Fundamental Medical Mycology Errol Reiss

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

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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 616.9^ꢀ6901–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

10 9 8 7 6 5 4 3 2 1

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

Contents

1.6.2 Investigating Outbreaks 1.6.3 Determining the Susceptibility to
Antifungal Agents 10
1.6.4 Estimating the Signiﬁcance of Fungi
Generally Considered to be Opportunists
10

Preface

xvii

Acknowledgments

xix or Saprobes
1.6.5 Types of Vegetative Growth

1.7 Sporulation 11 1.8 Dimorphism 11

1.8.1 Dimorphism and Pathogenesis
10
10

Part One Introduction to Fundamental Medical Mycology, Laboratory Diagnostic Methods, and Antifungal Therapy

12

1.9 Sex in Fungi

13
1.9.1 Anamorph and Teleomorph
Nomenclature

1.10 Classiﬁcation of Mycoses Based on the

1. Introduction to Fundamental Medical
Mycology

13

3

1.1 Topics not Covered, or Receiving Secondary
Emphasis
1.2 Biosafety Considerations: Before You Begin
Work with Pathogenic Fungi...

1.2.1 Biological Safety Cabinets (BSC)

Primary Site of Pathology

1.10.1 Superﬁcial Mycoses 1.10.2 Cutaneous Mycoses
13
13 13
3
3
1.10.3 Systemic Opportunistic Mycoses

1.10.4 Subcutaneous Mycoses 13 1.10.5 Endemic Mycoses Caused by Dimorphic
Environmental Molds 13

1.11 Taxonomy/Classiﬁcation: Kingdom
Fungi 14

1.11.1 The Phylogenetic Species Concept for
Classiﬁcation 15
1.11.2 The Higher Level Classiﬁcation of
Kingdom Fungi 15

1.12 General Composition of the Fungal
Cell 21

1.12.1 Yeast Cell Cycle 1.12.2 Hyphal Morphogenesis
13
4
1.2.2 Precautions to Take in Handling Etiologic
Agents that Cause Systemic Mycoses
1.2.3 Additional Precautions at Biosafety
4

Level 3 (BSL 3)

5

1.2.4 Safety Training

5

1.2.5 Disinfectants and Waste Disposal

5

1.3 Fungi Deﬁned: Their Ecologic Niche 1.4 Medical Mycology

5

1.5 A Brief History of Medical Mycology

6
1.5.1 Ancient Greece 1.5.2 Middle Ages
6
6
21

1.5.3 Twentieth Century

6

21
1.5.4 Endemic Mycoses in the Americas

1.5.5 Era of Immunosuppression in the
Treatment of Cancer, Maintenance of Organ Transplants, and Autoimmune
6

1.12.3 Cell Wall

22

1.13 Primary Pathogens

25
1.13.1 Susceptibility to Primary
Pathogens 26

1.14 Endemic V e rsus Worldwide Presence

Diseases
1.5.6 Opportunistic Mycoses 1.5.7 HIV/AIDS 1.5.8 Twenty-ﬁrst Century
7
26
7

1.15 Opportunistic Fungal Pathogens

26

7

1.15.1 Susceptibility to Opportunistic Fungal

8

Pathogens: Host Factors

26

1.6 Rationale for Fungal Identiﬁcation

1.6.1 Developing the Treatment Plan
99

1.16 Determinants of Pathogenicity

27

vii viii

Contents

General References in Medical

Selected References for Laboratory

Diagnostic Methods in Medical

Mycology

Selected References for Introduction to Fundamental Medical Mycology 28 Websites Cited 29 Questions 30

27

Mycology

69

Websites Cited

70

Commercial Manufacturers and Suppliers of Fungal Media, Stains, and Reagents 71 Packing and Shipping of Infectious Agents and Clinical Specimens 72 Questions 72

2. Laboratory Diagnostic Methods in Medical
Mycology

31

2.1 Who Is Responsible for Identifying

3A. Antifungal Agents and Therapy

3A.1 Introduction 75

75

Pathogenic Fungi?

2.1.1 Role of the Clinical Laboratorian 2.1.2 Role of the Physician 31

2.2 What Methods are Used to Identify
Pathogenic Fungi? 31

2.2.1 Culture and Identiﬁcation
31
31
3A.1.1 Major Antifungal Agents Approved for
Clinical Use 76
3A.1.2 Comparison of Antibacterial and
Antifungal Agents According to Their
31

Intracellular Targets

79

2.3 Laboratory Detection, Recovery, and
Identiﬁcation of Fungi in the Clinical
3A.2 Amphotericin B (AmB-deoxycholate)



(Fungizone , Apothecon Subsidiary of
Microbiology Laboratory

2.3.1 The Laboratory Manual 2.3.2 Specimen Collection 2.3.3 Direct Examination
33

Bristol-Myers-Squibb)

3A.2.1 Structure 80 3A.2.2 Mode of Action
80
33

33
34
80
3A.2.3 Indications 3A.2.4 Formulation
82
82
2.3.4 Histopathology 2.3.5 Culture 37
36
3A.2.5 Spectrum of Activity 3A.2.6 Clinical Uses 82 3A.2.7 Lipid Formulations of AmB 3A.2.8 Pharmacokinetics 84
82
2.3.6 Storage and Cryopreservation of Cultures

for QA and QC in the Clinical Mycology
83
Laboratory

2.3.7 Media and Tests for Yeast
Identiﬁcation 42
2.3.8 Methods Useful for Mold
Identiﬁcation 45
2.3.9 Microscopy Basics
41

3A.2.9 Interactions

85

3A.2.10 Adverse Reactions

85



3A.3 Fluconazole (FLC) (Diﬂucan ,
Pﬁzer) 86

3A.3.1 Structure and Mode of Action 3A.3.2 Indications 86 3A.3.3 Fluconazole Pharmacokinetics
53
86

87
2.3.10 Use of Reference Laboratories 2.3.11 Fungal Serology and Biochemical
59
Markers of Infection

2.4 Genetic Identiﬁcation of Fungi

2.4.1 Commercial Test 64 2.4.2 Peptide Nucleic Acid–Fluorescent In Situ
59

3A.3.4 Efﬁcacy

88

64

3A.3.5 Formulations 3A.3.6 Interactions
88
88

88



Hybridization (PNA-FISH)
2.4.3 PCR-Sequencing Method 2.4.4 Nuclear rDNA Complex 2.4.5 Genetic Tools for Species
64
64
64

3A.4 Itraconazole (ITC) (Sporanox , Janssen
Pharmaceutica Division of Johnson & Johnson)

3A.4.1 Action Spectrum 3A.4.2 ITC: Uncertain Bioavailability 3A.4.3 Properties 89 3A.4.4 Pharmacokinetics
89

Identiﬁcation

66

89

2.4.6 How Is the Genetic Identiﬁcation of an

89

Unknown Fungus Accomplished?
2.4.7 Growth of the Fungus in Pure Culture,
Extraction and Puriﬁcation of DNA
66
89

66

3A.4.5 Interactions

90

2.4.8 PCR of the Target Sequence

67

3A.5 Voriconazole (VRC) (Vfend ,
Pﬁzer) 90

90



2.4.9 PCR Cycle Sequencing

68

2.4.10 Assemble the DNA Sequence 2.4.11 Perform a BLAST Search
68
68
68
68
3A.5.1 Action Spectrum 3A.5.2 Pharmacokinetics 3A.5.3 Drug Interactions 3A.5.4 Adverse Reactions
90 90 91
91



2.4.12 The MicroSeq System 2.4.13 Other Sequence Databases

General References for Laboratory

Diagnostic Methods in Medical

3A.6 Posaconazole (PSC) (Noxaﬁl ,

Schering-Plough/Merck & Co.)

Mycology

69

91

Contents ix

3A.6.1 Action Spectrum 3A.6.2 Pharmacokinetics 3A.6.3 Drug Interactions 3A.6.4 Adverse Reactions
91 91 92
3A.14.1 Mode of Action 3A.14.2 Action Spectrum
99
99

3A.14.3 Indications

99

92

3A.14.4 Dosage Regimen

99

100

3A.7 Azole Resistance Mechanisms

3A.7.1 Alteration of Target Enzyme
(Lanosterol Demethylase)
92 92
3A.14.5 Metabolism 3A.14.6 Adverse Reactions

3A.15 Combination Therapy

99
100
3A.7.2 Overexpression of Target

3A.16 Suppressive or Maintenance
Therapy 100
3A.17 Prophylactic Therapy

3A.17.1 Bimodal Period of Risk 3A.17.2 Fluconazole and Alternatives for
Primary Prophylaxis 101
Enzyme
3A.7.3 Increased Efﬂux of Drug, CDR Efﬂux
Pumps 92
3A.7.4 Bypass Pathways
92
100
101
92

3A.7.5 Loss of Heterozygosity in
Chromosome 5 and Azole
3A.17.3 Prophylaxis in Patients During the

Pre-engraftment Period with a History of Invasive Mold Infections
3A.17.4 Prophylaxis in the Post-engraftment

Resistance

92

3A.7.6 Azole Resistance in Aspergillus
102

Species

3A.8 Echinocandins

93 93

Period

102

102
3A.8.1 Mode of Action 3A.8.2 Spectrum of Activity

3A.9 Caspofungin (CASF) (Cancidas ,
Merck) 94

3A.9.1 Action Spectrum 3A.9.2 Dosage 95
93

3A.18 Empiric Therapy

93



3A.19 Innately Resistant Fungi

103
3A.19.1 Innately Resistant Molds 3A.19.2 Innately Resistant Yeasts

General Reference for Antifungal Agents and Therapy 103 Selected References for Antifungal Agents

103 103
94

3A.9.3 Pharmacokinetics 3A.9.4 Drug Interactions 3A.9.5 Adverse Reactions
95 95
95

and Therapy

103



Websites Cited

105

3A.10 Micafungin (MCF) (Mycaminet , Astellas
Pharma, Inc.) 95
Questions

105
3A.10.1 Indications 3A.10.2 Dosage
95

3B. Antifungal Susceptibility Tests

107

95

3A.10.3 Metabolism

96

3B.1 Antifungal Susceptibility Tests

3A.10.4 Drug Interactions

3A.11 Anidulafungin (ANF) (Eraxis ,
Pﬁzer) 96

3A.11.1 Indications 3A.11.2 Invasive Candidiasis
96

Deﬁned
3B.2 National and International Standards for
AFS Tests 107
3B.3 Objective of AFS Tests 3B.4 Minimum Inhibitory Concentration (MIC) of an Antifungal Drug Deﬁned 107

107



96
107
96

3A.11.3 Molds 3A.11.4 Dosage
96
96

3B.4.1 MIC₅₀and MIC₉₀

108

3A.11.5 Metabolism

3A.11.6 Drug Interactions 3A.11.7 Adverse Reactions
96

3B.5 Broth Microdilution (BMD)

97
97



Method
3B.6 Clinical Indications for AFS
Testing 108
3B.7 Correlation Between the In Vitro
Determined MIC and the Clinical Efﬁcacy of Drug Therapy 108

108

3A.12 Terbinaﬁne (TRB) (Lamisil ,
Novartis) 97

3A.12.1 Mode of Action 3A.12.2 Action Spectrum 3A.12.3 Drug Synergy 3A.12.4 Metabolism 3A.12.5 Adverse Reactions
97
97
97
97
3B.7.1 How Are the Conditions of

“Susceptible” or “Resistant”
98

3A.13 5-Fluorocytosine (Flucytosine, 5FC)

Determined?
3B.7.2 What Are Breakpoints? 3B.7.3 Minimum Effective
109



(Ancobon , Valeant

109

Pharmaceuticals)

3A.13.1 Indications
98

98

Concentration

3B.8 AFS Methods Currently Available for Use in the Clinical Laboratory 110

3B.8.1 Broth Microdilution (BMD)
109
3A.13.2 Combination Therapy 3A.13.3 Metabolism 98 3A.13.4 Adverse Reactions

3A.14 Griseofulvin (Grifulvin V, Ortho
Pharmaceutical Corp.) 99

98
98
Method
3B.8.2 Etest
110
110



x

Contents

3B.8.3 Disk Diffusion Method

3B.9 Which Laboratories Conduct AFS
Tests? 110
3B.10 Principles of AFS Tests

3B.10.1 Standard Method for AFS Testing of
Yeasts 110
110

Selected References for Blastomycosis Questions

137
138
110

5. Coccidioidomycosis

141

3B.10.2 Modiﬁcations Suggested to Improve
Performance of the BMD Method for

5.1 Coccidioidomycosis-at-a-Glance 5.2 Introduction/Disease Deﬁnition

141
141

Yeast

112

5.3 Case Presentations 5.4 Diagnosis 143 5.5 Etiologic Agents 5.6 Geographic Distribution/Ecologic
Niche 144
5.7 Epidemiology

5.7.1 Incidence and Prevalence
142
3B.10.3 Commercial BMD Method with
Precoated Drug Panels: A
143
CLSI-Approved Method for AFS

Testing of Yeasts and Molds

113

3B.10.4 Standardization of AFS Tests for
147
Molds, M38-A2: Broth Microdilution

for Molds 114
3B.10.5 Etest (bioMe´rieux, Marcy l’Etoile,
France) 114
147
5.7.2 Effect of Weather on Annual Fluctuations in Prevalence of



Coccidioidomycosis

148

3B.10.6 Disk Diffusion AFS Tests

3B.10.7 VITEK 2 System for AFS
117

5.8 Risk Groups/Factors

149



5.9 Transmission 5.10 Determinants of Pathogenicity and
Pathogenesis 151

5.10.1 Allergic Findings
150
(bioMe´rieux, Marcy l’Etoile,

France)

118

3B.10.8 Flow Cytometry AFS Test

3B.11 Summary of the Current Status of
Antifungal Susceptibility Testing

118 119
151

5.10.2 Pathology

151

5.10.3 Host Factors 5.10.4 Pathogenesis
151 153

Selected References for Antifungal Susceptibility Testing Questions 121

120
5.10.5 Vaccine Development 5.10.6 Attenuated Live Chitinase Mutant

C. posadasii 154

5.11 Clinical Forms 5.12 Veterinary Forms 5.13 Therapy 156 5.14 Laboratory Detection, Recovery, and
Identiﬁcation 158

154
155
156

Part Two Systemic Mycoses Caused by Dimorphic Environmental Molds (Endemic Mycoses)

4. Blastomycosis

125

Selected References for Coccidioidomycosis
4.1 Blastomycosis-at-a-Glance

125
162

4.2 Introduction/Disease Deﬁnition

125

Websites Cited

163

4.3 Case Presentations 4.4 Diagnosis 127 4.5 Etiologic Agent 4.6 Geographic Distribution/Ecologic
Niche 128
4.7 Epidemiology 4.8 Risk Groups/Factors 4.9 Transmission 129 4.10 Determinants of Pathogenicity

126

Questions

164
127

6. Histoplasmosis

165

6.1 Histoplasmosis-at-a-Glance

165
129

6.3 Case Presentations 6.4 Etiologic Agents

166
169
130

6.5 Geographic Distribution/Ecologic
Niche 170
6.6 Epidemiology

4.10.1 Pathogenesis 4.10.2 Host Factors 4.10.3 Microbial Factors
130 130
171

130
6.6.1 Incidence and Prevalence

6.6.2 Risk Groups/Factors
171

4.11 Clinical Forms 4.12 Veterinary Forms 4.13 Therapy 133 4.14 Laboratory Detection, Recovery, and
Identiﬁcation 134 General Reference for Blastomycosis

131
171

133

6.7 Transmission

171

6.8 Determinants of Pathogenicity

172

6.8.1 Host Factors

172

6.8.2 Microbial Factors

173

137

6.9 Clinical Forms