16 Laboratory Patrick R

Home , Bloodstream infections, Lancefield grouping, Rapid plasma reagin, Viral culture

The Clinician and the Microbiology 16 Laboratory Patrick R. Murray*

The clinician and the microbiologist must actively work together to The responsibilities of the clinician, particularly of the infectious maximize the clinical value of diagnostic microbiology testing. Unfor- diseases physician, include acquiring a substantive understanding of tunately, the current trend to consolidate laboratory services and to the laboratory’s test menu, specimen collection and transport guide- move them off-site has made timely communication between labora- lines, and testing policies. The clinician should know the appropriate tory personnel and patient care providers more difficult.1 In addition, specimens and diagnostic tests for detection of specific pathogens and these changes potentially increase the time between specimen collec- be prepared to alert the laboratory personnel when a specific fastidious tion and laboratory processing, compromising the specimen integrity (e.g., Corynebacterium diphtheriae, Bartonella spp.) or highly patho- and delaying the availability of critical test results. These changes, along genic (e.g., Francisella tularensis, drug-resistant Mycobacterium tuber with other cost-cutting measures, make it even more important that culosis, Coccidioides immitis) organism is considered. The clinician the infectious diseases physician and the microbiologist work together. should also be prepared to prioritize test requests when only a limited The following expectations regarding the microbiologist’s and the clini- quantity of specimen can be collected. In this situation, discussions cian’s responsibilities should be recognized and addressed (Table 16-1). with the laboratory director can facilitate modification of the routine The menu of tests offered by the laboratory should be sufficient to laboratory practices to optimize the testing process (e.g., need for satisfy the patient care needs. Selection of these tests and the avail- routine stains, selection of media, and testing conditions to optimize ability of infrequently offered tests should be determined after discus- recovery of both bacteria and fungi on a limited number of media). sions with key members of the medical staff and should be revised as Communication with the laboratory director is also important when patient care needs change. If the performance of infrequent, expensive, the clinical testing needs are not satisfied by the available test menu or or sophisticated testing is shifted to an external laboratory, the micro- test turnaround time, when a laboratory policy causes patient care biologist should ensure that timely transport and appropriate test pro- problems, or when test results are inconsistent with a patient’s clinical cedures for these send-out tests are maintained so that test accuracy is presentation. not compromised. The microbiologist should provide the medical staff One inevitable difficulty that confronts both the microbiologist with a menu of the offered tests, including cutoff times for receipt of and clinician is the continuous revision of the taxonomic nomencla- specimens and turnaround times for test results. Guidelines for collec- ture, particularly for bacteria and fungi. Much of the taxonomic reor- tion of appropriate specimens, transport of the specimens to the testing ganization now under way is driven by our increased ability to classify site, and laboratory policies (e.g., limits on the number of specimens organisms by using gene sequencing and, more recently, mass spec- that can be submitted for a specific test, list of pathogens routinely trometry. The advantages of using these tools to classify and identify sought in stool cultures) should be available. The test menu and guide- organisms are greater precision than by using traditional phenotypic lines should be readily available on the hospital computerized informa- tests and a better understanding of the relationship between newly tion system and ideally integrated with electronic ordering of tests. The described organisms and disease. The disadvantages are the practical computer system should document receipt of specimens in the labora- challenges of remembering a rapidly expanding list of new names and tory, provide a record of testing in progress, and report preliminary the difficulties this presents for performing searches of the medical and final results. The microbiologist should coordinate with key literature. Fortunately, an up-to-date list of all validly published names members of the medical staff a policy of immediate telephone notifica- of bacteria, as well as the historical names of the organisms, is acces- tion of critical laboratory results, such as positive blood or other nor- sible on the Internet (www.bacterio.cict.fr/). The International Com- mally sterile fluid and tissue cultures, isolation of highly infectious mittee of Taxonomy of Viruses publishes a similar database for viruses pathogens, and significant positive stain results from clinical speci- (www.ictvonline.org). The scientific names of fungi and parasites mens. In addition, the microbiologist and medical staff should develop are defined by the International Code of Botanical Nomenclature guidelines for reporting antibiotic susceptibility results for bacteria, (www.bgbm.org/iapt/nomenclature/code/default.htm) and the Inter- mycobacteria, and fungi. Periodic publication of antimicrobial suscep- national Code of Zoological Nomenclature (www.iczn.org), respec- tibility patterns for the most common bacteria can be used to guide tively; however, up-to-date lists of validly named fungi and parasites empirical therapy.2 The laboratory must ensure that its operation meets do not currently exist. all current regulatory requirements (Clinical Laboratory Improvement Hospital epidemiology is an area of shared interest for the micro- Amendments), including initial verification and ongoing validation biology service and the hospital infection-control officers. Although a of procedures that are used by the laboratory.3,4 Participation in exter- hospital surveillance program can monitor infection rates and non- nal proficiency surveys, an effective program of quality control, and compliance with established infection prevention policies, the most the use of established quality assurance benchmarks can ensure the effective component to any infection control program is vigilance—on accuracy of the laboratory test results. Minimizing testing costs is the part of the microbiologist and clinician. The microbiologist has the essential for an efficient laboratory operation but must be done without unique perspective of seeing all the organisms isolated from patients significant compromise of the quality of results. Finally, a system of throughout the hospital and may be the first to recognize a small short-term storage of all specimens and long-term storage of important cluster of unique organisms. The clinician also has a unique isolates should be established to facilitate additional testing if required. perspective—the ability to assess the likelihood that an infection in his or her patient is nosocomial. Working in concert, the microbiologist *All material in this chapter is in the public domain, with the exception of any borrowed and clinician play an important role in guiding a potential outbreak figures or tables. investigation. 191 Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Chapter 16 The Clinician and the Microbiology Laboratory 191.e1 For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 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Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada culture; culture; laboratory diagnosis; molecular diagnosis; mycobacteriology; mycology; parasitology; serology; virology KEYWORDS antigen detection; bacteriology; clinical microbiology laboratory; Part I Basic Principles in the Diagnosis and Management of Infectious Diseases utrs o dtcin f atrma r ugma. irsoy s a is Microscopy fungemia). or bacteremia of detection for cultures specimens blood (i.e., of present transiently is number organism the adequate if collected an be should and pathogen, the maximize to of collected recovery be should specimen of volume adequate An the Ideally,specimens should be collected culture.before administration of antimicrobials. the of interpretation the confound the or overgrow pathogen or suppress either may that organisms avoid to taken be should care addition, in organisms; viable contain must specimen the then culture, in organism the of isolation is test diagnostic the If specimens. the of transport and collection for materials responsibility to provide appropriate, readily available instructions and care area adversely affect diagnostic testing. The microbiologist has the patient the remain in specimens while fashion.Delaystimely a in tory labora the to transported and appropriatecontainer the in collected are infection of site the of representative specimens ensure to sibility the responthe has ordered. physicianalso The be shouldthat has tests nostic physician the diag- the and pathogens Thus, potential carefully consider to biopsy). responsibility tissue fluids, aspirated (e.g., obtain to difficult specimen the or limited is quantity the if problem blood, urine, stool, expectorated sputum), this can present a significant (e.g., sufficient is specimen of quantity the if problem a pose not may this ordered.Althoughtests of number the and collected be mustthat specimens of types the increases which considered, usually are gens the differential diagnosis istypically notso limited. A variety ofpatho however, straightforward; is test diagnostic specific the for appropriate specimen the of selection then suspected, is pathogen specific a If organismresponsiblepatient’s for the ultimateobjective. the is disease an of detection the for test and appropriate specimen the of Selection General PROCESSING INITIAL AND TRANSPORT,COLLECTION, SELECTION, SPECIMEN 192 identify medically important organisms.important medically identify and detect and specimens process to used arethatprocedures general the presentationof a byfollowed summarized, are transport and tion, Establish an open communication with the laboratory director when testing when director laboratory the with communication open an Establish collected be can specimen of quantity limited a when requests test Prioritize or fastidious a (e.g., sought is organism specific a when laboratory the Alert and collection specimen and menu test laboratory the of knowledge Maintain Clinician’sResponsibilities of storage long-term and specimens all of storage short-term of system A standards regulatory to conforms that laboratory a Establish offered all of accuracy the ensures that control quality of program a Maintain most the for patterns susceptibility antimicrobial of publication Periodic of receipt acknowledging for system computerized effective an Maintain transport and collection specimen for guidelines Provide for times turnaround and requests test processing for times cut-off Provide infrequent,of performance for laboratory relationshipa external Establish an with physician to responsive is that menu test comprehensive appropriate, an Provide Microbiologist’sResponsibilities Partnership TABLE specimen is required is specimen a of handling special or menu test available the by satisfied not are needs organism) pathogenic highly guidelines transport required if testing additional facilitate to established be should isolates important tests institution the in bacteria isolated commonly system communication overall an of component a be should results critical of physician the to notification immediate results; of reporting and in-progress, testing specimens, results reporting site on performed be cannot that testing sophisticated or expensive, needs Selection Selection of the appropriate specimens depends on multiple factors. In the following sections, guidelines for specimen selection, collec followingInselection, for guidelinesthe specimen sections,

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and

Clinician

- - - - in the 48 hours48 the in before collection. blood treatedantibioticspatientshavewith in who been ofbacteria recovery fungal media. Use of resin-supplemented media significantly improves with supplementedresins to bind and brothsremove antibiotics, broths broths, for pediatric cultures, and anaerobic and aerobic including essentially is processing systems, these foravailable laboratoryare media culture of variety A eliminated. from resulting cultures the of doneintocontaminationwithoutprocess bottles, be the this can entry Because minutes. 20 blood to 10 monitor every growth microbial systems for bottles culture automated fully current The system. based broth- this of modifications of series a introducedcompanies mercial com of number a years, subsequent In growth. microbial of dioxide) carbon labeled radioactively (i.e., byproducts metabolic for broth the monitoring by cultures positive detected that developed was system automated first the 1970s, the In broths. nutrient containing bottles septicemic patients, a largeof volume of isblood traditionally culturedblood in the in present are fungi and bacteria few relatively Because laboratory.microbiology the by processed types most specimen important the of one considered is fungi and bacteria for cultured Blood Specimens Blood Initial Specific available.arechapter, sources this other of scope the For specimen. more extensive the guidelines or for of recommendations handling that are optimalbeyond ensuring for these procedures best For the guidelines. general under coveredinstances, the physician should contact laboratory personnel to be discuss can situations all separately.Not discussed be will these parasites, and viruses for used are systems transport specialized Because cultures. and stains fungal and bacterial for types specimen common of transport and collection often. less day or per once performed that are tests for time cutoff establishedan andbefore laboratory the collection in arrive they the ensure to specimens time of transport to important also is It program. assurance times should be transit monitored systematicallyThese latter.as part of the a diagnostic microbiologistquality the and to former responsibility the the minimize has physician The laboratory. the in receipt or collection after delays significant are there if wasted are systems) tube tory processing. Efforts to minimizetransit time (e.g., use of pneumatic the time receiptbetween of the specimen in the laboratory and labora delays the laboratory, between the collection of the specimen and to shipment to specimen the laboratory and a transport to required time the to delivery that laboratory is as Itoccurring expected. is important sure to include, with the make to should process and this delivered monitor be periodically should specimens often in what how and determine manner should laboratory The requests. urgent of dling han the in important particularly is which fashion, timely a in done andnotanafterthoughtas confronted when negative initial with tests. of samples should be coordinated carefully collections to optimizepathogens, their usefulness different for vary can levels these Because ies. level peak of antibodies or to demonstrate a significantrise in antibod the with coincide to properly timed is blood of collection the if useful chial lavage for culture and urine for antigen testing. Serologic tests are of diagnosis for specimens optimum the Likewise, meningitis. pneumococcal of diagnosis for collected be should urine and CSF both but meningitis, cryptococcal of diagnosis appropriate for be would (CSF) fluid cerebrospinalexample, For gen. patho suspected the by determined antigensmicrobialofis detection for specimen appropriate the of Selection techniques. microscopy or stains specialized using by examined be to need may specimens tiple of examination (e.g., test plasmodia, for smears diagnostic blood peripheral primary the is microscopy If of tests. diagnostic forother need the value with balanced microscopy mustbe the limited, is specimen of quantity the if Thus, pathogens. rapidbut insensitivetypically and nonspecificfor detection of method biologist and infectious disease specialists carefully consider the the consider carefully specialists medium. each ofdisadvantages andadvantages disease infectious and biologist should be routinely or selectively used should be made afterthe micro summarizes generally accepted guidelines for specimen specimen forTableguidelines accepted generally summarizes 16-2 be must laboratory microbiology the to specimens of Transport

Laboratory

Specimen

Pr Guidelines ocessing pneumonia are bron - are pneumoniaLegionella 6 Decisions about media which ), then mul- then BorreliaBabesia,),

and

5 ------

Chapter 16 The Clinician and the Microbiology Laboratory 193 Continued Neisseria is

Borrelia Ehrlichia Vibrio, Yersinia, Vibrio, Yersinia, and

Salmonella enterica will dilute marrow with mL

Bacterial

, Brucella, Escherichia coli). for

Submitted

Histoplasma, mycobacteria COMMENTS

Table . 16-5 Table are are needed. Do not inoculate blood culture bottles. serotype Typhi; aspiration serotype of Typhi; more than 2 peripheral blood. blood culture bottles for bacterial and yeast cultures unless also acceptable. pertinent information (e.g., surgical infection, trauma, bite wound). use of broths supplemented with resins designed to inactivate antibiotics. or processing. for more than 3 days unless approved by laboratory director; consider Clostridium difficile for hospitalized patients with diarrhea; multiple specimens per day are not indicated. Aeromonas, enterohemorrhagic suspected (inhibited by anticoagulant in bottles). because of the small amount of material collected. specimen is collected. burgdorferi. because of the small amount of material collected. Anesthetics may inhibit growth of some bacteria. vaginosis. of collection or immediately transport the syringe to the laboratory. anaerobes. because of the small amount of material collected. media at the time of collection or immediately transported to the laboratory; do not allow the swab to dry. the specific collectiondevice designed for the system. species. Send immediately; notify laboratory if anaerobic culture or acid-fast stains OTHER Culture Culture for yeasts and dermatophytes. If sufficient material can be collected, inoculate aerobic and anaerobic Specimen sent immediately in capped syringe after the air is excluded is Specify location of abscess for optimal processing; provide all other Optimal recovery of bacteria from patients receiving antibiotics requires All blood cultures should be transported immediately to start incubation Refer to Obtain for Do not process for bacterial pathogens if patient has been hospitalized Notify laboratory if specific pathogen is suspected (e.g., Drainage catheters may be superficially contaminated. The number of stains prepared and media inoculated should be limited Discuss the appropriate transport system with the laboratory before Growth Growth represents distal urethal microbes. The number of stains prepared and media inoculated should be limited Gram stain and not culture is recommended for the diagnosis of bacterial Transport rapidly Transport for smear preparation; test insensitive for rapidly Transport to prevent drying out. If the specimen is collected by aspiration, inoculate the media at the time Drainage collected from a perforated eardrum should not be cultured for The number of stains prepared and media inoculated should be limited Specimens collected for culture or stains should be inoculated onto If the specimen is processed by nucleic acid amplification technique, use Transport rapidly Transport for smear preparation; test insensitive for Specimens

* —darkfield; ransport T Haemophilus

, and for each blood culture;

10 for fungi and/or > mL/child

). Collection

and 1-3 Chlamydia trachomatis—DFA) and culture for bacteria, the

mL GUIDELINES

es aseptically; inoculate into blood culture bottle or mL/adults for

container. mycobacteria; transport in an anaerobic collection system. immediately to laboratory; transport in Cary-Blair holding medium if delayed in transport. 1.5-mL lysis-centrifugation tube. swabs usually have insufficient material for stains and culture. at margins of abscess. and anaerobic bacterial, fungal, and mycobacterial cultures. divide blood into two blood culture bottles, preferably an aerobic and an anaerobic bottle; collect two to three cultures per 24-hr period. should be transported immediately to the laboratory in the properly enclosed collection syringe. Klebsiella [Calymmatobacterium] granulomatis ducreyi—Gram stain, (H. ducreyi, C. trachomatis stains should be prepared and media inoculated at the time of collection. swab, sample the endocervical canal. place segment in sterile container. alcohol, then aspirate by syringe. and media inoculated at the time of collection. phlebotomy site with alcohol, followed by tincture of iodine or chlorhexidine; allow the disinfectants to dry. Clip affected areas; transport to laboratory in envelope or sterile dry, 1-2 5-10 for bacteria disease chronic for more fungi; or mycobacteria for optimum Refer to Send at least 2-5 Collect specimen directly in a sterile container and deliver Collect 1-2 Clean surface of closed abscess with 70% alcohol; collect specimens Aspirates in anaerobic transport tubes are acceptable for aerobic Collect 10-20 COLLECTION Aspirate of pus or fluid in anaerobic transport vial is preferred; Culture Culture of drainage catheter is not recommended. Because a limited amount of fluid can be collected by aspiration, it Collect exudate for microscopy pallidum (Treponema Not acceptable for culture Remove crusted debris and then firmly scrape or swab the outer ear. A sterile spatula should be used to scrape the surface of the cornea; Obtain secretions from the vaginal wall with a sterile swab. Collect material expressed from the urethra on a sterile swab. Remove mucus and secretions from the cervical os; using a fresh Prepare Prepare thick and thin smears from fingerstick or blood. EDTA For intact eardrum, clean ear canal with soap solution or 70% For ruptured eardrum, collect drainage with noncotton swab. Roll premoistened swab over conjunctiva; stains should be prepared Remove aseptically, Remove cut aseptically, at least a 2-inch segment from tip, and Prepare Prepare thick and thin smears from fingerstick or blood. EDTA Disinfect bottle tops with 70% isopropyl alcohol; disinfect and

Guidelines Stains

16-2

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Stains Guidelines

and Transport aspirated urine promptly to the laboratory.the to promptly urine Transportaspirated specimen. midstream a for Transportas contaminated be to likely is which urine, of portion first the Discard in submit promptly,or laboratory to transport and refrigerated Keep for urine first-voided Collect of portion first the discard foreskin, the retract to men Instruct of portion first the discard apart, labia hold to women Instruct sterile in transport collected; be may fluid of volume large A Acceptable for same tests as expectorated sputum; not screened for screened not sputum; expectorated as tests same for Acceptable Gram (e.g., microscopy to restricted generally is yeast of Detection of recovery the for saliva, avoiding pharynx, posterior the Swab anaerobic in submit suspected, are bacteria if aspiration; by Collect for appropriate swabs and washings Nasopharyngeal 1-2 swab premoistened Insert COLLECTION Decontaminate open lesion first. Aspirate if possible; if swabs must swabs if possible; if Aspirate first. lesion open Decontaminate use not laboratory.Do to rapidly transport and moist specimen Keep sterile in Place blood. avoid lesion; of edge active at skin Scrape 1 Collect 1 or container sterile in Transportspecimen Induced with sterile saline using a nebulizer.a using saline sterile with Induced flora; oral excess remove to water with gargle or rinse patient Have catheter. indwelling an colonized have that organisms or flora urethral with organisms. contaminating of overgrowth prevent to acid boric with tube urine tests. container. sterile a in portion midstream a collect and urine, voided container. sterile a in portion midstream a collect and urine, voided container. Neisseria meningitidis. Neisseria gonorrhoeae, Neisseria diphtheriae, Corynebacterium pertussis, acceptability such as induced sputa. induced as such acceptability stain). white calcofluor stain, meningitidis N. gonorrhoeae, N. diphtheriae, for appropriate pyogenes; Streptococcus system. transport mucosa. Identify type/location of wound. of type/location Identify culturing. anaerobic for suitable not are wounds superficial Open, cultures. and stains for sufficient is quantity that sure be used, be biopsy.of site on depending cultures, anaerobic and mycobacterial, fungal, routine, for specimens suitable are Biopsies formalin. or saline bacteriostatic lesion. of swabs than definitive more be may biopsy dish; Petri molds. and culture dry.to specimen the allow lower airways; collect and transport in a sterile container.sterile a in transport and collect airways; lower from secretions expectorate and deeply cough to patient instruct

Cultur

for bacterial culture; 5 culture; bacterial for for es—cont’d

GUIDELINES

the

Collection C. trachomatis C.

cm into nares and rotate against nasal against rotate and nares into

mL or more for mycobacterial for more or

and and

B. pertussis, C. pertussis, B. mL . sterile saline; do not do saline; sterile N. gonorrhoeae N.

T ransport Bordetella -

patients is 20 f lo ta i cultured. is that blood of volume the to related directly is culture blood positive a obtaining of organism/mL)patientsuccessaveragelow(an 1 is andthe than less of is collected. Typically, the number of organisms in the blood of a septic factor that influences recovery of organisms isthe volume of bloodthat *

of For most common, nonfastidious pathogens, the most important most the pathogens, nonfastidious common, most For

Specimens Urine is generally sterile, although transient colonization of the bladder the of colonization transient although sterile, generally is Urine culture. anaerobic for acceptable specimen urine only the is This infection. tract urinary iatrogenic an in result may Catheterization the of quality the improve consistently not does voiding before Cleansing sheathed special a unless culture anaerobic for submit not Do Contamination with oral bacteria is common; specimen is processed for processed is specimen common; is bacteria oral with Contamination considered. is pathogen bacterial fastidious a if laboratory the Notify bacterial if performed be should cultures bacterial anaerobic and Aerobic considered. is pathogen bacterial fastidious a if laboratory Notify of carriage detect to used Generally Transport in anaerobic transport media. transport anaerobic Transportin pathogen specific the indicate and tissue, of type the specify Always specific specify out; drying prevent to media transport in Transportswabs for processed be can Specimen for processed is specimen common; is bacteria oral with Contamination than other molds for specimens these process not will laboratories Some a flora; oral with contamination of indicative is cells epithelial of Presence of number the and collected, generally is specimen of volume small A OTHER may occur.may necessary.be may collection supervised and cleansing specimen, proper a provide to unable is patient the however,if specimen; of detection for specimen Preferred interpretation. guide to cultures bacterial quantitative perform laboratories some cultures; and stains from yield optimum for concentrated be should fluid used; is brush bronchoscopic bacteria despite the presence of epithelial cells. epithelial of presence the despite bacteria suspected. is infection fungal if performed be should culture and stain fungal suspected; is etiology laboratory. bacteria. (e.g., sought etc.). Mycobacterium, dermatophyte, (e.g., suspected is one if organism cells. epithelial of presence the despite bacteria dimorphics. molds. or mycobacteria for processed be can but culture, bacterial routine for unacceptable is specimen contaminated sensitivity.maximize to limited be should tests

COMMENTS of blood should be collected and distributed into two

if other than routine than other if Histoplasma) Bartonella, Nocardia, Submitted 7 The current recommendations for adult for recommendations current The Pneumocystis. Pneumocystis.

for ; specify for specify aureus; Staphylococcus

Bacterial Sporothrix,

and

Chapter 16 The Clinician and the Microbiology Laboratory

------10 195 Stenotroph 12,13 although other investiga other although 14 An effortto reduce the incu- spp., spp., Pseudomonas 11 Tsukamurella, ). Gordonia Tsukamurella, - Typi , rapidly growing mycobacteria). Fur Other methods include culture of sonicates sonicates of culture include methods Other 15 Culture of the tip in nutrient broth should not be done be done not should broth nutrient in the tip of Culture Aggregatibacter, Bartonella, Francisella, Campylobacter, Aggregatibacter, 16 Use of these definitions should help in the standardization standardization the in help should definitions these of Use 17 Patients frequently have catheters for intravenous access, including access, including intravenous for catheters have frequently Patients Blood cultures should be incubated routinely for a minimum of Blood culture systems have also been used to assess the sterility tors tors suggest a lower breakpoint, such as accurately. fivemore infection colonies, may identify Cerebrospinal, Cerebrospinal, Peritoneal, Pleural, Synovial, and Pericardial Fluids Laboratory processing of normally sterile body to manner a fluids collected in be should specimens The important. critically is considered avoid contamination and transported immediately to the laboratory. Upon receipt in the laboratory, processing these specimens should long-term percutaneous catheters and subcutaneous implanted devices implanted subcutaneous and catheters percutaneous long-term and short-term catheters used for a variety of limited vascular access needs. Although microbiologists have recommended that blood culture for should not be drawn through catheters, the the practicalonly access cases reality represents in and many done frequently is this is collectedis a catheter, through a culture If critically ill patients. for site then the the portbeaccess should before disinfected with 70% alcohol bacte a for source represent catheters Intravascular blood is removed. as well as surface skin the on organisms both with fungemia and remia (e.g., rods gram-negative opportunistic Intravenous Catheter Tips performed commonly most is tips catheter of culture Semiquantitative more or 15 of presence The plate. blood-agar a across the tip rolling by infection, catheter a of indicative is colonies omonas, omonas, Ochrobactrum, ) Methylobacterium and acid-fast organisms (e.g., rapidly growing mycobacteria, cally, blood cultures collected through a higher inoculum contaminated than line blood collected documented from have be can a This peripheral source. a the vein, is line which a if determine to used be can either by using a quantitative blood culture system, such as the lysis- earlier detection a of posi demonstrating by system, or centrifugation Thus, whenever possible, allblood culturessite. collected venipuncture from peripheral aa from catheter culture blood a include also should blood positive most that documented have studies of number A days. 5 cultures are detected during the first 2 daysof incubation,and exten tive tive culture collected from the catheter versus the peripheral site. sion of incubation beyond 5 days generally results in recovery of the skin contaminants. slow-growing more of of platelets and cell therapy products (i.e., processed human in cells vitro and and then tissues administered for therapeutic purposes). The BacT/Alert 3Dsystem (bioMérieux, Lyon, system France)currently approved for sterility testing of platelets, but valida the only is ments. ments. I believe that incubation for less than 5 because days a is small not justified number of significant detected pathogens (e.g., will likely not be thermore, the microbiologist should be willing to extend the incuba disease the infectious of the request at period selected tion cultures for specialist. tions studies have been done with BACTEC other 9240 (Becton culture Dickinson, systems; Sparks, MD) both and BacT/Alert the 3D systems have been validated for cell therapy products. bation bation period further has been are bottles driven culture If bottles. for capacity by limited a have systems culture the fact automated blood incubated for a periodlonger of time, fewer cultures can be processed in each instrument, thus increasing the need for additional instru Cryptococcus, Candida glabrata of segments or tips or culture intraluminal Quantitative segment. flushing catheter ofthe of inside thethe culture to catheter broth tip with performed by flushing, sonicating,or vortexing the catheter segment and then preparing and plating a set of dilutions for colony counts is catheter- of predictor accurate more bea may but intensive labor more sepsis. related and improvement of analyses of catheter-related infections. analyses catheter-related of of improvement and because this will result in false-positive cultures because of contamina of because cultures false-positive in result willthis because - Guide culture. before handling or removal tion theof during catheter lines have catheter-related of types been different for definitions established include these infection; for the prevention of infections. catheter-related

- - - - - wk

for C

in wk

Staphylococcus Staphylococcus examine broth Methods mo;

e If a collection system is 9 compounds (e.g., chocolate agar, BCYE agar) incubate for 7-10 days blood agar with pyridoxal bottle systems. Use fresh blood fungi; extend incubation for 4 up to 3 weekly by darkfield or phase- contrast microscopy agar; incubate 2-4 humidified chamber incubate for 7-10 days Campy agar; incubate in microaerophilic atmosphere may not be detected by monitoring system of automated blood culture instruments; stain broth with acridine orange for isolation of mycobacteria isolation of mycobacteria or of bottles for 7-10 days Subculture Subculture to media with sulfhydryl Blind subculture may be required; Subculture Subculture to chocolate agar or More sensitive and faster than Subculture onto BCYE agar Incubate in air at 28° to 30° COMMENTS May be most sensitive system Blind subculture may be required; Subculture to blood agar and Extend incubation for 7-10 days; Subculture to media for recovery of Subculture to broth and solid media Use media specifically designed for Incubate primary blood agar plates Propionibacterium Propionibacterium ). acnes True Cultur

or EMJH medium or bottle system METHOD Lysis-centrifugation Bottle systems Bottle systems Lysis-centrifugation Lysis-centrifugation semisolid Fletcher’s Lysis-centrifugation Bottle systems Bottle systems Bottle systems Lysis-centrifugation Lysis-centrifugation Lysis-centrifugation Lysis-centrifugation Special Corynebacterium spp., 16-3

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada Table 16-3). Table It is recognized that there are practical limits on the Blood cultures start with proper skin antisepsis before venipunc Granulicatella and Helicobacter and molds TABLE Francisella Legionella ORGANISM Abiotrophia and Bartonella Leptospira Mycobacterium Brucella Campylobacter Dimorphic fungi Nocardia BCYE, buffered charcoal yeast extract agar; EMJH, Elinghausen, McCullough, Johnson, and Harris medium for leptospires. rate venipunctures rate should venipunctures be performed for each culture to minimize (e.g., surface skin the from bacteria with contamination , epidermidis infections with these organisms are commonly associated with intra occurs,so allbacteremia persistent a setting, this In vascularcatheters. blood cultures should be positive. If only one of two or three cultures is positive, then this more culture during collection than sepsis. catheter-acquired likely represents contamination of the used, care must be taken to overfillingavoid the culture bottles. Sepa contaminated contaminated blood cultures significantly. volume volume of blood that can be collected from some should patients, be and considered this when interpreting the significance of negative blood cultures. blood culture bottles. Theroutine use of a singleblood culture bottle smaller proportionately children, and neonates For acceptable. never is volumes of blood should be collected.two or Ideally, three blood cul tures should be collected in a negative with 24-hour patients septic usefulfor not generally are bloodcultures period. Additional “routine” blood cultures, and alternative culture methods should be considered (see ture: The skin surface is first by cleansed 0.5% with chlorhexidine alcohol,70% gluconate followed (30 or 10% (1 Chlorhexidine is povidone-iodine minute), (2 the minutes). seconds), tincture of most iodine rapid, effective disinfectant. tube tube should also be wiped with 70% alcohol and allowed Blood to can air be collected dry. with a needle and syringe or directly into the used, is syringe needleand a If system. collection a with bottles culture the needle should not be changed between collection and inoculation of the blood culture system because this posesprocedure an unneces sary risk of a needlestick and has not been demonstrated to reduce Part I Basic Principles in the Diagnosis and Management of Infectious Diseases oe rsiaoy rc seies nld epcoae ad induced and expectorated include specimens tract of respiratory diagnosis lower the for submitted Specimens sinusitis. of diagnosis the substituteable submittedaspiratesforparanasal be notshouldforand unreli an are washings or swabs Nasopharyngeal effective. generally specific problematicbecausecases empiricaltherapy without culture is paranasal sinuses or the middle earof are submittedAspirates only occasionally scrapings.for or swabs cavity oral or mouth and washings, or swabs nasopharyngeal swabs,throatinclude specimens tract ratory egories: upper respiratory and lower respiratory. Common upper respi- Laboratories subdivide respiratory specimenstract into two major cat TractSpecimens Respiratory available. is diagnosis, clinical with ment assessment, analytical An tes between discrepanciesresolves which (FDA). Administration Drug and U.S. Food the by recently approved been has Salt 16-4) (Table UT) City,Diagnostics, Lake Biofire panel; Meningitis/Encephalitis FilmArray plexchainpolymerase reaction panel(PCR) for pathogens17 (BioFire and (neurosyphilis) for choice of test the is testing Serologic spira). (e.g., tion of otherDetection organisms culturein may require extendedincuba of sensitive the however, days; antibiotics. to exposure prior by decreased significantly be 2 culturecan to 1 within positive generally are neoformans C. monocytogenes, Listeriameningitidis, N. pneumoniae, (e.g., meningitis of causes common most the Cultures for specific. andsensitive aremans In situations. life-threatening in for tests antigen tests, bacterial the action with contrast of course safer a is testing, the most on appropriate based empirical therapycoverage, antibioticrather than ofin response initiationto antigen negative, is stain Gram rarely direct the If they meningitis. bacterial acute management for patient because affect setting hospital their in tests these maintaining any, if benefit, of real the determine to need Laboratoriestest. antigen reliable a of lack the and vaccine decreased with the introduction of the Haemophilusinfluenzae of youngmeningitisin childrensignificantly has incidence the because years recent in disfavor into fallen has CSF in bacteria for tests antigen direct ofUse properties. stain Gram their exposure to antibiotics can rapidly eliminate organisms in CSF or alter with (infection meningitis bacterial laboratory. Although microscopy is generally positive for patients with by be improved concentrating the specimen by centrifugation when it is can received in the microscopy of sensitivity The container. sterile a in laboratory the to submitted be should tests (NAA) amplification acid nucleic and testing, microscopy, antigen for fluid additional and collected, then aerobic is and anaerobic cultureblood fluid bottles should inoculated,be peritoneal or pleural of quantity large a If culture. 5 than more mycobacterial 2 infection is suspected, larger to volumes of fluid, 1 preferably only require may culture routine and staining Gram fluids. pericardial and CSF,synovial, with a problem not a pose generally may but is fluids, peritoneal This and pleural tests. with problem requested the for collected be should testing antigen and microscopy toshould the clinician. directly called be An adequate quantity of fluid of results the and priority, receive 196 then the laboratory should be notified so testing can be optimized. be can testing so notified be should laboratory the then of the specimen occasionally occurs. If a specific organism is suspected, processing and collection during contamination and because significance significant real considered assessing in be required is judgment must clinical immediately, reported fluids these from growth any should be carefully selected and prioritized a to maximize only yield. Although if addition, requestedtests the obtained, then be canquantity limited specimen of In performed. is culture and microscopy before volume of fluid is collected, it shouldbe concentrated by centrifugation large laboratory. a the If submitted to be fluids these of swab a should ing anaerobes, may be present in the specimen. Under no circumstance andand peritonealfluid collected, may be multiple organisms, includ is infection and available, is causedbyorganism. single a In contrast, large volumes offluid pleural fluid of volume small a only generally Processing synovial fluid and pericardial fluid is similar to CSF— to similar is fluid pericardial and fluid synovial Processing Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. Nocardia,(e.g., Mycobacterium media specialized or )

mL for each test, increase the likelihood of successful successful of likelihood the increase test, each for gop B] [group agalactiae Streptococcus (neuroborreliosis). A multi- A (neuroborreliosis). burgdorferi Borrelia For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.

mL eoye B serotype meningitidis Neisseria s h ecpin, prior exception), the is Listeria H. influenzae type B conjugated of fluid; however, if fungal or fungal if however, fluid; of 17a rather than assesses agree assesses than rather ts Treponemapallidum Cryptococcus neoforCryptococcus Streptococcus , Lepto - - - - - ) for nucleic acid amplification.acid nucleicfor for specimens calciumalginate swabsavoided should submittedisbe specimen the if collect to used be can swabs alginate calcium or rayon, Dacron, choice. of test the is amplification acid nucleic media, specialized on cultured be Bordetellacan pertussis testing is required to demonstrate diphtheria toxin. althoughLikewise, for tiveshouldused recoveryof media the be specialized organisms, culture or media alternative other test are methods For used. example, selec for required; is procedure testing ized Forsome organisms (i.e., than otheragents for tests because tularensis, haemolyticum, universally susceptible to penicillin. to susceptible universally routinelynotis testingondone susceptibility Antibiotic immunoassays. of generation new the with unnecessary be may assay, this acid althoughnucleic or culture tional tradi a with confirmed be negativeshouldantigentest a that mended significant be still low may organisms of very numbers therefore, process; sampling the of effectiveness the to ing ered in patients with disease, the quality of the specimen varies accord it would be thatexpected large numbers of organisms should be recov Although tests. antigen these of sensitivity the improved significantly matographic immunoassays, interpreted with small chro digital readers, has flow lateral of introduction recent The loworganisms. of very numbers detecting for especially sensitivity, their in vary tests these sive, and rapid, with results available within 10 to 20 minutes; inexpen however, relatively specific, are they because used widely are [EIAs]) immunoassays (enzyme tests Antigen hour. 1 approximately in able availareresultsand methods, culturecompared accuratewith highly of detection rapid the for used are tests antigen direct and assays NAA organism. scarlet fever–like rash, but a most laboratories do and Likewise, not screen for this pharyngitis uncommon isolates. cause these can haemolyticum reporting Arcanobacterium and for screening from ratories glomerulonephritis,labo deterredhaspoststreptococcal of associated the lack of proven rheumatic fever sequelae, as well as only agents.rare reports other for G and C group look to submitted that recognized is is it Although request specific a unless for processed only are be also collected. may biopsies lung open and aspirates lung Fine-needle sies. biop - and brushings, lavages, bronchial and aspirates, tracheal sputa, esd for cessed pro be can specimens tract lower contaminated ideal, not Although oral secretions (identified bythe presence ofsquamous epithelial cells). with contaminatedare expectorated that culture bacterial reject forsubmitted specimens and screen to required are laboratories because of contamination. Care should be taken to submit the proper specimen risk the increase only will cul specimens expectoratedrepeated and tures, bacterial for sufficient is specimen coughed single A container. cleanintolower herchesta directlyorexpectorate secretionsfromhis and deeply cough to patient the instructing by sputa expectorated in of minimized contamination be can however,aspirates; this to tracheal avoid and sputa induced difficult is It specimen. the contaminate can airways upper the colonizing pathogens because assess to difficult sometimes is isolatedarepathogensthat recovering potential of cance - signifi the obtained,readilyare specimens these Although infections. respiratorytract lowerof submitteddiagnosis forspecimens common organism. this immunochromatographic assaysavailableforare culturedalso forbacteria rapid the andof detection swabs nasal screening for tests NAA produced Commercially inoculated. are media agar the before broth are relatively insensitive unless the swab is incubated in an enrichment all although purpose, this for developed been have media differential Staphylococcus aureus (MRSA) using nares swabs. A variety of selective, f te aet o paygts r suh (e.g., sought are pharyngitis of agents other If pharyngitis bacterial with patients from submitted swabs Throat Expectorated and induced sputa and tracheal aspirates are the most Some laboratories screen high-risk patients for methicillin-resistant ) the laboratory must be notified be must laboratory the pneumoniaeMycoplasma) in throat swabs. The nucleic acid assays are assays acid nucleic The swabs. throat in pyogenes S. Legionella, 20,21 Neisseria gonorrhoeae, Corynebacterium diphtheriaegonorrhoeae,CorynebacteriumNeisseria -hemolytic streptococci) may also cause pharyngitis, cause also may streptococci) β-hemolytic (group A (groupStreptococcuspyogenes S. dysgalactiaeS. , Nocardia, 19 (members ofStreptococcus dysgalactiae(members because all isolatesremain all pyogenes because S. are not routinelydone.not are pyogenes S. ad molds and Mycobacterium, A. haemolyticumA. nospecial ) C. diphtheriae,C. and toxin It is currently recom currently is It . utr, but culture, Bordetella . dysgalactiae, S. Streptococcus) , A. F. 18 ------

Chapter 16 The Clinician and the Microbiology Laboratory

E. 197

E. coli,

E. coli, Continued spp., rotavirus C. difficile adenovirus F E. coli, Clinical

N. gonorrhoeae, C. enteropathogenic + respiratory syncytial virus Shiga toxin–producing gene, S. pneumoniae, Micrococcus /enteroinvasive Shigella/enteroinvasive mecA, vanA, vanB Parasites

Salmonella, Shigella, Campylobacter, , M. kansasii, M. avium, M. intracellulare, , Bordetella pertussis, Chlamydia and E. coli,

HPV CMV, enterovirus, EBV, HSV-1 and enterovirus, HSV-2, HHV-6, EBV, CMV, + respiratory syncytial virus iruses, E. coli K1, H. influenzae, L. monocytogenes, N. V S. aureus, S. lugdunensis, S. epidermidis, Streptococcus

C. difficile, Salmonella, Campylobacter, Shigella, E. coli, Giardia, Entamoeba, Cryptosporidium human papillomavirus, HIV-1, HCV HIV-1, HBV, HCV HIV-1, HBV, Campylobacter, Clostridium Campylobacter, difficile, Plesiomonas shigelloides, Streptococcus (mecA, vanA/B, KPC) Streptococcus, Enterococcus vanA Streptococcus, Clostridium difficile Streptococcus, C. difficile Cryptococcus neoformans/gattii Fungi,

E. coli, N. gonorrhoeae, C. trachomatis, Trichomonas Streptococcus, S. anginosus group, Listeria spp., and resistance markers , M. gordonae, Mycoplasma/Acholeplasma/Ureaplasma, Blastomyces Streptococcus, S. pneumoniae, Staphylococcus aureus, Listeria E. coli, Shiga-like toxin-producing Staphylococcus spp., Gardnerella vaginalis, vaginalis Trichomonas Campylobacter, Salmonella, Campylobacter, Shigella, Vibrio, Yersinia, Bacteria,

CMV, EBV, BK virus, EBV, HHV-6, HHV-7 CMV, , human metapneumovirus, adenovirus, influenza virus (A/H1, A/H3, A/H1N1 2009), , influenza virus (A, A/H1N1 2009), influenza virus (A, B) of

parainfluenza virus (1-3), influenza A/B metapneumovirus, rhinovirus, adenovirus) A, Norovirus Norovirus, Norovirus, rotavirus resistance genes Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, 40/41, astrovirus, Norovirus GI/GII, rotavirus A, sapovirus Candida, 3 resistance genes meningitidis, S. agalactiae, S. pneumoniae, human parechovirus, VZV, Pseudomonas Pseudomonas aeruginosa; aeruginosa/Klebsiella E. pneumoniae; coli/P. Candida albicans/C. glabrata; C. albicans/C. parapsilosis/C. krusei/C. glabrata/C. tropicalis monocytogenes, Mycobacterium tuberculosis complex M. avium complex dermatitidis, Coccidioides immitis, Histoplasma capsulatum spp., groups A and B Shiga toxin–producing metapneumovirus, human rhinovirus/enterovirus, influenza virus (A, A/H1, A/H3, A/H1-2009, B), parainfluenza virus (1-4),respiratory syncytial virus, pneumoniae, Mycoplasma pneumoniae Salmonella, enterocolitica, Yersinia Vibrio, enteroaggregative E. coli, enterotoxigenic enterotoxigenic trachomatis, influenza virus (A, A/H1-2009, B), enterovirus,respiratory syncytial virus, faecalis, E. faecium, HSV 1/2 C. difficile C. difficile, N. gonorrhoeae, C. trachomatis N. gonorrhoeae, C. trachomatis, Trichomonas, C. difficile Respiratory virus panel: influenza virus (A, A/H1, A/H3, B),respiratory syncytial virus, human Gastrointestinal pathogen panel: C. difficile Respiratory virus panel: influenza virus (A, H1, H3, H1N1 2009, B),respiratory syncytial virus (A, B) Enteric panel: Blood culture identification panel: 12 gram-positive bacteria, 8 gram-negative bacteria, 9 N. gonorrhoeae, C. trachomatis, Blood culture identification panel: 8 gram-positive bacteria, 11 gram-negative bacteria, 5 Multiplex meningitis/encephalitis panel: MRSA, MRSA/MSSA, group B HCV HIV-1, HBV, CMV, HCV HIV-1, HBV, HCV HIV-1, HBV, West Nile virus Multiplex: HIV-1 (M, HCV O), HIV-2, HBV, MRSA C. difficile TARGETS Staphylococcus aureus/CNS; Enterococcus faecalis/other Enterococcus; Escherichia coli/ Candida spp., Direct from specimen: group A Culture ID: group B Neisseria gonorrhoeae, Chlamydia trachomatis Blood culture ID: N. gonorrhoeae, C. trachomatis, MRSA, MRSA/MSSA, group B MRSA, MRSA/MSSA, group B Multiplex enteric parasite panel: Bacillus anthracis, Francisella tularensis Multiplex respiratory panel: adenovirus, coronavirus (HKU1, NL63, 229E, OC43), human Muliplex gastrointestinal panel: C. difficile M. tuberculosis

Detection

for

C. difficile ests T Anyplex ICEPlex BD ProbeTec BD Viper xTAG Verigene Prodesse Amplicor AmpliPrep/COBAS GeneXpert AmpliScreen COBAS TaqMan COBAS TaqScreen LightCycler Lyra Direct IMDx PRODUCT Affirm VPIII AccuProbe PACE Verigene PNA FISH Simplexa BD GeneOhm BD MAX RealTime PCR FilmArray Xpert MTB/RIF

(Gaithersburg, MD) MA) (Northbrook (Northbrook IL) Diagnostics (Pleasanton, CA) CA) CA) (Waltham, MA) (Sparks, MD) (San Diego, CA) (Northbrook, IL) MA) Diagnostics (Abbott Park, IL) Salt Technology; Lake UT) City, (Cypress, (Cypress, CA) Seegene PrimeraDx (Mansfield, BD Diagnostics Luminex (Austin, TX) Nanosphere Nanosphere Hologic Gen-Probe Roche Molecular Cepheid (Sunnyvale, Quidel (San Diego, IntelligentMDx BD Diagnostics Hologic Gen-Probe Nanosphere AdvanDx (Woburn, BD Diagnostics Abbott Molecular BioFire (formerly Idaho Focus Diagnostics MANUFACTURER Nucleic 16-4

Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. 16-4 , streptococci, staphylococci). Introduction of Introduction staphylococci). streptococci, Lactobacillus, M. tuberculosis, Pneumocystis jirovecii, M. pneumoniae, Nucleic MANUFACTURER Meridian Biosciences Meridian (Valencia,CA) Qiagen Healthcare Siemens Healthcare Siemens Gen-Probe Hologic Great Basin Scientific Basin Great (Lyon, bioMérieux (Cincinnati, OH) (Cincinnati, Diagnostics (Tarrytown,NY) Diagnostics UT) ValleyCity,(West France)

Acid–Based einla M pemna, Chlamydia, pneumoniae, M. Legionella, rRNA, ribosomal ribonucleic acid; VZV,virus. acid; varicella-zoster ribonucleic ribosomal rRNA, aureus; Staphylococcus For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. ; DNA, deoxyribonucleic acid; EBV, Epstein-Barr virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV,virus; HCV,C virus; hepatitis HBV,B EBV,virus; hepatitis acid; Epstein-Barr deoxyribonucleic DNA, Staphylococcus;

Illumigene Digene VERSANT VERSANT Aptima MTB Amplified PRODUCT Portrait NucliSens T ests 22

The value of this of value The for . pneumoniae, S.

Detection C. difficile, C. trachomatis, C. gonorrhoeae, N. HCV HIV-1, HCV trachomatis, C. gonorrhoeae, N. tuberculosis M. TARGETS C. difficile C. HIV-1

------ of group A group

Bacteria, complex in f re rnr tat neto my eur mr invasively more require may infection should culture tract mycobacterial for collected Urine specimens. obtained urinary true of tion yeasts determina so isolate, the the of significance the assessing in of useful not is Quantitation cultures. urine midstream routine from particularly significant. Yeast, ered ofcolonycounts populations,high nonenterococcal diphtheroids, lactobacilli, includedisregarded generally are thatnants and Enterococcus, Pseudomonas, Proteus, include organisms rapidly.These collected randomlyurines. with insensitive generally is methods rapid other screening of use Likewise, specimens. these for method screening ed ih h ol meso ln; n o mr ognss e oil per organisms more 10 or to equivalent one are field lens; immersion immersion is oil slide the The staining. with then read and drying, air slide, a onto urine fuged uncentri well-mixed, of devices. drop a placing filtration by prepared is stain and Gram A bioluminescence, methods, dipstick as such products, available commercially of variety a and stains Gram direct procedures. invasive other by or cystoscopy atrecommendation obtained specimens This to applies also reported. and such identified is as growth labeled all that clearly so be should suspected, is infection anaerobic an or difficult is urine clean-voided of assessment whom in patients other or infants for in laboratory primarily outside done an aspirates, Suprapubic to processing. shipped be to going impractical is is specimen this the although if done, be also can specimen the of tion Refrigera acid). boric (e.g., overgrowth with tubes bacterial inhibit specific that to agents transferred is urine the possible, not is this if or, collection of hours should 2 within Laboratories occurs delivery laboratory. specimen that ensure the to sent be should urine voided or gonorrhoeae N. by caused urethritis of diagnosis the for urine of collection is rule this to exception one The which flora. urethral specimen, the with contaminated catheterized be would or voided the of portion first the ing discard include would This bacteria. with contamination minimize be be greater than 50 staphylococci other than ae 10 have randomlyfromspecimens patientan frequentlyinfected collected will rn seie wl dces b 10-od r oe rm h first- the fromevening. the in more collected ones to specimen or morning voided 1000-fold by decrease will specimen urine a in organisms of number The flora. genital normal with tamination con indicates usually cells epithelial squamous of a amount or moderate types bacterial mixed of presence The (CFU/mL). milliliter per otntl, h ms cmo uiay rc ptoes grow pathogens tract urinary common most the Fortunately, ai sreig ehius o uiay rc ifcin include infection tract urinary for techniques screening Rapid group B group Streptococcus, 4 F/L r ee, rm tiig s rltvl insensitive relatively a is staining Gram fewer, or CFU/mL

Fungi, HPV HCV,HIV-1, HPV,

hmltc tetcci ad coagulase-negative and streptococci, α-hemolytic

mL . In this situation, the first- the situation, this trachomatisIn Chlamydia . V iruses, of a first-voided specimen, and a similar volume Streptococcus, B. pertussis, M. pneumoniae M. pertussis, B. Streptococcus, Staphylococcus saprophyticus. Trichomonasvaginalis

and Escherichia, Klebsiella, Enterobacter,Klebsiella,Escherichia,

may also be consid be also epidermidismay S. species, may be isolated be may species, Candida Parasites 5 r oe ooyfrig units colony-forming more or Contami- Staphylococcus.

Clinical Staphylococcus In some patient 23 Because Because

------

TABLE 16-5 Methods for the Detection of Selected Bacteria in Clinical Specimens* DETECTION METHOD† Antigen Nucleic Acid Antibody BACTERIA Microscopy Culture Detection Detection Detection COMMENTS Acinetobacter A A C C C Microscopy and culture tests of choice; characteristic microscopic morphology useful for preliminary identification Actinomyces A A C C C Microscopy and culture tests of choice; extended incubation in anaerobic atmosphere required for isolation Aggregatibacter A A C C C Extended incubation may be required for isolation in culture Anaplasma B C C A A NAA test of choice during active infection; serology phagocytophilum positive in >90% of patients but delayed response; Giemsa stain of buffy coat smears positive in 60% of patients Bacillus anthracis B A C B B Microscopy and culture used in most laboratories for initial detection; NAA tests commercially available (sensitive and specific); serologic assays for antibodies vs. protective antigen useful in culture-negative patients Bartonella B A C B A Microscopy only useful for early stage of CSD and in patients with bacillary angiomatosis; culture useful primarily for immunocompromised patients with bacteremia; direct immunofluorescent antibody tests used for patients with acute trench fever; NAA tests primarily used in reference laboratories; serology (IFA, EIA) test of choice for CSD, endocarditis, and bacteremia Bordetella pertussis B A C A A Microscopy (DFA) insensitive and nonspecific (if polyclonal antibodies used); culture insensitive and requires use of specialized media and prolonged incubation; NAA test of choice, sensitive and specific if appropriate controls used; serology (ELISA) useful in unvaccinated children if increases in IgG or IgA to pertussis toxin or filamentous hemagglutinin demonstrated Borrelia B B C A A Culture generally not performed except in laboratories with experience; microscopy useful only for relapsing fever—test of choice; NAA test used in reference laboratories for diagnosis of Lyme disease; serology test of choice for Lyme disease—patients screened with EIA or IFA and confirmed with immunoblotting Brucella B A C C A Culture is a sensitive test if prolonged incubation is used (e.g., 2 wk); microscopy generally not useful; antigen tests and NAA tests not available except in reference laboratories; serology helpful adjunct to culture; a significant rise in antibodies or titer ≥160 is highly suggestive but cross-reactivity occurs (e.g., Francisella, Vibrio, Yersinia) Burkholderia B A C C C Culture on selective media test of choice; microscopy cepacia complex sensitive but nonspecific Burkholderia B A B B B Culture test of choice; microscopy sensitive but pseudomallei nonspecific; antigen tests available with sensitivity 60%-85%; NAA tests and serology restricted to reference laboratories in endemic areas Campylobacter spp. B A A B C Culture is sensitive if fresh media are used; thin organisms difficult to detect by microscopy; commercial antigen tests sensitive (80%-95%) and specific; serology primarily restricted to epidemiologic investigations Chlamydia B C B B A Serology (MIF) is test of choice (positive IgM titer ≥16; IgG pneumoniae titer ≥512); NAA test sensitive and specific but not commercially available; microscopy (Giemsa, DFA) insensitive Chlamydia psittaci B C B B A Serology (MIF) is test of choice; NAA test sensitive and specific but not commercially available; microscopy (Giemsa, DFA) insensitive Chlamydia B B B A C Commercial NAA test of choice; culture sensitive but trachomatis infrequently used; microscopy and antigen tests have low sensitivity (70%-80%); serology (MIF test) sensitive and specific but increase in titer may not be detected in chronic or recurrent disease Clostridium B A B C C Diagnosis by clinical presentation and culture from food botulinum or stool/wound, or detection of toxin in serum, stool, or food; toxin assays insensitive (<60% positive) except in children (>90%) Clostridium difficile B A B A C NAA amplification of the toxin genes is the test of choice; culture is sensitive but additional tests required to determine toxin production; recent studies show antigen tests for toxins are insensitive Clostridium tetani B B C C C Diagnosis primarily by clinical presentation: culture and microscopy commonly negative (>50%) Continued

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Part I Basic Principles in the Diagnosis and Management of Infectious Diseases 200 Leptospira pylori Helicobacter Haemophilus Mycoplasma Mycobacterium Mycobacterium Listeria Legionella Klebsiella Kingella Haemophilus Francisella coli Escherichia Erysipelothrix Eikenella ewingii Ehrlichia chaffeensis Ehrlichia burnetii Coxiella Corynebacterium BACTERIA TABLE influenzae pneumoniae spp. other tuberculosis monocytogenes granulomatis ducreyi tularensis rhusiopathiae diphtheriae Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. 16-5 , Methods B C A B B C B Microscopy C A A A C A A B B A B A for For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. the B A B B A A A A C A A A A A A C C C A Culture Detection DETECTION A B C Antigen B C B C A C C C C B C C C C C C Detection of METHOD Selected B B B C B B C A A B B Detection Nucleic A B A C A C C C † Bacteria Acid A A C A C C C B A A C Detection Antibody A C A C B C C C in Clinical Culture requires specialized techniques with organisms with techniques specialized requires Culture (not conditions incubation specialized requires Culture for tests antigen choice; of tests culture and Microscopy Culture on selective media with prolonged incubation and incubation prolonged with media selective on Culture tests antigen tests; common most culture and Microscopy insensitive; generally microscopy choice; of test Culture extended choice; of tests culture and Microscopy serology specific infection; active for choice of test NAA (IFA)serology infection; active for choice of test NAA fourfold fever: Q acute choice; of (IFA)test Serology for test NAA choice; of tests culture and Microscopy COMMENTS NAA test of choice but not commercially available; commercially not but choice of test NAA performed; commonly most culture and Microscopy skin performed; commonly most culture and Microscopy probes molecular choice; of tests culture and Microscopy urine DFA); (including recommended not Microscopy choice; of test stain) Gram not stain, (Giemsa Microscopy extended choice; of tests culture and Microscopy ( insensitive Microscopy Specimens performed test; microagglutination titer microagglutination test; performed frequently most serology observer; experienced requires and insensitive (darkfield) microscopy thereafter; urine and illness of week first during blood in isolated 1 for persist may IgM and slowly develop may antibodies sensitive, less serology specific); and (sensitive choice other for pneumophila ( specific and sensitive antigens fecal for tests ELISA choice; of test detection antigen histopathology; to restricted microscopy performed); commonly laboratories reference in only available tests NAA child; vaccinated in useful less CSF in antigen capsular b serotype culture for media differential selective, detected within 1 within detected antibodies available; widely not tests NAA and antigen and insensitive, microscopy choice; of tests serology laboratories reference in available pathogens enteric for tests NAA specific; and sensitive toxin Shiga for laboratories reference in available tests NAA required be may incubation serology with reacts cross but available not IgG or titer in increase 4-fold serology: positive test; common most available commercially not but sensitive tests NAA antigen; I titer IgG titer IgM or antigen, II phase to titer increased laboratories reference in available is specimens clinical in directly and isolates in toxin diphtheria of detection may be delayed; cold agglutinin test not recommended not test agglutinin cold delayed; be may seroconversion used; commonly most (EIA) serology isolates cultured identify to used probes molecular isolates cultured identify to used assays probe molecular specific; and sensitive are specimens clinical in detection direct for tests NAA countries; high-risk in primarily available assays antigen tests; hypersensitivity delayed common most are QuantiFERON-TB) interferon- and (PPD) test isolates of identification for available delayed be may seroconversion and disease of stages early in insensitive is serology diagnostic; seroconversion ( sensitive tests antigen media conventional on grow not does required be may incubation insensitive) (too recommended not tests IgA practices; office in used are that tests IgG whole-blood than sensitive more tests IgG serum used; widely also tests breath urea (agglutination titer elevated an or seroconversion so years, to months yr or more or yr ≥ Legionella; 200; chronic Q fever: IgG titer IgG fever: Q chronic 200; ≥ serogroup 1 but insensitive or negative or insensitive but 1 serogroup 64 * ≥ 128) required 128) —cont’d wk of symptoms but persist for persist but symptoms of wk culture and NAA tests tests of tests tests NAA and culture < 50%) and nonspecific; use nonspecific; and 50%) > 80% for 80% γ assays (e.g., assays Legionella E. chaffeensis E. ≥ ≥ 200 or 200 800 to phase to 800 > 90%); ≥

50 and 50 Chapter 16 The Clinician and the Microbiology Laboratory 201

serotype 1. S. aureus and detection S. dysenteriae *—cont’d antigen tests used to detect group-specific antigen in specimens are not recommended 60%); (sensitivity, commercial NAA tests detect organism in specimens NAA tests for identification of of MRSA in clinical specimens Shiga toxin useful for test) insensitive and nonspecific—notrecommended seroconversion seroconversion delayed; NAA test sensitive but not commercially available but seroconversion delayed; NAA test sensitive but not commercially available sensitive; direct antigen tests provide rapid, sensitive (90%-100%) diagnosis incubation generally required for isolation incubation may be useful antigen are available in endemic areas; NAA tests available in reference laboratories incubation may be required incubation generally required for isolation sensitive in primary syphilis; NAA tests recently introduced for primary and secondary disease (sensitive and specific); serology is most commonly performed test and test of choice; nontreponemal and treponemal tests are sensitive and generally specific (refer to text) tests for capsular polysaccharides serum, in and CSF, urine less sensitive than microscopy and cross react with E. coli K1 antigen tests used to detect group-specific antigen in specimens 80%); (sensitivity, commercial NAA tests detect organism in specimens; serology used for diagnosis of poststreptococcal disease microscopy microscopy and culture are less sensitive compared with NAA antigen tests used to detect C polysaccharide in urine 50%-80%); (sensitivity, commercial NAA tests only for identification of isolates in culture Most laboratories also guidelines laboratories have that Most limit Specimens 24 Microscopy Microscopy and culture most commonly used tests: Microscopy Microscopy and culture tests of choice Microscopy Microscopy and culture most commonly used; commercial Microscopy Microscopy and culture tests of choice; antigen tests for Microscopy Microscopy and culture tests of choice; serology (Widal Serology Serology (IFA) using typhus group antigens sensitive but Serology Serology (IFA) using spotted fever group antigens sensitive culture, antigen Microscopy, tests, and NAA tests all Microscopy Microscopy and culture tests of choice; extended Microscopy and culture tests of choice; extended Microscopy Microscopy and culture tests of choice Microscopy and culture most common tests; tests for O1 Microscopy Microscopy and culture tests of choice; extended NAA test of choice, available in reference laboratories; Microscopy Microscopy and culture tests of choice; extended Unable to grow in culture; darkfield microscopy and DFA Microscopy Microscopy and culture tests of choice; antigen detection Microscopy and culture most commonly used tests: NAA test of choice; high sensitivity and good specificity; Microscopy and culture most commonly used tests; COMMENTS

Clinical

in C C C C C A A C

C C C C C C C A C B C C Antibody Detection

- Recommen quickly. laboratory the to transported be should and swabs minimizing unnecessary for rejection dations of include stool cultures bacterialroutine stool in cultures who patients beenhave hospitalized 3 for days or longer. specimen submissions to one per day and no more than three specimens specimens three than more no and day per one to submissions specimen gastroenteritis. acute of screening initial for days) successive (on Acid

Bacteria

† A C A B B B B A C B C B C A C B C A B A Nucleic Detection

Selected METHOD

C C C B C C C A C C C B C C C C B A B C Detection Antigen DETECTION Detection

A A A A A C C A A A A A A C A C A A A A Culture the

for

For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Cryptococcus, Cryptococcus, , Blastomyces, Histoplasma or A A A B B B B A A A A A A B A B A A A A Microscopy Methods 16-5

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada agalactiae (group B) maltophilia aureus prowazekii, R. typhi (typhus group) rickettsii (spotted fever group) enterocolitica whipplei pallidum meningitidis pyogenes (group A) pneumoniae gonorrhoeae A, Test A, is Test generally useful for the indicated diagnosis; B, test is useful under certain circumstances or for the diagnosis of specific forms of infection; C, test is seldom † CSD, cat-scratch cerebrospinal disease; fluid;CSF, DFA, direct fluorescent antibody test; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay;IFA, TABLE Streptococcus Streptococcus Stenotrophomonas Staphylococcus Shigella Salmonella Rickettsia Rickettsia akari, R. pestis Yersinia Rhodococcus equi Yersinia Pseudomonas Vibrio spp. Pasteurella spp. Tropheryma Nocardia spp. Treponema Neisseria Streptococcus BACTERIA Neisseria *Organisms not listed are generally detected primarily by culture and microscopy. . Coccidioides or or greater for recovery of Gastrointestinal Tract Gastrointestinal Specimens Tract bacteria, including etiologies, of variety wide a have can diarrhea Acute viruses, and parasites. Stool specimens are preferred to rectal useful for general diagnostic purposes. indirect fluorescent antibody test; IgA, IgG, and IgM, immmunoglobulin A, G, and M, microimmunofluorescence;respectively; MRSA, MIF, methicillin-resistant Staphylococcus aureus; NAA, nucleic acid amplification (includes polymerase chainreaction); PPD, purifiedprotein derivative. Part I Basic Principles in the Diagnosis and Management of Infectious Diseases utr mda s el s irsoy atgn et, n NA tests. NAA and tests, antigen microscopy, as well as media differential culture selective, of variety a of use the requires specimens fecal comprehensiverequired.antigenofNAAA or maybe tests workup of use the or methods culture selective because suspected are pathogens and Salmonella, the for look to minimum a at include should which agents, setup common a “routine” have laboratories all and defined, motn i te rgnl tde dmntaig hs raim in organism this demonstratingserology. or testingantigen by made is now diagnosis biopsies, gastric studies were original culture the and in microscopy important Although organism. this of absence or presence the of verification diagnostic for need the in resulted has the nonenterococcal with the enteric carrying bacteria reactivity however, specific; and genes sensitive the be detecting for available resistance this for areresponsible tests NAA commercial tive, of a commercial toxin-specific NAA test with high sensitivity and sensitivity high with specificity. test NAA toxin-specific commercial a of use is choiceof test diagnostic current the multisteptesting, with ated sensitivityingtestmustinherent andhigh the have delays associ very GDH sensitivity, the to screen a Because fromto100%. 75% reportedtovary hasbeen which relates approach this with controversy The positive GDH assay must confirmedbe with a toxin-specific NAA test. for marker C. difficile as well as some other bacteria. Because GDH is not a unique for glutamate dehydrogenase (GDH), a cytoplasmic enzyme present in and nonspecific and should not insensitive be performed. Controversy are surrounds use of assays immunoassays these but cytotoxin, and the of detection for available commercially are Various practical. immunoassaysclinically not is and days 5 to up for and enterotoxin cytotoxin; ofhowever, this assay production(“toxigenic culture”) demonstrate requires incubation and feces of culture by organism the isolate to is standard gold diagnostic The problematic. agar) supplemented with 6 supplementedwith agar) enterotoxigenic other for able tent with pathotype) confirmatorylaboratory tests are Bacterial notwidely avail isolates. culturedconsis - toxicity cellular of assessment the on (for culture cell and culture or be specimens can stool on (ELISAs) directly Commercially used assays toxin. immunosorbent Shiga the enzyme-linked of available detection or agar) sorbitol- (i.e., MacConkey media selective on culture by be can organism this of ofDetection colitis, stoolspecimen. frequently the in grosswith blood is which of common most the andlaboratory. the in examination specimen the of collection between delays during degraded are lactoferrin, not but cells, The markerforleukocytes. a as lactoferrin of Gram Although stains can to a used be moredetect leukocytes, reliable test is detection parasites). viruses, bacteria, toxin-producing (e.g., Campylobacter,Shigella,enteroinvasive (e.g., disease diarrheal inflammatory invasive between differentiating collected. is specimen the after medium transport Cary-Blair in mixed or laboratory the to sent of quantity immediately be an insufficient should Feces submitted. be not should and provide specimen swabs rectal Thus, used. be not mayororganisms may ofnumbers small detect brothstoEnrichment 202 (VRE). Selective media, such as bile azide esculin agar (Enterococcosel vancomycin-resistantenterococci with colonization fecal for patients previously recognized. community.Thus, the in organism the to exposed were who or hospitalization a during ously found in the community, either in individuals who were infected previ also are organism this of strains addition, In gastroenteritis. bacterial later). pathogenic h setu o bceil irha ptoes s eaiey well is relatively pathogens diarrheal bacterial of spectrum The The discovery of Shiga toxin–producing enterohemorrhagic for useful leukocytes of presence the found have physicians Some Some hospitals have established a program of screening high-risk high-risk screening of program a established have hospitals Some Clostridium difficile is the most common cause of hospital-acquired test. vanB Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. 26-28 and does not determine if toxin is produced, a produced, is toxin if determine not does and difficile C. EE] enteroaggregative [EPEC], coli E. strains responsible for gastroenteritis (including gastroenteritis for responsible strains coli E. [ETEC], enteroinvasive[ETEC], coli E. . The laboratory should be notified if other notified be should laboratory The Shigella. Helicobacter pylori and its association with gastritis 25 Documentation of disease is more widespread than widespread more is disease difficile C.

g/mL vancomycin, are effective. Alterna effective. vancomycin,are µg/mL vanB gene has compromised utility the of . The vanB). (vanA, For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. O157:H7, is an important cause important an is O157:H7, coli E. ) and secretory diarrhea secretory and coli) E. C. difficile has disease been E. coli (EHEC or STEC), [EIEC], entero- [EIEC], coli E. enterotoxindifficile C. [EAEC]) (see (see [EAEC]) coli E. test appears to appears test vanA Campylobacter, - - - - -

organism is suspected. media and extended incubation. Thelaboratory mustbe notified if this pathogen. likely the upon based tests and specimens appropriatediagnostic the associated with specifictypes be of genital infection;thus, to the physician known should select are pathogens Certain swabs. anorectal and vaginal or penile discharge, urine, genital ulcers, includeand urethral, cervical, specimens submitted Commonly granulomatis). bacterium] trachomatis, Haemophilus ducreyi, T. pallidum, Klebsiella [Calymmato (e.g., pathogens sexually-transmitted (e.g., exogenous population endogenous caused this be of may members infection by tract Genital including yeast. and organisms, anaerobes, cocci, many contains normally strepto - coagulase-negativecorynebacteria, staphylococci, lactobacilli, tract genital The Specimens Genital of bacteria, including bacteria, of mixture a proliferationof a the and lactobacilli in in decrease populationa with vagina bacterial normal the of disruption a from results because harbor women asymptomatic infection many candidal of diagnostic necessarily not is Candida of culture Although pseudohyphae. and yeast budding many show that smears this chapter. Vaginal candidiasis is most frequently diagnosed by direct vaginalis,monasCandida, of clinical importance remains in the hands of the clinician. the of hands the in remainsimportance clinical of EPEC, ofbasis accuratethe identification pathogen. the of determinationThe as such detectable, are andEAEC, ETEC,aswell asSTEC and others. Assays are approved now on practice clinical in ferentiated dif been commonly not have that pathogens because process disease the to result the of applicability the the determining for in remains challenge clinician The possible. now is platform PCR-based single a protozoan(see parasites)and bacteria, (viruses, gens recent or past of evidence provide infection. to used widely also is pylori H. to antibodies specificity (IgG) G and immunoglobulin sensitivity serum of high Detection a (>90%). have and specimens fecal in gens detecting for available are immunoassays Commercial have also been replaced by NAA tests, and tests for both both for tests and tests, andgonorrhoeae NAA by replaced been also have matis cultureAttempts to specimens. oropharyngeal or anorectal with used be also can tests NAA most and Culture delays. transport aretestslaboratoriesmore the sensitivebecause andnot influenced by most in culture replaced have tests NAA swabs. on laboratory the to should andnever be refrigerated. Specimens media should also not transport be appropriatetransported in laboratory the to promptly sent be should is specimen the then performed, organism is culture therefore,if labile; This epididymitis. and urethritis, salpingitis, cervicitis, because of the presence of other organisms, which can decrease the the specificity.test decrease can which organisms, other of presence the of because reliable remainsless be from to thought aredischarge specificity cervical of stains Gram specimens good. the although urethral examined, when are men decreases asymptomatic sensitivity The 95%. as The high as are setting discharge. this in smears urethral urethral of specificity and sensitivity of stain Gram a examining by made be can men. A rapid presumptive diagnosis of gonorrhea in symptomatic men acteristic fishy amine odor after addition of potassium hydroxide potassium of diagnosis. addition this support to used also (KOH)are after odor amine fishy acteristic char the and 4.5, greaterthan discharge,pH a the characterof watery ( cells epithelial on cobacilli (i.e., bacteria fusiform or curved and absent are lactobacilli) replaced with a prevalence of (i.e., delicate gram-negative and gram-variable rods gram-positive predominant normally the when vaginosis bacterial suggests secretions vaginal of presumed slow,pathogensdifficult, is unnecessary. and staining Gram these of identification and growth because culture bacterial on based Prevotella,and aiii wt acmayn dshre a b cue by caused be can discharge accompanying with Vaginitis The rapid andsensitive detection of multiple gastrointestinalpatho s mjr ahgn n eia ifcin sc as such infections genital in pathogen major a is gonorrhoeae N. eeto of Detection can be accomplished rapidly and easily, growth of easily,growth and rapidly accomplished be can Candida . The diagnosis of bacterial vaginosis is not is vaginosis bacterial of diagnosis AtopobiumThe . requires the use of specialized specialized of use the requires ducreyi Haemophilus can be performed on the same speci same the on performed be trachomatiscan C. Mobiluncus, Klebsiella granulomatis is an organism that does or bacteria. bacteria. or on “clue”The Gardnerellaon vaginaliscells). - coc gram-variableMobiluncus and ) Mycoplasma hominis, Gardnerella,hominis,Mycoplasma spp. Bacterial vaginosis Bacterial spp. Candida T.in later vaginalisdiscussed is 29 . ooroa, C. gonorrhoeae, N. or agalactiae) S. Tablewith 16-4) anti- pylori H. C. tracho C. Tricho N. - - - -

Chapter 16 The Clinician and the Microbiology Laboratory - - 203

) in blood Cryptococcus) ), and parasites Clostridium and ), and fungi Bacteria,

for

Babesia, Plasmodium Stains

Mycobacterium, Nocardia, Rhodococcus, Pneumocystis Toxoplasma, Table Table ). 16-5 An overview of detection microbiology. Used microbiology. for detection of fungal elements in tissues, although other organisms such as bacteria can be detected and nocardia will partially stain cysts and trophozoites Bacillus cysts and trophozoites and microsporidia Helicobacter, Campylobacter Helicobacter, Pneumocystis, microsporidia) and parasites (e.g., protozoa cysts) primarily to detect viruses in clinical specimens discharge, urine sediment for fungi and parasites; bacteria can be observed using darkfield microscopy and enables detection of fungi Bacillus anthracis) and fungi (e.g., (e.g., Gordonia, Tsukamurella, Legionella micdadei), microsporidia, and parasites (Cryptosporidium, Cyclospora, Cystoisospora, Sarcocystis) Borrelia, Ehrlichia, Anaplasma, Chlamydia), fungi (e.g., (e.g., microfilariae, Performed in histology labs rather than in Differential stain for bacteria and yeasts; mycobacteria Differential stain for detection of microfilariae in blood Differential stain for detection of intestinal protozoa Stain for detection of spores in Differential stain for detection of intestinal protozoa Used to detect bacteria, particularly thin ones (e.g., Used to detect acid-fast and partially acid-fast bacteria Nonspecific fluorescent stain for fungi (including Specific antibody-mediated, fluorescent stain used Used to directly examine aspirates, feces, vaginal KOH digests host cell components (e.g., skin cells) Contrasting dye to detect encapsulated bacteria (e.g., Contrasting dye for detection of protozoa in feces Contrasting dye for detecting bacteria and fungi Used to detect acid-fast and partially acid-fast bacteria Differential stain for detection of bacteria (e.g., APPLICATION Parasites Microscopic

and 16-6

stain (Delafield’s) stain (Tompkins- Miller method) modified trichrome stains stain stain stain antibody (KOH) (Ziehl-Neelsen, Kinyoun, modified Kinyoun) Wright-Giemsa stains TABLE Fungi, Methenamine silver Gram stain Iron-hematoxylin Iron-hematoxylin Spore stain and Trichrome Fluorescent Fluorescent Stains Acridine-orange Auramine-rhodamine Calcofluor white Direct fluorescent STAIN Wet Mount and Contrasting Stains Wet mount Potassium hydroxide India ink iodine Lugol’s Methylene blue Differential Stains Acid-fast stains Giemsa and to to give an immediate assessment of organism load. Because quantita DETECTION METHODS Five general approaches are used to bacte- of detection culture, detect microscopy, infections: specific to response organisms and the host rial antigens or nucleic acids, and against detection of the antibodies organism directed (see methods for bacteria is summarized in this section. Microscopy 16-6 Table is a summary of the stains used to detect bacteria, and fungi, parasites. Bacteria are detected most commonly with differential orange, (acridine stains fluorescent and Giemsa) acid-fast, Gram, (e.g., auramine-rhodamine stains). TheGram stain is a simple,yet reliable stain that can important provide preliminary on information whether a bacterial infection is caused by a gram-negative or a gram-positive organism and whether the organism is a rod or a coccus. An tive tive cultures are time and consuming not labor all intensive, laborato ries have procedures in availability of wound quantitative cultures should be checked by - con place for performing sultation with the laboratory before tests are ordered. these assays. The

------Capno S. aureus, Francisella, Francisella, Pasteurella Pasteurella multocida or infection can pallidum be T. made by Sporothrix Sporothrix schenckii, depending on the For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Bacteroides. Nocardia or mycobacteria are observed in a , among Acinetobacter others. Fungal, mycobacterial, CFU/g of tissue. Such colony counts are correlated 5 Bartonella are sought, the laboratory should be notified Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada Wound Wound infections and abscesses are caused by many different Whenever an ample amount of specimen is available, microscopy tissue or eschar, burn wound, quantitative settings, hospital some In and and anaerobic cultures must be specificallyrequested if theseorgan Specimens Specimens sent to the laboratory include tissue biopsies, aspirates or swabs of abscesses, wound débridement, and swabs, drainage material samples. obtained the of the site by limited is culture and The microscopy these by specimens by usefulness surgical of processing lesion. Lesions that communicate directly with the skin surfaces or mucosal are commonly contaminated with organisms. a For meaningful mixed test results, population laboratories prefer surgically of obtained tissue samples, aspirates of closed abscesses, and an aliquot of pus or fluidbacteria ratherWhen anaerobic than swab are samples. expected, the specimen should be inoculated into an - anaerobic trans port container and delivered promptly to superficial the skin ulcers, from laboratory. theskin Swabs surface of a of sinus tract,or from not yield mixed bacterial often do open abscesses and commonly flora reflect the organisms of true infectiousavoided. significance should and be strictand anaer anaerobic, facultatively aerobic, including organisms, the clinician for is important fungi. It bacteria,and obic mycobacteria, and the laboratory technician to are recognize often that associated certain with organisms particular An types infected of animal wounds bite or may yield abscesses. ; cytophaga a post-traumatic hand Mycobacterium marinum, or infection may yield Pseudomonas or isms are suspected. When fastidious organisms such Brucella, as or so that cultures can be set up appropriately and incubation held for as prolonged needed. Providing the or type laboratory of with wound, abscess, the or tissue location hasten is the recognition often of specific useful pathogens known becauseto it be associated may with a particular type of infection (e.g., abscess) brain cat bite, or ana tomic site. should be performed to obtain some preliminary infecting organism(s). indication If of antibiotic therapy the has been initiated, direct smear may be the only available guide to the etiology because growth may be smears inhibited. are Impression made by gentle pressing of a a freshly cut whichsurfaceslide, the can of then tissue onto be stained specimens of examination Direct fungi. and mycobacteria, bacteria, for by microscopy can preliminary provide about information the quality of the specimen (e.g., epithelial many cells in a wound specimen indi cates skin contamination), guides empirical therapy by the physician, and directs culture strategies for the microbiologist. For example, if organisms resembling additional pro culture, routine for specimen Gram-stained submitted cessing to recover these organisms can be performed. The organisms Dis results. culture with becorrelated should microscopy observed by otherwise not would be that identifyorganism an help may crepancies detected. rods staining faintly gram-negative example, that For do not grow on routine aerobic plates should suggest anaerobic the organism, such as possibility of an likelihood the predict help to usedbeen have cultures specimen biopsy sepsis, of wound burn infection, or the success of skin grafting. Quan of theweighing and requires culture careful specititative preparation men for serial dilutions to determine greater whether than 10 the colony count is Skin, Skeletal, and Soft Tissue Specimens source source of trauma; a postoperative wound infection might yield not not grow on media. conventional Diagnosis is made by observing the commonly more test a is This stain. Giemsa the with tissue in organism performed in the pathology anatomic laboratory rather than a micro Diagnosis biology of laboratory. (must microscopy darkfield collectedby specimen freshly a examining still are spirochetes the while collection after immediately examined be motile), a direct fluorescent antibody test (reagents available), or most commonly by serology. are not readily with with a greater likelihood of infection associated with wound closure. also specimen can be of used quantities known of smears Gram Direct Part I Basic Principles in the Diagnosis and Management of Infectious Diseases and specificity.and for tests DFA the and sensitivity; low for test DFA the available; widely not are reagents the for test DFA the tests; antigen by replaced been primarily has Streptococcus) nella,and (e.g., bacteria some selected forlaboratories in used still are tests DFA bacteria. than rather viruses of tion (DFA) stains are more commonly used for the detection and identifica antibody fluorescent direct present, At organisms. isolated of fication identi as well as specimens patient in bacteria of detection direct for both used are label fluorescent a to conjugated antiserum with stains (e.g., stain organisms thin the Gram arewith detect very to difficult certain when bacteria in positive blood cultures when the Gram stain is negative and acid-fast traditional stains and is equally than specific. The acridine-orange sensitive stain isused more to detect is stain This mycobacteria. detect to stain acid-fast the of variation the in used are dyes damine rho andAuramine background. black otherwise an in sensitive detect to easy most the are dyes fluorescent with generally stained bacteria because stains are microscopic stains Fluorescent organism. the areinsensitive, positivea rapidstainis confirmation the presenceof of stainsAlthough the specimens. and parasites, fungi wellasblood as in to primarily used detect bacteria stains ( differential are stains Wright-Giemsa and Giemsa The acid-fast. weakly or partially as to referred are and stains andgenera other than Tsukamurella, Rhodococcus, Nocardia, terium, responsiblegenerastains:retainacid-fast for human disease of number limited a Only wall. cell their in acids mycolic long-chain Gram the with stain. stain also may molds and yeasts, mycobacteria, dia, cocci. gram-positive resemble and stained when violet crystal quently arranged in pairs; and notrophomonas, staining]; [bipolar center the than rather ends the Enterobacteriaceae are gram-negative rods that stain more intensely at and moniae chains; in arranged cocci gram-positive are streptococci ters; clus in arranged cocci gram-positive are staphylococci example, For on based cells. the arrangements of organisms special or morphology in differences subtle classify further can microscopist experienced 204 bic atmosphere. Likewise, some organisms, such as obes requires that the inoculated agar plates be incubated in an anaero- incubation conditions. The obvious example is recovery of most anaer antimicrobial susceptibility performing tests. The success ofculture formethods is dependent onthe used Particularly are organisms. media these specialized of important recovery for developed been have media specialized so peroxides), and sulfides, acids, fattyinhibitorsof cysteine; and ironrequiresLegionella (e.g., requirements growth specific very have organisms Some agar). [XLD] xylose-lysine-deoxycholate onto inoculated specimen stool a gram- lactose-fermenting (e.g., negative organisms rods growing on MacConkey agar, specific of recognition facilitating substratescontain that media selective are typically media populationmixed a oforganisms Differentialpresentis specimen. a in if useful particularly are media These organisms. other of growth the organisms (e.g., gram-negative or gram-positive bacteria) and suppress agar, MacConkey (e.g., phenylethyl alcohol agar) are media designed to support the growth Selective of certain organisms. most of the growth support to designed are agar) chocolate agar, enriched blood (e.g., General media CSF). (e.g., recovered infrequently are anaerobes numbers of organisms or anaerobic organisms from wherea specimen small of detection the allows media broth of Use(Table 16-7). media specialized and differential, selective, enriched, general purpose: this for developed been agar, have with solidified media and media broth the ability to grow the organisms in vitro. A variety of media, including on based is bacteria about know we what of foundation much for The Culture The acid-fast stain is used to detect bacteria with medium- and medium- with bacteria to detect used is stain acid-fast The is sensitive and specific for primary syphilis, but syphilis, primary for specific and sensitive is pallidumT. Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. ); however,B.pertussis); DFA the testfor Campylobacter,Helicobacter fluorescent antibody Specific ). r ga-oiie oc arne i pairs; in arranged cocci gram-positive are Enterococcus 30 Borrelia) or bacterial inclusions Mycobacterium generally stain withweakly acid-fast r sal rmngtv rd fre rods gram-negative small are Burkholderia Acinetobacter are fat, that coccobacilli retain For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. requires media with media requires pertussis B. S. pyogenes, S. has low sensitivity low has pertussis B. Salmonella or (Anaplasma, Ehrlichia), S. pyogenesS. (group A Campylobacter and T.pallidum , Pseudomonas, The Gordonia. has Legionella Shigella from Mycobac S. pneu S. Nocar Legio Ste ------ pertussis, (e.g., incubationrequireextended andslowlygrow isms Helicobacter, grow only inamicroaerophilic atmosphere. Some organ- standard reference manualsreferencestandard using developed be specimens should protocols These sources. processingof variety a from for procedures detailed have should ratories - labo All specimen. the mishandlingavoid to microbiologist the with talk to clinician the for important is it then practical, not is material a as such biopsy or specimen normally sterile fluid a is collected, and If collection of pathogens. additional suspected the and specimen the source the tity. of clearly describe to clinician the for critical Thus, is it frequently must quanlimitedof specimen culturea decisionsaboutbest howmaketo microbiologist the Indeed, conditions. incubation numerous the in cultures the incubate and media available of variety large the ontospecimens all inoculate microbiologistto the for essary detection methods. detection characteristics of the individual assays and the performanceavailability the of alternativeon based assessed carefully be must tests antigen of value the Thus, NAAtests. laboratoriesby most in replaced been now anticipated that this technology will continue to be refined, the menu is the refined, be tocontinue It will technology this anticipatedthat IL). Northbrook, (Nanosphere, Verigene and (Cepheid, CA), GeneXpert Sunnyvale, FilmArray, BioFire MD), Sparks, Dickinson, (Becton- MAX BD include approach this of examples Leading gists. that kits sealed allow testing disposable, on simplified instruments of by minimallytrained technolo - use the to analysis product amplified and NAA, processing, specimen for instruments and rooms multiple have to need the from movingtransformation, a undergoing also are platforms testing the tests, of complexity the Despite tracts. intestinal respiratory,and the CSF of pathogenscommon most the of detection (see FDA the 16-4). Particularly noteworthy by is the development approved of multiplex are tests for that tests diagnostic of variety the have as increased, has diagnostics molecular of field the entered have that companies of number the years, few last the In specimens. clinical in fungi and bacteria selected of identification for probes fied (HBV),B virus and hepatitis (HCV); C virus and of use the nonampli - hepatitis (HIV), virus immunodeficiency human for products blood of detection and trachomatis, the C. to primarily restricted were tests Previously, book. reference this of edition last the since dramatically The commercial development of nucleic acid–based tests has increased Nucleic o bcei rsosbe o mnnii (i.e., meningitis for responsible bacteria for for assays (e.g., assays some of availability limited and organisms certain relatively for are specificity or and sensitivity poor performed are rapidly disadvantages The be inexpensive. can they are tests these of advantagesThe specimens. clinical in parasitesor viruses, fungi, teria, Tableavailable the summarizesantigen 16-8 ofbac fortestsdetection Antigen ain f n raim bevd n rm tiig A igoi of diagnosis A staining. by Gramcaused pneumonia on observed organism an of cation presumptivefor the however,identifiuseful stain; be assayscan these Gram a than sensitive more no are they because assay screening a as influenzae H. meningitidis, N. moniae, confirmed by either culture or a nucleic acid amplification assay. amplification acid a nucleic of or detection the for tests Antigen culture either be by also must confirmed assays negative used, widely are tests antigen tococcus negative however, assays in urine; must be confirmed byculture. Likewise, antigens although group specific of A detection by firmed should not be used. be not should however,stoolspecimens; testsare the insensitive andnonspecific and of detection for used widely also aretests Antigen organisms. these by caused infections diagnosing for assays sensitive by produced toxin Shiga or specimens stool in gens organisms will be associated with infections. with associated be will organisms unrecognized previously that likely is it expands, work this As isms. organ uncultured previously detect can that techniques sequencing nize the human body is rapidly evolving with the introduction of DNA It should be recognized that knowledge of the microbes that colo that microbes the of knowledge that recognized be shouldIt , Norovirus,Yersinia pestis, Brucella,Francisella, Legionella It). impracticalandis unnec

Acid–Based

Detection 31 n lncl pcmn; screening specimens; clinical in tuberculosis M. Immunoassays forImmunoassays or pneumoniae S. 5 and after discussions with clinicians. with discussions after and

T ). Antigen tests Antigen falciparumPlasmodium). ests Campylobacteror type B) are not recommended not are B) type C. trachomatishaveantigens C. can be con be can pneumophila L. . glcie S pneu S. agalactiae, S. C. difficilein toxinsC. Helicobacteranti - are rapid, are coli E. . gonorrhoeae , N. Bartonella, Strep Table B. ------

Chapter 16 The Clinician and the Microbiology Laboratory - wk 205

mo;

for up to 3 C

Methods

in microaerophilic atmosphere C

in microaerophilic atmosphere for 6 C

5 Biochemical

-cysteine; growth enhanced by iron; supplemented L by

or more incubation at 37° to 42° will not grow on routine media with antibiotics to suppress oral flora IsoVitaleX) bile, Tween bile, 40, Tween oleic acid, olive oil) surface Selective media; recovery optimal using multiple media; Selective, differential agar; will grow on routine media Selective, differential broths; will grow on routine media Selective, differential agar; will grow on routine media Selective, differential agar; will grow on routine media Selective, differential agar; will grow on most media Selective, differential agar; will grow on routine media Selective, differential agar; will grow on routine media Selective, differential agars; will grow on routine media Selective, differential agars; will grow on routine media Enriched medium; will grow slowly on routine media Selective media; will grow on chocolate agar but not blood agar Enriched media; will grow slowly on routine media COMMENTS Incubate at 30° to 33° Selective medium; incubation at 28° to 30° Requires Requires Incubate in microaerophilic atmosphere Selective, differential agar; stimulates sporulation Requires Requires sulfhydryl compounds (cysteine, cystine, thiosulfate, Selective, differential agar for presumptive identification Selective, differential agar; will grow on routine media Requires lipids for growth (e.g., whole milk, malt extract, ox InPouch TV is commercially available Selective medium; organisms will not grow on routine media Selective, differential media; will grow on routine media Selective, differential agar for presumptive identification Ameba digests bacteria as the protozoa moves over the agar Selective, differential agar; will grow on routine media Selective, differential agar; will grow on routine media Parasites

and

mation, mation, please refer to the individual chapters in microbiology reference book. this book or to a We believe that most readers of this chapter would either have minimal have either would chapter this of readers most that believe We interest in identificationmethods for bacteria,would consultspecific in chapters this book for an overview of the subject, or would refer to a microbiology reference book reason, the for covered in information this section is restricted to general detailed information. infor detailed more For For methods. identification concerning that comments Identification The microscopicmorphology and characteristics, such as colony size, are activity, hemolytic of absence or presence the and shape, and color, IDENTIFICATION IDENTIFICATION METHODS FOR BACTERIA Fungi,

Enterobacter - Pseudomonas cepacia E. coli or Bacteria,

Salmonella-Shigella (SS) agar; Salmonella-Shigella (SS) agar; of

selective agar; µg/mL methicillin (oxacillin); chromogenic agar

Recovery

µg/mL vancomycin

for

agar (CCDA) (PC) agar; oxidative-fermentative polymyxin B-bacitracin- lactose (OPBL) medium xylose-lysine-desoxycholate (XLD) agar xylose-lysine-desoxycholate (XLD) agar; chromogenic agar agar and 7H9 broth medium with rabbit serum 6 aerogenes Campy-CVA Campy-CVA medium, charcoal cefoperazone desoxycholate Bile esculin azide agar (Enterococcosel) supplemented with Lim broth; StrepB carrot broth CIN agar Thiosulfate citrate bile salt sucrose (TCBS) agar Mannitol salt agar (MSA); chromogenic agar Hektoen enteric (HE) agar; Hektoen enteric (HE) agar; BCYE agar Modified Thayer-Martin agar, New YorkModified New City Thayer-Martin agar, agar Burkholderia cepacia Dubos broth; Lowenstein-Jensen medium; Middlebrook Ellinghausen-McCullough-Johnson-Harris (EMJH), Fletcher’s Ellinghausen-McCullough-Johnson-Harris (EMJH), Fletcher’s BCYE agar Multiple enriched media Dermatophyte test medium (DTM) Chocolate agar; buffered charcoal yeast extract (BCYE) agar Chromogenic agar Novy-MacNeal-Nicolle (NNN) medium Barbour-Stoenner-Kelly medium Barbour-Stoenner-Kelly Sorbitol-MacConkey agar Agar medium supplemented with lipids Novy-MacNeal-Nicolle (NNN) medium complete Diamond’s medium; InPouch TV Regan-Lowe medium; Bordet-Gengou medium Cystine-tellurite blood agar; Tinsdale agar Birdseed (niger seed) agar Non-nutrient agar overlaid with MSA with 4 MEDIA Cefsulodin-Irgasan-novobiocin (CIN) agar Cycloserine-cefoxitin-fructose agar (CCFA) Media For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.

Enterococcus Selected 16-7

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada (VRE) TABLE Campylobacter spp. Vancomycin-resistant Vancomycin-resistant Streptococcus agalactiae (group B) Yersinia enterocolitica Yersinia Vibrio Vibrio cholerae, parahaemolyticus V. S. aureus, methicillin-resistant (MRSA) Staphylococcus aureus Shigella spp. Salmonella spp. Nocardia spp. Neisseria gonorrhoeae Burkholderia cepacia Mycobacterium spp. Leptospira spp. Legionella spp. Helicobacter spp. Dermatophytes Francisella tularensis Yeasts Trypanosoma Borrelia burgdorferi Escherichia coli O157 Malassezia furfur Leishmania Trichomonas Parasites Acanthamoeba Bordetella pertussis, B. parapertussis Corynebacterium diphtheriae Fungi Cryptococcus neoformans Bacteria Clostridium difficile ORGANISM Aeromonas spp. of approved tests expanded, and the cost and technical ease permitting easepermitting technical and cost the and expanded, tests approved of NAA tests laboratories. to be performed by most clinical microbiology cant increase in antibody titer may not months be after detected the until initial weeks presentation, persistence or antibodiesmake of it difficultto between differentiate may a recent and past a infection, and cross-reactions may compromise the specificity of the antibody response. In general, serology should be used to confirmother - diag nostic tests whenever possible. Serology bacteria, with infections confirm to historically used been has Serology fungi, and viruses that are difficult detectto difficulty other by methods. with The serology that is some immunocompromised will patients not mount an adequate antibody response to infection, a signifi Part I Basic Principles in the Diagnosis and Management of Infectious Diseases 206 Zygomycetes (not present) and present) (not Zygomycetes *Panfungal, beta- *Panfungal, PYR hydrolysis ( hydrolysis PYR Oxidase disk (P) Optochin Indole hydrolysis Hippurate Coagulase Catalase solubility Bile disk (A) Bacitracin BIOCHEMICAL Trichomonasvaginalis spp. Plasmodium (duodenalis) lamblia Giardia dispar histolytica/E. Entamoeba (hominis) parvum Cryptosporidium Parasites Rotavirus virus syncytial Respiratory Norovirus B A, virus Influenza metapneumovirus Human HIV-2) (HIV-1, virus immunodeficiency Human HSV-2)(HSV-1, virus simplex Herpes virus B Hepatitis virus Dengue Cytomegalovirus Adenovirus Viruses Panfungal* capsulatum Histoplasma neoformans Cryptococcus spp. Coccidioides albicans Candida dermatitidis Blastomyces spp. Aspergillus Fungi Yersiniapestis A) (group pyogenes Streptococcus pneumoniae Streptococcus toxin) (Shiga dysenteriae Shigella meningitidis Neisseria 1 serogroup pneumophila Legionella pylori Helicobacter influenzae Haemophilus toxin) (Shiga coli Escherichia difficile Clostridium trachomatis Chlamydia and jejuni Campylobacter Bacteria Urease TABLE Specimens Bacteria, TABLE

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Fungi, L -pyrrolidonyl-β-naphthylamide) D Common Antigen -glucan assay that detects cell wall antigen in all fungi except fungi all in antigen wall cell detects that assay -glucan

TEST type B type

V C. coli C. iruses, (low level). (low Cryptococcus

and

for

Parasites

Detection

Reactions A key reaction to help differentiate gram-negative rods (e.g., Enterobacteriaceae, (e.g., rods gram-negative differentiate help to reaction key A of identification presumptive is optochin to Susceptibility of identification presumptive for used test rapid A streptococci B group of identification presumptive for used is test positive A of identification for criterion key the is test coagulase positive A positive], [catalase staphylococci (e.g., cocci gram-positive of groups major defines that characteristic important An of differentiation rapid For of identification presumptive For A rapid test for presumptive identification of identification presumptive for test rapid A A rapid test for test rapid A PRIMARY are oxidase negative; oxidase are Stenotrophomonas negative]) [catalase enterococci and streptococci

of Clinical

USE

Used Proteus mirabilis Proteus

TEST to (which are bile soluble) from other streptococci other from soluble) bile are (which pneumoniae Streptococcus

Identify and ed i tcncly ipe o efr ad ae a e mnts to minutes few a is identification takes serologic which fororganisms of andExamplescomplete. perform to simple whole-organism technically is of beads use the suspensions with or latex agglutination by means of antibody Agglutinationbound to latex strains). monella of classification (e.g., purposes epidemiologic for bacteria typing sub for and organisms some of identification the confirm to antisera laboratories use identification, of methods biochemical to addition In Identification discussion detailed A a use laboratories later). systems. these combinationof most however, chapter; this of discussed scope is the systems beyond identification be commercial automated and manual (to the of tests tibility ria. These systems havealso been integratedwith antimicrobial suscep - bacte important clinically most identification of theoretical the allow that exist databases Extensive tests. these of interpretation and tion, incuba inoculation, the for systems automated the fully of development include improvements Further profile. individual metabolic and the reactions of interpretation the for instrumentation of duction that could interpretedbe after a few hours of incubationofreactions andthe intro selection included approach this of Refinements cation. identifi organism the for database established an with compared was negative a rapid testcoagulase to identify the isolate as gram-positive cocci found predominantly as in clustersappears will that be tested colonywith yellow light or white opaque an example, For tests. identification preliminary some and characteristics early these reliablepresumptive basis of a identificationthe on common bacteria of provide to able is often microbiologist experienced An procedure. used by the microbiologist in selection of the appropriate identification pattern of reactions could be converted into a “metabolic profile”“metabolic thata into converted be could reactions of The pattern plates. microwell or strips disposable in incorporated were that reactions of series a of consisted These 1970s. the in introduced were identification organism for kits commercial first The expensive. tively prohibiandapproach impractical this made performed be to that needed tests additional of number the expanded, organisms important numberclinically ofAs the tubes.test six reactionsbyin using riaceae Enterobacte family the of members known all identified laboratories most 1970s, early the until example, For bacteria. recognized monly com the of most identify could tubes test in performed tests numberthese of minimum A acids. fatty volatile of production or enzymes, specific of presence sources, carbon of use lactose), or glucose of tion fermenta (e.g., carbohydrates of oxidation or fermentation included patterns(Tablereactivity biochemicalof tests The biochemical 16-10). and anaerobic), aerobic, (i.e., tolerance oxygen spiral-shaped), rods, cocci, acid-fast; gram-negative, gram-positive, (e.g., properties scopic identify selected species of bacteria. of species selected identify to morphology, stain Gram and colony with along used, commonly , which is sensitive to low concentrations of bacitracin of concentrations low to sensitive is which pyogenes, Streptococcus The historical classification of bacteria was based on their micro- their on based was bacteria of classification historical The Helicobacter pylori Helicobacter

Bacteria Staphylococcus species. and Pseudomonas and pyogenes Streptococcus from urine specimens urine from coli Escherichia

Streptococcus pneumoniae Streptococcus Using are oxidase positive). oxidase are Burkholderia Staphylococcus aureus Staphylococcus

Specific describes key tests that arethat tests Tablekey describes 16-9 Enterococcus 32 , and Acinetobacter,

Antisera S. aureus or a coagulase- Sal ------

Chapter 16 The Clinician and the Microbiology Laboratory - - -

207 OTHER Anaplasma Chlamydia Coxiella Ehrlichia Mycoplasma Orientia Rickettsia Tropheryma Ureaplasma Selected

Identification SPIRAL/CURVED RODS Arcobacter Borrelia Campylobacter Helicobacter Leptospira Treponema

A, B, and C, G F, most common polysaccharides; polyvalent antisera used to identify S. ; pneumoniae subtyping used to select most common polysaccharides for vaccine development W135 most common clinical isolates 6 subtypes (A to F); subtype B most important flagella (H) proteins; O157:H7 most important polysaccharides into 45 subtypes serogroups; serogroup 1 most important COMMENTS Lancefield grouping based on cell wall carbohydrates; Subtyping based on more than 90 capsule Thirteen capsule polysaccharides; A, B, and C, Y, Capsule polysaccharides used to classify species into Subtyped by many cell wall (O) polysaccharides and Subtyped using more than 2500 cell wall (O) Cell wall (O) polysaccharides used to divide 4 species Polyvalent antisera used to subdivide species into 16 Serologic 16-11 Anaerobic Bacteroides Fusobacterium Leptotrichia Porphyromonas Prevotella Aerobic Acinetobacter Actinobacillus Aggregatibacter Bartonella Bordetella Brucella Burkholderia Capnocytophaga Citrobacter Comamonas Cronobacter Delftia Eikenella Enterobacter Escherichia Francisella Haemophilus Hafnia Kingella Klebsiella Legionella Moraxella Morganella Pantoea Pasteurella Proteus Providencia Pseudomonas Ralstonia Raoultella Salmonella Serratia Shigella Stenotrophomonas Vibrio Yersinia Rods The different methodsvary inease reproducibility, of per

33 β-hemolytic pneumoniae meningitidis influenzae pneumophila TABLE Bacteria BACTERIA Streptococcus, Streptococcus Neisseria Haemophilus Escherichia coli Salmonella enterica Shigella spp. Legionella mosomal mosomal DNA, and PCR-based methods, such as random amplifica tion of polymorphic DNA (RAPD) and multilocus sequence typing (MLST). (PFGE), (PFGE), restriction fragment length polymorphism (RFLP) of chro formance, formance, turnaround time, robustness, and discriminatory with PFGE power, considered by many the gold standard. It is important to GRAM-NEGATIVE - - - - Anaerobic Acidaminococcus Megasphaera Veillonella Aerobic Neisseria Cocci Table Table 16-4) Bacteria

- noncul the , pallidum T. Gordonia Mycobacterium Nocardia Rhodococcus Tsukamurella Acid-Fast

Common

of equence equence of screening confir and

s 32a Genomic

For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Actinomyces Bifidobacterium Clostridium Eubacterium Lactobacillus Mobiluncus Propionibacterium Anaerobic Aerobic Arcanobacterium Bacillus Brevibacterium Corynebacterium Erysipelothrix Listeria Rothia Streptomyces Rods Using GRAM-POSITIVE

. Infection with with Infection 16-11. Table Classification 16-10

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada DNA DNA sequence–based identification is another molecular - tech Whereas most subtyping of most organisms was previously per TABLE Anaerobic Anaerococcus Finegoldia Peptococcus Peptostreptococcus Peptoniphilus Abiotrophia Aerococcus Enterococcus Granulicatella Leuconostoc Pediococcus Staphylococcus Streptococcus Cocci Aerobic useful are listed in listed usefulare tivable cause of syphilis, is now frequently diagnosed with automated 16-12). The diagnostic (Table assays mation is discussed in Chapter 239. Identification Techniques Thefirst molecular identification techniques probe-based were tests for specific were that acid sequences nucleic of detection on relied that a particular genus or species. These probe assays (see became the method of choice for rapid, accurate variety identificationof bacteria, mycobacteria, of and fungi isolated in a culture. nique that can be used to improve both identification the of speed bacteria and that accuracy are of difficult identify to tests. Gene phenotypic by sequencing techniques that target ribosomal ribonucleic acid (rRNA) genes and various tech and housekeeping costs sequencing current the but organisms, of genes identification permit precise iden- proteomic Furthermore, use. widespread the limit demands nical methods sequencing replacing rapidly are later) (see methods tification and will likely restrict sequencing organisms and use for epidemiologic investigations. for characterization of selected formed using specific antisera, helps typing accurate this of Availability is techniques. molecular with plished now more efficiently accom and can in of help rapidly the more identification outbreaks to control methods The sources, carriers, genotypic main of spread. and patterns that are currently in use include pulsed-field gel electrophoresis Part I Basic Principles in the Diagnosis and Management of Infectious Diseases ue o bcei i ciia seies a urn lmtto with limitation identification.after more current a specimens, clinical MALDI-TOF mass spectrometry. Susceptibility test in results lag a day or bacteria of tures mix identify to used been has [REIMS]) spectrometry mass ization evaporative ion rapid - (e.g., systems spectrometry different mass with ment, and inexpensive solutionsinstru make cost Bruker per specimen low. the Research with plates, The reusable but results. expensive, are identification machines definitive to it time and improves testing, dramatically biochemical with associated delays stains, and Gram subcultures, for need the eliminating laboratory, microbiology the in workflow improves spectrometry mass MALDI-TOF use. local for base. Databases are particular to the manufacturer but can be expanded data the organismfrom the of absence to due be terminateresultcan results can result from insufficient number of organisms (usually 10 (usually organisms of number insufficient from result can results Negative reported. are results probable highly Only species. or only, genus indeterminate, negative, as reported are Results methods. be ous can organisms all previ all exceeds that accuracy hour, an of an is withthan less in achieved identification molds but and complex, more mycobacteria slightly Processing a spectrum. creating tube, protein vacuum a unique through move they as size by separated laser a beam, to the matrix ionizes exposure proteinsUpon present plate. in the sample, metal which are the then on spotted then is extract protein The acetonitrile. with extracted proteins the and acid formic with lysed sediment centrifuged the and ethanol in suspended be can organisms the Or, analyzed. then and matrix, formic a with with overlayed pretreated acid, plate, target a to transferred culture), blood or yeast are from selected a culture plate or positive broth culture (e.g., Bacteria inexpensive. and simple technically is method The yeast. and bacteria 193 identify to approved is NC) Durham, (BioMérieux, MS VITEK The yeast.and relevant clinical bacteria 280 identify clearedto FDA currently is MA) Billerica, Daltonics, (Bruker System SA typer Bio United MALDI approvedStates.The currentlythe are systems in cuaey efre, lncly eeat ad iey Mtos for Methods timely. and relevant, clinically performed, accurately and reproducibly be must tests the per laboratory: clinical tasks the complex in formed most the of one is testing susceptibility vitro In toxicity. drug or resistance antimicrobial of because for organisms limited other frequently are options treatment antibiotics, effective to susceptible predictably are organisms some Although agents. crobial antimi to pathogen presumed a of susceptibility the determine to is laboratory microbiology the of functions important most the of One TESTING SUSCEPTIBILITY ANTIMICROBIAL 10 or molds. and yeasts, mycobacteria, bacteria, of identification the for in StatesUnited the and Europe currently in use widespread method a spectrometry, mass (MALDI-TOF) time- of-flight desorption/ionization laser matrix-assisted by replaced being Thefundamental procedures for identification of organisms are rapidly Methods Identification methods. genotypic mated,it anticipatedis that genome replace earlier sequencingthe will auto further are techniques sequencing these investigations.As logic epidemio - in unattainable previously strain among in discrimination of improvements dramatic level a enabled the has sequencingwhole-genome techniques, sequencing With sequencing. whole-genome is this accomplishing for method precise more among A bacteria. composition of genetic strains in differences to or techniques similarities crude the relatively assess are methods these all that recognize 208 Enzyme Immunoassay Enzyme TECHNOLOGY Microhemagglutination Microhemagglutination Hemagglutination Rapid plasma reagin plasma Rapid TABLE 6 bacteria are required) or insufficient protein extract. An inde An extract. protein insufficient or required) are bacteria

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Using

Assays oteomic Trinity Biotech (Jamestown, NY) (Jamestown, TrinityBiotech MANUFACTURER Arlington Scientific (Springville, UT) (Springville, Scientific Arlington Fujirebio Diagnostics (Malvern, PA) (Malvern, Diagnostics Fujirebio Immucor (Norcross, GA) (Norcross, Immucor Arlington Scientific Arlington

for

34-36 the w MALDI-TOF Two

Diagnosis

------5 Syphilis

ehd te et gr lt i sabd ih standardized a with swabbed is plate agar test the method, tae ae re i mn ts pnl s te cn e trd o an for stored be can they so time. of period panelsextended test many in dried are strates antimicrobialsusceptibility tests. The antibiotics and sub biochemical bio with combined identification and of performancesimultaneous the be for tests chemical can and commercially available are biotics most in used widely clinical laboratories. Microdilution are panels with a large number of andanti companies of number a by developed been have tests microdilution Automatedthese tests.of versions MIC tion trays are very common. These are referred to as broth microdilution volumetesting. However, broth dilution tests microdiluin performed because the tests are technically cumbersome and not adapted to high- tests performed in test tubes are used primarily in research laboratories carries bacterium (e.g., antibiotic). the to resistance encoding genes present are mechanisms where resistance situations in special or doses therapeutic in achieved the concentrationthan greater is value MIC the when resistant and tract), urinary higher with concentratedantibiotic(e.g., is active the where sites achieved body in or doses antibiotic the of concentration the at is value the with achieved concentration the below usual recommended dose of or the antibiotic, intermediate at when the MIC is value MIC the results are expressed numerically.expressed areresults the because tests susceptibility quantitative or tests (MIC) centration aremethods determined.These referred inhibitory minimal to as con is organism the inhibits that antibiotic the lowest of concentration (minimum) period, incubation specific a after and, organism test the concentrationofstandardized a with inoculated then is dilution Each etc., 0.75, 1.5, 3.1, 6.25, 12.5, 25, 50, 100, (e.g., used is scheme dilutionTypically, twofoldmedium. a agar or liquid a either in pared pre areantibiotics of dilutions serial procedures, these In developed. Dilution methods were the first susceptibility test proceduresthat were Dilution resistance. encoding genes specific of presence of production as such mechanisms, resistance and overnight and rapidtoMethodsmeasuredeveloped methods. have specific been also methods, automated and manual methods, tive been have quantitative qualita methods, and diffusion anddilutioninto divided methods the Historically, bacteria. against antibiotics of clinician. andmicrobiologist the by made of decision final the antibiotics which reportedshouldfor be organismspecific a be should although organisms, specific against tested be to drugs of classes specific of selection the for guidelines provide also documents The reported. and be interpreted results should the and method performed test susceptibility each how detail exacting with define standards The (www.CLSI.org). agencies government and try, [NCCLS]), a consensus group representing Standards clinical laboratories, indus Laboratory Clinical for Committee National (CLSI, the Institute formerly Standards Laboratory and Clinical the by reviewed annually and published standards by defined are tests the performing fusion method. diffusion disk Kirby-Bauer the is method diffusion agar popular most The procedures. dilution tube broth and agar the to alternative a practical as developed was method diffusion agar The Agar tion. pretive criteria defined by CLSI foreach organism-antibiotic combina inter on resistant—based intermediate,interpretations—susceptible, gr iuin et promd n er dse ad rt dilution broth and dishes Petri in performed tests dilution Agar A number of different methods havebeen used to assessthe activity h qatttv vle cn e ovre it qualitative into be converted can values quantitative The PRODUCT Captia Syphilis (T.Syphilis pallidum)-G Captia ASI TPHA test TPHA ASI Beckman-Coulter (Olympus) PK TP system TP PK (Olympus) Beckman-Coulter TPHA Screen TPHA ASiManager-AT 38 n raim s eie a ssetbe o n niitc when antibiotic an to susceptible as defined is organism An

Dif

Methods

Methods 37 -lactamases or the the or β-lactamases SPECIMEN Serum, plasma Serum, Serum Serum, plasma Serum, Serum, plasma Serum, Serum, plasma Serum, 39 µg/mL). In this this In ------

Chapter 16 The Clinician and the Microbiology Laboratory ------is is C 209

Table 16-4). Table . E. This faecalis µg/mL for strep

40 Resistance to carbapenem carbapenem to Resistance β-lactam antibiotics except µg/mL for gentamicin. It is 38

ransport β-lactam and aminoglycoside mecC genes. Molecular-based T vanB genes. Commercial assays

and

mecA or vanA or vanA are widely available (see E. and less faecium in commonly β-lactamase inhibitors (i.e., sulbactam, clavulanic Collection

Issues

β-lactamase by All speciesoxacillin (or methicillin) are that of staphylococci resis Infections with enterococci are commonly treated with a treated combina commonly are with enterococci Infections ceftaroline,including carbapenems, regardless of the actual result. vitro For in either MIC or disk testing of staphylococcal susceptibility tion tion of interpretation of carbapenem resistance has led to conclusions the about same reporting by susceptibility com and single of based identification accurate the because mechanism on than MIC rather bination mechanisms of resistance to carbapenems is not within the capacity of many clinical laboratories. tant should be considered resistant to all antibiotics antibiotics in gram-negative rods is mediated by a number of β-lactamases, novel overexpression of β-lactamases, ampC– and changes in the mediated) pump (efflux from egress and changes) (porin into entry MICs elevated by detected initially is resistance This space. periplasmic to one or more carbapenems and in some cases can be confirmedby detection of the specificresistancegeneby molecular tests. The evolu Specimen beshould mycobacteria collected for culture smear and for Specimens and transported in closed, because leakproof, acceptable not are outside the sterile on specimen with contaminated containers. Containers to to oxacillin and vancomycin, a full 24 hours of incubation at 35° required to maximize the likelihood of detection of resistance except in automated broth dilution systems that use specialized interpretive algorithms that allow a definitive result resistance in is encoded 6 in the tohours. 8 Oxacillin definedby MIC values greater thanor equalto 2000 tomycin and greater than or equal to 500 MYCOBACTERIA acid are that bacteria rod-shaped nonmotile, aerobic, are Mycobacteria fast; that is, once stained with certain dyes, they resist decolorization with acid-alcohol because of their cell wall structure. Safety (5% sodium bleach hypo made, are direct smears concentrated When chlorite) can be used to inactivate mycobacteria that may be present in patient specimens. Non–aerosol-producing procedures, manipulating specimens for of preparation smears, such can be performed as using biosafety level (BSL)-2 practices and a biologic safety The cabinet. Centersfor Disease andControl Prevention (CDC) recommends that all other procedures that potentially generate aerosols should be restricted a access; facility includes Such performed in facility. a BSL-3 pressure; negative under laboratory the maintaining airflow directional and the use of special gowns, gloves, and masks. cefepime and piperacillin-tazobactam. One not entirely practical- solu piperacillin-tazobactam.entirely and One not cefepime dosing a schedules drug which cefepime, multiple for for tion adopted are available, is the adoption of reporting dependence), with the to alertintention the “SDD” prescriber to the need for (susceptible dose a dosing schedule to appropriate the MIC and site of infection. Prin cipally cipally useful for epidemiologic purposes, diffusionand dilution tests are used to detect ESBL production, report and results some interpreted as automated consistent systems with ESBL activity. aztre or cefotaxime, ceftriaxone, Reducedceftazidime, cefpodoxime, to activity onam is used to screen for ESBL production, and then inhibition of this cocci, particularly in for molecular testing for rare rare for true synergy testing between a to be performed. resistance Vancomycin is also common in entero can be measured by MIC tests, growth on media with or vancomycin, by molecular tests that measure for the presence of genes associated with vancomycin resistance, tion tion of an and a aminoglycoside cell Aminogly wall–active antibiotic. because alone used when enterococci against activity poor have cosides of poor uptake of the drug. high to Acquired corresponds ineffective) bewill resistance treatment combination to is, (that aminoglycosides MIC values for the aminoglycosides. Thus, high-level resistance is assays for one or both genes are widely used in clinical laboratories and and laboratories clinical in used widely are genes both or one for assays offer a rapidmethod for determiningresistance. acid) is usedtest. confirm to the screening

- - - - - β-lactamases (ESBLs). Bacteroides produce a [diameter [diameter of the paper disk] to mm

For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. -lactamase activity can be detected by measuring measuring by detected be can activity β-lactamase compared with compared dilution tests, which typically measure Methods

β-lactamase that will degrade most penicillins and selected mm),

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada One One variation of the Kirby-Bauer disk diffusiontest is the E test The dilutiontests are commonlyreferred to as quantitative tests, β-lactamases are the extended-spectrum losporins, losporins, and monobactams. The remains the clinicalsource of some debate, although current CLSI import guidelines of this no longer require that activity mechanisms of resistance be reported. Rather, susceptibility is reported by MIC. This modification extended-spectrum(oxyimino) of use the procedure to of relation in is supported best cephalosporins, such as ceftriaxone, Additional consideration to supporting clinical cefotaxime, information is - devel and ceftazidime. of this to to drugswith standard the regard oping applicability such as ESBL-producing organisms variably hydrolyze most penicillins, cepha penicillins, most hydrolyze variably organisms ESBL-producing Specialized methods have been developed to detect some specific Special this test, the antibiotic is in concentration a gradient extending commercially from the prepared top strip, to with the bottom a of the strip. When this is placed on a lawn of pattern bacterial of growth, inhibited an elliptic growth develops (greater inhibition with at a the higher end concentration of antibiotic). The strips so are that calibrated the area where the sponds to MIC bacterial value the for organism’s the antibiotic. growth The advantage meets the strip of corre the E test is that MIC results can be obtained easily for one or two antibiotics. However, when a large combinations number need of to be organism-antibiotic tested, the broth microdilution preferred. method is resistance mechanisms. An β-lactamase example production of by this both gram-positive organisms. is Thereare and constantrefinements to the gram-negative the methods by increase concomitant detection which a with organized, and identified are β-lactamases of in the number of enzymes that surpass clinical utility. Two examples willof these illustrate applications important tests. Organisms such as Staphylococcus, Neisseria, , Haemophilus and common The cephalosporins. the byproducts of these method degraded measures antibiotics. The degradation most of group important Another byproduct. red a produces nitrocefin, which common the chromogenic cephalosporin of about about 40 two to of five a concentrations drug inmost microdilution panels. So, the definitionof aor quantitative qualitativetest is determinedby the reporting method rather than the testing method. (bioMérieux, Lyon, France), which is a gradient diffusion test. In titative titative values (zone sizes from 6 concentration of concentration the test organism, and then paper disks a containing defined antibiotic concentration are placed on the lawnAfter bacteria.of overnight incubation, the diameter growth around of the disk (Mueller- the medium test is susceptibility zonethe including measured. variables, of inhibited of number Thiszone influenced is by a Hinton agar is the standard for bacterial tests), the concentration of the test the organism, of rate growth of the test the organism, concen tration of antibiotic in the disk, the diffusionof theantibiotic in the and agar, the susceptibility of the organism to the antibiotic. The first properly is test if the therefore, CLSI; by standardized are variables five performed, the size of the zone of inhibited growth is directly related to the susceptibility of the organism—the larger theexpected,the be would As antibiotic. the zone, to is organism the susceptible the more results of the dilution tests and diffusiontests are related. There is an inverse linear relationship between the size of the zone and the MIC value—the larger the zone of inhibited growth (more susceptible the to organism thethe antibiotic), smaller the MIC itvalue. Thus, is pos sible to extrapolate from the measured size of the inhibitory that criteria zone interpretive the to addition, In value. MIC corresponding the are applied to MIC tests apply to the diffusion tests. organism-antibiotic Thus, for tests, most the diffusion tests equally accurate and in predicting antimicrobial susceptibility. dilutiontests are whereas the diffusion tests are referred to is, as the qualitative tests. dilution That tests value, whereas are the diffusion usually test results reported are reported intermediate, or resistant. This as is as an oversimplification.Both tests susceptible, are a number in or quantitative; fact, the diffusiontests assess a greater rangeof MIC quan Part I Basic Principles in the Diagnosis and Management of Infectious Diseases is now available (see Chapter 251). Chapter (see availablenow is has not widelybeen used. A commer assayity)multiple has advantages resource-constrainedin butsettings ceptibility tests, the MODS (microscopic observation drug susceptibil concomitantsusmicrobrothand cording on based algorithm testing at least three first-morning specimensbe obtained andthat a minimum of overgrowth.bacterial For sputum and urine, it is recommended that likelihood the of because unacceptable are urine and sputum of tions Twenty-four–hourprotocol.collec laboratory on depending culture, may be inoculated directly into a special bottle for mycobacterial blood it or lysis-centrifugationtube, a in containsanticoagulantthator tube may be collected for mycobacterial culture, either in a collection blood appearance of individual cells and groups of cells should at best be be best at groupor species. ofspecies consideredcertain onlysuggestivea of should cells of groups microscopic and cells the individual However, of appearance parallel. axes long their with organisms of strands long of composed are that “cords” in together group often of cells medium, liquid a in grown when Particularly may appear coccobacillary, especially on whereas a smear appearance,from a liquid beaded medium. a than rather banded a with sometimesbroad, andlong be toexample, tends arrangementcells. orindividual ofwidth, length, the in others carbolfuchsinfrom differ mycobacteria regular of species a Some stain. with used a is uses than that step stain decolorizing acid-fast weaker modified a with visualized readily more areslowgrowersthan mayfast be and acid avidly less maybe bacteria myco growing Rapidly faster. done be can slides of reading smears, (100 lensimmersion oil the with (25 magnification lower of lens objective an with reliably ined In addition, because slides stained with a fluorescent dyecan be exam material. background from distinguished clearly be can and brightly organismsstain the because specimens, direct in particularly bacteria, myco of detection the for sensitive more are stainsFluorescent teria. mycobac of properties acid-fast the on based are but antibody an of use the involve not do stains these mycobacteria, of visualization for fluorescent compounds; although fluorescence microscopyrequired is use stains auramine-rhodamine and O auramine The stain. acid-fast been has and “cold”a is step which stain,Kinyoun the by heating a laboratories many in replaced requires stain Ziehl-Neelsen The red. mycobacteria staining carbolfuchsin, is stains Kinyoun and Neelsen (hence, retain decolorizer “acid fast”),an acid-alcohol unlike to most other Thebacteria. stainprimary intheZiehl- mycobacteria with of washing after ability dyes the certain on based are stains bacterial myco Specific specimen. the in outlines) rodlike (unstained negative images as appear may they or the gram-negative, of appearing rest organism the and (gram-positive) visible beads the only with rods mycobacteriavisibleat gram-positive mayall, appearbeaded, finelyas and (see constituents cellular other or acids nucleic myco bacterial specific of detection by or organisms stained of visualization through specimens patient in directly detected be may Mycobacteria Direct of midstream urine be processed for each culture. of 40 consideration. serious a is infection mycobacterial whenever 5 than CSF,more of case the (in 210 used by each of the laboratories they use. they laboratories the of each by used importantis awareitforproceduresandphysicianstories, the ofbe to for handling different significantly vary types specimen among labora would alter case management or tuberculosis (TB) control activities. activities. control (TB) tuberculosis or management case alter would a with suspected test (NAAT) should be performed on the first sputumfrom allpatients trations in sterile fluids maybe low,so a minimum ofseveral milliliters transport of sterile fluids, urine, and stool. However, organism concen teria. No special procedures are usually necessary for the collection and mycobac of isolation the for lesions tissue of specimens swab to able prefer are Biopsies handling. specimen optimal ensure to advance in that may be present; arrangements should be made with the laboratory ization soon after collection to ensurethe viability of any mycobacteria of the hazard to posed personnel. Gastric aspirates require pH neutral The Gram stain is insensitive for the detection of mycobacteria. If mycobacteria. of detection the for insensitive is stain Gram The The CDC currently recommends that a nucleic acid amplification acid nucleic a that recommends currently CDC The 16-6).

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neto fr hc te et result test the which for infection mL 42a cial cial product onbased the concept complex avium Mycobacterium if possible) should be obtained be should possible) if Mycobacteriumkansasii, M. tuberculosis M. Tables 16-4 Tables 41 Protocols 41 ) than ×) Blood Blood 42 for A ------ytm ae lo vial. eeto i bsd n uh etrs as features such (CO on based dioxide is carbon Detection available. also nonautomated are some for systems but instrument growth, organism an of use detection systemsautomated liquid-based Many considered. should uncultivable be is organism the that or used been not have ism organ the for conditions growth optimal that possibility the patient, growthobtainedissmear-positive from a fromanuntreated specimen no If enhanced. be may present mycobacteria any isolating of chance and mycobacteria growing kansasii); hence, rapidlyif media with and without ofantimicrobials are used, the strains some acid inhibit nalidixic may (e.g., mycobacteria some to these inhibitory of be some may contaminants;agents of overgrowth prevent help to solid media and liquid to added be may agents Antimicrobial sense. tional tradi the in growerrapid a necessarily is incubation of week a within detected organism an that assume cannot one used, been has media mycobacteria in even less time. Consequently, when one ofgrowing these liquid slowly other of isolation and weeks 2 within tuberculosis M. aeruginosa. oxalic the acid procedure for specimens heavily as contaminated with such used, occasionally are procedures other although of mixture a of use the entails proceduredecontamination and digestion used monly com most The concentrates. such from prepared are specimen a of smears and decontaminationprocedures, and digestion the of part as with liquid culture media. culture liquid with obtained be cannot quantitation but inoculation, direct for available automatedwith used culture thatbottlesbe canblood instruments are mycobacterial of types several Currently,determined. be can tration organismconcen blood which from planted media, onto solid be can method and lysis-centrifugation digestion a prior by require processed be Blood not decontamination. must do also sites but sterile plating before normally ground from obtained specimens Biopsies preparationplatingandwithout prior digestiondecontamination.and microorganisms as possible. other as microorganisms many as of elimination the mycobacteria maximizing of simultaneously loss while the minimize to struck be must balance a and processing somewhat inevitablyThese steps are toxic to mycobacteria, present. mycobacteria any and of overgrowth prevent digested to decontaminated be must flora, microbial normal considerable contain to expected be cansputum, asstool, such andwhich Specimensurine, Specimen et estvt i hg ol fr ma-oiie specimens. smear-positive for only high is sensitivity test available(see currently test FDA-approved one only is there recommendation, this Despite topee f prxmtl 8 CO 8% approximately of atmosphere number of mycobacterial species in clinical specimens. clinical in species mycobacterial of number the target, gene One testing. diagnostic for used be can they before dated amplification)- vali isothermal loop-mediatedbe must PCR, real-time sequencing, DNA (e.g., tests Laboratory-developed States.United the NAATs are available for research use only or for clinical testing outside nary and meningeal tuberculosis. meningeal and nary pulmo- both of diagnosis the for detection acid tuberculostearic than sensitive more be may amplification acid Nucleic available. generally not is testing such meningitis, tuberculous particularly tuberculosis, of diagnosis the for usefulness some have may acid tuberculostearic detection. organism of rapidity the and culture of sensitivity the both enhance may media liquid the practical; whenever specimens plating for used sciences, Cleveland, OH). Cleveland, sciences, suring pressure changes in the culture (VersaTREK,vial Magellan Bio Becton-Dickinson, [MGIT], Sparks, MD), and monitoring the rate of oxygen Tube consumption by mea Indicator Growth Mycobacteria and System 9000MB (BACTEC consumption oxygen France), Lyon, may have to be satisfied to ensure their isolation (Table isolation Given their 16-13 ). ensure to satisfied be to havemay that preferences or requirements growth different have mycobacteria of isolation lhuh iet eeto o bt mcbceil nies and antigens mycobacterial both of detection direct Although Mycobacterial cultures are generally incubated at 36° be medium, solid a to addition in liquid, a thatrecommended Itis gene, is useful for the detection and identification of a large a identification of and detection the for useful is gene, secA1 41,46 41 oee, eea seis f pathogenic of species several However, tuberculosis. M. Sterile fluids, such as CSF, maybe concentrated for smear Use of a liquid medium often results in the isolation of isolation the in results often medium liquid a of Use

Pr csen ad oim yrxd (NaOH), hydroxide sodium and l-cysteine N-acetyl- ocessing 2 pouto (aTAET D bioMérieux, 3D, (BacT/ALERT production ) ) for respiratory specimens, and the the Tableand respiratoryspecimens, for 16-4) 41 41 41 Concentration of the specimen occurs occurs specimen the of Concentration

and 45

2 te piu cniin for conditions optimum the , Plating 44

C Pseudomonas ± in an 1° 43

C Other Other M. ------

Chapter 16 The Clinician and the Microbiology Laboratory

------C.

M. M. 211 , HSP , and M. szulgai M. avium M. tubercu M. tuberculosis and and M. avium complex, M. tuberculosis complex is the demon M. tuberculosis but scotochromogenic at 36° at scotochromogenic but complex are nonpho are complex M. avium M. avium, M. intracellulare C

complex,” not “M. tuberculo complex,” M. tuberculosis It is currently recommended that all initial 50 esting T of of M. tuberculosis, M. africanum, M. bovis, M.

. M. tuberculosis Table Table 16-4). Thesechemiluminescent compound– M. tuberculosis complex should make it clear that M. tuberculosis complex be tested, as well as isolates from M. gordonae, M. intracellulare, M. kansasii, M. intracellulare, M. gordonae, Because more than 100 species of mycobacteria are recognized, use recognized, are mycobacteria speciesof 100 than Becausemore Some Some mycobacterial species that are closely and/or difficult related secA1 genes), followed by the use of species-specific probes or formed formed within a matter of hours from the time of organism isolation. Laboratories not doing further testing of isolates of the tificationof mycobacterial species includeamplification of portiona of the genome common to all mycobacteria (such as 16S rRNA, losis complex (see labeled probes are DNA probes for rRNA, which copies exists in in each multiple cell; they can be used only culture, not directly with on patient specimens. organisms Theprocedure grown can be in per complex and the their identifications have not gone beyond the level (e.g., they should report “ of complex the ” sis, unless additional appropriate testing such as niacin and acid–based iden been has Other methods nucleic reactions done). for nitrate or sequencing of the amplified material. Identification mycobacteria be of to reported been recently spectrometryhas mass MALDI-TOF with rapid and highly accurate. Conventional MALDI-TOF methods (see earlier) were modifiedfor mycobacteria by inactivationof isolates by heat treatment, followed by use of sonication with glass promising very beads is method in This acetonitrile. the and acid formic of presence because definitive identification of isolatescould be accomplished in less than 1 hour. ferent complexes, but these groupings are not so widely recognized. so widely not are these groupings but complexes, ferent of biochemical tests cannot reliably identify most isolates. A practical of identification preliminary method for stration of niacin production and reduction of nitrate. However, for the definitive of identification thisand other species, genomic orpro teomic methods should be used. to distinguish in the diagnostic laboratory are grouped into complexes. complexes. into grouped are laboratory diagnostic the in distinguish to The twomost frequently encountered complexes are complex (consisting , canettii and several other species primarily of animal origin) and avium complex (MAC, consisting of dif also placed into are growers ). Several speciesrapid of M. chimaera isolates isolates of or who of therapy 3months after positive still who culture are patients are clinically failing to respond. Theuse of liquidmedium is recom drugs mended the testing primaryfor (ethambu four antituberculous tol, isoniazid, pyrazinamide, and becauserifampin) results are usually within available a week, as opposed to the 3 weeks with required solid medium. In the proportion method, an isolate is considered resistant to a specific concentrationof antimicrobial if thenumber of colonies growing on the corresponding drug-containing quadrant of the plate is greater than 1% of the number of colonies on a drug-free quadrant. Testing that uses liquid media has been adjusted to provide results Susceptibility In the United States, susceptibility testing of agar 7H10 Middlebrook using method, proportion the by either done is or in a liquid medium. (a photochromogen) grow rapidly, and isolates of grow rapidly, (a photochromogen) marinum stimulation. Photochromogens produce pigment after light stimulation stimulation light after pigment produce Photochromogens stimulation. but not when in grown continuously the dark with no light exposure. whenin the grown as dark pigment well as produce Scotochromogens after light exposure. Nonphotochromogens do produce not pigment even after lightstimulation.not However, allisolates of somespecies will fall into the same category based on growth rate or - pigment pro of most isolates example, For duction. tochromogenic, but some are scotochromogenic; many isolates of Molecular Identification Methods Commercially available probes (AccuProbe [Hologic Gen-Probe; San Diego, CA]) are available for the identificationof , M. avium are usually photochromogenic at 25° at photochromogenic usually are M. complex, tuberculosis although by definitionnot amember of any impor is This nonphotochromogen. a fact in is groups, Runyoun the of tant because tant if is in acid-fast isolated a this colony culture, pigmented, effectively excludes - - M. para C

M. gordonae, a with

complex complex avium M. C

Characteristics It It is also important that

or C.

Specimens

Most isolates of but usually also grow well at 36° 47 Mycobacteria Mycobacterium avium subsp. Mycobacterium

C C

for initial growth 48 mo)

C om

Requir Isolation M. haemophilum, is suspected so that optimal

Identification fr

of but will at grow, least at slowly, 36°

Pathogenic Mycobacterium haemophilum, M. marinum, M. ulcer M. marinum, haemophilum, Mycobacterium C,

owth Gr

16-13

on acidified Middlebrook medium supplemented with blood and charcoal for supplementation with the use of newer media TABLE Unusual Rapidly Growing Mycobacteria Often grow best at approximately 30° May require approximately 30° Requires prolonged (up to 3 Mycobacterium xenopi Grows best at 42° Mycobacterium ulcerans Mycobacterium marinum May require approximately 30° Mycobacterium paratuberculosis May require many months to even grow, on special media Cultivation is not feasible in the diagnostic laboratory May be killed by routinely used digestion and decontamination procedures Grows Grows best in BD BACTEC media May grow on Middlebrook 7H11 medium supplemented with mycobactin J or Mycobacterium Mycobacterium haemophilum Requires ferric iron (provided as ferric ammonium citrate, hemin, or blood) Requires approximately 30° Mycobacterium genavense Mycobacterium Mycobacterium bovis Growth said to be enhanced by 0.4% pyruvate, but there is no apparent need sistent sistent clinical picture, requires consideration specimen of cross-contamination. the possibility of also be used in assessing the likelihood of cross-contamination. tuberculosis isolated from a one of several smear-negative specimens, especially in the absence histopathology or a of consistent highly con the preference the of preference several skin and subcutaneous pathogens to grow at Mycobacterial Categories Traditional and Methods aid their To further identificationto thespecies level,nontuberculous mycobacteria were divided into several groups by growers (Runyoun Runyoun. group IV) Rapid take less than 7 days subculture to for a solid medium from they a growth dilute suspension; however, after medium liquid in or solid on grow to days 7 than less take also generally on initial isolation. Slowly growing mycobacteria are further divided into photochromogens, scotochromogens, and nonphotochromogens ability their on depending respectively), III, and II, I, groups (Runyoun light to production pigment of relationship the and pigment produce to reported from some patients with Crohn’s disease the and Crohn’s with patients some from reported tuberculosis, disease in etiologic ruminants, agent of cannot reliably beJohne’s iso lated lated in the routine diagnostic laboratory. Significance Mycobacteria as such mycobacteria, of species certain of Isolation or a rapidly growing mycobacterium, may represent colonization or specimen contamination rather than active disease, but the potential significanceof repeated isolation of the samespecies from the same source requires careful evaluation. should should be incubated at 30° approximately the clinician notify the laboratory if a pathogen with specific growth requirements, such as culturing procedures can be used. 30° (including and rapid growers), at least at a growers), portion and ans, of rapid all skin specimens biopsy (and perhaps other specimen types obtained from cooler areas of the body such as the extremities) submitted for mycobacterial culture water organism, are regarded as contaminants; only rarely is it a proven proven a it is rarely only contaminants; as regarded are organism, water pathogen. Laboratory although rare, cross-contamination, fortunately has been well documented and requires detection and prevention. sustained vigilance for its Part I Basic Principles in the Diagnosis and Management of Infectious Diseases mycobacteria and aerobic actinomycetes. of species pathogenic other most of testing the forrecommendations standard for susceptibility testing of tive and should be confirmed by phenotypic tests. phenotypic by confirmed be should and tive detection of resistance genes, but these should be considered presump direct available forthe interpretivearealso Moleculartests guidelines. and procedural species-specific of with variety a use including growers, for rapid testing susceptibility bacterial for used procedure tion microdilu broth the of modification a describes document CLSI The notrespondtreatmentgenerallyforagents tothe used oftuberculosis. growingRapidlymycobacteriado useful. most maybe species various cussionsofcircumstances the susceptibility underwhich testing of the tions, are included with gram-positive anaerobic bacteria. In addition In bacteria. anaerobicgram-positive with included are tions, condi growth anaerobic for requirement or preference a have which genus the (including actinomycetes anaerobic similar morphologically The considerablenomenclatural occurring. changeis nomycetes. and aerobicmycobacteria,the are included with which often acti also corynebacteria both to respects certain aerobicconditions. in similar are They under best grow that branching) conspicuously and also beaded species most (in rods gram-positive are actinomycetes Aerobic ACTINOMYCETES AEROBIC CDC. the by publishedrecently havebeen test interpretation andthis of use the on of antigens secretory early to the similar peptides synthetic with incubation after individuals tized sensi of lymphocytes from blood whole heparinized fresh in released IFN- of detection the involve tests The 2008. in approved was MA) Marlborough,Immunotec,(Oxford test T-SPOTTB the and 2005, in A second-generation assay, QuantiFERON-TB Gold test, was approved test; Carnegie, Cellestis, Victoria, Australia) was approved by the FDA. IFN- first (IGRA) for the the 2001, detection of in uberculosis: and immunity, cell-mediated to sure expo assessing for method traditional the is test skin tuberculin The Laboratory Latent of isolates different of relatedness the to methods determine of variety a by performed be now can typing Molecular Epidemiology 0.1 is it media, liquid OH] is agar 7H10 in 0.2 isoniazid for concentration critical the (e.g., agent given a for media liquid and solid between somewhat differ maytions consideredis important.most drug These ofeach “critical” concentra standardized the with obtained those procedure using a solid medium. Testing with of the “critical accordanceconcentration” in are that 212 sequencing may rapidly render all other techniques obsolete. techniques other all renderrapidly may sequencing with epidemiologic investigation of bacterial outbreaks, whole-genome strain the of AsRAPD. and PFGE are evaluationnontuberculous mycobacteria ofrelatedness for used techniques molecular the Among ticable, such as for isolates with too Other few copies organism. of the of insertion IS the sequence. when strain used be can techniques one than more by caused is infection an strain,andisolated infecting determiningwhether strain newly or a to cross- situations, outbreak laboratory in determining whether a second episode of isdisease due to a previously possible infection of of sources tracing episodes contamination, investigating as purposes BG (n otat ih h tbrui si ts, hr i n cross- no is unreliable. is follow-uptest there skin where patientsfor and test, reactivity) skin tuberculin Calmette-Guérin the with bacillus contrast (in with (BCG) immunized previously patients for o eemn relatedness. determine the to and electrophoresis, gel by DNApatternsfragmentdifferentfromobtained isolates comparedare in each separated site one are sequence) at only insertion cleaves (which endonuclease specific a by chromosome the differentin in strains. The fragmentsDNA produced is found in 0 to approximately 25 copies scattered in different locations IS sequence insertion the on based is method used widely

g/mL; in BACTEC 12B and ESP II [Trek Diagnostics, Westlake,[TrekDiagnostics, II ESP and 12B BACTEC in µg/mL; Interferon- tuberculosis. M.

Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. T 53 52 The taxonomy of this group is under active revision, and revision, active under group is taxonomy this The of The CDC recommends that the IGRA tests can be used used be can tests IGRA the that recommends CDC The

Diagnosis 51 M. tuberculosis infection (QuantiFERON-TB

The procedure has been used for such for used been has procedure The g/mL). CLSI has published an approved an published has CLSI µg/mL).

Dtie guidelines Detailed tuberculosis. M. M. tuberculosis, which also includes (IFN- γ -based technique is not prac not is technique 6110-based 50 The document includes dis- i ciia i regulating in critical is γ) . tuberculosis M. ees assay release γ Actinomyces), which which 6110, A . γ ------

clinical significance of their isolation should be carefully assessed. isolationtheir be significance should of clinical cetes are very uncommon as laboratory contaminants,actinomy and aerobic the potential most However, very contamination. represent are to likely lesions, nonmycetomatous from and sites nonsterile from of colonies Single occur. can contamination specimen and colonizationenvironment, the in widespread are isms long, branched structures reminiscent of hyphae. Because these organ formto tendency relatively their their in rategrowthslowandin molds both resemble they because perhaps laboratory, the of section ogy mycol the in handled been organismstraditionallyhave these molds, positive and negative controls. Although they are andbacteria true not manceandinterpretation appropriatedonewith andshould always be perfor for expertise some requires stain The decolorization. for used are both or period, time shorter solution,a decolorizing weaker a that similar to the is Kinyoun actinomycetes stain for mycobacteriafor aerobic but differsparticularly in stain acid-fast modified The mycelium. of isolates only these, of tive; dococcus,and the in cultivatable not is routinelaboratory. genera the Organismsin disease, Whipple’s of agent causative the be necessary for isolation.for necessary be may biopsy abscess, brain as such infections, deep-seated cause may thatlikelihood the organism be detected. will these organismsBecause the enhancing for useful are procedures plating because and staining advisable special is laboratory the alerting suspected, is group this lose viability at low temperatures. low at viability lose some because avoided be should specimens of tion culturesadequateare also for aerobic the actinomycetes, but refrigera fungal and bacterial for suitable procedures transport and Collection Specimen is buffered charcoal-yeast extract (BCYE) agar, originally agar, (BCYE) extract charcoal-yeast buffered is dia that hasfound been forto particularly useful be the isolation of medium A thatflora. normal suppress the media to agentsantimicrobialcontain of use the with enhanced be may actinomycetes aerobic presentbe in low numbers and are relatively slow growing, isolation of may they Because culture. for from specimens mycobacterial flora rial they may not the survive specimen processing used to eliminate bacte actinomycetes, which grow on many differenttypes of media. However, processingrequired isspecimen foraerobicisolation No the of special Specimen necessary. be may stain, Giemsa a as for stained darkly such stain, too other some and detail, structural appearof visualizationadequate may organism the material, stained Gram- In filament. the of axis long the to perpendicular and parallel both occur that divisions with filaments branching produces golensis appearance. in coryneform more overall and dia, Tsukamurellaand unbranched,than muchshorterspp. be may be requested if always should smear acid-fast negativemodified asonly a images,and alone. morphology organism of basis the on certainty with made be cannot than other organism any of assignment genus definite a but gram-positive, avidly more and wider appear to tend mycetes), as such genera, other in isms Organ be noncontiguous tinybeads. of gram-positive mainly composed to appear may and long and thin especially be to tends Nocardia (see staining is patientspecimens in isms organ these of detection direct of means available generally only The tory should be notified in advancewhen such an infection is suspected. may greatly facilitate isolation of the etiologic agent; hence, the labora “grains” these of “grains”Detection material.as purulent scopically in macrovisible masses dense lesionsas organisms clinical maygrowin mycetoma, the as suchorganisms, these by caused infections some In Direct andStreptomyces, Tropheryma, Rhodococcus, Nocardiopsis, Nocardia, Gordonia, Dermatophilus, ura, includehumans for pathogenic species contain that cetes to and Corynebacterium may also be invisible on Gram stain or may appear may or stain Gram on invisible be also may Nocardia

Organism Tsukamurella- posi acid-fast arealwaysmodified almost

Nocardia infection is suspected. Collection Pr ocessing

, genera of aerobic actinomy aerobic of Mycobacteriumgenera , Detection - actino anaerobicStreptomyces the (and

regularly produce an aerial aerial an produce regularly Nocardia and Tsukamurella. and 53 If infection with an organism in organism an with infection If

T Plating ransport Tablestain,Gram On 16-6). Gordonia,Nocardia, Rho 53 especially especially Streptomyces, Gordonia, Rhodococcus,

Tropheryma whipplei,Tropheryma Dermatophiluscon strains Nocardia Dermatophilus Actinomad Nocar Nocar ------ Chapter 16 The Clinician and the Microbiology Laboratory - - - - - 213 Because Rhizomu 56 Table 16-14 Table Pseudallesche C.

and and hence are C

and and hence are not C

H. capsulatum, can be is named named is Scedosporium. True aseptate Ajellomyces Ajellomyces capsulatum, and Sporothrix Sporothrix schenckii grow as and and at 35° are now recognized, now are Coccidioides C

or imperfect state. In the past, past, the In state. imperfect or anamorph, Candida, also produce hyphal structures. 55 True . hyphae that True are septate suggest organ Coccidioides spp. and species most commonly grow in the in grow commonly most species Coccidioides Scedosporium apiospermum Scedosporium C. posadasii, and can be distinguished by molecular H. capsulatum is named Aspergillus, Aspergillus, Fusarium, and Issues

teleomorph, or perfect state; the form that results from asexual Many Many molds have a morphologic form that results from sexual Now, ria all boydii. Now, fungi will be given a single name, using historical precedent and common use. the sexual form of of sexualform the reproduction; reproduction; these differ from known is reproduction sexual the from results that form form The reproduction. that results from asexual as the the as known is reproduction each form has been given a different scientificname; for example,the sexual form of infectious spores are not present in patient specimens or in such as sputa patient present not are infectious spores biopsy specimens, these specimens are not considered hazardous by the aerosol route, but they would be infectious by accidental inocula labora significant be can molds dimorphic the of severalBecause tion. tory hazards when grown in mold phase, the laboratory personnel clinician whenever a should patient has alert a significant probability in the patient but grow as a mold in the laboratory at 30° at laboratory the in mold a as grow but patient the in isolated commonly more and significant more the of some summarizes genera and species according to the categories frequently used in the diagnostic laboratory. Of the clinically significant dimorphic molds, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, and yeasts or yeastlike structures in the dimorphic. thermally patient at 37° as patient thick-walled spherules and a require special medium that is to be in widely available not grown this in form otherwise,vitro; these species grow as a mold both at 30° species of thermally dimorphic. Two C. immitis and significant clinically any are there that clear not is it although methods, differences between them. Safety Virtually all molds release their reproductive structures (conidia spores) into or the air, and for many fungal pathogens, the tract is respiratory the initial portalit Thus, is of notentry. surprising that certain organisms, particularly Terminology it although terminologyuses mycologythat, laboratory diagnostic The both determining of terms in useful is significance, taxonomic little has clinical relevance and Yeastsidentification.are unicellular organisms that grow as relatively produce fuzzy smooth, colonies because creamy of the colonies, production of Some organisms may whereas initially aerial produce yeastlike colonies that become hyphae. molds more moldlike with continued incubation. as appear or round oval cells yeasts, by budding. that Many reproduce Microscopically, yeasts such as most species of Hyphae are morphologically subdivided into pseudohyphae and true resemble chains of sausages in Pseudohyphae hyphae. that the hyphae tend to be constricted at the septa, and each cell originates as often is a septum and a rounded, and small to be tends bud. tip the at cell The found at each branch point. In contrast, straighter, more true parallel walls hyphae without constrictions tend at the to septa have and usually without a septum at the branch point. True either septate hyphae or aseptate may be significant laboratory hazards. Containment facilities (e.g., laboratory for specimen handling, BSL-2 a initial isolation, and identification; BSL-3 is of for furtherneededare such agents) to propagation process specimens and to work with cultures of these organisms. isms isms such as hyphae hyphae may be broad and have a ribbon-like appearance; their pres ence suggests one of the Mucorales (aseptate molds) such as such molds) (aseptate the Mucorales of one ence suggests cor. Brown pigment in dematiaceous (black) mold; hyaline molds do not produce pigmented hyphae suggests that hyphae. the These simplified organism distinctions is are Candida species can absolute. not form a true hyphae, and molds the may “aseptate” some Thus, have an occasional septum. and Dermatophytes dimorphic molds are usually of separate considered categories mold distinct from the other hyaline septate molds. The likelihood thatan organism may be a der hair, skin, (e.g., source specimenthe by suggested usually is matophyte growing when they form are a nonmold have molds Dimorphic nails).

------Prelimi 53 Gordonia are best Table Table ); 16-7 the same Actinomycetes

Nocardia has become increasingly Legionella (see obic Nocardia should be held for 2 weeks, For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. spp., as performed in most labo most in performed as spp., Nocardia Aer

It may, however, prove to be the case that Rhodococcus and probably esting ), secA is the presently most methodreliable for 50 T of

53 is thought to be an achlorophyllous alga; achlorophyllous an be to thought is Prototheca R. equi, which is the principal pathogen in the genus Cultures for 53 . Nocardia formerly considered to be parasites, are now now are parasites, be to considered formerly Pneumocystis, typically have a powdery, whitish surface because surface whitish powdery, a have typically Nocardia Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada The genus genus The 54 Molecular Molecular methods, such as amplificationby PCRof a portionof Colonies Colonies of The taxonomy of the genus Rhodococcus, most commonly are somewhat mucoid and but initially is Therodlike ofisolate acquire incubation. 4 after days pink color a will evolve to a coccoid form with extended incubation. (and/ gene rRNA 16S the of sequencing DNA by followed genome, the or other genes such as medium medium with antimicrobial agents inhibitory to most other bacteria can be used for specimens such as sputum, which may normal also contain flora. complex as new pathogenic species are recognized and formerly rec ognized species are subdivided into additional species. Conventional for procedures identification are these and tests, phenotypic of number small a only involve ratories, in the species different the among accurately distinguish to insufficient genus. Differences in theresults of testing for susceptibility to certain may agents help in antimicrobial discriminating some among of these species, but such testing has limited value. identificationof aerobic actinomycetesto thespecies level. although although most isolates grow within 3 to 5 days. nary work using MALDI-TOF, with processing manner of similar the to isolate mycobacteria, in is a very become the identificationmethod of choice. promising and will likely isolates isolates in the genera tested tested for susceptibility by using the same procedures as for growing, rapidly aerobic bacteria. In the different past, susceptibility differentinvestigators testing used procedures; when attempting to cautioncompare published results obtained must by differing bemethodologies. used FUNGI bacteria, Unlike fungi are eukaryotic. is It estimated that there may be several million species of fungi, of reported which less to than be 500 have human been additional However, pathogenic or species are reported in animal the literature pathogens with as the regularly, result of both already-known organisms being recog any frequency. Because infection with aerobic actinomycetes generally occurs on sporadic basis, a epidemiologic are investigations only rarely needed. A variety of molecular techniques have been found useful for epidemio Epidemiology ratory ratory reproducibility. logic investigations. nized as pathogens and the description of new species. The members genus the of considered fungi, and recent taxonomic studies have that demonstrated members of the phylum Microsporidia belong to Fungi. the kingdom Susceptibility It is hoped that the susceptibility use testing of of the aerobic CLSI-recommended actinomycetes procedure will comparability for produce of greater results from different laboratories, with but the procedure is needed to ensure intralaboratory experienceand interlabo cobacillary fragments, with long, branching forms much less conspicu less much forms branching long, with cobacillaryfragments, ous than in direct patient material. Organisms stained from may culture be less acid fast than those in stains prepared from the original specimen. Growth on traditional mycobacterial media (e.g., - Middle brook agar, Lowenstein-Jensen medium) enhances acid-fastness. - Iso lates that form aerial hyphae and are acid fast can be identified as presumptively Identification of Colonies surface. reverse tan-to-orange a and mycelia aerial of production the of When stained from colonies, the organism oftenbreaks up into coc developed developed for the recovery of organisms in this can organisms be genus usually and are pathogenic in included discussions of fungi because they grow well on most media. fungal culture Part I Basic Principles in the Diagnosis and Management of Infectious Diseases aoaoy o ae diinl rcuin wt te utrs f such of cultures patients’specimens. the with precautions additional take to laboratory the allows notification advance Such infection. mold dimorphic a of this organism is suspected. Fungal cultures are generally incubated at incubated generally are cultures Fungal suspected. is organism this ofpresence the when laboratory the therefore notify shouldclinicians recover to media to added be must oil, olive as such ubr n qatttv PR i uuly ihr n netd than infected in higher usually is PCR, quantitative a in number *Often considered a dematiaceous mold. dematiaceous a considered *Often the isolation of all molds from blood (see (see blood frommolds isolationall of the capsulatumH. techniqueforand perhaps isbest fromtouse the blood of isolation the forprocedure sensitive most the be to considered still s A, lhuh C cno dsigih ooiain rm infec from tion. colonization distinguish cannot PCR although BAL, as undergo patients to invasiveill in benefit who are too procedures such detect can assay,which The infection. of suspicion clinical high a is there whom for negative are specimens sensitivea isfor assay patientsthat useful maywhom respiratory in be research tool. Real-time PCR for diagnosis of than other fungi of detection fordirect methods molecular of use organism; the the contained orin by produced substances of detection by or organisms the of alization Fungi are generally detected in clinical specimens either by direct visu Direct growing a laboratories. by of used number be to likely are pathogens, periods incubation shorter fungal these of most sought; being organisms the and specimen isolation of type the on the depending for suffice incubation of even or weeks,recently, until4 forand held generallywere they latumHowever, suspected. was shownthatshorterperiods been hasit as such growingmoldslowly a oflonger,presence the when 30° occur. to appears promptlyviabilitylaboratory,processed andfungal the ofby loss little ratory in an 200 anaerobic transport vial; to as long as (up the specimen is received urine and meningitis) considered optimal. Aspirated material chroniccan transportedbe to the labo for fungal are for specimens morning first suitable overgrowth; bacterial not of because areculture sputa or urine of collections four–hour 30 has specimen asfor collected, example,been of of cases CSF the in (up to 5 volume adequate an that sure be to important is It teria and yeasts, depending upon the specimen source (see ocessing bac suppressassociated and isolation fungal enhance to used be may bacterial for (see specimens of culture fungal for suffice collection will culture the for used procedures The and Specimen 214 detection of particular pathogens; these issues are dealt with in the the in organisms. specific to with pertain chaptersthat dealt are issues these the pathogens; for particular others of than detection useful more are types specimen Certain ratory. capsulatum, is suspected, it is useful for the clinician to notify the labo ie o te slto o yat fo blood. from yeasts of isolation the for tive available broth and biphasic systems have been shown to be quite sensi Trichosporon Rhodotorula Saccharomyces Malassezia Blastoschizomyces Cryptococcus Candida YEASTS TABLE Modified from Koneman EW, Roberts GD. EW, Roberts Koneman from Modified

C, 59

The quantity of quantity The Pr

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Collection, 57 Laboratory Blood culture performed by lysis-centrifugation is lysis-centrifugation by performed culture Blood Wangiella Exserohilum Exophiala Curvularia Cladophialophora Bipolaris Alternaria Dematiaceous 58 in a BAL, as assessed by cycle cycle by assessed as BAL, a in Pneumocystis

Thus, if a slowly growing mold, such as such mold, growing slowly a if Thus, Detection by traditional stains, but in but stains, traditional by Pneumocystis is still primarily a primarily still jiroveciiPneumocystisis For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.

ransport, Categorization 3rd ed. Baltimore: Williams & Wilkins; 1985. Wilkins; & Williams Baltimore: ed. Mycology.3rd Laboratory Practical Table16-5). ); special media media special 16-2); Table Trichophyton Microsporum Epidermophyton Dermatophyte 31 Pneumocystis pneumonia suc o fty acids, fatty of source A

57 spp.;Malassezia

of Commercially

mL).

Table 16-7). mL Selected H. capsuH. Twenty- oreven Sporothrix schenckii Sporothrix marneffei Penicillium brasiliensis Paracoccidioides capsulatum Histoplasma spp. Coccidioides dermatitidis Blastomyces Dimorphic H. ------

Clinically Hyaline and a variety of substances can cause a false-positive result. false-positive a cause can substances of variety a and such as those caused by infections, fungal invasive of variety a in positive be may β-d-glucan for test The studies. different in varied have specificity and that sensitivity in results test these of significance the interpreting in needed is are calcofluor white, toluidine blue O, methenamine silver, methenamine O, blue Giemsa, and monoclonal antibody stains. toluidine white, calcofluor are ovecii of cells the positive,whereas of phae (see stain alized directly by a Gram stain, a KOH wet mount, or a calcofluorwhite nicolaoustains. Inmicrobiology laboratory,the are fungi visu usually stains, such as the methenamine special silver, acid–Schiff, periodic and of Papa variety a by cytologically or histopathologically visualized be may Fungioverlap. extensive is there although patients, colonized to helpfulbe for the diagnosis of invasive aspergillosis. procedure. ink India the by cryptococci for CSF of nation exami is than meningitis sensitive cryptococcal of morediagnosis the for technique a be to demonstrated been has antigen cryptococcal of Detection fluids. body of variety a on performed be can tests These assessed. being and still are coccidioidomycosis cryptococcosis and blastomycosis of of sis diagnosis the for histoplasmosis. diagno tests the forthese Sensitivity and specificity of usefulness their strated and spp., oides specimen. the in presentareorganisms few relativelyif negative falsely be will and relativelyinsensitiveare stains these however, stains; other the with available not specificity provide stains antibody Monoclonal allow the visualization image. of both cysts and trophozoites negative of a as only appears cyst wall the but trophozoites, and structures intracystic of visualization be can which to cells, yeast similar stain morphologically also they that is stains these of back blueO, and methenamine silver stainsdraw a stainwall; only cyst the y hs y cn e edl vsaie wt te s o a fluorescence a of use the microscope. with visualized readily be can dye this by stainedorganisms fungi; all in found polysaccharides wall cell certain to binds compound fluorescent a stain, white calcofluor the In tions. prepara such of reading the for required are expertise and care able components, such as tissue blood vessels, can be addition,mistaken for fungal elements, out in so consider not stand background; do the from they prominently because discern to difficult quite be actually may organisms fungi, most of visualization allow mounts wet KOH and do not exhibit the width, folding often in found in aseptateregular molds.Although more and smaller be to tend molds septate hyaline whereas themselves, on fold to tend that width uneven rather of tures struc ribbon-like branching,broad, the as appear to tend molds specimens, aseptate clinical In invisible. Gram-stain or images, negative Aspergillus spp. or as such organisms of hyphae true The material. capsular presumablyis that halo orange an by surrounded be may and violet crystal with The detection of galactomannan in blood and BAL has been foundbeen has and BAL galactomannanblood in of detection The fortests detection Antigen Among the procedures that can be used for visualization of visualization for used be can that procedures the Among MOLDS

Septate species usually stain uniformly throughout as gram- as throughoutuniformly stain usually species Candida Significant ). With the Gram stain, yeast cells and pseudohyand cells Tableyeast stain, GramWith the 16-7). are available and have clearly demon clearly have and available are capsulatum H. Rhizopus spp. are gram-negative, visible as unstained Scedosporium prolificans Scedosporium apiospermum Scedosporium Penicillium Paecilomyces Fusarium Aspergillus Other

and/or Candida spp.; not all fungi contain yt. ima ti allows stain Giemsa cysts. Pneumocystis C. neoformansC. B. dermatitidisB. ,

Commonly * 25 Calcofluorwhite, toluidine * may be unevenly speckled speckled unevenly maybe C. neoformansC. ,

Isolated Saksenaea Rhizopus Rhizomucor Mucor Conidiobolus Cunninghamella Basidiobolus Apophysomyces Absidia Hyaline 42 P. jirovecii, and However, care

β-d-glucan, Aseptate Fungi Coccidi P.jir ------ Chapter 16 The Clinician and the Microbiology Laboratory

------63 215 Candida spp., but , may have an ana an have , may There may prove to prove Theremay be 61 ) that is morphologically Numerous caveats pertain to caveats Numerous Molecular methods, such as 63,64 44 Candida Candida krusei is assumed to be A. fumigatus. Neosartorya fischeri Neosartorya 64 demonstrated accuracy demonstrated to comparable gene 62 esting T Aspergillus Aspergillus fischerianus

For the echinocandins, the concept of (minimal effective 64 With regard to the relevance of the end points obtained in the 64 sequencing, sequencing, with results available in less than 1 hour. sequencing sequencing ribosomal genes or the internal transcribed spacer (ITS) region, are particularly useful for distinguishing accurately between such similar species. DNA sequencing is being used increasingly large by diagnostic microbiology laboratories to identify molds. As with spec mass MALDI-TOF useof the yeasts, and bacteria,mycobacteria, trometry promises to alter traditional approaches to mold identifica tion. Lau and coworkers in noninvasive disease has been found helpful allergic aspergillosis bronchopulmonary and aspergilloma. For for details the diagnosis of for and agents diagnostic methodologyoptimal different for regarding relating to problems see result interpretation, in chapters relevant this text that pertain to specific etiologicagents. VIRUSES The International with As viruses. of species Committee 2300 than more recognizes ) www.ictvdb.org on Taxonomy of Viruses (ICTV; Epidemiology fungal different to applied been have techniques molecular of variety A species for determination of strain relatedness. Such studies are avail able only in larger laboratories or in reference centers. Serology Theusefulness of serologic determinationsfor the diagnosisof infec tion has been investigated immunodif and for fixation complement as such many methodologies, different different fungi.Often, several fusion, beenhave developed for the same Kits for detection organism. of antibody to certain organisms are commercially available. Testing for the presence of antibody to disease has assist been used in for the various the dimorphic molds; such diagnosis testing of may be invasive particularly useful for the diagnosis histoplasmosis, and paracoccidioidomycosis. Antibody testing for of the coccidioidomycosis, gener pathogens fungal other by caused infection invasive of diagnosis ally has not been found to be useful. However, testing for antibody unfamiliar unfamiliar fungal name, the clinician should consult with the labora Susceptibility TheCLSI has published standardized fungi. filamentous and yeast of procedurestesting for susceptibility For itraconazole, the data interpretive are based solely on results with mucosal infections, and sufficient data are not breakpoints for available invasive infections. In provide the to case of filamentous fungi, the way the MIC should be read is and ketoconazole, for fluconazole pairs. example, antifungal agent For different for various organism– complete absence of growth at the MIC is not (nonder required. more or Rather, 50% “the approximately to corresponds allowed turbidity tory and/or search for relevant information on an Internet site such site as on an Internet information tory relevant search for and/or PubMed ). (www.pubmedcentral.nih.gov/ A further complication as such is teleomorphs, some that reduction isolates) to isolates) 80% (dermatophyte or more matophyte in growth compared with the growth in the control medium).” well (drug-free virtually indistinguishable virtually from indistinguishable testing testing of filamentous fungi, the CLSI documentstates, “The clinical of relevance testing this of fungaluncertain,group remains pathogens and breakpoints with proven relevance have yet to CLSI beor any regulatory agency.” identified by bial bial agent that leads to the growth of small, rounded, compact hyphal forms, compared with the hyphal growth seen in the growth control well.” concentration” is used; concentration” this is “the lowest of concentration antimicro the assessment of the clinical relevance of such results; these results; varythesuch of of clinical the relevance assessment with avail currently example, the infection. of For site drug,and organism, intrinsically resistant to fluconazole, scale. fluconazole and available the with interpreted be not should species MICs isolatesof that of able able interpretive guidelines are available only for CLSI document M27-S3 notes that differences in pathogenicityand antifungal susceptibility among such morphologically similar organisms. morph morph (in this case,

------C. A 60 Table Fusarium.

Penicillium om fr

Candida species, and and Curvularia H. capsulatum (see Fungi H. Use of capsulatum. these

Aspergillus Aspergillus ). flavus of

Aspergillus, is an area of active Identification of most molds is based is molds most of Identification ida from blood, with a mean time to d Candida Panel and T2DX (T2 BioSys 60a For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Can Aspergillus Aspergillus niger is less likely to be a - patho Fungi

Isolation

B. and dermatitidis of

Aspergillus spp. from the respiratory tract of an 1.0 hours. 1.0 ± of P. marneffei are common laboratory P. contaminants;

from pulmonary material other than a lung lung a than other material pulmonary from Candida C. neoformans may occasionally be a colonizer and Candida can be part of the normal upper respiratory Aspergillus Aspergillus fumigatus or Specimens

Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada The development molecularof methods for the diagnosis of B. B. dermatitidis, Coccidioides spp., and is also germ tube positive; however, because is it clear not is also germ tube however, positive; dubliniensis, that there is any clinically them. between distinguish significant to attempt not do differencelaboratories most species, between these two for Boththe systems are available identifi kits manual and automated lating lating molds are increasingly reported as agents of disease, especially which basidiomycetes, often are These hosts. in immunocompromised are difficult for clinical laboratories to speciate, basidiomycete but as report the a contaminant of a should be laboratory should reviewed never assume a carefully. particular The isolate is a contaminant and not report recovering the organism. Rather, the laboratory con cerns should be discussed with the ordering physician and the isolate reported with the contaminant. qualification that it is apossible laboratory cation of clinically significantyeasts. In addition, MALDI-TOF mass spectrometry is highly accurate species for level, making identificationthe need of for phenotypic yeasts tests unnecessary. at the Identification species yeast in clinical isolated , commonly the most albicans Candida laboratories, is identifiedby performing a simpletest, such as agerm tube test. It has recently been found that another species other than their isolation is almost never indicative of because disease. However, fungiofataredifferent many capable leastcausing occasional casesof infection, each needssituation to be assessed Nonsporu individually. fungal infection, particularly with research. Such methods are still largely investigational for specimens. direct use with immunocompromised patient, presents a particularly difficult inter pretive problem for which no unambiguous guidelines exist. In addi tion to the clinical and radiographic aspects of each situation, it may the in directly seen was organism the whether (1) consider usefulto be specimen, (2) the quantity of organism that grew in culture, (3) if the organism is isolated in multiple cultures, and (4) the particular genus or species recovered (e.g., gen than is Clinical It is often difficult to assess the significance of the isolation other than fungi of dimorphic molds from clinical specimens, particularly if they are isolated in small quantity from nonsterile sites, such respiratory tract. as is It usually impossible theto attach clinical significance of isolation the to tract flora. Even not a pathogen in the pathogens, respiratory such tract. as Isolation of opportunistic Significance biopsy because first-of-its-kind system, the T2 tems, Lexington, MA) is based on detection and speciation proprietary of magnetic resonance 4.4 of identification largely largely on the morphologic features of the organism, but for certain organisms, other features, such as the ability to grow at higher tem peratures or the need straight be may for genus particular a to specific assignment fungi, some nutritionalFor used. supplements, are also forward, whereas identification to examples question; in group particular the thein expertise with mycologist species level may require the genera to belong that a organisms include isolates of identification the for available are probes Chemiluminescent of As with similar probes for mycobacterial identification, they are are they identification, mycobacterial for probes similar with As 16-4). intended for use with isolates, not with direct patient specimens; the Theseprobes can ribosomal RNA. organism for probe is a probe DNA be used with either the yeast or mold phase, thus allowing early iden probes also the eliminates of need cultures manipulation for extensive of these hazardous organisms. As with other organisms, the number of recognized species is increasing When rapidly. confronted with an tification of isolates and obviating the need for conversion of themold theneed conversion for andobviating ofisolates tification to the yeast phase for Part I Basic Principles in the Diagnosis and Management of Infectious Diseases *Circular nucleic acid; all other viruses with linear nucleic acids. nucleic linear with viruses other all acid; nucleic *Circular zoster virus. zoster VZV, medium; varicella- transport viral VTM, virus; RSV, syncytial amplification; repiratory acid nucleic NAA, virus; choriomeningitis lymphocytic LCM, virus; simplex herpes HSV, respectively; 8, 7, 6, herpesvirus human -8, -7, HHV-6, acid; EDTA, ethylenediaminetetraacetic CMV, EBV,CSF, virus; fluid; cytomegalovirus; Epstein-Barr cerebrospinal formed with specimens collected from the site of active disease or, if or, disease active of site the from collected specimens with formed per Culture be can viruses. cell-free with specimens on performed be specimens with infected cells, whereas antigen tests and NAA tests can weeks later to moredemonstrate seroconversion.or Microscopy 2 requires collection of and disease of course the in early specimens blood of requires collection diagnosis For example,serologictest. diagnostic ransport the by is determined pathogen, site of infection, timing related toonset, disease and specific diagnosis viral for specimen appropriate The and Virology 216 human pathogens (Tablepathogenshuman 16-15). or helical (size, virion the icosahedral, enveloped or not). of Of the 87 families of viruses, structure26 contain and nucleus), or cytoplasm stranded, positive or negative type the strand, linear or circular), replicationdouble- (in or by single- RNA, or (DNA genome their classified of organization and are viruses and Specifically, morphologic properties. their by genomic classified are viruses fungi, and bacteria Blood fluid Amniotic BODY Parvoviridae Anelloviridae Single-Stranded Bone marrow Bone Urine Tissue Skin Respiratory Genital Feces Eye CSF,tissue brain TABLE TABLE DNA, deoxyribonucleic acid; RNA, ribonucleic acid. ribonucleic RNA, acid; deoxyribonucleic DNA,

SITES

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Specimen Specimen Classification Arbovirus, adenovirus, arenavirus, BK virus, coronavirus, CMV,coronavirus, virus, BK arenavirus, adenovirus, Arbovirus, CMV,virus HIV,rubella HSV,B19, parvovirus POTENTIAL CMV, EBV, HHV-6, parvovirus B19, rubella, VZV CMV,rubella, EBV,B19, parvovirus HHV-6, Adenovirus, BK virus, CMV, filovirus, HSV, enterovirus, measles virus, CMV, HSV, measles virus, filovirus, enterovirus, BK Adenovirus, CMV,viruses Adenovirus, HSV,other many rubella poxvirus, B19, parvovirus HSV,HHV-8,virus, Enterovirus, measles CMV, hantavirus, HSV,filovirus, coronavirus, arenavirus, Adenovirus, CMV,Adenovirus, HSV,retrovirus papillomavirus, CMV,Norovirus,coronavirus, enterovirus,astrovirus, Adenovirus, CMV,virus, HSV,A VZV coxsackie enterovirus, Adenovirus, enterovirus, virus, CMV,A arenavirus, coxsackie arboviruses, Adenovirus, DNA rubella virus, VZV virus, rubella retroviruses, rabies, B19, parvovirus virus, measles LCM, HHV-8,HHV-7, HSV, E), HHV-6, D, C, B, (A, virus EBV, hepatitis enterovirus, filovirus, mumps virus, rubella virus rubella virus, mumps VZV virus, VZV RSV,virus, rhinovirus, rubella parechovirus, virus, measles metapneumovirus, virus, parainfluenza virus, influenza sapovirus rotavirus, VZV retroviruses, virus, rabies prions, parechovirus, virus, mumps virus, measles EBV,LCM, HSV,HHV-6,virus, JC

VIRUSES

Collection

Collection Poxviridae Polyomaviridae* Papillomaviridae* Herpesviridae Hepadnaviridae* Adenoviridae Double-Stranded

V iruses

for

V iruses

Associated Togaviridae Retroviridae Picornaviridae Hepeviridae Flaviviridae Coronaviridae Caliciviridae Astroviridae RNA Positive-Strand Rhabdoviridae Paramyxoviridae Orthomyxoviridae Filoviridae Deltaviridae Bunyaviridae Bornaviridae Arenaviridae* RNA Negative-Strand Single-Stranded -

rvds gie o slcin f pcmn fr igoi of diagnosis for specimens infections. human with associated of viruses selection for guide a provides 16-16 confirmation. laboratory for collected the specimens microbiologist, appropriate clinical the with collaboration in and, specialist disease infectious patient’sor physician the by determined be should studies.” The most likely viruses responsible forthe patient’s symptoms “viral for request generalized the with laboratory virology the to sent colonizationor secondary (e.g., stool, Specimensurine). should not be respiratorytract) upper replication(e.g., initial of site the impractical, e hr lvd n muooptn ptet ad persistent and patients immunocompetent in lived short be be prolonged can human shedding even when the viruses, patient appears viral asymptomatic. Some C viral shedding may [HIV]), infections and virus chronic B immunodeficiency other hepatitis For [CMV], cytomegalovirus decreases. (e.g., rapidly then and appear symptoms before shortly begins shedding viral virus), WestNile rus, viral many For host. rhinovi virus, mumps the virus, measles virus, and influenza (e.g., infections virus the of properties the by mined

with The timing for collection of specimens for viral diagnosis is deter is diagnosis viral for specimens of collection for timing The

Collect blood for culture in sterile vacuum tube or EDTA tube; avoid EDTAtube; or tube vacuum sterile in culture for blood Collect without container sterile in transport and amniocentesis by Collect SPECIMEN Aspirate transferred to EDTA tube and transported to laboratory to transported and EDTAtube to transferred Aspirate Collect midstream specimen in sterile container; VTM not necessary not VTM container; sterile in specimen midstream Collect VTM in Place lesion; of base at cells scrape and vesicle in fluid aspirate or Swab children; from choice of specimen (NPA)are aspirates Nasopharyngeal VTM in transport and swabs on Collect VTM in swabs fecal container; leakproof in (preferred) specimens Fecal with moistened swab rayon or Dacron on collect swab Conjunctival in dilute not do container; leakproof sterile in tissue and CSF Collect Human using tube with heparin; blood for serology should be collected at collected be should serology for blood heparin; with tube using VTM whole blood or plasma for NAA tests NAA for plasma or blood whole 2-4 and symptoms of onset place fluid in VTM in fluid place infections tract respiratory lower of diagnosis for lavage bronchoalveolar adults; from collected swabs nasopharyngeal NPAor VTM without container sterile in placed fluid vitreous and aqueous VTM; in place and saline VTM RNA

VIRUSES

Infections

COLLECTION Reoviridae Picobirnaviridae Double-Stranded

wk later; consult laboratory for use of use for laboratory consult later; Prions OTHER Table - -

Chapter 16 The Clinician and the Microbiology Laboratory ------217 95%), and and >95%), Fluorescein-labeled antibodies 67 cytopathic ). effect cytopathic In addition, some viruses Table Table 16-8). The tests are technically easy, Many microbiology Many laboratories that culture viruses have replaced native tests. Direct antigen tests are the test of choice for enteric viruses, enteric for choice of test the are tests antigen Direct tests. native such as rotavirus, adenovirus serotypes 40 and 41, and noroviruses if NAA tests are not available. In addition, antigen tests for CMV have proved useful for monitoring patients, although this infections assay has been replaced by NAA tests in in many immunocompromised is the that assays use rapid of regarding caution A of note laboratories. most assays target a single viral pathogen and, if used alone, will not detect other viruses or mixed viral infections. a positive test result is particularly useful during the peak months of transmission; however, some of alter with beconfirmed thesemust tests negative and (6%-80%), insensitive respiratory virus assays are for the herpesviruses (CMV, HSV, VZV) and respiratory viruses (influ viruses respiratory and VZV) HSV, herpesviruses(CMV, the for enza humanvirusesmetapneumo A and B, parainfluenza virus, RSV, virus). test Theis DFA particularlywhere effectiveculture is for VZV, sensitive less is viruses other most for microscopy however, insensitive; than alternative detection methods. Antigen Detection are tests agglutination and EIA, ELISA, commercial of number large A currently available for viral antigen detection and are widely used in clinical laboratories (see inexpensive, and generally can be performed test at rapid a permitting the laboratory, the point in received is specimenof the when care or for detection provides of viral example, time. antigens For turnaround than B infections and A influenza and RSV of identification rapid more alternative test methods, with results available within 1 highly are specific ( hour tests The or laboratory. in the receipt after less Culture Viruses are strict intracellular pathogens, requiring host cells for their replication. In some infections, this can be a symbiotic relationship, where viral does host replication cell not compromise survival, and in other cases, viral replication leads to cell death. In vitro tissue culture systems were developed to mimic the natural environment for viral replication. Thesecell culture techniques allowdetection of viruses, range infections with including a of viruses. mixture Tissue of a wide (capable diploid times), few a only (divided primary be can cells culture to indefinitely). (able be maintained 20-50 of heteroploid passages), or diag viruses,all of replication the support will line cell one no Because nostic nostic virology use laboratories cell multiple lines. influ example, For enza and parainfluenza virusesreplicate inprimary cell linessuch as RhMK (primary rhesus monkey kidney) cells or cell heteroploid lines such as LLC-Mk2 (rhesus monkey kidney), whereas adenoviruses and carcinoma) lung (human A549 as such lines cell other in replicate RSV mix cell Cocultured carcinoma). larynx epidermoid (human HEp-2 or tures (e.g., R-Mix, H&V Mix, Super of range a broader E-Mix, of the growth support that system]) virus-inducible ELVIS [enzyme-linked viruses are also available. For some viruses, replication in traditional cell cultures may not be obvious for many days (cell death caused by viral is replication termed ies are used to identify the specific virus. Thishas the - limita inherent tion of only detecting viruses that are targeted by the antibodies. such such as influenzaand parainfluenza viruses,may produce little or no by viruses is respiratory of these of growth effect. Detection cytopathic staining cell cultures with against the viral antigens fluorescein-labeled or reactivity with erythrocytes antibodies that bind to directed their cell surface. on viral antigens hemagglutinating expressing cells traditional cell culture techniques with shell Thiscultures. system vialuses 1-dram vials(SVA) cell that contain culture spin amplification monolayers on 12-mm round coverslips immersed in a tissue culture medium. As with traditional viral cultures, multiple contain shell different vials cell lines that are required. Specimens are addedto monolayer, the centrifuged at low speed, and incubated. examination Instead for of an cytopathic effect, usedantibodies are viruses.to detect of replicating viralShell antigens fluorescein-labeled monoclonal vial cultures are incubated for up to 5 days, but most viruses can detected in be as little as 24 hours because visual cytopathic effectis not necessary for detection. A pool of monoclonal antibodies is firstused to detect the of a presence virus; then antibod individual monoclonal are are also used to identify infected cells in tissue culture.

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All specimens 65 http://www.cdc.gov/ Methods 66

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70° is to preferable freezing at Swab Swab and tissue specimens for viral culture should be placed in a − for for virologic testing should be kept processing or cold transport specimen in afterDelays collection laboratory. the to transport and during should be avoided because loss of viral viability frozen be and not possibly should antigen Specimens occur. may degradation acid nucleic or Freezing days. 5 than more by delayed is processing or transport unless at Microscopy Microscopy should be considered a supplementary test for viral diag Microscopy in immunocompromised patients (e.g., Norovirus, CMV, respiratory syncytial virus [RSV]). In general, collection of specimens diagnostic for tests, with most the exception of serology tests, should be at the bloodfor of collection is to this rule exception The symptoms. of onset serologic diagnosis. An acute-phase serum should be collected during the firstweek of illnessand a second, convalescent serum collected 2 to 4 weeks later. viral transport medium that is albumin, or gelatin), and buffered, antibiotics. Antibiotics are usually contains incorpo protein contaminating (serum,of growth the suppress to media transport viral in rated bacteria and fungi, so separate specimens from the same site must be collected if bacterial or fungal cultures are requested. of Certain swab types materials are not recommended; or specifically,cotton wooden swabs shafts may be aluminum toxic shaft swabs for may interfere culture, with culture and of certain calcium viruses, immunofluorescencetests, and alginate- NAA tests. Liquidspecimens such as media. transport viral in diluted be not should urine or fluid, BAL CSF, Blood should be collected in a sterile vacuum tube or anticoagulant tube ethylenediaminetetraacetic acid with or (EDTA) the citrate. The anticoagulant heparin should be avoided if NAA tests are performed because heparin is a nonspecific polymerase inhibitor. Detection for As with bacteria, fivegeneralare approaches used for the detectionof viruses: microscopy, culture, detection iruses of viral acids, and detection of antibodies 16-17). against the organism antigens (Table or nucleic In contrast with bacteria, microscopy and culture are useful generally than alternative less detection methods. In addition, the tests used for the detection of viruses are usually considered anddefinitive, sup plementary identificationtests are not performed. be viruses viruses,cannot individual of smallsize the Because of nosis. usedcurrently for are approaches seen with the Two microscope. light the microscopic detection of viruses: electron microscopy to observe individual viral particles and light to microscopy observe intracellular a provides Although electron microscopy viralor “inclusions.” clumps rapid detection relatively of viral particles, few diagnostic laboratories currently use this technique. The and equally rapid however, poxviruses, (e.g., rotavirus, polyomavirus); test is useful for certain more sensitive viruses antigen and NAA tests are available for these agents. Histologic examination of tissues for viral inclusions is a rapid diag nostic some viruses,test for such as measles virus (Warthin-Finkeldey giant cells), rabies virus (Negri bodies), herpes simplex skin virus of smears (HSV), Wright-Giemsa CMV. and (VZV), virus varicella-zoster vesicles, showing multinucleated giant cells, have been used to diag nose HSV or VZV (Tzanck preparation). Specificity of of examination microscopic tissues and fluids from lesionsis with improved stain ing techniques that use antibodies fluorescein-labeled directedagainst tests available are DFA for tests). Commercial reagents the virus (DFA Emerging Recent outbreaks of viral diseases with epidemic potential or epidemic actual spread have occurred from sources in Africa (Ebola virus), Middle East respiratory syndrome (South virus Pacific and (MERS), Latin and America). The Zika CDC has ([email protected]) been Network guidance to established on provide Laboratory virus Response testing for emerging threats. Individual references are Ebola, www.cdc.gov/vhf/ebola/healthcare-us/laboratories/safe-specimen-; management.html MERS, ; guidelines-clinical-specimens.html and Zika, zika/hc-providers/diagnostic.html Part I Basic Principles in the Diagnosis and Management of Infectious Diseases 218 Hepatitis E virus E Hepatitis Hepatitis D virus D Hepatitis Hepatitis C and G and C Hepatitis Hepatitis B virus B Hepatitis Hepatitis A virus A Hepatitis Hendra and Nipah and Hendra Hantaviruses Filoviruses and Filoviruses Epstein-Barr virus Epstein-Barr Human Enteroviruses and Enteroviruses Human Cytomegalovirus Herpesvirus 8 Herpesvirus Coronaviruses Herpesviruses 6 Herpesviruses Arboviruses Herpes simplex virus simplex Herpes Adenoviruses VIRUS TABLE viruses viruses arenaviruses metapneumovirus parechovirus virus immunodeficiency and 7 and

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Testing confined to reference laboratories. Diagnosis is Diagnosis laboratories. reference to Testingconfined Serology is used for diagnosis. Qualitative NAA tests NAA Qualitative diagnosis. for used is Serology Detection of specific viral antigens and antibodies and antigens viral specific of Detection Serology is the standard diagnostic test. False-positive test. diagnostic standard the is Serology Testing confined to specialized laboratories. Serology laboratories. specialized to Testingconfined Testing confined to specialized laboratories. Serology laboratories. specialized to Testingconfined Testing confined to specialized laboratories. Antigen laboratories. specialized to Testingconfined Serology test of choice for routine diagnosis. NAA tests NAA diagnosis. routine for choice of test Serology NAA is the test of choice for diagnosis. Shell vial Shell diagnosis. for choice of test the is NAA Enterovirus RNA detection preferred for CNS infection. CNS for preferred detection RNA Enterovirus Serology is primary diagnostic method; rapid antibody rapid method; diagnostic primary is Serology IHC for tissue specimens; shell vial culture rapid and rapid culture vial shell specimens; tissue for IHC Serology used to identify infected persons; sensitivity persons; infected identify to used Serology Positive SARS CoV NAA tests and antibody results antibody and tests NAA CoV SARS Positive NAA test is test of choice for diagnosis. MAbs available MAbs diagnosis. for choice of test is test NAA Serology is primary diagnostic method. Most method. diagnostic primary is Serology Shell vial used for rapid determination of viral of determination rapid for used vial Shell IHC for tissue specimens; IFA and culture widely used widely culture and IFA specimens; tissue for IHC Serology is the standard diagnostic test. False-positive test. diagnostic standard the is Serology COMMENTS IHC of biopsy tissue useful for diagnosis. for useful tissue biopsy of IHC infection. B hepatitis of presence the in only relevant Genotyping helps determine duration of therapy.of duration determine helps Genotyping therapy.to response monitor to used tests NAA Quantitative infection. active confirm to used infection. NAA tests used to monitor therapy.monitor to used tests NAA infection. of course the monitoring for and diagnosis for allows IgM problematic in low-prevalence areas. low-prevalence in problematic IgM developing antibody.developing without die may disease severe with Patients culture. for needed facility BSL-4 cases. fatal in used IHC diagnosis. for useful equally tests NAA and Isolation difficult. Isolation culture. for needed facility BSL-4 cases. fatal in used IHC diagnosis. for useful equally tests NAA and LCM diagnosed primarily by serology.by primarily diagnosed LCM antibody.developing without die may disease severe with Patients LCM. for except culture, for needed facility BSL-4 diagnosis. rapid to key tests NAA and tumor biopsy specimens. biopsy tumor on used be can ISH or IHC recipients. transplant of blood in load viral monitoring for useful tests NAA Quantitative tumors. virus-related for useful widely available. Conventional culture difficult. culture Conventional available. widely yet not reagents detection antigen and culture isolation or NAA tests. NAA or isolation virus through primarily detected Parechovirus therapy and monitor response. monitor and therapy guide to used tests RNA Quantitative infection. neonatal diagnose to used tests RNA plasma and DNA Proviral available. widely more becoming testing Serology primarily used to determine prior infection. prior determine to used primarily Serology therapy.to response and disease of risk assess to used antigenemia pp65 and tests NAA Quantitative useful. be may cells infected in inclusions of Detection specimens. nonblood for sensitive for KS. for tests NAA than specific more IHC risk. KS monitoring for useful be may blood of tests NAA values. cutoff setting in difficulty by hampered specificity and tests becoming more widely used for non-SARS CoV.non-SARS for used widely more becoming tests laboratory. NAA reference a by confirmation require can document primary infection in children. in infection primary document can Serology IHC. for and isolates virus differentiate to agents may require BSL-3 or BSL-4 facilities. BSL-4 or BSL-3 require may agents some of isolation cultured; readily are arboviruses infection. Serology used to determine prior infection. prior determine to used Serology infection. CNS for choice of test is test NAA specimens. tissue or lesions membrane mucous or skin in detection rapid for used IHC and IFAreplication. viral load in compromised hosts. compromised in load viral monitor to used tests NAA adenoviruses. enteric for used assays antigen Rapid specimens. respiratory for specific for acute infection but lacks sensitivity. lacks but infection acute for specific is test NAA areas. low-prevalence in problematic IgM Chapter 16 The Clinician and the Microbiology Laboratory 219 useful for determination of immunity. presumptive presumptive diagnosis of PML. BK virus DNA quantification in plasma or urine used for preemptive diagnosis of PVAN. IHC and EM useful for biopsy tissues. useful. Smallpox isolation requires BSL-3 or BSL-4 and should be attempted only in WHO Collaborating Centers. Vaccinia virus requires BSL-2 and grows readily in cell culture. for detection of inclusions in infected cells (Negri bodies). Serology used to confirm immunization status and confirm exposure in absence of RIG. NAA test is most useful for non-CNS samples when fresh CNS tissue not available. culture. NAA test is most sensitive. Serology useful only for epidemiologic studies. NAA tests are most sensitive. choice for diagnosis. EM useful in suitably equipped laboratories. of Isolation immunity. useful for postnatal rubella if attempted early (prodromal period to 4 days postrash). In CRS, virus can be isolated for weeks to months after birth. markers popular but lack Western specificity. blot for PrP performed in specialized laboratories. Human genome sequencing useful for diagnosis of disorders. Culture less sensitive than IFA or IHC. Serology most for virus identification inWestern HTLV blot–positive but untypeable specimens. suboptimal in sensitivity and specificity; IFA and rapid culture more sensitive. NAA test most sensitive. Serology useful for epidemiologic studies or retrospective diagnosis. of Isolation immunity. useful only if attempted early (prodromal period to 4 days after rash develops). Detection of viral inclusions (Warthin-Finkeldey giant cells) may be useful. determination of immunity. more sensitive than culture. differentiation. Cytopathology useful for diagnosis. Serologic diagnosis of exposure not available. immunocompetent individuals. NAA is test of choice for exposed fetuses and immunocompromised hosts. NAA test can be used to detect human bocavirus. JC virus DNA detection in cerebrospinal fluid useful for EM is the most useful routine test. NAA tests are also IFA performed on fresh and frozen tissue at the CDC Rapid antigen tests, especially IFA, more sensitive than Culture still most common test in clinical laboratories. Direct antigen detection or NAA tests are tests of Serology most useful for diagnosis and determination Histology most useful diagnostic test. Surrogate IFA and NAA tests are most commonly used rapid tests. COMMENTS Serology is primary diagnostic method. NAA test useful Rapid antigen tests widely used but some are Serology most useful for diagnosis and determination Serology used most commonly for diagnosis and NAA test of choice. Antigen testing also sensitive. IFA most common rapid detection method. NAA test NAA is test of choice for detection and genotype Serology used to diagnose parvovirus B19 in —cont’d * C A B C C C A C B Antibody Detection A B A A C C C A . 9th ed. Washington, DC: American Society for Microbiology Press; Viruses of Acid † A A A A A A C C A Nucleic Detection B A B C A A A A METHOD Identification Manual of Clinical Microbiology and C A B A C A C A C C B C Detection C A C C B Antigen DETECTION al, eds. C A C A A C B A C C C B Culture C A B B C Detection for For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. A C C C C B A B C C C C C C C B Electron Microscopy C Methods 16-17 Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada spongiform encephalopathy genetic agents virus viruses lymphotropic lymphotropic virus TABLE Varicella-zoster Varicella-zoster virus Transmissible Transmissible Rubella virus Rotaviruses Respiratory syncytial Rhinoviruses Rabies virus Poxviruses Polyomaviruses Papillomaviruses Parvoviruses Noroviruses Parainfluenza Mumps virus Measles viruses Influenza viruses VIRUS Human T-cell A, Test A, is Test generally useful for the indicated diagnosis; B, test is useful under certain circumstances or for the diagnosis of specific forms of infection; C, test is seldom amplification; PML, progressive multifocal prion leukoencephalopathy; protein; PVAN, polyomavirus-associated PrP, nephropathy; RIG, rabies immune globulin; RNA, ribonucleic acid; SARS, severe acute respiratory syndrome; WHO, World Health Organization. Modified from Murray PR, Baron EJ, Jorgensen JH, et 2007:1304. With permission from ASM Press. † useful for general diagnostic purposes. BSL, biosafety level; CDC, Centers for Disease Control and Prevention; CNS, central nervous coronavirus; system; CRS, CoV, congenital rubella syndrome; DNA, deoxyribonucleic acid; lymphotropic human virus; EM, T-cell IFA, immunofluorescenceelectron assay;microscopy; HTLV, IgM, immunoglobulin M; IHC, immunohistochemistry; ISH, in situ hybridization; KS, sarcoma; LCM, Kaposi’s lymphocytic choriomeningitis virus; MAb, monoclonal antibody; NAA, nucleic acid immunostaining for detection of viral antigens. Viral antigens can be detected by a variety of immunoassays, such as enzyme-linked immunosorbent assays, assays, agglutination immunofluorescence or immunoperoxidase techniques,and Viral immunochromatography. nucleic acids (DNA or RNA) can be detected and quantified by direct hybridization or by the performance of amplification methods, such as polymerase chainreaction. Electron microscopy involves the visualization of viral particles bystaining negative or immunoelectron microscopy or by thin-section techniques. Antibody detection involves measurement of total or class-specific immunoglobulins directed at specific viral antigens. *Virus isolation includes conventional cell culture with detection of viral growth by cytopathic effects or hemadsorption; shell vial centrifugation culture uses Part I Basic Principles in the Diagnosis and Management of Infectious Diseases screening of donors blood is important,also with testing performed to recommended.longer no are and insensitive be to known are tion, detec antibody serum or CSF and detection, antigen culture, cell as CSF,suchHSV, in of CMV,VZV detection and the for methods tory Traditionallabora diagnosis. the this establishing nowfor test is recommended PCR CSF specimens, biopsy brain of stains histologic as on the use of brain relied biopsy. HSV Because detection in CSF by by PCR is as sensitivecaused infection CNS of diagnosis definitive past, hrceitc cmaal wt lbrtr-ae testing. laboratory-based with comparable performance characteristics have assays screening the point-of-care up migrates These it membrane. as sample then patient the and in membrane antibodies with nitrocellulose interact a on immobilized are antigens target the because perform matographicto easy assaysare particularly immunochro The methods. assayimmunochromatographic or EIAs membraneusing by developed been also haveassays serologicof-care after early particularly infection; and sensitivity, the ability to detect both HIV-1 and increased HIV-2. Rapid point- specificity; have improved assays of generations succeeding The antibodies. and antigens HIV both of detection the for combinationassays to phase, solid a to bound recombinantantigens HIV to lysates,protein viral HIV of use The test. a supplementary screening with tests have undergone ofa series developments, moving from confirmation by followed test, ing infection is by HIV-2detection of antibodies in or the HIV-1 patient serum by a of screen diagnosis initial The infection. HIV of detection antibody an mount to able useful. be to testing serologic be for infection to response must patient the Finally, increase. the document to timed sufficiently be mustsamplesconvalescent and acute the of a collection the then documenting antibodies, specific by in rise defined significant is infection an if Likewise, reliably. able poor sensitivityhave if the sampleblood is beforecollected antibodies will are detect test collection—a sample the of timing the by mined specificity and insome cases sensitivity. Overall testsensitivity is deter purifiedviral antigens or recombinant antigens has improved with the assay lysates whole-virus of Replacement assays. nochromatographic ELISA, membrane EIA, indirect fluorescent antibody (IFA),capture and (IgM) immu M immunoglobulin including assays current common most the historically,with used been have formats assay of variety A immune competence of the host, and timing of the specimen collection. of viral serology is determined by the assay format, target viral antigens, specificity sensitivity and The manyviruses. fortest commonlyused a remainslaboratoriesand clinical most by used test diagnostic the was serology tests,NAA and testsantigen viral developmentof the Before Serology therapy. antiviral of effects followto initiationthe astherapy of well as decisionsformaking assist toin used load”be canreferred“viral as to Quantitativeassays, disease. with associated titers viral high and virus of choicemethod isthe (specifically PCR) for ofdetectionCSF. in the HSV NAA choice. of test diagnostic the represent tests these A, B, and [HAV, C viruses HBV, HCV]; CMV; human papillomavirus), other viruses. For many of the viruses (e.g., HIV-1 and HIV-2; commonhepatitis most (see the viruses for available assays commercial with diagnosis, Nucleic amplification acid–based tests have dramatically changed viral TestsAcid–Based Nucleic 220 used to differentiate between asymptomatic shedding of low levels of levels low of shedding asymptomatic between differentiate to used are assays home-brew These adenovirus. and virus, BK (EBV), virus assays have also been developed for other viruses, such as Epstein-Barr home-brew Quantitative standard. quantitative Organization Health Worlda of establishment the been has diagnostics CMV in advance progressionand ofresponse disease to antiviral therapy. Animportant the monitoringfor important are assays these HCV,CMV.of and All are specimens as laboratory. the technologists in received trained minimally by processed be can and perform to simple relatively are assays these of Many 16-4). (see available now are and meningitis/encephalitis viruses for and tests include pathogens pathogens enteric of detection the for panels Multiplex kits. commercial as available are viruses respiratory Currently, serology may play no more important role than in the the in than role important more no play may Currently, serology Commercial quantitative assays are available for HIV-1 and HIV-2, of number a of detection simultaneous the for assays Multiplex Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. ad oebe asy dvlpd o many for developed assays home-brew and 16-4) Table For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 70 Serologic 68 In the Table 69 ------hw gety mrvd performance. improved greatly shown havethird-generation tests and specificity,second- and but sensitivity lackedC hepatitis for tests ELISA generation of first The assays. cence immunofluoresand - ELISA are diagnosis serologic for methods used ( disease chronic with symptomatic or months), (<6 disease acute with symptomatic asymptomatic, but profile testingthat varies according topatient characteristics: exposure tories have to maketried hepatitis diagnosis easier by offeringpanel or Labora antigens. these patient’sto the response andantibody viruses testingfor numbera of different antigenic markersthe with associated serologic by made usually is infections these of diagnosis Laboratorymanifestation. clinical major their as disease liver cause that viruses antigen.nuclear capsid antigen, antibody to early antigen, and antibody to Epstein-Barr to diagnose EBV infection commonly include IgG and IgM to the viral performed to determine if infection exists. infection if determine to performed be should tests NAA and individuals,immunocompromised or nates neo for useful not is serology contrast, In assays. available mercially IgM both measured usingantibodiesandcom antibodiestotalbe can whom in infection, B19 parvovirus to immunocompetentindividuals antibodies. develop to fail maypatients ill acutely although ruses, filovi and arenaviruses, hantavirus, with infections documenting for helpful also is cross-Serology common. are because viruses these results between reactions serologic the of interpretation the in used be must However,care virus. Crosse La and virus, Dengue virus, (EEE) encephalitis equine Eastern virus, (SLE) encephalitis Louis St. virus, (WEE) encephalitis Westernequine virus, WestNile including ruses, arbovi - many for available are tests Commercial infection. arbovirus not onset. of time will the at antibodies patients detectable have many because symptoms of onset the after week 1 measured be should level IgM second a infection, of phase acute the nificant increase in IgG antibodies. If IgM antibodies are absent during sig a or antibodies IgM of detection by made is infection acute an of confirmation Thus, years. for persist then and period, 2-month to 1- a over titer in increase symptoms, of week first the after develop will months.few symptomsofnextIgG for antibodies the persist then and will infections acute the with developIgMdetectablefirst the the week after antibodieswithin onset Patients indicate infection. levels to antibody immune detectable is patient so antibodies, (IgG) G lin immunoglobupersistenthave will patientsImmunized viruses. these with infections acute VZV,as and well virus, as rubella virus, measles (HTLV-1 virus HTLV-2),and HBV, HCV.and lymphotropicT-cellhuman HIV-2, and HIV-1 with infections detect useful because maternal antibodies may be present. be may antibodies maternal because useful not is neonates in antibodies IgG of assessment setting, this In tories. labora most applicationin this for serology replaced have tests NAA to diagnose acute infection in neonates and pregnant women, although nation tests. The detection of IgM be used antibodies canto CMV also aggluti latex or ELISA with performed is and life-threateningdisease severe, developing for risk high at patients immunocompromised in disease preventiondonorsrecipientsimportantCMV and the forof is phoma of the brain and EBV involvement in other organ lesions. whereas molecular methods are used to diagnose EBV-associated lym carcinoma, nasopharyngeal ofmonitoring and screening for and tion ydoe hud rmt h pyiin o osdr et fr EBV- for tests CMV, antibodies, consider toxoplasmosis.specific or to physician the prompt should mononucleosissyndrome clinical a of presence the in test antibody heterophil negative A antigens. heterophil purified use that formats ELISA and particles, agglutination latex horseantigen-coated or with erythrocytes sheep reactionsor agglutination complement, of presence the erythrocytes in bovine of lysis of detection including formats, assay of variety a in detected are antibodies The heterologouserythrocytes. on antigensto reactive are that antibodies IgMare Heterophilantibodies test. antibody heterophil a mononucleosis—is infectious frequently, infection—most EBV primary of diagnosis the for test common most h hptts iue ae gop f neae DA and RNA DNA of unrelated a group are viruses hepatitis The eooy s h fudto fr sesn te mue tts of status immune the assessing for foundation the is Serology an document to way only the is serology laboratories, many In virus, mumps to immunity measure to used is testing Serologic Serologic methods are used for the diagnosis of primary EBV infec organ or blood from serum in CMV to antibodies of Detection 6 months). The most commonly most The months). >6 73 iia t HV testing, HIV to Similar 71 72 Specific antibodies used used antibodies Specific 72 The ------

Chapter 16 The Clinician and the Microbiology Laboratory

- - 221 77 77

ARTHROPODS Acarines: ticks and mites Anoplura: lice Diptera: flies and mosquitoes Hemiptera: kissing bugs Siphonaptera: fleas Collection

Clonorchis Fasciola Fasciolopsis Heterophyes Opisthorchis Paragonimus Schistosoma Trematodes Specimen

Other end viral for phenotypic points assays that have 76 Ancylostoma Ascaris Brugia Dirofilaria Dracunculus Enterobius Gnathostoma Loa Mansonella Necator Onchocerca Strongyloides Toxocara Trichinella Trichuris Wuchereria Nematodes Table 16-19 Table provides guidelines for specimen collection for HELMINTHS For convenience and historical purposes, we will discuss the T 78

Genotypic Genotypic assays have been developed for the rapid detection of only detect can they that is assays genotypic of drawback major The mutations mutations that confer antiviral-drug resistance. Results assays simply report the from presence or absence of these a mutation known to confer resistance to a specific antiviral agent. Most genotypic assays use PCR to amplify specific viralgenes,by followed direct sequencing to hybridization of orproductsspecificthe probes on amplified mem strips brane or gene chips to detect in changes the sequence known to be associated with resistance to antiviral agents. Genotypic assays for the detection of drug resistance in HSV and VZV kinase reveal mutations that confer resistance to thymidine pen famciclovir, acyclovir, - resis determining usedfor assays Genotypic valacyclovir. and ciclovir, mutations detect CMV in cidofovir and foscarnet, ganciclovir, to tance in the and phosphotransferase polymerase DNA genes. commercial and in-house Both mutations. known by caused resistance assays must be continually updated as new mechanisms mutations are or identified. resistance If resistance to an antiviral agent can caused be by differentgene mutations, multiple assays might beneeded for optimal detection. Strains with new or differentresistance genes would go undetected, so phenotypic assays will identify still new responsible for mutations antiviral resistance. be required to been developed in an effort to make phenotypic testing more rapid and and rapid more testing phenotypic make to effort an in developed been - hybrid DNA cells, (noninfected) viable by uptake dye include sensitive and ization, flowELISA, cytometry. plaque autoradiography, whole whole blood, buffy coat preparations, concentrated or blood.films Blood can be prepared typically from fresh blood with Eukaryotic organisms that are classified as parasites are organized into into organized are asclassified parasites are that organisms Eukaryotic three broad groups: protozoa, helminths, and arthropods. Within the protozoa are placed the amebae, flagellates,sporozoa, and ciliates; the helminths include cestodes (tapeworms), nematodes (roundworms), and trematodes (flatworms); arthropods including ticks, mites, lice, fleas, are flies, mosquitoes, and soforth (Table collectiona 16-18). One group that has been by omitted this classification scheme bugs of is the microsporidia, which is a collection of more than 100 this species. was group placed Historically, with the but recent more protozoa, genomic analysis demonstrates these fungi. “parasites” belong with the Parasitology and blood are studies parasitic for submitted specimens common most The and feces. Other specimens alsothat may be collected other are sterile skin and specimens, urogenital and respiratory ransport tissues, and fluids body biopsies. parasites associated with human infections. Blood Microscopic examination for parasites can be done by using PRA for PRA HSV. for PARASITES microsporidia microsporidia with the parasites in this section of the chapter. - - - - - Diphyllobothrium Echinococcus Hymenolepis Taenia Cestodes - Details con 73 Susceptibility 75 Balantidium Ciliates ) or a 95% inhibition 50 Parasites

of esting

Babesia Cryptosporidium Cyclospora Plasmodium Toxoplasma Sporozoa For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. PROTOZOA Dientamoeba Giardia Leishmania Naegleria Trichomonas Trypanosoma Flagellates Classification Susceptibility

16-18

Antiviral resistance is definedas a decrease in susceptibility Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada 74 ) in growth of the virus. Comparison of the results of phenotypic 95 Laboratory assays for antiviral susceptibility testing include pheno include testing susceptibility antiviral for assays Laboratory TABLE Amoebae Acanthamoeba Balamuthia Entamoeba (IC immunoblots immunoblots performed with detected sera positive confirm to used recombinant are [RIBAs]) assays immunoblot antigens (recombinant by ELISA. Third-generation RIBAtests were byapproved the FDA in 1999; these use synthetic c100 and results. RIBA indeterminant of in the c22 number reduction peptides, with a significant cerning the of results interpretation for hepatitis tests can be found in in the which chapters discussedacute are hepatitis and chronic and in individual for chapters each virus. Antiviral drugs are available for the management of viral infections, including those caused by HIV-1, resis of The emergence HSV HCV. and types HBV, Aandinfluenza B 1 virus,RSV, and 2, CMV, VZV, and frequently used are agents antiviral that in inevitable is viruses tant widely. Antiviral assays from different laboratories is difficult. The most commonly used commonly most The difficult. is laboratories different from assays phenotypic antiviral susceptibility assay is the plaque reduction assay (PRA), which measures the ability of varyingantiviral agent to concentrations inhibit of the viral an cytopathic effectas the endThe point. CLSI offers proposed a standard in effort an standardize to the to an antiviral agent established by in vitro testing (phenotypic testing) testing) (phenotypic testing vitro in by established agent antiviral an to and confirmed by genetic analysis oftesting) and biochemical analysis of the altered enzymes. Clinical fail the viral genome (genotypic ures may not always result from the presence of a drug-resistant virus. a theof from drug-resistant presence result always not may ures Factors to consider in a patient with a poor immunologic antiviral status and agent include the the phar patient’s clinical response to an macokinetics of the drug in the individual patient (e.g., dose or route of administration). The developmentof resistance is favoredby long- term recurrent suppressive therapy, intermittent and therapy, the use of suboptimal doses HSV of of failure situations: clinical following the for warranted is testing an antiviral agent. Antiviral or susceptibility VZV lesions to resolve ganciclo or during disease CMV of the progression acyclovir, with appearance treatment of new lesions during vir continued therapy, shedding or transmission of influenza A virus and rimantadine, or with amantadine prophylaxis or treatment during increased HIV-1 RNA plasma levels or decreased CD4 cell counts in HIV-infected patients during antiretroviral therapy. typic typic and genotypic assays. Phenotypic assays require growth of the virus in vitro; virusestherefore, for which in vitro culture systems are not available, such as hepatitis B virus, cannot assays. be Results from tested phenotypic assays may with not be these available within a time frame that is clinically relevant because they of propagation the virus allto attain a standard inoculum concentration, require initial by followed growth of the virus in the assay to obtain Afterresults. up been has assay the inoculum,once prepare to propagation of week a to results HSV for setresults bemay up, obtained in 48 CMV, For hours. may take 8 to 14 days after severalweeks of propagation.phenotypic assays Results are of reported as the drug concentration that causes a 50% [IC] inhibition (inhibition concentration tests tests can also be used to guide treatment of HCV infections. Part I Basic Principles in the Diagnosis and Management of Infectious Diseases for ova and parasite examination. parasite and ova for submitted been have specimens three after until avoided be should it so days, 10 to up for protozoa intestinal obscure will barium of ence pres - The examination. microscopic for be will specimens stool Most Stool (convalescent later weeks 4 phase). to 2 then and phase) (acute symptoms of onset the at collected be should testing serologic for Blood plasma. or serum either use can tests serology and tests,NAA andantigen for and (i.e., day the during periods specific onlyduring blood the in present are and rhythm circadian a have parasites some addition, In patient. the of pattern fever the with correlated be can this that so noted be should collection the of Timing lost. be may morphologic features of the parasites (e.g., stippling in malarial smears) should be prepared within 1 hour after collection ofthe blood, orsome Smears anticoagulant. an as (EDTA) acid ethylenediaminetetraacetic 222 acid–Schiff. periodic amplification; PAS, acid nucleic NAA, eosin; and hematoxylin H&E, acid; EDTA,CSF, ethylenediaminetetraacetic fluid; lavage; cerebrospinal alveolar bronchial BAL, Eye brain and fluid Cerebrospinal marrow Bone Blood fluid Amniotic BODY Urogenital tract Urogenital ulcers cutaneous and Skin Respiratory tract Respiratory Muscle spleen and Liver tract Intestinal Associated TABLE biopsy Brugia, nocturnal). collected Blood in EDTA tubes should be used

SITE/SPECIMEN

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with Guidelines

Human For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. Acanthamoeba, Loa, Acanthamoeba, Balamuthia, Acanthamoeba, Toxoplasma,Plasmodium, Leishmania, Loa, Leishmania, Brugia, Babesia, Toxoplasma Acanthamoeba, Leishmania, Acanthamoeba, Leishmania, Acanthamoeba, POTENTIAL Ancylostoma, Echinococcus, Ancylostoma, Echinococcus, Cryptosporidium, Ascaris, Echinococcus, Clonorchis, Capillaria, Blastocystis, Balantidium, Ascaris,

Infections Trypanosoma Taenia(cysticerci) Naegleria, microsporidia Echinococcus, Trypanosoma Trypanosoma,Wuchereria Plasmodium, Mansonella, Schistosoma, TrichomonasSchistosoma, microsporidia Onchocerca, Naegleria microsporidia, Toxoplasma ), Trichinella,Trypanosoma(cysticerci), TaeniaOnchocerca, Multiceps, microsporidia Gnathostoma, Toxoplasma,Trypanosoma Strongyloides, Paragonimus, microsporidia hookworm, ToxoplasmaOpisthorchis, microsporidia, Metagonimus, Leishmania, Heterophyes, Fasciolopsis, Fasciola, Entamoeba, Toxocara,Trichuris Taenia,Strongyloides, Schistosoma, microsporidia , Isospora, Hymenolepis, Giardia, Enterobius, Entamoeba, Diphyllobothrium, Cyclospora, Cryptosporidium, 79 for In addition, other substances can substances other addition, In

Specimen

diurnal; Loa, P ARASITES microsporidia hookworm,

Collection ,

, , , , Wuchereria ,

and

LABORATORY NAA tests: fluids or tissue for amebae, for tissue or fluids tests: NAA for tissue or fluid Culture: Microscopy: CSF stained with Giemsa (TrypanosomaGiemsa , with stained CSF Microscopy: for EDTAblood tests: NAA for EDTAblood Culture: parasites all for smears thin and thick Giemsa-stained for EDTAblood Microscopy: parasites to antibodies of detection for plasma or Serum detection: Antibody and malaria for EDTAblood tests: NAA antigens microfilarial and malarial of detection for EDTAblood detection: Antigen blood all for smears thin and thick Giemsa-stained for blood whole fresh Microscopy: for fluid aspirated test: NAA NAA tests: in-house assays but no commercial assays available assays commercial no but assays in-house tests: NAA for discharge urethral or vaginal Culture: for secretion prostatic and discharge, urethral discharge, vaginal Microscopy: for H&E or biopsy—Giemsa and scrapings, snips, skin aspirates, Microscopy: NAA tests: in-house assays but no commercial assays available assays commercial no but assays in-house tests: NAA for biopsy Culture: available assays commercial no but assays in-house tests: NAA NAA tests: scrapings or biopsy for microsporidia and microsporidia for biopsy or scrapings tests: NAA and amoebae for biopsy or Scrapings Culture: or (amebae) white calcofluor or Giemsa with stained biopsy or Scrapings Microscopy: Microscopy: sputum (expectorated, induced), nasal discharge, BAL (aspirate, BAL discharge, nasal induced), (expectorated, sputum Microscopy: available assays commercial no but assays in-house tests: NAA for silver,PAS,Gram) methenamine (H&E, stains tissue for biopsy Microscopy: panel: gastrointestinal Muliplex tests: NAA for aspirate or biopsy Culture: for aspirate or biopsy Microscopy: available assays commercial no but assays in-house tests: NAA of detection for used stool nonpreserved collected, freshly detection: Antigen of (detection smear wet direct by examined aspirates duodenal or stool Microscopy: Diagnostic (larval cestodes, (larval H&E or (microsporidia) stain Gram with stained tissue (microsporidia); trichrome modified or (amebae), trichrome (microsporidia), acid-fast (amebae), white trypanosomes and microfilariae motile of detection for coat buffy microfilariae; for smears hematoxylin-stained or Giemsa- parasites; trichrome, acid fast); urine sediment for sediment urine fast); acid trichrome, (modified microsporidia and (hematoxylin), microfilariae Trichomonas(Giemsa), Acanthamoeba histolytica, E. Leishmania, arthropods (e.g., arthropods microsporidia and protozoa, helminths, for silver,PAS,Gram) methenamine (H&E, stains tissue and (microsporidia), trichrome modified ), (Cryptosporidium ( Giemsa preparation), (unstained Echinococcus modified trichrome (microsporidia) trichrome modified brushing), and biopsy for biopsy and brushing), Trichinellaand lamblia Giardia histolytica, E. or mount) Cryptosporidium Giardia, histolytica, Entamoeba detect to used smears impression anal unstained Isospora Cyclospora, (Cryptosporidium, stains acid-fast modified or (microsporidia), trichrome modified (protozoa), hematoxylin iron or trichrome with stained and concentrated or eggs), helminth (protozoa, staining iodine protozoa), motile Regardless of the preservative used, care must be used to add the the add to used be must tests). care NAA used, or preservative antigen the (e.g., of testsRegardless additional of use and specimen by the methods in used the microbiology laboratory for examining the determined be will preservatives of selection The alcohol). polyvinyl Schaudinn’sacid-formalin,acetate-acetic sodium formalin,(e.g., fluid, Avarietyofridia. fixatives are available for stool specimens preserving microspo- or coccidia, larvae, and eggs helminth or cysts protozoan with problem a not is This deteriorate.rapidly will and fragile are ites protozoan because trophozo collection of time the atpreservatives in placed be should specimen the then anticipated, is transport in delay a If preservatives. in placed not and collection after immediately tory labora the promptlyto transportedbe should specimen the case, this In parasites. motile of presence the for examined be may Stool specimens time. later a at submitted be should specimens additional and suboptimal, was specimen the indicate should negative, if report, the then agents, these administering of weeks 2 within examined be must specimen the If agents. antimalarial and tetracyclines, metronidazole, bismuth, oil, mineral including protozoa, of detection with interfere (Giemsa) Leishmania Echinococcus

T Taeniasolium TEST Leishmania , botflies, ticks, lice) ticks, botflies, Demodex, ests and Leishmania

Acanthamoeba for and Leishmania Ascaris, Strongyloides, Paragonimus, Capillaria Paragonimus, Strongyloides, Ascaris, Toxoplasma Leishmania

cysticerci, Detection , or Echinococcus, histolytica, E. , and helminths Toxoplasmaand , Leishmania Trypanosomacruzi Trichomonas or microfilariae (wet mount) (wet microfilariae or Schistosoma Cryptosporidium, Cyclospora cayetanensis, Cyclospora Cryptosporidium, Echinococcus) , and , Toxoplasma Toxoplasma

of ; direct visualization for visualization direct Onchocerca; ), modified acid-fast Toxoplasmamodified ), (pinworm eggs) (pinworm Enterobius

Toxoplasma Parasites , amebae), calcofluor Toxoplasmaamebae), , (wet Capillaria

, and , ); - - Chapter 16 The Clinician and the Microbiology Laboratory

- - - - - J Mol Mol J 223 Table Clostrid Clin Infect Clin Infect T. vagi T. Br Br Med J. Clostridium Clostridium Micro Clin J J Clin J Micro Clin Toxoplasma Toxoplasma Toxoplasma Cryptosporidium, T. vaginalis T. directly toxin. toxin. difficile Clostridium Entamoeba Entamoeba , histolytica . 2006;12:2-18. Staphylococcus Staphylococcus aureus (MRSA) Bordetella pertussis Bordetella by PCR and . 2002;40:3838-3840. . 1993;95:601-607. . 2010;48:889-893. . Microbiol Clin J spp., Plasmodium and E. , histolytica . 2012;50:3365-3367. Am Am J Med in difficile fecal samples. Clostridium J Clin Microbiol Clin Microbiol Infect Clin Microbiol , are Trichomonas now components of ests T

. biol 2012;50:3089-3090. testing in the clinical laboratory by use of multi of use by laboratory clinical the in testing difficile ium algorithms. testing ple -associated -associated difficile disease in North America and Europe: review. ogy laboratory for diagnosing diarrheal diseases. Dis. 1996;23(suppl 1):S97-S101. the BD MAX MRSA assay and the BD GeneOhm achromopeptidase MRSA assay with direct and enriched culture techniques using clinical specimens detection for of MRSA. J Clin Microbiol . biol 2008;46:3285-3290. of detection for assays five of . Diag 2011;11:395-400. swab swab types on detection of culture. Xpert methicillin-resistant rapid for platform PCR real-time GeneXpert the using assay specimens. screening from MRSA of detection of bacterial diagnosis the for fluid lavage bronchoalveolar of pneumonia. bacterial colony counts in urinary1967;1:400-403. infection. a new molecular test, the BD Max Cdiff, toxigenic for detection of H K N C C R D C R L

24. microbiol clinical the of use Effective I. Nachamkin J, ines 25. uijper EJ, Coignard B, Tull P. Emergence of 26. al. et JM, Miller EM, Marlowe SM, ovak-Weekley 27. hapin Andrea KC, Dickenson SB. RA, F, Comparison Wu 19. Carroll Impact KC. of nasopharyngeal loud JL, W, Hymas 20. ossney A, Herra C, Brennan G, et al. Evaluation of the 21. alpke AH, Hofko M, Zimmermann Comparison S. of 22. antral DE, TG, Tape Reed EC, et al. Quantitative culture 23. oberts A, Robinson R, Beard R. Some factors affecting 28. of R,eGuern et al. HerweghB, Evaluation S, Grandbastien Giardia , lamblia Giardia

- - - - for

J Clin

panel Detection

Preclinical

Acid–Based Trichomonas Trichomonas vaginalis infections. Although trichomonal

al. Comprehensive Comprehensive PCR

et The presence of IgG antibodies to parasitea consistent is pathogens. . 2006; Transfusion

82 JA, . Giardia

. 1986;24: . Microbiol Clin J multiplex . 2002;35:113-125. system

and and for years. . The tests are generally more sensitive than microscopic examina microscopic than sensitive more generally are tests The . nalis tion and less subjective (better specificity).In geographic areas where parasitic infections are uncommon, some laboratories restrict routine testing their to immunoassays for Serology Serologic testing is rarely performed except in to a patient document from a infection nonendemic country who with infected has be been to suspected exposed patients or to parasite a particular . gondii with past exposure, and evidence of a recent seroconversion is sistent con with an acute infection. if However, seroconversion cannot be demonstrated, then a careful travel and exposure history is required to interpret the significanceIgM negative A of determined. are titers seropositive antibody IgG results. and IgM infections, For titer excludes acute infection because all patients with active disease which decreaseantibodies thedevelop, at IgM to have time symptoms nondetectable levels by 6 to 9 IgG months. antibody levels will persist Culture Culture is primarily performed exception: in research laboratories with vaginal of inspection microscopic by detected readily be can infections one secretions, commercial kits permit the growth of than sensitive more are systems These exudate. vaginal the culturing by microscopic examination of the exudate, although this may increase detection of patients with colonization. asymptomatic Antigen Antigen tests are commercially available for Cryptosporidium spp., Nucleic NAA tests for parasitic infections, previously restricted to reference and research laboratories, with the developed for exception the detection of of commercial perfor Easeof pathogens. tests enteric of diagnosis the for panels multiplex mance and rapidity of result will undoubtedly lead to the expansion of leadof willto the expansion result undoubtedly of rapidity and mance these products and their more widespread use in practice (see 16-4). Killpack

. 2007;13:709-716. nervous . 1997;35:928-936.

ES, . 2005;41:1677-1680. ------automated

Clin Infect Clin Dis Infect . 1977;296:1305-1309. fully central

Slechta a . 2002;35:1281-1307.

of 2016;54:785-787.

KE, . Clin Infect Clin Dis Infect J Clin Microbiol

N Engl J Med It is recom Clin Microbiol Infect Clin Microbiol detection 80

Report Report on Blood Collection and Resource Data Transfusion Blood in National MD: Bethesda, 2001. the in States United Center; 2003. lines for the diagnosis and management of group A strepto group of the lines for diagnosis management and coccal pharyngitis. assessment the therapy therapy products: methods with parallel method. a CFR-compliant comparison of automated method for identifying intravenous-catheter related infec culture of central vein catheter tips useful in the diagnosis bacteremia? catheter-associated of of vascular catheter-related bloodstream infection: a meta- analysis. determinants determinants of time-to-positivity in patients with bactere mia. clinically recovery of increase do not subculturing extensive blood automated standard from microorganisms significant cultures. 46:2071-2082. tion. 532-535. the prevention of intravascular catheter-related infections. Clin Dis Infect K M C S M B A B O Microbiol

13. huu H, Patel N, Carter C, et al. Sterility testing of cell 14. culture semiquantitative A HW. Sarafin CE, Weise DG, aki 15. ollignon PJ, Soni N, et Pearson IY, al. Is semiquantitative 16. iegman-Igra Anglim Y, AM, Shapiro DE, et al. Diagnosis 17a. Hanson 10. artinez J, Pozo L, Almela M, et al. Microbial and clinical 11. and incubation LS. Prolonged Tompkins Scott JD, EJ, aron 12. merican Association of Blood Banks. 18. isno AL, Gerber MA, Gwaltney JM, et al. Practice guide 17. for Guidelines al. et EP, Dellinger M, Alexander NP, ’Grady - Methods

Clin Clin Clin Clin . 2007; . 10th ed. Verification and Analysis and Analysis Pre 8,79 J Clin Microbiol For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. The specimens should becollected on 81 . 1999;107:119-125. Fed . Regist 2003;68:3639-3714. Fed Identification . 3rd ed. CLSI document M39-

Am Am J Med . JAMA 1990;264:2111-2112. and Manual Manual of Clinical Microbiology

Labora Microbiology Clinical the in Procedures of Validation tory. Cumitech 31. DC: Washington, American Society for Microbiology; 1997. sentation sentation of Cumulative Antimicrobial Susceptibility Data; Approved Test Guideline Clinical MA3. PA: and Wayne, Laboratory - Standards Insti tute; 2009. L L B V Z M E H P ized trial of povidone-iodine compared with iodine tincture iodine with tincture ized compared trial povidone-iodine of for venipuncture site disinfection: effectson ratesof blood culture contamination. needle when inoculating blood cultures. A no-benefit and high-risk procedure. two two blood culture media shows therapy. antimicrobial significant on patients for recovery bacterial differences in volume of blood cultured still a diagnosis of bloodstream infections? significant factor in the Infect Dis. Infect 2013;56:790-797. - person certain and systems quality to relating requirements Finalnel qualifications. rule. microbiology microbiology laboratories in the management and control of infectious diseases and the delivery of health care. Infect Dis. Infect 2001;32:605-611. Washington, DC: American Society Press; Microbiology for Washington, 2011. 45:2765-2769.

9. eisure M, Moore D, Schwartzman J, et al. Changing the 8. ittle JR, Murray PR, PS, Traynor Spitznagel E. A random 7. ouza E, Sousa D, Rodriquez-Creixems M, et al. Is the 5. ersalovic J, ed. 6. of R, Gottschall et DM, al. Comparson R,adroga Williams 3. edicare, Medicaid, and 4. CLIA Programs. Laboratory lder BL, Hansen SA, Kellogg JA, et al. 2. Barton indler M, JF, Callihan DR, et al. 1. eterson LR, Hamilton JD, Baron EJ, et al. Role of clinical mended mended that three specimens should be collected, to exclude reliably a parasitic infection. Examination of a single specimen sensitivity may of have 50% a to 60%. separate separate days and, if possible, spread over a week or more. References appropriate amount of specimen to the container and ensure complete complete ensure and the container specimen to of amount appropriate mixing of the stool specimen with the preservative. exam by detected readily be can parasites Many parasites. most of tion iodine; with staining following or directly specimen collected the ining the specimenof concentration requires sensitivity maximum however, and examination with specific stains to allow internal differentiation structures of the (e.g., trichrome of theis determinedparasite by Giemsa stain). Identification themor stain, iron hematoxylin phologic stain, features of the protozoa and the characteristics of the eggs, larvae, of Detailscestodes, or forms adult and trematodes. nematodes, of these features are discussed in the individual chapters in this book and in the cited reference texts. Microscopy Microscopy is the definitivemethod for the detectionand identifica As As with the other microorganisms approaches exist discussed for the in detection of this parasites: microscopy, antigen chapter, culture, or nucleic acid detection, five and antibody detection. In contrast with the other groups of organisms, microscopy is the primary detec Detection for mens mens collected for microscopic examination should not be placed in it is critical preservative; therefore, to the have laboratory examine the tests should be Specimens NAA for if refrigerated specimen promptly. immediate transport is not feasible. tion and identificationprocedure for most parasites.Culture is used for only a few parasites and, when available, is not the primary diag nostic method. Antigen testing is used for a limited number of para sites, and acid–basednucleic tests are primarily restricted to reference or research laboratories. with countries in value Serology little has but uncommon are is infections endemic useful for populations where a high incidence of infection. Other Specimens sterile Normally, body fluidsand tissuessubmitted for culture should Speci to transported the in laboratory a sterile container. be promptly Part I Basic Principles in the Diagnosis and Management of Infectious Diseases 44. 43. Martin 42a. 42. 41. 40. 39. 38. 37. 36. 35. 34. 33. Morshed 32a. 32. 31. 30. 29. 223.e1 laa-ul L Mlao , lsre-zued A e al. et A, Alastruey-Izquierdo E, Mellado L, Alcazar-Fuoli O’Sullivan CE, Miller DR, Schneider PS, Roberts GD. Evalu opportunis Defining al. et B, dePauw JH, Rex S, Ascioglu Palicova GE, F. Pfyffer Chosewood LC, Wilson DE, eds. Standards Laboratory Clinical for Committee National (CLSI). InstituteStandards Laboratory and Clinical Standards Laboratory Clinical for Committee National bacteria of Identification al. et Olma T, LC, Thomas J, Kok Wieser A, Schneider L, Jung J, Schubert S. MALDI-TOF MS microbial microbiology: clinical in new is P.MurrayWhat MaidenMCJ, Bygraves JA, Feil Multilocusal. et E, sequence Standards Laboratory Clinical for Committee National KuijperEuroEJ.WilcoxMH, MJT,OM, Crobach Dekkers Limited CA. Petti AR, Rhansalkar E, Billetdeaux RC, She Totten AmselR, PA, Spiegel Nonspecifical. et CA, vaginitis: Chemother identification. sequence-based and terns Asper 2002;40:1723-1727. laboratory. center care tertiary a in mens speci nonrespiratory and respiratory using by test direct amplified Gen-Probe of ation One of the 7-14. trans cell consensus.international stem an plants: immunocompromised hematopoietic and in cancer with infections patients fungal invasive tic 2011:472-502. Press;Washington, ASM In: DC: procedures. staining ed. J,Versalovic and detection, laboratory tics, 2007. Office; PrintingGovernment BiomedicalLaboratoriesand Wayne,M2-A10. 2009. PA:document NCCLS; Standard Approved Tests. Susceptibility (NCCLS). Wayne,MS23. PA:2013. CLSI; Supplement Informational Testing:18th Susceptibility Antimicrobial for Standards mance 2009. NCCLS; Wayne,PA: M7-A8. document NCCLS Standard. Aerobically Grow That Bacteria Testsfor (NCCLS). laser matrix-assisted using mass spectrometry. flight of broths time and Sepsityper culture desorption-ionization blood in nol review). (mini beyond and ganisms microor of diagnostics—identification microbiological in 419-423. MALDI-TOF. by identification A S U Sci Acad within populations of pathogenic clonesmicroorganisms. of identification the to approach portable a typing: 137-147. diagnosis 2002. NCCLS; ApprovedGuideline (NCCLS). 1053-1066. diagnosing for Clostridium difficile recommendations and review data eases: Dis Infectious and Microbiology Clinical of Society pean laboratory. biology tella applicability of direct fluorescent-antibody testing for Med J Am andmicrobialcriteria epidemiologicdiagnostic association. tuberculosis . 2012;93:965-974. . Hardy sp.and . 2014;9:e107258. gillus L, Downloaded for dr.Rahmat DaniSatria, M.Sc,Sp.PK ([email protected]) atUniversitas Gadjah Mada fromClinicalKey.com byElsevier onJuly 28,2018. . 1983;74:14-22. . TB eto Fmgt: niugl ucpiiiy pat susceptibility antifungal Fumigati: section Methods for Dilution AntimicrobialDilutionSusceptibility for Methods . 2008;52:1244-1251. . Abbreviated Identification of Bacteria and Yeast: and Bacteria Identification of Abbreviated of MG, efrac Sadrs o Atmcoil Disk Antimicrobial for Standards Performance Coronel Legionella MODS PLoS One PLoS syphilis. . 1998;95:3140-3145. . and Singh aul f lncl Microbiology Clinical of Manual infection. . NCCLS document M35-A. Wayne,M35-A. document NCCLS . PA: J ClinMicrobiolJ multi-drug J, Kit sp. specimens for the clinical micro clinical the for sp.specimens Faulx Mycobacterium AE. . 2011;6:e23285. . ln acn Immunol Vaccine Clin for . 5th ed. Washington, DC: U.S.Washington, DC: ed. 5th . Recent the D, Clin Microbiol Infect resistant yoatru tuberculosis Mycobacterium CS dcmn M100- document CLSI . rapid Biosafety in Microbiological et Ml Diagn Mol J . 2007;45:2212-2214. . al. Clin Infect Dis InfectClin ApplMicrobiol Biotech trends phenotypic : general characteris general : For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. A

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