Fundamental Medical

Fundamental Medical Mycology

Errol Reiss Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia H. Jean Shadomy Department of Microbiology and Immunology, Virginia Commonwealth University, School of Medicine, Richmond, Virginia G. Marshall Lyon, III Department of Medicine, Division of Infectious Diseases, Emory University, School of Medicine, Atlanta, Georgia

A JOHN WILEY & SONS, INC., PUBLICATION This book was written by Errol Reiss in his private capacity. No official support or endorsement by the Centers for Disease Control and Prevention, Department of Health and Human Services is intended, nor should be inferred.

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Library of Congress Cataloging-in-Publication Data:

Reiss, Errol. Fundamental medical mycology / Errol Reiss, H. Jean Shadomy, and G. Marshall Lyon III. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-17791-4 (cloth) 1. Medical mycology. I. Shadomy, H. Jean. II. Lyon, G. Marshall. III. Title. [DNLM: 1. Mycology–methods. 2. Mycoses–microbiology. 3. Mycoses–therapy. QY 110] QR245.R45 2012 616.96901–dc22 2011009910

Printed in the United States of America oBook ISBN: 978-1-118-10177-3 ePDF ISBN: 978-1-118-10175-9 ePub ISBN: 978-1-118-10176-6

10987654321 To our spouses, with gratitude: Cheryl (E. R.), “Shad” (H. J. S.), and Tabitha (G. M. L.)

Contents

Preface xvii 1.6.2 Investigating Outbreaks 10 1.6.3 Determining the Susceptibility to Acknowledgments xix Antifungal Agents 10 1.6.4 Estimating the Significance of Fungi Generally Considered to be Opportunists or Saprobes 10 1.6.5 Types of Vegetative Growth 10 Part One Introduction to Fundamental Medical 1.7 Sporulation 11 Mycology, Laboratory Diagnostic Methods, 1.8 Dimorphism 11 and Antifungal Therapy 1.8.1 Dimorphism and Pathogenesis 12 1.9 Sex in Fungi 13 1. Introduction to Fundamental Medical 1.9.1 Anamorph and Teleomorph Mycology 3 Nomenclature 13 1.10 Classification of Mycoses Based on the 1.1 Topics not Covered, or Receiving Secondary Primary Site of Pathology 13 Emphasis 3 1.10.1 Superficial Mycoses 13 1.2 Biosafety Considerations: Before You Begin 1.10.2 Cutaneous Mycoses 13 Work with Pathogenic Fungi... 3 1.10.3 Systemic Opportunistic Mycoses 13 1.2.1 Biological Safety Cabinets (BSC) 4 1.10.4 Subcutaneous Mycoses 13 1.2.2 Precautions to Take in Handling Etiologic 1.10.5 Endemic Mycoses Caused by Dimorphic Agents that Cause Systemic Mycoses 4 Environmental 13 1.2.3 Additional Precautions at Biosafety 1.11 Taxonomy/Classification: Kingdom Level 3 (BSL 3) 5 Fungi 14 1.2.4 Safety Training 5 1.11.1 The Phylogenetic Species Concept for 1.2.5 Disinfectants and Waste Disposal 5 Classification 15 1.3 Fungi Defined: Their Ecologic Niche 5 1.11.2 The Higher Level Classification of 1.4 Medical Mycology 5 Kingdom Fungi 15 1.5 A Brief History of Medical Mycology 6 1.12 General Composition of the Fungal 1.5.1 Ancient Greece 6 Cell 21 1.5.2 Middle Ages 6 1.12.1 Cell Cycle 21 1.5.3 Twentieth Century 6 1.12.2 Hyphal Morphogenesis 21 1.5.4 Endemic Mycoses in the Americas 6 1.12.3 Cell Wall 22 1.5.5 Era of Immunosuppression in the 25 Treatment of Cancer, Maintenance of 1.13 Primary Pathogens 1.13.1 Susceptibility to Primary Organ Transplants, and Autoimmune Pathogens 26 Diseases 7 1.5.6 Opportunistic Mycoses 7 1.14 Endemic Versus Worldwide Presence 26 1.5.7 HIV/AIDS 7 1.15 Opportunistic Fungal Pathogens 26 1.5.8 Twenty-first Century 8 1.15.1 Susceptibility to Opportunistic Fungal 1.6 Rationale for Fungal Identification 9 Pathogens: Host Factors 26 1.6.1 Developing the Treatment Plan 9 1.16 Determinants of Pathogenicity 27

vii viii Contents

General References in Medical Selected References for Laboratory Mycology 27 Diagnostic Methods in Medical Selected References for Introduction to Mycology 69 Fundamental Medical Mycology 28 Websites Cited 70 Websites Cited 29 Commercial Manufacturers and Suppliers of Questions 30 Fungal Media, Stains, and Reagents 71 Packing and Shipping of Infectious Agents 2. Laboratory Diagnostic Methods in Medical and Clinical Specimens 72 Mycology 31 Questions 72 2.1 Who Is Responsible for Identifying 3A. Antifungal Agents and Therapy 75 Pathogenic Fungi? 31 2.1.1 Role of the Clinical Laboratorian 31 3A.1 Introduction 75 2.1.2 Role of the Physician 31 3A.1.1 Major Antifungal Agents Approved for 2.2 What Methods are Used to Identify Clinical Use 76 Pathogenic Fungi? 31 3A.1.2 Comparison of Antibacterial and 2.2.1 Culture and Identification 31 Antifungal Agents According to Their 2.3 Laboratory Detection, Recovery, and Intracellular Targets 79 3A.2 (AmB-deoxycholate) Identification of Fungi in the Clinical  Microbiology Laboratory 33 (Fungizone , Apothecon Subsidiary of 2.3.1 The Laboratory Manual 33 Bristol-Myers-Squibb) 80 2.3.2 Specimen Collection 33 3A.2.1 Structure 80 2.3.3 Direct Examination 34 3A.2.2 Mode of Action 80 2.3.4 Histopathology 36 3A.2.3 Indications 82 2.3.5 Culture 37 3A.2.4 Formulation 82 2.3.6 Storage and Cryopreservation of Cultures 3A.2.5 Spectrum of Activity 82 for QA and QC in the Clinical Mycology 3A.2.6 Clinical Uses 82 Laboratory 41 3A.2.7 Lipid Formulations of AmB 83 2.3.7 Media and Tests for Yeast 3A.2.8 Pharmacokinetics 84 Identification 42 3A.2.9 Interactions 85 3A.2.10 Adverse Reactions 85 2.3.8 Methods Useful for  Identification 45 3A.3 Fluconazole (FLC) (Diflucan , 2.3.9 Microscopy Basics 53 Pfizer) 86 2.3.10 Use of Reference Laboratories 59 3A.3.1 Structure and Mode of Action 86 2.3.11 Fungal Serology and Biochemical 3A.3.2 Indications 86 Markers of Infection 59 3A.3.3 Fluconazole Pharmacokinetics 87 2.4 Genetic Identification of Fungi 64 3A.3.4 Efficacy 88 2.4.1 Commercial Test 64 3A.3.5 Formulations 88 2.4.2 Peptide Nucleic Acid–Fluorescent In Situ 3A.3.6 Interactions 88 3A.3.7 Adverse Reactions 88 Hybridization (PNA-FISH) 64  2.4.3 PCR-Sequencing Method 64 3A.4 (ITC) (Sporanox , Janssen 2.4.4 Nuclear rDNA Complex 64 Pharmaceutica Division of Johnson & 2.4.5 Genetic Tools for Species Johnson) 89 Identification 66 3A.4.1 Action Spectrum 89 2.4.6 How Is the Genetic Identification of an 3A.4.2 ITC: Uncertain Bioavailability 89 Unknown Fungus Accomplished? 66 3A.4.3 Properties 89 2.4.7 Growth of the Fungus in Pure Culture, 3A.4.4 Pharmacokinetics 89 Extraction and Purification of DNA 66 3A.4.5 Interactions 90 2.4.8 PCR of the Target Sequence 67 3A.4.6 Adverse Reactions 90 2.4.9 PCR Cycle Sequencing 68 3A.5 Voriconazole (VRC) (Vfend, 2.4.10 Assemble the DNA Sequence 68 Pfizer) 90 2.4.11 Perform a BLAST Search 68 3A.5.1 Action Spectrum 90  2.4.12 The MicroSeq System 68 3A.5.2 Pharmacokinetics 90 2.4.13 Other Sequence Databases 68 3A.5.3 Drug Interactions 91 General References for Laboratory 3A.5.4 Adverse Reactions 91 Diagnostic Methods in Medical 3A.6 Posaconazole (PSC) (Noxafil, Mycology 69 Schering-Plough/Merck & Co.) 91 Contents ix

3A.6.1 Action Spectrum 91 3A.14.1 Mode of Action 99 3A.6.2 Pharmacokinetics 91 3A.14.2 Action Spectrum 99 3A.6.3 Drug Interactions 92 3A.14.3 Indications 99 3A.6.4 Adverse Reactions 92 3A.14.4 Dosage Regimen 99 3A.7 Azole Resistance Mechanisms 92 3A.14.5 Metabolism 99 3A.7.1 Alteration of Target Enzyme 3A.14.6 Adverse Reactions 100 (Lanosterol Demethylase) 92 3A.15 Combination Therapy 100 3A.7.2 Overexpression of Target 3A.16 Suppressive or Maintenance Enzyme 92 Therapy 100 3A.7.3 Increased Efflux of Drug, CDR Efflux 3A.17 Prophylactic Therapy 100 Pumps 92 3A.17.1 Bimodal Period of Risk 101 3A.7.4 Bypass Pathways 92 3A.17.2 Fluconazole and Alternatives for 3A.7.5 Loss of Heterozygosity in Primary Prophylaxis 101 Chromosome 5 and Azole 3A.17.3 Prophylaxis in Patients During the Resistance 92 Pre-engraftment Period with a History 3A.7.6 Azole Resistance in of Invasive Mold Infections 102 Species 93 3A.17.4 Prophylaxis in the Post-engraftment 3A.8 Echinocandins 93 Period 102 3A.8.1 Mode of Action 93 3A.18 Empiric Therapy 102 3A.8.2 Spectrum of Activity 93 3A.19 Innately Resistant Fungi 103 3A.9 (CASF) (Cancidas, 3A.19.1 Innately Resistant Molds 103 Merck) 94 3A.19.2 Innately Resistant 103 3A.9.1 Action Spectrum 94 General Reference for Antifungal Agents 3A.9.2 Dosage 95 and Therapy 103 3A.9.3 Pharmacokinetics 95 3A.9.4 Drug Interactions 95 Selected References for Antifungal Agents 3A.9.5 Adverse Reactions 95 and Therapy 103 3A.10 Micafungin (MCF) (Mycaminet, Astellas Websites Cited 105 Pharma, Inc.) 95 Questions 105 3A.10.1 Indications 95 3A.10.2 Dosage 95 3B. Antifungal Susceptibility Tests 107 3A.10.3 Metabolism 96 3A.10.4 Drug Interactions 96 3B.1 Antifungal Susceptibility Tests 3A.11 Anidulafungin (ANF) (Eraxis, Defined 107 3B.2 National and International Standards for Pfizer) 96 3A.11.1 Indications 96 AFS Tests 107 3A.11.2 Invasive 96 3B.3 Objective of AFS Tests 107 3A.11.3 Molds 96 3B.4 Minimum Inhibitory Concentration (MIC) 3A.11.4 Dosage 96 of an Antifungal Drug Defined 107 3A.11.5 Metabolism 96 3B.4.1 MIC50 and MIC90 108 3A.11.6 Drug Interactions 97 3B.5 Broth Microdilution (BMD) 3A.11.7 Adverse Reactions 97 Method 108 3A.12 Terbinafine (TRB) (Lamisil, 3B.6 Clinical Indications for AFS Novartis) 97 Testing 108 3A.12.1 Mode of Action 97 3B.7 Correlation Between the In Vitro 3A.12.2 Action Spectrum 97 Determined MIC and the Clinical Efficacy 3A.12.3 Drug Synergy 97 of Drug Therapy 108 3A.12.4 Metabolism 97 3B.7.1 How Are the Conditions of 3A.12.5 Adverse Reactions 98 “Susceptible” or “Resistant” 3A.13 5-Fluorocytosine (Flucytosine, 5FC) Determined? 109 (Ancobon, Valeant 3B.7.2 What Are Breakpoints? 109 Pharmaceuticals) 98 3B.7.3 Minimum Effective 3A.13.1 Indications 98 Concentration 109 3A.13.2 Combination Therapy 98 3B.8 AFS Methods Currently Available for Use 3A.13.3 Metabolism 98 in the Clinical Laboratory 110 3A.13.4 Adverse Reactions 98 3B.8.1 Broth Microdilution (BMD) 3A.14 Griseofulvin (Grifulvin V, Ortho Method 110 Pharmaceutical Corp.) 99 3B.8.2 Etest 110 x Contents

3B.8.3 Disk Diffusion Method 110 Selected References for 3B.9 Which Laboratories Conduct AFS 137 Tests? 110 Questions 138 3B.10 Principles of AFS Tests 110 3B.10.1 Standard Method for AFS Testing of 5. 141 Yeasts 110 3B.10.2 Modifications Suggested to Improve 5.1 Coccidioidomycosis-at-a-Glance 141 Performance of the BMD Method for 5.2 Introduction/Disease Definition 141 Yeast 112 5.3 Case Presentations 142 3B.10.3 Commercial BMD Method with 5.4 Diagnosis 143 Precoated Drug Panels: A 5.5 Etiologic Agents 143 CLSI-Approved Method for AFS 5.6 Geographic Distribution/Ecologic Testing of Yeasts and Molds 113 3B.10.4 Standardization of AFS Tests for Niche 144 Molds, M38-A2: Broth Microdilution 5.7 Epidemiology 147 for Molds 114 5.7.1 Incidence and Prevalence 147 3B.10.5 Etest (bioMerieux,´ Marcy l’Etoile, 5.7.2 Effect of Weather on Annual Fluctuations France) 114 in Prevalence of 3B.10.6 Disk Diffusion AFS Tests 117 Coccidioidomycosis 148 3B.10.7 VITEK 2 System for AFS 5.8 Risk Groups/Factors 149 (bioMerieux,´ Marcy l’Etoile, 5.9 Transmission 150 France) 118 5.10 Determinants of Pathogenicity and 3B.10.8 Flow Cytometry AFS Test 118 Pathogenesis 151 3B.11 Summary of the Current Status of 5.10.1 Allergic Findings 151 Antifungal Susceptibility Testing 119 5.10.2 Pathology 151 Selected References for Antifungal 5.10.3 Host Factors 151 Susceptibility Testing 120 5.10.4 Pathogenesis 153 Questions 121 5.10.5 Vaccine Development 154 5.10.6 Attenuated Live Chitinase Mutant C. posadasii 154 5.11 Clinical Forms 155 Part Two Systemic Mycoses Caused by Dimorphic 5.12 Veterinary Forms 156 Environmental Molds (Endemic Mycoses) 5.13 Therapy 156 5.14 Laboratory Detection, Recovery, and 4. Blastomycosis 125 Identification 158 Selected References for 4.1 Blastomycosis-at-a-Glance 125 Coccidioidomycosis 162 4.2 Introduction/Disease Definition 125 Websites Cited 163 4.3 Case Presentations 126 Questions 164 4.4 Diagnosis 127 4.5 Etiologic Agent 127 4.6 Geographic Distribution/Ecologic 6. 165 Niche 128 6.1 Histoplasmosis-at-a-Glance 165 4.7 Epidemiology 129 6.2 Introduction/Disease Definition 165 4.8 Risk Groups/Factors 129 6.3 Case Presentations 166 4.9 Transmission 129 6.4 Etiologic Agents 169 4.10 Determinants of Pathogenicity 130 6.5 Geographic Distribution/Ecologic 4.10.1 Pathogenesis 130 Niche 170 4.10.2 Host Factors 130 6.6 Epidemiology 171 4.10.3 Microbial Factors 130 6.6.1 Incidence and Prevalence 171 4.11 Clinical Forms 131 6.6.2 Risk Groups/Factors 171 4.12 Veterinary Forms 133 6.7 Transmission 171 4.13 Therapy 133 6.8 Determinants of Pathogenicity 172 4.14 Laboratory Detection, Recovery, and 6.8.1 Host Factors 172 Identification 134 6.8.2 Microbial Factors 173 General Reference for Blastomycosis 137 6.9 Clinical Forms 175 Contents xi

6.10 Veterinary Forms 178 8.14 Laboratory Detection, Recovery, and 6.11 Therapy 179 Identification 208 6.12 Laboratory Detection, Recovery, and Selected References for Penicilliosis 212 Identification 180 Website Cited 212 Selected References for Questions 212 Histoplasmosis 184 Websites Cited 185 9. 215 Questions 185 9.1 Sporotrichosis-at-a-Glance 215 7. 187 9.2 Introduction/Disease Definition 215 9.3 Case Presentations 216 7.1 Paracoccidioidomycosis-at- 9.4 Diagnosis 218 a-Glance 187 9.5 Etiologic Agents 218 7.2 Introduction/Disease Definition 187 9.6 Geographic Distribution/Ecologic 7.3 Case Presentation 188 Niche 219 7.3.1 Oral Lesions in a Legionnaire (Horre´ 9.7 Epidemiology 219 et al., 2002) 188 9.7.1 Risk Groups/Factors 220 7.4 Etiologic Agent 188 9.8 Transmission 220 7.5 Geographic Distribution/Ecologic 9.9 Determinants of Pathogenicity 220 Niche 189 9.9.1 Host Factors 220 7.6 Epidemiology 189 9.9.2 Microbial Factors 221 7.6.1 Incidence 189 9.10 Clinical Forms 223 7.6.2 Risk Groups/Factors 189 9.11 Human–Animal Interface 224 7.7 Transmission 191 9.12 Therapy 225 7.8 Determinants of Pathogenicity 191 9.13 Laboratory Detection, Recovery, and 7.8.1 Host Factors 191 Identification 227 7.8.2 Microbial Factors 191 Selected References for 7.9 Clinical Forms 192 Sporotrichosis 230 7.10 Veterinary Forms 193 Website Cited 231 7.11 Therapy 194 Questions 231 7.12 Laboratory Detection, Recovery, and Identification 195 10A. Less Frequent Mycoses Caused by Selected References for Dimorphic Environmental Molds: Paracoccidioidomycosis 198 Adiaspiromycosis 233 Questions 199 10A.1 Adiaspiromycosis-at- 8. Penicilliosis 201 a-Glance 233 10A.2 Introduction/Disease Definition 234 8.1 Penicilliosis-at-a-Glance 201 10A.3 Case Summaries 234 8.2 Introduction/Disease Definition 201 10A.4 Diagnosis 235 8.3 Case Presentation 202 10A.5 Etiologic Agents 235 8.4 Diagnosis 203 10A.6 Geographic Distribution/Ecologic 8.5 Etiologic Agents 203 8.6 Geographic Distribution/Ecologic Niche 235 10A.7 Epidemiology/Risk Niche 203 Groups/Factors 235 8.7 Epidemiology 204 8.7.1 Incidence 204 10A.8 Transmission 235 8.7.2 Molecular Epidemiology 205 10A.9 Determinants of Pathogenicity 236 8.8 Risk Groups/Factors 205 10A.10 Clinical Forms 236 8.9 Transmission 205 10A.11 Veterinary Forms 236 8.10 Determinants of Pathogenicity 205 10A.12 Therapy 236 8.10.1 Host Factors 205 10A.13 Laboratory Detection, Recovery, and 8.10.2 Microbial Factors 205 Identification 236 8.11 Clinical Forms 206 Selected References for 8.12 Veterinary Forms 208 Adiaspiromycosis 238 8.13 Therapy 208 Questions 238 xii Contents

10B. Less Frequent Mycoses Caused by 11.10.2 Microbial Factors 276 Dimorphic Environmental Molds 11.11 Therapy 282 (Endemic Mycoses): 11.12 Laboratory Detection, Recovery, and (Jorge Lobo’sˆ Disease) 241 Identification 286 11.13 Less Common Opportunistic Yeast 10B.1 Lobomycosis-at-a-Glance 241 Genera 292 10B.2 Introduction/Disease Definition 242 11.13.1 Geotrichum capitatum 10B.3 Case Presentations 242 (Blastoschizomyces capitatus) 292 10B.4 Diagnosis 244 11.13.2 Rhodotorula Species 294 10B.5 Etiologic Agent 244 11.13.3 Saccharomyces cerevisiae 295 10B.6 Geographic Distribution/Ecologic Selected References for Candidiasis 297 Niche 244 Questions 301 10B.7 Epidemiology 244 10B.8 Risk Groups/Factors 244 12. 303 10B.9 Transmission 244 10B.10 Determinants of Pathogenicity 245 12.1 Cryptococcosis-at-a-Glance 303 10B.10.1 Host Factors 245 12.2 Introduction/Disease 10B.10.2 Microbial Factors 245 Definition 303 10B.11 Clinical Forms 245 12.3 Case Presentations 304 10B.12 Veterinary Forms 245 12.4 Etiologic Agents 307 10B.13 Therapy 246 12.5 Geographic Distribution/Ecologic 10B.14 Laboratory Detection, Recovery, and Niche 311 Identification 246 12.6 Epidemiology 312 Selected References for 12.7 Risk Groups/Factors 315 Lobomycosis 246 12.8 Transmission 316 Questions 247 12.9 Determinants of Pathogenicity 316 12.9.1 Host Factors 316 12.9.2 Microbial Factors 318 Part Three Systemic Mycoses Caused by 12.10 Clinical Forms 321 Opportunistic Yeasts and Pneumocystis 12.11 Veterinary Forms 323 12.12 Therapy 324 11. Candidiasis and Less Common Yeast 12.13 Laboratory Detection, Recovery, and Genera 251 Identification 326 General References for 11.1 Candidiasis-at-a-Glance 251 Cryptococcosis 329 11.2 Introduction/Disease Definition 251 Selected References for 11.3 Case Presentations 252 Cryptococcosis 329 11.4 Diagnosis 255 Websites of Interest 11.5 Etiologic Agents and their Ecologic or Cited 331 Niches 255 Questions 331 11.5.1 Classification of Candida Species 255 11.5.2 Less Common Candida Species of 13. 333 Clinical Importance 257 13.1 Pneumocystosis-at-a-Glance 333 11.6 Epidemiology 258 13.2 Introduction/Disease 11.6.1 Major Types of Candidiasis 258 Definition 333 11.7 Risk Groups/Factors 264 334 11.7.1 264 13.3 Case Presentation 11.7.2 Mucosal Candidiasis 264 13.4 Etiologic Agent 335 11.7.3 Cutaneous Candidiasis 265 13.5 Geographic Distribution/Ecologic 11.8 Transmission 265 Niche 336 11.9 Clinical Forms 266 13.6 Epidemiology 337 11.10 Determinants of Pathogenicity 273 13.7 Risk Groups/Factors 339 11.10.1 Host Factors 273 13.8 Transmission 340 Contents xiii

13.9 Determinants of 15.7 Transmission 401 Pathogenicity 340 15.8 Determinants of Pathogenicity 401 13.9.1 Host Factors 340 15.8.1 Host Factors 401 13.9.2 Microbial Factors 342 15.8.2 Microbial Factors 401 13.10 Clinical Forms 343 15.9 Clinical Forms 402 13.11 Therapy 346 15.10 Veterinary Forms 404 13.12 Laboratory Detection, Recovery, and 15.11 Therapy 405 Identification 348 15.12 Laboratory Detection, Recovery, and Selected References for Identification 407 Pneumocystosis 351 Selected References for Fusarium Questions 352 410 Website Cited 411 Questions 411

Part Four Systemic Mycoses Caused by 16. Pseudallescheria/Scedosporium Mycosis 413 Opportunistic Hyaline Molds 16.1 Pseudallescheria/Scedosporium Mycosis-at-a-Glance 413 14. 357 16.2 Introduction/Disease Definition 414 14.1 Aspergillosis at-a-Glance 357 16.3 Case Presentations 414 14.2 Introduction/Disease Definition 358 16.4 Diagnosis 416 14.3 Case Presentations 358 16.5 Etiologic Agents 416 14.3.1 Diagnosis 360 16.6 Geographic Distribution/Ecologic 14.4 Etiologic Agents 361 Niche 418 14.5 Geographic Distribution/Ecologic 16.7 Epidemiology 419 Niche 362 16.7.1 Risk Groups/Factors 421 14.6 Epidemiology and Risk 16.8 Transmission 421 Groups/Factors 363 16.9 Determinants of Pathogenicity 421 14.7 Transmission 369 16.9.1 Host Factors 421 14.8 Determinants of Pathogenicity 370 16.9.2 Microbial Factors 421 14.8.1 Host Factors 370 16.10 Clinical Forms 422 14.8.2 Microbial Factors 372 16.11 Veterinary Forms 423 14.9 Clinical Forms 375 16.12 Therapy 424 14.10 Veterinary Forms 378 16.13 Laboratory Detection, Recovery, and 14.11 Therapy 379 Identification 426 14.12 Laboratory Detection, Recovery, and Selected References for Identification 383 Pseudallescheria/Scedosporium Selected References for Mycosis 428 Aspergillosis 390 Website Cited 429 Websites Cited 393 Questions 429 Questions 393 Appendix 395 17A. 431 17A.1 Mucormycosis-at-a-Glance 433 15. Fusarium Mycosis 397 17A.2 Introduction/Disease Definition 434 15.1 Fusarium Mycosis-at-a-Glance 397 17A.3 Case Presentations 434 15.2 Introduction/Disease Definition 398 17A.4 Diagnosis 436 15.3 Case Presentation 398 17A.5 Etiologic Agents 436 15.3.1 Diagnosis 399 17A.6 Geographic Distribution/Ecologic 15.4 Etiologic Agents 399 Niche 437 15.5 Geographic Distribution/Ecologic 17A.7 Epidemiology and Risk Niche 399 Groups/Factors 440 15.6 Epidemiology 399 17A.8 Transmission 442 15.6.1 Incidence and Prevalence 399 17A.9 Determinants of Pathogenicity 442 15.6.2 Risk Factors 400 17A.9.1 Host Factors 442 xiv Contents

17A.9.2 Microbial Factors 442 17C.10 Determinants of Pathogenicity 470 17A.10 Clinical Forms 444 17C.11 Clinical Forms 470 17A.11 Veterinary Forms 445 17C.12 Veterinary Forms 471 17A.12 Therapy 446 17C.13 Therapy 471 17A.13 Laboratory Detection, Recovery, and 17C.14 Laboratory Detection, Recovery, and Identification 447 Identification 471 Selected References for Selected References for Mucormycosis 452 Entomophthoramycosis Caused by Websites Cited 454 Conidiobolus Species 472 Questions 454 Questions 473

17B. Entomophthoramycosis Caused by 457 Part Five Mycoses of Implantation 17B.1 Entomophothoramycosis Caused by Basidiobolus ranarum-at- 18. 479 a-Glance 457 17B.2 Introduction/Disease Definition 458 18.1 Chromoblastomycosis-at-a-Glance 479 17B.3 Case Presentations 458 18.2 Introduction/Disease Definition 479 17B.4 Etiologic Agent 460 18.3 Case Presentation 480 17B.5 Geographic Distribution/Ecologic 18.4 Diagnosis 480 Niche 461 18.5 Etiologic Agents 481 17B.6 Epidemiology 461 18.6 Geographic Distribution/Ecologic 17B.7 Risk Groups/Factors 461 Niche 482 17B.8 Transmission 461 18.7 Epidemiology and Risk 17B.9 Determinants of Pathogenicity 461 Groups/Factors 482 17B.9.1 Host Factors 461 18.8 Transmission 483 17B.9.2 Microbial Factors 461 18.9 Determinants of Pathogenicity 483 17B.10 Clinical Forms 462 18.9.1 Pathogenesis 483 17B.11 Veterinary Forms 462 18.9.2 Host Factors 483 17B.12 Therapy 462 18.9.3 Microbial Factors 484 17B.13 Laboratory Detection, Recovery, and 18.10 Clinical Forms 485 Identification 463 18.11 Therapy 485 Selected References for 18.12 Laboratory Detection, Recovery, and Entomophthoramycosis Caused by Identification 487 Basidiobolus ranarum 464 Selected References for Questions 465 Chromoblastomycosis 489 Questions 490 17C. Entomophthoramycosis Caused by Conidiobolus Species 467 19. Phaeohyphomycosis 493 17C.1 Entomophthoramycosis Caused by 19.1 Phaeohyphomycosis-at-a-Glance 493 Conidiobolus Species-at-a-Glance 467 19.2 Introduction 493 17C.2 Introduction/Disease Definition 467 19A Cutaneous–Subcutaneous 17C.3 Case Presentation 468 Phaeohyphomycosis 494 17C.4 Etiologic Agents 469 19A.1 Introduction/Disease Definition 494 17C.5 Geographic Distribution 469 19A.2 Case Presentations 494 17C.6 Ecologic Niche 469 19A.3 Etiologic Agents 497 17C.7 Epidemiology 469 19A.4 Geographic Distribution/Ecologic 17C.8 Risk Groups/Factors 470 Niche 497 17C.9 Transmission 470 19A.5 Epidemiology 497 Contents xv

19A.6 Risk Groups/Factors 497 Selected References for 19A.7 Transmission 497 522 19A.8 Determinants of Pathogenicity 497 Websites Cited 523 19A.8.1 Microbial Factors 498 Questions 523 19A.9 Clinical Form 499 19A.10 Therapy 499 19B Cerebral Phaeohyphomycosis 499 19B.1 Introduction/Disease Definition 499 19B.2 Case Presentation 499 Part Six and Dermatomycoses 19B.3 Etiologic Agents 500 (Superficial Cutaneous Mycoses) 19B.4 Geographic Distribution/Ecologic Niche 500 21. Dermatophytosis 527 19B.5 Epidemiology 501 21.1 Dermatophytosis-at-a-Glance 527 19B.6 Risk Groups/Factors 501 21.2 Introduction/Disease Definition 528 19B.7 Transmission 502 21.3 Case Presentations 529 19B.8 Determinants of Pathogenicity 502 21.4 Diagnosis 530 19B.9 Clinical Form 502 21.5 Etiologic Agents 530 19B.10 Therapy 502 21.6 Geographic Distribution/Ecologic 19C Fungal Sinusitis 503 Niche 534 19C.1 Introduction/Disease Definition 503 21.7 Epidemiology 534 19C.2 Clinical Forms 503 21.8 Risk Groups/Factors 537 19C.3 Transmission 506 21.9 Transmission 539 19C.4 Determinants of Pathogenicity 506 21.10 Determinants of Pathogenicity 540 19.3 Laboratory Detection, Recovery, and 21.10.1 Host Factors 540 Identification 506 21.10.2 Microbial Factors 541 Selected References for 21.11 Clinical Forms 542 Phaeohyphomycosis 509 21.12 Veterinary Forms 550 Websites Cited 510 21.13 Therapy 552 Questions 510 21.14 Laboratory Detection, Recovery, and Identification 554 20. Eumycetoma (Madura Foot, Selected References for Maduramycosis) 513 Dermatophytosis 563 20.1 Eumycetoma at-a-Glance 513 Website of Interest on the Subject of 20.2 Introduction/Disease Definition 513 Dermatophytosis 565 20.3 Case Presentation 514 Questions 565 20.4 Diagnosis 514 20.5 Etiologic Agent(s) 514 22. Dermatomycoses 567 20.6 Geographic Distribution/Ecologic Niche 515 22A Major Nondermatophytic Fungi from 20.7 Epidemiology 515 Skin and Nails 567 20.8 Risk Groups/Factors 516 22B Superficial Mycosis of the Caused 20.9 Transmission 516 by a Nondermatophyte Mold: Black 20.10 Determinants of Pathogenicity 516 569 20.10.1 Host Factors 516 22B.1 Introduction/Disease Definition 569 20.10.2 Microbial Factors 516 22B.2 Diagnosis 570 20.11 Clinical Forms (Fahal, 2004) 517 22B.3 Etiologic Agents 570 20.12 Veterinary Forms 518 22B.4 Geographic Distribution/Ecologic 20.13 Therapy 518 Niche/Epidemiology 570 20.14 Laboratory Detection, Recovery, and 22B.5 Risk Groups/Transmission 570 Identification 520 22B.6 Determinants of Pathogenicity 570 xvi Contents

22B.7 Clinical Forms 570 22C.4 Pityriasis Versicolor and Other Superficial 22B.8 Veterinary Forms 570 and Deep Mycoses Caused by Lipophilic 22B.9 Therapy 570 Yeasts: Species 576 22B.10 Laboratory Detection, Recovery, and 22C.4.1 Introduction/Disease Identification of 571 Definition 576 22C Superficial Mycoses Caused by Yeasts 22C.4.2 Case Presentation 577 and Yeast-like Fungi 571 22C.4.3 Diagnosis 577 22C.1 Onychia and 22C.4.4 Etiologic Agents 577 22C.4.5 Geographic Distribution/Ecologic 571 Niche 578 22C.2 571 22C.4.6 Epidemiology 578 22C.2.1 Introduction/Disease 22C.4.7 Risk Factors/Transmission 578 Definition 571 22C.4.8 Clinical Forms of Superficial 22C.2.2 Diagnosis 571 Mycoses Caused by Malassezia 22C.2.3 Etiologic Agents 572 Species 579 22C.2.4 Geographic Distribution/Ecologic 22C.4.9 Clinical Form of Deep-Seated Niche/Epidemiology 572 Malassezia Mycosis: 22C.2.5 Transmission 573 580 22C.2.6 Determinants of 22C.4.10 Veterinary Forms 580 Pathogenicity 573 22C.4.11 Determinants of 22C.2.7 Clinical Forms 573 Pathogenicity 581 22C.2.8 Veterinary Forms 573 22C.4.12 Therapy for Superficial and 22C.2.9 Therapy for White Piedra 573 Deep-Seated Mycoses Caused by 22C.2.10 Therapy for Systemic or Malassezia Species 582 Disseminated Species 22C.4.13 Laboratory Detection, Recovery, Infection in the and Identification of Malassezia Immunocompromised Host 574 Species 582 22C.2.11 Laboratory Detection, Recovery, 22D and Other Nonpathogenic and Identification of Trichosporon or Opportunistic Fungi Isolated from Species 574 22C.3 (Tinea Nigra Skin and Resembling in Culture 584 Palmaris) 575 22C.3.1 Introduction/Disease Selected References for Definition/Clinical Form 575 Dermatomycoses 585 22C.3.2 Diagnosis 575 Website Cited 587 22C.3.3 Etiologic Agent 575 Questions 587 22C.3.4 Geographic Distribution 575 22C.3.5 Ecologic Niche 575 Glossary 589 22C.3.6 Epidemiology 575 22C.3.7 Therapy 576 Answer Key 607 22C.3.8 Laboratory Detection, Recovery, and Identification of 576 Index 611 Preface

RATIONALE FOR THIS TEXT pathogens, and determinants of pathogenicity. The sec- ond chapter presents a systematic treatment of laboratory Medical mycology is a distinct subspecialty of medical diagnostic methods in medical mycology, including mor- microbiology and infectious disease. The field has pro- phologic, genetic, and nonculture methods. That chapter is gressed along with advances in both disciplines, informed structured and annotated for ease of use. This is followed by new knowledge from general mycology, immunology, by a chapter introducing antifungal therapy. The anti- and molecular biology. This textbook aspires to integrate fungal agents in current use are discussed with regard to that knowledge. It is designed to function as a reference their action spectrum and applications in clinical medicine. work for the clinical microbiology laboratory, a textbook This is followed by a subchapter on the specialized subject for a course in medical mycology, and for independent of antifungal susceptibility tests. These chapters set the reading and reference by physicians and research micro- stage for the disease-specific chapters which focus with biologists. greater granularity on the pertinent laboratory diagnostic Textbooks in medical mycology are few in number methods and therapy. and those that exist are, by-and-large, outdated. The text’s scope is balanced between medical and microbiologic ORGANIZATION OF THE knowledge of the fungi pathogenic for humans. It is DISEASE-SPECIFIC CHAPTERS designed to accompany an upper level course in medical mycology, e.g., a six-week elective consisting of twelve Each disease-specific chapter is aligned to the same for- 2-hour lectures. The material is sufficiently detailed so mat in order to direct the reader or course participant that it may also be presented as a semester course. The to sections of particular interest as outlined in the fol- chapters are organized by disease and contain numerous lowing section, Generic Format for the Mycotic Dis- illustrations and one-to-three case presentations. A series ease Chapters. The participant will become knowledge- of questions is appended at the end of each chapter able about mycotic diseases through case presentations, to reinforce learning. The text is annotated with an including the disease definition and differential diagnosis. extensive glossary. The bibliography emphasizes selected The etiologic agents are described according to their gen- references. The text assumes no prior knowledge of eral properties, taxonomic relationships, and their ecologic mycology but assumes a foundation in modern biology niche. Geographic distribution of each mycotic disease is and medical microbiology. presented along with a contemporary view of the epidemi- ology including incidence, prevalence, risk groups, risk factors, and disease transmission. Current knowledge of SCOPE OF FUNDAMENTAL the determinants of pathogenicity is reviewed from the MEDICAL MYCOLOGY vantage point of both host and microbial factors. The clinical forms of each disease are detailed according to Three cross-cutting chapters are followed by 19 disease- the organ system involved, clinical signs and symptoms, specific chapters. The introductory chapter is designed to and pathology. All phases of the laboratory detection, orient the reader to the spectrum of fungal diseases, taxon- recovery and identification are included for each fungal omy within the fungal kingdom, reproduction of fungi, the pathogen. Emphasis is placed on the use of direct exami- composition of the fungal cell, primary and opportunistic nation to achieve a rapid diagnosis. Specialized tests and xvii xviii Preface genetic identification are covered for those methods in GENERIC FORMAT FOR THE clinical use. MYCOTIC DISEASE CHAPTERS

This format facilitates reader’s comfort with the mate- OBJECTIVES OF FUNDAMENTAL rial because of the treatment given to each category with MEDICAL MYCOLOGY content tailored for each mycotic disease. 1. Mycosis-at-a-glance The clinical laboratory scientist will gain knowledge about the etiologic agents, determinants of pathogenicity, 2. Introduction/Disease Definition laboratory detection, recovery and identification. The 3. Case Presentation physician will refresh and extend knowledge of mycotic a. Diagnosis diseases through case presentations, epidemiology, 4. Etiologic Agents clinical forms, and therapy. There is good reason for both professions to cross-train in the areas of the other, in order 5. Geographic Distribution/Ecologic Niche to gain a more balanced view of the totality of fungal 6. Epidemiology diseases. a. Incidence and Prevalence 7. Risk Groups/Factors Target audiences: 8. Transmission a. Clinical laboratory scientists who are called upon to 9. Determinants of Pathogenicity identify fungi in the clinical laboratory e.g., medical 10. Clinical Forms technologists, clinical laboratory supervisors. 11. Veterinary Forms b. Physicians who wish to refresh and extend their 12. Therapy knowledge with emphasis on antifungal therapy. 13. Laboratory Detection, Recovery, and Identification c. Microbiologists who wish to become cross-trained in 14. Selected References medical mycology. 15. d. Microbiologists who wish to conduct research in med- Questions & Answers ical mycology. Errol Reiss,Ph.D. e. Medical students wishing to take an elective in med- H. Jean Shadomy,Ph.D. ical mycology. G. Marshall Lyon, III, M.D. f. Students registered in clinical laboratory science Atlanta, Georgia programs. September 2011 Acknowledgments

We express our gratitude to the following scientists who Stephen A. Moser Ph.D., School of Medicine, Univer- provided critiques of the book chapters: sity of Alabama, Birmingham, Alabama Raza Aly, Ph.D., University of California School of Marcio Nucci, M.D., Universidade Federal do Rio de Medicine, San Francisco, California Janeiro, Rio de Janeiro, Brazil Ruth Ashbee, Ph.D., University of Leeds, Leeds, Flavio´ Queiroz-Telles, M.D., Universidade Federal do England Parana,´ Curitiba, Brazil John W. Baddley M.D., M.P.H., School of Medicine, Wiley A. Schell, M.S., Duke University Medical Cen- University of Alabama, Birmingham, Alabama ter, Durham, North Carolina Monicaˆ Bastos de Lima Barros, M.D., Hospital Evan- Jerry D. Smilack, M.D., Mayo Clinic Hospital, dro Chagas-Fiocruz, Rio de Janeiro, Brazil Phoenix, Arizona Andrew M. Borman, Ph.D., Mycology Reference Lab- Deanna A. Sutton, Ph.D., University of Texas Health oratory, Bristol, United Kingdom Science Center, San Antonio, Texas Iracilda Z. Carlos, Ph.D., D. Pharm., Sao˜ Paulo State Carlos P. Taborda M.D., University of Sao˜ Paulo, Sao˜ University, Sao˜ Paulo, Brazil Paulo, Brazil John D. Cleary, Pharm. D., University of Mississippi Carolina Talhari M.D., Institute of Tropical Medicine School of Pharmacy, University, Mississippi Amazonas, Manaus, Brazil Garry T. Cole, Ph.D., University of Texas, San Anto- Uma M. Tendolkar, M.D., LTM Medical College, and nio, Texas LTM General Hospital, Mumbai, India Chester R. Cooper, Ph.D., Youngstown State Univer- Brian L. Wickes, Ph.D., University of Texas Health sity, Youngstown, Ohio Sciences Center, San Antonio, Texas Arthur F. Di Salvo, M.D., University of South Carolina Peter R. Williamson, M.D., Ph.D., School of Medicine, School of Medicine, Columbia, South Carolina University of Illinois, Chicago, Illinois E. Lopez-Romero,´ Ph.D., Universidad Autonoma de Jon P. Woods, Ph.D., University of Wisconsin School Guanajuato, Mexico of Medicine, Madison, Wisconsin Xiarong Lin, Ph.D., Texas A&M University, College Rosely Zancope-Oliveira,´ Ph.D., Fundac¸ao˜ Oswaldo Station, Texas Cruz, Rio de Janeiro, Brazil Ronald E. Garner, Ph.D., Mercer University School of Many laboratory scientists and physician-scientists Medicine, Macon, Georgia have contributed individual illustrations for this book and Cornelia Lass-Florl,¨ M.D., Innsbruck Medical Univer- they are acknowledged in the figure legends. A smaller sity, Innsbruck, Austria. group went to great lengths to supply several illustrations, Paul F. Lehmann Ph.D., The University of Toledo many previously unpublished, from their collections, and Health Sciences, Toledo, Ohio they deserve special mention and gratitude. Christine J. Morrison Ph.D., U.S. Centers for Disease George Barron, Ph.D., Ontario Agricultural College, Control and Prevention, Atlanta, Georgia University of Guelph, Ontario, Canada xix xx Acknowledgments

Edward P. Ewing, Jr. M.D., Public Health Image Lynne Sigler, M.S., Professor, University of Alberta, Library, Centers for Disease Control, Atlanta, Edmonton, Canada Georgia Robert Simmons, Ph.D., Director, Biological Imaging Jim Gathany, Scientific photographer, the Centers Core Facility, Department of Biology, Georgia State for Disease Control Creative Arts Branch, Atlanta, University, Atlanta, Georgia Georia Uma M. Tendolkar, M.D., LTM Medical College, and Rajesh T. Gandhi, M.D., Editor for Partners in Infec- LTM General Hospital, Mumbai, India tious Disease Images, www.idimages.org Robert L. Wick, Ph.D., Plant, Soil and Insect Sciences Department, University of Massachusetts, Amherst, Arvind A. Padhye, Ph.D., Centers for Disease Control, Massachusetts Atlanta, Georgia Calvin O. McCall, Jr., M.D., School of Medicine, Brian J. Harrington, Ph.D., University of Toledo Health Virginia Commonwealth University, Richmond, Sciences, Toledo, Ohio Virginia, provided expert advice on therapy for Christoph U. Lehmann, M.D., Johns Hopkins Uni- dermatophytosis. versity School of Medicine, Baltimore, Maryland Michael M. McNeil, M.D., Centers for Disease Con- and Chief Information Officer of DermAtlas, trol, Atlanta, Georgia, provided expert advice on ther- www.dermatlas.org apy for candidiasis. Tadahiko Matsumoto, M.D., Yamada Institute of Health We express our sincere appreciation to Dr. Karen E. and Medicine, Tokyo, Japan Chambers, Editor, Ms. Lisa Van Horn, Production Editor, Jorge Musa, Sr. MLT, Life Laboratories, Toronto, and Ms. Anna Ehler, Editorial Assistant, Wiley-Blackwell Canada Publishers. Stephen W. Peterson, Ph.D., National Center for E. R. Agricultural Utilization Research, U.S. Department H. J. S. of Agriculture, Peoria, Illinois G. M. L., III Part One Introduction to Fundamental Medical Mycology, Laboratory Diagnostic Methods, and Antifungal Therapy

Fundamental Medical Mycology, First Edition. By Errol Reiss, H. Jean Shadomy and G. Marshall Lyon, III.  2012 Wiley-Blackwell. Published 2012 by John Wiley & Sons, Inc. 1

Chapter 1

Introduction to Fundamental Medical Mycology

1.1 TOPICS NOT COVERED, OR have the serious responsibility to train all technolo- RECEIVING SECONDARY EMPHASIS gists and students in the safe manipulation of clinical specimens and pathogenic fungi. Before working with The Table of Contents is explicit but it is well to advise pathogenic microbes, including fungi, microbiologists readers that some topics are either outside the scope of should participate in their organization’s safety training Fundamental Medical Mycology or receive secondary program, be certified to work with pathogens, and, emphasis. Although caused by, or associated with, fungi it when questions about biosafety arise, consult the super- is not within the scope of this work to discuss mushroom visor and the CDC/NIH biosafety manual: Biosafety poisoning (ingestion of toxins present in mushrooms), in Microbiological and Biomedical Laboratories,5th mycotoxicosis (ingestion of a fungal toxin), or allergies, edition (BMBL). The manual is available online at the except when encountered as a complication of one of the URL http://www.cdc.gov/biosafety/publications/bmbl5/ fungal diseases discussed. index.htm. This will ensure a safe work environment where the workers will not be afraid to work with fungi • More information on mushroom poisoning but instead will have confidence that they are observing (mycetismus) can be found in Benjamin (1995). prudent precautions. • Allergies caused by fungi are discussed in Kurup Molds growing on Petri plates can produce far more and Fink (1993) and Breitenbach et al. (2002). The infectious propagules (conidia or spores) than an envi- health effects of exposure to molds, apart from infec- ronmental exposure! Therefore, mold cultures should be tion, may be found in Storey et al. (2005) and U.S. transferred to screw cap- or cotton-stoppered agar slants. Environmental Protection Agency publication 402K- Mold cultures on Petri plates should never be opened 01-001 (2001). on the open laboratory bench. All cultures of unknown • Environmental mycology is discussed as it relates to molds should be handled inside a biological safety the ecologic niche of the causative agents of mycoses. cabinet (BSC). Petri plates should be sealed with shrink • Veterinary medical mycology is covered in a concise seals, which are colorless transparent cellulose bands. section, “Veterinary Forms,” in each disease-specific “Occupational Hazards from Deep Mycoses” is a useful chapter. and cautionary article summarizing laboratory infections (Schwarz and Kauffman, 1977; Padhye et al., 1998). The BMBL should also be consulted for further infor- 1.2 BIOSAFETY CONSIDERATIONS: mation about selection of BSCs, and biosafety considera- BEFORE YOU BEGIN WORK WITH tions of work with pathogenic fungi. If further questions PATHOGENIC FUNGI... arise on matters of fungal biosafety, please contact the State Department of Health in the United States of Amer- Safety in the laboratory is of prime importance. Clin- ica or the CDC Mycotic Diseases Branch, which is the ical laboratory supervisors and principal investigators World Health Organization Center for Mycoses.

Fundamental Medical Mycology, First Edition. By Errol Reiss, H. Jean Shadomy and G. Marshall Lyon, III.  2012 Wiley-Blackwell. Published 2012 by John Wiley & Sons, Inc. 3 4 Chapter 1 Introduction to Fundamental Medical Mycology

1.2.1 Biological Safety laboratory infections, and they too have remained local- Cabinets (BSC) ized. Localized infections require systemic antifungal therapy. What are the characteristics of Class II Biological Safety Laboratory exposures to aerosolized conidia (spores) Cabinets? The Class II BSC is designed with inward air- have led to pulmonary infections and, in the case of Coc- flow velocity (75–100 linear feet/min) and is fitted with cidioides species, to serious or even fatal infections. Some HEPA high efficiency particulate air ( ) filters. This design cases of coccidioidomycosis have occurred in laboratories ensures that the workspace in the cabinet receives filtered, beyond the endemic area and resulted when the laboratory downward, vertical laminar airflow. These characteristics did not suspect the mold they had isolated was Coccid- protect personnel and the microbiologic work conducted ioides. in the BSC. Biosafety level 2 (BSL 2) practices, containment HEPA-filtered-exhaust air ensures protection of the equipment, and facilities are recommended for han- laboratory and the outside environment. All Class II dling and processing clinical specimens, identifying cabinets are designed for work involving microorgan- isolates, and processing animal tissues suspected of isms assigned to biosafety levels 1, 2, and 3.1. Fungi containing pathogenic fungi. BSL 2 is also sufficient pathogenic for humans are classed in biosafety level 2 for mold cultures identified as Blastomyces dermatitidis, and work with them should be conducted in the BSC, and , dermatophytes, Penicillium not on the open bench. Certain manipulations of fungal marneffei pathogens or environmental samples require biosafety ,and . In addition to these level 3 (please see below). agents, certain melanized molds have caused serious Class II BSCs provide a microbe-free work environ- infection in immunocompetent hosts following inhalation ment. Class II BSCs are classified into two types (A and or accidental penetrating injuries: Bipolaris species, B) based on construction, airflow, and exhaust systems. Cladophialophora bantiana, Wangiella (Exophiala) Type A cabinets are suitable for microbiologic work in the dermatitidis, Exserohilum species, , absence of volatile or toxic chemicals and radionuclides, Ochroconis gallopava, Ramichloridium mackenziei,and since air is recirculated within the cabinet. Type A cabinets Scedosporium prolificans. may be exhausted into the laboratory or to the outdoors via All manipulations of clinical specimens and culture a special connection to the building exhaust system. Type work are performed inside an annually inspected and B cabinets are hard-ducted to the building exhaust system certified, well-functioning, laminar flow biological safety and contain negative pressure plenums to allow work to cabinet (BSC), equipped with HEPA filtered exhaust. be done with toxic chemicals or radionuclides. A list of Workers should wear personal protective equipment products that meet the standards for Class II BSCs are (PPE). available from the National Sanitation Foundation Inter- national, Ann Arbor, Michigan. It is mission-critical that • Clothing: laboratory coats with fronts fastened and BSCs be tested and certified in situ at the time of installa- shoes with closed fronts. tion, at any time the BSC is moved, and at least annually • Eye protection: safety glasses, goggles, as recom- after that. mended by the supervisor. • Gloves: latex or plastic. 1.2.2 Precautions to Take • Respiratory protection: goggles, mask, face shield, in Handling Etiologic Agents or other splatter guards are used when the cultures that Cause Systemic Mycoses must be handled outside the BSC. Surgical masks are not respirators and do not provide protection The major known reasons for laboratory exposures to against aerosolized infectious agents. The N95 pathogenic fungi are dropped cultures, preparing soil sus- disposable respirator provides a level of protection. pensions and inoculating animals, opening Petri plates, Supervisors should consult the National Institute of and aerosols from needles and syringes (Padhye et al., Occupational Safety and Health (NIOSH) Publication 1998). The portals of entry for the fungi, resulting from No. 99–143: TB Respiratory Protection Program the above exposures, are minor skin wounds or the inhala- in Health Care Facilities to match the respiratory tion of fungal conidia. protection to their risk assessment at the URL Pathologists and veterinarians should be mindful that http://www.cdc.gov/niosh/docs/99-143. autopsies and necropsies have caused accidental hand wounds, which have become infected. These infections • Sharps jars should be provided for disposal of needles are localized to the wound and have not disseminated. and syringes. Needle stick injuries have also been the source of • All waste should be autoclaved before disposal. 1.4 Medical Mycology 5

1.2.3 Additional Precautions 1.2.5 Disinfectants and Waste at Biosafety Level 3 (BSL 3) Disposal BSL 3 conditions should be observed when working with For information on these topics for the laboratory please mold-form cultures identified as species and see Chapter 2, Section 2.3.1.1, Disinfectants and Waste according to the following spe- Disposal. cific situations.

• and C. posadasii.OnceCoccid- 1.3 FUNGI DEFINED: THEIR ioides species are identified in the clinical laboratory, ECOLOGIC NICHE biosafety level 3 practices, equipment, and facilities are required for manipulating sporulating cultures and What are fungi? Where are they found? The kingdom for processing soil or other environmental materials Fungi is composed of unicellular or multicellular, eukary- known to contain infectious arthroconidia. Coccid- otic, heterotrophic microbes. Each fungal cell contains a ioides species are subject to the regulations regard- full array of organelles and is bound by a rigid cell wall ing Select Agents: biological agents and toxins that containing chitin, glucan, and/or cellulose (Table 1.1). could pose a severe threat to public health and safety. Please also see Section 1.12, General Composition of the These regulations are discussed in “Appendix F” of Fungal Cell. the BMBL. Of the thousands of fungal species that are free-living • Clinical laboratory supervisors and principal inves- in nature or are pathogenic for plants, only a small group tigators should be aware of what to do if there is are known to be pathogenic for humans and animals. It ◦ a Coccidioides exposure in their laboratory (Stevens is also true that any fungus capable of growing at 37 Cis et al., 2009). a potential pathogen in a debilitated or immunocompro- • BSL 3 practices, containment equipment, and facil- mised host. ities are recommended for propagating sporulating Some fungi are primary pathogens (e.g., Coccid- cultures of H. capsulatum in the mold form, as well ioides species) and can cause disease in immune-normal as processing soil or other environmental materials persons. Severity of a fungal disease is related to host known or likely to contain infectious conidia. factors (immune status, general health status) and the number of infectious propagules (conidia or spores) The criteria for BSL 3 practices are numerous and are inhaled, ingested, or injected. Persons who are immuno- detailed in the CDC/NIH manual, Biosafety in Microbio- compromised, or otherwise debilitated, are prone to logical and Medical Laboratories. BSL 3 conditions may develop more serious disease and to be susceptible require facility reconstruction: for example, two doors to opportunistic fungi against which immune-normal should separate the laboratory from a public area; the persons have a high level of resistance. laboratory should be at negative pressure with respect to Fungi are ubiquitous in nature, being found in the the entrance; and air that enters the laboratory should be air, in soil, on plants, and in water, including the oceans, vented through the biological safety cabinet HEPA fil- even as a part of lichens growing on rock. There is essen- ter and then vented outside the building. Waste is to be tially no part of our earth where fungi are not found. A autoclaved before it leaves the BSL 3 laboratory, so that a few fungal species are adapted to live as commensals in room adjacent to the level 3 laboratory should be equipped humans but for most fungal pathogens humans are acci- with an autoclave. dental hosts. Of all the fungi with pathogenic potential most are opportunistic, whereas a select few are able to cause disease in otherwise healthy humans who have intact immune and endocrine systems. 1.2.4 Safety Training The U.S. Centers for Disease Control and Prevention sponsor the International Symposium on Biosafety. Short 1.4 MEDICAL MYCOLOGY courses at that conference cover biosafety practices in laboratories and in veterinary practice: for example, What is medical mycology? Medical mycology is a distinct “Infection Control, Biosafety in Research and Clinical discipline of medical microbiology concerned with all Settings” and “Moving from BSL 2 to BSL 3.” Topics aspects of diseases in humans and lower animals caused included are engineering controls, personal protec- by pathogenic fungi. tive equipment (PPE), hand hygiene, environmental What are the mycoses? Mycoses are diseases of disinfection, and waste disposal. humans and lower animals caused by pathogenic fungi. 6 Chapter 1 Introduction to Fundamental Medical Mycology

Table 1.1 Comparison of Structure/Function of Bacterial and Fungal Organelles Organelles Bacteria Fungi Cell wall Murein = peptidoglycan Glucan, mannan, chitin Cell membrane No sterols Ergosterol Metabolism Aerobic or anaerobic Aerobic Energy transduction Cell membrane Mitochondria Cytoplasm Proteins Glycoproteins, actin, tubulin, mitotic spindle, Golgi Gene structure Operons, no introns Single genes, introns, repetitive DNA Nuclear material Prokaryotic: a single chromosome; no Eukaryotic: multiple chromosomes contained in a nuclear membrane. nucleus bounded by a nuclear membrane; for example Candida albicans has 8 chromosomes Ribosome 30S + 50S = 70S 40S + 60S = 80S Extrachromosomal DNA Plasmids Mitochondrial DNA, dsRNA Capsular polysaccharide Several agents of meningitis One agent of meningitis: Cryptococcus neoformans

There is a broad spectrum of mycoses ranging from However, there are significant exceptions, for example, superficial skin diseases to deep-seated, multisystem dis- the dermatophytes. In the Middle Ages, children in Europe seminated diseases. Please see Section 1.10, Classification became infected with , a fungal disease of the scalp, of Mycoses Based on the Primary Site of Pathology. smooth skin, even nails, due to schoenleinii. It was devastating to these individuals because they were considered unclean, separated from their peers, and sent to 1.5 A BRIEF HISTORY OF MEDICAL separate schools. There was no specific treatment at that MYCOLOGY time. Favus was so disfiguring that it was mistaken for leprosy by artists of the Renaissance (Goldman, 1968). A Because the fruiting bodies of some fungi are large enough modern case of scalp ringworm is shown in Fig. 1.1. to see without the aid of a microscope, such as mush- rooms, they were the first microorganisms known. Cen- 1.5.3 Twentieth Century turies later, it was discovered that mushrooms are only the obvious structures of complex fungi with a vast network Examples of outbreaks from a single source are not of fungal cells found beneath the soil, tree bark, and so on. uncommon. In Witwatersrand, South Africa, from 1941 through 1944 nearly 3000 gold mine workers 1.5.1 Ancient Greece were infected with the subcutaneous fungal pathogen Sporothrix schenckii, which they acquired by brushing Fungi have caused a variety of maladies affecting our against mine timbers on which the fungus was growing quality of life for millennia. Hippocrates (460–377 b.c.e.), (Du Toit, 1942). Figure 1.2 depicts the classic appearance the father of medicine, recognized that persons with oral of lymphocutaneous sporotrichosis. thrush (due to Candida albicans) were already debilitated by other diseases. This thought was echoed in our own 1.5.4 Endemic Mycoses time when Professor Graham S. Wilson, Director of the in the Americas U.K’s Public Health Laboratory Service said: “Candida is a much better clinician than we are, in its ability to A review of thousands of induction center roentgenograms detect abnormalities earlier in the course of development of young men inducted into the armed forces in World of such abnormalities than we can with all our chemical War II noted the “incidence of calcified lesions presumed tests.” This comment was made before the advent of AIDS to represent healed tuberculosis corresponded to the now but now it is well known that oral– well-known pattern of regional differences in the U.S.” heralds the onset of that disease. (reviewed by Iams, 1950). Mycologic investigations, including large scale skin testing with histoplasmin, estab- 1.5.2 Middle Ages lished that delayed type hypersensitivity to Histoplasma capsulatum was widespread among residents of the major In general, the vast majority of fungal infections are not river valleys of the central United States, thus establishing spread from person to person (are not communicable). the boundaries of the histoplasmosis endemic area. 1.5 A Brief History of Medical Mycology 7

methotrexate, and used it to treat childhood leukemia. His report in 1948 in the New England Journal of Medicine was greeted with ridicule because, at the time, the medical community held that childhood leukemia was incurable and children so afflicted should be allowed to die in peace. Since then, other researchers discovered drugs that blocked different functions involved in cell growth and replication ushering in the era of chemotherapy. The first cure of metastatic cancer was obtained in 1956 when methotrexate was used to treat a rare tumor called choriocarcinoma.

1.5.6 Opportunistic Mycoses Cancer chemotherapy using cytotoxic drugs and systemic corticosteroids, by weakening the immune system, created opportunities for yeast and mold disease, principally can- didiasis and aspergillosis, but also a long list of fungi for- merly regarded as “saprophytes.” Members of the genus Figure 1.1 Head of a child with (scalp ringworm). A Aspergillus, consisting of common environmental molds, 9-year-old girl complained of cradle cap for over a year before have been known as pathogens since 1842, when one developing itchy red patches on the right parietal scalp. She shed most of the hair in these areas. A culture was positive for species was detected in the air sac of a bullfinch. Later, , and she was treated with oral griseofulvin Aspergillus fumigatus was identified in other birds, and with complete clearing of the tinea and regrowth of hair. in humans where infections have increased proportional Source: Copyright Bernard A. Cohen, M.D. Used with permission to the use of immunosuppressive therapy. Masses of the from Dermatlas; www.dermatlas.org. fungus have been found behind suspended ceilings in hos- pitals, in building materials, and outside windows of hos- pitals, especially during renovation or construction, and are the cause of single cases of aspergillosis as well as outbreaks in hospitalized patients.

1.5.7 HIV/AIDS The AIDS epidemic in the United States, beginning in 1981, was brought to the attention of infectious disease specialists when two previously rare diseases, Kaposi’s sarcoma and pneumocystosis, were encountered in men having sex with men (MMWR, 1981). An increase in (PCP) was noticed at the Cen- ters for Disease Control (CDC) in April 1981, by Sandra Ford, a drug technician, who reported a high number of requests for the drug pentamidine, used to treat PCP. Figure 1.2 Sporotrichosis of the arm. Lesions draining each According to Ford, “A doctor was treating a gay man lymph node, from a primary lesion on the hand. in his 20’s who had pneumonia. Two weeks later, he Source: Wilson and Plunkett (1965), used with permission from the called to ask for a refill of a rare drug that I handled. University of California Press. This was unusual, nobody ever asked for a refill. Patients usually were cured in one 10-day treatment or they died.” 1.5.5 Era of Immunosuppression As HIV, the cause of AIDS, and its role as a blood- in the Treatment of Cancer, borne and sexually transmitted pathogen was elucidated, Maintenance of Organ Transplants, AIDS brought to the forefront those opportunistic infec- and Autoimmune Diseases tions whose host defense was coupled to T-cell mediated immunity. In addition to pneumocystosis, other fungal The era of cancer chemotherapy began when Sidney opportunistic infections of AIDS were identified in quick Farber synthesized a folic acid antagonist, now called succession as oro-esophageal candidiasis, cryptococcosis, 8 Chapter 1 Introduction to Fundamental Medical Mycology endemic mycoses in the United States and, in Southeast against clinical yeast isolates, and is available for Asia, penicilliosis. investigational use to test amphotericin B, ketoconazole, voriconazole (VRC), posaconazole (PSC), and caspofun- 1.5.8 Twenty-first Century gin (CASF). The YeastOne method was also evaluated to test the susceptibility of molds to triazoles and to 1.5.8.1 Advances in Clinical Laboratory amphotericin B. Mycology Automated Spectrophotometric Microdilution Important milestones in clinical laboratory mycology  include (i) more rapid tests to identify Candida species Susceptibility The VITEK 2 (bioMerieux,´ Inc., in blood cultures, (ii) the wider availability of antifungal France) is FDA approved for testing FLC against susceptibility tests because of new commercial kits, Candida species. It has also been evaluated for testing and (iii) the transfer of technology into the clinical amphotericin B, VRC, and flucytosine. laboratory for sequence-based identification of fungi.  These are state-of-the-art tests appropriate for well- Disk Diffusion Neo-Sensitabs tablet (Rosco, Taas- resourced hospitals. In resource-limited countries, a lack trup, Denmark) is an easy to perform disk diffusion of training, proper reagents, supplies, and equipment method available in Europe for testing yeasts and molds impacts their laboratories’ ability to identify pathogens against polyenes, azoles, and echinocandins. and to detect antimicrobial resistance. Beginning in  2005, the American Society for Microbiology (ASM) Drug Gradient Strips Etest (AB Biodisk, Solna, International Laboratory Capacity Building Program Sweden) is not new but accommodates newer antifun- (URL www.labcap.org) began to strengthen and expand gal agents so it can be viewed as an improved method clinical microbiology services in those regions. for AFS testing of yeasts and molds. It is an agar diffu- sion method with each drug applied to a plastic strip in Rapid Results for Candidemia The Candida albi- a gradient of concentrations and printed with a minimum cans/ peptide nucleic acid fluorescent inhibitory concentration scale. The FDA has approved the in situ hybridization assay (PNA-FISH, AdvanDx, Inc. Etest for testing FLC, ITC, and flucytosine. Woburn, MA) was described in 2002 and is approved by the U.S. Food and Drug Administration (FDA) as a kit to Sequence-Based Identification of Fungi Unusual identify yeast directly from a newly positive blood culture yeasts and molds, fungi that are slow growing, or those (Shepard et al., 2008). Cost savings accrue because, by that fail to sporulate pose challenges to morphologic iden- ruling out C. glabrata, unnecessary echinocandin therapy tification. Sequence-based identification is moving from can be avoided. Please see Chapter 11, Section 11.12, Lab- the research laboratory to the clinical microbiology lab- oratory Detection, Recovery and Identification, for further oratories of tertiary care medical centers, aided by the information. availablility of kits for DNA preparation and purification, and biotechnology core facilities. Wider Availability of Antifungal Susceptibility As an indication of progress, the CLSI issued a guide- (AFS) Testing The availability of commercial AFS line, “Interpretive Criteria for Identification of Bacteria tests in good agreement with reference methods facilitates and Fungi by DNA Target Sequencing” (CLSI, 2008) to rapid test results, thus aiding in clinical treatment deci- address sequence analysis in clinical laboratory practice. sions. These methods are discussed in Chapter 3B and The guideline provides a standardized approach to identify are referred to here as important milestones in advancing fungi by DNA sequencing using the most common target, clinical laboratory mycology (Canton´ et al., 2009). The the ITS region of rDNA. Topics covered include primer tests listed below have good correlation with reference design, quality control of amplification and sequencing, methods standardized by the U.S. Clinical Laboratory and reference sequence databases. The progress-to-date and Standards Institute (CLSI). and remaining challenges of DNA sequence-based iden- tification of opportunistic molds are discussed by Balajee Microtitration Plates Precoated with Drugs et al., (2009). Sensititre YeastOne (Trek Diagnostic Systems Inc., Westlake, OH) is a broth microdilution method. Wells of 1.5.8.2 Advances in Antifungal Therapy microtitration plates come precoated with dried dilutions of antifungal agents. Because of that they are stable in Licensing of extended spectrum azoles, and a new class storage for prolonged periods, even at room temperature. of antifungal drugs, the echinocandins, in this century, YeastOne is approved by the U.S. FDA for testing expand the therapeutic choices to treat invasive mycoses. fluconazole (FLC), itraconazole (ITC), and flucytosine Extended spectrum triazole antifungal agents (VRC, PSC,