrichment broth (i.e., LIM broth) can be used to re­cover small num­bers of or­gan­isms. Microscopy is not help­ful for de­tec­tion of gen­i­tal car­riage. Numerous di­rect an­ti­ gen tests have been de­vel­oped for the de­tec­tion of gen­i­tal car­riage, but the test sen­si­tiv­ity is too low to jus­tify its use. Commercially avail­­able mo­lec­u­lar tests are widely avail­­able and used for the screen­ing of gen­i­tal car­riage in preg­nant women. Of note, most mo­ lec­u­lar meth­ods still re­quire pre­lim­i­nary en­rich­ment broth growth prior to test­ing. Multiplex PCR men­in­gi­tis/encephalitis pan­els now in­clude GBS for de­tec­tion in CSF.

Streptococcus pneumoniae. Microscopy and cul­ture are sen­si­tive de­tec­tion meth­ods, al­though the bac­te­ria can un­dergo spon­ta­ne­ous ly­sis and hence will not be re­cov­ered in spec­i­mens when pro­cess­ing Bacterial Diagnosis is de­layed. A va­ri­ety of tests have been de­vel­oped for de­tect­ing pneu­mo­coc­cal cap­su­lar an­ti­gens in ce­re­bro­spi­nal fluid (CSF) and urine. Urine tests are less sen­si­tive than CSF tests, and all ­an­ti­gen tests are gen­er­ally no more sen­si­tive than a Gram stain. Use of di­ rect spec­i­men an­ti­gen tests has largely been dis­con­tin­ued due to poor per­for­mance. Type-specific anticapsular an­ti­body ti­ters can be mea­sured to as­sess the re­sponse to vac­ci­na­tion but are not mea­sured for di­ag­nos­tic pur­poses. Multiplex PCR men­in­gi­tis/encephalitis pan­els now in­clude S. pneumoniae for de­tec­tion in CSF.

Aerobic Gram-Positive Rods Bacillus anthracis. Microscopy is use­ful if pos­i­tive, but the test is in­sen­si­tive and the cap­sule is gen­er­ally not seen when the Gram stain is used. Fluorescein-labeled anticapsular an­ti­bod­ies have been de­vel­oped. The or­gan­ism grows rap­idly in cul­ture and has a char­ ac­ter­is­tic col­ony mor­phol­ogy (large, ir­reg­u­lar, sticky, non­he­mo­lytic col­o­nies). Antigen tests and mo­lec­u­lar di­ag­nos­tic tests have been de­vel­oped and are avail­­able through state pub­lic health lab­o­ra­to­ ries. These tests are gen­er­ally spe­cific but lack sen­si­tiv­ity, es­pe­cially Bacterial Diagnosis 159 for asymp­tom­atic pa­tients ex­posed to B. anthracis. The se­ro­logic re­sponse to an­thrax toxin (i.e., pro­tec­tive an­ti­gen) can be used to as­sess the re­sponse to vac­ci­na­tion but not as a di­ag­nos­tic tool.

Corynebacterium diphtheriae. Diagnosis is based on clin­i­cal pa­ ram­e­ters. Microscopy is gen­er­ally not use­ful. The or­gan­ism grows read­ily on non­se­lec­tive sheep blood agar; se­lec­tive me­dia (cys­te­ ine-tellurite blood agar or Tinsdale me­dium) should also be used for pri­mary cul­tures. The Centers for Disease Control and Preven- tion (CDC) of­fer a di­rect PCR test for diph­the­ria tox­in; this test is rec­om­mended for con­firm­ing the di­ag­no­sis of diph­the­ria but should not be used alone. Immunoassays have been de­vel­oped to mea­sure the level of an­ti­bod­ies against C. diphtheriae toxin in pa­tients im­ mu­nized with tox­oid. These tests as­sess im­mu­nity and can­not be used for the di­ag­no­sis of diph­the­ria. Antitoxin lev­els of ≥0.01 IU/ ml are con­sid­ered pro­tec­tive. Lower lev­els in­di­cate that im­mu­ni­ za­tion with tox­oid may be re­quired.

Corynebacterium, Other spp. Microscopy and cul­ture are the most com­monly used de­tec­tion meth­ods. Some spe­cies are slow grow­ing un­less the iso­la­tion me­dia are sup­ple­mented with lip­ids. Some path­o­genic li­po­philic strains, such as C. jeikeium, C. macgin­ leyi, and C. kroppenstedtii, will grow bet­ter on sheep blood agar than on choc­o­late.

Erysipelothrix spp. Microscopy is gen­er­ally in­sen­si­tive, but the pres­ence of long, slen­der, Gram-positive rods in the tis­sue of a pa­ Bacterial Diagnosis tient sus­pected of hav­ing erysipeloid is help­ful. Growth on blood agar plates is slow, and incu­ ­ba­tion should be ex­tended for 7 days. Antigen and mo­lec­u­lar di­ag­nos­tic tests have not been de­vel­oped, and se­ro­logic test­ing is not use­ful be­cause pa­tients do not de­velop an­ti­bod­ies af­ter an ep­i­sode of erysipeloid.

Gardnerella vaginalis. Gram stains of vag­i­nal spec­i­mens are help­ful if thin Gram-variable coccobacilli or rods are seen. The or­ gan­ism grows poorly in cul­ture and may not be de­tected. It can of­ten be iso­lated on CNA agar.

Listeria spp. Microscopy is in­sen­si­tive for pa­tients with men­in­gi­ tis (gen­er­ally, small num­bers of or­gan­isms are pres­ent in CSF) and non­spe­cific (the or­gan­ism can be con­fused with Corynebacterium or Streptococcus). The or­gan­ism grows well on most non­se­lec­tive me­dia, but he­mo­ly­sis may not be ob­vi­ous on sheep blood agar. Special se­lec­tive agars have been de­vel­oped for the re­cov­ery of Listeria from stools and food prod­ucts. Antigen de­tec­tion kits have also been de­vel­oped for food prod­ucts but are not li­censed for 160 SECTION 4 clin­i­cal spec­i­mens. Multiplex PCR men­in­gi­tis/encephalitis pan­els now in­clude Listeria monocytogenes for de­tec­tion in CSF.

Acid-Fast and Partially Acid-Fast Gram-Positive Rods Mycobacterium avium com­plex (MAC). Microscopy and cul­ ture are sen­si­tive de­tec­tion meth­ods. Disseminated in­fec­tions are com­mon in im­mu­no­com­pro­mised pa­tients, with or­gan­isms re­cov­ ered in high con­cen­tra­tions in blood and many body tis­sues. MAC probes ex­ist for or­gan­ism iden­ti­fi­ca­tion from cul­tured iso­lates but are not used for di­rect de­tec­tion from spec­i­mens. Mycobacterium tu­ber­cu­lo­sis complex. Tuberculosis is most com­ monly di­ag­nosed by mi­cros­copy (ac­id-fast stain) and cul­ture. Myco- bacteria can also be de­tected di­rectly by us­ing mo­lec­u­lar meth­ods. The am­pli­fi­ca­tion meth­ods are use­ful for use with smear-positive re­spi­ra­tory spec­i­mens but can­not be used with nonrespiratory spec­ i­mens and have low sen­si­tiv­ity for smear-negative spec­i­mens (~60%), al­though some meth­ods are now ap­proved for smear-negative spec­i­ mens. Serologic meth­ods in ad­di­tion to the skin test can be used for the de­tec­tion of la­tent tu­ber­cu­lo­sis in­fec­tion (LTBI). Interferron gamma re­lease as­says (IGRA) QuantiFERON-TB Gold (QFT) and T-Spot have re­ceived U.S. Food and Drug Administration ap­proval for LTBI screen­ing. IGRA tests mea­sure the pa­tient’s im­mune re­ac­ tiv­ity to M. tu­ber­cu­lo­sis. Overall, per­for­mance of IGRAs is equiv­a­

Bacterial Diagnosis lent to the skin test. However, IGRAs have bet­ter spec­i­fic­ity than the skin test in pa­tients who have re­ceived the BCG vac­ci­na­tion or have had a prior my­co­bac­te­rial in­fec­tion. Nocardia spp. Diagnosis of nocardiosis re­lies on mi­cro­scopic de­ tec­tion of the or­gan­ism in clin­i­cal spec­i­mens and iso­la­tion in cul­ ture. Filamentous forms stain poorly with the Gram stain and weakly with the ac­id-fast stain (even when a weak de­col­or­iz­ing so­ lu­tion is used). Although the or­gan­ism grows on most non­se­lec­tive en­riched me­dia, re­cov­ery is best on buff­ered char­coal yeast ex­tract (BCYE) agar and Thayer-Martin agar. Serological test­ing meth­ods have been hand­i­capped by the an­ti­genic het­ero­ge­ne­ity of path­o­genic Nocardia spp., poor se­ro­logic re­sponse of the pa­tient, high lev­els of im­mu­no­re­ac­tiv­ity to Nocardia spp., in healthy in­di­vid­u­als, and cross re­ac­tiv­ity with other mi­cro­bial an­ti­gens. Rhodococcus spp. Rhodococcus spp. are weakly ac­id-fast, with rel­a­tively few cells stain­ing ac­id-fast un­less the or­gan­ism is cul­tured on Lowenstein-Jensen me­dium or Middlebrook agar. Bacteria grown in broth cul­tures in­cu­bated for a few hours will ap­pear as long rods, while those grown for lon­ger pe­ri­ods will ap­pear as cocci or coccobacilli. Prolonged in­cu­ba­tion of cul­ture may be re­quired for the iso­la­tion of this or­gan­ism. Bacterial Diagnosis 161 Aerobic Gram-Negative Cocci Moraxella catarrhalis. Microscopy and cul­ture are the most com­ monly used de­tec­tion meth­ods. Large num­bers of or­gan­isms as­so­ci­ ated with po­ly­mor­pho­nu­clear leu­ko­cytes and mu­cus are typ­i­cally ob­served in pa­tients with re­spi­ra­tory tract in­fec­tions.

Neisseria gonorrhoeae. Historically, mi­cros­copy and cul­ture were the di­ag­nos­tic tests of choice. For pa­tients with gen­i­tal in­fec­tions, the Gram stain has a sen­si­tiv­ity of 90 to 95% and a spec­i­fic­ity of 95 to 100% in symp­tom­atic males but a sen­si­tiv­ity of only 50 to 70% in symp­tom­atic fe­males and much lower in asymp­tom­atic fe­males. The or­gan­ism grows on choc­o­late agar, but se­lec­tive me­dia are used most com­monly to sup­press the ure­thral flora. For the most part, cul­ture has now been re­placed by mo­lec­u­lar tests, ini­tially with am­pli­fi­ca­ tion-based mo­lec­u­lar tests. Amplification as­says are more sen­si­tive than the mo­lec­u­lar probes that have his­tor­i­cally been used, but care must be taken to elim­i­nate in­hib­i­tors pres­ent in spec­i­mens (par­tic­u­ larly in urine) and pre­vent cross-contamination of spec­i­mens. Anti- body test­ing is not use­ful. Molecular tests are not ap­proved for use in non-genital tract spec­i­mens but data sug­gest there is util­ity to mo­lec­ u­lar test­ing of rec­tal, pha­ryn­geal, and oc­u­lar spec­i­mens.

Neisseria meningitidis. Microscopy and cul­ture are the most com­monly used de­tec­tion meth­ods. N. meningitidis is a com­mon col­o­nizer of the re­spi­ra­tory tract and is a well-described cause of con­junc­ti­vi­tis. Tests are avail­­able for de­tect­ing me­nin­go­coc­cal cap­ Bacterial Diagnosis su­lar po­ly­sac­cha­ride an­ti­gens in CSF, se­rum, and urine. The tests de­tect serogroups A, B, C, Y, and W135 (serogroup B an­ti­bod­ies cross-re­act with E. coli Kl an­ti­gen), and false-positive urine an­ti­gen tests have been re­ported. The test sen­si­tiv­ity ap­proaches 90% for serogroups A, C, Y, and Wl35 but is much lower for serogroup B. Multiplex PCR men­in­gi­tis/encephalitis pan­els now in­clude N. meningitidis for de­tec­tion in CSF.

Aerobic Gram-Negative Rods Acinetobacter spp. The or­gan­isms are Gram-negative coccobacilli, oc­ca­sion­ally ap­pear­ing Gram pos­i­tive, and are typ­i­cally ar­ranged in pairs. Growth on blood agar me­dia is usu­ally good. Most strains are ­able to grow on MacConkey agar (some com­mon clin­i­cal spe­cies will not), but there is no growth an­aer­o­bi­cally (strict aer­o­bic growth).

Aeromonas spp. Most spe­cies grow read­ily in cul­ture. Selective me­dia (e.g., blood agar with am­pi­cil­lin [20 µg/ml] and CIN agar) im­prove re­cov­ery from con­tam­i­nated spec­i­mens. Enrichment broth (e.g., al­ka­line pep­tone wa­ter) en­hances re­cov­ery, but its use 162 SECTION 4 is gen­er­ally not in­di­cated. Serological as­says are not com­monly used. Multiplex PCR gas­tro­en­ter­i­tis pan­els now ex­ist but thus far do not in­clude Aeromonas de­tec­tion.

Bartonella spp. Bacilli may be ob­served in clin­i­cal spec­i­mens from dis­eased pa­tients (e.g., those with cat scratch dis­ease [CSD], ba­cil­lary angiomatosis, or peliosis) with the Warthin-Starry sil­ver stain, al­though this is not com­monly used in clin­i­cal mi­cro­bi­ol­ogy lab­o­ra­to­ries. Some ref­er­ence lab­o­ra­to­ries of­fer PCR as­says for these bac­te­ria and are most use­ful from tis­sue spec­i­mens in those be­ing con­sid­ered for CSD and/or cul­ture neg­a­tive en­do­car­di­tis. PCR from pe­riph­eral blood is sig­nif­i­cantly less sen­si­tive than from tis­sue for in­fec­tious en­do­car­di­tis. Culture is not rec­om­mended for pa­tients with CSD but has been suc­cess­ful in other set­tings. Blood should be pro­cessed in the Isolator sys­tem, al­though some suc­cess has been achieved with broth-based blood cul­ture sys­tems. Prolonged in­cu­ ba­tion is re­quired. Tissues should be cul­tured on heart in­fu­sion agar sup­ple­mented with rab­bit or horse blood. These me­dia are pre­ferred to blood or choc­o­late agar. Cultures should be main­tained in a hu­ mid at­mo­sphere for 3 to 4 weeks. Serologic test­ing is the main­stay of di­ag­no­sis, par­tic­u­larly in pa­tients with CSD, with tests per­formed at the CDC, state lab­o­ra­to­ries, and some ref­er­ence lab­o­ra­to­ries. Commercial test kits are not avail­­able in the United States. Cross- reactions are ob­served in pa­tients with in­fec­tions caused by Coxi­ ella and Chlamydia spp.

Bacterial Diagnosis Bordetella per­tus­sis. Microscopy and cul­ture are rel­a­tively in­sen­ si­tive de­tec­tion meth­ods com­pared with PCR-based as­says. The or­gan­isms ap­pear as small Gram-negative coccobacilli. They are best ob­served us­ing a DFA test; com­mer­cial mono­clo­nal and po­ly­ clonal (for B. per­tus­sis and B. parapertussis) DFA tests (di­rected against cell wall lipooligosaccharides) are avail­­able. Both tests have a low sen­si­tiv­ity (30 to 70% com­pared with cul­ture) and spec­i­fic­ ity. B. per­tus­sis is a fas­tid­i­ous, strictly aer­o­bic or­gan­ism which does not grow on blood agar me­dia or MacConkey agar (B. parapertussis grows on blood agar and has var­i­able growth on MacConkey agar). Growth on Regan-Lowe me­dium is more re­li­able than on Bor­det- Gengou me­dium. Detection gen­er­ally re­quires a min­i­mum of 3 to 4 days of in­cu­ba­tion, which should be ex­tended for a week or more. The PCR as­say is clearly the most sen­si­tive method for de­tect­ing B. per­tus­sis. A va­ri­ety of tar­get genes have been used, in­clud­ing the per­tus­sis toxin pro­moter re­gion and the IS481 gene. IS481 is a mul­ti­ copy gene, and as­say tar­get­ing this gene ap­pears to be more sen­si­tive than those tar­get­ing the per­tus­sis toxin pro­moter re­gion. However, IS481 is also found in B. holmeseii and can there­fore lead to false pos­i­tive re­sults in pa­tients col­o­nized with this or­gan­ism. Positive tests are ob­served even af­ter 7 days of ef­fec­tive ther­apy. Many se­ro­ Bacterial Diagnosis 163 logic tests are avail­­able, with ELISA be­ing the method of choice. Immunoglobulin G (IgG) and IgA re­sponses to per­tus­sis toxin (PT) or fil­a­men­tous hem­ag­glu­ti­nin (FHA) are re­li­able in­di­ca­tors of in­fec­ tion. Antibodies to PT are spe­cific for B. per­tus­sis; an­ti­bod­ies to FHA are spe­cific to B. per­tus­sis and B. parapertussis (with cross-re­ ac­tions with other bac­te­ria). Serologic test­ing (IgG ver­sus PT) ap­ pears to be the most sen­si­tive mea­sure of B. per­tus­sis in­fec­tion in an un­im­mu­nized in­di­vid­ual, but se­ro­con­ver­sion must be dem­on­strat­ed.

Brucella spp. The or­gan­ism is a small coccobacillus, with cells ar­ranged typ­i­cally sin­gly or, less com­monly, in pairs and small chains. DFA stains are not avail­­able. The or­gan­ism is a strict aer­ obe, re­quir­ing com­plex me­dia con­tain­ing sev­eral amino ac­ids, thi­ a­mine, nic­o­tin­amide, and mag­ne­sium ions. The pres­ence of se­rum and a CO2-enriched at­mo­sphere en­hance growth, and pro­longed in­ cu­ba­tion is re­quired, al­though mod­ern blood cul­ture me­dia can grow Brucella spp. within the com­monly used 5-day in­cu­ba­tion pe­ riod. Various se­ro­logic as­says have been de­vel­oped, with the se­ rum ag­glu­ti­na­tion test be­ing the most com­monly used. A sin­gle ti­ter of >1:160 is sug­ges­tive of in­fec­tion (with B. abortus, B. suis, or B. melitensis). Cross-reactions are ob­served with F. tularensis, V. cholerae, and Y. enterocolitica. A rapid dip­stick as­say for IgM an­ ti­bod­ies has been de­vel­oped.

Burkholderia cepacia complex. Selective me­dia have been used to im­prove re­cov­ery from con­tam­i­nated spec­i­mens. B. cepacia se­ lec­tive agar is the most sen­si­tive and se­lec­tive me­dium. PCR as­ Bacterial Diagnosis says have been de­vel­oped but are used for or­gan­ism iden­ti­fi­ca­tion rather than de­tec­tion. Serologic tests are not avail­­able.

Burkholderia pseudomallei. The or­gan­isms ap­pear on di­rect Gram stain as small Gram-negative rods with bi­po­lar stain­ing. The cells re­sem­ble “safety pins,” and this char­ac­ter­is­tic can be used to make a pre­sump­tive iden­ti­fi­ca­tion. The or­gan­ism grows on blood agar and MacConkey agar, but bet­ter re­cov­ery is found on Ashdown me­dium. An en­rich­ment broth of Ashdown me­dium sup­ple­mented with co­lis­tin (in­cu­bated for 7 days be­fore sub­cul­ture) im­proves re­ cov­ery. A non­com­mer­cial in­di­rect hem­ag­glu­ti­na­tion as­say has been de­vel­oped; how­ever, cross-re­ac­tions with B. cepacia com­plex oc­cur, and high an­ti­body ti­ters are found in healthy in­di­vid­u­als liv­ing in ar­eas of en­demic in­fec­tion. A sin­gle ti­ter can­not be in­ter­preted, and so se­ro­con­ver­sion must be dem­on­strat­ed.

Campylobacter spp. The bac­te­ria are curved rods typi­­cally ar­ ranged in pairs (re­sem­bling “gull wings” or S-shaped). The bac­te­ria are thin and may not be ob­served in clin­i­cal spec­i­mens. Growth of most spe­cies re­quires a microaerophilic at­mo­sphere, and se­lec­tive 164 SECTION 4 me­dium is rec­om­mended for the re­cov­ery of C. jejuni and C. coli from fe­cal spec­i­mens. C. upsaliensis may be a com­mon en­ teric path­o­gen, but its re­cov­ery is com­pro­mised be­cause it is in­hib­ ited on most se­lec­tive me­dia for Campylobacters. C. fe­tus is more com­monly re­cov­ered in the blood. Serologic test­ing is use­ful for ep­i­de­mi­o­logic in­ves­ti­ga­tions but not for di­ag­nos­tic pur­poses. Mul- tiplex PCR as­says now ex­ist for the test­ing of stool and incl­ ude Campylobacter jejuni, C. coli, and C. upsaliensis.

Capnocytophaga spp. (in­cludes for­mer DF-1 and DF-2). The or­ gan­isms are fu­si­form; curved, coc­coid, and spin­dle-shaped forms

are also ob­served. Growth re­quires en­riched me­dia and CO2; good growth oc­curs in 2 to 4 days; ad­her­ent col­o­nies may be slightly yel­low and have ei­ther a reg­u­lar edge or a spread­ing edge. There is no growth on MacConkey agar; the or­gan­ism is a fac­ul­ta­tive an­aer­obe.

Cardiobacterium spp. Rods are ar­ranged sin­gly or in pairs, short chains, or ro­settes. The or­gan­ism is fac­ul­ta­tively an­aer­o­bic, with no growth on MacConkey agar. It is fas­tid­i­ous and slow grow­ing and

re­quires CO2. Eikenella spp. The or­gan­isms are slen­der, straight rods that are fac­ul­ta­tively an­aer­o­bic. There is no growth on MacConkey agar;

growth re­quires he­min and is en­hanced by CO2. Escherichia coli. Selective me­dia (e.g., MacConkey agar with Bacterial Diagnosis sor­bi­tol) can be used to de­tect enterohemorrhagic E. coli strains, and im­mu­no­as­says can be used to de­tect the Shiga tox­ins. Com- mercial as­says are also avail­­able to de­tect the heat-labile and heat- stable tox­ins of en­tero­toxi­genic E. coli strains. Molecular tests have been de­vel­oped to de­tect vir­u­lence fac­tors in these and other E. coli strains. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude the de­tec­tion of Enteropathogenic, Enteroaggregative, En- teroinvsasive, Enterotoxigenic, and shiga-like toxin pro­duc­ing E. coli. In ad­di­tion, mul­ti­plex tests have been de­vel­oped for the de­tec­tion of E. coli from CSF as well as pos­i­tive blood cul­tures.

Francisella spp. Microscopy is rel­a­tively in­sen­si­tive. The or­gan­ism is a very small coccobacillus that re­tains the saf­ra­nin coun­ter­stain poorly. A po­ly­clonal DFA re­agent is avail­­able, al­though the sen­si­tiv­ ity and spec­i­fic­ity are not well char­ac­ter­ized. Francisella spp. are fas­tid­i­ous, strict aer­obes that fail to grow on blood agar or MacCon- key agar. Growth re­quires me­dia sup­ple­mented with sulf­hy­dryl com­pounds (e.g., cys­te­ine, cys­tine, thio­sul­fate, and IsoVitaleX). Growth is slow but good on choc­o­late agar, Mueller-Hinton agar, and BCYE agar. Media should be in­cu­bated for up to 2 weeks. An- tigen and PCR tests have been de­vel­oped, but their sen­si­tiv­i­ties are Bacterial Diagnosis 165 low (an­ti­gen test, 106 bac­te­ria/ml; PCR, 102 bac­te­ria/ml). Serologic test­ing is the most com­monly used di­ag­nos­tic method. Antibodies are de­tected as early as 1 week af­ter the on­set of symp­toms and may per­sist for years. Tube ag­glu­ti­na­tion (TA) and microagglutination (MA) are the stan­dard as­says. A sin­gle TA ti­ter of >1:160 or MA ti­ter of >1:128 is con­sid­ered a pre­sump­tive pos­i­tive re­ac­tion. A four­ fold ti­ter change is con­sid­ered di­ag­nos­tic.

Haemophilus ducreyi. The Gram stain mor­phol­ogy of this or­gan­ ism has been de­scribed as “long chains (‘schools of fish’),” but this de­scrip­tion is more char­ac­ter­is­tic of in vi­tro cul­tures than of spec­ i­mens col­lected from gen­i­tal ul­cers. The Gram stain sen­si­tiv­ity is <50%. Direct fluo­res­cent-antibody (DFA) as­say re­agents have been pre­pared, but po­ly­clonal antisera have poor spec­i­fic­ity and mono­clo­nal antisera are not com­mer­cially avail­­able. Culture re­ quires the use of se­lec­tive me­dia (e.g., GC agar sup­ple­mented with van­co­my­cin, he­mo­glo­bin, fe­tal bo­vine se­rum, and IsoVitaleX) and has var­i­able sen­si­tiv­ity. PCR-based as­says have been de­vel­oped but are not widely avail­­able.

Haemophilus influenzae. The or­gan­isms are pleo­mor­phic rods (i.e., coc­coid, coccobacillary, or short rods); they are fac­ul­ta­tively an­aer­o­bic, with no growth on sheep blood agar or MacConkey agar. Detection of type-specific cap­su­lar an­ti­gen has been used to di­ag­ nose dis­sem­i­nated dis­ease (i.e., men­in­gi­tis); how­ever, this test is no more sen­si­tive than a Gram stain, and vac­ci­na­tion has dra­mat­i­cally re­duced the in­ci­dence of H. influenzae dis­ease. Multiplex PCR as­ Bacterial Diagnosis says now ex­ist for the de­tec­tion of H. influenzae in CSF. Serologic test­ing is gen­er­ally re­stricted to dem­on­stra­tion of a re­sponse to vac­ci­na­tion.

Helicobacter py­lo­ri. Microscopy is use­ful for ex­am­i­na­tion of tis­sue bi­opsy spec­i­mens but is per­formed pri­mar­ily in cy­tol­ogy lab­o­ra­to­ ries. Diagnosis is most com­monly made us­ing an­ti­gen tests, par­tic­u­ larly the de­tec­tion of ure­ase ac­tiv­ity in tis­sue bi­opsy spec­i­mens or by breath anal­y­sis. Stool an­ti­gen test­ing is also widely avail­­able and com­monly used. The tests vary widely in sen­si­tiv­ity and spec­i­fic­ity but gen­er­ally are >90% sen­si­tive and spe­cific. PCR as­says are avail­­ able but of­fer no more sen­si­tiv­ity than the an­ti­gen tests. Culture can also be per­formed (and is use­ful if drug re­sis­tance is sus­pected). The or­gan­ism grows best on freshly pre­pared non­se­lec­tive me­dia (e.g., bru­cella or brain heart in­fu­sion agars sup­ple­mented with horse blood) in­cu­bated in a microaerophilic at­mo­sphere for a min­i­mum of 5 days. A va­ri­ety of se­ro­logic tests are also avail­­able. Serum IgG as­ says are the tests of choice, al­though whole-blood as­says are used with in­creas­ing fre­quency in phy­si­cian of­fices (the tests have a sen­ si­tiv­ity of 80 to 90% com­pared with se­rum as­says). Serum IgA 166 SECTION 4 as­says have a low sen­si­tiv­ity but may prove use­ful as a fol­low-up as­say for pa­tients with neg­a­tive IgG as­says.

Kingella spp. The or­gan­isms are nu­tri­tion­ally fas­tid­i­ous, fac­ul­ta­ tively an­aer­o­bic short rods with square ends, ar­ranged in pairs or chains. They de­col­or­ize un­evenly on Gram stain­ing. They do not

re­quire CO2, but growth is en­hanced; there is no growth on Mac- Conkey agar. Some ev­i­dence sug­gests that bed­side in­oc­u­la­tion of bone and/or joint spec­i­mens can in­crease the yield of Kingella. In ad­di­tion, PCR has been shown to dem­on­strate su­pe­rior sen­si­tiv­ity to cul­ture for the di­ag­no­sis of Kingella sep­tic ar­thri­tis. It is hy­poth­ e­sized that sy­no­vial fluid may be in­hib­i­tory to the in vi­tro growth of the or­gan­ism.

Legionella spp. Legionella spp. are small, poorly stain­ing rods. If ob­served in clin­i­cal spec­i­mens, they typ­i­cally ap­pear as coccoba- cilli; long fil­a­men­tous forms can be seen in cul­ture. Monoclonal and po­ly­clonal DFA stains are com­mer­cially avail­­able; their sen­si­tiv­ity com­pared with cul­ture is poor (33 to 70%), and cross-re­ac­tions are ob­served with the mono­clo­nal re­agents (Bacillus ce­re­us) and po­ly­ clonal re­agents (Bacteroides fragilis, Pseudomonas spp., Stenotro­ phomonas spp., and Bordetella per­tus­sis). Urinary an­ti­gen tests for L. pneumophila serogroup 1 are avail­­able (Wampole Laboratories, Bartels) and Binax and Biotest mar­ket uri­nary an­ti­gen tests for non- serogroup 1 L. pneumophila and other Legionella spp. The sen­si­tiv­ ity of these as­says var­ies, but sen­si­tiv­i­ties are re­ported in the range of Bacterial Diagnosis 70 to 80%. The spec­i­fic­ity is good, al­though the Bartels test re­acts with S. pneumoniae, and false-positive re­ac­tions due to non­spe­cific pro­tein bind­ing have been re­ported for the Binax EIA. Reactivity per­sists in Legionella-infected pa­tients for weeks to months af­ter ef­ fec­tive ther­apy. PCR as­says are avail­­able but gen­er­ally are not more sen­si­tive than cul­ture. The or­gan­ism is fas­tid­i­ous, re­quir­ing me­dia sup­ple­mented with L-cysteine and iron salts. BCYE, sup­ple­mented with an­ti­bi­ot­ics to sup­press the growth of con­tam­i­nat­ing or­gan­isms, is the me­dium of choice. Incubation should be ex­tended for a week or more. IFA tests and ELISA are avail­­able to mea­sure an an­ti­body re­sponse to in­fec­tion. The test sen­si­tiv­ity and spec­i­fic­ity are 75 and 96%, re­spec­tively. A ti­ter of >1:256 is sug­ges­tive of cur­rent in­fec­ tion; how­ever, ti­ters at this level have been found in healthy in­di­ vid­u­als. A four­fold change in ti­ter is pre­sump­tive ev­i­dence of re­cent dis­ease.

Pasteurella spp. The or­gan­isms are coc­coid to coccobacillary, ar­ ranged sin­gly, in pairs, or in short chains. Growth does not re­quire

he­min or CO2, but some strains re­quire V fac­tor. The most com­ monly iso­lated spe­cies fail to grow on MacConkey agar; the or­ gan­ism is a fac­ul­ta­tive an­aer­obe. Bacterial Diagnosis 167 Pseudomonas aeruginosa. P. aeruginosa grows read­ily on a va­ri­ ety of lab­o­ra­tory me­dia. PCR am­pli­fi­ca­tion meth­ods have been de­ vel­oped for di­rect de­tec­tion of the or­gan­ism in re­spi­ra­tory spec­i­mens, par­tic­u­larly from cys­tic fi­bro­sis pa­tients, but these are not com­monly used or com­mer­cially avail­­able. The method is rapid but less sen­si­ tive than cul­ture. Serologic test­ing is not use­ful. Multiplex PCR as­ says now ex­ist for the test­ing of me­dia from pos­i­tive blood cul­tures and in­clude P. aeruginosa.

Salmonella enterica Serovar Typhi. Selective me­dia must be used to op­ti­mize de­tec­tion in fe­cal spec­i­mens. The Widal test mea­ sures ag­glu­ti­nat­ing an­ti­bod­ies to the O and H an­ti­gens of S. enterica server Typhi and is used for se­ro­di­ag­no­sis; how­ever, it lacks sen­si­tiv­ ity and spec­i­fic­ity. Tests us­ing other an­ti­gens (e.g., Vi an­ti­gen) have been de­vel­oped but are re­stricted to ep­i­de­mi­o­log­i­cal stud­ies.

Salmonella, Other Serovars. Selective me­dia must be used to op­ ti­mize de­tec­tion in fe­cal spec­i­mens. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude Salmonella.

Shigella spp. Selective me­dia must be used to op­ti­mize de­tec­tion in fe­cal spec­i­mens. Serodiagnostic as­says have been de­vel­oped for ep­i­de­mi­o­log­i­cal sur­veys but have not been used for di­ag­nos­tic test­ing. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude Shigella.

Stenotrophomonas spp. Microscopy and cul­ture are sen­si­tive de­ Bacterial Diagnosis tec­tion meth­ods. PCR as­says have been de­vel­oped but are not used for or­gan­ism de­tec­tion.

Streptobacillus spp. The or­gan­isms are rod shaped, but on ex­ tended in­cu­ba­tion they can form very long fil­a­ments (100 to 150 µm long) and bul­bous forms. Colonies de­velop slowly. The or­gan­ism is fac­ul­ta­tively an­aer­o­bic; no growth oc­curs on MacConkey agar. Iso- lation of the or­gan­ism is in­hib­ited by the pres­ence of SPS, which is found in most com­mer­cially avail­­able blood cul­ture me­dia.

Vibrio cholerae. The vi­a­bil­ity of V. cholerae is main­tained at an al­ ka­line pH but de­creases in formed stools or at an acidic pH. If cul­ture is de­layed, the spec­i­men should be stored in Cary-Blair trans­port me­ dium but not in buff­ered glyc­erol sa­line. The or­gan­ism grows well on blood agar, slowly on MacConkey agar (lac­tose-negative), and well on se­lec­tive me­dia (e.g., TCBS). A re­verse pas­sive la­tex ag­glu­ti­na­tion test is com­mer­cially avail­­able for the de­tec­tion of chol­era toxin (re­acts also with E. coli heat-labile en­tero­toxin). Reference lab­o­ra­to­ries are also ­able to mea­sure an­ti­body re­sponse to in­fec­tion. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude Vibrio cholerae. 168 SECTION 4 Vibrio, Other spp. Most path­o­genic Vibrio spp. grow well on blood agar and MacConkey agar. As with V. cholerae, spec­i­mens should be pro­cessed im­me­di­ately or trans­ported in Cary-Blair me­ dium. V. parahaemolyticus he­mo­ly­sin (Kanagawa toxin) can be de­ tected di­rectly in spec­i­mens by a com­mer­cial re­verse pas­sive la­tex ag­glu­ti­na­tion test. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude Vibrio parahaemolyticus as well as V. vulnificus.

Yersinia enterocolitica. The use of en­rich­ment meth­ods (e.g., stor­age of spec­i­mens in­oc­u­lated into phos­phate-buffered sa­line for up to 21 days at 4°C) for re­cov­ery of Y. enterocolitica is gen­er­ally not nec­es­sary for di­ag­no­sis of pa­tients with di­ar­rhea but has proven use­ful for di­ag­no­sis of pa­tients with ter­mi­nal il­e­i­tis or postinfec- tious ar­thri­tis. CIN agar is the pre­ferred se­lec­tive me­dium, and growth is bet­ter at 25 to 30°C than at 35°C. MacConkey agar (lac­ tose-negative col­o­nies) can also be used. PCR tests di­rected against plas­mid and chro­mo­somal vir­u­lence fac­tors have been de­vel­oped but are not widely avail­­able. Antibodies against serogroups 0:3, 0:9, 0:5, 27, and 0:8 can be de­tected by tube or mi­cro­ti­ter ag­glu­ti­na­tion tests. Titers of >1:40 or a four­fold rise in ti­ter is con­sid­ered sig­nif­ i­cant. Cross-reactions oc­cur with Brucella spp. These tests are avail­­able through spe­cialty lab­o­ra­to­ries but are not com­mer­cially avail­­able. Multiplex PCR as­says now ex­ist for the test­ing of stool and in­clude Yersinia enterocolitica.

Bacterial Diagnosis Yersinia pestis. A va­ri­ety of stains have been used (i.e., Gi­emsa, Wright, Wayson, meth­y­lene blue) in ad­di­tion to the Gram stain. The char­ac­ter­is­tic “bi­po­lar, safety pin” mor­phol­ogy is not ob­served with the Gram stain. A DFA stain di­rected against the cap­su­lar Fl an­ti­gen is per­formed by state health de­part­ment lab­o­ra­to­ries but is not com­ mer­cially avail­­able. The or­gan­ism can be iso­lated on non­se­lec­tive agar (sheep blood agar or brain heart in­fu­sion agar) or from con­tam­i­ nated spec­i­mens on MacConkey agar or CIN agar (with a re­duced cefsulodin con­cen­tra­tion [4 pg/ml]). Pinpoint growth is seen at 24 h. PCR di­rected against the genes for the plas­min­o­gen ac­ti­va­tor pro­tein (pla) and cap­su­lar Fl an­ti­gen (cafl) have been de­vel­oped but are less sen­si­tive than cul­ture and an ELISA for cap­su­lar Fl an­ti­gen. Passive hem­ag­glu­ti­na­tion tests and ELISAs, avail­­able through the CDC, have been de­vel­oped to de­tect an­ti­bod­ies di­rected against the Fl an­ti­gen. A ti­ter of >1:10 is pre­sump­tive ev­i­dence of dis­ease, and a four­fold rise or fall in the an­ti­body ti­ter is con­fir­ma­to­ry.

Anaerobic Bacteria Actinomyces spp. The or­gan­isms may grow slowly in an aer­o­bic at­mo­sphere, and some strains are dif­fi­cult to iso­late in cul­ture. Bacterial Diagnosis 169 ­Microscopic ex­am­i­na­tion of “sul­fur gran­ules” (mac­ro­scopic col­o­ nies pres­ent in clin­i­cal spec­i­mens) is help­ful for mak­ing the di­ag­ no­sis of ac­ti­no­my­co­sis.

Bacteroides fragilis group. The or­gan­isms typ­i­cally ap­pear as pleo­mor­phic rods in clin­i­cal spec­i­mens. Growth is rapid on most an­aer­o­bic me­dia, al­though se­lec­tive me­dia (e.g., laked kana­my­cin- vancomycin sheep blood agar and Bacteroides bile esculin agar) should be used with nonsterile spec­i­mens.

Clostridium bot­u­li­num. Microscopy is gen­er­ally of lit­tle value ex­cept when ex­am­in­ing im­pli­cated food prod­ucts. The CDC pro­ vi­des tests mea­sur­ing the level of an­ti­bod­ies against C. bot­u­li­num toxin (an­ti­toxin lev­els) in pa­tients im­mu­nized with tox­oid. These tests as­sess im­mu­nity and can­not be used for the di­ag­no­sis of bot­ u­lism. The ap­pro­pri­ate di­ag­nos­tic test for foodborne bot­u­lism is dem­on­stra­tion of bot­u­li­nal toxin in se­rum, fe­ces, gas­tric con­tents, or vom­i­tus or re­cov­ery of the or­gan­ism in the fe­ces of the pa­tient. Demonstration of the or­gan­ism or toxin in sus­pected foods pro­vi­ des in­di­rect ev­i­dence of bot­u­lism. The pres­ence of the or­gan­ism or de­tec­tion of toxin in wound ex­u­dates con­firms the di­ag­no­sis of wound bot­u­lism.

Clostridium dif­fi­cile. Microscopic ex­am­i­na­tion of fe­cal spec­i­ mens is of no clin­i­cal util­ity. Culture on se­lec­tive me­dia is a sen­si­ tive method for de­tect­ing the or­gan­ism but does not dif­fer­en­ti­ate be­tween col­o­ni­za­tion and clin­i­cally sig­nif­i­cant dis­ease. The di­ag­no­ Bacterial Diagnosis sis of C. dif­fi­cile in­fec­tion (CDI) is con­tro­ver­sial, and there are sev­ eral nonculture-based di­ag­nos­tic strat­e­gies that are em­ployed. PCR that de­tects the toxin gene (usu­ally toxin B) is con­sid­ered to be the most sen­si­tive of the com­monly used di­ag­nos­tics, but there is con­ cern that it may be too sen­si­tive and de­tect pa­tients who are col­o­ nized with C. dif­fi­cile but do not have CDI. EIA as­says that de­tect toxin are less sen­si­tive than PCR and may miss some pa­tients with CDI, but is less likely to be pos­i­tive in col­o­nized pa­tients with­out­ CDI. Glutamine de­hy­dro­ge­nase (GDH) is an an­ti­gen pro­duced by C. dif­fi­cile and can be used as a non­spe­cific marker to de­tect C. dif­fi­cile. GDH is non­spe­cific for CDI be­cause both toxin neg­a­tive and toxin pos­i­tive strains can be de­tected by GDH. These three as­says (PCR, Toxin EIA, and GDH) are the foun­da­tion for var­i­ous al­go­rithms that can be used to di­ag­nose CDI. Among the most com­mon in­clude the GDH/Toxin EIA al­go­rithm, which re­flexes to PCR if GDH is pos­ i­tive and the EIA is neg­a­tive. An al­ter­na­tive to this method is a PCR screen with pos­i­tive tests reflexing to EIA.

Clostridium perfringens. The mi­cro­scopic mor­phol­ogy is char­ac­ ter­is­tic (large, short, fat, rect­an­gu­lar cells with no spores ob­served). 170 SECTION 4 Growth on an­aer­o­bic sheep blood agar is rapid, with a dou­ble zone of he­mo­ly­sis typ­i­cally ob­served. Antigen tests for de­tec­tion of tox­ ins have not been de­vel­oped, and PCR as­says are re­stricted to re­ search lab­o­ra­to­ries.

Clostridium tetani. Microscopy and cul­ture are typ­i­cally of lit­tle use be­cause rel­a­tively small num­bers of or­gan­isms can cause clin­i­cal dis­ease. However, ob­ser­va­tion of the or­gan­isms in clin­i­cal spec­i­mens or re­cov­ery in cul­ture can be di­ag­nos­tic in the ap­pro­pri­ate clin­i­cal set­ting. Serologic test­ing is also not use­ful for di­ag­nos­tic pur­poses be­cause an­ti­bod­ies are not formed in pa­tients with clin­i­cal dis­ease. It can be use­ful for as­sess­ing the im­mune sta­tus of an in­di­vid­ual, with an­ti­toxin lev­els of >0.5 IU/ml gen­er­ally con­sid­ered pro­tec­tive.

Fusobacterium spp. Some spe­cies (e.g., F. nucleatum) have a char­ac­ter­is­tic thin, fu­si­form mor­phol­ogy. Culture may re­quire pro­ longed in­cu­ba­tion (5 days or more). F. necrophorum is a com­mon cause of Lemierre’s syn­drome, which is typ­i­cally di­ag­nosed with pos­i­tive blood cul­tures. In ad­di­tion, F. necrophorum is an un­der­ ap­pre­ci­ated cause of phar­yn­gi­tis be­cause an­aer­o­bic throats cul­tures are not com­monly per­formed.

Mobiluncus spp. Routine cul­ture of vag­i­nal spec­i­mens for Mobi­ luncus spp. is gen­er­ally not clin­i­cally use­ful. The pre­ferred di­ag­ nos­tic test is mi­cros­copy, with curved rods ob­served in vag­i­nal smears. Bacterial Diagnosis

Curved and Spiral-Shaped Bacteria Borrelia burgdorferi. Microscopic ex­am­i­na­tion of blood, CSF, and other spec­i­mens is not use­ful for pa­tients with Lyme dis­ease be­cause the level of spi­ro­chetes is be­low the de­tec­tion level. Culture on me­dia such as mod­i­fied Kelly me­dium can be per­formed, al­though ex­ tended in­cu­ba­tion must be used and the yield is low. ELISAs have been de­vel­oped to de­tect an­ti­gen in tis­sues, but the method is not rec­om­mended. PCR as­says have been de­vel­oped for the de­tec­tion of B. burgdorferi. The sen­si­tiv­ity of the as­say de­pends on the test­ing method, tar­get gene, and stage of ill­ness. The fol­low­ing test sen­si­tiv­ i­ties have been re­port­ed: skin, 50 to 70% for cul­ture or PCR; sy­no­ vial fluid, 50 to 70% for PCR, cul­ture sel­dom pos­i­tive; CSF, 10 to 30% for cul­ture or PCR; and urine, 0%. Serologic test­ing has been the method most com­monly used for the di­ag­no­sis of Lyme dis­ease. The early an­ti­body re­sponse is pri­mar­ily an IgM re­sponse and is di­ rected against outer mem­brane–as­so­ci­ated pro­tein OspC, p35, and fla­gel­lum sub­units p37 and p41. The an­ti­body ti­ters peak within a week of clin­i­cal on­set but may per­sist for months, even af­ter ef­fec­tive treat­ment. IgG an­ti­bod­ies ap­pear af­ter the first weeks of dis­ease, Bacterial Diagnosis 171 with re­ac­tiv­ity against p37, p41, and OspC early in dis­ease; against p39 and p58 in the early dis­sem­i­nated stage; and against a wide va­ri­ ety of an­ti­gen in the late dis­sem­i­nated stage. Reactivity against other an­ti­gens is also com­monly ob­served. Assay meth­ods in­clude IFA, EIA, and im­mu­no­blot­ting. The spec­i­fic­ity of IFA is im­proved by ad­ sorp­tion of sera with Treponema phagedenis son­i­cate. IFA ti­ters of >1:64 are re­garded as pos­i­tive. This as­say is dif­fi­cult to stan­dard­ize, and EIA is the pre­ferred test­ing method. Immunoblotting is used to iden­tify which an­ti­gens are re­ac­tive. The rec­om­mended ap­proach to se­ro­di­ag­no­sis is to screen se­rum or CSF for IgG and IgM an­ti­bod­ies with EIA. If the re­ac­tion is pos­i­tive or bor­der­line, IgG and IgM im­ mu­no­blot­ting is per­formed. The sen­si­tiv­ity of se­ro­logic test­ing is as fol­lows: stage 1 (early, lo­cal­ized dis­ease), 20 to 50% with IgM pre­ dom­i­nant; stage 2 (early, dis­sem­i­nated dis­ease), 70 to 90% with IgG pre­dom­i­nant; stage 3 (late, dis­sem­i­nated dis­ease), nearly 100% with IgG pre­dom­i­nant. Antibodies per­sist for months de­spite treat­ment.

Borrelia, Other spp. Patients with re­laps­ing fe­ver have large num­ bers of borreliae in their blood dur­ing fe­brile at­tacks. The di­ag­nos­ tic method of choice is mi­cros­copy, with the blood ex­am­ined by dark-field mi­cros­copy or stained with Gi­emsa. Small num­bers of bac­te­ria can be con­cen­trated in buffy coat prep­a­ra­tions. Culture on me­dia such as mod­i­fied Kelly me­dium can be per­formed, al­though the cul­tures should be in­cu­bated at 30°C for at least 6 weeks.

Leptospira spp. The or­gan­isms may be de­tected by dark-field mi­ cros­copy or DFA, al­though large num­bers of or­gan­isms (104/ ml) Bacterial Diagnosis must be pres­ent in the spec­i­men. Culture of blood and CSF dur­ing the first week of ill­ness and cul­ture of urine be­gin­ning in the sec­ ond week can be per­formed us­ing Ellinghausen-McCullough- Johnson-Harris me­dium. Cultures are in­cu­bated at 28 to 30°C and ex­am­ined weekly by dark-field mi­cros­copy for up to 13 weeks. PCR as­says have been de­vel­oped but are not used ex­ten­sively. Serologic test­ing is the most com­monly used di­ag­nos­tic method. Antibodies are de­tected by us­ing the mi­cro­scopic ag­glu­ti­na­tion test with blood 5 to 7 days af­ter on­set of symp­toms. Paired sera are re­quired to con­ firm the di­ag­no­sis, with a pre­sump­tive di­ag­no­sis be­ing made if a sin­gle se­rum has a ti­ter of >1:200. The test is tech­ni­cally com­plex, and other as­says have been de­vel­oped. The in­di­rect hem­ag­glu­ti­na­ tion as­say was shown to have a sen­si­tiv­ity and spec­i­fic­ity of 92 and 95%, re­spec­tively. Latex ag­glu­ti­na­tion as­says and ELISAs have also been de­vel­oped but have not been ad­e­quately eval­u­at­ed.

Treponema pallidum. When avail­­able, dark-field mi­cros­copy is very sen­si­tive for di­ag­no­sis based on a freshly sam­pled gen­i­tal chan­cre or sec­ond­ary-stage ex­u­dates. The DFA test for T. pallidum (DFA-TP) does not re­quire vi­a­ble spi­ro­chetes and can dif­fer­en­ti­ate 172 SECTION 4 be ­tween T. pallidum and non­patho­genic spi­ro­chetes. The test sen­ si­tiv­ity for pri­mary or sec­ond­ary syph­i­lis ap­proaches 100%. The or­gan­ism has not been grown in vi­tro, and an­ti­gen tests are not avail­­able. PCR meth­ods have been de­vel­oped but cur­rently are re­ stricted to re­search lab­o­ra­to­ries. Serologic test­ing by nontrepone- mal and trep­o­ne­mal as­says is the most com­mon di­ag­nos­tic method. The nontreponemal as­says in­clude the Venereal Disease Research Laboratory (VDRL) test, rapid plasma re­gain (RPR) card test, un­ heated se­rum re­gain (USR) test, and to­lu­i­dine red un­heated se­rum test (TRUST). Treponemal tests in­clude fluo­res­cent trep­o­ne­mal an­ti­ body-absorption (FTA-ABS) test, trep­o­ne­mal pallidum par­ti­cle ag­ glu­ti­na­tion (TP-PA) test, and EIA. The test sen­si­tiv­i­ties vary with the method and stage of dis­ease. In gen­eral, nontreponemal tests have a sen­si­tiv­ity of 72 to >90% for the pri­mary stage, 100% for the sec­ond­ ary stage, 95 to 100% for la­tent dis­ease, and <75% for the late stage, while trep­o­ne­mal tests have a sen­si­tiv­ity of 80 to >90% for the pri­ mary stage, 100% for the sec­ond­ary and la­tent stages, and >95% for the late stage. A num­ber of con­di­tions af­fect the test spec­i­fic­ity, with more false-positive re­ac­tions be­ing ob­served with nontreponemal tests. Historically, pa­tients have been screened with a nontreponemal test (typ­i­cally RPR) with pos­i­tive re­sults reflexing to trep­o­ne­mal tests for con­fir­ma­tion. A so-called re­verse al­go­rithm has re­cently been adopted by many labs, which screens with a trep­o­ne­mal test and re­flexes to the RPR for con­fir­ma­tory test­ing of pos­i­tive re­sults. Bacterial Diagnosis Mycoplasma spp. and Obligate Intracellular Bacteria Anaplasma spp. Gi­emsa or Wright stains of pe­riph­eral blood or buffy coat cells have a sen­si­tiv­ity ap­proach­ing 60%. As with Eh­ rlichia spp., PCR as­says have been de­vel­oped and have a wide range of sen­si­tiv­i­ties (50 to 86%). Their spec­i­fic­ity is re­ported to be 100%. IFA is the se­ro­logic method of choice. The typ­i­cal re­sponse dur­ing the acute phase of in­fec­tion is a rapid rise in an­ti­body lev­els, reach­ ing ti­ters of >1:640 within the first month of dis­ease. Antibodies can per­sist for many months to years. The test sen­si­tiv­ity is >90%. False-positive re­ac­tions have been en­coun­tered for pa­tients with in­ fec­tions caused by Rickettsia spp., Coxiella spp., and Ep­stein-Barr vi­rus. Patients with high Anaplasma ti­ters will also have el­e­vated ti­ters to B. burgdorferi.

Chlamydia trachomatis. Infections have his­tor­i­cally been con­ firmed by cul­ture, ob­ser­va­tion of el­e­men­tary bod­ies in spec­i­mens by DFA, or de­tec­tion of chla­myd­ial an­ti­gens (i.e., li­po­po­ly­sac­cha­ride and ma­jor outer mem­brane pro­teins) by EIA. DFA has a sen­si­tiv­ity and spec­i­fic­ity of 75 to 85 and 99%, re­spec­tively. The sen­si­tiv­ity of Bacterial Diagnosis 173 EIAs is re­ported to be 60 to 70% com­pared with that of nu­cleic acid am­pli­fi­ca­tion as­says. More re­cently, these meth­ods have been re­ placed by mo­lec­u­lar meth­ods, ini­tially probe tests and now am­pli­fi­ca­ tion tests. Amplification tests are the most sen­si­tive, al­though care must be taken to elim­i­nate in­hib­i­tors (par­tic­u­larly in urine) and to avoid cross-contamination of spec­i­mens. Two se­ro­logic as­says are in com­mon use: CF and microimmunofluorescence (mi­cro-IF). The CF test de­tects an­ti­bod­ies to C. trachomatis as well as to Chlamydophila psittaci and Chlamydophila pneumoniae. The mi­cro-IF test is type spe­cific. The CF test is pos­i­tive in vir­tu­ally all­ pa­tients with lym­pho­ gran­u­loma ve­ne­reum but gen­er­ally not pos­i­tive in pa­tients with oculo- genital in­fec­tions and tra­choma. A pos­i­tive CF ti­ter is >1:16. CF ti­ters for pa­tients with lym­pho­gran­u­loma ve­ne­reum gen­er­ally ex­ceed 1:128, whereas pa­tients with in­clu­sion con­junc­ti­vi­tis, cer­vi­ci­tis, or ure­thri­tis have an­ti­body ti­ters of <1:16. The mi­cro-IF test is more sen­si­tive than the CF test. Most pa­tients with chla­myd­ial in­fec­tions show pos­i­tive re­ac­tiv­ity (90 to 100% of pa­tients have de­tect­able IgG an­ti­bod­ies). However, an­ti­bod­ies to past in­fec­tions per­sist for years and are com­ monly de­tected in the as­say.

Chlamydophila pneumoniae. Most in­fec­tions are di­ag­nosed by ei­ther PCR-based as­says or se­ro­log­i­cal test­ing. Multiplex PCR as­ says for up­per re­spi­ra­tory tract in­fec­tion are now com­monly used and in­clude C. pneumophila. Micro-IF test­ing is avail­­able for di­ag­ nos­ing acute in­fec­tions. Cross-reactivity with other bac­te­ria is un­ com­mon. Serum spec­i­mens are gen­er­ally screened for IgM and IgG an­ti­bod­ies at 1:8, and those giv­ing pos­i­tive re­ac­tions are tested at in­ Bacterial Diagnosis creas­ing two­fold di­lu­tions. The di­ag­nos­tic cri­te­ria for acute in­fec­ tions in­clude at least a four­fold rise in ti­ter and a sin­gle se­rum IgM ti­ter of >1:16 and/or IgG ti­ter of 2*1:512.

Chlamydophila psittaci. Most in­fec­tions are di­ag­nosed by se­ro­ log­i­cal meth­ods, with the CF test be­ing the most com­mon.

Coxiella spp. C. burnetii can be re­cov­ered in cul­ture, al­though this is rarely at­tempted. PCR ap­pears to be sen­si­tive but is cur­rently re­stricted to re­search lab­o­ra­to­ries. A va­ri­ety of se­ro­logic meth­ods, in­clud­ing microagglutination, CF, IFA, and ELISA, have been used, with IFA cur­rently be­ing the method of choice. ELISA ap­pears to be more sen­si­tive than IFA, but in­ter­pre­tive stan­dards have not been de­ fined. Antigenic phase var­i­a­tion oc­curs with C. burnetii in­fec­tions. In acute self-lim­ited in­fec­tions, an­ti­bod­ies to the phase II an­ti­gen ap­ pear first and dom­i­nate the im­mune re­sponse. In chronic in­fec­tions, an­ti­bod­ies to the phase I an­ti­gen pre­dom­i­nate. Phase II an­ti­bod­ies ap­pear first and peak within 1 month at 1:1,024 or greater. Phase I an­ti­bod­ies ap­pear later and peak at 4 months. The ra­tio be­tween phase I and phase II re­sponses may be use­ful for dis­tin­guish­ing 174 SECTION 4 be­tween acute and chronic in­fec­tions. A phase I ti­ter of 1:800 or greater is di­ag­nos­tic of chronic Q fe­ver (e.g., en­do­car­di­tis).

Ehrlichia spp. Ehrlichia spp. have been suc­cess­fully cul­tured from blood, al­though this test is rarely used for di­ag­no­sis. Likewise, in­fected mono­cytes can be de­tected by use of Gi­emsa or Wright- stained pe­riph­eral blood or buffy coat cells, but this test has a sen­ si­tiv­ity of only <30%. PCR is a widely used di­ag­nos­tic test, with the 16S rRNA gene be­ing tar­get­ed; it has a sen­si­tiv­ity of 79 to 100% com­pared with se­ro­logic test­ing. Serological test­ing is also use­ful, with IFA be­ing the test of choice. Although a spe­cific an­ti­body ti­ ter has not been de­fined for sig­nif­i­cant dis­ease, a ti­ter of >1:64 is con­sid­ered pre­sump­tive ev­i­dence of past or cur­rent dis­ease.

Mycoplasma pneumoniae. Microscopy is not use­ful, and iso­la­ tion of M. pneumoniae in cul­ture is slow and in­sen­si­tive. For this rea­son, a va­ri­ety of an­ti­gen-directed tests in­clud­ing DFA, EIA, and im­mu­no­blot­ting have been de­vel­oped. These tests, as well as DNA probes, have poor sen­si­tiv­ity and spec­i­fic­ity. In con­trast, PCR as­ says are re­ported to be highly sen­si­tive and spe­cific. Specific se­ro­ logic tests in­clude com­ple­ment fix­a­tion (CF), ELISA, IFA, and la­tex ag­glu­ti­na­tion. CF de­tects pri­mar­ily IgM an­ti­bod­ies. Seroconversion is ob­served for about 60% of cul­ture-positive pa­tients, while 80 to 90% of pa­tients have a sin­gle ti­ter of >1:32. ELISA de­tects both IgM and IgG an­ti­bod­ies and ap­pears to be more sen­si­tive than CF. Spec- ificity can be im­proved by us­ing pu­ri­fied PI adhesin pro­tein as the Bacterial Diagnosis cap­ture an­ti­gen. Immunofluorescence IgG and IgM an­ti­body ti­ters of >1:10 are con­sid­ered pos­i­tive, with ac­tive dis­ease be­ing in­di­cated by a four­fold change in ti­ter. The la­tex ag­glu­ti­na­tion as­say de­tects IgG and IgM an­ti­bod­ies. A sin­gle ag­glu­ti­na­tion an­ti­body ti­ter of >1:320 or a four­fold change in ti­ter is in­dic­a­tive of ac­tive or re­cent in­fec­tion. The spec­i­fic­ity of each of these tests is a prob­lem be­cause cross-re­ac­tions with other Mycoplasma spp. have been ob­served. Upper re­spi­ra­tory tract in­fec­tion can be di­ag­nosed with mul­ti­plex PCR pan­els, many of which in­clude Mycoplasma pneumoniae.

Rickettsia rickettsii. Members of the spot­ted fe­ver group of rick­ett­ siae can be de­tected in tis­sue spec­i­mens by im­mu­no­flu­o­res­cence or PCR. The 17-kDa li­po­pro­tein gene is the prin­ci­pal tar­get of PCR, al­ though other tar­gets have also been used. The sen­si­tiv­ity of PCR is gen­er­ally thought to be lower than se­rol­ogy. A va­ri­ety of group- specific se­ro­logic tests have been de­vel­oped (e.g., IFA, CF, ELISA, RIA, la­tex ag­glu­ti­na­tion, and hem­ag­glu­ti­na­tion), with the IFA be­ing con­sid­ered the “gold stan­dard.” A di­ag­nos­tic ti­ter of >1:64 is usu­ally de­tected in the sec­ond week of ill­ness. The Pro­teus OX ag­glu­ti­na­tion test was used his­tor­i­cally but has been re­placed by the more spe­cific se­ro­logic tests.

Bacterial Diagnosis 175

μ g disk) g (100-

Furazolidone Furazolidone S S S R R

NT

(0.04-U disk) (0.04-U

Bacitracin Bacitracin S S S R R

μ g/ml) (0.4

Erythromycin Erythromycin S S R R

NT NT NT

μ g/ml) (200

Lysostaphin Lysostaphin S NT NT NT NT

­ ci Motility

+ R —S Oxidase a c NT Bacterial Diagnosis ve.

­ ti ­ i Growth in 6.5% NaCl 6.5% in Growth se-positive se-positive Gram-positive coc ve. ­ ti se pos ­ la ­ a ­ a ­ da ­ i se neg

­ la

­ a dherent to agar to dherent A cs cs of cat + — — — R ­ ti ­ is r ve ve or cat ­ te ­ ti ­ i

­ ac

and S. vitulus are ox ­obe aer Strict -- - - + + — + - - - + + - R +++ — — — + + + + — — — R se pos — ­ la ­ a fferential char . is weakly cat b Di ­ ed

Alloiococcus NT, notNT, test Staphylococcus lentus, S. scuiri, S. lentus, Staphylococcus a b c Genus Staphylococcus Macrococcus Micrococcus Planococcus Alloiococcus Kocuria Rothia Table 4.5

176 SECTION 4

Polymyxin B Polymyxin Novobiocin

SR Voges-Proskauer + S S NT

s β -Galactosidase ­ cie (+)

spe Urease

+ +

rnithine decarboxytase rnithine O NT

Staphylococcus

­sis ­ly ­dro hy ant PYR c a f­i­

­ ni

­ta ­ph phos Alkaline se a + – V + – + S R V + lly sig

­ ca ­ i

e ­as le ­c nu eat-stable Bacterial Diagnosis H clin – – + + V — + S V NT mon ­ mon

on

­ ti ­tor lmig fac Clumping ­ ac + + + — — V – + S R + + + – – – — — – – – + + + R S (+)

yed re

­ la Coagulase ––––––—+——–+SS– – – + +V+++–++–SS+ - V–++——V––SR ate de cs cs of most com ­ c ­ ti ­ di ­ is r s in ­ te ­ se e ­ ac ­ th ry) n ­ na ­ re ­ i ­ er le; pa ­ ab ­ cies ­ i ry) subsp. subsp. spe ­ na fferential char ­ i ; V, var ­ er Di ­ ed

­ us ­ re PYR, pyrrolidonyl arylamidase. a NT, NT, not test Staphylococcus S. au S. epidermidis S. haemolyticus S. lugdunensis S. saprophyticus saprophyticus S. hyicus (vet S. intermedius S. schleiferi schleiferi subsp. S. pseudintermedius (vet Table 4.6

Bacterial Diagnosis 177 Esculin hyrolysis Esculin

+

β - Glucuronidase e

V —

­sis ­ly ­dro hy

e Arginine Vancomycin S/R ve

­ ti Motility ­ a d

­ ci Satellite growth Satellite ve andve BGUR neg

­ ti ­ i

6.5% NaC1 6.5% Growth in in Growth + —+ — — S — R + — — S + — V + — — S + — — S V——R V——SV——S V — + S ———S ———SV — + — S + — is ARG pos Bacterial Diagnosis

c b

Morphology a Chains Chains Chains Chains Chains Chains Chains Chains Chains Clusters Clusters Clusters Clusters ve; G. elegans se-negative se-negative Gram-positive coc ­ ti ­ i ­ la

le.

­ a ­ ti

ami se ­da ­ti p ­pe o ­n

Leucine Leucine + + are mo cs cs of cat rs. ­ ti ­ te rs. ­ is

ve andve BGUR pos ­ te r

­ ti ­ te arylamidase ­ a

re.

­ ac

Pyrrolidonyl Pyrrolidonyl ­ tu +++ + + + + — + + + + + + + — VV + + — — —— ­ ranged in clus and E. gallinarum ­ ranged in clus is ARG neg is ar is ar y in broth cul ­ og fferential char ol ­ ph Di

Enterococcus casseliflavus Enterococcus Cellular mor Gemella haemolysans Gemella Granulicatella adiacens Facklamia languida Facklamia a b c d e Genus Enterococcus Granulicatella Facklamia Aerococcus viridans Aerococcus urinae Pediococcus Lactococcus Streptococcus Leuconostoc Abiotrophia Vagococcus Gemella Globicatella Table 4.7 178 SECTION 4 Hippurate , but can lead to false + - + - ­ gen CAMP ­ ti + — — + + V ——— — + + ——— ——— NT Voges- Proskauer ss the Lancefield B an ­ se NT NT Bacitracin do not pos - + + V- V- —— —— —— —— PYR a ­ ci c ­ co Large Large Small Colony ­ to ; (!), pseudoporcinus S. ­ ed Bacterial Diagnosis Large Large Small Large Large Large Large Alpha Variable Beta- hemolysis LargeLarge Large Large -hemolytic strep ­ ta ncefield ncefield cs of be ­ ti C, G B A E, P, NG1, NG1, E, P, nongrp, B (!) C A, C, F, G, A, C, F, nongrp La group E, P, U, V, V, U, E, P, nongrp G ­ is r ­ te ­ ac ns. ­ cies ­ tio fferential char subsp. dysgalactiae subsp. ­ ac spe Di group g g re

­ in e e typ ­ tiv ­ i PYR, pyrrolidonyl arylamidase; BGUR, U-glucuronidase; not NT, test a S. dysgalactiae equisimilis subsp. S. agalactiae S. pyogenes Streptococcus S. dysgalactiae S. anginosus S. pseudoporcinus S. porcinus S. canis pos Table 4.8 Bacterial Diagnosis 179 ­ + + + V V Esculin Hydrolysis of: Hydrolysis + V — — — Arginine ­ lis, S. sinensis, S. orisratti, S. oligofermen ­ tra + + + + — Voges-Proskauer ­ ci c ­ co Bacterial Diagnosis + V ­ to — — — iles. Sorbitol ­ ph ­ mo and S. alactoyticus. Acid from:Acid and S. ther cs cs of viridans strep + V — — — ­ ti ­ is r Mannitol ­ te ­ ac ­ nis, S. parasanguinis, S. gordonii, S. cristatus, S. oralis, S. infantis, S. peroris, S. aus ­ gui a S. anginosus, S. intermedius, S. constellatus. S. intermedius, S. anginosus, S. fferential char S. mitis, S. san S. equinus, S. gallolyticus, S infantarius, S gallolyticus, S. equinus, S. group Di group group group:

group: and massiliensis. S. S. mitis S. mutans group: S . mutans, S sobrinus. S. anginosus a S. bovis group: S. salivarius group: S. S. vestibularis, salivarius, S. mitis group S. mutans group Streptococcus S. salivarius S. anginosus S. bovis group tans, Table 4.9

180 SECTION 4

-glucoopyranoside - x Methyl-c D

on ­ti c ­du imn pro Pigment

Motility

Growth in 0.04% tellurite 0.04% in Growth

­sis ­ly ­dro hy Arginine

a Pyruvate

s Sucrose ­ cie

spe Raffinose Bacterial Diagnosis

nterococcus

Sorbitol E a Acid from:Acid

mon ­ mon

Mannitol a — — + + + + — — — + cs cs of com

­ ti ­ is Arabinose r ++VV+—+———— ++V++V+—+++ + + + — + — — — — + ++++++————+ ++—++—+—+—+ —++—++++——— ——————————— — ——++++————— —+———+——+—+ — — — V + — + — — — ­ te ­ ac ­ ci. ­ coc ­ tero ing ­ ing en fferential char llinarum ispar Di raffinosus faecium casseliflavus hirae

Species E. avium E. E. faecalis E. E. E. ga E. durans E. E. d E. cecorum Vagococcus fluvialis Vagococcus Key Key tests for group a Group I

II

III

IV V Table 4.10 Bacterial Diagnosis 181 Table 4.11 Differential char­ac­ter­is­tics of Gram-positive rods Catalase neg­a­tive Beta-hemolytic — Arcanobacterium Alpha-hemolytic — Erysipelothrix, Lactobacillus Nonhemolytic — Actinomyces

Catalase pos­i­tive Regular shape, spore for­mer — Bacillus, Paenibacillus Regular shape, non-spore for­mer — Listeria Irregular shape, pink pig­ment — Rhodococcus equi Irregular shape, or­ange pig­ment — Rhodococcus, Microbacterium, Nocardia

Irregular shape, yel­low pig­ment Oerskovia, Brevibacterium, Cellulomonas, Aureobacterium

Irregular shape, no pig­ment Bacillary — Corynebacterium Coccobacillary — Arthrobacter, Brevibacterium, Corynebacterium, Dermabacter, Rhodococcus Branching — Nocardia, Actinomyces, Propionibacterium, Rothia, Streptomyces, Turicella Bacterial Diagnosis 182 SECTION 4 ve, ve, ­ ti ­ i on

­ ti h, ­ es s ­ is ­ duc w ­ lo ­ sces ­ ed ­ ed ­ it ­ it e e pro e ­ as ­ hib ­ hib ­ ob ­ or al odor ­ r

CAMP CAMP in CAMP CAMP in Most strains yel some gray/blacksome Strong ure rose rose col CAMP and BGUR pos flo From breast ab Strict aer Comments

Xylose Mannitol

—— —— Sucrose

s Acid from:Acid Maltose ­ cie

spe

Glucose

hy ­sis ­ly ­dro Esculin Esculin + + V + V + + + Bacterial Diagnosis

orynebacterium C Urease

mon ­ mon

on ­ti ­duc irt re Nitrate

—— Lipophilism cs cs of com ——— — V + — + + V — — ­ ti

­ is

r

­ te oxidation ­ ac Fermentation/ F — — + — + + — — — F — + — — + — V — — F — + —F — + + — — — FF +F + VF — + V — —F —F — — V — + — V + + + V V — V + + + — V V V — V + — — — From may have eye; V OO + — — + + + — — — — — — — — — — — — O + — — — + V — — — ­ sis ­ lo fferential char ­ cu r Di ­ be

­ tu m ­ tu ­ do ­ a C. ulcerans C. pseu C. stri C. diphtheriae Species C. jeikeium C. urealyticum C. pseudodiphtheriticum C. amycolatum macginleyi C. C. minutissimum C. glucuronolyticum C. kroppenstedtii aurimucosum C. Table 4.12 Bacterial Diagnosis 183 ve ­ ti ­ a ve ­ ti ­ i s ­ ed nt ­ si ­ it le le to Gram neg ­ ly ­ me ­ ab ­ hib ­ i ­ mo

Weak he Coccoid to coccobacillary to Coccoid Yellow Yellow pig Ear; CAMP pos Cheese-like odor CAMP CAMP in Gram var Comments Xylose

+

Mannitol Sucrose

———

Acid from:Acid Maltose Glucose a + + + V V V + + + + + ­ ri

­ te

otility M + + V V + + —

rm bac ­sis ­ly ­dro hy

­ fo ­ e Esculin + V + V V V + Bacterial Diagnosis V —— — n

­ ry

­ti ­duc on re Nitrate Nitrate — cted co

­ le Catalase

V V V V + + V + + ———— —— cs cs of se oxidation

­ ti ­ is Fermentation/ F F — — V — + V V V + F + — + — + + + — V F + + + V + + + — + F + + + V + + + V + F + + F — — — — + + — — — F — — — — + + V — — F — — — — + + V — — F V + + — + + + — — F O + V V V V V V — — O + — — — — — — — — O—V——VVV——O + V O + r O/F ­ te ­ ac fferential char spp. spp. spp. Di spp. spp. spp.

rcanobacterium pyogenes Trueperella bernardiae A Dermabacter hominis Oerskovia turbata Organism Turicella otitidisTuricella Arthrobacter Brevibacterium Microbacterium Curtobacterium Leifsonia aquaticaLeifsonia Arcanobacterium bemardiae Arcanobacterium Arcanobacterium haemolyticum Gardnerella vaginalis Rothia dentocariosa Rothia Helcobacillus Cellulomonas Trueperella pyogenes Trueperella Table 4.13

184 SECTION 4

Salicn

Mannitol

Insulin Glycogen a Acid from:Acid

­ er Glycerol

VVVVV

-Arabinose D Arginine dihydrolase Arginine —— ted spore-forming gen

­ la

G s ­si ­ly o ­dr hy elatin

es and re ­sis ­ly ­dro aen hy Casein ­ ci VV V + — V + + V + V spe nce.

­ re Lecithinase (egg yolk) (egg Lecithinase r Bacterial Diagnosis — — ­ cu

Bacillus Spore Position Spore cted ates rare oc ­ c ­ le ­ di S, C, T s in

­ se 60°C e cs cs of se ­ th ­ ti n ­ is ­ re r

­ te 50°C l; pa ­ ac ­ na V — S, C — + + — + + + + + — — S, C + + + V — V + — — +

­ mi

Growth: Anaerobic + — — S, (C) + + + V — + + — + + + — — S, T — — — — — V + + + + +—— S +++———+——— +——+ + S — S, C ++++—++——+ — + + + — + + V + + V + + S, T — ——— al; T, ter ­ tr fferential char Di l; C, cen spp.

­ na ­ mi ­ us ­ ter e ­ r S, sub B. subtilis B. ce B. mycoides B. circulans B. Geobacillus Organism B. anthracis thuringiensis B. B. licheniformis Paenibacillus Table 4.14 Bacterial Diagnosis 185 me ­ zy ­ so + + + + V V V V V V — — — NT NT Growth in ly + + + — — — — — — — — — — — NT Growth 50°C at s ­ id + + + + + + V — — — — — — — — Mycolic Mycolic ac re a,b ­ tu tes ­ ce + W W W W — — — — — — — — — — ­ my Bacterial Diagnosis ­ no ­ ti Acid-fast na cted ac le. ­ le + + + + + ­ ab V V V — — — — — — — ­ i onidia C cs cs of se ; V, var ; V, ­ ti ­ ed ­ is r ­ te ­ phae ­ ac + + + + + + + V V — — — — — — Aerial Aerial hy fferential char ally acid fast; not NT, test ­ ti Di

W, weak or parW, b Actinomadura Amycolatopsis Corynebacterium Dermatophilus Mycobacterium Nocardiopsis Rhodococcus Saccharomonospora Saccharopolyspora Streptomyces Thermoactinomyces Tsukamurella Nocardia Gordonia Dietzia Table 4.15

186 SECTION 4

Tobramycin

Imipenem Gentamicin le.

­ ab ­ i Clarithromycin

s. V, var Ciprofloxacin ­ cie

Susceptibility to: Ceftriaxone on of most spe ­ ti

­ ca

­ fi

Linezolid ­ ti Amikacin a ired for iden s SSRSVSRV ­ qu

­ cie Xanthine g is re spe ­ in Bacterial Diagnosis r test

­ la Tyrosine ­ u c Nocardia ­ le

cted d; mo ­ le Casein ­ fie ­ i ++—VSVRRVVV++—VSVSSVVV ———SSSRRVRV Hydrolysis of: Hydrolysis

cs cs of se Acetamide vely mod ­ ti ­ si +—+—SSRSRRSR +———SSRRRVSV ­ is n r ­ te ­ te

­ ac Arylsulfiatase (14 day) (14 Arylsulfiatase +————SSSVSVSV — ————— ———— — —— s has been ex ­ nu fferential char ex Di ­ pl

omy ­ omy of this ge om ­ on c ­ va The tax a N. no N. farcinica N. otitidiscaviarum N. N. brasiliensisN. pseudobrasiliensisN. N. cyriacigeorgica N. Species abscessus N. Table 4.16

Bacterial Diagnosis 187

irt re Nitrate on ­ti ­duc – – – – – – – – – V V

(continued)

Pyrazinamide

Susceptibility to to Susceptibility –– –– + – + +

g/ml) μ (10

b Growth on T2H T2H on Growth

NANANA NA NA NA NANANA NA NA NA NANANA NA NA NA NA NA NA NA Niacin – s ­ cie

spe

Pigmentation d –––––– ––– ––– –––––– ––– –––– –– – + +

– (7) – (88) –

­gy ­o ­ph ol mor

Mycobacterium c

­on y sa col Usual S S S S S R R R R R Sm S/R Smt Smt/R Smt/R

Bacterial Diagnosis Optimal temp (°C) temp Optimal 35 37 37 37 37 37 37 30 30 ct, slow-growing 28–32 25–37 30–37 30–37 33–42 40–45 ­ le cs cs of se c ­ ti ex ­ is s ­ pl r ­ si ­ te a ­ lo ­ r e ­ ac ­ cu BCG e com ­ ma ­ ra ­ ber . tu M. avium M. intracellulare M. chi haemophilum M. M. bovis M. M. bovis M. M. celatum M. ulcerans M. ter M. malmoense M. shimoidei M. genavense M. Species M M. caprae M. M. xenopi M. fferential char ns Di ­ ge

­ mo ­ plex Non- chro Descriptive term TB com Table 4.17

188 SECTION 4

irt re Nitrate on ­ti ­duc

– – – + + +

Pyrazinamide Susceptibility to to Susceptibility . ­ es ­ ag nt

­ ce g/ml) μ (10

b Growth on T2H T2H on Growth

NA NA NA NA NA NA NA NA NANA NA NA Niacin (continued) s s lt islt based on these per ­ cie ­ su

spe

Pigmentation d + – + V + – + V ++ – – s, and the test re ­ se e

t

­ th

n ­gy ­o ­ph ol

mor ­ en

Mycobacterium c

r ­ re

­on y sa col Usual ­ pa S S R S/R S/R S/R

Bacterial Diagnosis Optimal temp (°C) temp Optimal 37 37 37 30 ct, slow-growing 35–37 30–37 ­ le ss; Smt, smooth and trans ve inve each test is given in pa ­ ti ­ i ­ ne cs cs of se ­ ti de. ­ is r ­ zi a e in rough ­ te ­ dr ­ at f strains pos ­ ac ­ di r. ­ me ­ age o r ­ to nt ­ te ­ ce M. szulgai M. simiae M. M. scrofulaceum M. M. kansasii M. marinum Species gordonae M. fferential char e. Thee. per ­ bl n as growth fac Di ­ ca ­ mi

­ pli R, rough; S, smooth; S/R, in T2H, thiophere-2-carboxylic acid hy Requires he NA, not ap a b c Descriptive term Chromogens Table 4.17 Bacterial Diagnosis 189 Table 4.18 Differential char­ac­ter­is­tics of clin­i­cally rel­e­vant, rap­idly grow­ing, Mycobacterium spe­cies rn ence ­ te on pe hsp 65 ­ ti lete) ­ qu ­ ty ­ p ­ no ­ duc

Species Pigment pro Unique phe Unique PRA pat Unique (com 16S se M. abscessus subsp. abscessus - - + + M. chelonae - + + + M. fortuitum - + + + M. abscessus subsp. bolletii ---- M. abscessus subsp. massiliense ---- M. bacteremicum + - + + M. boenickei - - + + M. canariasense - - + + M. cosmeticum + + + + M. goodii + - + + M. houstonense ---- M. immunogenum - - + + M. mageritense - + + + M. mucogenicum - + + + M. neoaurum + - + + M. peregrinum - - + - M. porcinum - - + + M. senegalense - - + + M. smegmatis + - + + Bacterial Diagnosis

190 SECTION 4 Fructose

+ +

V — — — — — — — — NT NT NT Sucrose

+ + V V — — — — — — — — — — Lactose + — — — — — — — — — — — — — Acid from:Acid

te agar, blood BA, agar. Maltose ­ la ­ o + + + + + + — — — — — — — — s

­ cie spe Glucose + + + + + + + + V V — — — — Neisseria

nt nt ase ­t c ­du re

­ va Bacterial Diagnosis ­ e Nitrate + + + + V V V — — — — — — — lly rel

­ ca ­ i

Media

a Selective Selective

+ + + + V V — — — — — Growth on on Growth NT NT NT cs cs of med ­ ti ­ is , and nitroreducens r ­ te

and subflava. ­ ac Morphology , glycolytica C C R C C C C C C C C R CR CR elongata flava, perflava, s s s s fferential char ­ cie ­ cie d Thayer-Martin agar, ML, Martin-Lewis agar; City agar, NYC, York CHOC, New choc e e Di ­ fie ­ sp ­ sp ­ i b c

­ sa ­ er ­ co MTM, mod Includes Includes sub Includes Includes sub a b c N. elongata N. subflava N. weav N. cinerea N. sicca N. Species N. animaloris bacilliformisN. flavescens N. gonorrhoeaeN. lactamica N. meningitidis N. polysacchareaN. N. zoodegmatis N. mu Table 4.19 Bacterial Diagnosis 191 r-like ­ la l ­ pi ­ er nt ­ me -hemolysis nge nge in cat ­ ta ­ ra nts ­ me ­ a

Produces violacein Produces Pleomorphic rods Weak be Yellow pig Cells ar fil

Mannitol

Maltose Sucrose

Acid from:Acid

and the of Cardiobacteriaceae Glucose

VVVV

se ­ta a ­ph phos lkaline A

NT

­sis ­ly ­dro sui hy Esculin

Neisseriaceae — Ornithine decarboxylase Ornithine

rs of the Arginine dihydrolase Arginine

Bacterial Diagnosis ­ be

NT NT

se ­ta ­duc irt re Nitrate cted mem Indole

­ le Growth on MacConkey agar MacConkey on Growth

cs cs of se ­ ti Catalase ­ is r

­ te Oxidase ­ ac +——+—————+++++ —+———————+ —+———+————+———+———————+ V+———————————— — ++———V————+——— — —+——+————++++— — + — + +—+— — +——— — — — — — Slender rods; agar may pit V++V++——++V—— . ­ ed Differential char

le; NT, notle; test NT, ­ ab ­ i Species hominis Cardiobacterium valvarum Cardiobacterium Chromobacterium violaceum kingae Kingella denitrificans Kingella oralisKingella potusKingella corrodens Eikenella Simonsiella muelleri Suttonella indologenes V, var V, Table 4.20 192 SECTION 4 e from ­ at ­ ti ­ en r and Pasteurella es es ­ fe ­ ni ­ ni ­ o ­ o s t t col ­ cie ­ en ­ her

Coccobacillus Coccobacillus Comments ad Adherent col Urease can dif Aggregatibacter Xylose

V — — Trehalose

(+) NT NT Sucrose

and Pasteurella spe + + + Melibiose

NT NT NT

Mannitol

Acid from:Acid Maltose

+ — Lactose

(+)

se ­ co Gas from glu from Gas ———— — + — —

Bacterial Diagnosis ONPG + + V — V + V V + V V — — — + — — + — + — V — + V — — — V Coccobacillus; star-shaped, NT NT

Aggregatibacter, Actinobacillus, Aggregatibacter, ndole I + + NT NT NT NT

cted

­sis ­ly ­dro ­ le hy Esculin

NT NT Urease

cs cs of se

­ ti Growth on MacConkey agar MacConkey on Growth ­ is r

­ te Catalase

­ ac + — — — Oxidase + + — — + V V + V + + — — V—V—— W –/ —V——— fferential char spp. Di

P. canis Species Aggregatibacter actinomycetemcomitans A. aphrophilus Actinobacillus multocida P. A. segnis Table 4.21 Bacterial Diagnosis 193 ic ic ­ ph ­ ph ­ mor ­ mor nts ­ me s ­ a ­ cie

spe Fusiform; pleo Fusiform; pleo Comments Strawberry-like odor Strawberry-like Typically formTypically very long fil Melibiose

NT NT NT Xylose — —

and Streptobacillus Sucrose

Acid from:Acid Lactose

+ V V + Glucose

NT NT ONPG

Bacterial Diagnosis

irt re Nitrate se ­ta duc ­ , and C. haemolytica Capnocytophaga, Dysgonomonas, Capnocytophaga, VV V + ­ sa ­ lo ­ u cted

­ le Indole —

cs cs of se ­ ti Catalase ­ is r ­ te

­ ac Oxidase + + ——— — — V — + + + + + + — — — — — + — — — and C. cynodegmi

) a b ­ ra Differential char )

­ ra n flo ­ ma Includes C. ochracea,Includes C. sputigena, C. gingivalis, C. gran Includes C.canimorsus Includes a b (hu Species Capnocytophaga Capnocytophaga D. capnocytophagoides D. S. moniliformis (dog and(dog cat flo Table 4.22

194 SECTION 4 Mannose

+ + — Lactose

— — —— Sucrose

——— Acid from:Acid NT Glucose

++ + — Catalase

W

+ NT

Indole

decarboxylase

Ornithine Ornithine Urease + — + ————V s NT NT NT

­ cie Bacterial Diagnosis Hemolysis a spe b

t:

r V) r ­ to (fac NAD +++ —+ + V — + ++ V V + — — V V — V + + V + — — + V — V — — + — + + — + + — — — — — — — + + + + + — — + + — — — + + —— ­ men Haemophilus ire ­ qu cs cs of ­ ti

­ is

r r X) r ­ to (fac Hemin ­ te + + + + — — — — — ­ ac Growth Growth re rs in some strains. ­ cu s oc ced on sheep and horse blood. ­ si ­ du Differential char ­ ly on. ­ ti

­ mo ­ ac Delayed he Hemolysis pro a b w, w, weak re H. ducreyi H. pittmaniae H. influenzae H. haemolyticusH. parahaemolyticusH. parainfluenzae H. paraphrohaemolyticusH. aegyptiusH. Species H. sputorum H. Table 4.23

Bacterial Diagnosis 195

Xylose Maltose

(continued)

Adonitol

Dulcitol Mannitol

Acid from:Acid

Sucrose

Lactose

Glucose

Motility

Ornithine decarboxylase Ornithine

Arginine dihydrolase Arginine

Lysine decarboxylase Lysine

se ­na ­i ­am Enterobacteriaceae de Phenylalanine Urease

rs of the

ion ­t ­za ­li irt uti Citrate

Bacterial Diagnosis ­ be Voges-Proskauer

++——+—++++++—+++ Methyl red Methyl cted mem

—

­ le

nt ­me pig Yellow

—

on ­ti ­duc cs cs of se pro Indole +++——————+++VV+——++ +—+————+—+++——+—+++ +—+————+VV+++V+V—++ +—+—+V——V+++VV+V+++ +—+——++——+++——————— +—+————+—+++—————+— +—V+++—+———++++V+++ +—+————+++++V—————— ­ ti V—V—VV——V—++V++——++ — —+—++—V—+++++++——++ ———++V——+++++++VV++ ——V+++—+———++++V+++ ­ is r ­ te ­ ac ex sakazakii ­ pl ) a e com ­ ca ­ a Differential char Enterobacter (

Escherichia hermannii Escherichia fergusonii Escherichia coli Cronobacter Enterobacter clo Citrobacter koseri Enterobacter aerogenes Morganella morganii Species Citrobacter freundii Edwardsiella tarda Klebsiella oxytoca Klebsiella Klebsiella pneumoniae Plesiomonas shigelloides Table 4.24

196 SECTION 4

Xylose

Maltose

Adonitol

Dulcitol Mannitol

Acid from:Acid

Sucrose

Lactose

Glucose

(continued) Motility

Ornithine decarboxylase Ornithine

Arginine dihydrolase Arginine

Lysine decarboxylase Lysine

se ­na ­i ­am Enterobacteriaceae de Phenylalanine Urease

rs of the

ion ­t ­za ­li irt uti Citrate

Bacterial Diagnosis ­ be

Voges-Proskauer Methyl red Methyl cted mem

­ le

nt ­me pig Yellow

on ­ti ­duc cs cs of se pro Indole +—+—+++———++—V+—+—— +—+—V++———++—+———++ +—+—+V+———V+—+————— ­ ti V—+——V———+—+—++——VV ——V————————+——+——V+ ——+++V—+—+++—++—V+— ——+—+——+V+++——++—++ ——+VV++——+++—V————+ ——+++——+—+++—++——++ ——+——————+—+——+——+— ­ is r ­ te ­ ac s s s ­ cie ­ li Differential char ­ ri ­ bi

­ ga spe ­ ra us vul ­ te ­ teus mi Providencia rettgeri Yersinia pestis Pro Salmonella Serratia marcescens Yersinia enterocolitica Table 4.24 Species Pro Providencia stuartii Providencia Serratia liquefaciens Serratia Shigella sonnei Bacterial Diagnosis 197 + — — — — — — — — — — Adonitol + + + V V V — — — — — Melibiose Acid from:Acid + + + V V — — — — — — Dulcitol + V V V V V — — — — — Sucrose r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th ­ te a s + + + + + — — — — — — ­ cie Bacterial Diagnosis Malonate spe b + + + + + + — — — — — Citrobacter ODC ll Funke KC, G, Landry ML, Rich ­ ro cs cs of ­ ti ­ is r + + + + ­ te V V V V — — — ­ ac Indole ­ to) n, D.C., 2015. n, D.C., Differential char ­ to g

­ thine decarboxylase. (sensu stric ­ in ­ ni . koseri ODC, or Adapted from Jorgensen JH, Pfaller MA, Car a b C C. gillenii C. youngae C. freundii C. C. farmeri C. werkmanii C. braakii C. sedlakii Species C. amalonaticus C. rodentium C. murliniae Press, Wash Table 4.25

198 SECTION 4

nt ­me a pig Yellow s —-

­ cie

spe Melibiose

Esculin

α de ­si ­co glu -Methyl Rhamnose

Acid from:Acid

Sorbitol

Adonitol Sucrose

r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th

­ te Voges-Proskauer

Bacterial Diagnosis Ornithine decarboxylase Ornithine Enterobacter, Pluralibacter, Cronobacter, Kosakonia, Cronobacter, Pluralibacter, Enterobacter, and Pantoea

ct Arginine dihydrolase Arginine ­ le ++++V++VV+—

ll Funke KC, G, Landry ML, Rich Lysine decarboxylase Lysine cs cs of se ­ ro +—+++++++++— +—+++——+—++— —V+++——+V——— —++++——+++++ ——V++——+V++ —++—+—+++V+— —V+—+—+—++—— ———VV—VV—VVV —+++———+—+—— — ———++—++—++V ­ ti ­ is r ­ te ­ ac ­ cae ­ a subsp. hormaechei subsp. n, D.C., 2015. n, D.C., Differential char biogroup 1 bsp. clo bsp. ­ to g

su ­ in ­ cae ­ a Adapted from Jorgensen JH, Pfaller MA, Car a C. sakazakii C. E. hormaechei E. amnigenus E. aerogenes Species E. kobei E. asburiae P. agglomeransP. E. cancerogenus E. clo K. cowanii Pluralibacter gergoviae Press, Wash Table 4.26

Bacterial Diagnosis 199 44°C + + + — — NT NT NT on.

­ ti Growth at: 10°C ­ ca ­ fi + + + ­ ti — — — NT NT m iden

­ fir ONPG + + + + + + V — ired to con ­ qu g is re ­ in

enc Malonate ­ qu + + + + + + + — a s ­ cie gene se r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., 11th

­ te Voges-Proskauer but rpoB + + + + + V — — Bacterial Diagnosis

and Raoultella spe Ornithine decarboxylase Ornithine + — — — — — — — Klebsiella ll Funke KC, G, Landry ML, Rich on that the strain is variicola K. ­ ro ­ ti cs cs of ­ ti ca ­

­ is ­ di

r ­ te Indole + + V — — — — — ­ ac on may be an in ­ ti ­ ac n, D.C., 2015. n, D.C., ­ to g ­ in Differential char b

­ tive adonitol re ­ a Adapted from Jorgensen JH, Pfaller MA, Car A neg a b Species K. oxytoca K. rhinoscleromatis R. ornithinolytica R. planticola R. terrigena K. ozaenae K. K. pneumoniae K. variicola ASM Press, Wash Table 4.27

200 SECTION 4

Inositol myo-

Trehalose D-Arabftol

Acid from:Acid D-Adonitol a s

­ cie

Maltose

decarboxylase

Ornithine Ornithine r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., 11th

­ te Urea

Bacterial Diagnosis

2 S us, Providencia, and Morganella spe H ­ te Pro

ll Funke KC, G, Landry ML, Rich Indole ­ ro cs cs of ++ — — — — — — — — + — — — — — — — + V + — + — — + — ++ — — + V — — — — + — + — — + + + + — + + — — — — — + V + — + — — — — V — + + — — — + — ­ ti — — — — V + + — V — + + + — — — + — — V + — + — — V — ­ is r ­ te ­ ac n, D.C., 2015. n, D.C., ­ to g ­ in Differential char s s ­ li

­ ri ­ bi ­ ga ­ ra organii sibonii subsp. organii morganii subsp. auseri enneri tuartii ettgeri vul mi ­ teus P. h P. alcalifaciens P. heimbachae P. rustigianii P. P. r P. P. s P. P. p P. M. m P. P. M. m Adapted from Jorgensen JH, Pfaller MA, Car a Organism Pro

Morganella

Providencia ASM Press, Wash Table 4.28

Bacterial Diagnosis 201 Sorbose

NT Melibiose

—— — Cellobiose + — + — + — NT

Acid from:Acid Rhamnose — + — + (+)

a Sucrose

—— (+)

decarboxylase

Ornithine

on at 25°C for 48 h for on at 25°C ­ ti Citrate + + + — + — + ­ ba r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th

­ cu

­ te r in Esculin ­ te (+)

es af Bacterial Diagnosis ­ ci Indole + — — + + + + + + —

spe

Proskauer

Voges- — — + — — — — — — NT Yersinia ll Funke KC, G, Landry ML, Rich

cs cs of ­ ro ­ ti Urease le. ­ is + — — + — — — r —- ­ ab ­ i ­ te

­ ac Motility ++++++++++—+ + ++VV——++—+—+ ++++————++—+—— + ++———+++—++—+++——++—+——— +++++++++++++++————+——+—— —+ — +—+——+++—+—— + — + + + + ++——++——+———+ + V—————+————— ve; V, var ve; V, — —+———————+—— ­ ti ­ i ­ sis ­ lo n, D.C., 2015. n, D.C., ­ cu Differential char r ; (+), weak; (+), pos ­ to g

­ be ­ ed ­ in ­ tu s ­ do ­ li ­ mi Adapted from Jorgensen JH, Pfaller MA, Car a notNT, test Y. Y. pseu Y. frederikseniiY. Species Y. pestis kristensenii Y. Y. mollaretii aleksiciae Y. pekkanenii Y. massiliensisY. Y. enterocoliticaY. Y. bercovieriY. Y. rohdei aldovae Y. intermediaY. Y. nurmii Y. ruckeriY. Y. Y. si Y. entomophagaY. Press, Wash Table 4.29

202 SECTION 4

nce ­ta s ­si re Ampicillin

Cellobiose

Sucrose Lactose

Acid from:Acid

— — — + Rhamnose Manual of Clinical Microbiology, ed., ASM 11th

—

Gas from glu from Gas se ­ co Lipase (corn oil) test oil) (corn Lipase + + — — — V + ­ ter SS, Warnock (ed.), DW

a

s ­ cie Indole Bacterial Diagnosis + V — — V + + V + — V — — V + — ++

spe Voges-Proskauer + + + + V V + — + + + + + — — + V + + V + + V V — + V + V + + V — + + V + V V + — — — V V — — V + + + — — + + V V — V V + Aeromonas cs cs of ­ roll Funke KC, G, Landry ML, Rich ­ ti ­ is r ­ te ­ dia ­ ac Species A. hydrophila A. bestiarum A. salmonicida A. caviae A. me A. eucrenophila A. veronii A. jandaei A. A. schubertii A. trota n, D.C., 2015. Differential char Complex ­ to g

­ in e. Complex ­ abl ­ i Adapted from Jorgensen JH, Pfaller MA, Car a V, V, var Complex A. hydrophila A. caviae A. veronii Complex Table 4.30 Press, Wash

Bacterial Diagnosis 203

decarboxylase

Ornithine

Lysine decarboxylase Lysine

Arginine dihydrolase Arginine

fer on n ­ti ­ta ­me

Inositol myo-

ONPG

Motility Indole

r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th

­ te Oxidase a Bacterial Diagnosis s

­ cie

+ 6% NaCl 6% +

Nutrient broth broth Nutrient + + — + V — + — — + + + — — — — + — + + — V V — + — — + +++ — + + + V + + + + + — — — — — V — — — + + V + + + — V — — + V — V +V + — + — + — + — V — — — — + V

Vibrio spe + 0% NaCl 0% + ll Funke KC, G, Landry ML, Rich

Growth in: ­ ro cs cs of

Nutrient broth broth Nutrient + V + + + + — — + + + V + + + + — — + + — — — — — — — — — —V—VVVVVV— ­ ti ­ is r ­ te ­ ac damsela ) ) hollisae Vibrio fferential char ( n, D.C., 2015. n, D.C., Di ­ to Vibrio g

( ­ in e. ­ abl ­ i Adapted from Jorgensen JH, Pfaller MA, Car a V, var V. furnissii V. V. mimicus V. Grimontia V. harveyi V. fluvialis Species choleraeV. melschnikoviiV. cincinnatiensis V. Photobacterium alginolyticus V. parahaemolyticus V. V. vulnificus Press, Wash Table 4.31

204 SECTION 4 Xylose

+ Mannitol

V + Maltose

Acid from:Acid NT NT —- Glucose

+ Starch

NT Gelatin

— Esculin + V — + + V + — + — + V V — ——————— ——————V — V — + + + + NT

Hydrolysis of: Hydrolysis Acetamide — NT NT NT NT NT r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th ­ te

a

s Cetrimide growth Cetrimide ­ cie V NT

spe Bacterial Diagnosis

Arginine dihydrolase Arginine Nitrate to gas to Nitrate

Pseudomonas

irt re Nitrate se ­ta duc ­ ll Funke KC, G, Landry ML, Rich ——— ­ ro cs cs of ­ ti

­ is

r ­ te 42°C at Growth

+ V — + — V

­ ac Oxidase +V++—————+++++ + — — — + + +———++————+——— + — + + + +———+V————+VV+ + V V — — V +++++V————+——V+ + + — V V +—V—++——+—+—V+ +++++++—V—+ — — . ­ ed n, D.C., 2015. n, D.C., Differential char ­ to g

­ in le; not NT, test ­ ab ­ i Adapted from Jorgensen JH, Pfaller MA, Car V, var V, a P. stutzeriP. P. mosselii P. montelliiP. P. veroniiP. P. putida P. P. luteola P. P. oryzihabitansP. P. alcaligenes P. P. pseudoalcaligenes P. P. mendocina P. fluorescens Species aeruginosaP. Press, Wash Table 4.32 Bacterial Diagnosis 205 a s ­ cie a odor ­ ni spe n, D.C., 2003.n, D.C., ­ mo ­ to g -tan -orange ­ in nt nt es; am ­ low ­ low ­ ni ­ me ­ me ­ o es ­ ni pig pig ­ o ­ ble ­ ble ­ u ­ u and Stenotrophomonas

Brown-tan col Yellow, Yellow, sol Colonies may be yel Lavendar-green col Yellow Yellow sol Colonies may be yel Comments

Xylose

Mannitol Maltose

Acid from:Acid

Glucose

Urea

Gelatin Citrate

Bacterial Diagnosis Hydrolysis of: Hydrolysis

Lysine decarboxylase Lysine Arginine dihydrolase Arginine

Acidovorax, Brevundimonas, Delftia, Comamonas, Brevundimonas, Acidovorax, Nitrate to gas to Nitrate

cs cs of on ­ti ­duc re Nitrate ­ ti ­ is r

­ te 42°C

­ ac MacConkey

Growth: Oxidase ++ V—————V—V——— +——+—+——+++—+++++++————V+—V—++V+———+————+— ++ V +———V—————— ++V+—+—+—++—V++VV—————V—V+—V V+VV——+V+—V+—V y PR, Baron EJ, Jorgensen JH, Pfaller MA, RH Yolken (ed.), Manual of Clinical Microbiology, 8th ed., ASM Press, Wash ­ ra Differential char spp.

Adapted from Mur a B. diminuta A. facillis A. temperans D. acidovorans Species A. delafieldii B. vesicularis Comamonas S. maltophilia Table 4.33

206 SECTION 4 MacConkey agar MacConkey

- - + + + +

­bi ­l agar a um Co + + +

Growth on: RL Medium RL NT NT NT

+ (1 day)+ (1 + + (3 days)+ (3 - + (2 days) V Pigment low - - - - own Br Yel s

­ cie Motility spe - - - + + V Bacterial Diagnosis

Bordetella Catalase + + + + + + cted ­ le

cs cs of se ­ ti Oxidase - - - + + + ­ is r ­ te ­ ac s ­ si s ­ tu Differential char

Organism Bordetella per Bordetella parapertussis Bordetella holmesii Bordetella bronchiseptica trematum Bordetella Bordetella ansorpii Table 4.34

Bacterial Diagnosis 207

OF-xylose OF-maltose

—— OF-mannitol

NT OF-sucrose

— Acid from:Acid OF-lactose

NT

D-Glucose

se ­ta ­duc re itrate N — + ve, ve, Gram-negative rods

­ ti ­sis ­ly ­dro hy Esculin

­ da NT ­ i

on ­ti ­duc noe pro Indole Urease —— se-negative, ox Bacterial Diagnosis

­ da ­ i Lysine decarboxylase Lysine NT

cted ox ­ le dihydrolase Arginine

— Motility ics of se ­ t NT ­ is r

­ te

Catalase

­ ac Growth on MacConkey agar MacConkey on Growth ++—V—V———+V——V++++—V—+V+VV+++++V——+———V——V—VV+V————+—+++—+++++—+V—VV+VV—+V +++V—V———+VV+++— + es Differential char ­ ci

Genus spe or Acinetobacter Chryseomonas Roseomonas Sphingomonas Stenotrophomonas Flavimonas Francisella Table 4.35

208 SECTION 4

Xylose

Mannitol

l ­co tyee gly Ethylene

Acid from:Acid Glucose

- + - - - + - - + + V +

nt ­ta s ­si oitn re Colistin

NT NT NT Indole

------+ ------V--

2 S (On KIA) (On S H

NT NT NT NT NT NT NT

se ­ta a ­ph phos Alkaline

se ­ta ­duc re Nitrite

se ­ta ­duc irt re Nitrate

- + - + + - + - - + + - + -+ - - V V-V Esculin

NT NT NT NT NT NT NT

Ur ease - - - - se-positive, se-positive, nonfermentative, Gram-negative rods + Bacterial Diagnosis

­ da

­ i

­gy ­o ol ­ph mor stain Gram C + C- CB CB CB CB CB

cted ox nt ­me lxrbn pig Flexirubin ­ le

NT NT NT NT NT NT NT Motility cs cs of se ­ ti

­ is r agar MacConkey on Growth ------V + - V- V- ­ te ­ ac fferential char Di

Organism catarrhalis Moraxella Moraxella nonliquefaciens Moraxella osloensis Moraxella lacunata ureolyticaOligella urethralisOligella Paracoccus yeei Table 4.36Table Bacterial Diagnosis 209 ++ + + + + - + - + - - + - ++ - - + - - - - - + + - - - - + + + - - + NTNT NT NT NT NT NT NT NT NT NT NT Bacterial Diagnosis . ­ ed le; not NT, test ­ ab ­ i - ---B---- B + - - - - - B - + - V + - B - - + - V-B--V-+--VV+-V s; V, var s; V, - - - - - +V-BV------++-++++-B+-++----++V+++-B+-+V---V++++++-B-+++++-V-+--++-B-+++++--V+-- V-+B++-++--+----VV - - V - B B - - + + V - V V ­ lu l ­ ci spp. ­ gae ­ coid; CB, coccobacillus; ba B, C, coc Inquilinus limosus Inquilinus odoratus Myroides Ochrabactrum anthropi radiobacter Rhizobium Shewanella al Shewanella putrefaciens Sphingomonas Balneatrix alpica Bergeyella zoohelcum Chryseobacterium indologenes meningoseptica Elizabethkingia Weeksella virosa Sphingobacterium mizutaii

210 SECTION 4 OF-xylose

— OF-mannitol NT

Acid from:Acid Glucose

—

2

H S on TSI on S a

NT

Nitrate to gas to Nitrate

se ­ta ­duc re Nitrate

on ­ti ­duc noe pro Indole

Urease

Peiitrichous

r ­la po >2

se-positive, se-positive, nonoxidative, Gram-negative rods Flagella

Bacterial Diagnosis

­la r

­ da po 1–2 ­ i Motility cted ox

­ le Catalase + — — — — — — — — +——————V—————

cs cs of se ­ ti agar SS —+++————+————— —+++——V—V——V— + ­ is r

­ te ­ ac agar MacConkey ++++——+————————++++——+——++————++++——++—+—————+—+———————————— V—+————+—+V—+— + V V V—V++——+—+———— + —— —— Growth on: ­ cies ­ cies s ­ si spe s s spe ­ tu Differential char ­ cie

spe Brucella Bordetella per Species felts Aflpia faecalisAlcaligenes xylosoxidansAlcaligenes Bordetella bronchiseptica Campylobacter Methylobacterium Moraxella atlantae catarrhalis Moraxella Table 4.37 Bacterial Diagnosis 211 —- ——— NT ++ — V — — — + — — — Bacterial Diagnosis +————————————+————++——————+—————+—————— —+——————++—+—— —+++——+—V——VV V ++++——++—++V+V++—+————————————+—+++——V————V—— + V—V—————————+—— V V—+———————————— V—+——————V—————V—+————+—V—————V—+V——V+—+V———— —— —————————————————————— ——+———————— s ­ cies ­ sa ­ cie ­ co spe spe ­ ple sugar iron. TSI, tri a Neisseria sicca Neisseria mu Bartonella Neisseria flavescens Moraxella osloensis Moraxella lacunata Moraxella phenylpyruvica Ochrobactrum anthropi ureolyticaOligella urethralisOligella diminuta Pseudomonas Roseomonas corrodens Eikenella denitrificans Kingella Weeksella virosa Weeksella zoohelcum

212 SECTION 4 1% Glycine 1%

+ 42°C + — — + —— — + NT

Growth: 25°C + V V + — V + — + — + + — + V — + + — + + — s NT NT NT

­ cie

ϒ se ­a er ­f spe trans -Glutamyl

NT NT NT NT NT NT NT NT NT NT

s ­si ­ly ­dro hy ate ­t ­e ac Indoxyl

iprt hy Hippurate s ­si ­ly o dr and Helicobacter ­

+ + V + — + — + — + — + —— ——

se ­ta a ­ph laie phos Alkaline

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT Urease + V — — — — — — —

Bacterial Diagnosis

se ­ta ­duc irt re Nitrate — + + — — + Campylobacter, Arcobacter, Campylobacter, NT NT NT NT NT NT NT NT

cted ­ le Catalase + + — — + — + — + + — — — — + + + + + + — — — — — V V V — NT NT cs cs of se ­ ti ­ is r ­ te ­ ac s C. lari concheus subsp. ­ tu / Differential char

m subsp. jejuni subsp. subsp. doylei subsp. ­ ru ­ ri subsp. fe s subsp. ­ lo ­ lo ­ tu . jejuni . jejuni Species C C. upsaliensis H. fennelliae H. heilmannii C H. pul A. butzleri C. coli A. cryaerophilus C. fe H. py C. lari lari subsp. H. cinaedi H. Table 4.38 Bacterial Diagnosis 213 a nic ­ ci ; S, suc ts (GLC) ­ served. ­ id c ­ uc ­ tic a ­ larly ob ­ u L, (a), (s) A, L s, 1 iv, A, P, S, L, a S, L, a S, L, a S, L, a S, L, a A S, A, (L) A, P, iv, s, 1 iv, A, P, s, 1 iv, A, P, (A), (B) A, P, iv, s, 1 iv, A, P, s, 1 iv, A, P, (A, L,(A, S) L,(A, S) ­ reg ; L, lac ­ id oic ac Metabolic prod ­ ids are ir ­ pr on ; C, ca ­ ti ­ id ­ duc cate that the ac + — — — — — — — — — — — — — — — — ­ di ­ ­ ses in ­ the Indole pro ; IC, isocaproic ac ­ ren ­ id c c, c, Gram-positive rods se ­ bi ­ o ­ ric a ­ ta r ­ le ­ ters in pa ­ ae ­ duc + + + + + + V V V — — — — — — — — ; V, va ; V, ­ id Nitrate re ­ nor peak, and let Bacterial Diagnosis + + + + + + V ; IV, isovaleric; IV, ac — — — — — — — — — — cate a mi ­ id ­ di ­ Catalase ric ac ct non-spore-forming, an ­ ty ­ ters in e ­ le ­ ob ; B, bu; B, ­ aer ­ case let ­ id + + + + + + + ­ er V V V — — — — — — — cs cs of se ­ ti ­ is r Strict an ­ te ­ ac ; IB, isobutyric ac ­ jor acid peak, low ­ id s ic ac ­ ne ­ on cate a ma ­ pi ­ di ­ fferential char spp. Di ­ ters in id; P, pro ­ id; P,

­ tic ac A, ace a Mobiluncus Eubacterium Actinomyces neuii Neuii subsp. Actinomyces turicensis Genus Actinomyces spp. Actinomyces israelii odontolyticus Actinomyces Actinobaculum Bifidobacterium Lactobacillus Propionibacterium acidifaciens Propionibacterium propionicum Propionibacterium ac Propionibacterium avidum Eggerthella lenta Propionibacterium granulosum Eggerthella sinensis Table 4.39 acid. Capital let

214 SECTION 4

s (GLC) ts ­uc prod Metabolic Metabolic

A, IB, B, IV, (P),A, (V), IB, IV, B, (IC), PP A, B, IV, ib (p, ic, l, s) ic, (p, ib A, IV, B, A, (PP) A, IB, B, IV, (P),A, (V), IB, IV, B, (IC), PP

Sucrose

Lactose Maltose

Acid from:Acid Glucose + + + + A, L, (PY) + + + + A, B + + + + A, (L), B, (PP) s

­ cie Indole

+ + + — — A, IC, (P), (IB), (IV) + + + — — A, IC, (P), (TB), (IV), PP + + + + — A, F ————— — — — — + + — + A, B — + + — — — + + — — (V) B, A, P, — + — — — IC, (P), PP V, A, IB, IV, B, — + + + + A, (L) B, — + + + — A, B — + + + + A, (P), B, (L) — +————— + — —

spe Spore Location Spore

T

ST ST ST ST ST ST ST ST ST ST ST ST ST ST ST Bacterial Diagnosis Clostridium digestión Milk cted

­ le Gelatinase + — T + — — — — (PP) B, A, P, + — + — + +

cs cs of se ­ ti Lipase + + + ­ is ——— r ­ te

­ ac Lecithinase + — + + + — + + + + + — + — — — + — + — ———— ———— —— — —— —— — —————— — + + V T — + V — — B A, P, — + + + Egg yolk agar Differential char A, F B, B, E, B, F — + + — C and D

um um um, ­ n le ­ n ­ n i i i ­ l ­ l ­ l ­ um ­ ci ­ u ­ u ­ u ­ ti ­ fi C. bot C. bot C. bot C. subterminale — — + + C. sordellii C. ramosum C. C. bifermentans C. butyricum C. tetani C. novyi A C. dif C. ter C. baratii C. septicum Species C. perfringens C. sporogenes C. histolyticum C. sphenoides Table 4.40

Bacterial Diagnosis 215

­gy ­o ol ­ph mor Cell l ­ ra

Cocci Long, thin, pointed ends Straight Coccoid Short Short Variable Short Curved Pleomorphic Long, thin, pointed ends Long, spi Coccobacillary

imn pro Pigment on ­ti c ­du

Catalase Indole s.

­ nie

­ o

irt re Nitrate se ­ta duc ia ­ ­ r ­ te nted col

­ me Motility lop pig

­ ve Growth in 20% bile 20% in Growth Bacterial Diagnosis

c c Gram-negative bac μ g) (10 Colistin ­ bi ­ o sce red and then de

­ aer ­ re

μ g) (5 Vancomycin ally fluo cs cs of an

Susceptibility to: ­ ti

­ ti

­ is μ g) (1,000 anamycin K S R S — — — + — — S R S — — + — V — S R S + — + — + — SRV—————— SSRS————V— RS S R V V — V — + + — — — — — — RRRV——VVV RR RRRV———V—VR S R R R + R — —R + — — S — — V — R V — — V V — V + + — V — V + ­ ter spp. ini ­ ac fferential fferential char a and some Prevotella Di

a spp. Porphyromonas a Fusobacterium nucleatum Veilonella Alistipes Prevotella Bilophila Dialister Species Bacteroides Porphyromonas Parabacteroides Fusobacterium necrophorum Leptotrichia Anaerospirillium Desulfovibrio Table 4.41

216 SECTION 4 Xylose

- + + + + + + + + + + Trehalose ------+ + W NT

Acid from:Acid Arabinose

- - - - + + + + + + W

α -Fucosidase - - - + + + + + + + W group

Bacterial Diagnosis Catalase ------+ NT NT NT Bacteroides fragilis cs cs of the

­ ti ­ is Indole r ------+ + + + NT ­ te ­ ac Differential char

Species B. caccae B. cellulosilyticus B. coprocola B. coprophilus B. finegoldii B. dorei B. fluxus B. fragilis B. clarus B. faecis B. eggerthii Table 4.42 Bacterial Diagnosis 217 - + + + + + + + + + + + ------+ + + + - - - + + + + + + + + + - - - + + + + + + + + V Bacterial Diagnosis ------+ + + V NT - - - - + + + + + + + + . ­ ed on; not NT, test ­ ti ­ ac W, weak re W, B. intestinalis B. B. massiliensis B. oleiciplenus thetaiotaomicron B. B. xylanisolvens B. ovatus B. plebeius B. uniformis B. nordii B. vulgatus B. stercoris B. salyersiae

SECTION 5 Viral Diagnosis

Ge­ne­ral Comments 220 Table 5.1 Detection Methods for Viruses 221 Table 5.2 Cells Used for Viral Isolation 224 RNA Viruses 225 DNA Viruses 232 Transmissible Spongiform Encephalopathies 239

doi:10.1128/9781683670070.ch5 220 SECTION 5 Historically, vi­ral in­fec­tions have been di­ag­nosed by cul­ture in pri­ mary or con­tin­u­ous cell lines and by de­tec­tion of an­ti­body re­sponses to in­fec­tion. However, these meth­ods are in­sen­si­tive and slow, such that re­sults are rarely avail­­able in a clin­i­cally rel­e­vant time frame. Molecular meth­ods or nu­cleic acid am­pli­fi­ca­tion test­ing (NAAT) have re­placed cul­ture due to their su­pe­rior sen­si­tiv­ity and more rapid turn­around time. In ad­di­tion, an­ti­gen cap­ture im­mu­no­as­says, which al­low for the di­rect de­tec­tion of vi­ruses in clin­i­cal spec­i­mens, have also been widely adopted. Despite these tech­no­log­i­cal ad­vances, se­ rol­ogy re­mains an im­por­tant di­ag­nos­tic tool for many vi­ral in­fec­ tions. Initially, many se­ro­log­i­cal tests were con­ducted with the la­bor-intensive com­ple­ment fix­a­tion and neu­tral­i­za­tion tests. How­ ever, those meth­ods have largely been re­placed by com­mer­cially pre­ pared en­zyme im­mu­no­as­says. This tech­ni­cal change al­lows many smaller lab­o­ra­to­ries to per­form tests that were pre­vi­ously avail­­able only in ref­er­ence lab­o­ra­to­ries. This sec­tion sum­ma­rizes the tests cur­ rently avail­­able for the lab­o­ra­tory di­ag­no­sis of the most com­mon vi­ ral in­fec­tions. For ad­di­tional in­for­ma­tion, the reader is re­ferred to the ASM Manual of Clinical Microbiology and Clinical Virology Man- ual by Specter et al. (see the Bibliography). Viral Diagnosis Viral Viral Diagnosis 221 cs ­ ti s ­ no g (continued) ­ a B C C B B B B B B A A A A A A A A A A Molecular di n n ­ tio ­ tec B C B B C A A A A A A A D A A A A A A ds ­ o on by: ­ ti c meth Antibody Antibody de ­ te n n ­ tio ­ tec C C C C C C C A D D D D D D D D D A A ds ­ o Usefulness of de meth Antigen Antigen de C C C C C B D D D D D D D D D D D D D Microscopy re Viral Diagnosis Viral ­ tu C C C C B C C C C B B D D A D D D A A a ­ es s Cell cul ­ ru s for vi ­ rus ­ vi ­ od I ­ rus ­ rus ­ rus I tection meth s - s II - ­ ru De s ­ ru ­ e and nd nd

I us - ­ r I a I - b TLV epatitis C vi C epatitis epatitis E vi E epatitis nfluenzavirus types A to C Arenavirus Alphavirus Bunyaviruses Astrovirus, calicivirus, ro ­ ta Hantavirus Coronavirus Coxsackievirus types A and B Enterovirus Flavivirus vi A Hepatitis H Filoviruses H H HIV I Parainfluenzavirus types 1 to 4 Measles vi Mumps vi RNA vi Virus Table 5.1 222 SECTION 5 cs ­ ti s ­ no g ­ a B B B B A A A A A Molecular di n n ­ tio ­ tec B C C C D A A A A ds ­ o on by: ­ ti c meth Antibody Antibody de ­ te n n ­ tio ­ tec C B C D D A D A D ds ­ o Usefulness of de meth Antigen Antigen de C C C B B C A D D Microscopy re Viral Diagnosis Viral ­ tu C B B C A A A A D (continued) a ­ es s Cell cul ­ ru s for vi ­ od s ­ ru al vi ­ ti ­ cy ­ rus tection meth s s rr vims ­ ru De s ­ ru ­ e Ba

- us in ­ r b ­ ste Respiratory syn Poliovirus Rabies vi Rhinovirus Rubella vi Adenovirus Cytomegalovirus Ep Hepatitis B vi B Hepatitis DNA DNA vi Virus Table 5.1 Viral Diagnosis 223 s; ­ rie ­ to ­ ra ­ o C B D D A A A D A A ce lab ­ en ­ er le in ref ­­ ab B B B B D D D D A A s but may be avail ­ si ­ no ­ ag al di ­ er B C D D D D D D D D m used gen for ­ do s. ­ ru C C C C C C B C A D ; C, test is sel ­ es ncy vi anc ­ st ­ cie ­ fi m ­ de ­ cu Viral Diagnosis Viral ­ no in cir ­ mu C B B D A D D D ­ ta n im s. r cer ­ si ­ ma ­ de ­ no ­ ag l un ­ fu s) D ry di ­ ru ­ to s; HIV, hu ­ ra ­ ru ­ o

a vi s, vi JC ­ mi ­ ru hy hy agent l; test B, is use ­ ke s types I and II A form ­ form ­ t ­ a ­ fu s 6 ­ ru p ­ ru ll leu ­ lo ob agent ­ a ­ vi ce ly usely s h - ex viex y y agents ster vims ­ al Jak ly ly not used for lab ­ pl ­ pe - ­ th ­ er ­ cep ­ al zo ­ a dt - ­ er p ­ man T ­ fel ­ lo ­ a h ­ cep Kuru agent Varicella Creutz Bovine Bovine en Polyomavirus vi (BK Herpes sim Papillomavirus Parvovirus (B19) Orthopoxvirus Human her A, test is gen HTLV, HTLV, hu a b en Transmissible ­ gi spon D, D, test is gen 224 SECTION 5 Table 5.2 Cells used for vi­ral iso­la­tiona Type of cell Tissue of or­i­gin Viruses iso­lat­edb Primary cell lines Af­ri­can green Kidney HSV, VZV, mumps vi­rus, mon­key ru­bella vi­rus CBMC, Human HIV-1, HIV-2, HTLV-1, PBMCc HTLV-2, HHV-6 Neonatal, Kidney HSV, VZV, ad­e­no­vi­ruses, hu­man mumps vi­rus Rabbit Kidney HSV Rhesus or Kidney Enteroviruses, in­flu­enza vi­ruses, cy­no­mol­gus para­in­flu­enza vi­ruses, RSV, mon­key mumps vims, mea­sles vi­rus Low-passage/finite cell lines Foreskin Human HSV, CMV fi­bro­blasts Lung Human em­bryo HSV, CMV, VZV, rhi­no­vi­ fi­bro­blasts ruses, co­ro­na­vi­rus Kidney Human fe­tus Coronavirus, HSV, rhi­no­vi­rus fi­bro­blasts WI-38, Human fe­tal lung HSV, VZV, CMV, ad­e­no­vi­ MRC-5 ruses, en­tero­vi­ruses, RSV, rhi­no­vi­rus­es Continuous cell lines 293 Human kid­ney Adenoviruses (types 5, 40, and 41) A549 Human lung Adenoviruses (ex­cept types 40 and 41), HSV HeLa Human cer­vix Poxviruses, RSV, rhi­no­vi­ruses, en­tero­vi­rus­es HEp-2 Human lar­ynx Adenoviruses, RSV, mea­sles Viral Diagnosis Viral vi­rus MDCK Canine kid­ney Influenza vi­ruses, para­in­flu­ enza vi­rus­es Mink lung Mink lung HSV RD Human Enteroviruses (coxsackievirus rhab­do­myo­sar­co­ma type A), co­ro­na­vi­rus, po­lio­vi­rus

RKI13 Rabbit kid­ney Rubella vi­rus, pox­vi­rus­es BGMK, Af­ri­can green HSV, VZV, en­tero­vi­ruses, Vero, CV-1 mon­key kid­ney mea­sles vi­rus, pox­vi­ruses, ru­bella vi­rus, RSV, para­in­flu­ enza vi­rus­es aAdapted from Jorgensen JH, Pfaller MA, Car­roll KC, Funke G, Landry ML, Rich­ter SS, Warnock DW (ed.), Manual of Clinical Microbiology, 11th ed., ASM Press, Wash­ing­ton, D.C., 2015. bCMV, cy­to­meg­a­lo­vi­rus; HHV, hu­man her­pes­vi­rus; HIV, hu­man im­mu­no­de­fi­ ciency vi­rus; HSV, her­pes sim­plex vi­rus; HTLV, hu­man T-cell leu­ke­mia vi­rus; RSV, re­spi­ra­tory syn­cy­tial vi­rus; VZV, var­i­cel­la-zoster vi­rus. cCBMC, cord blood mono­nu­clear cells; PBMC, pe­riph­eral blood mono­nu­clear cells. Viral Diagnosis 225 RNA Viruses Alphavirus (Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Ven­e­zu­e­lan Equine Encephalitis ­Virus, Chikungunya Virus). Viruses grow in a va­ri­ety of cell lines in­clud­ing Vero, A549, and MRC-5 cells. Virus can be found in blood at the time of clin­i­cal on­set but is typ­i­cally cleared when neu­ro­log­i­cal symp­toms de­velop. Additionally, an­ti­gen de­tec­tion as­says and re­verse tran­scrip­tase PCR (RT-PCR) tests have been de­vel­oped for some mem­bers of this group (e.g., Ven­e­zu­e­lan equine en­ceph­a­li­tis vi­rus, Chikungunya). Of note, PCR as­says quickly be­ come neg­a­tive shortly af­ter symp­toms de­vel­op; thus, neg­a­tive re­sults for pa­tients who have been symp­tom­atic for >7 days do not rule out­ dis­ease. The most sen­si­tive se­ro­log­i­cal as­says de­tect vi­rus-specific im­mu­no­glob­u­lin M (IgM) an­ti­bod­ies by cap­ture en­zyme-linked im­ mu­no­sor­bent as­say (ELISA). Some se­ro­log­i­cal cross-re­ac­tiv­ity has been ob­served be­tween Chikungunya and the closely re­lated Dengue vi­rus. IgM an­ti­bod­ies are de­tected in se­rum and ce­re­bro­spi­nal fluid (CSF) within the first 7 to 10 days of clin­i­cal ill­ness. Because IgM an­ti­bod­ies may per­sist for months, se­ro­con­ver­sion should be dem­on­ strated. Virus-specific as­says are avail­­able for Chikungunya, West­ ern, and Ven­e­zu­e­lan equine en­ceph­a­li­tis vi­rus. However, due to a high de­gree of cross-re­ac­tiv­ity, pos­i­tive re­sults should be con­firmed with neu­tral­i­za­tion as­says.

Arenavirus (Lymphocytic Choriomeningitis Virus, Lassa ­Virus, Ju­nin Virus, Machupo Virus). Arenaviruses are a fam­ily of 43 named vi­ruses di­vided into two groups: Old World com­plex (e.g., lym­pho­cytic cho­rio­men­in­gi­tis [LCM] vi­rus, Lassa vi­rus) and New World or Tacaribe com­plex (e.g., Ju­nin vi­rus, Machupo vi­rus).

Indirect fluo­res­cent-antibody (IFA) tests have been used with pe­ Diagnosis Viral riph­eral blood and urine sed­i­ment for de­tec­tion of Ju­nin vi­rus. An­ tigen cap­ture ELISAs have also been de­vel­oped for de­tec­tion of Lassa vi­rus an­ti­gens in blood as well as for the iden­ti­fi­ca­tion of ­vi­rus grown in cell cul­tures. The RT-PCR as­say is a use­ful test for the rapid, de­fin­i­tive di­ag­no­sis of LCM and Lassa vi­rus in­fec­tions. The test sen­si­tiv­ity is ap­prox­i­ma­tely 80% for Lassa vi­rus; this re­ flects the ge­netic var­i­a­tion among Lassa vi­rus strains. Cell cul­ture is a rel­a­tively sen­si­tive di­ag­nos­tic method for Lassa and re­lated vi­rus­es; how­ever, PCR has now re­placed it as the most sen­si­tive method. The vi­ruses grow in Vero cells (and other cell lines), with vi­ral an­ti­gens de­tected by im­mu­no­flu­o­res­cent-antibody (IFA) stain­ing of in­oc­u­ lated cell cul­tures or ELISA. LCM vi­rus also grows in cell cul­ture, but in­tra­cra­nial in­oc­u­la­tion of wean­ling mice is a more sen­si­tive di­ ag­nos­tic pro­ce­dure. ELISA can be com­bined with se­ro­logic test­ing (ELISA) for IgM and IgG for a sen­si­tive and rapid de­tec­tion of Lassa vi­rus in­fec­tion. The di­ag­no­sis of in­fec­tion can be con­firmed for most pa­tients at the time of clin­i­cal on­set. ELISAs have also been 226 SECTION 5 de­vel­oped for de­tect­ing an­ti­body re­sponses to other ar­e­na­vi­ruses and have re­placed neu­tral­i­za­tion and IFA se­ro­logic tests. RT-PCR as­says fol­lowed by ge­nome anal­y­sis are re­plac­ing the above-men­ tioned di­ag­nos­tic meth­ods and are ad­van­ta­geous be­cause it in­ac­ti­ vates the vi­rus, which would oth­er­wise re­quire BSL4 lab­o­ra­tory fa­cil­i­ties. Astrovirus, Calicivirus, and Rotavirus. Eight se­ro­types of astro­ viruses have been iden­ti­fied, with se­ro­type 1 be­ing the most com­ mon hu­man path­o­gens, but all­ eight have been re­ported to cause hu­man in­fec­tion. Caliciviruses are sub­di­vided into five gen­era with Norovirus and Sapovirus be­ing re­spon­si­ble for the ma­jor­ity of hu­man in­fec­tions. Rotaviruses have been sub­di­vided into seven an­ti­ genic groups A to G, with most hu­man in­fec­tions caused by group A. All of these vi­ruses can be de­tected in stool spec­i­mens by elec­tron mi­cros­copy. Only ro­ta­vi­ruses can be iso­lated in cell cul­ture; how­ ever, growth is slow and cul­ture is not gen­er­ally per­formed. RT- PCR is a sen­si­tive method for de­tec­tion of these vi­ruses, al­though in­hib­i­tors in fe­cal spec­i­mens can cause false-negative re­ac­tions. A wide range of ELISAs and la­tex ag­glu­ti­na­tion tests have been de­vel­ oped for the de­tec­tion of ro­ta­vi­ruses in fe­cal spec­i­mens, and an ELISA is com­mer­cially avail­­able for astroviruses. Some stand- alone PCR as­says for the de­tec­tion of Norovirus are now com­mer­ cially avail­­able. In ad­di­tion, some mul­ti­plex-PCR as­says in­clude Norovirus, Sapovirus, and Rotavirus. Bunyavirus (Bunyamwera Virus, Cal­i­for­nia Encephalitis ­Virus, La Crosse Virus, Hartland Virus, Rift Valley fe­ver, Cri­ mean Congo hem­or­rhagic fe­ver). Virus can grow in Vero and BHK-21 cell lines; how­ever, at­tempts to iso­late vi­rus from clin­i­cal spec­i­mens are gen­er­ally un­suc­cess­ful. Serologic test­ing (e.g., neu­ Viral Diagnosis Viral tral­i­za­tion, hem­ag­glu­ti­na­tion in­hi­bi­tion [HI], com­ple­ment fix­a­tion [CF], and ELISA) is pri­mar­ily used to es­tab­lish in­fec­tion. Most pa­ tients are se­ro­pos­i­tive by IgM ELISA at the time of on­set of ill­ ness. Neutralizing an­ti­bod­ies are de­tected at the end of the first week of ill­ness and per­sist for life. In con­trast, HI an­ti­bod­ies are de­tected at the end of the first week, and CF an­ti­bod­ies de­velop a few weeks lat­er; both an­ti­bod­ies dis­ap­pear within 1 year. Coronavirus. Although co­ro­na­vi­ruses are rec­og­nized as a com­mon source of up­per re­spi­ra­tory dis­ease, in­ter­est in this group of vi­ruses has been stim­u­lated by the on­set of se­vere acute re­spi­ra­tory syn­ drome (SARS) and more re­cently, Middle Eastern re­spi­ra­tory syn­ drome (MERS). The vi­ruses are dif­fi­cult to grow in cul­ture, so di­ag­no­sis has pri­mar­ily de­pended on RT-PCR and se­ro­logic test­ing. Historically, RT-PCR has only been avail­­able through pub­lic health lab­o­ra­to­ries, ter­tia­ry-care cen­ters, and com­mer­cial lab­o­ra­to­ries, but co­ro­na­vi­ruses have now been in­cluded in most mul­ti­plex re­spi­ra­ tory path­o­gen RT-PCR pan­els. Notably, these as­says do not de­tect Viral Diagnosis 227 SARS or MERS; thus, pos­i­tive RT-PCR re­sults do not sug­gest SARS or MERS, and sim­i­larly, neg­a­tive re­sults do not rule out­ in­ fec­tion with SARS or MERS. If SARS or MERS are sus­pected, test­ing should be co­or­di­nated with pub­lic health lab­o­ra­to­ries. Enterovirus (Cox­sackie A and B Viruses, Echovirus, Parechovirus, Enterovirus, Poliovirus). Isolation in cul­ture is the method of choice for some en­tero­vi­ruses and for spec­i­mens from which RT-PCR can­not be per­formed. Some se­ro­types of coxsackie A vi­rus fail to grow in cul­ture. These se­ro­types can be re­cov­ered by in­oc­u­la­tion of suck­ling mice, but the pro­ce­dure is not usu­ally per­formed in clin­i­cal lab­o­ra­to­ries. In ad­di­tion, parecho­ viruses re­quire a com­bi­na­tion of hu­man and pri­mate cell lines. Hu­man rhab­do­myo­sar­coma cells, WI-38, and hu­man em­bry­onic lung cells are best for coxsackie A vi­rus and mon­key (e.g., Buf­ falo green, rhe­sus, and cy­no­mol­gus) kid­ney or HeLa cells are best for coxsackie B vi­rus. Isolation in cul­ture is the method of choice for en­tero­vi­rus and po­lio­vi­rus, with growth be­ing ob­ served in a wide range of cell lines. RT-PCR is also a use­ful as­say, par­tic­u­larly for CSF sam­ples, for which this as­say is as sen­si­tive as cul­ture. Serological test­ing is re­stricted pri­mar­ily to re­search lab­o­ra­to­ries. Filovirus (Ebola Virus, Mar­burg Virus). Filoviruses are bio­ safety level 4 (BSL-4) path­o­gens, and so all­ work with the vi­ruses is re­stricted to BSL-4 fa­cil­i­ties. Virus can be cul­tured from se­rum at the time of clin­i­cal on­set. Vero cells are per­mis­sive. Antigen cap­ture ELISA has been used to de­tect vi­ral an­ti­gens in se­rum. Filovirus- specific IgM cap­ture and IgG ELISA are used to as­sess the se­ro­log­ i­cal re­sponse to in­fec­tion. IgM and IgG ap­pear 8 to 10 days af­ter on­set of dis­ease. IgM an­ti­body lev­els de­crease over the first few months of in­fec­tion, but IgG an­ti­bod­ies will per­sist for 2 years or Diagnosis Viral more. For pa­tients who have been symp­tom­atic for a short time (<3 days), Ebola vi­rus nu­cleic acid may not be de­tect­able in se­rum. Patients with­out­ an al­ter­na­tive di­ag­no­sis should be re­tested at a later time to rule out­ Ebola in­fec­tion. Ebola vi­rus nu­cleic acid may be de­tected in nonblood spec­i­mens, and pub­lic health lab­o­ra­to­ries should be con­sulted to de­ter­mine whether test­ing is ap­propr­ i­ate. Flavivirus (Yellow Fever Virus, Dengue Virus, St. Louis En- cephalitis Virus, West Nile Virus). Yellow fe­ver vi­rus an­ti­gen can be de­tected by an­ti­gen cap­ture as­says or RT-PCR; how­ever, these as­says are not com­mer­cially avail­­able. Most in­fec­tions are di­ ag­nosed by IgM cap­ture ELISA, with a pre­sump­tive di­ag­no­sis be­ing based on the pres­ence of IgM an­ti­bod­ies and the di­ag­no­sis be­ing con­firmed by the dem­on­stra­tion of a sig­nif­i­cant rise in an­ti­ body lev­els. Dengue vi­rus in­fec­tions are di­ag­nosed on the ba­sis of clin­i­cal pre­sen­ta­tion and de­tec­tion of vi­ral RNA by RT-PCR. Sero­ logical is of great­est value in pa­tients who have been symp­tom­atic for 228 SECTION 5 lon­ger than 5 days. Therefore, neg­a­tive IgM test re­sults should be con­firmed by test­ing a con­va­les­cent spec­i­men. Although an­ti­gen cap­ture as­says and RT-PCR tests have been de­vel­oped for St. Louis en­ceph­a­li­tis vi­rus, se­ro­logic test­ing is the most sen­si­tive di­ag­nos­tic test. Cross-reactivity with West Nile vi­rus and Jap­a­nese en­ceph­a­li­tis vi­rus oc­curs, and so neu­tral­i­za­tion as­says must be per­formed to dem­on­strate which vi­rus is re­spon­si­ble for the in­fec­tion. Likewise, West Nile vi­rus in­fec­tions are di­ag­nosed pri­mar­ily by se­ro­logic test­ ing, ex­cept in im­mu­no­com­pro­mised pa­tients, for whom RT-PCR as­ say may re­main pos­i­tive. Positive se­ro­logic tests must be con­firmed by neu­tral­i­za­tion as­says, and a four­fold change in an­ti­body lev­els must be dem­on­strated be­cause IgM and IgG an­ti­bod­ies can per­sist for months to years. Flavivirus (Hepacivirus [Hepatitis C Virus]). Diagnosis of in­fec­ tions caused by hep­a­ti­tis C vi­rus (HCV) is by ei­ther se­ro­logic test­ing or nu­cleic acid am­pli­fi­ca­tion test­ing (NAAT). Current se­ ro­log­i­cal as­says used for screen­ing blood do­nors and pa­tients are di­rected against a va­ri­ety of an­ti­gens, in­clud­ing core, NS3, NS4, and NS5 an­ti­gens. Seroconversion is de­tected by 10 weeks af­ter ex­ po­sure. False-positive re­ac­tions oc­cur at a low rate. To im­prove the test spec­i­fic­ity, a strip im­mu­no­as­say (re­com­bi­nant im­mu­no­blot as­ say [RIBA]) was de­vel­oped. However, the per­for­mance of the RIBA was sim­i­lar to the ini­tial screen­ing as­says and is no lon­ger of­fered in lieu of more de­fin­i­tive NAATs. In the set­ting of acute hep­a­ti­tis, qual­i­ta­tive and quan­ti­ta­tive NAATs have been de­vel­oped for de­tect­ ing vi­ral nu­cleic ac­ids in se­rum or plasma. However, pa­tients are of­ ten asymp­tom­atic in acute in­fec­tion, and thus most pa­tients are not di­ag­nosed un­til the chronic in­fec­tion has been es­tab­lished. In the set­ ting of chronic hep­a­ti­tis, EIAs are highly sen­si­tive and spe­cific, and Viral Diagnosis Viral con­fir­ma­tion by NAA tests is not nec­es­sary. For pa­tients pre­sent­ing with acute hep­a­ti­tis, qual­i­ta­tive NAA tests are used to con­firm ac­tive in­fec­tion. Quantitative NAA tests can be used to mon­i­tor the re­ sponse to ther­apy or the pro­gres­sion of dis­ease. Hantavirus (Hantaan Virus). Hantaviruses are dif­fi­cult to grow in cul­ture. Diagnosis is most com­monly made by se­ro­logic test­ing, and nearly all­ pa­tients who de­velop se­vere symp­toms from Hantaan vi­rus in­fec­tion will have high IgM ti­ters at or near the time of symp­tom de­vel­op­ment. IgG an­ti­body is also com­monly de­tect­able dur­ing the acute phase of dis­ease. RT-PCR as­says have also been de­ vel­oped and are incr­ eas­ingly be­ing used to di­ag­nose Hantaan vi­rus in­fec­tion. Viral RNA can be de­tected from blood and plasma, as well as from lung and kid­ney tis­sue. Hepatitis A Virus. Hepatitis A vi­rus (HAV) is dif­fi­cult to cul­ture, and so this is done only in re­search lab­o­ra­to­ries. Commercially avail­­able as­says for an­ti-HAV IgM are the meth­ods of choice for di­ag­no­sis of acute type hep­a­ti­tis, with sol­id-phase an­ti­body cap­ture Viral Diagnosis 229 im­mu­no­as­say be­ing the most com­monly used method. Antibodies are de­tected at the time of on­set of symp­toms and have dis­ap­peared by 6 months fol­low­ing in­fec­tion. EIAs are used to mea­sure to­tal an­ti-HAV an­ti­body lev­els (IgM, IgG, and IgA), which incr­ ease dur­ ing acute in­fec­tions and then per­sist in­def­i­nitely. Detectable an­ti- HAV an­ti­bod­ies in the ab­sence of IgM an­ti­bod­ies are in­dic­a­tive of past in­fec­tion and im­mu­nity. RT-PCR can be used to de­tect vi­re­ mic pa­tients in the early stages of dis­ease, but these as­says are not widely avail­­able and are not com­monly used. Hepatitis E Virus. Although hep­a­ti­tis E vi­rus (HEV) has been grown in cul­ture, this is in­ef­fi­cient and is per­formed only in re­ search lab­o­ra­to­ries. The method of choice for di­ag­no­sis of acute HEV in­fec­tions is de­tec­tion of IgM an­ti­bod­ies, which are de­tect­ able at the time of on­set of symp­toms and dis­ap­pear within sev­eral weeks af­ter symp­toms re­solve. IgG an­ti­bod­ies are also short-lived, typ­i­cally be­com­ing un­de­tect­able within sev­eral months of res­o­lu­ tion of symp­toms. Acute HEV in­fec­tions may also be di­ag­nosed by de­tect­ing HEV RNA in se­rum or plasma. This NAA as­say re­mains pos­i­tive for 2 to 7 weeks af­ter on­set, al­though vi­ral RNA may be de­tected in some in­di­vid­u­als for a more pro­longed pe­ri­od. Human Immunodeficiency Virus Types 1 and 2. Human im­mu­ no­de­fi­ciency vi­rus (HIV) in­fec­tions can be di­ag­nosed by cul­ture, an­ ti­gen or an­ti­body de­tec­tion, and NAA meth­ods. Most in­fec­tions are made ini­tially by screen­ing with a com­bined ap­proach that de­tects HIV-specific an­ti­bod­ies and the p24 an­ti­gen which are pro­duced within a few weeks af­ter in­fec­tion. It is cur­rently rec­om­mended that pos­i­tive screen­ing as­says be con­firmed with an HIV 1 and 2 dif­ fer­en­ti­a­tion as­say. Rapid im­mu­no­as­says and tests de­signed for home di­ag­no­sis are also avail­­able. Quantitative NAA meth­ods are avail­­able for mon­i­tor­ing the vi­ral load, which has prog­nos­tic im­ Diagnosis Viral pli­ca­tions. The com­plex­ity of the avail­­able di­ag­nos­tic tests pre­ cludes a de­tailed dis­cus­sion here; the user of this Pocket Guide is re­ferred to the ASM Manual of Clinical Microbiology. Human T-Cell Lymphotropic Virus Types 1 and 2. Amplifica­ tion of pro­vi­ral DNA is the pre­ferred method of di­ag­nos­ing HTLV in­fec­tion. NAATs can also be used to dis­tin­guish be­tween the four HTVL groups. EIA mea­sur­ing the se­ro­logic re­sponse to hu­man ­T-cell lymphotropic vi­rus type 1 (HTLV-1) and HTLV-2 in­fec­tion is the pri­mary di­ag­nos­tic test, and a WB as­say is used to con­firm the di­ag­no­sis. It is rec­om­mended that a test giv­ing an ini­tial pos­i­tive EIA re­sult be re­peated. If both tests are pos­i­tive, the band pro­file ob­served in the WB as­say is used to dis­tin­guish be­tween HTLV-1 and HTLV-2. Influenza Virus (Types A to C). Influenza in­fec­tion is typ­i­cally di­ag­nosed in one of two ways. Rapid an­ti­gen de­tec­tion is a com­ monly used method, but these as­says suf­fer from poor per­for­mance 230 SECTION 5 and are dis­cour­age by most mi­cro­bi­ol­o­gists. With the ad­vance­ment of rapid PCR as­says that can be per­formed with min­i­mal hands- on-time, and yield high sen­si­tiv­ity and spec­i­fic­ity, rapid an­ti­gen tests are less com­monly used. RT-PCR de­tec­tion of in­flu­enza vi­ ruses can be done in sev­eral for­mats. Stand-alone as­says typ­i­cally in­clude de­tec­tion and dif­fer­en­ti­a­tion of in­flu­enza A and B with­out­ subtyping. Multiplex PCR as­says will also de­tect in­flu­enza A and B, but also sub­type in­flu­enza A. The Madin-Darby ca­nine kid­ney (MDCK) cell line is most com­monly used for iso­la­tion of in­flu­enza vi­ruses, al­though growth is ob­served in a va­ri­ety of cell lines (e.g., Vero, MRC-5, and baby ham­ster kid­ney cells). Cytopathic ef­fect (CPE) is typ­i­cally ob­served within 2 to 3 days, but neg­a­tive cul­ tures should be tested by hemadsorption. Immunologic stain­ing of in­fected cells at 1 and 2 days (shell vial as­say) is more com­mon than tra­di­tional tube cul­ture. DFA can be per­formed with na­so­pha­ryn­ geal washes, al­though this test has a sen­si­tiv­ity of only 80 to 90% com­pared with cul­ture. Specific EIAs for ei­ther in­flu­enza A vi­rus or in­flu­enza A plus B vi­ruses are avail­­able but have lim­ited util­ity in the di­ag­no­sis of ac­tive in­fec­tion. Serologic tests are used pri­mar­ ily for ep­i­de­mi­o­log­i­cal sur­veys. Measles Virus. Measles vi­rus can be iso­lated from the con­junc­tiva, na­so­phar­ynx, and blood dur­ing the late pro­dro­mal pe­riod and early stage of rash de­vel­op­ment. Viremia clears within 2 to 3 days of the rash, but vi­rus can be de­tected in urine for up to 7 days. B95-8 (B-lymphoblastoid) cells are used for iso­la­tion of vi­rus. However, few clin­i­cal lab­o­ra­to­ries at­tempt to cul­ture the vi­rus. Virus-infected cells can be de­tected by cy­to­logic ex­am­i­na­tion (de­tec­tion of intra­ cytoplasmic and intranuclear in­clu­sions and gi­ant cells), and RT- PCR is of­fered by ref­er­ence and pub­lic health lab­o­ra­to­ries. The Viral Diagnosis Viral rec­om­mended lab­o­ra­tory method for con­fir­ma­tion of in­fec­tion is a se­rum-based IgM EIA. Commercial as­says are avail­­able. Serum can be col­lected at the time of rash on­set or up to 4 weeks later. IgG as­says are also avail­­able and, in com­bi­na­tion with IgM as­says, can be used to as­sess im­mu­nity as well as pri­mary dis­ease. Mumps Virus. Mumps is di­ag­nosed by vi­ral iso­la­tion, NAA, or se­ro­logic test­ing. Mumps vi­rus is cul­tured most com­monly in pri­ mary rhe­sus mon­key kid­ney cells and hu­man neo­na­tal kid­ney cells. Cells are ex­am­ined for CPE for 14 days, and neg­a­tive cell cul­ tures are tested by hemadsorption with guinea pig eryth­ro­cytes. Rapid an­ti­gen tests are in­fre­quently used be­cause mumps in­fec­tions are un­com­mon in vac­ci­nated pop­u­la­tions. Mumps vi­rus can be de­ tected by RT-PCR, al­though this tech­nique is not widely used as there are no com­mer­cially avail­­able as­says. Serologic test­ing can be used to de­fine an acute in­fec­tion or im­mu­nity. A sin­gle pos­i­tive IgG test is suf­fi­cient to iden­tify an im­mune pa­tient; se­ro­con­ver­sion is nec­es­sary to iden­tify a pri­mary in­fec­tion. EIAs are avail­­able for Viral Diagnosis 231 mea­sur­ing IgM and IgG an­ti­bod­ies with whole vi­rus, son­i­cated vi­ rus, or pu­ri­fied vi­ral an­ti­gens (e.g., HN or nu­cle­o­cap­sid [NP]) used in the as­says. Serologic test­ing is of lim­ited util­ity in di­ag­nos­ing pos­si­ble in­fec­tion in vac­ci­nated in­di­vid­u­als as IgM is only weakly pro­duced in a sec­ond­ary im­mune re­sponse. Parainfluenza Virus. RT-PCR is the pre­ferred method of di­ag­ nos­ing para­in­flu­enza vi­rus in­fec­tion. Most mul­ti­plex re­spi­ra­tory path­o­gen PCR pan­els in­clude para­in­flu­enza 1-4 and per­form with high sen­si­tiv­ity and spec­i­fic­ity. Primary hu­man em­bry­onic kid­ney and pri­mary mon­key kid­ney cells are the most sen­si­tive cell line for cul­ture of para­in­flu­enza vi­rus (PIV). Other cell lines can sup­ port the growth of PIV but are not rec­om­mended for pri­mary iso­ la­tion. Cultures are ex­am­ined for CPE for 10 to 14 days, with 50% of pos­i­tive cul­tures be­ing de­tected at 5 days. Positive cul­tures can also be de­tected as early as 48 h af­ter in­oc­u­la­tion if cul­tures have been stained with vi­rus-specific fluo­res­cent an­ti­bod­ies. In most clin­i­cal lab­o­ra­to­ries, cul­ture has been re­placed with the shell vial as­say, which has com­pa­ra­ble sen­si­tiv­ity and is more rapid. Direct and in­di­rect im­mu­no­flu­o­res­cent an­ti­body tests are com­monly used to ex­am­ine re­spi­ra­tory spec­i­mens for vi­rus-infected cells. Speci­ mens are typ­i­cally ex­am­ined with pooled re­agents (for in­flu­enza A and B vi­ruses, PIV-1 to PIV-3, re­spi­ra­tory syn­cy­tial vi­rus [RSV], and ad­e­no­vi­ruses), and those giv­ing pos­i­tive re­ac­tions are tested with vi­rus-specific re­agents. A va­ri­ety of se­ro­logic as­says (CF, HI, IFA, neu­tral­i­za­tion, and EIA) have been de­vel­oped. Cross-reactions with mumps vi­rus limit the util­ity of these tests. Rabies Virus. The pre­ferred method for the di­ag­no­sis of ra­bies in an­i­mals is the DFA test for ra­bies vi­rus an­ti­gen in brain tis­sue. Fluorescein iso­thio­cy­a­nate-labeled an­ti­ra­bies an­ti­bod­ies can be pre­ pared against whole ra­bies vi­ruses or pu­ri­fied RNA-nucleoprotein Diagnosis Viral com­plex or nu­cle­o­pro­teins (N pro­teins). For the di­ag­no­sis of ra­bies in hu­mans, the fol­low­ing spec­i­mens are col­lect­ed: sa­liva (col­lected with an eye drop­per and placed in a ster­ile con­tainer with no pre­ser­ va­tives), neck bi­opsy spec­i­men (col­lected from the hair line and of suf­fi­cient depth to in­clude cu­ta­ne­ous nerves and placed in a ster­ile con­tainer with no pre­ser­va­tives), 0.5 ml of se­rum (not whole blood) or CSF, and brain bi­opsy spec­i­men (only if the spec­i­men was col­ lected for other di­ag­nos­tic pro­ce­dures). The fol­low­ing tests are rec­ om­mend­ed: sa­liva, RT-PCR and cul­ture; neck bi­opsy spec­i­men, RT-PCR, and IFA; se­rum and CSF, se­ro­logic test­ing; brain bi­opsy spec­i­men, RT-PCR, and IFA. Serologic test­ing can be used to as­sess the re­sponse to vac­ci­na­tion. The neu­tral­i­za­tion test is most com­monly used, al­though a ra­bies sur­face gly­co­pro­tein (G)-specific ELISA is avail­­able in Eu­rope. Respiratory Syncytial Virus. NAAT are now the pre­ferred di­ag­ nos­tic method for RSV in­fec­tion. These as­says ex­ist as stand-alone 232 SECTION 5 as­says or as part of mul­ti­plex pan­els. Both forms of NAAT have high sen­si­tiv­ity and spec­i­fic­ity. Rapid an­ti­gen tests are avail­­able and are com­monly found in smaller lab­o­ra­tory set­tings or phy­si­ cian of­fices. Like the in­flu­enza rapid an­ti­gen tests, RSV rapid an­ti­ gen tests suf­fer from poor per­for­mance. Viral in­fec­tiv­ity is rap­idly lost at room tem­per­a­ture, and so spec­i­mens for cul­ture should be pro­cessed promptly. The most sen­si­tive cell lines for cul­ture are HEp-2 and HeLa; less sen­si­tive cells in­clude pri­mary mon­key kid­ ney and hu­man fi­bro­blast cell lines. CPE is ob­served on av­er­age at 4 to 5 days. The shell vial as­say is slightly more sen­si­tive, and pos­ i­tive cul­tures are de­tected at 1 to 2 days. Direct an­ti­gen de­tec­tion tests (IFA and EIA) have a sen­si­tiv­ity equiv­a­lent to that of cul­ture, are more rapid, and are not ad­versely af­fected by spec­i­men trans­ por­ta­tion prob­lems. Serologic test­ing is use­ful for ep­i­de­mi­o­log­i­cal sur­veys but is not as sen­si­tive as cul­ture or an­ti­gen tests. Rhinovirus. Multiplex re­spi­ra­tory path­o­gen PCR pan­els com­ monly in­clude Rhinovirus de­tec­tion. These as­says per­form with high sen­si­tiv­ity but are typ­i­cally un­able to dif­fer­en­ti­ate Rhinovi­ rus from Enterovirus. The cell lines used most com­monly for growth of rhi­no­vi­ruses are WI-38 and MRC-5. Cultures should be in­cu­bated at 33°C, with CPE seen as early as 1 to 2 days af­ter in­ oc­u­la­tion. Negative cul­tures should be held for 7 days or more. EIAs are in­sen­si­tive be­cause there are a large num­ber of se­ro­types of rhi­ no­vi­ruses and no com­mon an­ti­gen ex­ists. The large num­ber of se­ ro­types also makes se­ro­logic test­ing im­prac­ti­cal. Rubella Virus. Throat swabs and na­so­pha­ryn­geal spec­i­mens are re­li­able sources of ru­bella vi­rus, with pos­i­tive cul­tures de­tected a few days be­fore the rash de­vel­ops to up to 4 days af­ter on­set. The

Viral Diagnosis Viral vi­rus grows in a va­ri­ety of cell lines (e.g., Vero, BHK21, AGMK, and RK-13). Cultures are main­tained for 1 week and then pas­saged. Viral growth is de­tected by IFA or RT-PCR. RT-PCR as­says have also been used for pri­mary de­tec­tion of vi­rus but are re­stricted pri­ mar­ily to re­search lab­o­ra­to­ries and are not used rou­tinely for clin­ i­cal di­ag­no­sis. Detection of ru­bella vi­rus-specific IgM is the fast­est and most ef­fi­cient method to di­ag­nose re­cent post­na­tal in­fec­tion. However, only 50% of in­fected new­borns are IgM pos­i­tive on the day of symp­tom on­set. By 8 days af­ter the on­set of rash, the in­fant should be pos­i­tive for both IgM de­tect­able by IgM cap­ture ELISA and IgG de­tect­able by in­di­rect ELISA. False-positive tests can oc­ cur; care must be ex­er­cised in in­ter­pret­ing the test re­sults.

DNA Viruses Adenovirus. Syndromic mul­ti­plex NAATs are now the pre­ferred method of de­tec­tion for ad­e­no­vi­rus-caused up­per re­spi­ra­tory tract in­fec­tion and gas­tro­en­ter­i­tis. In ad­di­tion, quan­ti­ta­tive and qual­i­ta­ tive Adenovirus PCR as­says are com­monly used for the di­ag­no­sis Viral Diagnosis 233 and mon­i­tor­ing of dis­sem­i­nated in­fec­tion, typ­i­cally in im­mu­no­com­ pro­mised pa­tients. All ad­e­no­vi­ruses ex­cept types 40 and 41 rep­li­cate and pro­duce CPE in cell cul­tures (e.g., HeLa, KB, A549, HEp-2, and HEK cells). CPE usu­ally ap­pears in 2 to 7 days, but pas­sage of cell cul­tures for up to 1 month is rec­om­mended for neg­a­tive cul­tures. Shell vial as­says are as sen­si­tive as tra­di­tional cul­ture and more rapid (tak­ing 2 to 5 days). Clinical spec­i­mens can also be ex­am­ined by IFA, but this is sig­nif­i­cantly less sen­si­tive than cul­ture. Commercial EIAs are also avail­­able and are par­tic­u­larly use­ful for de­tect­ing types 40 and 41 in pa­tients with gas­tro­en­ter­i­tis. Serologic tests are used pri­mar­ily for ep­i­de­mi­o­log­i­cal pur­poses. A se­ro­con­ver­sion must be dem­on­strated to con­firm a cur­rent in­fec­tion, be­cause seroreactivity to ad­e­no­vi­rus is com­mon. Cytomegalovirus. NAATs are the pre­ferred method of di­ag­nos­ing and mon­i­tor­ing dis­sem­i­nated in­fec­tion in im­mu­no­com­pro­mised pa­tients. NAATs can also be done from urine or sa­liva in cases of sus­pected con­gen­i­tal in­fec­tion. In ad­di­tion, cul­ture is a sen­si­tive method for de­tect­ing cy­to­meg­a­lo­vi­rus (CMV) in re­spi­ra­tory spec­ i­mens, urine, and anticoagulated whole blood (leu­ko­cytes). Inter­ mittent shed­ding in urine is pos­si­ble, and so mul­ti­ple spec­i­mens should be pro­cessed. Recovery of CMV from leu­ko­cytes is a bet­ ter in­di­ca­tor of symp­tom­atic in­fec­tion. Human fi­bro­blast cell lines (e.g., WI-38, MRC-5, and IMR-90) are best, but growth is typi­­cally slow and may re­quire se­rial pas­sage of the cells and pro­longed in­cu­ ba­tion (for up to 6 weeks). Results of this test com­pare fa­vor­ably with the quan­ti­ta­tive de­tec­tion of CMV DNA in leu­ko­cytes or plasma by NAA meth­ods (PCR). A va­ri­ety of se­ro­logic tests are avail­­able (e.g., EIA, IFA, and pas­sive la­tex ag­glu­ti­na­tion), in­clud­ing IgM- and IgG- specific tests. IgM re­sults must be in­ter­preted with cau­tion be­cause IgM an­ti­body is found in both pri­mary and re­ac­ti­vated in­fec­tions Diagnosis Viral and can per­sist for months. Demonstration of IgG se­ro­con­ver­sion is di­ag­nos­tic of pri­mary in­fec­tion. Serologic test­ing is im­por­tant in as­sess­ing or­gan do­nors and re­cip­i­ents but is not use­ful in di­ag­nos­ing in­fec­tions in im­mu­no­com­pro­mised pa­tients. Ep­stein-Barr Virus. Ep­stein-Barr vi­rus (EBV) can be cul­tured in hu­man cord blood lym­pho­cytes, but this is rarely done for di­ag­ nos­tic pur­poses. Indirect, di­rect, and anticomplement im­mu­no­flu­ o­res­cence are the main meth­ods used for the de­tec­tion of EBV an­ti­gens in tis­sues and cell cul­tures. Quantitative PCR as­says are com­monly per­formed from whole blood and plasma, but these as­ says are not in­tended to be used for the di­ag­no­sis of EBV in­fec­ tion. Rather, these as­says are used to mon­i­tor the de­vel­op­ment of post-transplant lymphoproliferative dis­ease in trans­plant pa­tients. EBV-positive CSF is sig­nif­i­cantly as­so­ci­ated with pri­mary lym­ phoma in HIV-positive in­di­vid­u­als and with en­ceph­a­li­tis in im­mu­ no­com­pe­tent in­di­vid­u­als. The di­ag­no­sis of acute EBV in­fec­tion 234 SECTION 5 (in­fec­tious mono­nu­cle­o­sis) is es­tab­lished through the de­tec­tion of het­ero­phile an­ti­bod­ies (non­spe­cific an­ti­bod­ies) or de­tec­tion of EBV- specific se­ro­log­i­cal mark­ers (pre­ferred method). For in­ter­pre­ta­tion of se­ro­logic test re­sults, re­fer to Table 5.3. Erythrovirus (B19 Virus). B19 vi­rus, a mem­ber of the Parvoviri- dae, is dif­fi­cult to grow in vi­tro. Viral par­ti­cles or DNA can be de­tected in blood about 6 days af­ter in­fec­tion, with peak vi­re­mia oc­cur­ring 2 to 3 days later. Viral ti­ters de­crease, but B19 DNA can be de­tected by PCR for up to 2 months. Serologic tests to de­tect an­ti­bod­ies are com­mer­cially avail­­able and are the most com­monly used meth­ods for di­ag­no­sis of acute in­fec­tions and im­mune sta­tus. In im­mu­no­com­pe­tent in­di­vid­u­als, IgM an­ti­bod­ies de­velop 2 weeks af­ter in­fec­tion and per­sist for up to 30 weeks. In pa­tients with aplas­ tic cri­sis, an­ti­bod­ies ap­pear sev­eral days af­ter on­set of clin­i­cal symp­toms. In pa­tients with fe­tal hydrops, de­tec­tion of IgM an­ti­bod­ ies at the time of clin­i­cal on­set is more var­i­able. In im­mu­no­com­pe­ tent pa­tients, IgG an­ti­bod­ies ap­pear sev­eral days af­ter IgM an­ti­bod­ies and per­sist for years. The pres­ence of IgG an­ti­bod­ies is con­sis­tent with im­mu­nity. In im­mu­no­com­pro­mised pa­tients, IgG and IgM an­ti­ body re­sponses are un­pre­dict­able, and so se­ro­logic test­ing is not used for these pa­tients. NAA and PCR are the most com­mon meth­ ods used to de­tect B19 DNA. Serologic di­ag­no­sis of re­cent in­fec­tion is gen­er­ally per­formed by IgM cap­ture EIAs. If IgM as­says are neg­ a­tive for im­mu­no­com­pro­mised pa­tients, DNA de­tec­tion meth­ods should be used. B19 IgG an­ti­bod­ies de­tected by EIA in the ab­sence of se­ro­con­ver­sion are in­dic­a­tive of past in­fec­tion. See Table 5.5 for a de­scrip­tion of di­ag­nos­tic meth­ods for the var­i­ous pre­sen­ta­tions of par­vo­vi­rus in­fec­tion. Hepatitis B Virus. Diagnosis of hep­a­ti­tis B vi­rus (HBV) in­fec­ Viral Diagnosis Viral tions is based pri­mar­ily on the de­tec­tion of vi­rus-specific an­ti­gens and an­ti­bod­ies. A va­ri­ety of as­says have been de­vel­oped to de­tect early and late an­ti­gens and the an­ti­body re­sponse to each. Refer to Table 5.4 for in­ter­pre­ta­tion of these as­says in spe­cific clin­i­cal pre­ sen­ta­tions. NAATs are used for the quan­ti­ta­tion of HBV DNA, which can be used for the ini­tial eval­u­a­tion of in­fec­tion as well as mon­i­tor­ing the pro­gres­sion of chronic in­fec­tion dur­ing treat­ment. Herpes Simplex Virus Types 1 and 2. Culture is a sen­si­tive method for de­tect­ing vi­rus in mu­co­cu­ta­ne­ous, gen­i­tal, and oc­u­lar le­sions. Viral growth, as in­di­cated by a CPE, is rapid in most cell lines (95% of spec­i­mens are pos­i­tive by 5 days). Some cell lines (e.g., mink lung cells) are bet­ter than oth­ers (e.g., MRC-5 and Vero cells). Culture is in­sen­si­tive for CSF in­fec­tions, for which PCR is the rec­ om­mended test. DFA and IFA tests are avail­­able and pro­vide a rapid re­sult if pos­i­tive, but they are rel­a­tively in­sen­si­tive com­pared with cul­ture and PCR. DFA and IFA can be used to dis­tin­guish be­tween her­pes sim­plex vi­rus type 1 (HSV-1) and type 2 (HSV-2) when the Viral Diagnosis 235 — — — ++ nt; EA/R, y, therey, are rs; +++, an­ ­ ne ­ it ­ te v ­ po ­ ti EBNAI, IgG ­ a g um li ­ ne ­ di se com ­ ro ­ fu t in me — — — ++ —dif ­ en EBNA, Ig ­ gen res : ­ ti c pe ­ ies p ys beys kept in mind. d ons apart from se ­ to ­ ti ­ wa ­ bo ­ di ­ ti n — — ++ ++ ­ co 1. Manualof Clinical Microbiology, ed., ASM 11th s must al ; EA/D, early an ­ si ­ gen EA/R, IgG rs; ++, an ­ ti n and Ig iso ­ no ­ te ­ gen ­ ti ­ ge ­ ag ­ ti ear an d an ­ cl ­ si t in ti low y is given. For all — — ++ ++ rr nu ­ it s to an od a di of ­ en l cap v ­ ie ­ ho ­ ra ­ ti Ba res - d ­ li EA/D, IgG ­ ac e ein ­ bo ­ ies p ­ st ­ ti d ­ tic r r SS,r Warnock (ed.), DW ­ is ­ bo ­ te r ­ ti g the like rs. VCA, vi ­ te ­ in ­ te ­ ac rt — — ++ ­ po a,b ted ti ; EBNAI, Ep rs; an +, ­ te ­ va ­ e ons ­ gen rs sup Presence of an ­ ti ­ ti ­ te ­ e ­ di m uded. The char ear an ­ ra t in ti low ­ cl ­ cl t con ­ en — — ry pa ­ en Viral Diagnosis Viral rr nu r t in strongly el ­ to ­ en ­ fe ­ ra Ba - ­ o nt or pres pres ein ­ se r dif llFunke KC, G, Landry ML, Rich ­ st b ­ ies ­ ro ­ de d ­ ies a ­ bo d g other lab — ++ ­ ti +++ ++++ ++ +++ ++ ­ in ++++ ++ +++ +++ ­ bo es un s and Ig isotypes are in ­ ti ud VCA, IgG VCA, IgM VCA, IgA ­ fil ­ cl ­ gen nt; EBNA, Ep ­ ti ­ ne nt; ±, an ­ po rs; ++++, an on ­ se n gic pro ­ te ­ ti oyed anoyed c ­ lo ­ tio ­ pl on ­ fe c ­ ti ­ ro ted ti c ­ fe on etely ab ­ va ­ ti ­ fe ricted com ­ e c ­ pl ­ st V se ­ fe ry ry in ently em n, D.C., 2015. n, D.C., ry in ­ qu ­ to EB t in el —re ­ ma ve ve EBV in g ry in ­ ti ­ en ­ ies com ­ ma

­ in ­ gen ­ ti ­ ma ­ bod pres ­ ti ons to the rule. Clinical data in ­ ti ­ ies p d The most fre Adaptedfrom Jorgensen JH, Pfaller MA, Car −, an−, ­ ce a b c ­ bo Condition Seronegative Ongoing Ongoing pri Recent pri Past pri Chronic ac ex Table 5.3 Press, Wash ti 3 early an 236 SECTION 5 + — — — — — — + or ++ + + — — — — — — — + or — rs. ­ te Anti-HBe Anti-HBs . + ­ gen t in very high ti ­ ti IgM + or — + or — ­ en pres a,b,c ­ ies Anti-HBc d nce s B core an ­ bo ­ ti ++ +++ +++ ­ ce ­ ti ­ ti Total s ­ a ­ le ­ va rs; +++, an ; HBc, hep ­ te + + + + — ­ gen r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th HBeAg ­ ti ­ te on and con ­ ti ely ely high ti c s Be an ­ at ­ ti ­ fe ­ er ­ ti ­ a + + or — — — + + + ———— ———— — — + + — — + — HBsAg t in mod ­ en pres ; HBeAg, hep t stages in of Viral Diagnosis Viral ­ en ­ ies ­ gen r d ­ ti + + + + — — — — ­ fe ­ bo ­ ti + or — — — ce an HBV DNA ll Funke KC, G, Landry ML, Rich ­ fa ­ ro s in dif t; ++, an ­ er s B sur ­ en ­ ti ­ ti ­ a pres s mark ­ ies d on + or — ­ ru ­ ti ­ bo c ­ ti ­ fe ­ er s; HBsAg, hep ­ ri ­ ru nt; +, an ­ se patitis B vi on on on n, D.C., 2015. n, D.C., ­ ti n on on s B vi ­ ti ­ ti on ­ to He c ­ ti ­ ti ­ ti c ­ ies ab g ­ ti ­ tio c ­ ti ­ fec ­ fe c ­ a ­ ba ­ fe ­ in

­ fe egative acuteegative in ­ ba u ­ fe ­ bod n u ­ c - ­ ti ­ c , an HBV, HBV, hep Adapted from Jorgensen JH, Pfaller MA, Car — a b c Stage of in Vaccination re ­ sponse Early in Late in Acute Acute in HBsAg Chronic in Healthy HBsAg car Recent in Remote in Table 5.4 Press, Wash Viral Diagnosis 237 c c ­ oti e ­ ni ­ su PCR Positive Positive Positive Positive Positive Positive Positive am fluid or tis ve ­ ti ­ i IgG Positive Positive Negative/weakly pos t ­ se a s ­ si r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th ­ te ­ no ve ­ ti ­ ag ­ i sitive IgM Positive within 3 month on of Negative/positiveNegative/positive Negative/positive Negative/positive Po Negative/weakly pos Negative/positive Positive ome ­ dr on Viral Diagnosis Viral ­ ti a/pure red ­ ta /congenital /congenital ­ mi ­ lis s B19 and method di of s B19 ­ sen ­ ta ll Funke KC, G, Landry ML, Rich ­ ru ­ ease ­ ro ­ vi ia ­ vo ­ m Fifth dis Polyarthropathy syn Petechial or pupruic rash TAC Disease pre Persistent ane cell aplasia Hydrops Hydrops fe ane ses par of ­ ea ents ­ ti eased nt pa inical dis ­ cr ­ te n, D.C., 2015. n, D.C., ­ to Cl is ­ pe ­ dren ­ dren g ­ s ­ e m

­ in i 20 weeks) 20 ­ co ­ po < ­ no ­ ro ­ mu Adapted from Jorgensen JH, Pfaller MA, Car a Host(s) Healthy chil Healthy adults Healthy chil Patients with in or Immunodeficient im eryth Fetus ( Press, Wash Table 5.5 238 SECTION 5 vi­ruses are iso­lated in cul­ture. An EIA has also been de­vel­oped but is less sen­si­tive than cul­ture, par­tic­u­larly for spec­i­mens from asymp­ tom­atic pa­tients. PCR as­says can de­tect all ­strains of HSV and can dis­tin­guish be­tween HSV-1 and HSV-2. Rapid PCR as­says are now com­mer­cially avail­­able for the test­ing of le­sions from skin and mu­co­cu­ta­ne­ous spec­i­mens. Type-specific IgG as­says are also avail­­able com­mer­cial­ly. Human Herpesvirus 6. Human her­pes­vi­rus 6 (HHV-6) causes ro­se­ola in­fan­tum (ex­an­them subitum) in chil­dren and op­por­tu­nis­ tic in­fec­tions in im­mu­no­com­pro­mised pa­tients. HHV-6 can be iso­ lated from pe­riph­eral blood mono­nu­clear cells by co­cul­ti­va­tion with hu­man cord blood lym­pho­cytes; how­ever, this test is not com­ monly per­formed. Quantitative PCR as­says have been de­vel­oped and have proved use­ful for mon­i­tor­ing vi­ral con­cen­tra­tions in pe­ riph­eral blood mono­nu­clear cells. A va­ri­ety of se­ro­logic as­says (e.g., neu­tral­i­za­tion, im­mu­no­blot, IFA, and ELISA) are avail­­able for mea­ sur­ing IgG an­ti­bod­ies to HHV-6. Some cross-re­ac­tiv­ity with HHV-7 and CMV oc­curs. Seroconversion can be used to de­fine a pri­mary in­fec­tion. Orthopoxvirus (Vaccinia Virus, Smallpox Virus, Monkeypox Virus). Orthopoxviruses can grow in a va­ri­ety of es­tab­lished cell lines (BSC-1, CV-1, and LLCMK-2 cells, mon­key kid­ney cells, hu­ man em­bry­onic lung fi­bro­blasts, HeLa cells, chicken em­bryo fi­bro­ blasts, and MRC-5 cells). Growth is rapid, with most cul­tures be­ing pos­i­tive within 48 h. PCR-based NAA tests are also avail­­able for de­tect­ing vi­rus in se­rum and ve­sic­u­lar le­sions. NAA tests are of­ fered by the CDC and WHOCC. Virus- and fam­i­ly-specific as­says have been de­vel­oped. Neutralizing an­ti­bod­ies can be de­tected as early as 6 days af­ter in­fec­tion or vac­ci­na­tion. The ab­sence of an­ti­ Viral Diagnosis Viral bod­ies does not de­fine sus­cep­ti­bil­ity to in­fec­tion, be­cause the level of an­ti­bod­ies re­quired for pro­tec­tive im­mu­nity is not known. Papillomavirus. Human pap­il­lo­ma­vi­rus (HPV) can­not be grown in cul­ture. Late-structure an­ti­gens have been de­tected in tis­sue bi­ opsy spec­i­mens by us­ing vi­rus-specific po­ly­clonal an­ti­bod­ies. This as­say is spe­cific but in­sen­si­tive and is rarely used for di­ag­nos­tic pur­ poses. HPV DNA can be de­tected by us­ing type spe­cific DNA or RNA probes in a va­ri­ety of hy­brid­iza­tion tech­niques. The use of tar­get am­pli­fi­ca­tion to in­crease the sen­si­tiv­ity of this method has made this the di­ag­nos­tic test of choice. In ad­di­tion, mo­lec­u­lar as­says now of­fer the abil­ity to sub­type HPV into high- and low-risk ge­no­ types. Serologic tests are used for ep­i­de­mi­o­log­i­cal stud­ies be­cause type-specific an­ti­bod­ies can be de­tect­able for many years af­ter ex­po­sure. Polyomavirus (JC Virus, BK Virus). Viral cul­ture is not rou­ tinely used for clin­i­cal di­ag­no­sis be­cause JC vi­rus and BK vi­rus have long growth cy­cles and a lim­ited range of host cells. Serologic Viral Diagnosis 239 test­ing has been used pri­mar­ily for ep­i­de­mi­o­log­i­cal stud­ies. Most in­di­vid­u­als are in­fected at a young age. IgM an­ti­bod­ies de­velop ini­ tially, as mea­sured by EIAs. The role of this an­ti­body re­sponse in re­cur­rent in­fec­tions is not well char­ac­ter­ized. NAA tests are the pri­ mary tests used to doc­u­ment in­fec­tion with JC vi­rus and BK vi­rus. JC vi­rus DNA is de­tected in pa­tients with pro­gres­sive mul­ti­fo­cal leukoencephalopathy, and BK vi­rus DNA is de­tected in the blood and urine of re­nal trans­plant re­cip­i­ents. Varicella-Zoster Virus. Diploid hu­man cell lines (e.g., hu­man fe­ tal dip­loid kid­ney [HFDK] and hu­man fe­tal dip­loid lung [HFDL] cells) are the most sen­si­tive cells for iso­la­tion of var­i­cel­la-zoster ­vi­rus (VZV). Other cell lines can sup­port the growth of VZV but are much less sen­si­tive. CPE is gen­er­ally ob­served in the first week of in­cu­ba­tion, but pro­longed in­cu­ba­tion may be re­quired. DFA tests are avail­­able and are much more sen­si­tive than cul­ture (98 and 50%, re­spec­tively) be­cause the vi­rus is highly la­bile. Cellular ma­te­rial from the base of a ve­sic­u­lar le­sion must be col­lect­ed; ves­i­cle fluid alone is un­sat­is­fac­tory. PCR is also more sen­si­tive than cul­ture. The value of se­ro­logic test­ing (many tests are com­mer­cially avail­­able) is lim­ited be­cause in­creases in the ti­ter of het­ero­typic an­ti­body to VZV may oc­cur in HSV-infected pa­tients who have had a prior in­ fec­tion with VZV. Serologic test­ing is used pri­mar­ily to as­sess im­ mu­nity in un­vac­ci­nated health care work­ers ex­posed to pa­tients with doc­u­mented VZV in­fec­tions. Transmissible Spongiform Encephalopathies Bovine Encephalopathy. Diagnosis is based on clin­i­cal his­tory and his­to­path­o­logic ex­am­i­na­tion of brain tis­sues. Early di­ag­no­sis has also been made on the ba­sis of ex­am­i­na­tion of ton­sil­lar and ap­pen­dix bi­opsy spec­i­mens. Diagnosis Viral Creutz­feldt-Jakob Disease. Diagnosis is based on clin­i­cal his­tory and his­to­path­o­logic ex­am­i­na­tion of brain tis­sues. Kuru. Diagnosis is based on clin­i­cal his­tory and his­to­path­o­logic ex­am­i­na­tion of brain tis­sues.

SECTION 6 Fungal Diagnosis

Mycology Specimen Collection and Transport Guidelines 242 Table 6.1 Methods for the Identification of Fungi 245 Microscopy 246 Table 6.2 Characteristic Fungal Elements Seen by Direct Examination of Clinical Specimens 248 Primary Plating Media 254 Table 6.3 Mycology Plating Guide 256 Specific Diagnostic Tests 258 Aspergillus Species 258 Blastomyces dermatitidis 258 Candida Species 259 Coccidioides Species 259 Cryptococcus Species 260 Histoplasma capsulatum 260 Malassezia Species 261 Paracoccidioides brasiliensis 261 Talaromyces (Penicillium) marneffei 262 Pneumo­ cys­ tis­ jiroveci 262 Sporothrix schenckii 262 Zygomycetes 262 Identification Tables 264

doi:10.1128/9781683670070.ch6 242 SECTION 6 Mycology: Specimen Collection and Transport Guidelines Ge ­ne­ral Guidelines 1. Although the re­cov­ery of some fungi in clin­i­cal spec­i­mens is al­ways con­sid­ered sig­nif­i­cant (e.g., der­ma­to­phytes, di­mor­phic fungi, Cryptococcus neoformans), most fungi are part of the pa­ tient’s nor­mal flora (e.g., Candida spp.) or found in the en­vi­ron­ ment (e.g., most dematiaceous and moniliaceous fungi). Specimens must be care­fully col­lected to avoid con­tam­i­na­tion with in­dig­e­nous or ex­og­e­nous fun­gi. 2. Bacteria can rapid­ ly over­grow fungi, so care must be used in clean­ing the site where the spec­i­men will be col­lected (e.g., skin sur­face, nail beds). Transport con­di­tions must be se­lected to min­i­ mize the risk of bac­te­rial over­growth. 3. Microscopic ex­am­i­na­tion of spec­i­mens is im­por­tant for the rapid de­tec­tion of a fun­gal in­fec­tion and for as­sess­ing the sig­nif­i­ cance of an iso­late.

Dematiaceous Fungi Specimens sub­mit­ted for mi­cros­copy and cul­ture in­clude as­pi­rates, scrap­ings, and tis­sues. Cotton-wrapped swabs should not be used be­cause an in­ad­e­quate quan­tity of ma­te­rial is re­cov­ered and des­ic­ ca­tion oc­curs. Flocked swabs, al­though not of­fi­cially ap­proved for use in col­lec­tion of spec­i­mens for fun­gal cul­ture, may suf­fi­ciently cap­ture fun­gal el­e­ments so as to be ac­cept­able for spec­i­men col­ lec­tion. Unpublished data sug­gest that this is the case, but more ev­i­dence is needed to con­firm that these spec­i­mens can be rec­om­ mended for use. Transport me­dium is un­nec­es­sary if the spec­i­men is pro­cessed im­me­di­ate­ly. Serologic test­ing for some of the ther­mally di­mor­phic fungi is avail­­able, but cross-re­ac­tiv­ity may ex­ist be­tween Blastomyces and Histoplasma. In ad­di­tion, di­rect de­tec­tion of fun­gal an­ti­gens is Fungal Diagnosis avail­­able and com­monly used for sev­eral fungi. Urine an­ti­gen test­ ing is com­monly per­formed for the di­ag­no­sis of Blastomyces and Histoplasma in­fec­tion, but like se­ro­logic test­ing, there is a high de­ gree of cross-re­ac­tiv­ity be­tween the two path­o­gens. Cryptococcal an­ti­gen test­ing is com­monly used to di­ag­nose dis­sem­i­nated in­fec­ tion and can be per­formed from both se­rum and ce­re­bral spi­nal flu­id.

Dermatophytes (Epidermophyton, Microsporum, and Trichophyton spp.) Collect in­fected hairs with ster­ile for­ceps (guided by the use of a Wood’s lamp if the sus­pected der­ma­to­phyte is fluo­res­cent). Endo­ Fungal Diagnosis 243 thrix fungi may re­quire the use of a ster­ile scal­pel to col­lect the hair root. Sample skin le­sions at the ac­tive bor­der of the le­sion with a ster­ile scal­pel to col­lect the sam­ple. Disinfect nails with al­co­hol be­ fore col­lect­ing the sam­ple by clip­ping or scrap­ing. Do not place hair, skin, or nail sam­ples in closed tubes. The high hu­mid­ity fos­ ters over­growth of con­tam­i­nat­ing bac­te­ria. If pos­si­ble, di­rectly in­ oc­u­late the sam­ple to ap­pro­pri­ate me­dia.

Dimorphic Fungi (Blastomyces, Coccidioides, Histoplasma, Paracoccidioides, and Sporothrix spp.) Process spec­i­mens (e.g., re­spi­ra­tory spec­i­mens and wound as­pi­ rates) promptly to avoid over­growth of con­tam­i­nat­ing bac­te­ria. Do not use swabs, be­cause these or­gan­isms are sus­cep­ti­ble to des­ic­ca­tion. Histoplasma capsulatum can be re­cov­ered in fun­gal blood cul­tures, par­tic­u­larly from pa­tients with AIDS and other im­mu­no­sup­pres­sive dis­eases. The ly­sis-centrifugation sys­tem (Isolator [Wampole]) and the MycoF/Lytic blood cul­ture bot­tle (Becton Dick­in­son) are use­ful for the iso­la­tion of di­mor­phic molds from blood.

Eumycotic Mycetoma Agents Examine pus, ex­u­date, or bi­opsy ma­te­rial for the pres­ence of gran­ ules (scle­ro­tia) con­sist­ing of the eumycotic agents and ma­trix ma­ te­rial. Wash the gran­ules with sa­line con­tain­ing an­ti­bi­ot­ics (e.g., pen­i­cil­lin and strep­to­my­cin), and then cul­ture them. Organisms can be vi­su­al­ized by ex­am­in­ing crushed gran­ules mi­cro­scop­i­cal­ly.

Moniliaceous Fungi Process spec­i­mens (e.g., bi­opsy spec­i­mens, lower re­spi­ra­tory se­cre­ tions, nails, eye spec­i­mens) promptly to avoid over­growth of con­ tam­i­nat­ing bac­te­ria. Do not trans­port spec­i­mens on swabs, be­cause or­gan­isms are sus­cep­ti­ble to des­ic­ca­tion. Specimens should be ex­ am­ined mi­cro­scop­i­cally and cul­tured. Fusarium spp. are among the few fil­a­men­tous fungi that can be re­li­ably re­cov­ered in blood cul­ tures and will oc­ca­sion­ally grow in rou­tine bac­te­rial blood cul­tures. Fungal Diagnosis Collect blood spec­i­mens in the ly­sis-centrifugation sys­tem (Isola­ tor) and/or the MycoF/Lytic blood cul­ture bot­tle. Serologic tests are avail­­able for some of these fun­gi.

Pneuo ­m ­cys­tis (ca­ri­nii) jiroveci Respiratory spec­i­mens should be lim­ited to in­duced sputa or bron­ chos­copy spec­i­mens. Patients can only rarely ex­pec­to­rate spu­tum, and throat wash­ings are in­sen­si­tive. Collect first morn­ing spec­i­mens when pos­si­ble. A 24-h col­lec­tion is un­ac­cept­able. The pres­ence of oral con­tam­i­na­tion, sig­ni­fied by squa­mous ep­i­the­lial cells, does not in­val­i­date the spec­i­men. 244 SECTION 6 Yeast Yeasts are rel­a­tively easy to iso­late from clin­i­cal spec­i­mens, al­ though over­growth of con­tam­i­nat­ing bac­te­ria should be avoided. Yeasts are iso­lated com­monly from blood spec­i­mens. Lysis cen­tri­ fu­ga­tion, bi­phasic sys­tems, and the MycoF/Lytic blood cul­ture bot­ tle are re­li­able meth­ods for iso­lat­ing yeasts from blood. Automated con­tin­u­ous-monitoring sys­tems are re­li­able for com­mon yeasts but less re­li­able for Cryptococcus neoformans. Direct de­tec­tion in­ cludes mi­cros­copy (Gram stain, KOH, In­dia ink, Calcofluor white) and an­ti­gen tests. Serologic test­ing is avail­­able for an­ti­bod­ies to Candida spp. but is not com­monly used. Fungal Diagnosis Fungal Diagnosis 245 le c­ b n ­ fe ­­ ab ­ tio nts and c ­ te ­ me ­ op l B C B D D D D D D D D D D D D D D ­ ve l de ­ ca ­ ni ses but may be avail ic fungi are highly in Nucleic Nucleic acid de ­ po ­ ph r ur er tech ­ mo ­ th n ­ tic p s ­ tio ­ no t, fur ­ tec ­ ag ­ en B C C B C B D D D A D D D D D A D al di ­ er : al form these of di a ­ li ons. At pres ­ ti l for genl for ­ ce c ­ fu ­ fe n Antibody de l in m use ­ tio ­ ga ­ do ­ tec dia the of my Applicability of Applicability C B C B C A A D D A A D D D D D D ­ ni s of funs of ­ si ­ no ­ ag Antigen Antigen de on. ; C, test is sel ­ ti c ­ es ­ fe cted. The co anc ­ pe ­ st m s of in B A A A A A A A A A A A A A D A A ­ si ulture ­ cu C ­ no ry tools the for di ­ ag in cir ­ ta ­ ma ry di ­ py ­ to o r cer ­ c ­ ra ­ gi ­ de ­ o os ry if this agent is sus re as pri ­ cr ­ to ­ tu l un A A A A A A A A A A A A A A A A A ­ ra fu ­ ­ o on of fun ­ ti Direct Direct mi ­ ca fy the lab on. ­ ti ­ fi ­ ti ly ly not used for lab Fungal Diagnosis a ­ ti ­ al s; test B, is use ­ iz l y be used in the fu ­ si ­ er ­ so ­ bl ­ no spp.) ­ a ­ ag ses, please no e by aero ­ po l for dil for . ­ bl c c ­ fu Aspergillus ­ si ­ ed c s s; D, test is gen ques will prob ­ mi ­ rie ­ ni thods for the iden ma agents ly usely ­ gi ­ to c ­ al ­ to ­ ra are need ­ o ­ er Me ­ ce spp. spp. y trans spp. spp. s ­ il ­ tory safety pur ­ ies ­ ti spp.

spp. ­ ra ys ce lab ­ o spp. ­ c ­ en ­ mo al stud ­ er ­ c ­ i A, test is gen Nucleic acid tech For lab a b c Aspergillus Organism Candida Cryptococcus Trichosporon dermatitidis Blastomyces capsulatumHistoplasma brasiliensis Paracoccidioides marneffei Talaromyces schenckii Sporothrix Moniliaceous fungi (not Dematiaceous fun Dermatophytes Coccidioides Zygomycetes Eumycotic my Pneu Malassezia clin Table 6.1 in ref tious and eas 246 SECTION 6 Microscopy Acridine Orange Stain Acridine or­ange stains fungi red-orange, but the back­ground ma­ te­rial stains green-yellow. For stain de­tails, see sec­tion 4.

Calcofluor White Stain Calcofluor white is a non­spe­cific fluo­ro­chrome that binds to cel­lu­ lose and chi­tin in the cell walls of fungi. The dye can be mixed with 10% po­tas­sium hy­drox­ide so that mam­ma­lian cells can be dis­ solved, thus fa­cil­i­tat­ing vi­su­al­i­za­tion of fun­gal el­e­ments. Fungi (in­clud­ing Pneu­mo­cys­tis jiroveci) ap­pear green or blue against a dark back­ground when the stained slide is ex­am­ined un­der UV il­ lu­mi­na­tion. Care must be used to dis­tin­guish spe­cific stain­ing from stained de­bris. Optimal de­tec­tion of fluo­res­cence re­quires the use of a 400- to 500-nm ex­ci­ta­tion fil­ter and 500- to 520-nm bar­rier fil­ter.

Fluorescent-Antibody Stain Direct and in­di­rect fluo­res­ce­in-conjugated mono­clo­nal an­ti- Pneumocystis an­ti­bod­ies are used for im­mu­no­flu­o­res­cence as­says and tar­get a fam­ily of sur­face gly­co­pro­teins that con­tain both com­ mon and dis­tinct epi­topes within and among Pneu­mo­cys­tis spe­ cies. Depending on the mono­clo­nal an­ti­body sup­plied with the kit, stain­ing may tar­get only the cyst form or may tar­get all­ forms of the or­gan­ism. The typ­i­cal fluorophore that is con­ju­gated to the an­ ti­body or used in an in­di­rect as­say is fluo­res­cein isothiocynate, which pro­duces a bril­liant ap­ple green color. The stain­ing re­ac­tion shows a dif­fuse pat­tern dis­trib­uted over the sur­face of the en­tire clus­ter of or­gan­isms and of­ten over the ma­trix in which the or­gan­ isms are em­bed­ded. Single cysts usu­ally ap­pear with a dis­tinc­tive rim of fluo­res­cence and a duller in­te­rior fluo­res­cence.

Gi ­emsa Stain The Gi­emsa stain com­bines meth­y­lene blue and eo­sin. It is use­ful Fungal Diagnosis for the de­tec­tion of Histoplasma capsulatum in bone mar­row, pe­ riph­eral smears, and touch prep­a­ra­tions, as well as intracystic bod­ ies and tro­pho­zo­ites of Pneu­mo­cys­tis jiroveci in in­duced spu­tum, bron­chos­copy spec­i­mens, and lung tis­sue. H. capsulatum ap­pears as tiny blue-purple bud­ding yeast cells. The cyst wall of P. jiroveci ap­pears as a clear ring around spores or intracystic bod­ies. The nu­ clei stain red-purple, and the cy­to­plasm gen­er­ally stains light to dark blue.

Gram Stain The Gram stain de­tects most fungi if pres­ent. Most yeast ap­pear Gram pos­i­tive; how­ever, Cryptococcus and Malassezia spp. Fungal Diagnosis 247 stain weakly and in some in­stances ex­hibit only stip­pling. The hy­phae of molds gen­er­ally ap­pear Gram-negative. Pneu­mo­cys­tis pro­duces a neg­a­tive (pink) re­ac­tion with poorly de­fined or­gan­ism mor­phol­o­gy.

In­dia Ink Stain (Nigrosin) The use of In­dia ink is not tech­ni­cally a stain­ing method. Detec­ tion of en­cap­su­lated fungi (i.e., Cryptococcus neoformans) is made pos­si­ble by ex­clu­sion of the ink par­ti­cles by the po­ly­sac­cha­ride cap­ sule of the or­gan­ism. Care in in­ter­pre­ta­tion is re­quired be­cause ar­ti­facts (e.g., leu­ko­cytes, eryth­ro­cytes, pow­der, and bub­bles) may be con­fused with yeast cells. The mor­pho­logic char­ac­ter­is­tics of the yeast cells must be rec­og­nized be­fore the prep­a­ra­tion can be in­ter­ preted. Although a rapid de­tec­tion method for en­cap­su­lated yeasts, the In­dia ink pro­ce­dure is an in­sen­si­tive method for the de­tec­tion of Cryptococcus neoformans; cryp­to­coc­cal an­ti­gen test­ing (la­tex or en­zyme im­mu­no­as­say) is rec­om­mend­ed.

Kinyoun Stain Some as­co­my­ce­tous fungi pro­duce as­co­spores when grown on a me­dium that pro­motes their for­ma­tion. Ascospores are ac­id-fast and stain red, whereas the as­co­my­cete cell wall and cy­to­plasm ap­pear blue. (For stain de­tails, see sec­tion 4.)

Potassium Hydroxide (KOH) A 10 to 15% so­lu­tion of po­tas­sium hy­drox­ide can be used to dis­ solve cel­lu­lar and or­ganic de­bris and fa­cil­i­tate the de­tec­tion of fun­ gal el­e­ments, which are not af­fected by strong al­kali so­lu­tions (al­though fun­gal el­e­ments dis­solve af­ter ex­po­sure for a few days). The hy­phae of dematiaceous fungi can be dis­tin­guished from those of hy­a­line molds by their brown mel­a­nin pig­ment on these di­rect prep­a­ra­tions. Ink (e.g., per­ma­nent blue-black Par­ker Super Quick Ink) can be added as a con­trast­ing agent to aid the de­tec­tion of fungi. Lactophenol cot­ton blue (i.e., Poirrier’s blue) can also be added to the KOH. The an­i­line blue stains the outer cell wall of Fungal Diagnosis fungi, and the lac­tic acid is a clear­ing agent.

Toluidine Blue-0 Stain Toluidine blue-0 stain is used pri­mar­ily for the de­tec­tion of Pneu­ mo­cys­tis jiroveci in re­spi­ra­tory spec­i­mens. P. jiroveci cysts stain red­dish blue to dark pur­ple against a light blue back­ground. Tro­ phozoites do not stain by this method. This stain­ing method is rapid and in­ex­pen­sive, but some skill is re­quired to rec­og­nize P. jiroveci cysts (usu­ally pres­ent in clumps). Many lab­o­ra­to­ries pre­fer the di­ rect fluo­res­cent-antibody test for the de­tec­tion of P. jiroveci, even though the stain is more ex­pen­sive. 248 SECTION 6 a Illustration le le nly ­ su e or ­ mo ­ gl m n found ­ te ­ co ns ­ me ­ i es aped, sin nt; pseudohyphal ly roundly to oval; buds ns sh es ­ cyt ­ al ­ de - ­ i ­ me t; yeast un io ­ i ng cells;ng of ­ t l spec ­ tur ­ gar ­ en ­ di ­ ca ­ i l spec e and “pinched off”; cap ­ ca ­ i ­ gl e buds pres on of clin ly sinly ­ pl ered within his ­ ti ­ t ­ ti ­ al ­ na ­ i Characteristic Characteristic fea Oval to round bud clus mul Cells vary in size; usu usu may or may not be ev forms rare seen in clin Oval to round to ci ­ am ct ex ­ re m) μ Diam range ( 2–5 2–15 2–6 nts seen by di ­ me ­ e Fungal Diagnosis l el ­ ga rothrix schenckii rothrix ganism(s) yptococcus Or Histoplasma capsulatum Spo Cr neoformans ­ gal aracteristic fun Ch

­ ture found Morphologic fun struc forms Yeast Table 6.2 Fungal Diagnosis 249 (continued) a Illustration in ­ ma y t bl ­ ­ sule ­ en ­ monly ­ gle or l, dou m) thatm) ­ ten found r’s r’s wheel ­ com ­ ca ­ i ­ ne unded smaller by e but may re ­ i ­ mens ­ ro ­ gl ­ i may be pres shaped, sin ­ cytes ­ ally round to oval; buds ­ dent; pseudohyphal - ­ i y (mar ly sinly ­ mens ­ i ­ er ­ al ­ tures t cells broad by base ­ gar ­ ent; yeast un ­ ding cells; of ­ cal spec ph ­ en ­ i ­ ri ­ tum ­ cal spec ­ i ly largely and spher ly largely andsur ­ gle and “pinched off”; cap H. capsulatumH. ­ al ­ al ce); small cells (2–5 μ sep e e le; buds usu ­ an cted to par ­ bl ­ ti ple ­ ple buds pres ­ tral ar ­ tion of clin m ­ ti ac ­ ne ­ ally sin ­ pe ­ se ­ fr ­ na ­ i ap re Characteristic Characteristic fea con Fission yeast, do not bud; round to with oval a cen Cells usu Cellsusu buds around pe forms rare Oval to round bud ­ teredclus within his ­ tio Oval to round to ci mul Cells vary in size; usu usu may or may not be ev re seen in clin ­ am ­ rect ex m) μ Diam range ( 3 8–15 5–60 2–5 2–6 2–15 ­ ments seen by di ­ e Fungal Diagnosis ­ gal el Organism(s) Talaromyces marneffei Blastomyces dermatitidis Paracoccidioides brasiliensis Histoplasma capsulatum schenckii Sporothrix Cryptococcus neoformans ­ gal Characteristic fun

­ ture found struc Morphologic fun forms Yeast Table 6.2 250 SECTION 6 a Illustration ry ­ ta ­ i icted ae, ­ str ­ ph in (continued) ng; ­ ta ­ di ns al yeast cells that t, are con nts may be ­ me l walls ­ i e bud ov ­ en - ­ le ­ me ­ gl ­ e ched; true hy ­ al es ­ ta l spec al el ­ tur bit bit sin ae may be found in cav ­ ca in at ­ ph ­ i ­ hi ­ ph ­ ma te end t, have part, have ly ly ex ­ si ­ en ­ al ­ po ores; hy t along with round on of clin ­ sp ns ­ en ­ ti ­ do ­ sio ­ na ­ i Cells usu Characteristic Characteristic fea are round at one end and a flat collarette have at the op Short, curved hy pseudohyphae, when pres at ends and re when pres Spherules vary in size; some con en le pres ­ am ct ex ­ re m) μ 3–4 (yeast forms), 5–10 (pseudohyphae) 3–4 (yeast 2.5–4 forms), (pseudohyphae) 10–200 Diam range ( nts seen by di ­ me ­ e spp. Fungal Diagnosis spp. l el spp. ­ ga ndida Ca Malassezia Organism(s) Coccidioides aracteristic fun Ch re found

­ tu ­ phae Morphologic fun­ gal struc forms andYeast pseudohyphae or true hy Spherules Table 6.2 Fungal Diagnosis 251 (continued) g

es red or ­ in ­ gl ars ng; ­ tu in ­ di ­ pe ­ ta a are es ­ gi n ­ ul n frac ly ly ap ­ ra a con ­ te ­ al ­ gi n ly at rightly an spher ­ ra ae, of re spo ­ al ­ tu ­ ph g usu lled spo ­ in ke hy ­ li wa n - ­ bo or of adiacondium usu ­ ty ­ ri ­ te Large, round, thick walled, no bud in emp Large, thick Large, rib twisted, branch sporangiospores; ma larger than Coccidioides 20–140 10–30 6–300 Fungal Diagnosis spp. Emmonsia crescens Zygomycetes, Pythium Rhinosporidium seeberi te or ­ ta te ­ ta ­ sep ae ­ ph Rarely sep Sporangium Adiaconidia Wide non hy 252 SECTION 6 a Illustration r g ­ cu ­ in (continued) bit 45° 45° bit ae, dark ­ hi ns ­ ph e branch ­ me ­ i ­ gl te hy ae; chains of es ­ ta ­ ph l spec ­ tur te and may ex ­ ca ­ i te hy ­ ta ­ ta us) and 90° an gmented sep ­ mo pi ­ o - ng yeastlikeng forms may also oc on of clin ot ­ ti ­ di ­ ch ­ na ­ i Brown Characteristic Characteristic fea (di Hyphae are sep Branched, sep arthroconidia may be seen bud ­ am ct ex ­ re m) μ Diam range ( 2–6 3–12 3–15 nts seen by di g ­ in ­ me ­ e Fungal Diagnosis l el ­ line ­ a ­ ga ganism(s) oulds; Aspergillus, rganisms caus Or O phaeohyphomycosis Other hy m Fusarium Dermatophytes ­ gal aracteristic fun te Ch ­ ta

­ phae ­ ture found ae ­ tate hy ­ ph Morphologic fun struc Hyaline sep hy Dematiaceous sep Table 6.2 Fungal Diagnosis 253 l ­ ca ­ ti rs ­ cur n, D.C., 2002.n, D.C., ­ ing ­ te psed ­ to g nted and ­ la ­ in l and ver hibit 45° ­ hibit 45° ­ me ­ ta n ­ phae, dark ­ zo ­ gle branch ­ i ound ­ tate hy ­ gr ­ phae; chains of ar in small clus ­ pe ­ tate and may ex s are brown pig ­ tate hy ­ ie ­ mous) and 90° an pigmented sep ­ o - nt formsnt ap ­ ce ­ ding yeastlike forms may also oc ­ chot Brown Hyphae are sep (di Branched, sep arthroconidia may be seen bud Sclerotic bod thick walled and hor have Nonbudding, round, or col ovoid, cres against a foamy back septations 2–6 3–12 3–15 3–5 5–12 g ­ ing ­ in s s Fungal Diagnosis ­ ti ­ line ys ­ c i) jiroveci ­ mo ­ ni ­ ri rganisms caus Organisms caus phaeohyphomycosis Other hy ­ a moulds; Aspergillus, Fusarium Dermatophytes O Pneu (ca chromoblastomycosis ­ ies ­ tate s ­ phae ­ ite ­ zo o rotic ­ rotic bod ­ tate hy ­ ph ­ phae Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a hy Hyaline sep Dematiaceous sep Dematiaceous scle Cysts and tro 254 SECTION 6 Primary Plating Media Birdseed Agar Birdseed (also called niger seed) agar is used for the se­lec­tive iso­ la­tion and iden­ti­fi­ca­tion of Cryptococcus neoformans. The agar me­dium con­tains an ex­tract of Guizotia abyssinica seed, caffeic acid. C. neoformans pro­duces phe­nol ox­i­dase, and dark brown col­ o­nies de­velop in the pres­ence of caffeic acid. The me­dium con­ tains chlor­am­phen­i­col to sup­press the growth of bac­te­ria. Brain Heart Infusion Agar (BHI) Brain heart in­fu­sion agar is a nu­tri­tion­ally en­riched me­dium that can be used for the iso­la­tion of a va­ri­ety of fas­tid­i­ous bac­te­ria, yeast, and molds. It is pre­pared with in­fu­sions of calf brains and beef hearts, pep­tones, glu­cose, so­dium chlo­ride, and di­so­dium phos­ phate. Supplementation with 5 to 10% sheep blood can en­rich the me­dium, and the ad­di­tion of an­ti­bi­ot­ics (e.g., gen­ta­mi­cin, chlor­am­ phen­i­col, and pen­i­cil­lin) can make this me­dium se­lec­tive for fun­gi. CHROMagar Candida CHROMagar Candida is a se­lec­tive, dif­fer­en­tial agar me­dium for the iso­la­tion and pre­sump­tive iden­ti­fi­ca­tion of Candida albicans, C. krusei, and C. tropicalis. The me­dium con­sists of pep­tones, glu­ cose, chlor­am­phen­i­col, and “chro­mo­genic mix.” The an­ti­bi­otic in­ hib­its the growth of most bac­te­ria. C. albicans forms green col­o­nies, C. krusei forms pink col­o­nies, and C. tropicalis forms pur­ple col­o­nies. Dermatophyte Test Medium (DTM) Dermatophyte test me­dium is a se­lec­tive agar me­dium used for the iso­la­tion and iden­ti­fi­ca­tion of der­ma­to­phytes. It con­sists of di­gests of soy­bean meal sup­ple­mented with glu­cose, cy­clo­hex­i­mide, chlor­ tet­ra­cy­cline, gen­ta­mi­cin, and phe­nol red. The an­ti­bi­ot­ics sup­press

Fungal Diagnosis the growth of bac­te­ria, sap­ro­phytic yeasts, and molds. Dermato­ phytes grow­ing on this me­dium pro­duce al­ka­line by-prod­ucts that change the phe­nol red in­di­ca­tor from yel­low to red. This color change may be ob­scured when grossly con­tam­i­nated spec­i­mens (e.g., nails) are pro­cessed on this me­dium. The pig­ment pro­duced by der­ma­to­phytes, which is used for their iden­ti­fi­ca­tion, is ob­scured by the in­tense red color pro­duced on this me­di­um. Inhibitory Mold Agar (IMA) Inhibitory mound agar is an en­riched, se­lec­tive me­dium that is used for the iso­la­tion of path­o­genic fungi other than der­ma­to­phytes. It con­sists of di­gests of an­i­mal tis­sue and ca­sein, yeast ex­tract, Fungal Diagnosis 255 dex­trin, starch, glu­cose, salts, and chlor­am­phen­i­col. Contami­ nating bac­te­ria are in­hib­ited by chlor­am­phen­i­col. Mycosel (Mycobiotic) Agar Mycosel (Mycobiotic) agar is a se­lec­tive me­dium used for the iso­ la­tion of path­o­genic fungi from con­tam­i­nated spec­i­mens. Mycosel and Mycobiotic (BD Diagnostic) agars con­sist of di­gests of soy­bean meal sup­ple­mented with glu­cose, cy­clo­hex­i­mide, and chlor­am­phen­ i­col. Cycloheximide-susceptible fungi, in­clud­ing Cryptococcus neoformans, Pseudallescheria boydii, the zygomycetes, many spe­ cies of Candida and Aspergillus, Trichosporon spp., and most sap­ ro­phytic or op­por­tu­nis­tic fungi, do not grow on this me­di­um. Sabouraud Agar–Brain Heart Infusion (SABHI) Sabouraud agar–brain heart in­fu­sion (SABRI), an en­riched agar me­dium, is a var­i­a­tion of Sabouraud dex­trose agar (de­scribed be­ low). The me­dium con­sists of in­fu­sions of beef heart and calf brains, pep­tones, salts, glu­cose, blood, and chloromycetin (chlor­ am­phen­i­col). It is used for the cul­ti­va­tion of der­ma­to­phytes and other path­o­genic and non­patho­genic fun­gi. Sabouraud Dextrose Agar (SDA) Sabouraud dex­trose agar is an en­riched agar me­dium used for the iso­la­tion of sap­ro­phytic and path­o­genic fungi. The orig­i­nal for­mu­ la­tion of SDA con­sists of di­gests of ca­sein and an­i­mal tis­sue sup­ ple­mented with 4% glu­cose and ad­justed to pH 5.6. The Emmons mod­i­fi­ca­tion is pre­ferred by many my­col­o­gists. It con­tains a re­ duced con­cen­tra­tion of glu­cose (2%) and is buff­ered to neu­tral­ity (pH 6.9). Yeast, der­ma­to­phytes, and other fil­a­men­tous fungi grow on these me­dia. The orig­i­nal for­mu­la­tion of SDA was acidic to sup­ press the growth of bac­te­ria. This prob­lem can be cir­cum­vented by the ad­di­tion of an­ti­bi­ot­ics (e.g., cy­clo­hex­i­mide and chlor­am­phen­i­ col) to the me­dia. However, cy­clo­hex­i­mide-susceptible fungi (re­fer to Mycosel agar above) do not grow on this me­di­um. Yeast Extract-Phosphate Agar Fungal Diagnosis Yeast ex­tract-phosphate agar is a se­lec­tive me­dium used for the iso­ la­tion of path­o­genic fungi such as Histoplasma and Blastomyces spp. It con­sists of yeast ex­tract and phos­phate buffer sup­ple­mented with chlor­am­phen­i­col to sup­press the growth of bac­te­ria. The pH is ad­justed to 6.0. 256 SECTION 6 e,

­ in ems ms; for ms. ­ am ­ t ­ is ­ is ­ gan ­ gan ge, ge, ex r r ted sys ­ fu i e oil to plates. on at 2,000 x g on at 2,000 x g ­ ma ­ tr ­ ti ­ ti g C or X marks. ­ iv ­ gal o ­ gal o ated loop onto a ­ to a. ­ in ­ ga ­ ga ­ br ­ i ­ di ­ fu ­ fu i i ­ tr ­ tr ate fun ate fun on; au ctly us ­ tr ­ tr ­ ti n n ­ re ­ ga t, wash, cen n), and ESP if (Trek); cted, add ol ­ ce ­ ce es. ­ fu ­ en ­ so i ­ pe a di ­ ul ­ tr ­ in on or cen on or cen ­ di ­ ti ­ ti on of urines, uncentrifuged urine sus a a ­ ti ­ tr ­ tr es pres ­ ca ­ ul ­ fi ­ ti ude BacT/Alert (bioMerieux),ude BacT/Alert BACTEC ­ cl 2 ml, fil Malassezia >2 ml, fil Lysis Lysis and cen in (Becton Dick min 10 for to con plate noninhibitory of me Inoculate me > Comments quan can be streaked with a cal min 10 for to con If gran and crush gran act, act te ­ tr ­ tr ­ pha MA, Mycosel IMA, Mycosel, yeast ex phos I Selective IMA, Mycosel, yeast ex a) ted ­ di ­ ma ­ to × × × × × × ems me ­ t sys × (or au Enriched SABHI, SDA, BHI b × MAF for Nocardia Fungal Diagnosis guide

a ing ­ O N O O O N O m ­ a cology plat cology Direct Direct ex (wet mount,(wet Calcofluor KOH) white, My e ­ va

s ­ ti

­ ag ­ row ­ id nc neal lid, ­ ju ­ id rapings itreous Cor Sc Eye con V flu Body flu

Bone mar Eye Source Blood Exudates, pus, drain Table 6.3 Fungal Diagnosis 257 ry est is s ­ sa ­ qu ­ si a, please ­ es ­ di opic ct ­ sc m ctly sed on ­ re o ss the re ­ tu re me ­ re ­ cr ­ no ­ le

­ tu ­ er ­ ag h

d di ­ ing ct mi cted, add ­ ac sed on the ba ­ be al cul tic agent; nec ­ re ly ly di ced spu ­ pe ms and di ­ ri rmed un ­ no ­ ly ­ al on of ­ te ­ to ­ du ­ fo ­ ag ­ ti ­ co sus c on. ­ ti ­ te e bac ly dily ­ at ­ na l symp s is usu i ­ i ­ al ms and di ­ si on for in ­ pr ­ ca ­ to ­ a ­ i o ­ ti ­ am spp. ­ di ­ pr ), or use stom of ­ ga s ­ di ­ ti ­ fu i on. ys l symp ­ ti ­ tr g should not be per ns for the de ­ c opic opic ex s of clins of ­ ca ­ in ­ i ­ na ­ si ­ sc ­ i ­ me ­ mo ry. For apry. o ­ i e oil to plates. ­ to ­ cr ­ am ­ iv ­ ra ­ o of of clin ex Candidiasis is usu Cut into small pieces and em into agar; if Malassezia ol Liquefication with a mu with cen spec grind (zygomycetes), Mincing ( Histoplasma Vaginal can Pneu the ba mi ; N, stain ­ ed y lab est ­ og l ­ qu act, act,

IMA IMA ­ co I MA ­ tr ­ tr ate, ate, ed ed ­ ph ­ ph ­ se ­ se rmed if re CHROMagar Candida, IMA, Mycosel, DTM CHROMagar Candida, IMA, Mycosel, yeast ex phos bird CHROMagar Candida, IMA, Mycosel, yeast ex bird CHROMagar Candida, phos ­ fo ed through the my ­ su × × × nly pur ­ mo R or Gram stain onal and should be per ­ ti × × g is op res are com ok. ­ in ­ tu ­ bo Fungal Diagnosis rmed; stain O, ­ fo on this 4 of hand um, stains and cul ­ ti ­ ri R R ­ te nely per ­ ti ian. ­ c ­ si is a bac , O (TCP stain) R R, O (PCP stain, lung) R, Gram stain s Nocardia ­ ing ­ ing should be rou ­ tions ssed with the phy gina ­ cre ­ cu R, stain Although a b Mouth Nails, hair, skin scrap Respiratory se Tissue Sinus Va see Primary Plating Media in sec dis 258 SECTION 6 Specific Diagnostic Tests Abbreviation Guide. CF, com­ple­ment fix­a­tion; CIE, count­er- immunoelectrophoresis; EIA, en­zyme im­mu­no­as­say; ID, im­mu­ no­dif­fu­sion; IFA, in­di­rect fluo­res­cent-antibody test; LA, la­tex ag­glu­ti­na­tion; RIA, ra­dio­im­mu­no­as­say; TA, tube ag­glu­ti­na­tion; TP, tube pre­cip­i­tin. Aspergillus Species. Microscopy and cul­ture are sen­si­tive de­tec­ tion meth­ods for Aspergillus spe­cies. Molecular meth­ods are be­ing de­vel­oped for both di­rect spec­i­men and Aspergillus spp. iden­ti­fi­ ca­tion. EIA and RIA for Aspergillus an­ti­gens and CF, CIE, and ID tests for an­ti­bod­ies have been de­vel­oped. Antigen tests are used pri­mar­ily to di­ag­nose in­va­sive as­per­gil­lo­sis. Commercial EIAs de­tect galactomannan (GM) in se­rum and re­spi­ra­tory spec­i­mens. The tests have a sen­si­tiv­ity be­tween 71 and 95% (higher when mono­clo­nal an­ti­bod­ies are used) and good spec­i­fic­ity. Antibody tests are most sen­si­tive for im­mu­no­com­pe­tent pa­tients with al­ler­ gic bronchopulmonary as­per­gil­lo­sis (ABPA), pul­mo­nary aspergil­ loma, and in­va­sive as­per­gil­lo­sis (IA). The sen­si­tiv­i­ties of the ID and CIE tests are com­pa­ra­ble, while ID is more spe­cific. The CF test is more spe­cific but less sen­si­tive than ID. The sen­si­tiv­ity of ID is im­ proved by the use of mul­ti­ple an­ti­gens from A. fumigatus, A. fla­ vus, A. niger, and A. terreus. Precipitins are pres­ent in more than 90% of pa­tients with aspergillomas, 70% of pa­tients with ABPA, and fewer pa­tients with IA. A four­fold con­cen­tra­tion of se­rum and re­test­ing is rec­om­mended for pa­tients with sus­pected IA and a neg­ a­tive ID re­sult. The ID is highly spe­cific, with false-positive pre­ cip­i­tins de­vel­op­ing only against C-re­ac­tive pro­tein. A com­ple­ment fix­a­tion an­ti­body ti­ter of 1:8 is con­sid­ered pos­i­tive. Skin test re­ac­ tiv­ity to Aspergillus an­ti­gen ex­tracts is use­ful for pa­tients with sus­ pected al­ler­gic bronchopulmonary as­per­gil­lo­sis, atopic der­ma­ti­tis, or al­ler­gic asthma sen­si­tized to as­per­gil­li. Blastomyces dermatitidis (Blastomycosis). Demonstration of broad-based bud­ding yeast cells and cul­ture are the most re­li­able Fungal Diagnosis de­tec­tion meth­ods for B. dermatitidis. This or­gan­ism can be iden­ ti­fied by us­ing mi­cro­scopic and mac­ro­scopic mor­phol­ogy as well as mo­lec­u­lar probe and exoantigen meth­ods. Due to the lack of sen­ si­tiv­ity and spec­i­fic­ity of avail­­able se­ro­logic tests (ID, EIA, CF, and RIA), these tests are gen­er­ally not help­ful for di­ag­nos­ing blas­ to­my­co­sis. The use of the A an­ti­gen, ob­tained from yeast cul­ture fil­trates, has im­proved the spec­i­fic­ity of these tests. The com­mer­ cially avail­­able EIA is more sen­si­tive but less spe­cific than ID. An EIA ti­ter of 1:32 or greater is con­sid­ered di­ag­nos­tic for blas­to­my­ co­sis. Titers of 1:8 to 1:16 should be con­firmed with ID or cul­ture be­cause cross-re­ac­tiv­ity with Histoplasma an­ti­bod­ies can oc­cur at this level. ID has a sen­si­tiv­ity of ap­prox­i­ma­tely 80% and a spec­i­ Fungal Diagnosis 259 fic­ity ap­proach­ing 100%. Antibodies are de­tected within 1 month of on­set, and the level de­clines with suc­cess­ful treat­ment. RIA has a sen­si­tiv­ity and spec­i­fic­ity sim­i­lar to ID but is rarely used. A CF an­ti­body ti­ter of ≥1:8 is con­sid­ered pos­i­tive. This test is rel­a­tively in­sen­si­tive and non­spe­cific for blas­to­my­co­sis and has gen­er­ally been re­placed by EIA and ID. Blastomyces urine an­ti­gen test­ing can be use­ful in di­ag­nos­ing in­fec­tion, but there is near com­plete cross-re­ac­tiv­ity in cases of Histoplasma in­fec­tion, and thus pos­i­ tive re­sults do not con­firm Blastomyces in­fec­tion. Candida Species (Candidiasis). Candida spe­cies can be de­ tected by di­rect ex­am­i­na­tion with Gram stain, KOH, or Calcofluor white prep­a­ra­tions. Candida spe­cies grow well on most cul­ture me­ dia. The iden­ti­fi­ca­tion of Candida spe­cies can be pur­sued through use of color pro­duc­tion on dif­fer­en­tial agars, germ tube pro­duc­tion, sugar as­sim­i­la­tion and/or fer­men­ta­tion, tem­per­a­ture tol­er­ance, cy­ clo­hex­i­mide tol­er­ance, and urea and ni­trate test­ing. Currently, yeast iden­ti­fi­ca­tion us­ing nu­cleic ac­ids is not widely prac­ticed. LA, ID, and CIE tests for an­ti­bod­ies and EIAs for an­ti­gens have been de­ vel­oped for the di­ag­no­sis of can­di­di­a­sis. In gen­eral, the sen­si­tiv­ity and spec­i­fic­ity of avail­­able tests are low. Coccidioides Species (Coccidioidomycosis). Microscopy and cul­ture are re­li­able de­tec­tion meth­ods for Coccidioides spe­cies. There are two spe­cies of Coccidioides: C. immitis and C. posada­ sii. C. immitis can be iden­ti­fied us­ing mi­cro­scopic and mac­ro­scopic mor­phol­ogy as well as Accuprobe and exoantigen meth­ods, while mo­lec­u­lar meth­ods are re­quired to de­fin­i­tively iden­tify C. posada­ sii. CF, TP, ID, and EIA have been de­vel­oped for de­tec­tion of an­ti­ bod­ies against C. immitis. The test an­ti­gens, called coccidioidin, are pre­pared from fil­trates of my­ce­lial cul­tures. Two pri­mary an­ti­ gens are used: a heat-stable 120-kDa gly­co­pro­tein (fac­tor 2 an­ti­ gen) lo­cated in the walls of arthroconidia and spher­ules, and a heat-labile 110-kDa chitinase en­zyme (F an­ti­gen). The for­mer pro­ tein is de­tected in the TP test, and the lat­ter is de­tected in the CF

test. Both an­ti­gens can be de­tected in the ID test and EIAs. An­ Fungal Diagnosis other an­ti­gen, spherulin, is pre­pared from spher­ules of C. immitis and has been used in CF tests. Factor 2 an­ti­gen is not spe­cific for C. immitis and is also found in mor­pho­log­i­cally sim­i­lar sap­ro­phytic fungi. The TP test is used to de­tect early dis­ease (80% pos­i­tive at 2 to 3 weeks, dis­ap­pear­ing by 6 months), and the CF test de­tects per­sis­tent an­ti­bod­ies. TP re­mains pos­i­tive in pa­tients with dis­sem­ i­nated dis­ease. The com­bi­na­tion of CF and TP tests is pos­i­tive in more than 90% of in­fected pa­tients. ID is com­pa­ra­ble to CF and TP. The com­mer­cially pre­pared EIA mea­sures both im­mu­no­glob­ u­lin M (IgM) and IgG an­ti­bod­ies. Both tests must be per­formed for max­i­mum sen­si­tiv­ity. A pos­i­tive EIA re­sult must be con­firmed by ID. CF an­ti­body ti­ters of 1:2 to 1:4 usu­ally in­di­cate early, re­sid­ual, 260 SECTION 6 or men­in­geal dis­ease. Antibody ti­ters of 1:16 in­di­cate dis­sem­i­nated dis­ease. Negative ti­ters do not ex­clude the dis­ease. Coccidioidin skin tests are of lim­ited use­ful­ness, al­though fail­ure to de­velop a pos­i­tive skin test has been as­so­ci­ated with poor re­sponse to ther­a­py. Cryptococcus neoformans (Cryptococcosis). Microscopy and cul­ture are use­ful de­tec­tion meth­ods for C. neoformans. Crypto­ coccus grows read­ily in cul­ture but is in­hib­ited by cy­clo­hex­i­mide. Cryptococcal an­ti­gens can be mea­sured by LA and EIA. EIA is more sen­si­tive for cap­su­lar glucuronoxylomannan po­ly­sac­cha­ride, and the method is suit­able for test­ing of mul­ti­ple spec­i­mens. Titers are gen­er­ally de­ter­mined us­ing the LA method. A ti­ter of 1:8 or greater in se­rum or ce­re­bro­spi­nal fluid (CSF) is con­sid­ered di­ag­ nos­tic. Titers of 1:4 or less may be in­dic­a­tive of early dis­ease or non­spe­cific re­ac­tions (prozone, pa­tients with rheu­ma­toid ar­thri­tis, syn­er­e­sis fluid, plat­i­num wire loops, Capnocytophaga [DF-1], Trichosporon beigelii, dis­in­fec­tants, and soaps). These non­spe­cific re­ac­tions have been doc­u­mented by LA only. Titers in CSF can be help­ful in mon­i­tor­ing ther­apy when the ti­ters are tested over ap­pro­ pri­ate in­ter­vals (at least 2 weeks). Interpretation of fol­low-up ti­ters is some­times dif­fi­cult be­cause the an­ti­gen is har­bored within the body; there­fore, the de­fin­i­tive de­ci­sion of­ten de­pends on the re­sults of cul­ture. More than 99% of pa­tients with cul­ture-confirmed cryp­ to­coc­co­sis have pos­i­tive an­ti­gen tests. IFA, EIA, and LA have been de­vel­oped for mea­sur­ing cryp­to­coc­cal an­ti­bod­ies. These tests are not use­ful for di­ag­no­sis be­cause cap­su­lar po­ly­sac­cha­ride may in­hibit an­ti­body syn­the­sis or mask the pres­ence of an­ti­body. Anti­ body test­ing may have prog­nos­tic value dur­ing the re­cov­ery of non- AIDS pa­tients. Histoplasma capsulatum (Histoplasmosis). Definitive di­ag­no­sis of his­to­plas­mo­sis re­quires growth of the fun­gus. Mycelial forms ma­ture within 20 days and dis­play di­ag­nos­tic tu­ber­cu­late macro­ conidia. Identification is per­formed by mound-yeast con­ver­sion, Ac­

Fungal Diagnosis cuprobe, and exoantigen meth­ods. CF, EIA, LA, and ID have been de­vel­oped to mea­sure an­ti­bod­ies to H. capsulatum. RIA and EIA have been de­vel­oped to de­tect Histoplasma an­ti­gens in urine and se­rum. The CF test is sen­si­tive (more than 90% of cul­ture-confirmed pa­tients have an­ti­bod­ies), but cross-re­ac­tions can oc­cur in pa­tients with blas­to­my­co­sis, coccidiodomycosis, other my­co­ses, and leish­ man­i­a­sis. Two an­ti­gens are used in the CF test: yeast phase an­ti­gen and my­ce­lial phase an­ti­gen (histoplasmin). CF an­ti­bod­ies de­velop within 4 weeks af­ter ex­po­sure in pa­tients with pul­mo­nary in­fec­ tions, with an­ti­bod­ies against the yeast phase be­ing de­tected first and those against histoplasmin de­vel­op­ing later. Patients with chronic his­to­plas­mo­sis gen­er­ally have higher ti­ters to histoplasmin. Antibody ti­ters be­tween 1:8 and 1:32 are con­sid­ered pre­sump­tive Fungal Diagnosis 261 ev­i­dence of his­to­plas­mo­sis; how­ever, high ti­ters can be ob­served in pa­tients with other dis­eases, so se­rol­ogy should be con­firmed by cul­ture. Antigen EIA and RIA pro­vide rapid di­ag­no­sis, but cross- re­ac­tiv­ity oc­curs with other my­co­ses. Cross-reactivity does not de­ tract from the value of these tests be­cause, de­pend­ing of the se­ver­ity of the clin­i­cal pic­ture, an­ti­fun­gal ther­apy is es­sen­tially the same. If these tests are used, the re­sults must be con­firmed by ID tests. The ID test can de­tect as many as six pre­cip­i­tin bands when histo­ plasmin is used as the test an­ti­gen. Two bands, H and M, have di­ ag­nos­tic value. The M band gen­er­ally ap­pears first and is an in­di­ca­tor of early dis­ease. The pres­ence of both the M and H bands is in­dic­a­tive of ac­tive dis­ease, past dis­ease, or re­cent skin test­ing. The pres­ence of both M and H bands is con­sis­tent with ac­tive dis­ ease. The LA test is used to de­tect acute his­to­plas­mo­sis, with re­ac­ tiv­ity oc­cur­ring 2 to 3 weeks af­ter ex­po­sure. Positive re­ac­tiv­ity should be con­firmed with the ID test. A heat-stable po­ly­sac­cha­ride an­ti­gen can also be de­tected in se­rum, urine, and CSF spec­i­mens in pa­tients with dis­sem­i­nated his­to­plas­mo­sis (90% sen­si­tiv­ity), as well as lo­cal­ized pul­mo­nary dis­ease (<50% sen­si­tiv­ity). The urine test is the most sen­si­tive for dis­sem­i­nated dis­ease, but false-positive re­ac­tions have been re­ported with other dis­eases (e.g., Coccidioido­ mycosis paracoccidioidomycosis, penicilliosis, and blas­to­my­co­ sis). Positive re­ac­tions should be con­firmed with cul­ture. Malassezia Species. Direct ex­am­i­na­tion and cul­ture are the meth­ods of choice for the de­tec­tion of Malassezia spe­cies. All spe­ cies are li­po­philic (ex­cept M. pachydermatitis) and re­quire the ad­ di­tion of long-chain fatty ac­ids (e.g., ster­ile ol­ive oil) to cul­ture me­dia for growth. Malassezia ex­ists both as a skin com­men­sal and as an eti­o­log­i­cal agent of cu­ta­ne­ous and sys­temic dis­ease. Differ­ entiation of li­po­philic spe­cies is not gen­er­ally per­formed. Paracoccidioides brasiliensis. Diagnosis is es­tab­lished when di­ rect ex­am­i­na­tion dem­on­strates the or­gan­ism’s char­ac­ter­is­tic “ship’s wheel.” Mycelial forms ma­ture within 21 days, but their pres­ence is

not di­ag­nos­tic. Mold-yeast con­ver­sion or exoantigen test­ing is nec­ Fungal Diagnosis es­sary for de­fin­i­tive iden­ti­fi­ca­tion. CF, ID, EIA, and CIE have been de­vel­oped to mea­sure an­ti­bod­ies to P. brasiliensis. The CF test de­ tects an­ti­bod­ies (ti­ter of 1:32 or greater) in at least 80 to 95% of pa­ tients with paracoccidioidomycosis, while pos­i­tive se­ro­logic test re­sults are re­ported for 98% of pa­tients when both the CF and ID tests are used. Cross-reactivity with H. capsulatum can oc­cur in the CF test. Declining CF ti­ters are con­sis­tent with a re­sponse to ther­ apy, and the pres­ence of per­sis­tently high ti­ters in­di­cates a bad prog­ no­sis. One to three unique pre­cip­i­tin bands are ob­served in the ID test. Antigen 1 has been char­ac­ter­ized as a 43-kDa gly­co­pro­tein. This an­ti­gen has also been used in EIA. Both the CF and ID tests are avail­­able through the CDC. 262 SECTION 6 Talaromyces (Penicillium) marneffei. The di­ag­nos­tic test of choice is dem­on­stra­tion of fis­sion yeast cells in di­rect ex­am­i­na­tions and the re­cov­ery of T. marneffei in clin­i­cal spec­i­mens. These in­ fec­tions are usu­ally dis­sem­i­nated, with mul­ti­ple-organ in­volve­ment in­clud­ing lymph­ad­e­ni­tis, sub­cu­ta­ne­ous ab­scesses, bone le­sions, ar­ thri­tis, spleno­meg­aly, and le­sions in the lungs, liver, or bow­el. Pneu­mo­cys­tis jiroveci. Demonstration of the or­gan­ism in clin­i­cal spec­i­men by mi­cros­copy is di­ag­nos­tic. P. jiroveci grows poorly in cell cul­ture, and re­li­able an­ti­gen and an­ti­body tests have not been de­vel­oped. The pres­ence of the PCR prod­uct has not been strictly cor­re­lated with dis­ease. Toluidine blue 0, Calcofluor white, and me­ the­na­mine sil­ver stain the cyst wall; Gram Weigert and Papani­ coalou stain the intracystic bod­ies and faintly stain troph­ o­zo­ite forms; Gi­emsa and fluo­res­ce­in-labeled an­ti­bod­ies (IFA, DFA) stain both cysts and tro­pho­zo­ites. Sporothrix schenckii (Sporotrichosis). Isolation and mold-yeast con­ver­sion are re­quired for the di­ag­no­sis of spo­ro­tri­cho­sis. Al­ though an­ti­gen and an­ti­body tests are avail­­able, they are not widely used. EIA, LA, and TA can be used re­li­ably to de­tect an­ti­ bod­ies to S. schenckii, while the CF and ID tests are less re­li­able and are not rec­om­mended. Antibodies to at least two cell wall an­ ti­gens (40- and 70-kDa an­ti­gens) are de­tected. EIA ti­ters of at least l:16 in se­rum and l:8 in CSF are con­sid­ered di­ag­nos­tic. Elevated ti­ters can be ob­served, which de­cline with suc­cess­ful ther­apy. LA ti­ters of 1:4 or greater are con­sis­tent with dis­ease, al­though non­ spe­cific re­ac­tions can oc­cur at ti­ters of 1:8. Antibody ti­ters in the LA test do not change pre­dict­ably with ther­apy, so they can­not be used for prog­nos­tic pur­pos­es. Zygomycetes (Zygomycosis, Mucormycosis). EIA and ID have been de­vel­oped to de­tect an­ti­bod­ies in pa­tients with ac­tive zygo­ mycosis. The tests have a sen­si­tiv­ity of ap­prox­i­ma­tely 70% and a spec­i­fic­ity of greater than 90%. They are rarely used be­cause the eti­o­logic agents of zygomycosis grow rap­id­ly. Fungal Diagnosis Biomarkers of Invasive Fungal Infection Galactomannan. This is a di­ag­nos­tic test that de­tects cir­cu­lat­ing galactomannan in se­rum (a com­po­nent of Aspergillus cell wall) as a marker of in­va­sive as­per­gil­lo­sis (IA). The di­ag­no­sis of IA is of­ ten very dif­fi­cult due to the low yield of cul­ture, non­spe­cific symp­ toms, and the fact that those pa­tients at great­est risk for IA are of too un­sta­ble to un­dergo di­ag­nos­tic bi­opsy pro­ce­dures. The per­for­ mance of galactomannan de­tec­tion in se­rum yields rel­a­tively high sen­si­tiv­ity and spec­i­fic­ity, al­though false pos­i­tive re­sults have been noted in pa­tients re­ceiv­ing piperacillin/tazobactam. In ad­di­tion to Fungal Diagnosis 263 test­ing the se­rum, there is ev­i­dence that test­ing of re­spi­ra­tory spec­ i­mens may be use­ful in the di­ag­no­sis of pul­mo­nary as­per­gil­lo­sis. 1, 3 Beta-D-Glucan. Detection of cir­cu­lat­ing be­ta-D-glucan (BDG) is used as a marker of in­va­sive fun­gal in­fec­tion. BDG is a non­spe­cific com­po­nent of the cell wall of many fun­gal spe­cies. Im­ portantly, Cryptococcus and the Zygomycetes do not pos­sess this el­e­ment of their cell wall and there­fore neg­a­tive BDG re­sults do not rule out­ in­fec­tion with these or­gan­isms. In ad­di­tion, BDG is con­ tro­ver­sial due to its over­all poor per­for­mance. In par­tic­u­lar, false pos­i­tive re­sults (poor spec­i­fic­ity) are a sig­nif­i­cant prob­lem with this test, as a wide range of pre­dis­pos­ing fac­tors can lead to falsely pos­ i­tive re­sults. Notably, pa­tients ex­posed to gauze or med­i­ca­tions that have been fil­tered through a cel­lu­lose-containing mem­brane are among the most com­mon causes of false pos­i­tive re­sults.

The fol­low­ing ta­bles sum­ma­rize or­gan­isms de­scribed in the eighth edi­tion of the Manual of Clinical Microbiology. They are or­ ga­nized in par­al­lel with the dis­cus­sions of the or­gan­isms within the man­ual. Due to the de­pen­dence on phe­no­typic growth char­ac­ ter­is­tics for the iden­ti­fi­ca­tion of molds, the in­for­ma­tion sum­ma­rized in­cludes col­ony mor­phol­ogy, line draw­ings, and key dif­fer­en­tial char­ac­ter­is­tics. All line draw­ings are used with the au­thor’s per­mis­ sion and come from the book Medically Important Fungi, a Guide to Identification, fourth edi­tion (2002), by D. H. Larone. For fur­ ther or­gan­ism in­for­ma­tion, please re­fer to the eighth edi­tion of the Manual of Clinical Microbiology and the fourth edi­tion of Medi­ cally Important Fungi, a Guide to Identification. Fungal Diagnosis

264 SECTION 6

se ­da ­i ox henol P

3 KNO ­tion a ­z ­li uti

—— Urease

——— +* Trehalose

F——— Galactose

F* F* F* Lactose

F————

ab Sucrose ns

F* ­ me Maltose

­ i

———————— Fermentation of: Glucose

W—W——W——— F*

l spec Dulcitol

­ ca

+* ­ i

Trehalose Raffinose

+ — F — F —+—FF——FF——— Xylose

—————————— +* +* ted from clin Inositol

— +* ­ la Cellobiose

+* Melibiose

ntly ntly iso Galactose

+ + + — + + + ­ que

Assimilation of: Assimilation Lactose

—+———+—+—FF——FF——— —+—+—+—+—FFF— +* Sucrose

+*

Maltose Glucose

+ + +

cs cs of yeasts fre

a Ink a ­ di ­ ti In Capsule

­ is

r ­ te tubes Germ

­ ac Chlamydospores

− — −

ae ­ ph Pseudo—or true hy true Pseudo—or l char

­ ca ­ i Broth in Pellicle Fungal Diagnosis + + −—− +———————+———F———————— −+−+−———+——— − − − −+−—−++——+———+———

em Growth at 37°C at Growth + −—−—− + +————————+—F————F——— +−+−—−+—+++— + − — − — − + + + + + −+−+++−+++—+——— + + ++−+−—−+++—+++—++++F—F— − ++ + ++ ++−+−—−+++—+—+—+—+—F—F—FF——— + −—−+−+−—−+++—+———+—+—F———————— + −—− +———————————F—————+ +—————————————————+—— + + ­ ch +* +* ltural and bio e d Cu d,e c

c ­ sa ­ go C. krusei C. glabrata C. kefyr C famata C lambica C. ru Candida albicans C. catenulata dubliniensis C C. guilliermondii C. lipolytica C. lusitaniae parapsilosisC C. pintolopepsii C. tropicalis Species Table 6.4 Fungal Diagnosis 265 − − − − − — — ——— F* on; R, rare; the sugar F, is fer­ ­ ti ­ ac te re ­ si + + — — — — + — + — — — — — — + + — ­ po and to 37°C 33 C. for lipolytica. − − − − − − + + − —FFF—F — — — — — — — + + +* —* Manualof Clinical Microbiology, ed., ASM 11th + +——————+—— + +———————+— +* +* —* − +* +* +* tes may give the op ­ la + + −+−W−−−−−−−++ —+ + +———————+—— ———+—————————— +* +* + + + +* +* —* —* + + + + + + − — + + + + —+—+++———————++— — — +* r SS,r Warnock (ed.), DW res are C. for krusei to 45°C 43 ­ te ­ tu ­ a +* +* on; *, someon; iso *, r —* —* ­ ti ­ pe ­ ac + + − + +* +* +* +* ve reve — +* ­ ti ­ a +* + + + — + — + + + — ly doesly not. ­ al ol;—, neg ­ tr −* −* usu ly doesly not. ­ al ve conve ­ ti usu ­ a llFunke KC, G, Landry ML, Rich − − − + + + − on. ­ ti ­ ro tropicalis does not. Maximum growth tem ­ ta ++−−−++++++++++++−−−−−+−− − − − + + +−−−+++++++++++−−−−−−+−− + − — − + — — — n +* ­ me Fungal Diagnosis tropicalis se; C. − — − — + + − — − — + + + + + + − — − — + + + + + + −—− +———+———+———————————— krusei − − − + −* NT NT NT ­ no ­ i * +−—−—−+++—+————+ + − — − — + − — − — +−R−—++++—+—+++++−−−−−+−−−+−−−−−+−−−−−−−−− + ++ −—−+ +———+——————————————— — —−—−—++++—++++++++——————+—— — − — − — + + + + — +* +* +* se; C. — —* —* ­ no ced); W, weakced); fer W, ­ lates L—arab tes rham ­ i tes erythritol; C. ­ la ­ du ­ la ­ i ­ i ­ sim m m ­ si as ­ si n, D.C., 2015. n, D.C., as ­ to as g ­ in Symbols: growth +, greater than that the of neg C. lusitaniae Adaptedfrom Jorgensen JH, Pfaller MA, Car C. parapsilosis C. lipolytica a b c d e C. terreus S. cerevisiae C. luteolus R. rubra C. laurentii R. glutinus C. albidus G. candidumG. C. zeylanoides neoformans Cryptococcus uniguttulatus C. C. peliculosa B. capitalus wickerhamii Prototheca salmonicolor Sporobolomyces asahii Trichsoposon mucoides Trichosporon Trichosporon ovoides mented gas (i.e., is pro Press, Wash 266 SECTION 6 + + − + − + − + V V — — — — — T. ovoides T. − + + + + + + + + + − + W W NT T. mycotoxinivorans T. + + − + + + + + + + + + − + — T. mucoides T. - NT T. loubieri r SS, Warnock (ed.), DW Manual of Clinical Microbiology, ed., ASM 11th ­ te − + − V + + T. inkin T. a s ­ cie spe − ++++ —+ —+ — — V — + — + V V + −+ V + — + − —— V — + + —— — + + T. cutaneum ll Funke KC, G, Landry ML, Rich ­ ro − + + − + V V V V cted Trichosporon Fungal Diagnosis T. asteroides T. ­ le + + — + − + V V —— —V ———— —— T. asahii aracteristics se of n, D.C., 2015. n, D.C., t cells − ­ to Ch g ­ an

­ in litol elibiose alactitol ibitol affinose –Arabinitol rbitol L–Rhamnose M R R Xy L G Adapted from Jorgensen JH, Pfaller MA, Car a Appresoria Fusiform gi Assimilation of: Cycloheximide0.01% Cycloheximide0.1% Characteristic So Growth at 37°CGrowth at 42˚CUrease + − Press, Wash Table 6.5 Table Fungal Diagnosis 267 rse ­ ve (continued) wdery, ack, re po - bl - ­ low Biseriate Biseriate Smooth, straight long, A. niger Velvety buff yel h ­ is ­ low rse to gold wdery, rk yel ­ ve po - da wn - bro - ­ low Biseriate Biseriate Rough A. flavus Velvety green, re red yel n ta - wdery, po - een to gray, gr - erse white ­ v Velvety A. fumigatus blue re Unseriate Unseriate Smooth cs ­ ti a y y s ­ is ­ g ­ g r ­ o ­ o ­ cie ­ te ol ol ­ ac ­ ph ­ ph spe Diagnostic char Colony mor Seriation Conidiophore Microscopic mor Fungal Diagnosis aracteristics Aspergillus of Ch

Illustration Table 6.6 268 SECTION 6 een w, w, e gr - ­ lo ­ abl ­ i se se var ­ ver Green to gray or tan with patches pinkof or yel re Biseriate Biseriate Smooth A. versicolor n ­ mo w to ­ lo ­ na n, D.C., 2002.n, D.C., rse yel ­ to g ­ ve ­ in tan Velvety, tan to cin Velvety, brown, re Biseriate Biseriate Smooth A. terreus rown, rown, b - ish rse buff to deep ­ pl ­ ve Biseriate Biseriate Smooth, short, brown re Velvety, dark green toVelvety, pur red A. nidulans cs ­ ti y y ­ is ­ g ­ g r ­ o ­ o ­ te (continued) ol ol a ­ ac s ­ ph ­ ph ­ cie Seriation Conidiophore Microscopic mor Diagnostic char Colony mor spe Fungal Diagnosis aracteristics Aspergillus of Ch

Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a Illustration Table 6.6 Fungal Diagnosis 269

- ­ low ss orssyel (continued) ­ le r ­ o rsecol ange ­ ve Trichoderma or Fluffy green, re erse white ­ v Gliocladium Fluffy dark green, re ly ly se se ­ al on ­ ver ­ si

- ­ ca ack; re ack; bl - r ly ly tan, oc ­ e ­ al nnamon or dark gray Scopulariopsis brown to Powdery, cream Powdery, ci usu dark h h, ­ is ­ is ­ low een; een; ite, pink gr se w or pale brown a - wh ­ lo - ­ gi ­ ver Paecilomyces yel re Velvety, Velvety, yel brown or mauve, brightnever green or blue off rse Fungal Diagnosis ­ ve nt, rule ­ me arneffei g pig ­ in s P. m ­ ­ fu Talaromyces out Velvet, green, reVelvet, white to cream; if red dif portunistic moniliaceous fun cs Op ­ ti y ­ is

­ g ­ g r ­ o ­ o ­ te ol ­ ac ­ phol ­ ph

Diagnostic char Microscopic mory Colony mor Table 6.7 270 SECTION 6 a­ ­ tr ­ lus il rse white; der ­ si ­ ve Fluffy cream to pink; re also con Engyodontium Beauveria n, D.C., 2002.n, D.C., The Paecilomyces range; range; imy, pinkimy, to o ­ to - g sl - ­ in ­ on rse pink or tan ­ ve re salm Moist Lecythophora w or ­ lo nt ff or ­ me rse light with bu - ­ ve eam, yel ng pig cr ­ i - s ­ fu green; re pale wine brown; green some have dif Flat, spready white Phialemonium h; s, s, ­ is ­ les ­ or ltlike, white, es are se se col fe w, or pinkw, ­ ci - ­ lo (continued) ­ ver a re ­ gi ­ se; tan, lightor pale gray, ro pale yel some spe dematiaceous Glabrous Acremonium le le ­ ab rse ­ i Fungal Diagnosis ­ ve der ­ si ce var ­ fa le (white tole dark on. t, or pink), re ­ ab ­ ti ­ i ­ le ­ o pink); also con Cylindrocarpon var Talaromyces Cottony sur in color (white, cream, vi portunistic moniliaceous fun cs Op ­ ti y

­ is ­ g ­ g r ­ o ­ o ­ te ol ­ ac ­ phol ­ ph Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a Diagnostic char Microscopic mory Colony mor Table 6.7 tion is from the third edi Fungal Diagnosis 271 (continued) erse white ­ v Fluffy dark gray, re 40°C Syncephalastrum rse ­ ve Fluffy re gray, white 45–50°C Lichtheimia erse white ­ v Fluffy gray to dark brown, re 54–58°C Rhizomucor own, br - erse white ­ v re Fluffy gray 45–50°C Rhizopus

- Fungal Diagnosis rse white ­ ve a rown, re Fluffy gray to gray b 37°C Mucor gomycetes cs Zy ­ ti y ­ is ­ g ­ g

r ­ o ­ o ­ te ol ­ ac ­ ph ­ phol Colony mor Diagnostic char Microscopic mory Maximum temp growth Table 6.8 Table 272 SECTION 6

ed with ry erse white ­ er ­ v ­ de g cov ­ in um, re ­ li m ­ ce abrous flat cream ­ co 35°C Conidiobolus be Gl my a white pow te ­ li n, D.C., Fungaln, D.C., Diagnosis, 2002 ­ el ­ to g ­ ia ­ in d ­ ni own; sat es formed by br - ­ ni ­ o 37°C Flat, buff waxy, to gray Basidiobolus col ejected co rse al ­ ci ­ ve nce - w, or w, ­ ha ­ lo n ­ tio own; re ­ la br a to en ­ u - ­ di llow; usellow; spe 42°C spor Apophysomyces Cream, yel gray white to pale ye me rse al ­ ve ­ ci nce ­ ha n ­ tio ­ la a to en ­ u ­ di <37°C Saksenaea Fluffy white, re me spor white; use spe Fungal Diagnosis (continued) a erse white ­ v 42°C Cunninghamella Fluffy gray, re gomycetes cs Zy ­ ti y ­ is ­ g ­ g

r ­ o ­ o ­ te ol ­ ac ­ phol ­ ph Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a Diagnostic char Microscopic mory Maximum temp growth Colony mor Table 6.8 Table Fungal Diagnosis 273 g own ce ­ in br ke ­ fa - m li (continued) - ­ co ed; sur re, be se se cream ­ kl ­ tu ­ ver re wrin Moist yeast tex Sporothrix schenckii ed floccose, ­ kl Glabrous brown to wrin beige or white Paracoccidioides brasiliensis le le se se ­ ib s ­ ver ­ fu ; ; re ange or ange or - ­ ery nt e or - ­ pl iph ­ me ­ r pig pe buff, red dif pur Talaromyces marneffei Green with gray spp. own g tan; ay, br ­ in gr - y, y, - se se m ­ vet ­ co ­ ver Glabrous to vel white be re cream own g tan; br ­ in - ty, ty, white, se se m ­ ve ­ co ­ ver Histoplasma Histoplasma capsulatum Coccidioides Glabrous to vel be re cream Fungal Diagnosis ay, gr a - own g tan; br ­ in - ty, white se se m ­ ve ­ co ­ ver Glabrous to vel Blastomyces dermatitidis be cream re morphic molds cs Di at ­ ti ­ is

­ am r ­ ogy ­ te ol ­ ac ­ ph Direct ex 25°C Diagnostic char Colony mor Table 6.9 274 SECTION 6 spp. Acrodontium Sporothrix schenckii on n, D.C., 2002.n, D.C., ct exam, ­ to g ­ re ­ in Blastomyces dermatitidis Emmonsia; di Paracoccidioides brasiliensis

ng ng ­ i c ­ du nt ­ me Other Penicillium spp. pro red diffusible pig Talaromyces marneffei spp. Arthrographis, Arthrographis, Geotrichum, Malbranchea, Trichosporon Emmonsia, Sepedonium Histoplasma Histoplasma capsulatum Coccidioides Fungal Diagnosis (continued) a monsia, Em Chrysosporium, Scedosporium apiospermum Blastomyces dermatitidis morphic molds cs Di ­ ti ­ is

r ­ ­ te ­ ac ­ am Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a Diagnostic char Microscopic ex Rule out Table 6.9 Fungal Diagnosis 275 g ­ in es, ­ ni ­ o own + –/V br - rse white to ­ low ­ ve T. verrucosum T. Very slow grow white to cream heaped col re yel ay ty gr ­ ve - rse pale, – + ­ low ­ ve +/+— n, D.C., 2002.n, D.C., ­ to ry to vel g ce, re ­ de ­ in ­ fa White to cream pow sur T. terrestre T. slightly yel

- w ­ lo ­ on ty own ­ ve br - rse lem + + ­ ve –/+ ry or vel ce, re ­ de ­ fa llow or red sur ye White to creamy yel pow T. tonsurans T. a s ­ cie le. ­ ab ny ny spe ­ i ­ to rse deep + — ­ ve 4+/4+ wn bro - white; re red Variable, cot T. rubrum T. Trichophyton m growth; var V, ­ mu ­ i mon ­ mon rse ­ ve w Fungal Diagnosis r, r, ­ lo ­ la ­ u ny, ny, ­ to + + 4+/4+ ly ly white; re ­ al ty or gran ­ er ­ ve ricted growth; 4+, max ­ st white to pale yel T. mentagrophytes T. vel Variable cot gen Characteristics com of cs ­ ti

y ­ is ­ g ­ g r b ­ o ­ o ­ te ol ­ ac ­ phol ­ ph —, no growth; re +, Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a b Diagnostic char Microscopic mory Urease Trichophyton agars 1/4 Growth at 37°C Colony mor Table 6.10 276 SECTION 6 ex ­ pl com ; ­ or ly palely to brown ­ al rse usu ­ ve ranular, sandy col G re Microsporum gypseum Microsporum w; ­ lo der ­ si var. canis a s w; also con ty, pale toty, yel ­ cie ­ lo ­ ve ­ cox rse yel ­ ve Flat to vel re M. prae Microsporum canis Microsporum w ­ lo mon Microsporum­ mon spe r, sandyr, to and com ­ la ­ u rse pale to yel ­ ve Fungal Diagnosis own; re br - e ­ iv Flat, slightly gran ol Epidermophyton floccosum Epidermophyton floccosum cs ­ ti

y ­ is ­ g ­ g r ­ o ­ o ­ te ol ­ ac ­ ph ­ phol Diagnostic char Colony mor Microscopic mory Table 6.11 Fungal Diagnosis 277 own br - ish ­ d se se red ­ ver Microsporum nanumMicrosporum sandy color, Powdery, re n, D.C., 2002.n, D.C., ­ to g ­ in ty; ­ ve rse wine red ­ ve Microsporum cookei Microsporum Granular to vel re h ­ is rse ­ ve ty; re Fungal Diagnosis ­ ve Microsporum audouiniiMicrosporum Flat to vel pale salmon to pale brown cs ­ ti y ­ is ­ g ­ g r ­ o ­ o ­ te ol ­ ac ­ phol ­ ph Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a Diagnostic char Microscopic mory Colony mor 278 SECTION 6 Helminthosporium Exserohilum a res ­ tu Drechslera c Fungal Diagnosis Bipolaris b Dematiaceous fungi with macroconidia or other struc cs ­ ti

­ is ­ g r ­ o ­ te ­ ac ­ phol Diagnostic char Microscopic mory Table 6.12 Fungal Diagnosis 279 Phoma n, D.C., 2002.n, D.C., ­ to on. g ­ ti ­ a ­ in ­ ti ­ en r ­ fe Chaetomium ; a germ tube test is needed dif for Curvularia ly calledly Drechslera ­ en k ­ ta Fungal Diagnosis were mis g, andg, shades green of and gray to black. Reverse is dark. ­ in Alternaria ly growly ­ id tes Bipolaris of ­ la b cs ­ ti ­ is ­ g r ­ o ­ te ­ ac ­ phol Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash Colonies are rap woolly, In the past, most iso a b c Diagnostic char Microscopic mory 280 SECTION 6 Botrytis ­ sider Rhinocladiella amichloridium Also con R a ­ ia d Phialophora verrucosa ni ­ Fungal Diagnosis Fonsecaea pedrosoiFonsecaea b Dematiaceous fungi with small co cs ­ ti

­ is ­ g r ­ o ­ te ­ ac ­ phol Diagnostic char Microscopic mory Table 6.13 Fungal Diagnosis 281 - ly yeastly ­ al are usu Stachybotrys n, D.C., 2002.n, D.C., , and Phaeoannellomyces ­ to g ­ in ve; growth at le Wangiella ­ ti ­ i , ­ ab ­ i Hortaea werneckii Nitrate pos 40°C var ve; growth at ­ ti ve ­ a ­ ti ­ i Exophiala dermatitidis Exophiala Nitrate neg 40°C pos spp. Fungal Diagnosis g, andg, shades green of and gray to black. Reverse is dark. Exophiala ve; growth at ­ in le ­ ti ­ i ­ ab ­ i Exophiala ly growly ­ id Nitrate pos 40°C var b cs ­ ti y y and ­ is ­ g al test r ­ o ­ og ­ ti l ­ te ­ en ­ ac r ­ phol ­ pho ­ fe Colonies are rap woolly, Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a b ke when young. dif Diagnostic char Microscopic mory Colony mor li 282 SECTION 6 c + — — Variable bit growthbit at 40°C; some iso­ ­ hi Cladophialophora bantiana Cladophialophora onsex ­ si n, D.C., 2002.n, D.C., alle ­ to g ­ br ­ in ­ re from ce + — — — a s ­ cie spe Cladophialophora carrionii Isolates bantiana C. of own or black. Reverse is black. br - e spp. ­ iv Xylophyphaernmonsii. nd Cladophialophora + + a — — d as ay toay ol gr ­ fie - e ­ si ­ iv ) do not grow above 37°C. Cladosporium ny, ol ny, sly sly clas ­ to ­ ou Fungal Diagnosis ­ vi X. ernmonsii ty or cot tes pre ­ ve b ­ la d as ­ fie ­ tics ­ si g, vel g, ­ is ­ in udes iso ­ ter ­ cl ­ ac sly sly clas is ce ­ s ly growly ­ ou Differentiation Cladosporium of ­ ly ­ an

­ id ­ vi now in ­ er

y ­ dro ­ g ­ o ol ­ ph C. bantiana C. Colonies rap Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a b C mor Diagnostic char Microscopic Gelatin hy 15% 15% salt tol Growth at 37°C Growth at 42°C lates (those pre Table 6.14 Fungal Diagnosis 283

-

e ­ iv nt rse dark (continued) (not ­ ve ­ me sh brown, or ­ di le pig ack; re ­ ib bl s - ­ fu ay, red ay, Dactylaria constricta var. constricta incovered MIF) Woolly and dark,Woolly ol gr gray with a red to brown dif

- e ­ iv nt rse dark ­ ve ­ me gallopava sh brown, or ­ di le pig ack; re ­ ib bl s - ­ fu ray, red ray, Ochroconis Woolly and dark,Woolly ol g gray with a red to brown dif rse gray ­ ve Scedosporium prolificans Cottony, lightCottony, gray to black; re to black rse a s ­ ve ­ cie spe exual S t ­ en Fungal Diagnosis g gray or black spp. complex ­ in nd Dactylaria m a ­ co

Graphium may be pres Asexual white, be Scedosporium Cottony white to gray or brown; re Scedosporium cs ­ ti

y ­ is ­ g ­ g r ­ o ­ o ­ te ol ­ ac ­ phol ­ ph Diagnostic char Microscopic mory Colony mor Table 6.15 284 SECTION 6

(not + — covered incovered MIF) Dactylaria constricta var. constricta n, D.C., 2002.n, D.C., ­ to g gallopava ­ in + — Ochroconis — NA 45°C Scedosporium prolificans (continued) a s ­ cie

spe de) ed ­ it b ­ mi l stage ­ i ­ hi x ­ ua ­ he NA 37°C Fungal Diagnosis ­ clo spp. complex the sex nd Dactylaria may be in by cy + ( a Scedosporium Scedosporium cs ­ ti

­ is s in <7 r ­ si ­ te ­ ly ­ ac ­ ance o ­ er ­ dr Illustrations from H. Larone, D. Medically Important Fungi, a Guide to Identification, 4th ed., ASM Press, Wash a days Diagnostic char Gelatin hy Cycloheximide tol Growth at 37–45°C Table 6.15 SECTION 7 Parasitic Diagnosis

Ge­ne­ral Comments 286 Table 7.1 Detection Methods for Parasites 287 Microscopy 290 Specific Diagnostic Tests 291 Free-Living Amebae 291 Intestinal and Urogenital Protozoa 292 Blood and Tissue Protozoa 294 Microsporidia 297 Helminths: Nematodes 297 Helminths: Trematodes 299 Helminths: Cestodes 300 Identification Tables and Figures 302 Table 7.2 Trophozoites of Common Intestinal Amebae 302 Table 7.3 Cysts of Common Intestinal Amebae 305 Figure 7.1 Intestinal Amebae of Humans 308 Table 7.4 Trophozoites of Flagellates 309 Table 7.5 Cysts of Flagellates 311 Figure 7.2 Intestinal and Urogenital Flagellates of Humans 312 Table 7.6 Morphological Characteristics of Ciliates, Coccidia, Microsporidia, and Tissue Protozoa 313 Table 7. 7 Morphological Characteristics of Protozoa Found in Blood 315 Table 7.8 Morphological Characteristics of Blood and Tissue Nematodes 317 Table 7.9 Morphological Characteristics of Helminths 318 Figure 7.3 Relative Sizes of Helminth Eggs 320

doi:10.1128/9781683670070.ch7 286 SECTION 7 Diagnosis of most par­a­sitic in­fec­tions has tra­di­tion­ally been made by the mi­cro­scopic ex­am­i­na­tion of clin­i­cal ma­te­rial, ne­ces­si­tat­ing that highly trained tech­nol­o­gists spend a sig­nif­i­cant amount of time ex­am­in­ing in­di­vid­ual spec­i­mens. In most de­vel­oped coun­tries, the prev­a­lence of par­a­sitic in­fec­tion is very low, and as a re­sult, many lab­o­ra­to­ries send their par­a­sitic test­ing to large re­fer­ral lab­o­ra­to­ ries where ex­per­tise is cen­tral­ized and com­pe­tency can more eas­ily be main­tained. For the de­tec­tion of more com­mon par­a­sites, im­mu­ no­as­says have been de­vel­oped (e.g., Entamoeba histolytica, Giardia duodenalis, and Cryptosporidium parvum). However, these tests are ad­juncts to the mi­cro­scopic ex­am­i­na­tion of spec­i­mens for ova and par­a­sites and can rarely re­place mi­cros­copy. Likewise, a num­ ber of tests have been de­vel­oped to de­tect par­a­site-specific nu­cleic ac­ids, and re­cently, these as­says have been FDA cleared for di­rect de­tec­tion from stool. This sec­tion sum­ma­rizes the tests cur­rently avail­­able for the lab­o­ra­tory di­ag­no­sis of the most com­mon par­a­sitic in­fec­tions. For ad­di­tional in­for­ma­tion, the reader is re­ferred to the Manual of Clini- cal Microbiology, 11th ed., 2015, and Gar­cia’s Diagnostic Medical Parasitology, 4th ed., 2001. Parasitic Diagnosis Parasitic Parasitic Diagnosis 287 s ­ tic ­ nos (continued) ­ ag B B C C C C C B B B C C D D D A olecular di M ­ tion ­ tec B C C B D D A A D D D D D D D D Antibody Antibody de ­ tion ­ tec C C B D D D D A D A A A D D D D Antigen Antigen de B A A A A A A A A A A A A A A A Microscopy a es B C C B C A D D D D D D A D D D ­ sit ­ a Culture for for par ­ zoa ­ to ­ ods ­ zoa ­ tal pro ­ i ­ to Parasitic Diagnosis Parasitic ­ gen pro ection meth ­ li ­ sue Det

abesia Acanthamoeba Blastocystis hominis Cryptosporidium parvum Cyclospora cayetanensis Dientamoeba fragilis Entamoeba histolytica/dispar duodenalis Giardia vaginalis Trichomonas gondii Toxoplasma Balantidium coli Trypanosoma Leishmania Plasmodium Naegleria Isospora bel B Parasite Free-living ame ­ bae Intestinal and uro Blood and tis Table 7.1 288 SECTION 7 s ­ tic ­ nos ­ ag C C C C C C D D D D D D D D olecular di M ­ tion ­ tec C C C C D D D D D D D A A D Antibody Antibody de ­ tion ­ tec C C C C D D D D D D D D D D Antigen Antigen de A A A A A A A A A A D A A A Microscopy (continued) a es D D D D D D D D D D D D D D ­ sit ­ a Culture for for par ­ ods ­ lus Parasitic Diagnosis Parasitic ­ todes ­ vu ­ a ection meth Det ­ era

spp. trongyloides stercoralis Many Many gen lumbricoides Ascaris Capillaria philippinensis Dracunculus medinensis canis Toxocara spiralis Trichinella Ancylostoma duodenale Onchocerca vol Mansonella perstans Necator americanus Brugia Enterobius vermicularis Loa loa S Parasite Microsporidia Helminths—nem Table 7.1 Parasitic Diagnosis 289 ; ­ ries ­ to ­ ra ­ o e lab C C D D D D D D D D D D D D D ­ enc ­ er e in ref ­­ abl ut may be avail C C C A D D D D D D A A D D D ­ sis b ­ no ­ ag l di ­ era sed gen for C C D D D D D D D D D D D D D ­ dom u , test is sel ­ es; C nc A A A A A A A A A D D D A A A ­ sta ­ cum n cir ­ tai er D D D D D D D D D D D D D D D ­ sis ­ der c ­ no n ­ ag ful u ­ y di ­ tor ­ ra ­ o ; B, test; B, is use ­ ful Parasitic Diagnosis Parasitic ­ todes ­ a ­ ca ­ i y use ­ todes y not used for lab ­ all spp. ­ pat ­ er ­ all ­ er Trichuris trichiura Trichuris sinensis Clonorchis buskiFasciolopis westermani Paragonimus caniumDipylidium granulosus Echinococcus multilocularis Echinococcus diminuta Hymenolepis Hymenolepis nana saginata Taenia Diphyllobothrium latumDiphyllobothrium Wuchereria bancrofti Fasciola he solium Taenia Schistosoma A, test is gen a D, D, test is gen Helminths—trem Helminths—ces 290 SECTION 7 Acid-Fast Trichrome Chromotrope Stain The ac­id-fast trichrome chromotrope stain is used to de­tected mi- crosporidia, Cryptosporidium, Cyclospora, and Isospora. Speci- mens are stained with carbol fuch­sin fol­lowed by Didier’s trichrome so­lu­tion (Chromotrope 2R, an­i­line blue, and phosphotungstic acid in ace­tic acid) and then washed with ac­id-alcohol fol­lowed by 95% eth­a­nol. Cryptosporidium, Cyclospora, and Isospora stain bright pink or vi­o­let, and microsporidia ap­pear pink. Calcofluor White Stain Calcofluor white binds to cel­lu­lose and chi­tin; it fluo­resces best when ex­posed to long-wavelength UV light. Free-living ame­bae (i.e., Acanthamoeba, Balamuthia, and Naegleria) and lar­vae of Di- rofilaria fluo­resce. Delafield’s Hematoxylin Stain Delafield’s he­ma­tox­y­lin stain is used for thin and thick blood films for the de­tec­tion of mi­cro­fi­laria. Structural de­tail (e.g., nu­clei and sheaths) may show greater de­tail than with Gi­emsa or Wright’s stains. This stain is not com­mer­cially avail­­able and so is typ­i­cally used only in spe­cialty lab­o­ra­to­ries. Direct Fluorescent-Antibody Stain A va­ri­ety of or­gan­isms (e.g., Cryptosporidium parvum and Giar- dia duodenalis) are de­tected di­rectly in clin­i­cal spec­i­mens by us­ing spe­cific fluo­res­ce­in-labeled an­ti­bod­ies. The la­beled an­ti­bod­ies bind to the or­gan­isms and fluo­resce green un­der UV light. The sen­si­tiv­ ity and spec­i­fic­ity of the stain are de­ter­mined by the qual­ity of the an­ti­bod­ies used in the re­agents. Optimal de­tec­tion of fluo­res­cence re­quires the use of ei­ther a 420- to 490-nm (wide band) or 470- to 490-nm (nar­row band) ex­ci­ta­tion fil­ter and a 510- to 530-nm bar­ rier fil­ter. Gi­emsa Stain Gi­emsa stain and Wright’s stain are mod­i­fi­ca­tions of Romanowsky stain, which com­bines meth­y­lene blue and eo­sin. Both stains are used for the de­tec­tion of blood par­a­sites (e.g., Plasmodium, Babe- sia, and Leishmania). A pro­to­zoan tropho­ ­zo­ite has a red nucleu­ s and gray-blue cy­to­plasm.

Parasitic Diagnosis Parasitic Iron Hematoxylin Stain Iron he­ma­tox­y­lin stain is used for the de­tec­tion and iden­ti­fi­ca­tion of fe­cal pro­to­zoa. Helminth eggs and lar­vae gen­er­ally re­tain too much stain and are more eas­ily iden­ti­fiedwith wet-mount prep­a­ra­ tions. Iron he­ma­tox­y­lin stain can be ap­plied to ei­ther fresh stool spec­i­mens or ones pre­served with po­ly­vi­nyl al­co­hol or a sim­i­lar pre­ser­va­tive. Formalin-fixed spec­i­mens can­not be used. Parasitic Diagnosis 291 Lugol’s Iodine Stain Iodine is added to “wet” prep­a­ra­tions of par­a­si­tol­ogy spec­i­mens to en­hance the con­trast of the in­ter­nal struc­tures (e.g., nu­clei and gly­ co­gen vac­u­oles). One dis­ad­van­tage of this method is that pro­to­zoa are killed by the io­dine and hence mo­til­ity can­not be ob­served. Modified Acid-Fast Stain Acid-fast stains are used for de­tect­ing Cryptosporidium, Cy- clospora, and Isospora. Because the pro­to­zoa can be read­ily de­col­ or­ized, a weak ac­id-alcohol so­lu­tion is used for re­mov­ing the ba­sic carbol fuch­sin from non-acid-fast or­gan­isms. Organisms that re­tain this mod­i­fied tains are re­ferred to as be­ing par­tially ac­id-fast. Modified Acid-Fast Stains (Weber Green, Ryan Blue) The trichrome stain has been mod­i­fied pes ­cif­i­cally for the de­tec­tion of microsporidia. A higher con­cen­tra­tion of dye and lon­ger stain­ing time are used to fa­cil­i­tate the stain­ing of microsporidia. Weber Green stains the or­gan­isms pink with a green back­ground, whereas the Ryan Blue also stains the or­gan­isms pink but with a blue back­ground. Trichrome Stain The trichrome stain, like the iron he­ma­tox­y­lin stain, is a per­ma­nent stain that is used for the de­tec­tion and iden­ti­fi­ca­tion of pro­to­zoa. The stain con­sists of a so­lu­tion of three dyes (Chromotrope 2R, light green SF, and fast green FCF) in phosphotungstic acid and ace­tic acid. When stain­ing is done prop­erly, the spec­i­men back­ground is green and the pro­to­zoa have a blue-green to pur­ple cy­to­plasm with red or pur­ple-red nu­clei, chromatoid bod­ies, eryth­ro­cytes, and bac­te­ria. Parasite eggs and lar­vae usu­ally stain red. Wright’s Stain Wright’s stain is a po­ly­chro­matic stain that con­tains a mix­ture of meth­ y­lene blue, azure B (from the ox­i­da­tion of meth­y­lene blue), and eo­sin Y dis­solved in meth­a­nol. The eo­sin ions are neg­a­tively charged and stain the ba­sic com­po­nents of cells or­ange to pink, whereas the other dyes stain the acidic cell struc­tures var­i­ous shades of blue to pur­ple. Specific Diagnostic Tests Free-Living Amebae Parasitic Diagnosis Parasitic Acanthamoeba. Chronic gran­u­lo­ma­tous ame­bic en­ceph­a­li­tis, caused by sev­eral spe­cies of Acanthamoeba, can be di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of Gi­emsa- or trichrome-stained brain tis­ sue and, rarely, ce­re­bro­spi­nal fluid (CSF). Acanthamoeba ker­a­ti­tis is di­ag­nosed by di­rect mi­cro­scopic ex­am­i­na­tion of cor­neal scrap­ings or by cul­ture of the spec­i­men. Nucleic acid am­pli­fi­ca­tion (NAA) tests and se­ro­logic test­ing have been used only in re­search lab­o­ra­to­ries. 292 SECTION 7 Naegleria. Primary me­nin­go­en­ceph­a­li­tis, caused by Naegleria fowleri, is di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of Gi­emsa- or trichrome-stained brain tis­sue or de­tec­tion of mo­bile tro­pho­zo­ites in CSF. Gi­emsa or trichrome stain­ing can be per­formed on CSF, but Gram stains are un­re­li­able (giv­ing false-positive and false-neg- ative re­sults). NAA tests and se­ro­logic test­ing have been used only in re­search lab­o­ra­to­ries.

Intestinal and Urogenital Protozoa Balantidium coli. B. coli is best de­tected by wet mount ex­am­i­na­ tion of stool spec­i­mens. The or­gan­ism tends to overstain with tri- chrome stains and may be mis­iden­ti­fied. B. coli cil­i­ated and the tropho­ ­zo­ites have a rapid ro­tary mo­tion, which makes them easy to miss as they move across a field of view. Blastocystis hominis. The role of B. hominis in hu­man dis­ease re­mains con­tro­ver­sial. The pro­to­zoa can be de­tected by mi­cro­ scopic ex­am­i­na­tion (io­dine wet mount, trichrome, or di­rect fluo­ res­cent an­ti­body [DFA] as­say) or an­ti­gen tests (en­zyme im­mu­no­as­say [EIA]) of fe­cal spec­i­mens col­lected from symp­tom­atic and asymp­ tom­atic in­di­vid­u­als. Serologic test­ing is not use­ful be­cause ­pro­longed ex­po­sure is re­quired be­fore an an­ti­body re­sponse is de­tect­ed. Cryptosporidium parvum. C. parvum in­fec­tions are di­ag­nosed by ex­am­in­ing fe­cal spec­i­mens. This pro­to­zoon does not stain ad­e­ quately with io­dine or with per­ma­nent stains (trichrome or iron he­ma­tox­y­lin). It can be rec­og­nized by us­ing a wet mount; how­ever, mod­i­fiedac ­id-fast stains or the DFA test is more sen­si­tive and spe­ cific. EIAs and lat­eral flow as­says now com­mer­cially avail­­able for the rapid de­tec­tion of C. parvum have sen­si­tiv­i­ties and spec­i­fic­i­ties ap­proach­ing 100%. Multiplex, PCR-based, syndromic pan­els now in­clude the de­tec­tion of C. parvum. Some lab­o­ra­to­ries are adopt­ing syndromic gas­tro­in­tes­ti­nal pan­els to re­place rou­tine stool cul­ture. This prac­tice is some­what con­tro­ver­sial but may have the ben­e­fit of de­tect­ing more C. parvum dis­ease, which has his­tor­i­cally been dif­ fi­cult to de­tect with­out­ the or­der of a spe­cific di­ag­nos­tic test. Sero- logic test­ing has not been used for di­ag­nos­tic pur­pos­es. Cyclospora cayetanensis. C. cayetanensis is de­tected by the mi­cro­ scopic ex­am­i­na­tion of fe­cal spec­i­mens. The pro­to­zoon does not stain well with io­dine, Gi­emsa, trichrome, or chromotrope stains. It is most Parasitic Diagnosis Parasitic com­monly de­tected by us­ing a mod­i­fied ac­id-fast stain, al­though even the ex­pe­ri­enced par­a­si­tol­o­gist can find it dif­fi­cult to iden­tify this or­ gan­ism with the ac­id-fast stain. Cyclospora will also autofluoresce un­der UV epifluorescence (green with 450- to 490-nm ex­ci­ta­tion fil­ ter; blue with 365-nm ex­ci­ta­tion fil­ter), which can greatly en­hance one’s abil­ity to de­tect the or­gan­ism. PCR-based NAA tests are now com­mer­cially avail­­able but thus far only ex­ist on large mul­ti­plex pan­ Parasitic Diagnosis 293 els de­signed to be tested from stool spec­i­mens. Serologic test­ing is not used for di­ag­nos­tic pur­pos­es. Dientamoeba fragilis. Microscopic ex­am­i­na­tion of a con­cen­ trated, per­ma­nently stained spec­i­men is the method of choice to di­ag­nose D. fragilis in­fec­tions. Stool an­ti­gen de­tec­tion as­says have been de­vel­oped but are not com­monly used. This may be due to the his­tor­i­cal con­tro­versy over whether D. fragilis is a true path­o­gen. Entamoeba histolytica/dispar. Microscopy can­not re­li­ably dif­fer­en­ ti­ate be­tween E. histolytica (path­o­genic) and E. dispar (non­patho­ genic) un­less eryth­ro­cytes are de­tected in the cy­to­plasm of E. histolytica tro­pho­zo­ites (an un­com­mon find­ing). These two pro­to­zoa are de­tected by ex­am­in­ing clin­i­cal spec­i­mens (e.g., fe­ces, tis­sue bi­ opsy spec­i­mens, and ab­scess as­pi­rates) us­ing wet mount or per­ma­ nent stains. A num­ber of an­ti­gen de­tec­tion tests (EIAs) can be used to iden­tify E. histolytica. These tests are now more sen­si­tive and spe­ cific than mi­cros­copy. PCR-based NAA tests are also avail­­able for the de­tec­tion and iden­ti­fi­ca­tion of E. histolytica. Thus far, com­mer­ cially avail­­able NAA tests are un­able to dif­fer­en­ti­ate be­tween E. histolytica and E. dispar. Tests that in­clude these or­gan­isms are typ­i­cally mul­ti­plexed to vary­ing de­grees. E. histolytica/dispar can be found on both large gas­tro­in­tes­ti­nal pan­els, which in­clude bac­te­ ria, vi­ruses, and par­a­sites, or on smaller pan­els which in­clude on par­ a­sites. Serologic test­ing is valu­able for the di­ag­no­sis of extraintestinal in­fec­tions be­cause cysts or tro­pho­zo­ites may not be de­tected in stool spec­i­mens. Indirect hem­ag­glu­ti­na­tion (IHA) is the ref­er­ence test. The Centers for Disease Control and Prevention (CDC) rec­om­mend the use of 1:256 as a cri­te­rion for a pos­i­tive IHA se­ro­logic re­sult. This level iden­ti­fies 5%9 of pa­tients with extraintestinal in­fec­tions, 70% of pa­tients with ac­tive dis­ease lo­cal­ized to the in­tes­tines, and 10% of asymp­tom­atic in­tes­ti­nal car­ri­ers. Positive ti­ters may per­sist for years af­ter suc­cess­ful ther­apy. EIA is a sen­si­tive as­say, which iden­ti­fies sig­nif­i­cantly more pa­tients with he­patic dis­ease than does IHA. No cross-re­ac­tions with other amoe­bas are ob­served. Detection of im­ mu­no­glob­u­lin M (IgM) an­ti­bod­ies is in­sen­si­tive, even for pa­tients with ac­tive in­va­sive dis­ease (pos­i­tive in only 65% of these pa­tients). Giardia duodenalis. Microscopic ex­am­i­na­tion of fe­cal spec­i­mens (wet mount, per­ma­nent stain, or DFA test [cyst spe­cific]) for G. duodenalis tro­pho­zo­ites and cysts is used to es­tab­lish in­fec­tion. Antigen de­tec­tion by EIA and lat­eral flow are used ex­ten­sively and are more sen­si­tive and spe­cific than mi­cro­scopic meth­ods. Antigen de­tec­tion as­says have the Diagnosis Parasitic ben­e­fit of de­tect­ing both cysts and tropho­ ­zo­ites (pre­ferred test). PCR- based NAA meth­ods have been de­vel­oped. Giardia can be found on both large gas­tro­in­tes­ti­nal PCR pan­els, which in­clude bac­te­ria, vi­ruses, and par­a­sites, or on smaller pan­els which in­clude par­a­sites. Cystisospora bel­li. As with Cryptosporidium and Cyclospora, the most com­mon method used to de­tect C. bel­li in fe­cal spec­i­mens is 294 SECTION 7 the mod­i­fied ac­id-fast stain. NAA tests are re­stricted to re­search lab­o­ra­to­ries, and se­ro­logic test­ing is not use­ful for di­ag­no­sis. Trichomonas vaginalis. Trichomonas vaginalis is now rec­og­ nized as one of the most com­mon sex­u­ally trans­mit­ted dis­eases. Historically, T. vaginalis in­fec­tions have most com­monly been di­ ag­nosed by mi­cro­scopic ex­am­i­na­tion (wet mount, DFA) of vag­i­nal and ure­thral dis­char­ges, pros­tatic se­cre­tions, and urine sed­i­ments. The sen­si­tiv­ity of a mi­cro­scopic ex­am­i­na­tion is be­tween 50 and 70%. If mi­cros­copy is neg­a­tive, cul­ture can be per­formed to en­ hance sen­si­tiv­ity (>80%) and is con­sid­ered the “gold stan­dard.” A lim­i­ta­tion of mi­cro­scopic and cul­ture-based di­ag­nos­tic meth­ods is that rapid trans­port is re­quired to main­tain or­gan­ism vi­a­bil­ity. An- tigen de­tec­tion with lat­eral flow as­says is an al­ter­na­tive di­ag­nos­tic method that ob­vi­ates the need for rapid spec­i­men trans­port be­cause or­gan­ism vi­a­bil­ity is not re­quired. More re­cently, NAA tests have been de­vel­oped and are be­ing adopted due to the su­pe­rior sen­si­tiv­ ity and rapid turn­around times. Serologic test­ing is not use­ful. Blood and Tissue Protozoa Babesia spp. Babesia in­fec­tions are most com­monly di­ag­nosed by de­tect­ing par­a­sit­ized eryth­ro­cytes in Gi­em­sa-stained thin films of pe­riph­eral blood. For pa­tients with low-grade parasitemia or in­con­ clu­sive pe­riph­eral smears, se­ro­logic test­ing can be help­ful, al­though in prac­tice this is rarely per­formed. Antibody ti­ters in the im­mu­no­ flu­o­res­cent-antibody (IFA) test rise rap­idly dur­ing the first weeks of dis­ease to 1:1,024 or higher and then grad­u­ally de­cline over the next 6 months. Low but de­tect­able ti­ters may per­sist for 1 year or more. Elevated an­ti­body ti­ters may be pres­ent in healthy in­di­vid­u­als liv­ing in ar­eas of en­demic in­fec­tion. Therefore, a pos­i­tive se­ro­logic test re­sult should be con­firmed by de­tec­tion of the par­a­site in blood smears. Cross-reactivity among Babesia spe­cies is var­i­able; there­ fore, re­gional dif­fer­ences in se­ro­logic re­ac­tiv­ity may be ob­served. Leishmania spp. Leishmaniasis is di­ag­nosed by de­tec­tion of amastigotes in clin­i­cal spec­i­mens or promastigotes in cul­ture. Spec- imens should be col­lected from the mar­gin of the le­sion by as­pi­ra­ tion, scrap­ing, or punch bi­opsy. Tissue is used to make touch prep­a­ra­tions and is sub­mit­ted for his­to­path­o­logic ex­am­i­na­tion. Amastigotes are found in mac­ro­phages in Gi­em­sa-stained prep­a­ ra­tions. PCR-based NAA tests have been de­vel­oped to iden­tify spe­ Parasitic Diagnosis Parasitic cific Leishmania spe­cies in tis­sue bi­opsy spec­i­mens. Specimens can also be cul­tured in Schneider’s Drosophila me­dium sup­ple­ mented with 30% fe­tal bo­vine se­rum. Although this is a sen­si­tive pro­ce­dure, cul­tures must be held for 4 weeks or lon­ger. Serologic tests, in­clud­ing the IFA test, en­zyme-linked im­mu­no­sor­bent as­ say (ELISA), and im­mu­no­blot (IB) test, have been de­vel­oped for di­ag­no­sis but are avail­­able only in ref­er­ence lab­o­ra­to­ries and at the CDC. Parasitic Diagnosis 295 Plasmodium spp. Malaria is most com­monly di­ag­nosed by de­ tect­ing par­a­sit­ized eryth­ro­cytes in Gi­em­sa-stained thick and thin films of pe­riph­eral blood. If blood is col­lected with an­ti­co­ag­u­lant, EDTA but not hep­a­rin should be used. Examination of thick films is the most sen­si­tive mi­cro­scopic method, but iden­ti­fi­ca­tion of the Plas- modium spe­cies re­quires ex­am­i­na­tion of thin films. Acridine or­ange has also been used to stain blood films. This method is sen­si­tive, but spe­cies iden­ti­fi­ca­tion is dif­fi­cult. PCR-based NAA tests have been de­vel­oped and can iden­tify Plasmodium at the spe­cies level. These NAA tests are at least as sen­si­tive as ex­am­i­na­tion of thick films but are cur­rently re­stricted to ref­er­ence lab­o­ra­to­ries. Antigen de­tec­tion tests spe­cific for P. fal­cip­a­rum his­ti­dine-rich pro­tein 2 (HRP-2) and par­a­site lac­tate de­hy­dro­ge­nase (LDH) spe­cific for P. fal­cip­a­rum and non-P. fal­cip­a­rum plas­mo­dia are com­mer­cially avail­­able. These as­ says have poor sen­si­tiv­ity for low-parasitemia in­fec­tions such as those typ­i­cally seen with non-falciparum in­fec­tion. However, de­spite lim­i­ta­tions in sen­si­tiv­ity, an­ti­gen test­ing can of­fer a rapid pre­lim­i­ nary re­sult in set­tings where ex­pe­ri­enced par­a­si­tol­o­gists are not avail­­able. Although spe­cies-specific se­ro­logic tests have been de­vel­ oped, there is ex­ten­sive cross-re­ac­tiv­ity among Plasmodium spe­cies, and these tests have not been used for di­ag­nos­tic pur­pos­es. Toxoplasma gondii. Microscopic ex­am­i­na­tion of tis­sues and flu­ids is gen­er­ally unrevealing, al­though tachyzoites and cysts may be ob­ served in Gi­em­sa-stained spec­i­mens. Additionally, par­a­sites can be re­cov­ered by in­oc­u­lat­ing mice or cell cul­tures, but this also has a low yield. EIA an­ti­gen tests are in­sen­si­tive and are not rec­om­mended. A PCR test has been de­vel­oped and is use­ful for con­fir­ma­tion of con­ gen­i­tal in­fec­tions and for test­ing the CSF of pa­tients with char­ac­ter­ is­tic ring-enhancing le­sions seen on im­ag­ing. This test is less use­ful for other forms of toxo­plas­mo­sis and is typ­i­cally only avail­­able through ref­er­ence lab­o­ra­to­ries. Serologic test­ing is the method of choice for the di­ag­no­sis of toxo­plas­mo­sis. A va­ri­ety of com­mer­cial tests are avail­­able (IFA test, EIA, and ag­glu­ti­na­tion) for mea­sur­ing the IgM and IgG re­sponse to toxo­plasma. Care must be used when tests us­ing dif­fer­ent as­say meth­ods are com­pared. For the di­ag­no­sis of an acute ac­quired in­fec­tion, an IgG IFA test or EIA should be per­ formed. If the test is neg­a­tive in an im­mu­no­com­pe­tent per­son, the di­ag­no­sis is ex­cluded. Detection of IgM an­ti­bod­ies or a four­fold or greater in­crease in the level of IgG an­ti­bod­ies (rarely ob­served) is con­sis­tent with an acute in­fec­tion. The fol­low­ing de­scribes se­ro­log­i­ Diagnosis Parasitic cal test­ing for Toxoplasma in sev­eral im­por­tant clin­i­cal sce­nar­ios. Pregnancy: Immunocompetent women who have de­tect­able IgG an­ti­body be­fore be­com­ing preg­nant are es­sen­tially im­mune, and there is low risk of trans­mit­ting the or­gan­ism to the fe­tus. Seronega- tive women are at risk for trans­mit­ting the or­gan­ism to the fe­tus and in some coun­tries are test­ing monthly for the de­vel­op­ment of IgG an­ti­ body. If a woman is tested for the first time af­ter be­com­ing preg­nant 296 SECTION 7 and has IgG an­ti­body, she should be tested for IgG and IgM avid­ity to de­ter­mine whether the acute in­fec­tion oc­curred dur­ing preg­nan­cy. Newborns: An at­tempt to iso­late the or­gan­ism from the pla­centa should be made as ~95% of pla­cen­tas of un­treated, con­gen­i­tally in­ fected new­borns. The child’s se­rum should be tested for to­tal an­ti­ body as well as IgG, IgM, and IgA spe­cific an­ti­bod­ies. CSF should also be an­a­lyzed with se­rol­o­gies as well as di­rect ex­am­i­na­tion for T. gondii tachyzoites. Persistent in­creas­ing IgG ti­ters in the in­fant as com­pared to the mother are di­ag­nos­tic for con­gen­i­tal in­fec­tion. Detection of par­a­site-specific DNA by PCR in am­ni­otic fluid is also de­fin­i­tive ev­i­dence of dis­ease. Ocular in­fec­tions: Ocular in­fec­tions can be di­ag­nosed by dem­ on­strat­ing lo­cal pro­duc­tion of an­ti­body or de­tec­tion of par­a­site DNA. Immunocompromised pa­tients: Most in­fec­tions in im­mu­no­com­ pro­mised pa­tients rep­re­sent re­ac­ti­va­tion dis­ease. IgM an­ti­body is usu­ ally not de­tected, and IgG an­ti­body ti­ters are con­sis­tent with chronic in­fec­tions. Diagnosis is typ­i­cally con­firmed by de­tec­tion of par­a­sites or Toxoplasma DNA in tis­sue bi­opsy spec­i­mens or as­pi­rated flu­ids. Trypanosoma brucei. Af­ri­can try­pano­so­mi­a­sis is di­ag­nosed by de­ tec­tion of trypanomastigotes in blood, lymph node as­pi­rates, ster­ num bone mar­row, or CSF. Parasites are pres­ent in the blood dur­ing fe­brile pe­ri­ods but are found in only small num­bers when the pa­tient is afe­brile. Thick and thin films, as well as buffy coat cells, should be ex­am­ined us­ing the Gi­emsa stain. CSF should be con­cen­trated be­fore ex­am­i­na­tion. ELISA has been used to de­tect par­a­sitic an­ti­ gens in se­rum and CSF. PCR-based NAA tests have also been de­vel­ oped in ref­er­ence lab­o­ra­to­ries. Serologic tests (IFA, ELISA, IHA, and ag­glu­ti­na­tion) are used for ep­i­de­mi­o­logic stud­ies but not for di­ag­no­sis. Trypanosoma cruzi. Amer­i­can try­pano­so­mi­a­sis (Chagas dis­ ease), caused by T. cruzi, is di­ag­nosed dur­ing the acute phase of ill­ness by de­tec­tion of trypanomastigotes in Gi­em­sa-stained pe­riph­ eral blood (thick film, thin film, or buffy coat cells). Blood smears are less re­li­able for de­tec­tion of con­gen­i­tal in­fec­tions and chronic dis­ease. Immunoassays for par­a­sitic an­ti­gens in sera and urine have been used for these in­fec­tions. PCR-based NAA tests have been de­ vel­oped but are used pri­mar­ily in re­search lab­o­ra­to­ries. Aspirates, Parasitic Diagnosis Parasitic blood, and tis­sues can be cul­tured with sam­ples in­cu­bated for 4 weeks or lon­ger. Serologic tests are avail­­able in ref­er­ence lab­o­ra­to­ ries and at the CDC. These tests inclu­ de com­ple­ment fix­a­tion (CF), IFA test, IHA, and ELISA. Most tests use an epimastigote an­ti­gen, and cross-re­ac­tions oc­cur with other try­pano­somes, Leishmania, and Toxoplasma. An el­e­vated ti­ter can­not be used to dis­crim­i­nate be­tween ac­tive and past dis­ease. Parasitic Diagnosis 297 Microsporidia As many as 140 gen­era have been de­scribed for the phy­lum Micro- sporidia, with at least 7 be­ing im­pli­cated in hu­man dis­ease. Diag- nosis is most com­monly made by ex­am­i­na­tion of fe­cal spec­i­mens or by cy­to­logic or his­to­path­o­logic test­ing. Fecal smears are pre­pared on glass slides (con­cen­tra­tion of spec­i­mens re­sults in a loss of or­ gan­isms) and then stained with chromotrope-based stains or che- mofluorescent agents (Calcofluor white). Immunofluorescent stains have been de­vel­oped but are not widely used. Microsporidial spores have been de­tected by cy­to­logic ex­am­i­na­tion of con­cen­trated flu­ ids such as bronchoalveolar la­vage fluid, bil­i­ary as­pi­rates, du­o­de­ nal as­pi­rates, and CSF. Histologic ex­am­i­na­tion of bi­opsy spec­i­mens has also been use­ful. NAA tests have been de­vel­oped but are re­ stricted to re­search lab­o­ra­to­ries. Serologic test­ing is not use­ful for the di­ag­no­sis of hu­man in­fec­tions.

Helminths: Nematodes Ancylostoma duodenale. Hookworm in­fec­tions are di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens with a di­rect smear for char­ac­ter­is­tic eggs. Heavy in­fec­tions (e.g., >25 eggs per cov­er­slip) are as­so­ci­ated with ane­mia. Delays in ex­am­in­ing the spec­i­men should be avoided be­cause eggs can hatch in unpreserved spec­i­ mens and re­lease lar­val forms that can be mis­iden­ti­fied as Stron- gyloides. Infection with other spe­cies of Ancylostoma (and other hook­worms and Strongyloides spe­cies) can cause cu­ta­ne­ous larva mi­grans, where filariform lar­vae mi­grate through the skin lay­ers and stim­u­late an in­flam­ma­tory re­sponse. This dis­ease is di­ag­nosed on the ba­sis of clin­i­cal pre­sen­ta­tion. Ascaris lumbricoides. Roundworm in­fec­tions are di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens for char­ac­ter­is­tic eggs (fer­til­ized, de­cor­ti­cated, and un­fer­til­ized eggs). Fertilized eggs can be de­tected in a di­rect fe­cal smear or in con­cen­trated spec­i­mens. Unfertilized eggs are not con­cen­trated in floa­ta­tion con­cen­tra­tion meth­ods. Adult worms may also be passed in fe­ces or re­gur­gi­tat­ed. Brugia spp. Infections are de­tected by ex­am­in­ing blood for the pres­ ence of mi­cro­fi­laria. Most in­fec­tions con­sist of rel­a­tively few mi­cro­fi­ laria in the blood, so that a large vol­ume must be ex­am­ined by ei­ther thick films or, more ap­pro­pri­ately, con­cen­tra­tion on a mem­brane fil­ter (Knott tech­nique). The worms are stained with Gi­emsa or he­ma­ Parasitic Diagnosis Parasitic tox­y­lin. Identification of the spe­cific microfilariae is based on their mor­phol­ogy (size, nu­clear ar­range­ment in the tail, and pres­ence or ab­sence of sheath). Antigen, an­ti­body, and PCR-based NAA tests have also been de­vel­oped for the de­tec­tion of mi­cro­fi­lar­ial in­fec­tions. These tests are gen­er­ally avail­­able through the CDC and re­search lab­ o­ra­to­ries. Microfilariae cir­cu­late in blood in well-de­fined pe­ri­odic cy­ cles cor­re­spond­ing to the bit­ing hab­its of the in­sect vec­tor. 298 SECTION 7 Capillaria philippinensis. Diagnosis is made on the ba­sis of mi­ cro­scopic de­tec­tion of char­ac­ter­is­tic eggs in fe­cal spec­i­mens. Lar- vae and adults are oc­ca­sion­ally de­tect­ed. Dracunculus medinensis. Infections with the “Guinea worm” are di­ag­nosed by re­cov­ery of the adult fe­male worm when it mi­ grates from the sub­cu­ta­ne­ous tis­sues to the skin sur­face. Adult male worms are small and are only rarely de­tect­ed. Enterobius vermicularis. Pinworm in­fec­tions are di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of par­a­site eggs col­lected from the peri- anal folds. Eggs are col­lected with cel­lu­lose tape or a com­mer­cial pad­dle, trans­ferred to a mi­cro­scope slide, and ex­am­ined di­rectly or af­ter ex­po­sure to one drop of tol­u­ene or xy­lene. Multiple spec­i­mens may have to be ex­am­ined. Loa loa. Refer to Brugia. L. loa has a di­ur­nal pe­ri­od­ic­ity and spec­i­ mens should be col­lected at mid­day. Adult worms may be de­tected when they mi­grate through the con­junc­ti­vae. Mansonella perstans. Refer to Brugia. M. perstans has no pe­ri­od­ic­i­ty. Necator americanus. Refer to Ancylostoma duodenale. Onchocerca vol­vu­lus. Adult worms live in sub­cu­ta­ne­ous tis­sues and de­posit mi­cro­fi­laria in the skin tis­sue. Diagnosis is made by de­tect­ ing the mi­cro­fi­laria in skin snips sus­pended in sa­line so­lu­tions. Skin snips should be col­lected from the scap­u­lar re­gion or the il­iac crest. Care must be used to not con­tam­i­nate the spec­i­men with blood. Strongyloides stercoralis. Strongyloidiasis is di­ag­nosed on the ba­sis of mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens for char­ac­ter­is­tic lar­ val forms. Eggs are rarely ob­served, and lar­vae may be scarce even in con­cen­trated spec­i­mens, par­tic­u­larly in those from pa­tients with chronic in­fec­tions. Techniques de­vel­oped to de­tect light in­fec­tions in­clude the Baermann pro­ce­dure (fe­cal ma­te­rial is placed in a fun­nel with wa­ter, the lar­vae are al­lowed to mi­grate into the wa­ter, and the spec­i­men is ex­am­ined mi­cro­scop­i­cally) and the agar plate method (fe­cal ma­te­rial is placed on an agar plate and then ex­am­ined af­ter 1 to 3 days for tracks of lar­vae mi­grat­ing from the fe­cal mass). Multi- ple spec­i­mens may be needed to make the di­ag­no­sis. Adult worms, eggs, and lar­vae may be ob­served by his­to­path­o­logic test­ing. Sero- logic tests (EIA and IB anal­y­sis) are avail­­able through the CDC.

Parasitic Diagnosis Parasitic EIAs have a re­ported sen­si­tiv­ity be­tween 84 and 92%. Cross-reac- tions can oc­cur in pa­tients with other nem­a­tode in­fec­tions. Titers may per­sist, so se­ro­logic test­ing there­fore can­not be used re­li­ably to dif­fer­en­ti­ate be­tween cur­rent and past in­fec­tions. Polymicrobial blood cul­tures re­sem­bling stool flora in im­mu­no­com­pro­mised pa­ tients may be an in­di­ca­tion that the pa­tient has Strongyloides hyper- infection. In these cases, the lar­vae mi­grate through the in­tes­ti­nal wall into the blood stream, car­ry­ing with them in­tes­ti­nal flo­ra. Parasitic Diagnosis 299 Toxocara canis. Human in­ges­tion of T. canis eggs leads to vis­ ceral larva mi­grans, char­ac­ter­ized by hypereosinophilia, he­pa­to­ meg­aly, fe­ver, and pneu­mo­ni­tis. Diagnosis is based on clin­i­cal find­ings and se­ro­logic test­ing (EIA). The test sen­si­tiv­ity and spec­ i­fic­ity can­not be pre­cisely as­sessed be­cause al­ter­na­tive meth­ods to dem­on­strate in­fec­tion have not been de­vel­oped. However, the test sen­si­tiv­ity is es­ti­mated to vary from 70 to 80%, and the spec­i­fic­ity is es­ti­mated to be >90%. Trichinella spiralis. Trichinosis is di­ag­nosed by dem­on­stra­tion of en­cap­su­lated lar­vae in bi­opsy spec­i­mens of skel­e­tal mus­cle, par­tic­ u­larly del­toid and gas­troc­ne­mius mus­cles. Detection of lar­vae may be im­proved by di­ges­tion of mus­cle tis­sue with an acidic so­lu­tion. Detectable an­ti­bod­ies do not de­velop un­til 3 to 5 weeks af­ter in­fec­ tion (af­ter the acute phase of dis­ease); their lev­els peak in the sec­ ond or third month and then de­cline slowly for sev­eral years. Antibodies are de­tected ear­lier by EIA than by other meth­ods, but EIA is less spe­cific. Positive EIA re­sults can be con­firmed by floc­ cu­la­tion tests. Trichuris trichiura. Diagnosis in pa­tients with heavy in­fec­tions is made by mi­cro­scopic ex­am­i­na­tion of a di­rect wet mount prep­a­ra­ tion of a fe­cal spec­i­men. Concentration meth­ods may be re­quired to de­tect eggs in light in­fec­tions. Wuchereria bancrofti. Refer to Brugia. W. bancrofti has a noc­ tur­nal pe­ri­od­ic­i­ty.

Helminths: Trematodes Clonorchis sinensis. Infections with the Oriental liver fluke are di­ ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens for char­ac­ ter­is­tic eggs. Fasciola he­pat­i­ca. Infection with the in­tes­ti­nal fluke, F. he­pat­i­ca, is di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens for char­ac­ter­is­tic eggs. Serologic tests (EIA and m as­say) are avail­­able through the CDC. EIA uses the ex­cre­to­ry-secretory an­ti­gens. Spe- cific an­ti­bod­ies ap­pear within 2 to 4 weeks af­ter in­fec­tion. Sensitiv- ity is ex­cel­lent (95%); how­ever, cross-re­ac­tiv­ity with Schistosoma may oc­cur. This can be re­solved by us­ing IB as­says. Antibody ti­ters fall rap­idly fol­low­ing treat­ment and can be used to pre­dict the re­ sponse to ther­a­py. Fasciolopis buski. Infections with the liver fluke, F. buski, are di­ Diagnosis Parasitic ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens for char­ ac­ter­is­tic eggs. Paragonimus spp. Infections with the lung fluke, Paragonimus, are di­ag­nosed by mi­cro­scopic ex­am­i­na­tion of fe­cal spec­i­mens and, less com­monly, spu­tum for char­ac­ter­is­tic eggs. Serologic tests (EIA and m as­say) are avail­­able through the CDC. EIA has a high sen­si­tiv­ity 300 SECTION 7 and spec­i­fic­ity, and an­ti­body ti­ters can be mon­i­tored to as­sess the re­sponse to ther­a­py. Schistosoma spp. The three most im­por­tant blood flukes that in­fect hu­mans are S. mansoni, S. japonicum, and S. haematobium. They pro­duce mor­pho­log­i­cally char­ac­ter­is­tic eggs that can be de­tected in fe­cal spec­i­mens (S. mansoni and S. japonicum) or urine (S. haemato- bium). In chronic S. mansoni and S. japonicum in­fec­tions, eggs ac­cu­ mu­late in the walls of the in­tes­tine, rec­tum, and liver and may be scarce in fe­cal spec­i­mens. Biopsy of the rec­tum or ce­cum may be re­ quired to make a di­ag­no­sis. Likewise, bi­opsy of the blad­der wall may be re­quired to di­ag­nose S. haematobium in­fec­tion. Antigen (EIA) and an­ti­body (EIA and IB as­say) tests are avail­­able through the CDC. The tests have a high sen­si­tiv­ity for S. mansoni in­fec­tions but a lower sen­ si­tiv­ity for S. japonicum and S. haematobium in­fec­tions. IB anal­y­sis is used to dis­crim­i­nate among the Schistosoma spe­cies.

Helminths: Cestodes Diphyllobothrium latum. Fish tape­worm in­fec­tions are di­ag­ nosed on the ba­sis of de­tec­tion of char­ac­ter­is­tic eggs or pro­glot­tids in fe­cal spec­i­mens. Dipylidium caninum. Infections with the dog tape­worm, D. caninum, are di­ag­nosed on the ba­sis of de­tec­tion of pro­glot­tids or egg pack­ets in fe­cal spec­i­mens. Echinococcus granulosus. Diagnosis of uni­loc­u­lar hy­da­tid in­fec­ tion is dif­fi­cult but is made by de­tect­ing cysts in tis­sues by us­ing im­ag­ing tech­niques (e.g., X-ray anal­y­sis, ul­tra­sonic scan­ning, and com­puted to­mog­ra­phy). Aspiration of the cyst con­tents is not rec­ om­mended. Serologic test­ing (IHA, IFA tests, and EIA) is also use­ful. The test sen­si­tiv­ity ranges from 60 to 90% and is im­proved when a com­bi­na­tion of tests is used. Antibody re­ac­tiv­ity in pa­tients is influ­enced by the lo­ca­tion and in­teg­rity of the cyst. Detectable an­ti­bod­ies are more com­mon in pa­tients with cysts in the bones and liver than in those with cysts in the lungs, brain, and spleen. Sero- reactivity is al­ways lower in pa­tients with in­tact cysts. False-posi- tive re­ac­tions may oc­cur in per­sons with other hel­min­thic in­fec­tions, can­cer, col­la­gen vas­cu­lar dis­ease, and cir­rho­sis. Echinococcus multilocularis. As with E. granulosus, in­fec­tion with E. multilocularis (multilocular hy­da­tid in­fec­tion) is dif­fi­cult. Parasitic Diagnosis Parasitic Definitive di­ag­no­sis is made by his­to­logic ex­am­i­na­tion of he­patic tis­sue. Serologic tests (EIA) have also been de­vel­oped for di­ag­no­ sis of in­fec­tions with E. multilocularis. Purified an­ti­gens are used, which has im­proved the test sen­si­tiv­ity and spec­i­fic­i­ty. Hymenolepis diminuta. H. diminuta (mouse tape­worm) in­fec­ tions are di­ag­nosed by find­ing char­ac­ter­is­tic eggs in fe­cal spec­i­ mens. Proglottids are rarely ob­served. Parasitic Diagnosis 301 Hymenolepis nana. H. nana (rat tape­worm) in­fec­tions are di­ag­ nosed by find­ing char­ac­ter­is­tic eggs in fe­cal spec­i­mens. Proglot- tids are rarely ob­served. Taenia saginata. Beef tape­worm in­fec­tions are di­ag­nosed by find­ing char­ac­ter­is­tic eggs or pro­glot­tids in fe­cal spec­i­mens. Taenia solium. Infections with the pork tape­worm fol­low­ing in­ ges­tion of cys­ti­cerci are di­ag­nosed by find­ing char­ac­ter­is­tic eggs or pro­glot­tids in fe­cal spec­i­mens. T. solium eggs are also in­fec­tious for hu­mans. Ingestion of eggs leads to cys­ti­cer­co­sis. Cysticerci can de­velop in any tis­sue, with di­ag­no­sis made on the ba­sis of de­tec­tion of the par­a­site in his­to­logic prep­a­ra­tions or on the ba­sis of a se­ro­logic re­sponse (EIA and ben­ton­ite floc­cu­la­tion). Seropositivity is re­ported in 50 to 70% of pa­tients with a sin­gle cyst, 80% of pa­tients with mul­ ti­ple cal­ci­fied le­sions, and >90% of pa­tients with mul­ti­ple, noncalci- fied le­sions. EIAs are less sen­si­tive than the IB as­say and cross-re­act with an­ti­bod­ies spe­cific for other hel­minth in­fec­tions. Current tests do not dif­fer­en­ti­ate be­tween ac­tive and in­ac­tive in­fec­tions. Parasitic Diagnosis Parasitic 302 SECTION 7 n s, s, s n , are ­ tai ­ tai ; ; no ­ tic ­ cyte ­ cyte ­ ent ­ ism ­ ria ­ ria ­ ro ­ ro ­ nos ­ te ­ te ay ay con ­ gan ay ay con ­ ag m eryth Noninvasive or bac if pres di Inclusions M bac eryth

­ ­ a

m; t, m ­ ti s ­ ally ­ en ­ lar ­ lar, ­ en ­ plas r ­ u ­ ole ­ u ­ plas of ­ u ­ fe ­ do ­ to “ground “ground clear glass,” dif Finely gran tion ec and en if pres vac are usu small Finely gran Cytoplasm

ed lly t; ­ cat ­ tra ­ ally ­ pac c c t; lly lo ­ tri ­ tri ed or ll, usu ­ pac ­ tra ually small ­ cen ­ cen aryosome ­ cat ppearance of stained: Sma com cen but may also be ec K may be cen lo Us and com ec A

m ­ ed

­ tin ­ for ­ tin ­ ogy y ­ ed ­ ma

­ ed; ­ all ­ ma ­ ut ­ phol s, uni ib r bead r as solid ­ tr ­ ule , chro ­ pea ­ pea ­ lar gh mor ­ i E. histolytica, ay ay ap ­ thou Nucleus may stainNucleus more darkly than that of al Fine gran in size and usu evenly evenly dis m is sim Peripheral chro may ap ring rather than bead ­ ty) ­ i

t a ons ons y not ­ bil ­ i ­ ti ­ ti ­ cul ned ned ­ all ­ ra ­ ra ­ fi ­ bae ­ a ­ a ­ stai ­ stai dif ame 1; usu 1; seen in un prep Nucleus (no. and vis 1; to see in un prep ­ nal e

­ ti e ­ siv ­ id s ­ tes ­ dia; ­ lin n ­ a ke ­ po ­ gre ­ li r ­ do ­ pro ­ ge mon ­ mon i Usually non Progressive, with hy fin pseu may be rap Motility

(diam 20 μm b ­ sive Parasitic Diagnosis Parasitic ­ va 5–60 μm; usual range 15–20 μm; in forms may be > μm;5–12 usual range μm 8–10 Size or length)or hozoites hozoites of com Trop

ntamoeba Entamoeba histolytica hartmanni E Organism Table 7.2 Parasitic Diagnosis 303 n ­ tai ­ bris ed ­ ria teria, teria (continued) ­ te ay ay con ­ gest yeast cells, other de Bac in M bac Bac ­

­ a m; ­ ed ­ ed m m ­ ti

­ tle ­ lat ­ lat ­ lar ­ en ­ plas r ­ o ­ o ­ u ­ plas ­ ally ­ u ­ u ­ fe n into ­ do ­ to nely Granular, with lit dif usu tio ec and en vac Fi gran Granular, vac ly

­ ed ­ lar ­ cat ­ u ar ­ ally or e and ; may ­ cle ­ reg c; may t; may or ns ns are E. lly lly lo ­ fus ­ tri r r “blotlike”; ­ tio ­ pac ­ mon ­ a ­ tra ­ i ­ cen ­ pea may not be ec be dif Large, not com darkly stained Small, usu cen Large, ir many nu com mimic hartmanni Dientamoeba fragilis shaped; may may shaped; ap var l ­ era may ­ ar h ne ged on ­ tin ­ cle ­ rip ­ bra s; may be e ­ ran ­ ma may be ; nu ­ ule ­ abl ­ i ne; may also rsed with large s; evenly ­ tin ­ tin r as solid dark nly ar ged on mem ­ bra ­ spe ­ ule ­ ma ­ ma ne; chro ­ eve ­ pea ­ ran ­ ter mem ap gran ar May beMay clumped and un ring with no beads or clumps Fine gran in bra also be clumped at edge mem of Usually no pe chro quite var chro in ­ ­ n n ons on ons ble ­ ble ­ ti ­ ti ­ ti ­ i in ­ sio ­ sio ned ned ­ ra ­ ra ­ ra ­ ca ­ ca ­ ten ­ a ­ a ­ a ble ­ ble ­ i y seen y vis ­ stai ­ stai is 1; 1; oc un 1; 1; of v prep all on a wet on a wet prep 1; 1; oc prep all un e h nal, ­ ­ siv s s ­ gis ­ tio

­ gre ­ gre ­ lar ­ rec ­ u ­ di ­ pro ­ pro ­ ally e, slug Usually non siv non Sluggish, non with blunt, gran pseudopodin Sluggish, usu Parasitic Diagnosis Parasitic 10–12 μm 15–50 μm; usual range, 8–10 6–12 μrn; usual range, μm 8–10 dolimax dolimax ntamoeba polecki E En Entamoeba coli nana 304 SECTION 7 ­ bris teria, yeast cells, other de Bac Inclusions

; ­ ed es t ­ lat ­ lar ­ lar, ­ o ­ ol ­ u ­ u ­ en ­ u ­ u Coarsely gran may be highly vac Cytoplasm vac may be pres Finely gran . e e s that are ”) t to see s of 4–8s of nded by es ­ til ­ ket ­ us ­ ule ­ ul ­ cul e ­ ter yosome ge; may be ­ rou ­ fi ded in Table 7.4 ­ cl aryosome ­ frac u ppearance of stained: Lar dif sur re gran (“bas n K Kar clus gran ­ clu A l ­ tin . Also in ­ era ­ ma h ­ ble ­ rip , D.C., 2001. , D.C., l ­ ton ­ era h ­ ing ­ tin ­ tin n in this ta ­ rip ­ ma ­ ma ­ sio e 1 to 2 um less). ­ clu ­ sur Usually no pe chro No No pe Peripheral chro chro y mea ­ ty) ­ i (continued) (continued) ­ all a ons y not ­ bil ­ i ­ ti in ned s usu ­ all ­ bae ­ ra ­ a 0%) or 2 ble ­ ble ­ i ­ ism ­ stai ame un vis 1; usu 1; prep Nucleus (no. and vis 1 (4 (60%) ­ gan d an amoeba, thus its in ­ nal e ­ ere ­ ti ­ sid ­ siv ­ tes s ­ dia n t stains, or ­ lar

­ po ­ gre ­ gu ly conly ­ nen ­ do ­ pro ­ ally ­ cal ­ ma mon ­ mon i ­ i e e an ­ tor Sluggish, usu non Nonprogressive, Nonprogressive, pseu ar Motility ts (in per e, but his ­ men e ­ lat (diam , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash b ­ el ­ sur Parasitic Diagnosis Parasitic ­ cia hozoites hozoites of com 8–20 μm; range, usual 12–15 μm Size 5–15 μm 5–15 or length)or d a flag Trop ­ ere

­ sid c Data from L. Gar S. Wet-preparation mea Now conNow a b c Iodamoeba butschlii Table 7.2 Organism Dientamoeba fragilis Parasitic Diagnosis 305 c in tin ­ tin , as in e or e cyst; t in ­ ma ­ ent ­ ent (continued) ss may be ­ tur ­ fus ycogen ­ sen pres May beMay dif ab ma clumped chro ma early cysts Gl May orMay may not be pres E. histolytica ; y ies ­ ies ­ ent ­ es; ­ es; ­ all with with

usu t; t; bod ­ gate ­ gate ­ en ­ ies ­ ies ­ ally ually ay beay round ay beay round su May beMay pres bod Cytoplasm chromatoidal bod elon rounded, blunt, smooth edg m or oval Us pres u elon rounded, blunt, smooth edg m or oval y y ­ all c t, t,

ly ly ly ly on ­ tri ed but ­ ed ­ si ll, ­ pac ­ pac ­ ally ­ ally ­ tral ­ tral ­ ca ­ cen aryosome ­ cat ­ cat ppearance of stained: ec Sma com usu cen lo oc K usu lo Small, com cen A s ­ tin ar ar ­ ma ble a ­ ble ­ i ­ cle ­ cle s evenly s evenly cs may not cs may be nu m ­ ti ­ ti d on ­ ule ­ is ­ is ­ for ­ ite ­ ed; ne; nu s, evenly t to see ­ ter ­ ter ­ zo ­ ut ­ ute ­ bra ib ib ­ ule ­ ac ­ ac ­ cul ­ tr ­ tr ­ fi ­ pho ine gran Fine, uni char gran dis be as clearly vis Peripheral chro tro F dis mem char dif s e, e, ­ tic ­ is on ­ tur ­ tur d ­ ti t to ­ ty) ­ i ­ ter ­ ra ­ ate ­ ma ­ ma ­ a ­ mon ­ ac ­ cul ­ bil ­ i ­ fi ­ cle ature cyst, ature cyst, a Nucleus (no. and vis 4; im 1 or 2; char M dif see on wet prep M cysts very com 4; im 1 or 2; 2 nu ­ bae ame ­ cal ­ cal ­ i ­ i ­ nal ­ ti ­ tes n Shape Usually spher Usually spher mon ­ mon i Parasitic Diagnosis Parasitic (diam b ts of com or length)or Size 10–20 μm; usual range, 12–15 μm 5–10 μm;5–10 usual range, 6–8 μm Cys

Organism Entamoeba histolytica Entamoeba hartmanni Table 7.3 306 SECTION 7 y y c ­ all t on e or e cyst; e cysts t in ­ si ­ en ­ tur ­ tur ­ fus ycogen ­ ca ­ sen Gl May beMay dif ab ma clumped mass oc seen in ma orMay may not be pres e ­ ent ­ lik ntly ointed haped ay beay ­ que t ­ ter s ­ lar p ­ en ­ ies ­ ies m y be pres undant, undant, ­ gu less fre Cytoplasm chromatoidal bod Ma ( than in E. histolytica ); splin with rough, pointed ends Ab an ends; thread chromatoidal bod pres y y t ­ ite ­ all c; c; ­ pac ly ly ­ zo on ­ tri ­ ed ­ si ­ tral ­ pho ­ ca ­ cen aryosome ­ cat imilar to ppearance of stained: K Large, may or may not be com and/or ec oc cen lo tro S A ­ tin ­ ite ; ar ­ zo ­ ma ­ lar ged on ­ cle ­ u d as in ­ pho cs not as ­ ran ; may ­ ti ­ fine E. histolytica ­ is

­ ite ne; nu ­ ter ­ zo ­ ble nly ar ­ bra ­ ac ­ pho ­ eve ­ sem imilar to tro Peripheral chro may be clumped and un mem re char clearly de tro Coarsely gran S 2 ­ y y n ons; ­ all e ­ ti ­ sio in wet ­ ty) ­ i on

­ tur 16; ­ ra ­ ca ­ si ­ a ≥ ­ bil ble ­ ble ­ i ­ i ­ ma (continued) ­ clei ­ clei ­ ca ature cyst, ature cyst, a Nucleus (no. and vis 8; oc M im cysts with ≥ nu oc seen ally rarely 2 or 4 nu prep M may be 1; vis ­ bae ; ame ­ cal ­ cal ­ i ­ i o ­ nal t stained , or e e ­ ti ay beay ­ ing t ­ tion

­ lar ­ nen ­ tes ­ quat rted on ­ tra ­ gu ­ e n ­ tive ­ e ­ ma ­ er; m ­ to ­ a ­ an ­ ad pen slide ow in fix Shape Usually spher may be oval, tri oth per dis Usually spher mon ­ mon i Parasitic Diagnosis Parasitic (diam b ts of com or length)or Size μm;10–35 usual range, μm 15–25 5–11 μm 5–11 Cys

Organism Entamoeba polecki Table 7.3 Entamoeba coli Parasitic Diagnosis 307 ­ ent t, fined ­ fined t se se if ­ pac ­ en ally ­ fu ass ot ot pres dif Usu pres Large, com well-de m N l ­ tes l y y ­ na ­ i ­ era ­ tra h ­ all

s are ­ tin t on ­ rip ­ ture ­ nal ­ si ­ ule ­ ma ­ en ­ ca ­ ter n No No pe chro None; small gran oc pres i struc Large cen body dom

y e e ­ all ­ ites c c e e d ”) ­ er

­ zo es ­ til ­ som ­ som

­ tri ­ ket ­ us ­ ul e ger, ­ ally ­ yo ­ yo ­ pho ­ serve ­ cl ­ nus ­ cen ­ frac u Entamoeba Smaller than kar larger than those of ge seen in tro but gen usu re gran may be on one side of kar (“bas n Lar ec Not ob ns ­ sio y seen; ; small on , D.C., 2001. , D.C., ­ clu l ay beay ­ all hromatoi- ­ ent n ­ ton ble ­ ble ­ i ­ era rved h ­ sio ­ ing s or in ­ ies m ­ tin ­ ear c ­ se ­ ca ­ rip ­ ule ­ ma e 1 to 2 pm less). ­ sur are oc Rarely pres well-stained smears gran fine lin dal bod faintly vis No No pe chro Not Not ob i

y mea -

e e ­ cle ­ all cysts ­ ing a ­ tral ­ tur nd s usu ble ­ ble ­ ma ­ rou ­ ism Enteromo ultiple nu ­ sem Mature cyst, 4; im cysts, 2, very rarely seen and may re of hominis nas Mature cyst, 1 M sur body large large cen ­ gan

se ­ cal ­ ole ­ i ­ u ­ lap t stains, or vac ­ nen o large ­ ma ­ gen ­ co ­ ing t e. Usually oval, may be round May varyMay from tooval round; may col cyst ow gly space Usually spher ts (in per ­ din ­ men e , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash Parasitic Diagnosis Parasitic ­ sur ­ cia 5–10 μm;5–10 usual range, 6–8 μm 5–20 μm; usual range, 10–12 μm 6–40 μm ­ dish-brown with io - Data from L. Gar S. Stains red Wet-preparation mea a b c Endolimax Endolimax nana Iodamoeba butschlii tis hominis Blastocys 308 SECTION 7 rom Jorgensen JH,rom Jorgensen Pfaller ­ tion. F ­ por .C., 2015. .C., e pro Manual of Clinical Clinical (ed.), of Manual WarnockS, DW ­ tiv ­ a ­ ton, D ­ ter S s. (Top row) Trophozoites. (Middle Cysts. Trophozoites. row) row) (Top s. ­ ing ­ man u i, shown ini, shown rel ­ cle ­ bae h of estinal ame 11th ed., ASM Press, ed., ASM Wash 11th Int l KC, Funkel KC, G, Landry ML, Rich

­ rol Figure 7.1 (Bottom row) Trophozoite nu Trophozoite (Bottom row) MA, Car Microbiology, Parasitic Diagnosis Parasitic Parasitic Diagnosis 309 e; ­ lar ­ u ­ fac ­ ing ed with ­ py (continued) ­ lat l sur , yeasts, mear ­ o ­ cu s; may be ­ u ­ tra ­ ria roove acrossroove n in size and ­ te ­ bri ­ gle s /3–1/2 length of ­ tio ­ face ­ tures ­ a ­ ral g ­ i sur ­ ing 1 ed bac d ­ tral toplasm finely gran ­ ten ­ gest ucking disk oc /2–3/4 ven of nd may be vac Other Other fea a pear shaped from front, spoon shaped from side Prominent cytostome ex body; spi ven Cy in and other de great var shape on sin S 1 ­ la b ­ gel ­ la a

­ gel

r, 1 in r, l, l, ­ ble fl ­ i ­ rio ­ tra ­ dal ­ era ­ te . of fla No No vis 3 an cytostome 4 lat 2 ven 2 cau ;

ned ned ­ tin n ns; r of ­ stai ­ stai ­ ma ­ tio ­ te s have s have ble i ­ ble ­ ra ­ i ­ a n un n un ­ ism l chro es ­ gan ble i ­ ble e is clus ble i ­ ble ­ era ­ i ­ i ­ ul h ­ ty) us and 60% have i; not vis ­ i ned prep ­ som ­ rip ­ cle ­ cle ot vis ­ bil ­ yo ­ i ­ stai ercentage may vary, but ; not vis un Nucleus (no. and vis P 40% or of 1 nu 2 nu no pe kar 4–8 gran mounts 1; n 1; 2 mounts ­ ent ed, e; are ­ par ­ rat ­ siv s is in ­ ry ­ dia may be ­ ta , ser trans t to see if ­ po ­ gre ­ ism ­ ity ­ lar ­ do ­ cul ­ pro ­ til ­ cus ­ fi ­ gu ­ gan ­ most a Motility pseu an Usually non or broad lobed and al Stiff, Stiff, ro or Falling-leaf mo mu dif es ­ lat ­ el ; 5–15 μm; 5–15 ­ bae Parasitic Diagnosis Parasitic Shape and size Shaped like ame (usual range, 9–12 μm) Pear shaped; 6–24 μm long (usual range, μm10–15 long), 4–8 μm wide Pear shaped; 10–20 μm long; μm5–15 wide hozoites hozoites of flag Trop

Organism Dientamoeba fragilis Chilomastix mesnil Giardia Giardia duodenalis Table 7.4 310 SECTION 7 ; ­ ly ds ds ed; ­ lum ­ al y 1/2 ­ ten ­ ten ­ er ­ ten ­ gel ex ex ­ tel ane ane ­ br ­ br een ­ ma r fla ­ i ­ lum ­ lum x y or lat ­ rio ­ ily s ­ gel ­ gel ­ orl ­ te d end body of ­ pro ­ tures ­ ri r fla r fla ­ te ­ yon lating mem lating mem ds ap ds length body; of ds 1/2 length body; of ­ rio ­ rio ­ te ­ te e side of bodye side of flat ­ ten ­ ten ­ ten ominent cytostome Other Other fea On pos Pr ex length body of Undu ex pos Undu ex no free pos axostyle eas free pos free be b

­ la ; ,

­ or ­ gel r, r, r, ­ or ­ or ­ or ­ or ­ rior ­ ri ­ ri ­ ri ­ ri ­ ri ­ te ­ te ­ rio ­ rio ­ te ­ te ­ te ­ te ­ te ­ te . of fla No 3–5 an 3–5 an 3 an 1 pos 1 an 1 pos 1 pos 1 pos ned ned ned ned , D.C., 2001. , D.C., ­ stai ­ stai ­ stai ­ stai ­ ton n un n un n un n un ­ ing ble i ­ ble i ­ ble i ­ ble i ­ ble ­ i ­ i ­ i ­ i ­ ty) ­ i ot vis ot vis ­ bil ­ i Nucleus (no. and vis n 1; mounts n 1; mounts not vis1; mounts not vis1; mounts ­ id ­ id (continued) a Motility Jerky, rap Jerky, rap Jerky Jerky es ­ lat ­ el , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash Parasitic Diagnosis Parasitic ­ cia Shape and size Pear shaped; μm5–15 long (usual range, 7–9 μm long), μm wide 7–10 Pear shaped; μm7–23 long (usual range, μm) μm13 5–15 wide Oval, μm 4–10 (usuallong range, 8–9 μm long), 5–6 μm wide Pear shaped or oval; 4–9 μm long (usual range, 6–7 μm long), 3–4 μm wide hozoites hozoites of flag t to see. ­ cul Trop ­ fi

Data from L. Gar S. Usually dif a b Organism Trichomonas hominis Trichomonas vaginalis Enteromonas hominis Retortamonas intestinalis Table 7.4 Parasitic Diagnosis 311 in nts la ­ gel ­ age ble ­ ble ­ i n e e of e of e of cyste of n ­ dia e of ­­ lin s; there is ­­ sid d’s crook” d’s y vis ­ ing r t ds above me ­ sid ls or fla ble i ­ ble s ­ ber ­ all ­ i ­ her fi t ­ ten ­ dra ­ bri ­ ing ­ ple ­ hy ­ nal ­ men ls ex l along ls, usu ­ di sam ge t around out ­ tu ­ bri ­ bri ­ bri cyst; fi sm pullssm from cyst away fi ­ cal ­ gi ­ i ­ ran ­ fec f ­ pla cyst; shadow out ­ ing ­ ing fi l ar red to as “shep t t ­ to aused de by lo” e ­ lo” ­ bri ­ fer ns; deeply stain ­ por ­ por s in cysts may be vis n; curved fi ­ age c nd cy ­ tio ed in clin y re ­ ra ­ tio s ­ ber ­ ter ­ a ­ all n ­ ra y lie across lon ­ a ­ age, a ­ all ­ ture ­ cou bird beak fi usu hrink y not seen ned prep ­ us; ­ ies ­ all her her fea ­ stai ­ cle ­ ten s arely en un bod of wall; may also be “ha wall due to shrink Cytostome with sup stained prep cytostome usu nu R Longitudinal fi cytostome with sup Ot NA ChilomastixResembles usu Endolimax nana Endolimax Resembles red ned ned t ­ ets ns , D.C., 2001. , D.C., ­ fer y ­ stai ­ stai ­ tio ­ ton ted into ­ all ­ ing a pack ­ ra s, re ned mounts ­ a ­ ing n un ­ tin ­ ule ­ men ct in ct in un ­ stai y 2 ly ble i ­ ble ­ ma rag ­ tin ­ i tin ­ ons; usu ­ all ­ ti ­ ty) e ends cyst; of not at one end in un ­ i ned prep ­ ra ­ sit ­ ten f nct gran ­ a ­ bil ble ­ ble ­ i ­ ti ­ i ­ stai ­ po ­ cated 1; not dis1; un lo prep 4; not dis 2; of dis to as chro Nuclei (no. and vis NA 1–4; usu not vis1; mounts op vis l, ­ da r ­ soi ­ rio ­ lip ­ te e knob ­ lin b und ­ a Lemon shaped with an hy or round Ro Oval, el Shape NA Elongate or oval Pear shaped or slightly lemon shaped ­ ter ­ e a

­ am es ­ lat ­ el , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash Parasitic Diagnosis Parasitic ­ cia 6–10 μm6–10 long (usual range, 7–9 μm long), 4–6 μm wide 8–19 μm8–19 long (usual range, μm long), 11–14 μm wide 7–10 (usual range, 6–8 μm long), 4–6 μm wide Size 4–7 μm in di stage cyst No μm4–10 long 4–9 μm long (usual range, 4–7 μm long), 5 μm wide . ts of flag ­ ble ­ ca Cys ­ pli

Data from L. Gar S. NA, not ap a b Chilomastix mesnili Giardia Giardia duodenalis Species Dientamoeba fragilis, Trichomonas hominis Enteromonas hominis Retortamonas intestinalis Table 7.5 312 SECTION 7

­ has re ­ roll KC, s. (Top row) Trophozoites. Trophozoites. row) (Top s. ­ man Manual of Clinical Microbiology, es of hu esof ­ lat s shown; a cyst stage for D. stage for fragiliss shown; a cyst ­ el ite i ­ ite flag ­ zo ­ tal .C., 2015. .C., ­ i ­ pho S, Warnock DW (ed.), (ed.), DW Warnock S, ­ gen tro ­ ton, D ­ ter S ­ ing estinal and uro Int

(Bottom row) Cysts. D.(Bottom fragilis row) Figure7.2 here. JH, From Jorgensen shown not Pfaller is but MA,been found Carcently Funke G, Landry ML, Rich 11th ed., ASM Press, ed., ASM Wash 11th Parasitic Diagnosis Parasitic Parasitic Diagnosis 313 ­ 1 ­ nu ble ­ ble s ­ i ­ us; sm sm ­ ro ­ sue (continued) ­ cle ­ pla s of s of rs t to see ned ­ to ­ nu n young ­ der ­ opsy d with cilia, ­ pea ­ cul ­ ro ­ stai ­ fi ­ ere ble i ­ ble ­ i e ap r clear, round, and us may be vis t to see; mac in un ­ tur ­ cle ­ pea age in stool. Various ­ cul ble ­ ble ruc ­ fi ­ i ­ nu s are vis -shaped mac ­ ro tract) and other tis ­ tic st an be seen in bi ­ ole ­ nal st ear cytostome; cy us dif us vis tract). ­ ney ­ u us, which is dif ­ nal ­ nos ns; body is cov ­ ter ­ ti ­ cle ­ cle t to see within wall. cyst ­ ary ­ ag ­ cle cle c ­ cle ­ i ­ tio a ­ ger n ­ tes ­ nu ­ nu e vac ­ nu ­ cul fast stains; ap ­ ra d in trichrome stains. ­ fi ­ ro ­ a ­ til ­ zoa ­ cro ­ ed. c ­ id- ­ cro ­ kle s ­ to ­ lat ­ tra ­ o ro ­ u y y tract, bil ns; mi ­ ture s taken from GI tract (brush bor t wrin ned prep l cells in in ; cilia dif ­ tor ­ tio y with ac sue ­ sue p ­ lia ­ lar ­ ra ­ ra ­ men ­ wha ­ stai ­ u ­ i ­ a ­ abl ­ i ­ the us and con ­ spi semble nonrefractile spheres in wet-preparation ­ i yst: 1 large mac pec n un other stages in life cy Oocyst is the usual di s ep (re Other Other fea Trophozoite: 1 large, kid small, round mi may be vac Re smears; autofluoresce with epifluorescence; stain var some even in stainedeven smears; mac which tend to be lon C i prep gran cle cysts; in older cysts, in e fast ­ tur ­ id- 0–100 μm ­ or; 5 ­ ri ia, microsporidia, and tis ­ te ­ er n ­ cid ­ ing a s, coc s, which may or may not be ­ per ­ ate ­ ite ­ i spp., but larg spp., ­ zo ­ ro y round, 8–9 μm in diam; ac ­ all r oval; 50–70 μm in diam (usual s of cil y round, 4–5 μm in diam; each ma ­ er ns spo ­ tic ­ all ­ cal o ­ is ­ i ­ er ­ tai n ­ ter ­ ac e ­ bl ­ i cyst co­ cyst vis oo long; 40–70 μm wide (usual width range, 40–50 μm) Cyst: spher range, 50–55 μm) like Cryptosporidium Shape and size withTrophozoite: ta ovoid Oocyst gen Organisms gen Parasitic Diagnosis Parasitic phological char Mor

Species Balantidium coli Cryptosporidium parvum Cyclospora cayetanensis Table 7.6 314 SECTION 7 y s ­ ite ­ tor is ­ zo e by n stool. ­ ro ­ cal ­ nos ­ cal h ­ i ­ cur i ­ ag c with 4 spo t to di ded. Enteric ded spores. for y seen in fe n. ­ cysts o ­ cul ­ all ­ ro ­ tio ­ fi ­ cysts ­ men ­ men fast, trichrome, and ­ ro ­ fec ­ om ­ om s. ­ id- re usu ntly based on clin nts dif void spo void ­ men ­ tie (continued) ­ i ­ que ce of in ns 2 spo a ­ cysts a n not rec ­ tai ­ den ­ zoa ­ i ­ tio on ts vary; ac cyst or ­ cyst o s e oo ­ to ­ la ­ u ­ sul ro ­ tur ic ic ev stool spec ­ oc ­ ture cyst ­ cyst c s. ­ ma ­ log ns in AIDS pa ­ ing ­ ro sue ­ sue p ­ in ­ men ­ tio ­ i ­ am ­ fec istology re pec ex each; im H Animal in in Calcofluorwhite stains rec Diagnosis is most fre Thin-walled oo Other Other fea Mature oo s and se e , D.C., 2001. , D.C., , ­ sur d ­ ton ­ ere ­ ing ­ cysts ntly ut meaut ­ cov ­ ro ­ que e spo re ­ cysts b ia, microsporidia, and tis ; 200 μm to 1 mm ­ tur ­ cal ­ cid ­ i cyst ­ cyst f t shaped; 4–6 μm of oo­ of ­ cen s; oo ns 2 ma s, coc y spher , each 9–1 6 μm and long ­ ite ­ tai ­ ate ­ all ­ zo ­ i ­ er ­ ro ­ cysts sual size, 20–30 μm μm 10–19 long, ­ ro arely seen out 4 spo ­ gans. mely smallmely and been have re s of cil r ­ cyst; u ­ tre ­ tic ­ ing ­ cysts r n ­ is ­ ro ­ tai ­ ter ­ body o es; ovoid spoes; ovoid ­ ac ­ tur each con in diam 7.5–12 μm wide 7.5–12 rup Oocyst with thin wall con Shape and size Ellipsoidal oo Spores are ex Trophozoite (tachyzoite): cres wide; spo 9–11 μm from all 2–3 μm by long wide. Cyst (bradyzoite); gen , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash Parasitic Diagnosis Parasitic ­ cia phological char ­ nal. ­ ti Mor ­ tes spp. ­ in

­ tro Data from L. Gar S. GI, gas a b Species Table 7.6 Cystisospora bel ­ li Microsporidia gondii Toxoplasma Sarcocystis Parasitic Diagnosis 315

c ­ ti ­ ont n of n of t- e ­ tio ­ nos ic inic the ­ tur (continued) ­ cen ts young ­ ag ­ cep e schiz ­ fec ­ dem n n di ­ tur ­ tio ­ es; i uid in later stages ­ ra nts; en ­ u l blood; ma ­ tie ­ fig ­ era ­ nal fl d edg e of bande of forms and h pi l blood; ma e RBCs ­ ate ed pa ­ rip ­ enc ages, with rare ex ­ era ­ tur ­ bri h ne; lymph nodes and blood ­ ine s ­ miz ­ rip ­ to ­ am ­ bra ts ma ­ ing st e cross” con em ­ nec ­ op ; ex ­ fec ­ tes ­ vel ­ ful es seen in pe l blood; pres t; in ­ ing m ­ era , “Mal ­ lat h ­ stag es seen in pe ­ men n help ­ ent ­ du ig ­ rip n ­ tio ng (rareng clefts); Maurer’s rings and cres b ­ stag ­ tra ­ pli ­ ial p ­ cal u ­ i ings); seen in sple ­ cen ­ lar r y); y); if pres l blood (no other de ­ sar ­ rum es seen in pe ­ a ­ es ­ era , with typ h s; RBCs may be and oval fim have ts young RBCs ­ cip ­ stag Cs ; lots ma of ­ ite a ­ rip ­ der ­ fec ry nec ­ zo ­ ont ­ RB e of Schiiffner’se of dots; all P. P. fal ­ o ­ to ng; all s; in /accole forms; no stip ­ enc ble ­ ble e of Schiiffner’se of dots; all ­ pli ts all ­ ite e schiz ­ zo ­ que ­ fec ­ enc ­ sem ­ o s seen in pe ­ tur ­ pli ); in ns 8–10 mer ns 8–10 ­ cyte found found in blood d; microhematocrit tube con ­ to ­ ont ­ tai ­ ple on ­ me ­ zoa ­ to ­ tion ns 16–18 mer e schiz ­ ont c te-shaped ma ­ fec eboid rings; pres ­ tai ltiple rings; ap ­ tur nameboid rings; pres ngs arengs thick; no stip ­ set of of in can be sam United States (no travel his Ri ro Mu shaped ga ma Ring forms only (re Trypomastigotes and long slen Diagnostic stage RBCs schiz No Am con s of pro ­ tic ­ is ­ ter n ­ ac ­ ia) ­ nant ­ tia ­ lar ­ lig (ma

­ nign ter s) ­ rum ­ a ­ nes (ovale ma (be Parasitic Diagnosis Parasitic ­ le ­ ia) ­ vax phological char sick ­ ia) es ­ lar ­ ing ­ sit ­ lar Mor ­ a ma

ma ­ ia) spp. ­ tan n sleep ­ lar ­ tian ­ ca r ­ ri (quar te Plasmodium Plasmodium vi Plasmodium ova malariaePlasmodium Plasmodium fal ­ cip ma  Trypanosoma brucei gambiense (West Af Babesia Malaria par Organism   Table 7.7 316 SECTION 7 us n; ­ lar , etc.; ­ tio ­ cle ­ lu u ­ row ­ fec ­ cel ­ ing n ­ tra n ­ tai uid in later stages e of ine of c ­ ti d early in in ­ nal fl ­ enc pi ­ ple ­ nos orms con ­ ag m into amastigote form ne; lymph nodes and blood ­ ine s ­ lar f ­ for st di nes; pres ­ am ­ bra ­ lu ­ pla ­ bra ­ cel em ; ex ­ o ­ tra ­ ful ­ net ­ ing m m and in spleen, liver, bone mar s mem ­ te ­ lat e of ine of n help c ­ cou ­ du ­ ti n ­ tio l sys ­ enc us and ki ­ lia ­ nos ­ tra ­ cle ­ cal u ­ ag ­ i ­ the nu urved in C shape; blood sam ­ cen ­ do st di heart, tract) GI* and trans ­ ing ­ en ­ ten c n , D.C., 2001. , D.C., ­ pla ­ lo ­ o ­ u ­ tai cle ( ­ cle ­ ton , with typ ­ tic ­ net ges of skinges of and mu ges of skin;ges of pres (continued) ­ ing a ­ der ­ re d mus ­ pha ­ pha ­ out ­ ro ­ ro ­ ate c forms con ri ­ ti us and ki ­ lar ­ nos ­ cle ­ lu ­ ter st ­ ag nu ­ cel found found in blood d; microhematocrit tube con ­ tra ­ ing st di n ­ ple ­ pla ­ tai ­ zoa ­ o ­ to ­ tion e of in ­ net ­ fec ­ enc astigotes found through orms con of of in can be sam Amastigotes found in mac pres Diagnostic stage Trypomastigotes and long slen f Am Trypomastigotes short and of stumpy, trypomastigotes en Amastigotes found in mac and ki s of pro ­ tic ­ is y ) ­ ­ all ­ ter e, es. ­ tu ­ ne ­ ac (East ) ­ ta ­ eas ­ cyt e but L. ­ cu ­ ro ­ sis ­ a ­ sit ­ al) ­ co ­ a ; not ac ­ mi ted for ­ cer with ­ so s) ­ ous (mu o ­ sen ­ ne ­ son , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash (vis ­ nes ­ i Parasitic Diagnosis Parasitic ­ ta ­ pan (Chagas’’ dis (Chagas’’ ­ cia ; ; RBCs, eryth ­ par phological char Leishmania donovani Leishmania try sick ­ nal y a blood par ­ ti e but pre ­ ing ­ can ­ all Mor ­ sit with ­ tes ­ i spp. (cu ­ a ­ tu ­ in

) ­ son ­ tro ­ i n sleep ted for com ­ ca ­ par ; not ac ­ sen ­ ri Data from L. Gar S. GI, gas a b donovani ous pre Organism Trypanosoma brucei rhodesiense cruzi Trypanosoma Leishmania braziliensis donovani Leishmania Af South Amer a blood par Leishmania com Table 7.7 Parasitic Diagnosis 317 en i not ­ twe i not i i ­ cle ail with ­ cle ­ cle ­ cle ­ der t ed; nu ed; nu ed; nu ed; nu ed; gap be ­ per ­ per ­ per ­ per ­ per i ­ cle athed; slen long, athed; tail ta nu athed; tail ta ­ she ­ she ­ nal ­ she ­ mi and ter ­ nal ­ mi ds to tip tail of ds to tip tail of i at tip ­ ter ­ cle ­ ten ­ ten ex ex sub at tip tail of 230–250 6–8 by μm; sheathed; tail ta 177–230 by 5–6 by 177–230 μm; sheathed; tail ta nu at tip tail of Microfilaria 3–4 by 163–203 μm; un 5–9 by 304–315 μm; un 244–296 μm; sheathed; 7–10 by tail ta es es es ­ tod ­ a ­ mal ­ mal em es 50–70 mm by es 33–50 mm by sue ­ sue n ­ males 80–100 mm ­ mal ­ mal es 70–80 mm μm 120 by 190–200 4 μm; by un ­ mal s of blood and tis ­ tic ­ is ­ ter ­ ac Males 30–35 mm 350–430 by μm; fe μm 500 Males mm 45 60 by μm; fe 43–55 mm by 130–170 μm mm43–55 130–170 by Adult worm Threadlike; males 13–25 mm 70–80 by μm; fe Threadlike; males 24–28 mm 70–80 by μm; fe Males 19–42 μm; mm 130–210 by fe Threadlike; males 40 mm μm; 100 by fe 250 by μm 32–62 mm μm 130–160 by μm 270–400 Parasitic Diagnosis Parasitic ­ lus phological char ­ vu Mor

Loa loa Mansonella perstans Parasite Brugia malayi ozzardi Mansonella Onchocerca vol Wuchereria bancrofti Table 7.8 318 SECTION 7 ; ; . ­ tic ed ­ tic ­ vae ­ tum ­ tat ­ nos ­ nos ­ ti d (tend en shell ­ ag ­ ag ­ ize ­ twe ic testsic racks re e with large, ­ til s. ­ log ­ fer ­ sul ed soil; lar ­ ing t ­ ro c tests. ­ vae a ­ men ­ at ­ ti ­ i o or oncosphere); ­ nat o or oncosphere); ide) are di ­ i ide) are di r cap ­ nos pec ­ bry ­ bry ns, lar ­ tam an be found in spu ­ gle s ­ ag ­ ity o f eye; sef eye; ­ gle s di ­ cal s ­ tio ­ i av ­ vae c ­ fec e shell) and un ­ cal ­ cer o ­ i ar ­ cal c ­ lat an be found in stool (slit in tail). ­ log ­ cu –hooked em -hooked em ­ o ­ ing l ed with con t ­ bi ­ ber ­ vae c ­ gra ­ gest ­ ing 6 ­ ing 6 en for canen for ­ cro n n 2 branches on sin 2 branches on sin mi tak ­ 1 e) lare) ­ tai ­ tai 1 ggs on perianal skin. < ed or tu d) eggsd) found in stool. Adult worms: in. 10–12 > ­ ed. ns), mi ­ tiv ­ cal ­ lat ­ ti ­ ate ­ tio hrough outer skin, of layer cre ­ tid ( ­ its e ­ fec ­ tid ( t ggs are in t ­ mil ­ ger ­ fec ­ pos ­ glo ­ der t ­ ag ­ glo ­ rid e e in d shell, con d shell, con ­ ca ­ ver y found in stool; short buc ­ ate ­ ate ye); canye); be mis lugs. Adult worm:lugs. rarely seen. Eggs should be quan ­ all ed Adult side. worm: about 3/8 in. (ca. white 1 cm) long, with ­ lar p ­ ing e ­ ia l ns may not be treat d ­ ten e) usue) ­ tio ­ phi ­ clu ; oval with; oval broadly rounded ends, thin shell, and clear space be ­ o ­ tiv s) ands) gravid pro lia). There are no prac ­ fec s) ands) gravid pro , and/or filariform(in ith bumpy shell ex ­ cal ­ sin ­ let s (in ­ fec ­ ti ­ let tes from anus and de ­ phi ­ in ­ tum ­ o osts. Larvae will wan osts. Dog or cat as ­ sue ­ gra n; eo ­ sin ­ gate, w o (8–16 cello (8–16 stage). Adult worms: rarely seen in clin non s iden spp. only spp. (thick, stri spp. only spp. (thick, stri ­ tal h ­ tal h ­ tio ith hook ­ cie ­ bry l packet cells of (“short and sexy”). In very heavy in d (oval tod (oval round with thick, mam , no hook a ­ ma ­ vae ( ­ den ­ den m and eo ­ ci ­ ci aenia aenia ­ fir ­ ize ­ ers ­ dia ­ ers w ths T T ­ til l shaped with one flat y approx 12 ft (ca. 3.7 m) long m) fty approx 12 (ca. 3.7 y approx 12 ft (ca. 3.7 m) long m) fty approx 12 (ca. 3.7 ­ ing e ­ ing y found in spu ­ mor ­ all ­ all ­ min ­ bal ­ op ­ cate ­ cate ri hrough deep tis ­ all ­ rel shaped with two clear, po n ­ di ­ di ­ vel l for con e itch ­ sio ­ tal p ­ fu ­ der t ­ i ­ ver ­ ca an s of hel gen oc pointed tail. Female mi Egg: two eggs spe of and de Rhabditiform lar Diagnostic stage Egg: both fer to be more or oval elon (ca. 25–30 cm), found in stool. Rarely (in se Egg: bar etc.), since(rare, light few, in Egg: foot Humans are ac (se Humans are ac w help Scolex (4 suck (4 Scolex eggs eggs in Scolex (4 suck (4 Scolex worm usu eggs eggs in worm usu ­ tic ­ is ­ ter ­ ac

Taenia saginata Taenia ms) Parasitic Diagnosis Parasitic ms) ­ wor phological char Toxocara canis or Toxocara ­ wor Mor

a solium a ylostoma duodenale, Necator ocara cati ocara erobius vermicularis ongyloides stercoralis stodes (tape americanus Anc Str Trichuris trichiura Trichuris Ent Ancylostoma braziliensis Tox Taeni  Nematodes (round Helminth lumbricoides Ascaris

Ce

Table 7.9 Parasitic Diagnosis 319 ­ r s ­ va es es ith o or o or es in ­ tiv ­ ser ­ tiv ts ­ man ­ tum o ­ va ­ ces. ­ tur ­ va ­ bry ­ bry ­ cer w ­ li ­ ser gs) ­ ser ­ lum fi n liver) when n liver) eg ­ cu e struc d in spu ic canic ­ ca ­ ily i ­ i ­ tiv ­ ere ­ stat ­ a ts -hooked em -hooked em ­ pat ted with no pre ­ mar ­ duc ­ cov n liver) when hu n liver) ­ lec ­ pro ­ ing 6 ­ ing 6 ted with no pre ­ lum fi ­ ily i ted with no pre sts and many sco n n op (pri ­ lec ­ cu ­ lec ­ tai ­ tai ­ mar ­ vel s into which oper ­ ter cy ); can be re e ­ der ­ ets ike that F. of he ­ tin sts de y. op (pri s should be col ­ tes e ­ vel ­ ger ns daugh ­ ogy l o and egg shell ­ tid cy l ­ tin s should be col ­ men ms. Cyst grows like met wider than re long, ­ i ­ tai s should be col ­ da o and egg shell ­ tes ­ bry sts de s into which oper ­ pho ­ men ­ wor ­ tid ( ­ i ed, with shoul t ­ der ­ bry ” = egg pack ­ men ­ der ­ i ­ lat ­ tid cy en em ­ glo ­ ing ­ cu e tape ­ da ed (mor en em ­ twe d from those F. of buski e ­ vor ­ lat ms; con cyst l spine); spec ­ twe ­ ni ­ ate iron fil ed from at cyst sur pine); spec ­ cu e ­ ti , D.C., 2001. , D.C., ­ wor ­ era ­ ing b ­ rat ­ en l spine); spec ­ ton ed, with shoul ­ ish “ ­ nal s ­ pi es (fox or wolf);es (fox hy ­ ing b es (dog); hy ­ fer ts ly 35 um);35 oper ­ lat ­ era ­ ing ­ ed ­ mi < ­ vor ; worms in veins small of in ­ vor ­ cu ­ lat ts ly ­ ni uid as e dif brown ­ men ­ ni ­ ty) ggs from car ­ i ­ cu ­ a l ; worms in veins blad of ; worms in veins large of in ­ men ­ bil ­ a rooves) androoves) gravid pro ­ ine fl ­ a ­ not b l ­ tum ( ly seen;ly egg round to with oval thin shell, con seen;ly egg round to with oval thin shell, con ­ ty) ­ ty) ­ i ­ i ­ gest e ­ lar fi ­ am ggs from dog tape ­ bil ­ bil ne. ­ mal ­ mal ­ ing g ­ lar fi ­ a ­ a ly inly ­ bra ed); eggs oper n egg vi ­ tal ­ gest e e”); eggs oper ­ tai ­ low ­ den mem n egg vi n egg vi ­ ered in stool (large lat ­ ered in stool small (very lat ­ ered in urine (large ter ­ eral suck ­ sett ­ ci ly inly ­ tai ­ tai ­ ing ­ tal ­ cov ­ cov ­ cov s ac “ro ­ it ­ den ­ ter, gs found in stool; can es (to main ­ man ­ ci aboratory should ex Scolex (latScolex Adult worm not nor oncosphere with po Adult worm not nor oncosphere with no po Adult worm found only in car ac Adult worm found only in car hu Eggs found in stool; very small ( Eggs coughed up in spu stool (if swal Eggs re Eggs re Eggs re Eggs found in stool; very large and oper Eg cen no lim tiv L (to main (to main , Diagnostic Medical Parasitology, 4th ed., ASM Press, Wash Parasitic Diagnosis Parasitic Fasciolopsis bushFasciolopsis ­ cia ­ ca ­ i ­ pat ciola he Diphyllobothrium latumDiphyllobothrium Hymenolepis nana diminuta Hymenolepis granulosus Echinococcus multilocularis Echinococcus Clonorchis (Opisthorchis) sinensis westermani Paragonimus Fas Schistosoma mansoni Schistosoma haematobium Schistosoma japonicum Data from L. Gar S. a

(flukes) Trematodes

320 SECTION 7 Parasitic Diagnosis Parasitic Parasitic Diagnosis 321 ▼ Parasitic Diagnosis Parasitic

Figure 7.3 Relative sizes of hel­minth eggs (from CDC). Schisto- soma mekongi and Schistosoma intercalatum have been omit­ted. From M. Brooke and D. Melvin, Morphology of Diagnostic Stages of Intestinal Parasites of Humans, 2nd ed., U.S. Department of Health and Human Services pub­li­ca­tion (CDC) 84-8116, Centers for Disease Control and Prevention, At­lanta, GA, 1984.

SECTION 8 Vaccines, Susceptibility Testing, and Methods of Organism Identification

Ge­ne­ral Comments 324 Table 8.1 Recommended pe­di­at­ric im­mu­ni­za­tion sched­ule 325 Table 8.2 Recommended adult im­mu­ni­za­tion sched­ule 328 Table 8.3 Clinical and Laboratory Standards Institute (CLSI) doc­u­ments re­lated to an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing from hu­mans 330 Table 8.4 Summary of CLSI an­ti­mi­cro­bial sus­cep­ti­bil­ity test meth­ods for se­lect bac­te­ria, my­co­bac­te­ria, and fun­gi 332 Table 8.5 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­mi­cro­bial agents 337 Table 8.6 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­fun­gal agents 342 Table 8.7 Routes of ad­min­is­tra­tion and drug class for se­lect an­ti­par­a­sitic agents 343 Table 8.8 Antibacterial agents for spe­cific bac­te­ria 345 Table 8.9 Intrinsic re­sis­tance of se­lected Gram-negative bac­te­ria 354 Table 8.10 Intrinsic re­sis­tance of se­lected Gram pos­i­tive bac­te­ria 356 Table 8.11 Important mech­a­nisms of mul­ti­drug re­sis­tance in bac­te­ria. 358 Table 8.12 Organisms in­cluded in CLSI and EUCAST breakpoint ta­bles 361 Table 8.13 Guide to in­ter­pre­tive cri­te­ria for se­lect or­gan­isms for com­monly tested an­ti­mi­cro­bi­als 364 Table 8.14 Summary of MALDI-TOF MS iden­ti­fi­ca­tion of bac­te­ria, my­co­bac­te­ria, and fun­gi 374 Table 8.15 Gene se­quenc­ing tar­gets for or­gan­ism iden­ti­fi­ca­tion 380

doi:10.1128/9781683670070.ch8 324 SECTION 8 Two im­por­tant con­trol mea­sures for in­fec­tious dis­eases are vac­ci­na­ tion to pre­vent in­fec­tion and use of an­ti­mi­cro­bial ther­apy to erad­i­cate in­fec­tions. This sec­tion pro­vi­des in­for­ma­tion for both ap­proaches. Tables 8.1 and 8.2 sum­ma­rize im­mu­ni­za­tion rec­om­men­da­tions for pe­di­at­ric and adult pa­tients. These rec­om­men­da­tions are pub­lished pe­ri­od­i­cally in the Morbidity and Mortality Weekly Report and at the Centers for Disease Control and Prevention (CDC) web­site (hppt://​ www.​cdc.​gov/​nip). The ta­bles are a sum­mary of the rec­om­men­da­ tions of the Advisory Committee on Immunization Practices (ACIP), the Amer­i­can Academy of Family Physicians (AAFP), the Amer­i­can College of Obstetricians and Gynecologists (ACOG), the Amer­i­ can College of Physicians-American Society of Internal Medicine (ACP-ASIM), and the Infectious Diseases Society of Amer­ica (IDSA). Information re­gard­ing an­ti­mi­cro­bial agents is in­tended to be used as a quick ref­er­ence guide for the prac­tic­ing clin­i­cal mi­cro­bi­ ol­o­gist. Included within are ta­bles that out­line com­mon meth­ods of an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing, key in­for­ma­tion re­gard­ing clin­i­cal breakpoint cri­te­ria as pre­sented by the Eu­ro­pean Commit- tee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI). These ta­bles should help the clin­i­cal mi­cro­bi­ol­o­gist eas­ily de­ter­mine what break- points ex­ist for com­monly en­coun­tered or­gan­isms as well as com­ monly used an­ti­mi­cro­bi­als. Lastly, sev­eral ta­bles re­gard­ing in­trin­sic mech­a­nisms of re­sis­tance and im­por­tant mech­a­nisms of re­sis­tance have been in­cluded. A no­ta­ble dif­fer­ence from pre­vi­ous edi­tions of the Pocket Guide is the omis­sion of antibiogram ta­bles. Due to the re­gional dif­fer­ences seen in an­ti­mi­cro­bial re­sis­tance, it was de­ter­ mined that it would be im­pos­si­ble to pres­ent gen­er­al­iz­able data that would be rel­e­vant to all­ us­ers of this Pocket Guide. Readers are en­ cour­aged to con­sult con­tem­po­rary data pub­lished by the nu­mer­ous on­go­ing sur­veil­lance pro­grams that pres­ent antibiogram data that is or­gan­ism, pa­tient, and re­gion-specific. Antimicrobial Agents Antimicrobial Vaccines, Susceptibility Testing 325 al ­ v r ­ te (continued) ore next dose ­ f 4 wk 4 wk 4 wk 8 wk Minimum Minimum in be 4 wk 4 wk 8 wk 4 wk 4 wk 6 mo 6 mo al ­ v er ­ t ore next dose ­ f 8 wk 8 wk 1–4 mo 2–17 mo be 2 mo 2 mo 6–9 mo Recommended in 2 mo 2 mo 6–12 mo 3 yr a le 2–4 mo 4–6 mo 6–9 mo Birth-2 mo 1–4 mo 6–18 mo 2 mo 4 mo 6 mo 12–15 mo Recommended age thisfor dose 2 mo 4 mo 6 mo 15–18 mo 4–6 yr ­ u ion ion sched ­ t a ­ z ­ ni u ­ m 6 wk Birth 6 wk Minimum age for first dose 6 wk ic ic im ­ r t ­ a ­ di type b ecommended ecommended pe R

Antimicrobial Agents Antimicrobial First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose Fourth dose First dose Second dose Third dose o Fourth dose dose Fifth Rotavirus Hepatitis B Table 8.1 Vaccine Diphtheria-tetanus-pertussis influenzae Haemophilus 326 SECTION 8 al ­ v r ­ te ore next dose ­ f Minimum Minimum in be 4 wk 4 wk 4 wk 6 yr 4 wk 4 wk 8 wk al ­ v er ­ t ore next dose ­ f be 3–5 yr Recommended in 2 mo 2–14 mo 3.5 yr 2 mo 2 mo 6 mo (continued) a le 12–15 mo 4–6 yr Recommended age thisfor dose 2 mo 4 mo 6–18 mo 4–6 yr 2 mo 4 mo 6 mo 12–15 mo ­ u ion ion sched ­ t a ­ z ­ ni u ­ m 12 mo Minimum age for first dose 6 wk 6 wk ic ic im ­ r t ­ a ­ di a ­ l el ­ b ­ vi ­ rus ­ lio ecommended ecommended pe R

Antimicrobial Agents Antimicrobial First dose Second dose First dose Second dose Third dose Fourth dose First dose Second dose Third dose Fourth dose Measles, mumps, ru Vaccine Inactivated po Pneumococcal Table 8.1 Vaccines, Susceptibility Testing 327 ­ ber 2017 ­ tem ­ tion. Sep 4 wk 6 mo 12 wk 4 wk 12 wk ­ za ­ ni ­ mu ­ tices im for ­ ral best prac ­ ne 4 wk 6–18 mo 3–5 yr 8 wk 4 mo 12–23 mo 12–23 18 mo 12–15 mo 4–6 yr 11–12 yr yr mo) (+2 11–12 yr (+6 mo) 11–12 6 mo 12 mo 12 mo 9 yr

Antimicrobial Agents Antimicrobial First dose Second dose First dose Second dose First dose Second dose Third dose Data from Centers Disease for Control and Prevention, Advisory Committee on Immunization Practices: Ge a Influenza Hepatitis A Varicella Papillomavirus Human 328 SECTION 8 ry ­ e y ­ l l ­ a u ­ n eive PCV13 PCV13 eive er PCV13 ­ c ­ t owed by PPSV23 at least PPSV23 by owed yr ­ l

>65 10 One dose an Same as 19–49-yr for age group Same as 19–49-yr for age group Immunocompetent adults should re fol 1 year af ry 10 yrry 10 One booster dose ev ­ e y ­ l l ­ a u ­ n Age group (yr) 50–64 One booster dose ev One dose an Same as 19–49-yr for age group Same as 19–49-yr for age group Same as 19–49-yr for age group ent. ­ ­ d s ain ­ i ­ t al, en al, ­ c in ­ i ­ p ­ c ween ­ i ­ t ­ m ng onng ­ i ered first ­ t s ine de al be ­ i end ­ c ­ v a ­ p in er ry 10 yrry 10 le ­ t ­ m ­ e ional, ional, or other ional, ional, or other ­ u ­ t ­ t ons with med y ­ s ­ l ons with med ­ pa ­ pa l ­ s u u ­ a ions. One dose of ­ c ­ c ­ t u s. i ­ n ­ e ­ d ng theng same visit. There ­ i ion ion sched ­ t ions. Timing vac a oral, oc oral, oc ­ tions ­ t al con ­ i ­ i als) for per ­ z a ­ c i ­ v ine) for per ­ i ­ ca ­ c ­ c ­ n i i er ­ hav ­ hav ­ d ­ d ­ t u ered dur 19–49 One booster dose ev One dose an and 2,PCV13 or 3 doses 1, PPSV23. of shouldPCV13 be ad Recommended those for with cer med and should PPSV23 not be ad should be a 5-year in in t in Three doses and (at 0, 1, 6 mo in be PPSV23 PPSV23 dos Two or three dosesTwo (de vac be ­ m ecommended adult im R

Antimicrobial Agents Antimicrobial Vaccine Tetanus/diphtheria Influenza Pneumococcal (PCV13 and PPSV23) Hepatitis B Hepatitis Hepatitis A Hepatitis Table 8.2 Vaccines, Susceptibility Testing 329 ​ es ­ p https:// ess of es ­ l ­ p rd d d ­ ga ­ e ­ e end end d ­ e ine re ­ m ­ m ­ c ode of her m m ­ s ­ cat ­ i i ­ o ­ o ­ d er. ­ t ­ ter vac eive one dose hereive of ­ c Not Not rec zos Same as 19–49-yr for age group Not rec Adults >60 yr should re zos whether had they a have prior ep Not Not in Same as 19–49-yr for age group eive eive ­ c er ­ t es ­ p d d es zos ­ e ­ e ess of ­ p ­ l rd end end ­ m ­ m ­ ga ode of her ated >60 for yr m m ­ s ­ c ­ i i ­ o ­ o ­ d ine re er. ­ c ­ t Not Not rec Same as 19–49-yr for age group Not Not rec Adults >60 yr should re one dose her of zos vac whether had they a have prior ep Up to 60Up same yr, as for 19–49 yr age group. inNot Same as 19–49-yr for age group ble ble ion, ­ t ­ a i a ­ l ­ n ). i ­ c ons with df ­ s ­ a re ​ p ory is ­ t ut ons with ­ o ­ s ­ tions ase, vac ­ ca ­ e chedule. ion hision i ​ s ­ t ­ d raphic, or other a ­ g ­ n d i ­ e ons (with ­ c al dis ponse) ­ s ­ r ­ s ­ u end ombined- ­ m ​ c le le per ­ b ional, geo ody re ­ o i l l or other in ­ t ble; two doses per for ­ t ­ b dult- ­ tions a ­ a i i ​ a ­ t ­ ca ­ p ­ l ­ i ep ­ ca u e i ­ c ­ tory of nat ­ r ­ c ­ d dult/ ​ a Three doses through yr at 0, age 21 and mo, 6 months1–2 One or more dose per for med Three doses through age 26 yr at 0, and mo, 6 months1–2 Not Not m rec un oc in Two doses (1 to doses 2 mo apart) (1 for Two sus his or an ownloads/ ​ d ide ­ r ha ­ c ac ­ s ​ schedules/ y ­ l accines/ ​ v ale ­ m ov/ ​ g dc. Antimicrobial Agents Antimicrobial ​ c Datafrom Centers Disease for Control and Prevention, Recommended Immunization Schedule Adults for or Years AgedOlder, United 19 States, ( 2017 ww. a HPV—male (MenACWY or MPSV4) Meningococcal po HPV—fe Measles-mumps-rubella (MMR) One dose if vac Herpes Zoster Varicella w 330 SECTION 8 Table 8.3 Clinical and Laboratory Standards Institute (CLSI) doc­u­ments re­lated to an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing from hu­mansa No. Title M2-A12 Performance Standards for Antimicrobial Disk Suscep­ tibility Tests (2015) M7-A10 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically (2015) M11-A8 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria (2012) M23-ED4 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters (2016) M24-A2 Antimycobacterial Susceptibility Testing of Mycobacte- rium, Nocardiae, and Other Aerobic Actinomycetes (2011) M26-A Methods for Determining Bactericidal Activity of Antimicrobial Agents (1999) M27-ED4 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts (2017) M38-ED3 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi (2017) M39-A4 Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Data (2014) M43-A Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas (2011) M44-A2 Method of Antifungal Disk Diffusion Susceptibility Testing of Yeasts (2009) M45-ED3 Methods for Antimicrobial Dilution and Disk Susceptibil- ity Testing of Infrequently Isolated or Fastidious Bacteria (2016) M51-S1 Performance Standards for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi (2010) M52-ED1 Verification of Commercial Microbial Identification and Antimicrobial Susceptibility Testing Systems (2015) M57-ED1 Principles and Procedures for the Development of Epidemiological Cutoff Values for Antifungal Susceptibil- ity Testing (2016) M59-ED1 Epidemiological Cutoff Values for Antifungal Susceptibility Testing (2016) M60-ED1 Performance Standards for Antifungal Susceptibility

Antimicrobial Agents Antimicrobial Testing of Yeasts (2017) Vaccines, Susceptibility Testing 331 Table 8.3 Clinical and Laboratory Standards Institute (CLSI) doc­u­ments re­lated to an­ti­mi­cro­bial sus­cep­ti­bil­ity test­ing from hu­mansa (continued) No. Title

M61-ED1 Performance Standards for Antifungal Susceptibility Testing of Filamentous Fungi (2017) M100- Performance Standards for Antimicrobial Susceptibility S27 Testing (2017) SC21-L Susceptibility Testing (col­lec­tion of doc­u­ments: M2, M7, M11, M21, M24, M27, M31, and M100) aDocuments avail­able from CLSI (950 West Valley Road, Suite 2500, Wayne, PA 19087; tele­phone, 610-688-0100; FAX, 610-688-0700; web­site, http://​www.​clsi.​org). Antimicrobial Agents Antimicrobial 332 SECTION 8 ions a ­ t i i ­ g ­ d ; 20–24 h; 35°C ; 20–24 h; 35°C 2 2 ia, and fun ­ r e ­ t 5% CO 5% CO Air; 16–18 h (24Air; and h CONS 16–18 35°C cefoxitin); Air; 16–20 vanco: h (oxac, 24 h); 35°C Air; 24 h; 35°C Air; 16–18 h (vanco:Air; 24 h); 35°C 16–18 Air; 16–20 h (vanco: 24 h); 35°C Air; 24 h; 35°C Air; 24–48 h; 35°C Air; 24 h; 35°C Incubation con Air; 20–24 h; 35°C Air; 20–24 h; 35°C c ­ ba o ­ c ia, my ­ r e ­ t Direct Direct Direct Direct Direct Direct Direct Direct; broth Direct; broth Direct; broth Direct; broth Direct; broth Inoculum ect bac ­ l ent ent

­ m ­ m le le ­ p ­ p ent ds for se ­ m ­ o ent w/ le /ml) ­ m ­ p g le /ml) μ ­ p /ml) g g μ μ ium daptomycin) for ium daptomycin) for ium daptomycin) for ­ c ­ c ­ c ium daptomycin) for ­ c ty test meth ­ i il ­ b i g/ml cal ­ t g/ml cal g/ml cal µ µ ep g/ml cal ­ c MHA CAMHB/MHA + 2%(oxac NaCI) (sup w/ 50 µ MHA + 2% NaCI + oxac MHA + 5% sheep blood CAMHB + 2–5% LHB (sup w/ 50 MHA CAMHB/MHA (sup 50 µ BHIA (500 + gent MHA + 5% sheep blood CAMHB + 2–5% LHB (sup w/ 50 Medium BHIA + vanco (6 BHIA + strep (2,000 ial sus ­ b ion ion ­ t ­ t ro u u ­ c ­ l ­ l i ion ion ion ion ­ m ion ion ­ o ­ s ­ s ­ s ­ s ) ­ t ­ t i ­ t s u u ­ l ­ l ­ fu ­ fu ­ fu ­ fu ­ u ­ re S. au Disk Disk dif Broth/agar di Agar screen ( Disk Disk dif Broth di Disk Disk dif Agar screen Broth/agar di Disk Disk dif Broth di Test Test d meth spp. other ummary of CLSI an spp. S

Antimicrobial Agents Antimicrobial (M100) (M100) (M100) spp. (M100) Organism (Document) Staphylococcus Streptococcus pneumoniae Streptococcus, Enterococcus Table 8.4 Vaccines, Susceptibility Testing 333 (continued) ; 20–24 h; 36°C ; 20–24 h; 36°C ; 20–24 h; 35°C ; 20–24 h; 35°C h; 35°C ; 16–18 2 2 2 2 2 5% CO Air, 16–18 h; 35°C Air, 16–18 Air, 16–20 h; 35°C Air; 20–24 h; 35°C Air, 16–18 h; 35°C Air, 16–18 Air, 16–20 h; (Yersinia pestis, 24 h); 35°C h; 35°C Air, 16–18 Air, 16–20 h; 35°C h; 35°C Air, 16–18 Air, 16–20 h; 35°C 5% CO 5% CO 5% CO Air; 20–24 h; 35°C 5% CO Direct Direct Direct; broth Direct; broth Direct Direct; broth Direct; broth Direct; broth Direct; broth Direct; broth Direct; broth Direct Direct Direct Direct ent ent ­ m ­ m le le ­ p ­ p HTM agar HTM broth MHA CAMHB/MHA CAMHB + 2–5% LHB sup GCA + 1% sup GCA + 1% MHA CAMHB/MHA MHA CAMHB/MHA MHA CAMHB/MHA MHA + 5% sheep blood CAMHB + 2–5% LHB MHA + 5% sheep blood ion ion ion ion ion ­ t ­ t ­ t ­ t ­ t u u u u u ­ l ­ l ­ l ­ l ­ l ion ion ion ion ion ion ion ion ion ­ s ­ s ­ s ­ s ­ s ­ s ­ s ion ­ t ­ t ­ t u u ­ l ­ l u ­ fu ­ fu ­ fu ­ fu ­ fu ­ fu ­ fu ­ l Disk Disk dif Broth di Disk Disk dif Broth/agar di Disk Disk dif Broth di Disk dif Disk dif Disk dif Agar di Broth/agar di Broth/agar di Broth/agar di Disk Disk dif Broth/agar di

ng ­ i lud ­ c spp. spp. spp. (in Antimicrobial Agents Antimicrobial (M100) (M100) Neisseria gonorrhoeae (M45) Listeria monocytogenes (M45) Haemophilus Enterobacteriaceae (M100) choleraeV. (M45) Pseudomonas aeruginosa (M100) Acinetobacter Vibrio Neisseria meningitidis (M45) 334 SECTION 8 ions ­ t (continued) i a ­ d ­ gi ds) ds) ­ o ­ o ; 20–24 h; 35°C 2 eth eth ­ m ­ m ­ ria, and fun Incubation con Air, 24–48 h; 35°C Air; 20–24 h; 35°C (all Air; 20–24 h; 35°C (all Air, 16–18 h; 35°C Air, 16–18 Air, 16–20 h; 35°C Air; 20–24 h; 35°C 5% CO ­ te ­ bac ­ co ­ ria, my Inoculum Direct Direct; broth Air; 16–20 h; 35°C Direct; broth Direct: broth Direct; broth Direct: broth Direct Direct Direct ­ te ­ lect bac ent w/ ­ m le ­ p ide ­ r hlo ­ c ium daptomycin) for ro ­ c ­ d ­ ity test meth ­ ods for se ­ bil ­ ti oxal hy g/ml cal ­ d ­ i ­ cep Medium CAMHB + 2–5% LHB (sup 50 µ CAMHB/MHA MHA CAMHB/MHA CAMHB + 2–5% LHB +0.001% pyr MHA CAMHB/MHA MHA CAMHB CAMHB + 2–5% LHB ­ bial sus ion ion ion ­ t ­ t ­ t u u u ­ cro ­ l ­ l ­ l ion ion ion ion ion ion ion ­ mi ­ o ­ s ­ s ­ s ­ t ­ t ­ t ­ t ­ ti u u u u ­ l ­ l ­ l ­ l ­ fu ­ fu ­ fu Test Test d meth Broth/agar di Disk dif Broth di Broth/agar di Broth di Broth/agar di Disk Disk dif Broth di Broth di Disk Disk dif

era spp. spp. Summary of CLSI an spp. and spp. spp. spp.

lex Antimicrobial Agents Antimicrobial Enterobacteriaceae ­ p Abiotrophia Other Non- Organism (Document) (M100) cepacia Burkholderia Stenotrophomonas maltophilia (M100) Granulicatella Corynebacterium and Coryneform­ gen (M45) (M45) com (M45) Aeromonas Aerococcus (M45) (M100) Table 8.4 Vaccines, Susceptibility Testing 335 (continued) ; 24–48 h; 35°C ; 24–48 h; 35°C ; 20–24 h; 35°C 2 2 2 5% CO Air, 20–24 h; 35°C Microaerophilic; 24 h; 42°C Microaerophilic; 24 h; or 42°C 48 h; 36–37°C Microaerophilic; 3 days; 35°C Air, 20–24 h; 35°C h; 35°C Air, 16–18 Air, h; 18–24 35°C 5% CO 5% CO Air; 20–24 h; 35°C Direct Direct Direct Direct(72 h fromhold BAP) Direct Direct Direct Direct in K, ent w/ ­ m a ­ m ­ t le ­ p ium daptomycin) for ­ c in, and 5% LHG g/ml cal µ ­ m CAMHB + 2–5% LHB (sup 50 CAMHB MHA + 5% sheep blood CAMHB + 2–5% LHB MHA + 5% aged sheep blood CAMHB + 2–5% LHB BMHA CAMHB + 2–5% LHB CAMHB + 2–5% LHB Or HTM or Brucella broth with vi MHA CAMHB he ion ion ion ion ion ion ion ion ion ion ­ s ­ s ­ s ion ­ t ­ t ­ t ­ t ­ t ­ t ­ t ­ t u u u u u u u ­ l ­ l ­ l ­ l ­ l ­ l ­ l u ­ fu ­ fu ­ fu ­ l Broth di Agar di Broth di Disk Disk dif Broth di Disk Disk dif Broth di Broth di Disk dif Broth di Broth di i ­ r o ­ l spp. spp. spp. Antimicrobial Agents Antimicrobial (M45) (M45) (M45) Campylobacter jejuni/ coli (M45) Helicobacter py (M45) Pasteurella Erysipelothrix rhusiopathiae (M45) HACEK group (M45) Lactobacillus catarrhalis Moraxella (M45) Micrococcus 336 SECTION 8 in; ­ c

y ­ m o ­ t spp.); ions ­ t (continued) i a ­ d ­ gi Cryptococcus, rs); 35°C ­ e ­ cin; strep, strep i ure (broth). ­ m ­ t a ­ t ­ ria, and fun Incubation con Air; 24–48 h ( h); 35°C 70–74 Air; 16–24 h (zygomycetes); 24–48 h ( Aspergillus 48–72 h (oth Air, 16–20 h; 35°C to CLSIRefer M24 46–48 h (broth); 36°C ­ te in; gent, gen ­ bac ­ l il sm in a broth cul ­ co ­ i ­ c ­ a an ­ g ide; LHB, lysed horse blood; BHIA, brain heart ­ r rose agar ­ t ­ tato ­ ria, my Inoculum Direct Direct from po dex Direct Direct; broth Air; 3–5 days; 35°C to Refer CLSI M24 Direct; broth Anaerobic; 42–48 h (agar) ­ te i; oxac, ox ­ c ium chlo ­ d oc ­ c o ­ l ­ y ­ lect bac er growth the of or ­ t /ml), in ect) or af g ­ r ­ m μ ase-negative staph ­ l ­ u g ­ a in K (1 in K (1 ­ m a ­ t ­ ity test meth ­ ods for se ­ bil ­ ti ies on an agar plate (di m; CONS, co ­ n ­ u ­ cep i ­ o ­ d on-adjusted Mueller-Hinton broth; NaCI, so ­ i Medium Brucella broth/agar + he pg/ml),(5 vi RPMI broth 1640 MHA CAMHB CAMHB 5% lysed sheep blood ated col ­ l ­ bial sus ion ­ t ­ u ­ cro ­ l ion ion ion ion ion ion ­ mi ­ o ­ s ­ s ­ t ­ t ­ t ­ t ­ ti u u u u ­ l ­ l ­ l ­ l ­ fu ­ fu group) Bacteroides bes) bes) di ectly with iso ­ o ­ r er y)/agar (all ­ l ­ a Broth di an Test Test d meth Broth di to CLSIRefer M24 to CLSI Refer M24 Disk dif Broth di Broth di Broth ( fragilis on Disk dif her di ­ t ared ei ­ p etes ­ c ) ­ my in. ­ c Summary of CLSI an y ­ no i ­ m ­ t

o ­ c spp. Antimicrobial Agents Antimicrobial ion agar, GCA, GC agar; HTM, Haemophilus test me ­ s u Inoculum can be pre ­ f a (M45) Bacillus (not B. anthracis Organism (Document) (M24) Mycobacteria (M24) Fungi (molds) (M51) Anaerobes (M100) Fungi (yeasts) (M27) Aerobic Aerobic ac in Abbreviations: MHA, Mueller-Hinton agar, CAMHB, cat Table 8.4 vanco, van Vaccines, Susceptibility Testing 337 Topical (continued) ion ­ t ra ­ t X X X X X X X X X X X X X s IV ­ i in ­ m X X X X X X X X IM Route Route of ad a X X X X X X X X X X PO ial agents ­ b ro ­ c i ­ m i ­ t or or or ­ t ­ t ­ t ­ i ­ i ­ i ib ib ib ­ h ­ h ­ h ect an ­ l -lactamase in -lactamase in -lactamase in β β β -lactam/ -lactam/ -lactam/ Cephem Cephem Cephem Penicillin Penicillin β Macrolide Penicillin Monobactam β β Drug class Aminoglycoside Cephem Cephem Penicillin Penicillin Cephem Cephem Cephem Cephem Cephem ion ion and drug class for se ­ t ra ­ t s ­ i in ­ m ­ um ­ di outes of ad R

Antimicrobial Agents Antimicrobial Cefonicid Azlocillin Aztreonam Amikacin Cefmetazole Cefamandole Carbenicillin Carbenicillin indanyl so Carbenicillin Aztreonam-avibactam Azithromycin Ampicillin-sulbactam Amoxicilin-clavulanate Antimicrobial agent Cefazolin Cefaclor Ampicillin Amoxicillin Cefepime Cefdinir Cefadroxil Cefditoren Cefixime Table 8.5 338 SECTION 8 Topical ion ­ t ra ­ t X X X X X X X X X X X X X X X X s IV ­ i in ­ m X X X X X X X X X IM Route Route of ad (continued) a X X X X X PO ial agents ­ b ro ­ c i ­ m i ­ t or or or ­ t ­ t ­ t ­ i ­ i ­ i ib ib ib ­ h ­ h ­ h ect an ­ l -lactamase in -lactamase in -lactamase in β β β -lactam/ -lactam/ -lactam/ Cephem β Cephem β β Cephem Cephem Drug class Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem Cephem ion ion and drug class for se ­ t ra ­ t s ­ i in ­ m outes of ad R

Antimicrobial Agents Antimicrobial Cefuroxime axetil Ceftolozane-tazobactam Ceftobiprole Ceftazidime-avibactam Ceftaroline-avibactam Cefpodoxime Cefoperazone Antimicrobial agent Cephalexin Ceftriaxone Ceftibuten Ceftazidime Cefprozil Cefotaxime Cephalothin Cephapirin Cefuroxime Ceftizoxime Ceftaroline Cefotetan Cefoxitin Cefpirome Table 8.5 Vaccines, Susceptibility Testing 339 X (continued) X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X teroidal Fluoroquinolone Fluoroquinolone S Macrolide Macrocyclic Penicillin Macrolide Fluoroquinolone Lincosamide Lipopeptide Lipopeptide Fluoroquinolone Macrolide Fluoroquinolone Phenicol Quinolone Cephem Aminoglycoside Fluoroquinolone Fluoroquinolone Fosfomycyin Carbapenem Tetracycline Carbapenem Carbapenem Aminoglycoside ­ id Antimicrobial Agents Antimicrobial Colistin Daptomycin Delafloxacin Cinoxacin Levofloxacin Gemifloxacin Erythromycin Fidaxomicin Dicloxacillin Dirithromycin Clinafloxacin Clindamycin Clarithromycin Ciprofloxacin Chloramphenicol Cephradine Gentamicin Gatifloxacin Fleroxacin Fosfomycin Fusidic ac Doripenem Doxycycline Ertapenem Imipenem Kanamycin 340 SECTION 8 X Topical ion ­ t ra ­ t X X X X X X X X X X X X X s IV ­ i in ­ m X X X X IM Route Route of ad (continued) a X X X X X X X X X X X X X PO ial agents ­ b ro ­ c i ­ m i ­ t or ­ t ­ i ib ­ h ect an ­ l d ­ i -lactamase in β nolone -lactam/ Qui Nitrofurantoin Pseudomonic ac Penicillin Aminoglycoside Fluoroquinolone Nitroimidazole Penicillin Tetracycline β Fluoroquinolone Drug class Oxazolidinone Fluoroquinolone Penicillin Cephem Fluoroquinolone Lipoglycopeptide Penicillin Carbapenem Fluoroquinolone Penicillin ion ion and drug class for se ­ t ra ­ t s ­ i in ­ m outes of ad R ­ id

Antimicrobial Agents Antimicrobial Mupirocin Nafcillin Nalidixic ac Netilmicin Mezlocillin Minocycline Linezolid Nitrofurantoin Moxifloxacin Metronidazole Meropenem-vaboractam Lomefloxacin Norfloxacin Methicillin Loracarbef Ofloxacin Oritivancin Oxacillin Meropenem Pefloxacin Penicillin Table 8.5 Antimicrobial agent Vaccines, Susceptibility Testing 341 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X or or ­ t ­ t ­ i ­ i ib ib ­ h ­ h or or or ­ t ­ t ­ t ­ i ­ i ­ i ib ib ib ­ h ­ h ­ h ay ay in ay ay in ay ay in -lactamase in -lactamase in ­ w ­ w ­ w β β -lactam/ -lactam/ Fluoroquinolone Glycopeptide Folate path β Ketolide Oxazolidinone Glycopeptide Aminoglycoside Folate path Fluoroquinolone Aminocyclitol Lipopeptide Streptogrammin Ansamycin β Penicillin Folate path Glycylcline Nitroimidazoles Aminoglycoside Tetracycline Penicillin ous. ­ n e ­ v ra ­ t ar; IV, in ­ l u ­ c us ­ m ra ­ t Antimicrobial Agents Antimicrobial Clinical and Laboratory Standards Institute, M100–S27. PO, oral; IM, in a b Vancomycin Tedizolid Teicoplanin Rifampin Trovafloxacin Trimethoprim Ticarcillin-clavulanate Telithromycin Streptomycin Sulfonamindes Sparfloxacin Spectinomycin Polymyxin B Quinupristin-dalfopristin Piperacillin-tazobactam Piperacillin TMP-SMX Tigecycline Tinidazole Tobramycin Tetracycline Ticarcillin 342 SECTION 8 X X X X X Topical X X X X X X X IV ion ­ t IM ra ­ t s ­ i in ­ m al agents ­ g un X X X X X X X X X ­ f PO Route Route of ad i ­ t ect an ­ l ine ­ d ­ i ­ rim ous. ion andion drug se class for ­ t ­ n e ra ­ v ­ t ra s Drug class Polyene Fluorinated py Azole Azole Azole Azole Azole Azole Echinocandin Echinocandin Echinocandin Allylamine ­ t ­ i in ­ m ar; IV, in ­ l u ­ c us ­ m ra ­ t outes ad of R

Antimicrobial Agents Antimicrobial PO, oral; IM, in Flucytosine Fluconazole Micafungin Ciclopirox Terbinafine Antimicrobial agent Amphotericin B Ketoconazole Itraconazole Voriconazole Isavuconazole Posaconazole Caspofungin Anidulafungin Griseofulvin Table 8.6 Vaccines, Susceptibility Testing 343 X Topical (continued) ion ­ t ra ­ t s X X X IV ­ i in ­ m Route Route of ad X X IM X X X X X X X X PO itic itic agents ­ s ­ a ar ­ p i ­ t

ect an ­ l spp., is is, Trypanosoma ­ t ­ t ys ­ cys ­ c spp. o o ­ m ­ m orm, microsporidia, ­ w ion ion and drug class for se ­ t Malaria pin Malaria Microfilaria, Antihelminthic, Strongyloidiasis, Bayliascaris Louse Indication Antihelminthic, Echinococcus, Bayliascaris Acanthamoeba spp., Leishmania spp., Naegleria fowleri, Malaria Extraintestinal Malaria, Amebiasis Babesia Microfilaria Acanthamoeba spp., Balamuthia spp., Leishmania Pneu brucei gambiense Pneu ra ­ t s ­ i in ­ m outes of ad R

Antimicrobial Agents Antimicrobial Doxycycline Antimicrobial agent Albendazole Amphotericin B Atovaquone-proguanil Chloroquine Clindamycin Diethylcarbamazine Pentamidine Artemether Ivermectin Permethrin Dapsone Table 8.7 344 SECTION 8 Topical ion ­ t ra ­ t s X X IV ­ i in ­ m Route Route of ad IM X X X PO X X X X X itic itic agents (continued) ­ s ­ a ar ­ p i ­ t ect an ­ l , spp. , Amebiasis, spp. , Giardiasis, is ­ t ­ cys o ­ m ous. ion ion and drug class for se ­ t ­ n Malaria, Babesia Malaria Malaria Indication Antihelminthic Malaria, Toxoplasmosis Antihelminthic, Microfilaria Balantidium coli Dracunculus Trichomonas Cyclospora, Isospora Pneu e ra ­ v ­ t ra s ­ t ­ i in ­ m ar; IV, in ­ l u ­ c us ­ m ra ­ t outes of ad R

Antimicrobial Agents Antimicrobial PO, oral; IM, in Quinine Antimicrobial agent Praziquantel Pyrimethamine Mebendazole Metronidazole Trimethoprim/ Sulfamethoxazole Malarone Mefloquine Table 8.7 Vaccines, Susceptibility Testing 345 lines ­ c y (continued) ­ c a ­ r ors ors ­ t ­ t ­ i ­ i ib ib ­ h ­ h line y ­ c ­ t ­ i y ­ c iv a ­ t ­ r -lactamase in -lactamase in β β -lactam– -Lactam- Clindamycin, macrolides Unpredictable ac Penicillins Macrolides, aminoglycosides, tet Chloramphenicol Sulfonamides, Sulfonamides, tet Clindamycin, carbapenems, cefoxitin, cefoxitin, carbapenems, Clindamycin, β β Antibiotics in ­ c i ­ m a ­ t , ins ­ r o ones ­ sp ones ­ l ones, gen ­ l ­ l o ­ o in, aminoglycosides, ­ l ­ o ­ o ­ p n ­ a ­ rins, carbapenems, in in uin ­ ci am ­ q ­ a ­ qu ­ f ­ qu line, broad-spectrum ­ spo o ­ c o ­ ro o ox ­ r ­ r a ­ l ­ o , ri ­ o ­ fl ­ o ­ cy ­ a ive ­ ro ­ t , carbapenems, macrolides, ­ rins n, n, macrolides, clindamycin, ceph cip , doxy ­ ci lines lines, flu y ­ spo ia ­ c ­ c o ­ r ­ m ­ l y e ­ a ­ cy ­ c ­ t ­ co a ­ ra ­ r ceph Generally ac Cephalosporins Broad-spectrum ceph carbapenems, flu Doxycycline Penicillin, van Penicillin, Metronidazole Penicillin Carbapenems tet Vancomycin clindamycin, flu tet ific bac ­ c group ntibacterial spe agents for spp. s spp. ­ u A spp. ­ re

Antimicrobial Agents Antimicrobial Organism Acinetobacter Actinobacillus Actinomyces Aeromonas hydrophila Anaplasma phagocytophila Arcanobacterium haemolyticum Bacillus anthracis Bacteroides fragilis Bacillus ce Table 8.8 346 SECTION 8 ones, ­ l ­ o in ins, carbapenems, ­ qu ­ r o y ­ r ­ t ­ i po ­ o ­ s iv o ­ t ­ l ­ a rim-sulfamethoxazole ­ p ­ o eth ­ m Fluoroquinolones, carbapenems Fluoroquinolones Cephalosporins Unpredictable ac Ampicillin, macrolides Fluoroquinolones, Tigecycline Penicillins, ceph aminoglycosides, flu tri Fluoroquinolones ­ Antibiotics o ­ l ­ a -lactamase β ­ cline ­ lins rim-sulfamethoxazole, il ­ p ­ cy lines ­ c ­ o ­ i ­ c ­ cy a eth ins, ins, macrolides -lactam– ­ l ­ ra ­ m l , doxy ive , aminoglycosides , β ­ t ­ ci , ­ i ones , tri , , tet ­ l ­ o rim-sulfamethoxazole ­ p in , , pen (continued) ia ­ o ­ r ­ qu ­ tors ­ i e o ­ t eth ­ r ­ rins ib ­ o ­ m ­ h po s in Generally ac Trimethoprim-sulfamethoxazole Doxycycline or broad-spectrum or Doxycycline ceph Erythromycin flu Doxycycline tri Carbapenems, pen Clindamycin Carbapenem Macrolides Macrolides ific bac ­ c ­ p com lex us is ­ t ­ s spp. us ntibacterial spe agents for ­ t A ­ rins

­ spo ­ l Antimicrobial Agents Antimicrobial ­ a Borrelia burgdorferi Organism Bartonella henselae Brucella spp. cepacia Burkholderia pseudomallei Burkholderia Fluoroquinolones, broad-spectrum cepho Capnocytophaga Bordetella per Campylobacter jejuni Campylobacter fe Table 8.8 Vaccines, Susceptibility Testing 347 lines ­ c y ­ c (continued) a ­ r Fluoroquinolones Fluoroquinolones Fluoroquinolones, macrolides, tet Trimethoprim-sulfamethoxazole ­ ­ o ones ones in ­ l ­ l ­ o ­ o ­ qu lines, ­ c o , , ­ r uin uin ­ o ­ cy ­ q ­ q a ins ins o o ­ r ­ r ­ r ­ r ­ r ­ o ­ o o o in ones ones in, flu ­ sp ­ sp n ­ l ­ l ­ c ­ c o o ­ o ­ o ­ ci ­ l ­ l y ins, tet y lin, ­ lin, clindamycin, ­ a ­ a ­ my ­ rins, carbapenems, ­ r ­ m il uin uin o ­ m o ­ c ­ c ­ q ­ q po ­ i ­ c ­ ro ­ spo ­ s ­ ro ­ ro o o ­ l ­ l ­ o ­ o ­ a ­ a , van , , pen , , eryth , macrolides, flu , macrolides, flu ins, van ­ r , , ceph , ceph po line ­ s ­ c o ­ l ­ a ­ cy ­ ra ones, aminoglycosides Broad-spectrum ceph Penicillin Doxycycline Doxycycline Doxycycline carbapenems, flu Penicillin Metronidazole Penicillin clindamycin Penicillin Erythromycin Vancomycin Vancomycin Broad-spectrum ceph tet carbapenems, flu ceph l ­ ria ­ nia he ­ t ­ mo ­ num ­ li ­ cile ­ u ­ fi Antimicrobial Agents Antimicrobial Citrobacter freundi Corynebacterium diph Cardiobacterium hominis Cardiobacterium trachomatis Chlamydia Chlamydophila pneu Chlamydophila psittaci Clostridium bot Clostridium dif perfringens Clostridium tetani Clostridium jeikeium Corynebacterium Corynebacterium urealyticum Citrobacter koseri 348 SECTION 8 in with ­ c y ­ m o ­ c ins, ins, ­ r ­ r po po ­ s ­ s ones o o ­ l ­ l ­ l y ­ o ­ t ­ a ­ a ­ i in in, or van iv ­ l ­ t il ­ qu ­ c o i ­ r ­ p ­ o ones ones ­ l ­ l ­ o ­ o , am in in in ­ c i ­ qu ­ qu ­ m o o a ­ r ­ r ­ t ­ o ­ o Aminoglycosides Unpredictable ac Broad-spectrum ceph Chloramphenicol Chloramphenicol 3 flu Broad-spectrum ceph flu Imipenem, flu Penicillin gen Antibiotics ins, ­ r ones ones ­ l ­ l in in with ­ o po ­ o ­ c ­ s y o in ­ l uin ­ m ­ a ­ q ­ qu o o o ­ c ­ r ­ r ­ o ­ o ones ­ l ­ o ­ rins, carbapenems, in ­ qu lin, ­ lin, or van ­ spo o o il ­ r a ­ l ­ c ­ o ­ a i ­ p ive ­ t , macrolides, flu , broad-spectrum ceph , ceph in ­ c i lines, flu (continued) ia ­ c ­ r ­ m y e ­ c ­ t ­ ta a ­ r Generally ac Carbapenems Doxycycline Doxycycline Doxycycline Penicillin tet Carbapenems Penicillin Penicillin, Penicillin, am macrolides, clindamycin, flu gen Oxazolidinones ific bac ­ c ae ­ c ­ a ntibacterial spe agents for A

Antimicrobial Agents Antimicrobial Enterobacter aerogenes Organism burnetii Coxiella Ehrlichia chaffeensis ewingii Ehrlichia corrodens Eikenella Erysipelothrix rhusiopathiae Enterobacter clo Enterococcus faecalis Enterococcus faecium Table 8.8 Vaccines, Susceptibility Testing 349 ors, ­ t ­ i ib ­ h (continued) ins ­ r po ­ s o -lactamase in ­ l β ­ a -lactam– β rim-sulfamethoxazole ­ p lines, lines, ­ o ­ c ­ cy eth ­ ra ­ m tet Tetracyclines Penicillins, tri Broad-spectrum ceph Ampicillin Clindamycin - ­ ­ o ins in, ­ r ­ l eth il

po , ­ m ­ c ­ s ­ i , o ­ l ors, ors ­ a ­ t ­ t ins ­ i ­ i ­ r ins, tri ib ib o ­ r ­ h ­ h in, carbapenems, ­ sp ins, carbapenems, ­ c po o ­ l i ­ s ­ l il o ­ a ­ m ­ l ­ c a ­ i ­ a ­ t , carbapenems, pen or gen , macrolides, trimethoprimsul macrolides, , -lactamase in -lactamase in ones ones, macrolides ones ones, broad-spectrum ones, broad-spectrum β β ­ l ­ l ­ l ­ l ­ l ­ rins ­ rins ­ o ­ o ­ o ­ o ­ o in in in in ­ spo ­ spo uin o o ­ qu ­ q ­ qu ­ qu ­ qu ­ l ­ l ­ a ­ a o o o o o ­ r ­ r ­ r ­ r ­ r ­ o ­ o ­ o ­ o ­ o -Lactam– -Lactam– rim sulfamethoxazole rim Metronidazole clindamycin Streptomycin flu β Penicillin, broad-spectrum ceph Broad-spectrum ceph flu Broad-spectrum ceph p Cephalosphorins, pen aminoglycosides, macrolides Tetracyclines famethoxazole, aminoglycosides, flu β flu ceph flu ceph ­ za ­ en ­ flu spp. Antimicrobial Agents Antimicrobial Escherichia coli Francisella tularensis Fusobacterium Hafnia alvei Haemophilus aphrophilus ducreyi Haemophilus Haemophilus in kingae Kingella Klebsiella granutomatis 350 SECTION 8 ones ­ l ­ o ins ­ r in po ­ s ­ qu ins, ins, aminoglycosides o o ins ­ l ­ r y ­ r ­ r ­ t ­ a ­ o ­ i po po ­ s iv ­ s ­ t o o ­ l ­ l ­ a ­ a ­ rins ­ spo o ­ l ­ a Penicillins, Penicillins, ceph Aminoglycosides, flu Penicillin, Penicillin, ceph Unpredictable ac Broad-spectrum ceph Fluoroquinolones, broad-spectrum ceph Antibiotics in ­ p ins ­ r

, am po ­ f ­ s ins, ins o ­ l ones, ­ r ­ r ­ l ­ a o o ­ o in ­ sp ­ sp ones, ri o o ­ l ­ l ­ l ­ qu ­ o ­ a ­ a o lines, macrolides ­ r in with aminoglycoside in with aminoglyco- in ­ c lines ­ l ­ l ­ o y ­ c line, ceph il il ­ qu ­ c , carbapenems, ­ c ­ c ­ c o a ­ cy i i a ­ r ­ r , , flu ­ p ­ p ­ cy n ­ o ­ ra , aminoglycosides, ive ­ ci ­ t y ones, ones, carbapenems ones, ones, carbapenems , , flu , tet ­ l ­ l ­ m ­ o ­ o , doxy or am or am ­ co lines (continued) ia uin uin ­ c ­ r ­ q ­ q e ­ cy ­ t ­ ro ­ ro ­ o ­ o ­ ra Generally ac Penicillin carbapenems, tet Broad-spectrum ceph Fluoroquinolones Broad-spectrum ceph flu Macrolides Cephalosporins Carbapenems Macrolides Penicillin Penicillin Carbapenems tet flu side, side, van ific bac ­ c ­ nia ­ mo ntibacterial spe agents for spp. A spp.

Antimicrobial Agents Antimicrobial Leptospira interrogans Morganella morganii Listeria monocytogenes Organism oxytoca Klebsiella ozaenaeKlebsiella Klebsiella pneu Lactobacillus pneumophila Legionella Leuconostoc catarrhalis Moraxella Mycoplasma pneumoniae Table 8.8 Vaccines, Susceptibility Testing 351 (continued) ins ins ins, ­ r ­ r ­ r po po po ­ s ­ s ­ s o o o ­ l ­ l ­ l ­ a ­ a ­ a lines, macrolides, ­ c ­ cy a ­ r ones ones ­ l ­ l ­ rins ­ o ­ o in in ­ spo o ­ qu ­ qu ­ l ­ a o o ­ r ­ r ­ o ­ o Broad-spectrum ceph Broad-spectrum ceph Macrolides, clindamycin Macrolides, Broad-spectrum ceph flu Penicillin, tet flu Fluoroquinolones, broad-spectrum ceph - β ins, ­ r ins ­ r po ­ s po ­ s o ­ l o ones ­ a -lactam– ­ l ­ l ­ a ­ o ins ­ r in o ­ rins, carbapen - ­ rins, carbapen - ­ qu ­ sp o lines ­ r o ­ c ­ spo ­ spo ­ l ­ o y o o ­ a ­ l ­ l ­ c ­ rins, carbapenems, ­ a ­ a a ­ r ­ spo o ones ones ­ l ors, flu ­ l ­ l , carbapenems, β ­ t ­ a ­ i ­ o ­ o , carbapenems, clindamycin , carbapenems, clindamycin , carbapenems, amikacin, ib in in ­ h ones, tet ­ l , broad-spectrum ceph ­ qu ­ qu ­ o , , ceph , , broad-spectrum ceph o o ­ r ­ r in ­ o ­ o ­ qu o ­ r ­ o Broad-spectrum ceph ems, flu Ampicillin carbapenems Broad-spectrum ceph ems, flu lactamase in Penicillin Cephalosporins Carbapenems Metronidazole Metronidazole Broad-spectrum ceph flu Penicillin Sulfonamides linezolid es ­ d ­ i it ­ g spp. is ­ l n is i ­ i ­ r ­ b spp. ­ ga ­ ra spp. spp. Antimicrobial Agents Antimicrobial eus vul eus mi ­ t ­ t Providencia Pro Prevotella Pro Neisseria gonorrhoeae multocidaPasteurella Plesiomonas shigelloides Porphyromonas aeruginosa Pseudomonas Neisseria men Nocardia 352 SECTION 8 in ­ l il ­ c ­ pi lines ­ c y ­ c a ­ r in, in, ­ l ­ l ones ­ l il il ­ o ­ c ­ c ­ i ­ i y uin ­ t ­ i ­ q iv ­ ro ­ t ­ o rim-sulfamethoxazole ­ p ­ o eth ­ m Erythromycin Unpredictable ac Macrolides, Macrolides, clindamycin, tet Fluoroquinolones Carbapenems, flu Chloramphenicol, amox trimethoprimsulfamethoxazole Chloramphenicol, amox tri Aminoglycosides, clindamycin, macrolides clindamycin, Aminoglycosides, Trimethoprim-sulfamethosxazole, am Antibiotics - ins, ­ r , po ­ s ins o ­ l ­ r ­ a o n, n, aminoglyco ones ­ sp ones ­ l ­ l o ­ ci ­ o ­ o ­ l y ­ a ­ rins, carbapenems, in ­ m uin in, ceph ­ q ­ c ­ qu ­ co ­ spo y , broad-spectrum , broad-spectrum o ones ­ ro o ­ r ­ l a ­ l ­ m ­ o ­ o ­ o ­ a o , , van ­ c in ive ­ t , , flu ones ­ qu ­ l ­ rins ­ rins o ­ o in , , ceph ­ r , van ­ c ­ o in ­ spo ­ spo (continued) ia o o ­ r ­ my ­ qu ­ l ­ l e ­ a ­ a o o ­ t ­ r ­ c ­ o Doxycycline Generally ac Carbapenems Penicillin Trimethoprim-sulfamethoxazole Oxacillin Vancomycin Fluoroquinolones ceph Fluoroquinolones ceph van sides, sides, flu Broad-spectrum ceph carbapenems, flu Fluoroquinolones, azithromycin imipenem, macrolides, clindamycin, flu ific bac ­ c in-susc.) in-res.) ­ l ­ l il il ­ c ­ c ­ i ­ i serovar Typhi serovar spp. (meth spp. (meth ntibacterial spe agents for A spp.

spp. spp. Antimicrobial Agents Antimicrobial Rickettsia Organism equi Rhodococcus Rothia mucilaginosa enterica Salmonella Staphylococcus Staphylococcus Stenotrophomonas maltophilia Serratia marcescens Salmonella Shigella Table 8.8 Vaccines, Susceptibility Testing 353 line ­ c y ­ c n, n, doxy ins ­ r ­ ci ­ a po ­ s ox o ­ l ­ fl ­ a ­ ro rim-sulfamethoxazole ­ p ­ o eth ­ m Trimethoprim-sulfamethoxazole Tetracyclines, Tetracyclines, tri Macrolides Aminoglycosides Broad-spectrum ceph Chlorampenicol, cip Penicillin (drug choice of if susc.) Penicillin (drug choice of if susc.) in in ­ c ­ c y y ins, ­ r ­ m ­ m po ­ co ­ co ­ s o ­ l ­ a in ones ­ c ­ l i ­ o ­ m ­ rins, carbapenems, ­ rins, carbapenems, ­ rins, carbapenems, in a ­ t ­ qu lines ­ spo ­ spo ­ spo , o ­ c o o o ­ r ­ l ­ l ­ l , , carbapenems, van , , carbapenems, van ­ o ­ a ­ a ­ a ­ cy , aminoglycosides, or gen ­ ra , , flu with ceftazidime with ones ­ l ­ o in in in , , tet , broad-spectrum ceph , , ceph , , ceph , , ceph rim-sulfamethoxazole ­ c ­ c ­ c ­ p in lines ­ o ­ c ­ qu ­ my ­ my ­ my o o o o ­ cy eth ­ r ­ c ­ c ­ c ­ o ­ ra ­ m Carbapenems flu Doxycycline Penicillin Cephalosporins Penicillin tet Doxycycline Fluoroquinolones tri Streptomycin Penicillin Penicillin Cephalosporins Penicillin van van van group (group B) (group A) group spp. Antimicrobial Agents Antimicrobial Therapy choice of in bold type. a Vibrio cholerae Yersinia enterocolitica Yersinia pestis Streptobacillus moniliformis Streptococcus agalactiae Streptococcus, anginosus Streptococcus, mitis pneumoniae Streptococcus pyogenes Streptococcus pallidum Treponema Tsakamurella vulnificusVibrio

354 SECTION 8 Fosfomycin

Chloramphenicol

Cotrimoxazole

Aminoglycosides

Nitrofurantoin

Polymixin B Polymixin

Colistin Colistin Tigecycline

RRR Tetracycline

RRRRRRRRRRRR

Aztreonam Ertapenem

a Imipenem ia * * * * ­ r

e ­ t Piperacillin

R

Cephalosporin

Generation Generation 3

rd

Cephalosporin

2 Generation Generation nd

ected Gram-negative bac

­ l

Cephalosporin

1 Generation Generation

st ance of se Ampicillin ­ t RRR RRRR RRRR RRRRRRRRR is ­ s ae ­ c ­ a is ­ l is i ­ r ntrinsic re ­ b I ­ ga ­ ra

lex Antimicrobial Agents Antimicrobial eus mi eus penneri eus vul ­ p ­ t ­ t ­ t com Escherichia coli Enterobacter clo Organism Citrobacter freundii Citrobacter koseri Hafnia alvei Klebsiella pneumoniae Morganella morganii Pro Pro Pro Table 8.9 Vaccines, Susceptibility Testing 355 R R RR RRR R ** RRR RR RRRR RRR RR * ion. ­ t uc ­ d RRRRR RRRR *** *** *** isms other than carbapenemase pro ­ n ­ a RRRRRR RRR R RRR RRR ne but not ceftazidime.

­ o x ­ a ri ­ t lex ­ p ated MICs mech by ­ v ­ e spp. com spp. lex Antimicrobial Agents Antimicrobial ­ p Clinical and Laboratory Standards Institute, M100-S27. *May have el have *May a **Resistant to most aminoglycosides but not amikacin. ***Resistant to cef Providencia stuarti Providencia Yersinia enterocolitica cepacia Burkholderia Pseudomonas aeruginosa Stenotrophomonas maltophilia Salmonella Shigella Serratia marcescens Acinetobacter baumannii com

356 SECTION 8 Cotrimoxazole

R** R**

dalfopristin Quinupristin-

R

Clindamycin Aminoglycosides

R** R** R** R**

Vancomycin Cephalosporins R* R** R** ­ tance. a

is

­ s ia ­ r Acid Fusidic e ­ t R R ic ic re ­ s

rin

­ t ive ive bac Fosfomycin

­ t

­ i R Novobiocin There is no in RRRR R ected Gram pos ­ l spp. ance of se ­ t is ­ s ­ us ntrinsic re I

Antimicrobial Agents Antimicrobial S. cohnii S. xylosus capitis S. Enterococcus faecalis Organism Staphylococcus au­ re Staphylococcus lugdunensis epidermidis Staphylococcus Methicillin Resistant Staphylococcus S. saprophyticus Enterococcus faecium Table 8.10 Vaccines, Susceptibility Testing 357 R** R ins such as ceftaroline. ­ r po ­ s o ­ l ­ a R** R** R RRR R R*** R R R** R ept for the so-called antistaphylococcal ceph ­ c ins ex ­ r po ­ s o ­ l ­ a y. ­ l in. ­ ceph ­ cal ­ c ­ i y ­ m o ­ c ive ive clin ant to all ­ t ­ t ec is ­ f ­ s ant to van ­ t is ­ s occi are re ­ c o ­ l ­ y ro but are not ef ies are re ­ t ­ c pe in vi ­ , s ant staph ive ive ­ t ­ t is ­ s spp. spp. spp. spp. ear ac ­ p Antimicrobial Agents Antimicrobial Clinical and Laboratory Standards Institute, M100-S27. ***Some, but not***Some, all **May **May ap *Methicillin re a Enterococcus gallinarum/E. casseliflavus innocuumClostridium Erysipelothrix rhusiopathiae Leuconostoc Pediococcus Lactobacillus Clostridium 358 SECTION 8 ar ­ l in and ed hodge ed hodge ar, and ar ­ l ­ u ­ l ­ l ­ fi ­ fi il ­ i ­ i ­ u ­ u ec ­ c ­ l ­ a ec ec ­ l ­ l ed carbapenemase ­ fi on on method (mCIM), ­ i orms poorly), ar ­ ti ­ f ­ l a ion ion of PBP2a’ ­ u ­ v ­ t ­ ti ec ­ l ec c ­ t ­ a None Phenotypic (ox Phenotypic (mod Molecular Phenotypic (mod Phenotypic, mo cefoxitin), mo test, mod in carbaNP), mo test per de mo Detection le ­ fi ors ­ t ­ i ept ept ept ept ept ­ c ­ c ­ c ­ c ­ c ble ble ib ­ a ­ h ­ i ance pro ­ t ­ ty ­ i is ­ s iv ­ t a-lactams ex a-lactams ex a-lactams. New a-lactams ex a-lactams ex a-lactams ex ­ t ­ t ­ t ­ t ­ t ­ t ­ tance to cefepime ibit ac is ­ ta-lactamase in ­ h ­ s cefepime ceftaroline cephamycins and carbapenems, var aztreonam cefepime and carbapenems All be re Typical Typical re All be All be (such as avibactam) in All be All be All be be ia. ­ r e ­ t sm(s) ­ i spp. spp., spp., spp., spp., ­ gan spp. ance in bac ­ t is ­ s Acinetobacter spp. Enterobacteriaceae, Pseudomonas Primary or Staphylococcus Enterobacteriaceae, Pseudomonas Acinetobacter spp. Enterobacteriaceae Enterobacteriaceae, Pseudomonas Acinetobacter spp. Enterobacteriaceae, Pseudomonas Acinetobacter spp., Aeromonas rug re ­ d i ­ t s ­ e ­ g isms of mul ­ n ­ a Beta-lactamase and cell wall chang Category Altered tar Beta-lactamase et Beta-lactamase Beta-lactamase Beta-lactamase

­ tance ­ sis mportant mech I

(Penicillin Binding Binding (Penicillin Antimicrobial Agents Antimicrobial AmpC + Porin Mutation New DelhiNew Metalo Protein 2a’ (PBP2a’)) Extended Spectrum AmpC Mechanism of re Klebsiella pneumoniae Carbapenemase (KPC) Beta-Lactamases (ESBL) Beta-Lactamase (NDM) mecA Table 8.11 Vaccines, Susceptibility Testing 359 ar (continued) ­ l ­ u ec ­ l Molecular Molecular Molecular Phenotypic (D-test) Phenotypic, mo Molecular Molecular ­ ta-lactams ­ ta-lactams ­ ta-lactams All be All be All be lincosamides,Macrolides, streptogrammins Glycopeptides Polymyxins Oxazolidinones, lincosamides,Macrolides, streptogrammins, phenicols, pleuromutilin - E. spp., spp. (rare) spp. spp., spp., spp. spp., s only ­ u spp. e ­ m ­ r ), Vancomycin ), Vancomycin Enterobacteriaceae, Pseudomonas Acinetobacter spp. Enterobacteriaceae, Pseudomonas Acinetobacter spp. Bacteroides Staphylococcus faecium Resistant Staphylo coccus au Streptococcus Enterococcus (most com Enterobacteriaceae Staphylococcus Enterococcus ­ ­ a l ­ y ng site ­ i omal ­ s o se ­ b ive ive meth ­ t ­ a u ­ t er i ng site ­ f ­ i ­ st ion ion of ri Beta-lactamase Beta-lactamase Beta-lactamase Inducible or con t bind Altered bind Phosphoethanolamine trans Methyltransferase Antimicrobial Agents Antimicrobial VIM cfiA erm vanA/B mcr IMP cfr 360 SECTION 8 Molecular Molecular Molecular Detection Molecular le ­ fi ones, ­ l ­ o ­ ta-lactams, ance pro ­ t is ­ s lines, quin ­ c ­ cy a ­ r Aminoglycosides Typical Typical re Oxazolidinones, Phenicols Macrolides, be tet aminoglycosides Quinolones ia. (continued) ­ r e ­ t sm(s) ­ i spp. spp., ­ gan sms ­ i ance in bac ­ t an is ­ g ­ s Gram-positives, Gram-positives, Gram-negatives, Mycobacteria Primary or Predominantly Gram-negative or Enterococcus Staphylococcus Gram-negatives rug re ­ d i ­ t ion r ­ t ­ e a ­ c ­ fi ort ­ i ­ p on ­ ti a ­ v isms of mul ­ ti ­ n c ­ a ­ a Enzymatic in Category pumps Efflux Target mod ABC trans ­ tance mportant mech ted ymes I ­ a ­ z is i ­ s ­ d

­ i one re ance ­ l ­ f Antimicrobial Agents Antimicrobial ­ t ­ o ­ i ­ sis (AME) quin Plasmid me Mechanism of re pumps Efflux Aminoglycoside mody ng en optrA Table 8.11 Vaccines, Susceptibility Testing 361 (continued) ow) ow) ­ l ­ l les be les be ­ p ­ p y) ­ l m m ­ a ­ a on ific ex ific ex ­ c ­ c ean Committee on Antimicrobial ­ p o ­ r Y Y Y H. influenzae Y ( H. Y Y Y Y Y Y Y Y Y Y (see spe Y Y Eu (EUCAST) Testing Susceptibility Y (Groups A, C, B, and G) Y Y Y (see spe Y er) ­ b les ­ b Y (M100) Y (M100) Y (M100) Y (M100) Y (M100) Y (M100) Y (M45) Y (M100) Y (M100) Y (M100) Y (M100) Y (M100) Clinical and Laboratory Standards Institute (CLSI) (Document num Y (M45) Y (M100) Y (M100) Y (M45) Y (M100) Y (M100) luded in CLSI andbreakpoint EUCAST ta spp. spp. ­ c and parainfluenzae H. Enterobacteriaceae i ­ r ­ aer ­ obes ­ cile o rganisms in ­ l Streptococcus spp. ­ fi O spp. spp. spp.

Antimicrobial Agents Antimicrobial Helicobacter py Organism Enterobacteriaceae Pseudomonas Acinetobacter cepacia Burkholderia Stenotrophomonas maltophilia Miscellaneous non- Staphylococcus Enterococcus influenzae Haemophilus pneumoniae Streptococcus Beta-hemolytic Gram-positive an ­ aer ­ obes Clostridium dif Gram-negative an multocidaPasteurella Neisseria gonorrhoeae Viridans group Streptococcus Listeria monocytogenes Anaerobes Neisseria meningitidis Table 8.12 362 SECTION 8 ) (see above) and y) ­ l on ean Committee on Antimicrobial ­ p o ­ r Y N Y Y Y N N N N Kingella kingae N (Except Kingella for N Eu (EUCAST) Testing Susceptibility Aerococcus sanguinicola Y ( Aerococcus S. urinae N Y er) ­ b (continued) les ­ b Y (M45) Y (M45) Y (M45) Y (M45— See HACEK group) Y (M45) Y (M45) Y (M45) Y (M45) Y (M45) Y (M45) Y (M45) Clinical and Laboratory Standards Institute (CLSI) (Document num Y (M45) Y (M45) Y (M45) , spp. spp., luded in CLSI andbreakpoint EUCAST ta ­ c spp. spp., corrodens Eikenella rganisms in O spp. spp. spp. spp. spp. and spp. Granulicatella spp. spp.

spp. spp. (not anthracis) Antimicrobial Agents Antimicrobial and Kingella Organism catarrhalis Moraxella Corynebacterium kingae Kingella Erysipelothrix rhusiopathiae Cardiobacterium Lactobacillus Lactococcus Leuconostoc Aerococcus Aeromonas Abiotrophia Gemella HACEK group: Aggregatibacter Campylobacter jejuni and coli Bacillus Table 8.12 Vaccines, Susceptibility Testing 363 N N N N N N Y N N N N N N N N Y N N Y N N Y (M45) Y (M45) Y (M45) Y (M45) Y (M45) N Y (M24) Y (M24) Y (M24) Y (M24) Y (M45) N Y (M45) Y (M45) Y (M45) Y (M27) Y (M24) Y (M24) N Y (M24) Y (M24) ia ­ r e ­ t ac ­ b o ­ c ) lex ­ ria ng ng my ­ p ­ i is ­ te ­ s o ­ l u ­ bac ­ gi V. choleraeV. ­ c ­ co and B. pseudomallei er ng etes ­ i ­ b ­ lei ­ c lud ­ my ­ c spp. spp. spp. ­ no i spp. ­ t spp. spp spp. spp. (in Antimicrobial Agents Antimicrobial Micrococcus Pediococcus Rothia mucilaginosa Bacillus anthracis Burkholderia mal Francisella tularensis Cryptococcus Other Filamentous fun Rapidly ­ ing grow my Vibrio Yersinia pestis Brucella Mycobacterium tu Mycobacterium avium com Miscellaneous slowly grow Aerobic ac Nocardia Aspergillus Candida Mycobacterium kansasii Mycobacterium marinum 364 SECTION 8 ble ble ge ge ­­ a ­ a t. ­ ser MIC and DD breakpoints avail in the pack FDA in Comments ion ­ t ra ­ t ­ tory ls ­ i en ­ a ­ c i ib ­ b ­ h Y Y Y Y Y N N Y Y Y Y Y Y Y Y con Minimum in ro ­ c i ­ m i ion ­ t EUCAST ­ s u ­ f Y Y Y Y Y N N Y Y Y Y Y Y Y Disk dif only tested an ion ­ m ­ t ra ­ t ­ tory ­ i en ­ c ib ­ h Y Y Y Y Y Y Y Y Y Y Y Y Y Y Minimum in con sms for com ­ i CLSI ion an ­ s ­ g u ­ f ect or ­ l Y Y Y Y Y Y Y N Y Y Y Y Inferred Inferred Inferred Y Y Disk dif ia ia for se ­ r e ­ t ive ive cri ­ t re ­ p er ­ t Ceftazidime Cefotaxime Ceftriaxone Cefepime Ceftaroline Cefazolin (Urine) Cefazolin Cefazolin Ceftolozane-tazobactam Ticarcillin-clavulanate Piperacillin-tazobactam Piperacillin Amoxicillin-clavulanate Amoxicillin Ampicillin-sulbactam Ampicillin Antibiotic uide to in G

Antimicrobial Agents Antimicrobial Enterobacteriaceae Organism Table 8.13 Vaccines, Susceptibility Testing 365 ble ble ble ble ge ge ge ge ­­ a ­­ a ­ a ­ a (continued) t. t. ­ ser ­ ser MIC and DD breakpoints avail in the pack FDA in in the pack FDA in MIC and DD breakpoints avail Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y a Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y Y N Y Y Y Y N Y a Y Y Y Y Y Y Y N Y Y Y N Y Y Y Y Y N

Ciprofloxacin Amikacin Tobramycin Gentamicin Imipenem Nitrofurantoin Ertapenem Colistin Meropenem Trimethoprim- Sulfamethoxazole Doripenem Tigecycline Aztreonam Minocycline Cefuroxime Tetracycline Levofloxacin Ceftazidime-avibactam Antimicrobial Agents Antimicrobial 366 SECTION 8 ble ble ­­ a ge ge ­ a ion ion ion MICion and ­ s ­ t u u ­ f ­ l t. ­ ser Agar di breakpoints avail from pack FDA disk dif in Comments ion ­ t ra ­ t ­ tory ls (continued) ­ i en ­ a ­ c i ib ­ b ­ h Y Y Y Y Y Y Y Y Y Y Y Y Y con Minimum in ro ­ c i ­ m i ion ­ t EUCAST ­ s u ­ f c N Y Y Y Y Y Y Y Y Y Y Y Y Disk dif only tested an ion ­ m ­ t ra ­ t ­ tory ­ i en ­ c ib ­ h b Y Y Y Y Y Y Y Y Y Y Y Y Y Minimum in con sms for com ­ i CLSI ion an ­ s ­ g u ­ f ect or ­ l b N Y Y Y Y Y Y Y Y Y Y Y Disk dif Y ia ia for se ­ r e ­ t ive ive cri ­ t re ­ p er ­ t Colistin Imipenem Meropenem Doripenem Aztreonam Cefepime Ceftazidime Ticarcillin-clavulanate Ceftolozane-tazobactam Piperacillin-Tazobactam Piperacillin Chloramphenicol Antibiotic Fosfomycin uide to in G

Antimicrobial Agents Antimicrobial Pseudomonas Pseudomonas aeruginosa Organism Table 8.13 Vaccines, Susceptibility Testing 367 (continued) Y Y Y N N Y Y Y Y Y Y Y N N Y N Y N Y N Y N Y Y Y Y N N Y Y Y N Y Y Y N N Y N Y N Y N Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Trimethoprim- Sulfamethoxazole Levofloxacin Ciprofloxacin Minocycline Doxycycline Amikacin Tobramycin Gentamicin Colistin Imipenem Meropenem Doripenem Ceftriaxone Cefepime Levofloxacin Ceftazidime Ciprofloxacin Ticarcillin-clavulanate Amikacin Piperacillin-Tazobactam Tobramycin Piperacillin Gentamicin spp. Antimicrobial Agents Antimicrobial Acinetobacter 368 SECTION 8 Comments ion ­ t ra ­ t ­ tory ls (continued) ­ i en ­ a ­ c i ib ­ b ­ h Y Y Y Y Y Y Y N con Minimum in ro ­ c i ­ m i ion ­ t EUCAST ­ s u ­ f d N N N N Y Y Y Y Disk dif only tested an ion ­ m ­ t ra ­ t ­ tory ­ i en ­ c ib ­ h e N Y Y Y Y Y Y Y Minimum in con sms for com ­ i CLSI ion an ­ s ­ g u ­ f ect or ­ l e Y Inferred Inferred Inferred Inferred InferredInferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred N Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred N Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred N Disk dif N Y N Y ia ia for se ­ r e ­ t ­ rins

­ spo d o ive ive cri ­ rins ­ l ­ t ­ a d d re ­ spo ­ p o ­ l er ­ a ­ t Ceftaroline Parenteral ceph Carbapenems Oral ceph Piperacillin-Tazobactam Oxacillin Dalbavancin Ampicillin-Sulbactam Nafcillin Televancin Clavulanate Cefoxitin Amoxicillin- Methicillin Vancomycin Oritavancin Antibiotic Penicillin uide to in spp. G

Antimicrobial Agents Antimicrobial Staphylococcus Organism Table 8.13 Vaccines, Susceptibility Testing 369 ert. ­ s ble ble ble ble in ­­ a ge ge ­­ a ­ a ge inge (continued) ­ a ion ion ion MICion and ­ s ­ t u u ­ f ­ l t. ­ ser Agar di breakpoints avail from pack FDA disk dif in MIC and DD breakpoints avail the pack FDA Y Y Y Y Y Y Y Y Y/Inferred Y Y Y Y Y Y Y Y Y d d d d d N N/Inferred Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y N N Y Y Y Y Y N Y Y Y Y Y Y Y Y N Y

Fosfomycin Tedizolid Linezolid Quinipristin- dalfopristin Rifampin Chloramphenicol Ciprofloxacin Tigecycline Cindamycin Gentamicin Doxycycline Nitrofurantoin Tobramycin Amikacin Tetracycline Levofloxacin Trimethoprim- Daptomycin Sulfamethoxazole Antimicrobial Agents Antimicrobial 370 SECTION 8 - ert for ­ s y. ­ l ble inble the ­ a ge inge on ­ a in-susceptible ­ c y ­ m o ­ c MIC and break DD points avail van FDA packFDA E. faecalis Comments ion ­ t ra ­ t ­ tory ls (continued) ­ i en ­ a ­ c i ib ­ b ­ h Y N N N N N N N Y Y Y Y Y N con Minimum in ro ­ c i ­ m i ion ­ t EUCAST ­ s u ­ f Y N N N N N N N Y Y Y Y N Disk dif only tested an ion ­ m ­ t ra ­ t ­ tory ­ i en ­ c ib ­ h N Y Y Y Y N Y Y Y Y Y Y Minimum in con sms for com ­ i CLSI ion an ­ s ­ g u ­ f ect or ­ l N Y Y Y N N N N Y Y Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred Inferred Y Y Disk dif ia ia for se ­ r e ­ t ive ive cri ­ t re ­ p er ­ t Tigecycline Minocycline Doxycycline Tetracycline Daptomycin Dalbavancin Televancin Oritavancin Teicoplanin Vancomycin Imipenem Piperacillin-Tazobactam Amoxicillin Ampicillin Penicillin Antibiotic uide to in G spp

Antimicrobial Agents Antimicrobial Enterococcus Organism Table 8.13 Vaccines, Susceptibility Testing 371 (continued) ­ lin il ­ c ­ a f g g g h Y Y Y N Y N Y Inferred N N Inferred from ox Y Y Y Y Y ­ lin il ­ c ­ a f g g g Y Y Inferred Inferred Y Y Inferred Inferred N Inferred Inferred Y N Y Y Y N N Y Y Inferred from ox Y /Inferred f f g g h h Y Y Y Y Y/Inferred Y Y Y/Inferred Y N Y Y N N Y Y Y Y ­ lin il ­ c ­ a f g g N Inferred Y Y Inferred Y Y Y Y Inferred Y N Y Y Y N N Inferred from ox Inferred Inferred

Tedizolid Cefepime Linezolid Nitrofurantoin Amoxicillin/ Clavulanate Quinipristin- dalfopristin Gentamicin Levofloxacin Fosfomycin Amoxicillin Chloramphenicol Amikacin Ciprofloxacin Rifampin Streptomycin Tobramycin Trimethoprim- Sulfamethoxazole Penicillin Ampicillin Antimicrobial Agents Antimicrobial Streptococcus pneumoniae 372 SECTION 8 Comments ion ­ t ra ­ t ­ tory ls (continued) ­ i en ­ a ­ c i ib ­ b ­ h Y Y Y Y Y Y Y Y Y Y Y con Minimum in ro ­ c i ­ m i n ion ­ t EUCAST ­ s ­ ci u y h ­ f ­ m ­ lin ­ lin ­ lin ­ lin ­ lin ­ lin ­ lin ­ ro il il il il il il il ­ c ­ c ­ c ­ c ­ c ­ c ­ c ­ a ­ a ­ a ­ a ­ a ­ a ­ a Inferred from eryth Y Y Inferred from ox Inferred from ox Inferred from ox Y Inferred from ox Inferred from ox Inferred from ox Inferred from ox Disk dif only tested an ion ­ m ­ t ra ­ t ­ tory ­ i en ­ c ib /Inferred /Inferred ­ h h h Y Y N Y Y Y Y Y Y/Inferred Y Minimum in Y con sms for com ­ i CLSI ion an ­ s ­ g u ­ f ect or ­ l Y Y N N N N Y Disk dif Inferred Inferred N Inferred ia ia for se ­ r e ­ t ive ive cri ­ t re ­ p er ­ t Azithromycin Tetracycline Teicoplanin Ertapenem Imipenem Doripenem Vancomycin Antibiotic Cefuroxime Ceftaroline Meropenem Ceftriaxone uide to in G

Antimicrobial Agents Antimicrobial Organism Table 8.13 Vaccines, Susceptibility Testing 373 ble ble ge ge ­­ a ­ a t. ­ ser in the pack FDA MIC and DD breakpoints avail in nce. ­ a N Y N Y Y Y Y Y N Y ia. ­ r e ies-specific guid ­ t ­ c ent for spe N Y N Y Y Y Y Y N Inferred ­ m ­ u ent doc ­ r N Y Y Y Y Y Y Y N Y ent up-to-date for breakpoint cri ­ m ­ u ies. Consult cur ­ c ine doc ­ l occi spe ­ c o ­ l N Y Y Y Y Y Y Y N Y ­ y ent guide ­ r y. ­ l spp. s on us staph ­ u ­ o ­ i e ­ r

st. Consult cur S. au ­ i y. ­ l Salmonella ant ­ t ions for var ­ t is ­ s a ­ cept ­ d ex en in re ­ l ­ m ary tract on Tedizolid Linezolid Quinipristin- dalfopristin Clindamycin Rifampin Chloramphenicol Trimethoprim- Sulfamethoxazole Levofloxacin Tigecycline Doxycycline ­ n m ­ cil ­ i ­ o ific breakpointsex y. ­ l ­ c y. ­ l ific rec on y. ­ c ­ l from the uri on ­ i ­ e E ­ ­ p Antimicrobial Agents Antimicrobial There are spe For all For E. coli nterobacteriaceae For syn rgy on Infection-site spe For E. faecalis For E. coli For re ort ng on meth a b c d e f g h 374 SECTION 8 ed ­ fi s. i ­ e ­ t as ­ b den ­ i a

­ t

al trial ion. ­ c ­ t ­ i a ­ c ­ fi i ­ t ei and ­ l nt innt some da ­ e A. hydrophila A. xylosoxidans ases. be May mis ates in clin ­ l ­ b l. a ­ e ­ t een een een een uce an iden ­ tw ­ tw ­ d lude B. mal ­ c i te te be te te be ­ g spp. ­ a ­ a i i ­ t ­ t luded in da n n ­ c ­ e ­ e a ­ t ­ fer ­ fer ed to Genus lev ­ fi i ­ t ia, and fun ­ r den ­ i e ­ t c Comments Cannot Cannot dif Databases do not in B. pseudomallei. Cannot Cannot dif Minimal data. not May be pres and A. caviae Frequently fails to pro Minimal Minimal da Minimal data. One iso 12 of notMay be in as Ochrabactrum mis and A. rhulandii. ­ ba o ­ c ia, my es level es level ­ r e ­ ci ­ t ion − + + + + + + + ­ t +/− +/− +/− +/− +/− a ­ c ­ fi i ­ t ion ion of bac ­ t Reliable spe Reliable iden a ­ c ­ fi i ­ t s level s level ­ nu ion ­ t + + + + + + + + + + a +/− +/− ­ c ­ fi i ­ t Reliable ge iden ­ ria

i ­ l ­ te ­ o spp. ­ i spp. spp. ummary of MALDI-TOF MS iden spp. spp. S spp. spp. spp.

spp. lex Antimicrobial Agents Antimicrobial ­ p Organism Gram-negative bac Aeromonas Achromobacter Acinetobacter Aggregatibacter faecalisAlcaligenes cepacia Burkholderia Campylobacter hominis Cardiobacterium Chryseobacterium sakazakii Cronobacter com Bacteroides Brucella Burkholderia glad Citrobacter Table 8.14 Vaccines, Susceptibility Testing 375

(continued) nt nt ween ­ t ­ e

lex. ­ p te be ­ a i ween ­ t ­ t n com ­ e Neisseria spp. er spp. ae ­ f te be ion. Not pres Not ion. ­ c ­ a ­ t i ­ a a ­ t enic ­ c n ­ g s. ­ fi ­ e ly ly dif i ­ e ­ i ­ t er Shigella ­ f as E. clo atho ­ b is. ­ p a ­ r ­ t ­ ga te with te from ­ a ­ a s. i i uce an iden ­ e ­ t ­ t and non ­ d n n as ­ e ­ e ­ b a. ­ t er er a ­ t ­ f ­ f nt nt in some da ­ tems may not read ­ tems may not dif ­ e Minimal da Some sys meningitidis N. Not Not pres Cannot dif May failMay to pro in some da P. penneriP. and P. vul Some sys Cannot dif + + + + + + + + + + + + + + +/− +/− +/− +/− +/− + + + + + + + + + + + + + + + + + + +/− spp. spp. spp. spp. spp. spp. (non E. spp. coli) spp. spp. spp. spp. spp. spp. spp. eus Antimicrobial Agents Antimicrobial ­ t Neisseria spp. Escherichia Francisella Klebsiella Morganella Pandorea Pro Prevotella Edwardsiella corrodens Eikenella Enterobacter Fusobacterium Haemophilus limosus Inquilinus kingae Kingella catarrhalis Moraxella urethralisOligella agglomerans Pantoea Porphyromonas Pasteurella Escherichia coli 376 SECTION 8 ion. ­ t a ­ c ­ fi i ­ t ­ les. ion. ­ e ­ t ­ a i ­ t n uce an iden ­ e ­ d s. er ­ f ­ e as ­ b ain S. anoph a ­ t ­ t TYPHI ies dif (continued) ­ c S. lude Y. pestis. i ­ c te ­ g ­ a i ­ t n ­ e a ­ t er ­ f nt nt in some da ­ e ia, and fun ­ r e ­ t c Comments Databases may not con Not Not pres Cannot dif Minimal da Databases do not in Mucoid strains may fail to pro Minimal data for spe ­ ba o ­ c ia, my ­ r es level es level e ­ t ­ ci ion + + + + + + + + + + + + + ­ t +/− +/− +/− a ­ c ­ fi i ­ t ion ion of bac ­ t Reliable spe Reliable iden a ­ c ­ fi i ­ t s level s level ­ nu ion ­ t + + + + + + + + + + + + + + + + a ­ c ­ fi i ­ t Reliable ge iden

­ ria group ­ te ummary of MALDI-TOF MS iden S spp. spp. spp.

spp. spp. spp. spp. spp. Antimicrobial Agents Antimicrobial Roseomonas Providencia Table 8.14 Organism aeruginosa Pseudomonas putida Pseudomonas fluorescens Pseudomonas Ralstonia pickettii Spingomonas Stenotrophomonas maltophilia Gram-positive bac Vibrio Yersinia spp. Abiotrophia ­ tive de ­ fec group group Actinomyces spp. Aerococcus Salmonella Serratia Raoutella Vaccines, Susceptibility Testing 377 and from (continued) ance. s group ­ m ­ u ween the

­ t S. pyogenes or ­ re ­ f S. mitis/oralis te be ies. ween ­ a B. ce te i. ­ t ween ­ c i ­ t ­ a ­ c ­ t i ble ble per n ­ t B. anthracis. ­ a oc ­ e n i ­ c ­ l ­ e te be er te be e o ­ a ­ f ­ l ­ r i er ­ a ­ t i ­ f ­ y ­ t ludes n n ­ c ­ e ­ e er ­ f er ot dif te within the ­ f ests un ­ n and other spe ­ a ­ g i ­ t ive ive staph n ­ t ly ly dif ­ e ­ a ­ i a. er ays ays dif ­ t ­ f ­ w ­ tems may not dif ­ tems can sms, which in ­ i n ase neg ­ l ­ u ­ ga g ­ a Minimal data sug Does not read L. monocytogenes Some sys co Minimal da Some sys May not al S. dysgalactiae. May May not dif S. pneumoniae. of of or - + + + + + + + + + + + + + +/− +/− +/− +/− +/− + + + + + + + + + + + + + + + + + + +/− ­ o spp. spp. spp. spp. ­ na spp. Viridans Large col ny, spp. spp. spp. spp. spp. spp. spp. spp. spp. Antimicrobial Agents Antimicrobial a-hemolytic ­ t Gordonia Clostridium Listeria Gemella Group be Arcanobacterium Corynebacterium Enterococcus Finegoldia mag Lactobacillus Micrococcus Peptostreptococcus Propionibacterium Rothia mucilaginosa Staphylococcus Streptococcus, Streptococcus, otitidisTuricella Nocardia Bacillus 378 SECTION 8 lex. ­ p ies within the MTB com ­ c (continued) te spe i ­ a ­ g i ­ t n ­ e er ­ f ia, and fun ­ r e ­ t c Comments Does not dif ­ ba o ­ c ia, my ­ r es level es level e ­ t ­ ci ion + + + + + + + + + + + + + + + + ­ t a ­ c ­ fi i ­ t ion ion of bac ­ t Reliable spe Reliable iden a ­ c ­ fi i ­ t s level s level ­ nu ion ­ t + + + + + + + + + + + + + + + + a ­ c ­ fi i ­ t Reliable ge iden ­ p lex ­ p lex ­ p lex ­ p ­ p com lex ummary of MALDI-TOF MS iden lex S ­ p is com com lex ­ s com

o ­ l u ­ c er ­ b Antimicrobial Agents Antimicrobial M. marinum com simiae M. M. szulgai xenopi M. fortuitum M. Table 8.14 Organism Mycobacteria—Slow Growers M. tu haemophilum M. scrofulaceum M. Mycobacteria—Rapid Growers chelonae M. abscessus M. mucogenicum M. immunogenum M. smegmatis M. M. avium com M. gordonae M. M. kansasii Vaccines, Susceptibility Testing 379

ed as C. duobushaelmulonii C. neoformans and C. gattii. ­ fi i ­ t een een den ­ i ­ tw te te be ­ a i ­ t n ­ e may be mis ­ fer C. auris C. Can Can dif and C. haelmulonii. a a a a a a a a a a ­ t ­ t ­ t ­ t ­ t ­ t ­ t ­ t ­ t ­ t + + + + + + + + + No da No da No da No da No da No da No da No da No da No da + + + + + + + + + + + + + + + + + + +/- spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. spp. Antimicrobial Agents Antimicrobial Curvularia Candida Mucor Bipolaris Fungi Alternaria dermatitidis Blastomyces Cladosporium Cryptococcus Histoplasma capsulatum Paecilomyces Rhodotorula Saacharomyces Scopulariopsis Trichosporon Trichophyton Aspergillus Penicillium Rhizopus Fusarium spp. 380 SECTION 8

spp., ive ive ­ t

­ a group, occi, ­ c spp. o ase neg ­ l ­ l ­ y ­ u g ­ a staph Streptococcus pneumoniae/mitis group, S. bovis group, S. salivarius Corynebacterium Bacillus Gram-negative Klebsiella, Enterobac- Citrobacter,ter, Pantoea, Escherichia, Shigella, Yersinia, Salmonella, Acinetobacter Limitations in Species-Level Differentiation Gram-positive co ion ion ­ g ­ g rpoB gyrB 16S-32S spp.— ets dnaJ, sodA, ­ g or Tt dnaJ, tuf, tuf ­ t spp.— spp.—16S-23S re spp.—16S-23S gyrB ion, ­ g Alternative tar Corynebacterium Staphylococci— tuf, rpoB Streptococci— rpoB, gyrB Enterococci— Bacillus Enterobacteriaceae—gyrB, Elongation fac Acinetobacter, Haemophilus, Aggregatibacter— re Pseudomonas Bordetella, Burkholderia—recA Bordetella, re Neisseria spp.—16S-32S a

ies ies ies ion ­ c ­ c ­ c ion ion ion ion ion ion ­ t ­ t ­ t ­ t ity with ity with ity with a ­ t a a a ­ t ­ t ­ r ­ r ­ r ­ c spe

­ a ­ a ­ a etes— ­ fi i ­ c - >99.4% ­ t y ­ m ­ ty i o ­ t ­ n ­ ti Criteria for Species-level Identification from other spe >99.6% iden >0.4% sep from other >99.0% iden >0.8% sep For Klebsiella, Enterobacter, Pantoea- Citrobacter, >99.5% iden >0.5% sep Campylo bacter— from other spe iden Aerobic ac sm sm iden ­ i an ­ g ­ ty i ­ t ets for or ­ g Criteria for Genus-level Identification >97.0% iden ng ng tar ­ i uenc ­ q Organisms Identified Bacteria ene se G

Antimicrobial Agents Antimicrobial Gene Target 16S rRNA Table 8.15 Vaccines, Susceptibility Testing 381 lex ­ p spp. M. kansasii, com spp., lex, ­ p Aerobic ActinomycetesAerobic Nocardia equi Rhodococcus M. tuberculosus com Phaeoacremonium, Fusarium spp., zygomycetes, Alternaria M. marinum, M. ulcerans, M. chelonae, M. abscessus, M. fortuitum n ­ li u ­ b gyrB i—D1/D2 n rpoB ion ­ g ­ t ­ li cpn60 a lex— u beta beta tu ­ g ­ p ­ b lex— ­ p secA1 spp.— beta beta tu com spp.— F) (E

i—D1/D2 or ­ g ­ t Campylobacter Brucella—recA, gyrB Nocardia equi—choE Rhodococcus M. kansasii—gyrB, rpoB, secA1, dnaA, hsp65, ITS M. marinum, M. ulcerans—gyrB, hsp65, secA1, dnaA fortuitum M. M. chelonae, M. abscessus—rpoB, ITS secA1, hsp65, Fusarium spp.—elon fac and yeast-likeYeast fun DimorphicZygomycetes, fun Aspergillus— Dermatophytes—28S D2 M. tuberculosus com Phaeoacremonium— y ­ t i ­ t 100% iden ­ ty i ­ t 99.0–99.9% iden Mycobacteria Fungi Antimicrobial Agents Antimicrobial Clinical and Laboratory Standards Institute—MM18AE. Interpretive Criteria Identification for Bacteriaof andTarget Fungi DNA by Sequencing; Approved a Guideline 16S rRNA Intergenic Spacer Region (ITS)

Index

A Administration routes Abiotrophia, 5, 145, 334 an­ti­fun­gal agents, 342 Abiotrophia defectiva, 22, 35 an­ti­mi­cro­bial agents, Abscess 337–341 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 an­ti­par­a­sitic agents, 343–344 spec­i­men col­lec­tion and Advisory Committee on trans­port, 84, 112 Immunization Practices Acanthamoeba spp., 15, 39, 40, 41, (ACIP), 324 43, 71, 118, 119, 122 Aerobic ac­ti­no­my­cetes, di­ag­nos­tic tests, 287, 291 an­ti­mi­cro­bial sus­cep­ti­bil­ity Acholeplasma laidlawii, 22 test meth­ods, 336 Achoromobacter, 7 Aerococcus spp., 5, 334 Acidaminococcus fermentans, 22 Aerococcus christensenii, 22 Acid-fast stain, 139, 291 Aerococcus sanguinicola, 54 Acid-fast trichrome chromotrope Aerococcus urinae, 22, 54 stain, 290 Aerococcus viridans, 22 Acidovorax, 7, 205 Aeromonas spp., 8, 22, 44 Acinetobacter spp., 8, 22, 47, 51, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 345 meth­ods for, 334 an­ti­mi­cro­bial sus­cep­ti­bil­ity test di­ag­nos­tic tests, 161–162 meth­ods for, 333 dif­fer­en­tial char­ac­ter­is­tics of, com­monly tested an­ti­mi­cro­bi­als, 202 367 Aeromonas hydrophila, 52, 345 di­ag­nos­tic tests, 161 Aggregatibacter spp., 8, 192 Acinetobacter baumannii, 22, Aggregatibacter actinomycetem- 355 comitans spp., 22 Acremonium spp., 67, 270 Agrobacterium, 6 Acridine or­ange stain, 139, 246 Alcaligenes, 7 Actinobacillus spp., 8, 130, 192, Alistipes spp., 22 345 Alloiococcus, 5 Actinobacteria, 3–4 Alloiococcus oti­tis, 22 Actinobaculum, 3 Alloscardovia, 4 Actinomyces spp., 3, 22, 40, 41, 45, Alphaproteobacteria, 6–7 46, 48, 50, 107, 345 Alphavirus, 221, 225 di­ag­nos­tic tests, 168–169 Alphavirus, 11, 40 Actinomycetes, dif­fer­en­tial Alternaria spp., 69. 279 char­ac­ter­is­tics of, 185 Amer­i­can Academy of Family Acute men­in­gi­tis, 38–39 Physicians (AAFP), 324 Adenoviruses, 36, 39, 40, 42–45, Amer­i­can College of Obstetricians 48–51, 54, 109, 222, and Gynecologists 232–233 (ACOG), 324 384 Index Amer ­i­can College of Physicians– bac­te­ria, my­co­bac­te­ria and Amer­i­can Society of fungi, 332–336 Internal Medicine CLSI docu­ ments­ for, 330–331 (ACP–ASIM), 324 Antimicrobial ther­apy, 324 Amer­i­can Tru­deau Society Antiparasitic agents, ad­min­is­tra­ me­dium, 154 tion routes and drug class, Amniotic fluid, spec­i­men 343–344 guide­lines for par­a­si­tol­ogy, Aphthovirus, 10 123 Apicomplexans, path­o­gens, 73–74 Amoebae, path­o­gens, 71 Apophysomyces, 272 Amphotericin, 144 Apophysomyces elegans, 66 Ampicillin, 348, 351, 364, 370, Arachnida, 59–60 371 Arcanobacterium spp., 3, 22 Anaerobes Arcanobacterium haemolyticum, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 48, 345 meth­ods, 336 Arcobacter spp., 7, 212 de­tec­tion meth­ods, 131, Arenavirus, 12, 36, 40, 221, 168–170 225–226 Anaerococcus spp., 6, 22 Arthritis, 34 Anaplasma spp., 7, 105, 132, 172 Arthrobacter, 4 Anaplasma phagocytophila, 345 Arthropod vec­tors, med­i­cally Anaplasma phagocytophilum, 55 im­por­tant dis­eases, 58–60 Anaplasmosis, 55, 59 Ascaris lumbricoides, 16, 51, 75, Ancylostoma braziliensis, 317 121, 318, 320 Ancylostoma duodenale, 16, 75 mor­pho­log­i­cal char­ac­ter­is­tics mor­pho­log­i­cal char­ac­ter­is­tics of, 318 of, 318 par­a­sitic di­ag­no­sis, 288, 297 par­a­sitic di­ag­no­sis, 288, 297 Ascomycota, 13 Angiostrongylus cantonensis, 39, Ashdown me­dium, 141 40 ASM Manual of Clinical Animalia, 15–16 Microbiology, 82, 220, 229, Anisakis spp., 75 263, 286 Anisomycin, 143 Aspergillus spp., 34, 35, 36, 37, 41, Anthrax, 54 43, 46, 47, 49, 50, 52, 53, Antibacterial agents, 345–353 68, 255 Antibiotics char­ac­ter­is­tics of, 267–268 agents for spe­cific bac­te­ria, di­ag­nos­tic tests, 258 345–353 di­rect ex­am­i­na­tion, 252 in­ter­pre­tive cri­te­ria for se­lect Aspergillus fumigatus, 51, 267 or­gan­isms, 364–373 Astrovirus, 11, 44, 221, 226 Antibody de­tec­tion Atopobium spp., 4, 22 bac­te­ria, 129–132 Auramine-rhodamine stain, 139 fungi, 245 par­a­sites, 287–289 Antifungal agents, ad­min­is­tra­tion B routes and drug class, 342 Babesia spp., 15, 73, 117 Antigen de­tec­tion di­ag­nos­tic tests, 287, 294 bac­te­ria, 129–132 mor­pho­log­i­cal char­ac­ter­is­tics fungi, 245 of, 315 par­a­sites, 287–289 Babesia microti, 15, 38 Antimicrobial agents, routes of Babesiosis, 57, 59 ad­min­is­tra­tion and drug Bacillus spp., 5–6, 22, 140 class, 337–341 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Antimicrobial sus­cep­ti­bil­ity meth­ods, 336 test­ing dif­fer­en­tial char­ac­ter­is­tics of, 184 Index 385 Bacillus anthracis, 52, 54, 129, BK vi­rus, 54, 109, 223, 238–239 158–159, 345 Blastocystis hominis, 15, 23, 308 Bacillus ce­re­us, 43, 44, 345 cysts of, 307 Bacteria di­ag­nos­tic tests, 287, 292 an­ti­bac­te­rial agents for, 345–353 Blastomyces spp., 242, 255 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Blastomyces dermatitidis, 35, 37, meth­ods for, 332–336 46, 51, 53, 62 mech­a­nisms of mul­ti­drug re­sis­ char­ac­ter­is­tics of, 273, 274 tance in, 358–360 di­ag­nos­tic tests, 258–259 pri­mary plat­ing me­dia, 141–154 di­rect ex­am­i­na­tion, 249 screen­ing spec­i­mens, 137 Blastomycosis, di­ag­nos­tic tests, tax­o­nom­i­cal clas­si­fi­ca­tion of, 258–259 2–3 Blastoschizomyces capitatus, Bacteroides spp., 4, 22, 23, 41, 148 23, 61 Bacteroides bile-esculin (BBE) Blood agar, 141 de­tec­tion meth­ods for pro­to­zoa, Bacteroides fragilis, 23, 38, 47, 48, 287, 294–296 50, 131, 141 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 Bacteroides fragilis group, 345 my­col­ogy plat­ing guide, 256 di­ag­nos­tic tests, 169 spec­i­men col­lec­tion and trans­ dif­fer­en­tial char­ac­ter­is­tics of, port, 85–86, 112–113 216–217 spec­i­men guide­lines for Bacteroidia, 4 ­par­a­si­tol­ogy, 117–118 Balamuthia, 118 vi­rol­ogy guide­lines, 108 Balamuthia mandrillaris, 15, 71 Blood agar, 142 Balantidium coli, 15, 287, 292, Body cav­ity flu­ids, Gram stain and 313 plat­ing me­dia, 133 Bartonella spp., 6, 35, 52, 53, 105, Bone in­fec­tions, 34–35 162 Bone mar­row Bartonella henselae, 42, 46, 346 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 Bartonella parapertussis, 162–163 my­col­ogy plat­ing guide, 256 Bartonella per­tus­sis, 162–163 spec­i­men col­lec­tion and trans­ Basidiobolus, 272 port, 87, 113 Basidiobolus ranarum, 66 spec­i­men guide­lines for par­a­si­ Basidiomycota, 13 tol­ogy, 118 Bayliascaris procyonis, 16 vi­rol­ogy guide­lines, 108 Bayliasciaris spp., 39 Bordetella spp., 7, 206 Beauveria, 270 Bordetella parapertussis, 135, 142 Bergeyella, 5 Bordetella per­tus­sis, 50, 55, 135, Bergey’s Manual of Systematic 142, 346 Bacteriology, 3 Bor­det-Gengou agar, 142 1,3-Beta-D-glucan, bio­marker of Borrelia spp., 9, 40, 105, 171 fun­gal in­fec­tion, 263 Borrelia burgdorferi, 34, 36, 39, Betaproteobacteria, 7 42, 55, 59, 105, 132, 346 Bifidobacterium spp., 4, 23 di­ag­nos­tic tests, 170–171 Bile-esculin agar, 141 Botrytis, 280 Bilophila wadsworthia, 23, 141 Botulism, 54 Biopsy spec­i­men, my­co­bac­te­rial Bovine en­ceph­a­lop­a­thy agent, 223, iden­ti­fi­ca­tion, 138 239 Bipolaris spp., 70, 278 Brachyspira, 8 Birdseed agar, 254 Brain ab­scess, 41 Bismuth sul­fite agar, 142 Brain heart in­fu­sion agar, Bite wound, spec­i­men col­lec­tion 142–143, 254 and trans­port, 85 Branhamella, 8 BK poly­oma­vi­rus, 9 Brevibacterium spp., 4, 23 386 Index Brevundimonas spp., 6, 205 di­ag­nos­tic tests, 259 Brilliant green agar, 143 di­rect ex­am­i­na­tion, 250 Broad-spectrum ceph­a­lo­spo­rins, Candida albicans, 24, 37, 41, 43, 345, 345–353 44, 47, 48, 54, 254 Bronchitis, 49–50 Candidiasis, 259 Brucella spp., 6, 34, 35, 39, 46, Cantipeda periodontii, 24 54, 105, 131, 135, 140, 163, Capillaria spp., 16 346 Capillaria he­pat­i­ca, 75, 121 Brucella agar, 143 Capillaria philippinensis, 75, 288, Brucellosis, 54, 105 298 Brugia spp., 288, 297 Capnocytophaga spp., 5, 24, 260, Brugia malayi, 16, 75, 317 346 Brugia timori, 16, 75 di­ag­nos­tic tests, 164 Buffered char­coal-yeast ex­tract dif­fer­en­tial char­ac­ter­is­tics of, (BCYE) agar, 143 193 Bunyamwera vi­rus, 12 Carbapenems, 345, 346, 348, 350, Bunyaviruses, 40, 221, 226 351, 352, 353 Burkholderia, 7 Cardiobacteriaceae, 191 Burkholderia cepacia, 150, 163 Cardiobacterium spp., 8, 164 Burkholderia cepacia com­plex, Cardiobacterium hominis, 24, 23, 131, 346 347 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Catheter, spec­i­men col­lec­tion and methods­ for, 334 trans­port, 87, 103–104 in­trin­sic re­sis­tance of, 355 Cat scratch fe­ver, 105 Burkholderia cepacia se­lec­tive Cedecea, 7 agar (BCSA), 144 Cefamandole, 143, 337 Burkholderia pseudomallei, 46, Cefoperazone, 144, 338 131, 141, 163, 346 Cefsulodin-Irgasan-novobiocin Burn, spec­i­men col­lec­tion and (CIN) agar, 144 trans­port, 87 Cell lines, vi­ral iso­la­tion, 224 Butyrivibrio fibrisolvens, 23 Cellulitis, spec­i­men col­lec­tion and trans­port, 88 Cellulomonas, 4 C Centers for Disease Control and Calcofluor white stain, 246, 290 Prevention (CDC), 293, Calicivirus,44, 221, 226 298, 299, 300, 324 Cal­i­for­nia en­ceph­a­li­tis vi­rus, 12, Central ner­vous sys­tem 226 in­fec­tions, 38–41 Campylobacter spp., 7, 23, 24, 44, spec­i­men guide­lines for par­a­si­ 94, 131, 140, 143, 152 tol­ogy, 118–119 di­ag­nos­tic tests, 163–164 Cephalosporins, 345, 346, 347, dif­fer­en­tial char­ac­ter­is­tics of, 349, 350, 353 212 Cephalothin, 144 se­lec­tive me­dium, 144 Cervicitis, 45 Campylobacter coli, 335 Cestodes Campylobacter fe­tus, 38, 346 de­tec­tion meth­ods, 289, Campylobacteriosis, 54 300–301 Campylobacter jejuni, 23, 335, mor­pho­log­i­cal char­ac­ter­is­tics of, 346 318–319 Candida spp., 24, 34, 35, 36, 37, path­o­gens, 79–80 38, 39, 41, 42, 43, 46, 47, Chaetomium, 279 52, 53, 54, 61, 116, 244, Chagas dis­ease, 316 254, 255 Chancroid, 54 cul­tural and bio­chem­i­cal char­ac­ Chickungunya vi­rus, 11, 34, 56 ter­is­tics, 264–265 Chikungunya vi­rus, 225 Index 387 Chilomastix mesnili, 14, 24, 71, Clostridium bot­u­li­num, 54, 131, 312 169, 347 cysts of, 311 Clostridium dif­fi­cile, 44, 107, 135, tro­pho­zo­ites of, 309 143, 347 Chlamydia, 5, 54, 102, 140 di­ag­nos­tic tests, 169 Chlamydia trachomatis, 42, 45, spec­i­men col­lec­tion and trans­ 46, 47, 50, 54, 96, 132, 140, port, 92, 93 347 Clostridium perfringens, 36, 43, di­ag­nos­tic tests, 172–173 44, 131, 169–170, 347 Chlamydophila spp., 5, 36, 50 Clostridium tetani, 55, 170, 347 Chlamydophila pneumoniae, 48, Coagulase neg­a­tive staph­y­lo­cocci 49, 50, 132, 173, 347 (CONS), 157 Chlamydophila psittaci, 35, 55, Coccidioides spp. 132, 173, 347 char­ac­ter­is­tics of, 273, 274 Chocolate agar, 145 di­ag­nos­tic tests, 259–260 Cholera, 54 di­rect ex­am­i­na­tion, 250 Chopped-meat broth, 145 Coccidioides immitis, 34, 35, 37, CHROMagar Candida, 254 39, 41, 46, 51, 53, 57, 63 Chromista, 15 Coccidioides posadasii, 63 Chromogenic me­dia, 144–145 Coccidioidomycosis, 57, 259–260 Chronic men­in­gi­tis, 39–40 Colistin-nalidixic acid (CNA) Chryseobacterium, 5 agar, 145 Chryseomonas, 8 Col­o­rado tick fe­ver vi­rus, 12, 38 Ciliates, path­o­gens, 73 Coltivirus, 12 Citrobacter spp., 7, 37, 51, 197 Co­lum­bia agar, 145–146 Citrobacter freundii, 24, 347, Comamonas spp., 7, 205 354 Community-acquired pneu­mo­nia, Citrobacter koseri, 24, 347, 354 50–51 Cladophialophora spp., 41, 70, Conidiobolus, 272 282 Conidiobolus coronatus, 67 Cladosporium spp., 70, 282 Conjunctivitis, 42 Clindamycin, 346 Coriobacteria, 4 Clinical and Laboratory Standards Coronavirus, 11, 44, 48, 49, 50, Institute (CLSI), 324, 221, 226–227 330–331 Corynebacterium spp., 3, 24, 25, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 35, 43, 47 meth­ods, 332–336 an­ti­mi­cro­bial sus­cep­ti­bil­ity test in­ter­pre­tive cri­te­ria for tested meth­ods for, 334 ­an­ti­mi­cro­bi­als, 364–373 di­ag­nos­tic tests, 159 in­trin­sic re­sis­tance of Gram-​ dif­fer­en­tial char­ac­ter­is­tics of, negative bac­te­ria, 354–355 182 in­trin­sic re­sis­tance of Gram-​ Corynebacterium diphtheriae, 25, positive bac­te­ria, 356–357 36, 42, 48, 52, 55, 135, 146, or­gan­isms, 361, 362–363 149, 153–154, 347 Clinical Microbiology Laboratory, di­ag­nos­tic tests, 159 2 Corynebacterium jeikeium, 25, Clinical Virology Manual 347 (Specter), 220 Corynebacterium kroppenstedtii, Clonorchis sinensis, 17, 78, 319, 25, 46 289, 299, 320 Corynebacterium macginleyi, 25, Clostridium spp., 6, 24, 41, 47, 52, 42 131, 140 Corynebacterium ulcerans, dif­fer­en­tial char­ac­ter­is­tics of, 25, 48 214 Corynebacterium urealyticum, 25, in­trin­sic re­sis­tance of, 357 53, 54, 347 388 Index Coryneform bac­te­ria, dif­fer­en­tial Cyclospora spp., 120 char­ac­ter­is­tics of, 183 Cyclospora cayetanensis, 15, 45, Cowpox vi­rus, 10 57, 73 Coxiella spp., 8, 106, 132, 173–174 di­ag­nos­tic tests, 287, 292–293 Coxiella burnetii, 35, 40, 46, 50, mor­pho­log­i­cal char­ac­ter­is­tics 55, 348 of, 313 Coxsackievirus, 36, 39, 48, 110, Cyclosporiasis, 57 221, 227 Cysticercus cellulosae, 43 Creutz­feldt-Jakob agent, 223, 239 Cystine tellurite blood agar, 146 Cri­me­an-Congo hem­or­rhagic Cystitis, 53–54 fe­ver vi­rus (CCFV), 12, 56, Cystoisospora spp., 120 60, 226 Cystoisospora (Isospora) bel­li, 15, Cronobacter spp., 7, 198 73 Cryptococcosis, 260 Cysts Cryptococcus spp., 61 of flag­el­lates, 311, 312 Cryptococcus albidus, 25 in­tes­ti­nal ame­bae, 305–307, 308 Cryptococcus gattii, 39, 51, 61 Cytomegalovirus, 10, 38, 44, 45, Cryptococcus neoformans, 35, 36, 46, 49, 51, 54, 108, 109, 37, 39, 40, 41, 46, 51, 54, 110, 222, 233 61, 242, 244, 247, 254 di­ag­nos­tic tests, 260 di­rect ex­am­i­na­tion, 248 D Cryptoisospora (Isospora) bel­li, Dactylaria constricta var. 45 constricta, 283–284 Cryptosporidiosis, 57 Decubitis ul­cer, spec­i­men Cryptosporidium spp., 120, 124 col­lec­tion and trans­port, 89 Cryptosporidium parvum, 15, 45, Delafield’s hematoxylic stain, 57, 73 290 di­ag­nos­tic tests, 287, 292 Delftia spp., 7, 205 mor­pho­log­i­cal char­ac­ter­is­tics Deltaretrovirus, 12 of, 313 Dematiaceous fun­gi Cryptosporidium viatorum, 73 spec­i­men col­lec­tion and trans­ CSF (ce­re­bro­spi­nal flu­id) port, 242 spec­i­men col­lec­tion and trans­ with macroconidia, 278–279 port, 88–89, 115 with small conidia,­ 280–281 vi­rol­ogy guide­lines, 108–109 Dengue vi­rus, 11, 34, 40, 56, Culture 227–228 bac­te­ria de­tec­tion, 129–132 Dental cul­ture, spec­i­men fungi de­tec­tion, 245 col­lec­tion and trans­port, par­a­site de­tec­tion, 287–289 89–90 Cunninghamella, 272 Department of Health and Human Cunninghamella bertholletiae, 67 Services (HHS), 21 Cupriavidis, 7 Dermabacter, 4 Curved bac­te­ria, de­tec­tion Dermabacter hominis, 25 meth­ods, 132, 170–172 Dermacoccus, 4 Curvularia spp., 67, 279 Dermacoccus nishinomiyaensis, Cutaneous spec­i­men col­lec­tion 25 and trans­port, 113 Dermatophytes, 242–243, 252 Cutaneous ul­cers, spec­i­men Dermatophyte test me­dium guide­lines for par­a­si­tol­ogy, (DTM), 254 119 Desulfomonas pigra, 25 Cycloheximide, 143, 144 Detection meth­ods Cycloserine-cefoxitin-egg clin­i­cal spec­i­mens for bac­te­ria, yolk-fructose agar (CCFA), 129–132 146 par­a­sites, 287–289 Index 389 Diagnostic Medical Parasitology Double-stranded, en­vel­oped RNA 4th edit­ion, 286 vi­ruses, 12 Diagnostic tests Double-stranded, nonenveloped fungi, 258–262 DNA vi­ruses, 9 par­a­sites, 291–301 Doxycycline, 345, 346, 347, 348, path­o­gens, 157–174 352, 353 Dialysis-associated peri­to­ni­tis, 47 Dracunculus medinensis, 16, 76, Dicrocoelium dendriticum, 288, 298 17, 78 Drechslera, 278 Dientamoeba fragilis, 14, 71, 124, Drug class 125, 312 an­ti­fun­gal agents, 342 cysts of, 311 an­ti­mi­cro­bial agents, 337–341 di­ag­nos­tic tests, 287, 293 an­ti­par­a­sitic agents, 343–344 tro­pho­zo­ites of, 304, 309 Dubos broth, 155 Dietzia, 3 Dysgonomonas spp., 4, 25, 193 Dimorphic fungi, spec­i­men col­lec­tion and trans­port, 243 E Dimorphic molds, 273–274 Ear, spec­i­men col­lec­tion and Dipetalonema streptocerca, 122 trans­port, 90–91, 113 Diphtheria, 55 East Af­ri­can sleep­ing sick­ness, Diphtheria-tetanus-pertussis, 316 im­mu­ni­za­tion sched­ule, Eastern equine en­ceph­a­li­tis (EEE) 325 vi­rus, 11, 40, 56, 225 Diphyllobothrium spp., 120 Ebo­la vi­rus, 11, 40, 54, 226 Diphyllobothrium latum, 17, 45, Ebo­la-like vi­rus, 11 79, 320 Echinococcus spp., 48, 118, 121 mor­pho­log­i­cal char­ac­ter­is­tics Echinococcus granulosus, 17, 80, of, 319 289, 300, 319 par­a­sitic di­ag­no­sis, 289, 300 Echinococcus multilocularis, 17, Dipylidium caninum, 17, 79, 289, 80, 121, 289, 300 300 Echinococcus vogeli, 80 Direct fluo­res­cent-antibody stain, Echinostoma hortense, 78 140, 290 Echoviruses, 36, 39, 227 Dirofilaria spp., 121 Edwardsiella spp., 7, 154 Dirofilaria immitis, 16, 76 Edwardsiella tarda, 44 Dirofilaria repens, 122 Eggerthella, 4 Diseases, ar­thro­pod vec­tors of, Eggerthella lenta, 25 58–60 Egg yolk agar, 146 Disseminated in­fec­tions with Ehrlichia spp., 7, 39, 106, 132, 174 cu­ta­ne­ous man­i­fes­ta­tions, Ehrlichia chaffeensis, 348 53 Ehrlichia ewingii, 348 DNA vi­rus­es Ehrlichiosis, 55, 106 de­tec­tion meth­ods, 222–223 Eikenella spp., 7, 130, 164 diag­ noses­ of, 232–234, 238–239 Eikenella corrodens, 25, 34, 348 dou­ble-stranded, en­vel­oped, Elizabethkingia, 5 9–10 Ellinghausen-McCullough-​ dou­ble-stranded, nonenveloped, Johnson-Harris me­dium, 9 146–147 single-stranded,­ nonenveloped, 9 Emmonsia crescens, 251 Dolosicoccus, 5 Empedobacter, 5 Dolosigranulum, 5 Empyema, 50 Donovanosis, 105 Encephalitis, 40, 56, 59 Double-stranded, en­vel­oped DNA Encephalitozoon spp., 42 vi­ruses, 9–10 Endocarditis, 35 390 Index Endolimax nana, 15, 25, 71, 308 Enteromonas hominis, 312 cysts of, 307 cysts of, 311 tro­pho­zo­ites of, 303 tro­pho­zo­ites of, 310 Endophthalmitis, 43 Enterovirus, 10, 40, 42, 49, 51, Entamoeba bangladeshi, 71 109, 110, 221, 227 Entamoeba coli, 15, 26, 71, 308 Entomophthoromycotina, 13 cysts of, 306 Eosin-methylene blue (EMB) agar, tro­pho­zo­ites of, 303 147 Entamoeba dispar, 15, 71, 125, Epidermophyton floccosum, 26, 287, 293 64, 276 Entamoeba gingivalis, 15, 26, 71 Epsilonproteobacteria, 7 Entamoeba hartmanni, 15, 26, 71, Ep­stein-Barr vi­rus, 10, 38, 46, 48, 308 109, 222 cysts of, 305 di­ag­no­sis of, 233–234 tro­pho­zo­ites of, 302 se­ro­logic pro­files, 235 Entamoeba histolytica, 15, 37, 45, Erwinia, 7 48, 71, 121, 122, 124, 125, Erysipelothrix spp., 6, 159 308 Erysipelothrix rhusiopathiae, 35, cysts of, 305 52, 348 di­ag­nos­tic tests, 287, 293 an­ti­mi­cro­bial sus­cep­ti­bil­ity test tro­pho­zo­ites of, 302 meth­ods, 335 Entamoeba polecki, 15, 71, 308 in­trin­sic re­sis­tance of, 357 cysts of, 306 Erysipelotrichia, 6 tro­pho­zo­ites of, 303 Erythromycin, 346, 347 Enterobacter spp., 7, 26, 37, 51, Erythrovirus, 9 53, 54, 147, 149, 198 Erythrovirus (B19 vi­rus), 234 Enterobacter aerogenes, 348 Escherichia, 7, 41, 147, 149 Enterobacter clo­a­cae, 348, 354 Escherichia coli, 26, 34, 37, 38, Enterobacteriaceae, 140, 144 44, 47, 48, 51, 53, 54, 130, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 349 methods­ for, 333 di­ag­nos­tic tests, 164 com­monly tested an­ti­mi­cro­bi­als, spec­i­men col­lec­tion and trans­ 364–366 port, 93 dif­fer­en­tial char­ac­ter­is­tics of, Esophagitis, 44 195–196 Eubacterium spp., 26 Enterobius vermicularis, 16, 76, Eumycotic my­ce­toma agents, 243 320 Eu­ro­pean Committee on mor­pho­log­i­cal char­ac­ter­is­tics Antimicrobial Susceptibil- of, 318 ity Testing (EUCAST), 324 par­a­sitic di­ag­no­sis, 288, 298 in­ter­pre­tive cri­te­ria for tested Enterococcosel agar, 147 ­an­ti­mi­cro­bi­als, 364–373 Enterococcus spp., 5, 35, 38, 47, or­gan­isms in breakpoint ta­bles, 48, 51, 54, 129 361, 362–363 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Eustrongyloides spp., 76 methods­ for, 332 Ewingella americana, 26 com­monly tested an­ti­mi­cro­bi­als, Exophiala spp., 41, 46, 47, 281 370–371 Exophiala dermatitidis, 281 di­ag­nos­tic tests, 157 Exophiala jeanselmei, 68 dif­fer­en­tial char­ac­ter­is­tics of, Exserohilum, 278 180 Eye Enterococcus faecalis, 26, 43, 348, Gram stain and plating­ media,­ 356 133 Enterococcus faecium, 26, 348, in­fec­tions, 42–44 356 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 Enterococcus gallinarum, 26, 357 my­col­ogy plat­ing guide, 256 Index 391 spec­i­men col­lec­tion and trans­ char­ac­ter­is­tic el­e­ments by di­rect port, 91–92, 114 ex­am­i­na­tion, 248–253 spec­i­men guide­lines for par­a­si­ di­ag­nos­tic tests, 258–262 tol­ogy, 119 meth­ods for iden­ti­fi­ca­tion of, vi­rol­ogy guide­lines, 110 245 my­col­ogy plat­ing guide, 256–257 F op­por­tu­nis­tic moniliaceous, Facklamia, 5 269–270 Fasciola gigantica, 17 pri­mary plat­ing me­dia, 254–255 Fasciola he­pat­i­ca, 17, 78, 289, tax­o­nomic clas­si­fi­ca­tion of, 13 299, 319, 320 Fusarium spp., 37, 43, 47, 53, 69, Fasciolopsis buski, 17, 78, 299, 243, 252 320 Fusobacterium spp., 6, 26, 27, 47, Feces 48, 131, 170, 349 spec­i­men col­lec­tion and trans­ port, 92–94 vi­rol­ogy guide­lines, 110 G Fibribacteria, 5–6 Galactomannan, bio­marker of Filovirus, 40, 221, 227 fun­gal in­fec­tion, 262–263 Finegoldia, 6 Gammaproteobacteria, 7–8 Finegoldia magnus, 26 Gangrenous cel­lu­li­tis, 52 Flagellates Gangrenous tis­sue, spec­i­men cysts of, 311, 312 col­lec­tion and trans­port, 95 path­o­gens, 71–72 Gardnerella, 4 tro­pho­zo­ites of, 309–310, 312 Gardnerella vaginalis, 27, 46, 159 Flavimonas, 8 Gastric, spec­i­men col­lec­tion and Flaviviruses, 36, 40, 221 trans­port, 95 Flavobacterium, 5 Gastric wash­ing, my­co­bac­te­rial Fletcher me­dium, 147 iden­ti­fi­ca­tion, 138 Fluids Gastrodiscoides hominis, 78 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 Gastrointestinal in­fec­tions, 44–45 my­col­ogy plat­ing guide, 256 Gastrointestinal tract, Gram stain spec­i­men col­lec­tion and trans­ and plat­ing me­dia, 133 port, 94, 114, 115 Gemella spp., 5, 27 Fluorescent-antibody stain, fun­gal Genital di­ag­no­sis, 246 fe­male spec­i­men col­lec­tion and Fluoroquinolones, 350, 352, 353 trans­port, 95–98 Fonsecaea spp., 46 Gram stain and plat­ing me­dia, 134 Fonsecaea pedrosoi, 280 in­fec­tions, 45–46 Foot-and-mouth dis­ease vi­rus, 10 male spec­i­men col­lec­tion and Francisella spp., 8, 106, 131, 140, trans­port, 98–99 143, 164–165 ul­cers and skin nod­ules, 45 Francisella tularensis, 42, 46, 52, vi­rol­ogy guide­lines, 110–111 55, 60, 135, 349 Geographic dis­tri­bu­tion Free-living ame­bae fun­gal path­o­gens, 61–70 de­tec­tion meth­ods, 287 par­a­sitic path­o­gens, 71–80 di­ag­nos­tic tests, 291–292 Geotrichum spp., 27, 61 Fungal path­o­gens, and geo­graphic Giardia spp., 120, 125 dis­tri­bu­tion, 61–70 Giardia duodenalis, 44, 72 Fungi Giardia lamblia, 14, 57, 124, 290, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 312 meth­ods for, 336 cysts of, 311 bio­mark­ers of in­va­sive in­fec­tion, di­ag­nos­tic tests, 287, 293 262–263 tro­pho­zo­ites of, 309 392 Index Giardiasis, 57 Guanarito vi­rus, 56 Gi­emsa stain, 246, 290 Guizotia abyssinica, 254 Gliocladium, 269 Globicatella, 5 Gnathostoma spp., 76 H Gnathostoma spinigerum, 16 HACEK group, 35, 335 Gonorrhea, 55 Haemophilus spp., 8, 27, 145 Gordonia, 3, 139 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Gram-negative ba­cilli, 20 meth­ods for, 333 Gram-negative bac­te­ria dif­fer­en­tial char­ac­ter­is­tics of, 194 dif­fer­en­tial char­ac­ter­is­tics of Haemophilus test me­dium (HTM) an­aer­o­bic, 215 agar, 147–148 in­trin­sic re­sis­tance of, 354–355 Haemophilus aegyptius, 42 Gram-negative broth, 147 Haemophilus aphrophilus, 349 Gram-negative cocci, aer­o­bic, Haemophilus ducreyi, 45, 52, 54, de­tec­tion meth­ods, 130, 136, 165, 349 161 Haemophilus influenzae, 38, 41, Gram-negative rods 49, 50, 51, 55, 130, 349 dif­fer­en­tial char­ac­ter­is­tics of di­ag­nos­tic tests, 165 ox­i­dase-negative, ox­i­da­tive, im­mu­ni­za­tion sched­ule for type 207 b, 325 dif­fer­en­tial char­ac­ter­is­tics of ox­ Hafnia, 8 i­dase-positive, nonfermen- Hafnia alvei, 27, 349, 354 tative, 208–209 Hajna, 152 dif­fer­en­tial char­ac­ter­is­tics of ox­ Hansen dis­ease, 55 i­dase-positive, nonoxida- Hantaan vi­rus, 12, 228 tive, 210–211 Hantavirus, 12, 221, 228 Gram-negative rods, aer­o­bic, Hantavirus pul­mo­nary syn­drome, de­tec­tion meth­ods, 56 130–131, 161–168 Hartland vi­rus, 226 Gram-positive bac­te­ria, in­trin­sic Ha­ver­hill fe­ver, 106 re­sis­tance of, 356–357 Hektoen en­teric agar, 148 Gram-positive coc­ci Helcobacillus, 4 dif­fer­en­tial char­ac­ter­is­tics of Helcococcus, 6 cat­a­lase-negative, 177 Helcococcus kunzii, 27 dif­fer­en­tial char­ac­ter­is­tics of Helicobacter spp., 7, 27, 140, 212 cat­a­lase-positive, 175 Helicobacter py­lo­ri, 95, 131, 136 Gram-positive cocci, aer­o­bic, an­ti­mi­cro­bial sus­cep­ti­bil­ity test de­tec­tion meth­ods, 129, meth­ods, 335 157–158 di­ag­nos­tic tests, 165–166 Gram-positive rods Helminthosporium, 278 ac­id-fast, de­tec­tion meth­ods, Helminths 129, 160 de­tec­tion meth­ods, 288–289, aer­o­bic, de­tec­tion meth­ods, 129, 297–302\301 158–160 mor­pho­log­i­cal char­ac­ter­is­tics of, dif­fer­en­tial char­ac­ter­is­tics, 181 318–319 dif­fer­en­tial char­ac­ter­is­tics of rel­a­tive sizes of eggs, 320 an­aer­o­bic, 213 Hemolytic ure­mic syn­drome, 55 Gram stain, 140 Hendra vi­rus, 12 fun­gal di­ag­no­sis, 246–247 Henipavirus, 12 rec­om­men­da­tions, 133–136 Hepacivirus, 11, 228 Granulicatella spp., 5, 27, 145, 334 Hepatitis A vi­rus, 38, 56, 221, Granulicatella adiacens, 35 228–229 Granuloma in­gui­nale, 105 adult im­mu­ni­za­tion sched­ule, 328 Granulomatous in­fec­tions, 46–47 im­mu­ni­za­tion sched­ule, 327 Index 393 Hepatitis B vi­rus, 34, 38, 56, 222, Human pap­il­lo­ma­vi­rus, 9, 238 234, 236 adult im­mu­ni­za­tion sched­ule, adult im­mu­ni­za­tion sched­ule, 329 328 im­mu­ni­za­tion sched­ule, 327 im­mu­ni­za­tion sched­ule, 325 Human para­in­flu­enza vi­rus, 12 Hepatitis C vi­rus, 11, 38, 56, 221, Human par­vo­vi­rus B19 vi­rus, 9 228 Human re­spi­ra­tory syn­cy­tial vi­rus Hepatitis D vi­rus, 38 (RSV), 12 Hepatitis E vi­rus, 221, 229 Human T-cell leu­ke­mia vi­rus, 38 Hepatovirus, 10 Human tis­sue, cells for vi­ral Herbaspirillum, 7 iso­la­tion, 224 Herpes sim­plex vi­rus, 39, 44, 45, Human T-lymphotropic vi­rus type 48, 94, 109, 110, 111 1 (HTLV–1), 12, 221, 229 Herpes sim­plex vi­ruses (type I and Human T-lymphotropic vi­rus type II), 223, 234, 238 2 (HTLV–2), 12, 221, 229 Herpesvirus, 36, 40, 42, 43, 51 Human torovirus, 11 Herpes zos­ter, adult im­mu­ni­za­tion Human vi­ruses, tax­o­nomic sched­ule, 329 clas­si­fi­ca­tion of, 9–12 Heterophyes heterophyes, 17, 78, Hydrogen sul­fide, 148 320 Hymenolepis spp., 120 Histoplasma spp., 242, 255 Hymenolepis diminuta, 17, 80, 320 Histoplasma capsulatum, 35, 37, mor­pho­log­i­cal char­ac­ter­is­tics 39, 40, 43, 45, 46, 51, 63, of, 319 243, 246 par­a­sitic di­ag­no­sis, 289, 300 char­ac­ter­is­tics of, 273, 274 Hymenolepis nana, 17, 80, 289, di­ag­nos­tic tests, 260–261 301, 320 di­rect ex­am­i­na­tion, 248 Hyphomycetes, 49 Histoplasmosis, 260–261 Hookworm, 320 Hortaea werneckii, 281 I Hospital-acquired pneu­mo­nia, 51 Iadomoeba butschlii, 308 Human ad­e­no­vi­ruses, 9 Immunization sched­ule Human astrovirus, 11 adult, 328–329 Human co­ro­na­vi­rus, 11 pe­di­at­ric, 325–327 Human gran­u­lo­cytic ehr­lich­i­o­sis, In­dia ink stain, fun­gal di­ag­no­sis, 105 247 Human hep­a­ti­tis A vi­rus (HHAV), Indirect hem­ag­glu­ti­na­tion (IHA), 10 293, 300 Human her­pes­vi­rus 1 (HHV–1), 10 Infectious Diseases Society of Human her­pes­vi­rus 2 (HHV–2), Amer­ica (IDSA), 324 10 Inflammatory di­ar­rhea, 44–45 Human her­pes­vi­rus 3 (HHV–3), 10 Influenza Human her­pes­vi­rus 4 (HHV–4), 10 adult im­mu­ni­za­tion sched­ule, 328 Human her­pes­vi­rus 5 (HHV–5), 10 im­mu­ni­za­tion sched­ule, 327 Human her­pes­vi­rus 6 (HHV–6), Influenza-associated pe­di­at­ric 10, 108, 223, 238 mor­tal­ity, 56 Human her­pes­vi­rus 7 (HHV–7), 10 Influenza A vi­rus, 11, 36, 37, 56, Human her­pes­vi­rus 8 (HHV–8), 10 221, 229–230 Human im­mu­no­de­fi­ciency vi­rus, Influenza B vi­rus, 11, 36, 37, 221, 34, 38, 39, 40, 44, 49 229–230 Human im­mu­no­de­fi­ciency vi­rus Influenza C vi­rus, 11, 221, type 1 (HIV–1), 12, 229 229–230 Human im­mu­no­de­fi­ciency vi­rus Influenza vi­rus, 42, 48, 49, 50, 51 type 2 (HIV–2), 12, 229 Inhibitory mold agar (IMA), Human in­dig­e­nous flora, 22–33 254–255 394 Index Inquilinus, 6 K Insecta, 58–59 Kanamycin-vancomycin laked International Air Transport blood agar, 148 Association (IATA), Keratitis, 43–44 Dangerous Goods Kidney, cells for vi­ral iso­la­tion, Regulations, 83 224 International Civil Aviation Kingella spp., 7, 27, 130, 166 Organization (ICAO), Kingella kingae, 34, 349 83 Kinyoun stain, fun­gal di­ag­no­sis, International Code of Botanical 247 Nomenclature (ICBN), 2, Klebsiella spp., 8, 27, 37, 38, 41, 13 53, 147, 149, 199 International Code of Nomencla- Klebsiella granulomatis, 45, 105, ture (ICN), 13 349 International Code of Nomencla- Klebsiella oxytoca, 350 ture of Bacteria, 2 Klebsiella ozaenae, 350 International Committee on Klebsiella pneumoniae, 47, 50, 51, Taxonomy of Viruses 54, 350, 354 (ICTV), 2 Kluybera, 8 International Journal of System- Kocuria spp., 4, 27 atic and Evolutionary Kosakonia spp., 198 Microbiology, 3 Kuru agent, 223, 239 Intestinal pro­to­zoa Kytococcus, 4 cysts of in­tes­ti­nal ame­bae, Kytococcus sedantarius, 27 305–307, 308 de­tec­tion meth­ods, 287 di­ag­nos­tic tests, 292–294 L tro­pho­zo­ites of in­tes­ti­nal La Crosse vi­rus, 12, 40, 56, 226 amebae,­ 302–304, 308 ß-Lactam-ß-lactamase in­hib­i­tors, Intestinal tract, spec­i­men 349 guide­lines for par­a­si­tol­ogy, Lactobacillus spp., 5, 27, 350 120 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Intra-abdominal in­fec­tions, meth­ods, 335 47–48 in­trin­sic re­sis­tance of, 357 Invasive pneu­mo­coc­cal Lactococcus spp., 6, 27 dis­ease, 55 Larone, D. H., 263 Iodamoeba buetschlii, 15 Laryngitis, 49 Iodamoeba butschlii, 71 Laryngotracheobronchitis, 49 cysts of, 307 Lassa vi­rus, 12, 40, 56, 225 tro­pho­zo­ites of, 304 Leclercia, 8 Iron he­ma­tox­y­lin stain, 290 Leclercia adeocarboxylata, 27 Isospora bel­li Lecythophora, 270 di­ag­nos­tic tests, 287, 293–294 Legionella spp., 8, 50, 55, 136, mor­pho­log­i­cal char­ac­ter­is­tics 140, 143, 166 of, 314 Legionella micdadei, 139 Legionella pneumophila, 350 Legionellosis, 55 J Leifsonia, 4 James ­town Canyon vi­rus, 56 Leishmania spp., 14, 38, 47, 52, Jap­a­nese en­ceph­a­li­tis (JE) vi­rus, 72, 117, 118, 121 11, 40 di­ag­nos­tic tests, 287, 294 JC poly­oma­vi­rus, 9 mor­pho­log­i­cal char­ac­ter­is­tics, JC vi­rus, 109, 223, 238–239 316 Joint in­fec­tions, 34–35 Leishmania braziliensis, 14, 44, Ju­nin vi­rus, 12, 56, 225 316 Index 395 Leishmania donovani, 42, 316 Mansonella perstans, 16, 76 Leminorella spp., 27 mor­pho­log­i­cal char­ac­ter­is­tics Lentivirus, 12 of, 317 Leprosy, 55 par­a­sitic di­ag­no­sis, 288, 298 Leptospira spp., 9, 39, 40, 106, Mansonella streptocerca, 76 132, 136, 147 Mar­burg-like vi­ruses, 11 di­ag­nos­tic tests, 171 Mar­burg vi­rus, 11, 40, 56, 227 Leptospira interrogans, 53, 350 Martin-Lewis agar, 149 Leptospirosis, 55, 106 Mastadenovirus, 9 Leptotrichia, 6 Matrix-assisted la­ser de­sorp­tion/ Leptotrichia bucalis, 28 ionization time-of-flight Leuconostoc spp., 5, 28, 350, 357 mass spec­trom­e­try Lichtheimia, 271 (MALDI-TOF MS), 20 Lichtheimia (Absidia) corymbifera, Measles-mumps-rubella (MMR) 66 adult im­mu­ni­za­tion sched­ule, LIM broth, 148 329 Listeria spp., 5, 129, 159–160 im­mu­ni­za­tion sched­ule, 326 Listeria monocytogenes, 28, 38, 40, Measles vi­rus, 12, 39, 40, 42, 43, 41, 46, 53, 55, 150, 333, 350 45, 46, 51, 56, 221, 230 Listeriosis, 55 Mechanisms of re­sis­tance, Liver, spec­i­men guide­lines for bac­te­ria, 358–360 par­a­si­tol­ogy, 121 Medically Important Fungi, a Loa loa, 16, 42, 76, 119 Guide to Identification mor­pho­log­i­cal char­ac­ter­is­tics (Larone), 263 of, 317 Megasphaera elsdenii, 28 par­a­sitic di­ag­no­sis, 288, 298 Meningococcal dis­ease, 55 Loff­ler me­dium, 149 Meningococcal po­ly­sac­cha­ride, Lowenstein-Gruft mod­i­fi­ca­tion, 155 adult im­mu­ni­za­tion Lowenstein-Jensen (LJ) me­dium, sched­ule, 329 155 Metagonimus yokogawai, 78, 320 Lugol’s io­dine stain, 291 Metapneumovirus, 12 Lujo vi­rus, 56 Metorchis conjunctus, 78 Lyme dis­ease, 55, 59, 105 Metronidazole, 345, 347, 349, 351 Lymphocryptovirus, 10 Microbacter, 4 Lymphocytic cho­rio­men­in­gi­tis Microbacterium, 4 (LCM) vi­rus, 12, 34, 40, Micrococcus spp., 4, 28, 335 225 Microscopy Lyssavirus, 11 bac­te­ria de­tec­tion, 129–132 fun­gal di­ag­no­sis, 246–247 fungi de­tec­tion, 245 M meth­ods, 139–141 McBride Listeria agar, 150 par­a­site de­tec­tion, 287–289 McClung-Toabe agar, 146 Microsporidia, 44 MacConkey agar, 149 de­tec­tion meth­ods, 288, 297 Machupo vi­rus, 12, 40, 56, 225 mor­pho­log­i­cal char­ac­ter­is­tics Macrolides, 346, 350 of, 314 Malaria, 57, 117 Microsporum spp., 28, 64, 65, Malaria par­a­sites, mor­pho­log­i­cal 276–277 char­ac­ter­is­tics of, 315 Middlebrook 7H9 broth, 155–156 Malassezia spp., 28, 37, 38, 41, 62, Middlebrook 7H10 broth, 156 256, 257 Middlebrook 7H11 broth, 156 di­ag­nos­tic tests, 261 Middlebrook 7H13 broth, 156 di­rect ex­am­i­na­tion, 250 Middle Eastern re­spi­ra­tory Mannitol salt agar, 149 syn­drome (MERS), 11, Mansonella ozzardi, 16, 76, 317 226–227 396 Index Mitchison’s mod­i­fi­ca­tion of Mycobacterium avium com­plex, Middlebrook 7H11 broth, 39, 48, 129, 160 156 Mycobacterium bovis, 156 Mitsuokella multiacidus, 28 Mycobacterium intracellulare, 156 Mobiluncus spp., 3, 28, 46, 131 Mycobacterium leprae, 55 di­ag­nos­tic tests, 170 Mycobacterium marinum, 34, 52 Modified ac­id-fast stain, 291 Mycobacterium tu­ber­cu­lo­sis, 34, Modified Thayer-Martin (MTM) 36, 39, 40, 41, 45, 48, 50, agar, 149, 153 53, 54, 55, 129, 160 Moellerella wisconsensis, 28 Mycobacterium ulcerans, 45 Molds Mycobactosel mod­i­fi­ca­tion, LJ an­ti­mi­cro­bial sus­cep­ti­bil­ity test me­dium, 155 meth­ods for, 336 Mycology di­mor­phic, 273–274 col­lec­tion and trans­port guide­ Molecular di­ag­nos­tics lines, 112–116 bac­te­ria, 129–132 plat­ing guide, 256–257 par­a­sites, 287–289 spec­i­men col­lec­tion and trans­ Mollicutes, 9 port, 242–244 Molluscipoxvirus, 10, 45 Mycoplasma spp., 9, 28, 29, 140 Molluscum contagiosum vi­rus, 10 de­tec­tion meth­ods, 132, Moniliaceous fungi, 243 172–174 Monkeypox vi­rus, 10, 238 Mycoplasma genitalium, 28, 45 Moraxella, 8 Mycoplasma hominis, 28, 34, 46 Moraxella catarrhalis, 28, 42, 49, Mycoplasma pneumoniae, 29, 36, 50, 51, 130, 350 40, 48, 49, 50, 132, 174, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 350 meth­ods, 335 Mycosel (Mycobiotic) agar, 255 di­ag­nos­tic tests, 161 Myocarditis, 36 Morbidity and Mortality Weekly Report, 21, 324 Morbillivirus, 12 N Morganella spp., 8, 200 Naegleria, 287, 292 Morganella morganii, 28, 54, 350, Naegleria fowleri, 14, 39, 40, 71 354 Nails and hair, my­col­ogy plat­ing Mouth, my­col­ogy plat­ing guide, guide, 257 257 Nairovirus, 12 Mucor spp., 67, 271 Nanophyetus salmineola, 78 Mucorales, 51 Necator americanus, 16, 76 Mucormycotina, 13 mor­pho­log­i­cal char­ac­ter­is­tics Mueller-Hinton agar, 150 of, 318 Multidrug re­sis­tance, mech­a­nisms par­a­sitic di­ag­no­sis, 288, 298 in bac­te­ria, 358–360 Negativicutes, 6 Mumps vi­rus, 12, 34, 36, 39, 40, Neisseria spp., 7, 29, 36, 190 46, 56, 109, 221, 230–231 Neisseriaceae, 191 Muscle, spec­i­men guide­lines for Neisseria gonorrhoeae, 34, 42, 43, par­a­si­tol­ogy, 122 45, 47, 48, 54, 55, 94, 96, Myclplasma spermatophilum, 29 102, 130, 136, 149, 351 Mycobacteria spp., 139 an­ti­mi­cro­bial sus­cep­ti­bil­ity test an­ti­mi­cro­bial sus­cep­ti­bil­ity test methods­ for, 333 meth­ods for, 336 di­ag­nos­tic tests, 161 pri­mary plat­ing me­dia, 154–156 Neisseria meningitidis, 29, 36, 38, pro­cess­ing spec­i­mens, 138 45, 50, 55, 130, 351 Mycobacterium spp., 3, 46, 52, 54, an­ti­mi­cro­bial sus­cep­ti­bil­ity test 187–188, 189 meth­ods for, 333 Mycobacterium avium, 156 di­ag­nos­tic tests, 161 Index 397 Nematodes P de­tec­tion meth­ods, 288–289, Paecilomyces spp., 69, 269 297–299 Paenibacillus, 5 mor­pho­log­i­cal char­ac­ter­is­tics Pandoraea, 7 of, 318 Pantoea spp., 8, 29, 198 path­o­gens, 75–77 Papillomavirus, 9, 42, 45, 223, 238 Neobalantidium (Balantidium) Parabacteroides, 4 coli, 45, 73 Parabacteroides distasonis, 29 Neodiplostomum seoulense, 78 Paracoccidioides brasiliensis, 46, Neotestudina rosatii, 68 51, 63 New World ar­e­na­vi­rus, 56 char­ac­ter­is­tics of, 273, 274 New York City agar, 150 di­ag­nos­tic tests, 261 Nipah vi­rus, 12 di­rect ex­am­i­na­tion, 249 Nocardia spp., 3, 39, 41, 46, 50, Paracoccus, 6 52, 53, 129, 136, 139, 143, Paragonimus spp., 121, 289, 351 299–300 di­ag­nos­tic tests, 160 Paragonimus kellicotti, 17 dif­fer­en­tial char­ac­ter­is­tics of, Paragonimus westermani, 17, 51, 186 79, 319, 320 Nocardiopsis, 4 Parainfluenza vi­rus, 48, 49, 50, 51, Nodular le­sions, 52 221, 231 Non-Enterobacteriaceae, 334 Parapoxvirus, 10 Noninflammatory di­ar­rhea, 44 Parasites Norovirus, 10, 226 de­tec­tion meth­ods for, 287–289 Nor­walk vi­rus, 10 di­ag­nos­tic tests, 291–301 Nucleic acid, fungi de­tec­tion, and geo­graphic dis­tri­bu­tion, 245 71–80 pro­cess­ing stool spec­i­mens for, 124–126 O tax­o­nomic clas­si­fi­ca­tion of, Obligate in­tra­cel­lu­lar bac­te­ria, 13–17 de­tec­tion meth­ods, 132, Parasitology, spec­i­men guide­lines, 172–174 117–123 Ochrabactrum, 6 Parastrongylus (Angiostrongylus) Ochroconis gallopava, 283–284 cantonensis, 16, 75 Oerskovia, 4 Parastrongylus (Angiostrongylus) Oligella spp., 7, 29 costaricensis, 16, 75 Onchocerca vol­vu­lus, 16, 42, Parechovirus, 227 43, 77, 122, 288, 298, Parvovirus B19, 34, 38, 108, 223, 317 237 Opisthorchis spp., 17, 78, 320 Pasteurella spp., 8, 29, 53 Orbivirus, 39 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Orf vi­rus, 10 meth­ods, 335 Orientia, 7 di­ag­nos­tic tests, 166 Orientia tsu­tsu­ga­mu­shi, 36, dif­fer­en­tial char­ac­ter­is­tics of, 39, 60 192 Orthobunyavirus, 12 Pasteurella multocida, 34, 351 Orthohepadnavirus, 10 Pediatric im­mu­ni­za­tion sched­ule, Orthopoxvirus, 9, 223, 238 325–327 Osteomyelitis, 34–35 Pediococcus spp., 5, 357 Oxacillin, 352 Penicillin, 345, 346, 347, 348, 350, Oxazolidinones, 348 351, 352, 353, 368, 370, 371 Oxidative-fermentative po­ly­myxin Penicillium spp., 69 B-bacitracin-lactose Pentatrichomonas (Trichomonas) (OFPBL) agar, 150 hominis, 14, 29, 72 398 Index Peptococcus spp., 6, 29 Porphyromonas spp., 4, 30, 148, Peptoniphilus spp., 6, 29 351 Peptostreptococcus spp., 6, 29, 41, Potassium hy­drox­ide (KOH), 47 fun­gal di­ag­no­sis, 247 Pericarditis, 36–37 Prevotella spp., 4, 30, 148, 351 Peritonitis, 47 Primary plat­ing me­dia Pertussis, 55 bac­te­ria, 141–154 Petragnani me­dium, 156 fungi, 254–255 Phaeoannellomyces (Exophiala) my­co­bac­te­ria, 154–156 wemeckii, 65 Primary pyo­der­mas, 52 Phaneropsolus bonnei, 79 Prokaryotes, tax­o­nom­i­cal Pharyngitis, 48–49 clas­si­fi­ca­tion of, 2–3 Phenylethyl al­co­hol (PEA) blood Propionferax innocuum, 31 agar, 150 Propionibacterium spp., 3, 31, 43, 47 Phialemonium, 270 Propionibacterium ac­nes, 31, 39 Phialophora spp., 46 Prostatic fluid, 114 Phialophora verrucosa, 280 Prostatitis, 54 Phlebovirus, 12 Prosthodendrium molenkampi, 79 Phoma, 279 Pro­teus spp., 8, 41, 54, 147, 148, Piedraia hortae, 65 149, 200 Plague, 55, 58 Pro­teus mi­ra­bi­lis, 31, 37, 43, 51, Plasmodium spp., 38, 57, 117, 287, 53, 54, 351, 354 295 Pro­teus penneri, 31, 354 Plasmodium fal­cip­a­rum, 15, 40, Pro­teus vul­ga­ris, 31, 351, 354 73, 315 Protozoa, 14–15, 71–74 Plasmodium knowlesi, 15, 74 Providencia spp., 8, 31, 200, 351 Plasmodium malariae, 15, 74, 315 Providencia stuartii, 31, 200, 355 Plasmodium ova­le, 15, 74, 315 Pseudallescheria boydii, 41, 46, Plasmodium vi­vax, 15, 74, 315 68, 255 Plating me­dia, rec­om­men­da­tions, Pseudomonas spp., 8, 37, 156, 204 133–136 Pseudomonas aeruginosa, 31, 34, Platyhelminthes, 16–17 35, 38, 41, 42, 43, 47, 49, Pleisiomonas, 8 50, 52, 53, 155, 351 Plesiomonas shigelloides, 44, 351 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Pluralibacter spp., 198 meth­ods for, 333 Pneumococcal vac­cine com­monly tested an­ti­mi­cro­bi­als, adult im­mu­ni­za­tion sched­ule, 366–367 328 di­ag­nos­tic tests, 167 im­mu­ni­za­tion sched­ule, 326 in­trin­sic re­sis­tance of, 355 Pneu­mo­cys­tis jiroveci, 51, 62, 121, Pseudozyma spp., 62 243, 246–247 Psittacosis, 55 di­ag­nos­tic tests, 262 Pyelonephritis, 53–54 di­rect ex­am­i­na­tion, 253 Pygidiopsis sum­ma, 79 Pneumovirus, 12 Pyrenochaeta romeroi, 68 Poliomyelitis, 56 Pythium spp., 13, 251 Poliovirus, 36, 222, 227 Poliovirus (in­ac­ti­vated), im­mu­ni­za­tion sched­ule, Q 326 Q fe­ver, 55, 106 Poliovirus in­fec­tion, 56 Polymyxin, 144 Polymyxin B, 143 R Polyomavirus, 238–239 Rabies vi­rus, 11, 40, 56, 222, 231 Polyomavirus (BK vi­rus, JC Ralstonia, 7 vi­rus), 223 Raoultella spp., 8, 199 Index 399 Rat bite fe­ver, 106 Rotavirus, 221 Regan-Lowe agar me­dium, 151 im­mu­ni­za­tion sched­ule, 325 Relapsing fe­ver, 105 vi­ral di­ag­no­sis, 226 Renal cal­culi, 54 Rotavirus (types A, B, C), 12, 44 Respiratory Rothia, 4 Gram stain and plat­ing me­dia, Rothia dentocariosa, 31 134, 135 Rothia mucilaginosa, 31, 35, 352 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 Rubella vi­rus, 11, 34, 36, 40, 42, my­col­ogy plat­ing guide, 257 43, 56, 109, 222, 232 spec­i­men col­lec­tion and trans­ Rubivirus, 11 port, 99–102, 114–115 Rubulavirus, 12 vi­rol­ogy guide­lines, 109 Ruminococcus productus, 31 Respiratory syn­cy­tial vi­rus, 42, Ryan Blue stain, 291 48, 49, 50, 51, 222, 231–232 Respiratory tract S in­fec­tions, 48–51 Sabia vi­rus, 12, 56 spec­i­men guide­lines for par­a­si­ Sabouraud agar-brain heart tol­ogy, 121 in­fu­sion (SABHI), 255 Respirovirus, 11 Sabouraud dex­trose agar (SDA), 255 Retortamonas intestinalis, 14, 31, Saccharomyces spp., 31 312 St. Louis en­ceph­a­li­tis (SLE) vi­rus, cysts of, 311 11, 40, 56, 227–228 tro­pho­zo­ites of, 310 Saksenaea, 272 Rhadinovirus, 10 Saksenaea vasiformis, 67 Rhinocladiella, 280 Salmonella spp., 8, 34, 35, 36, 37, Rhinosporidium, 13 44, 55, 143, 144, 147–149, Rhinosporidium seeberi, 251 152, 154, 352 Rhinovirus, 10, 42, 48, 49, 50, 51, di­ag­nos­tic tests, 167 222, 232 in­trin­sic re­sis­tance of, 355 Rhizobium, 6 Salmonella enterica serovar Rhizomucor, 271 Typhi, 53, 55, 130, 142, Rhizomucor pusillus, 67 167, 352 Rhizopus spp., 67, 271 Salmonella-Shigella (SS) agar, 151 Rhodococcus spp., 3, 129, 139, 160 Salmonellosis, 55 Rhodococcus equi, 41, 50, 352 Sapovirus, 10, 226 Rhodotorula spp., 62 Sap­poro vi­rus, 10 Rickettsia spp., 7, 39, 106, 140, 352 Sarcinia, 6 Rickettsia rickettsii, 36, 40, 55, 60, Sarcocystis spp., 74, 314 132, 174 Sarcocystis hominis, 15 Rifampin, 144 Scedosporium spp., 41 Rift Valley fe­ver vi­rus, 12, 40, 226 Scedosporium spp. com­plex, RNA vi­rus­es 283–284 de­tec­tion meth­ods, 221–222 Scedosporium prolificans, di­ag­noses of, 225–232 283–284 dou­ble-stranded, en­vel­oped, 12 Schaedler’s agar, 151 sin­gle-stranded, neg­a­tive-sense, Schistosoma spp., 37, 45, 47, 120, en­vel­oped, 11–12 289, 300 sin­gle-stranded, pos­i­tive-sense, Schistosoma haematobium, 16, 54, en­vel­oped, 11 79, 123, 319, 320 sin­gle-stranded, pos­i­tive-sense, Schistosoma intercalatum, 16 nonenveloped, 10–11 Schistosoma japonicum, 16, 79, Romanowsky stain, 290 319, 320 Roseola vi­rus, 10 Schistosoma mansoni, 16, 79, 319, Roseomonas, 6 320 400 Index Schistosoma mekongi, 16, 79 in­fre­quently en­coun­tered bac­te­ Scopulariopsis spp., 69, 269 ria, 105–106 Secondeary skin in­fec­tions, 52–53 pro­cess­ing guide­lines for par­a­ Selenite broth, 151 sites, 124–126 Selenomonas spp., 31 Sphingobacteria, 5 Sen­dai vi­rus, 12 Sphingobacterium, 5 Sepsis, 37 Sphingomonas, 7 Serratia spp., 8, 31, 35, 37 Spiral-shaped bac­te­ria, de­tec­tion Serratia marcescens, 31, 37, 51, meth­ods, 132, 170–172 352, 355 Spirochaetes, 8–9 Severe acute re­spi­ra­tory syn­drome Spirometra mansoni, 80 (SARS), 11, 226–227 Spleen, spec­i­men guide­lines for Severe acute re­spi­ra­tory syn­drome par­a­si­tol­ogy, 121 as­so­ci­ated co­ro­na­vi­rus Spore stain, 140–141 dis­ease (SARS-CoV), 56 Sporothrix schenckii, 34, 39, 42, Shewanella, 8 46, 52, 64 Shiga tox­in-producing Escherichia char­ac­ter­is­tics of, 273, 274 coli (STEC), 55 di­ag­nos­tic tests, 262 Shigella spp., 8, 44, 55, 94, 130, di­rect ex­am­i­na­tion, 248 143, 147, 148, 149, 154, 352 Sporotrichosis, 262 di­ag­nos­tic tests, 167 Spotted fe­ver rickettsiosis, 55 in­trin­sic re­sis­tance of, 355 Spotted fe­vers, 106 Shigellosis, 55 Sprillium, 7 Simplexvirus, 10 Sputum, rou­tine bac­te­rial cul­ture, Sindbis vi­rus, 11 137 Single-stranded, neg­a­tive-sense, Stachybotrys, 281 en­vel­oped RNA vi­ruses, Staphylococcus spp., 5, 31, 32, 37, 11–12 352 Single-stranded, nonenveloped an­ti­mi­cro­bial sus­cep­ti­bil­ity test DNA vi­ruses, 9 methods­ for, 332 Single-stranded, pos­i­tive-sense, com­monly tested an­ti­mi­cro­bi­als, en­vel­oped RNA vi­ruses, 11 368–369 Single-stranded, pos­i­tive-sense, dif­fer­en­tial char­ac­ter­is­tics of, nonenveloped RNA 176 vi­ruses, 10–11 in­trin­sic re­sis­tance of, 356 Sin Nombre vi­rus, 12 Staphylococcus au­re­us, 31, 34–38, Sinusitis, 49 41–44, 47–52, 129, 144, Skin 149, 157 in­fec­tions, 52–53 Staphylococcus saprophyticus, 53, spec­i­men guide­lines for par­a­si­ 54 tol­ogy, 122 Stenotrophomonas spp. vi­rol­ogy guide­lines, 111 di­ag­nos­tic tests, 167 Skirrow bru­cella me­dium, 152 dif­fer­en­tial char­ac­ter­is­tics of, Slackia, 4 205 Smallpox vi­rus, 10, 56, 238 Stenotrophomonas maltophilia, Sneathia, 6 51, 352 Soft tis­sue in­fec­tions, 52–53 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Sorbitol-MacConkey agar, 152 meth­ods for, 334 South Amer­i­can try­pano­so­mi­a­sis, in­trin­sic re­sis­tance of, 355 316 Stomatococcus, 4 Specimen col­lec­tion, 82–83 Stool spec­i­mens bac­te­ri­­ol­ogy, and trans­port my­co­bac­te­rial iden­ti­fi­ca­tion, 138 guide­lines, 84–104 pro­cess­ing for par­a­sites, guide­lines for an­aer­o­bic cul­ture, 124–126 107 rou­tine bac­te­rial cul­ture, 137 Index 401 StrepB car­rot broth, 152 mor­pho­log­i­cal char­ac­ter­is­tics Streptobacillus spp., 6, 106 of, 318 di­ag­nos­tic tests, 167 par­a­sitic di­ag­no­sis, 289, 301 dif­fer­en­tial char­ac­ter­is­tics of, 193 Talaromyces, 269–270 Streptobacillus moniliformis, 32, Talaromyces (Penicillium) 34, 53, 353 marneffei, 63, 262 Streptococcal toxic shock, 55 Talaromyces marneffei, 46 Streptococcus spp., 6, 32, 33, 35, char­ac­ter­is­tics of, 273, 274 41, 42, 48, 94 di­rect ex­am­i­na­tion, 249 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Tannerella, 4 meth­ods for, 332 Tetanus, 55 dif­fer­en­tial char­ac­ter­is­tics of Tetanus/diphtheria, adult be­ta-hemolytic, 178 im­mu­ni­za­tion sched­ule, dif­fer­en­tial char­ac­ter­is­tics of 328 viridans, 179 Tetracyclines, 349 Streptococcus, Group A, Tetrathionate broth, 152 di­ag­nos­tic tests, 157–158 Thayer-Martin (mod­i­fied) agar, Streptococcus, Group B, 153 di­ag­nos­tic tests, 158 Thioglycolate broth, 153 Streptococcus agalactiae, 32, 38, Thiosulfate cit­rate bile salts 42, 53, 144–145, 353 su­crose (TCBS) agar, 153 Streptococcus anginosus, 32, 47, 48 Tin­dale agar, 153–154 Streptococcus bovis, 32, 35 Tissierella praeacuta, 33 Streptococcus pneumoniae, 33–36, Tissue 38, 41–43, 47, 49–51, 55, de­tec­tion meth­ods of pro­to­zoa, 129, 145, 150, 353 287, 294–296 com­monly tested an­ti­mi­cro­bi­als, Gram stain and plat­ing me­dia, 371–373 134 di­ag­nos­tic tests, 158 my­co­bac­te­rial iden­ti­fi­ca­tion, Streptococcus pyogenes, 33, 36, 138 41–43, 48–50, 52, 53, 55, my­col­ogy plat­ing guide, 257 353 spec­i­men col­lec­tion and trans­ Streptococcus se­lec­tive me­dium, 152 port, 102, 115 Streptomyces, 4 vi­rol­ogy guide­lines, 110 Streptomycin, 349, 353 Todd-Hewitt broth, 148 Strongyloides spp., 120 Toluidine blue-O stain, fun­gal Strongyloides fuelleborni, 16 di­ag­no­sis, 247 Strongyloides stercoralis, 16, 45, Torovirus, 11 47, 51, 77, 121 Toxic shock syn­drome, 55 mor­pho­log­i­cal char­ac­ter­is­tics Toxocara spp., 43, 47, 77 of, 318 Toxocara canis, 16, 42, 318 par­a­sitic di­ag­no­sis, 288, 298 par­a­sitic di­ag­no­sis, 288, 299 Succinivibrio dextrinosolvens, 33 Toxocara cati, 16, 318 Sulfonamides, 351 Toxoplasma spp., 119 Suppurative throm­bo­phle­bi­tis, 38 Toxoplasma gondii, 15, 36, 37, 38, Suttonella, 8 40, 41, 43, 47, 51, 74, 118 Syncephalastrum, 271 di­ag­nos­tic tests, 287, 295–296 Syphilis, 55 im­mu­no­com­pro­mised pa­tients, 296 mor­pho­log­i­cal char­ac­ter­is­tics T of, 314 Taenia spp., 120, 320 new­borns, 296 Taenia multiceps, 80 preg­nancy, 295–296 Taenia saginata, 17, 80, 289, 301 Transfusion-associated sep­sis, Taenia solium, 17, 80, 118 37–38 402 Index Transmissible spon­gi­form Trypanosoma cruzi, 14, 38, 72 en­ceph­a­lop­a­thy agents, di­ag­nos­tic tests, 287, 296 223, 239 mor­pho­log­i­cal char­ac­ter­is­tics, Trematodes 316 de­tec­tion meth­ods, 289, Tryptic(ase) soy agar (TSA) and 299–300 broth (TSB), 154 mor­pho­log­i­cal char­ac­ter­is­tics Tsukamurella spp., 3, 139, 353 of, 319 Tuberculosis, 55 path­o­gens, 78–79 Tularemia, 55, 59, 106 Trematosphaeria spp., 68 Turicella, 3 Treponema spp., 9, 33, 140 Turicella otitidis, 33, 42 Treponema pallidum, 39, 40, 45, Typhoid fe­ver, 55 46, 48, 52, 55, 98, 132, 353 Typhus, 106 di­ag­nos­tic tests, 171–172 Trichinella spp., 122 Trichinella spiralis, 16, 36, 45, U 57, 77 Ureaplasma, 9 par­a­sitic di­ag­no­sis, 288, 299 Ureaplasma parvum, 33 Trichinosis, 57 Ureaplasma urealyticum, 33, 34, Trichoderma, 269 45, 54 Trichomonas hominis, 312 Urethritis, 45 cysts of, 311 Urinary tract in­fec­tions, 53–54 tro­pho­zo­ites of, 310 Urine Trichomonas tenax, 14, 33, 72 Gram stain and plating­ media,­ Trichomonas vaginalis, 14, 45, 46, 135 72, 123, 312 my­co­bac­te­rial iden­ti­fi­ca­tion, 138 di­ag­nos­tic tests, 287, 294 rou­tine bac­te­rial cul­ture, 137 tro­pho­zo­ites of, 310 spec­i­men col­lec­tion and trans­ Trichophyton spp., 33, 65, 66, 275 port, 102–104, 116 Trichosporon spp., 33, 37, 62, 255, vi­rol­ogy guide­lines, 109–110 266 Urogenital sys­tem Trichosporon beigelii, 260 de­tec­tion meth­ods of pro­to­zoa, Trichostrongylus spp., 77, 320 287 Trichrome stain, 291 di­ag­nos­tic tests for pro­to­zoa, Trichuris spp., 120 292–294 Trichuris trichiura, 16, 77, 320 spec­i­men guide­lines for par­a­si­ mor­pho­log­i­cal char­ac­ter­is­tics tol­ogy, 123 of, 318 U.S. Department of Agriculture par­a­sitic di­ag­no­sis, 289, 299 (USDA), 21 Trimethoprim, 144 U.S. Department of Transporta- Trimethoprim-sulfamethoxazole, tion, 83 346, 352, 365, 367, 371, 373 Tropheryma, 4 Tropheryma whippelii, 46 V Trophozoites Vaccination, 324 of flag­el­lates, 309–310 adult im­mu­ni­za­tion sched­ule, of in­tes­ti­nal ame­bae, 302–304, 328–329 308 pe­di­at­ric im­mu­ni­za­tion sched­ Trueperella, 3 ule, 325–327 Trypanosoma spp., 15, 36, 40, 44 Vaccinia vi­rus, 10, 238 Trypanosoma brucei, 287, 296 Vagina, my­col­ogy plat­ing guide, Trypanosoma brucei gambiense, 257 14, 72, 315 Vaginal, spec­i­men col­lec­tion and Trypanosoma brucei rhodesiense, trans­port, 116 15, 72, 316 Vaginitis, 46 Index 403 Vagococcus, 5 W Vancomycin, 143, 144, 345, 347, Weber Green stain, 291 348, 368, 370, 372 Weeksella, 5 Vancomycin-intermediate Weeksella virosa, 33 Staphylococcus au­reus Weissella, 5 (VISA), 55 West Af­ri­can sleep­ing sick­ness, Vancomycin-resistant Staphylo- 315 coccus au­reus (VRSA), Western equine en­ceph­a­li­tis 55 (WEE) vi­rus, 11, 40, 56, Varicella, 56 225 adult im­mu­ni­za­tion sched­ule, West Nile en­ceph­a­li­tis, 56 329 West Nile vi­rus, 11, 40, 227–228 im­mu­ni­za­tion sched­ule, 327 Wirtz-Conklin spore stain, Varicella-zoster vi­rus (VZV), 10, 140–141 108, 109, 111, 223, 239 Wolbachia, 7 Varicellovirus, 10 Wright’s stain, 290, 291 Variola vi­rus, 10 Wuchereria bancrofti, 16, 42, 77 Veillonella spp., 6, 33 mor­pho­log­i­cal char­ac­ter­is­tics Ven­e­zu­e­lan equine en­ceph­a­li­tis of, 317 (VEE) vi­rus, 11, 40, par­a­sitic di­ag­no­sis, 289, 299 225 Vibrio spp., 8, 44, 136, 153 an­ti­mi­cro­bial sus­cep­ti­bil­ity test X methods­ for, 333 Xylose-lysine-deoxycholate (XLD) di­ag­nos­tic tests, 168 agar, 154 dif­fer­en­tial char­ac­ter­is­tics of, 203 Vibrio cholerae, 54, 167, 353 Y Vibrionaceae, 55 Yeast, 244 Vibriosis, 55 an­ti­mi­cro­bial sus­cep­ti­bil­ity test Vibrio vulnificus, 52, 353 meth­ods for, 336 Viral hem­or­rhagic fe­ver, 56 cul­tural and bio­chem­i­cal char­ac­ Virology spec­i­men guide­lines ter­is­tics of, 264–265 blood, 108 di­rect ex­am­i­na­tion, 248–250 bone marro­ w, 108 Yeast ex­tract-phosphate agar, CSF, 108–109 255 eyes, 110 Yellow fe­ver vi­rus, 11, 56, 227 fe­ces, 110 Yersinia spp., 8, 201 gen­eral, 107–108 Yersinia enterocolitica, 37, 44, 48, gen­i­tal, 110–111 130, 136, 144, 145, 353 re­spi­ra­tory, 109 di­ag­nos­tic tests, 168 skin, 111 in­trin­sic re­sis­tance of, 355 spe­cific, 108–111 Yersinia pestis, 55, 130, 168, 353 tis­sue, 110 Yokenella, 8 urine, 109–110 Viruses cells for viral­ isola­ tion,­ 224 Z de­tec­tion meth­ods for, Zika vi­rus, 11, 59 221–223 Zygomycetes, 49, 51, 52, 271–272 tax­o­nomic clas­si­fi­ca­tion of hu­ di­ag­nos­tic tests, 262 man, 9–12 di­rect ex­am­i­na­tion, 251 Visceral ab­scesses, 48 Zygomycosis, 262