Pocket Guide to Mycological Diagnosis 1st Edition

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 Pocket Guide
to Mycological
Diagnosis

Edited by
Rossana de Aguiar Cordeiro
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742

© 2020 by Taylor & Francis Group, LLC

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Contents

Preface vii
Editor ix
Contributors xi
List of Abbreviations xiii

1 Mycological Diagnosis: General Principles 1

Rossana de Aguiar Cordeiro

2 Antifungal Drugs and Susceptibility Testing of Fungi 19

Débora de Souza Colares Maia Castelo-Branco, Glaucia Morgana de
Melo Guedes, and Marcos Fábio Gadelha Rocha

3 Candida spp. 29
Silviane Praciano Bandeira, Glaucia Morgana de Melo Guedes, and
Débora de Souza Colares Maia Castelo-Branco

4 Cryptococcus 39
Luciana Trilles, Márcia dos Santos Lazéra, and Bodo Wanke

5 Trichosporon 47
João Nobrega de Almeida Júnior

6 Malassezia 53
Reginaldo Gonçalves de Lima-Neto, Danielle Patrícia Cerqueira
Macêdo, Ana Maria Rabelo de Carvalho, Carolina Maria da Silva, and
Rejane Pereira Neves

7 Rhodotorula spp. 63
Rejane Pereira Neves, Ana Maria Rabelo de Carvalho, Carolina
Maria da Silva, Danielle Patrícia Cerqueira Macêdo, and Reginaldo
Gonçalves de Lima-Neto

8 Dermatophytes 69
Germana Costa Paixão, Marcos Fábio Gadelha Rocha, Débora de
Souza Colares Maia Castelo-Branco, Raimunda Sâmia Nogueira
Brilhante, and José Júlio Costa Sidrim

9 Aspergillus spp. 83
Reginaldo Gonçalves de Lima-Neto, Patrice Le Pape, and Rejane
Pereira Neves

v
vi    Contents

10 Mucorales 91
Rejane Pereira Neves, André Luiz Cabral Monteiro de Azevedo
Santiago, and Reginaldo Gonçalves de Lima-Neto

11 Sporothrix spp. 99
Anderson Messias Rodrigues, Rosane Orofino-Costa, and Zoilo Pires
de Camargo

12 Histoplasma capsulatum 115

Rosely Maria Zancopé-Oliveira, Claudia Vera Pizzini, Marcos de
Abreu Almeida, and Rodrigo de Almeida Paes

13 Paracoccidioides Complex 125

Zoilo Pires de Camargo and Anderson Messias Rodrigues

14 Coccidioides 135
Rossana de Aguiar Cordeiro

15 Pneumocystis jirovecii 145

Rosely Maria Zancopé-Oliveira, Fernando Almeida-Silva, Rodrigo de
Almeida Paes, and Mauro de Medeiros Muniz

Index 153
Preface

In the past three decades, the epidemiology of fungal infections has changed dramatically.
Along with the AIDS pandemic, extensive medical progress has led to an increase in the
global population of severely immunocompromised patients, who are vulnerable to fungal
pathogens. Since proper diagnosis is considered a pivotal factor for patient management,
routine microbiology laboratories have been overloaded with requests for mycological tests.
This situation has compelled many microbiologists to seek ongoing education in medical
mycology—a field of knowledge neglected in many health courses.

In addition, the science of medical mycology has witnessed tremendous changes since the
1980s. The description of new species and species complexes, the discovery of fungal biofilms
and their importance in pathogenesis, the continuous reports of refractory infections and
antifungal resistance, and the increasing list of opportunistic fungal species have highlighted
the importance of mycological diagnosis.

Cellular and molecular techniques, immunological methods, and more accurate microscopy
equipment are now available to mycology laboratories. Furthermore, information regarding
medical mycology, including identification of specific fungal pathogens, is widely available on
the World Wide Web. Mycologists have to face the challenge of systematizing all this body of
knowledge and applying it in routine diagnosis.

Therefore, this book aims to provide concise and useful information for microbiologists
and professionals interested in the diagnosis of the most relevant fungal species of medical
importance. Potential contributors were chosen based on their personal experience and previous
authorship of widely cited publications on the subject.

We hope this book inspires young mycologists worldwide and contributes to enhancing the
detection of fungal pathogens in routine laboratories.

Professor Rossana de Aguiar Cordeiro

Federal University of Ceará, Brazil

vii
Editor

Rossana de Aguiar Cordeiro is a biologist and undertook her postgraduate training in

Microbiology at the Federal University of Minas Gerais and Federal University of Ceará, both in
Brazil. During the past two decades, she has dedicated her efforts to Medical Mycology, focusing
on emerging fungal infections in Brazil, mainly coccidioidomycosis. She is the first author of more
than 40 original research and review articles and, as an author, has contributed to the books
Molecular Detection of Human Fungal Pathogens and Manual of Security Sensitive Microbes and
Toxins, both published by CRC Press.

ix
Contributors

Fernando Almeida-Silva Zoilo Pires de Camargo

National Institute of Infectology Laboratory of Emerging Fungal Pathogens,
Fundação Oswaldo Cruz Cell Biology Division
Brazil Department of Microbiology, Immunology
and Parasitology
Silviane Praciano Bandeira Federal University of São Paulo
Specialized Medical Mycology Center Brazil
Department of Pathology and Legal Medicine
Federal University of Ceará Ana Maria Rabelo de Carvalho
Brazil Department of Mycology
Center for Biosciences
Raimunda Sâmia Nogueira Brilhante
Federal University of Pernambuco (UFPE)
Specialized Medical Mycology Center
Cidade Universitária
Department of Pathology and Legal Medicine
Recife, Brazil
Federal University of Ceará
Brazil
Mauro de Medeiros Muniz
Danielle Patrícia Cerqueira Macêdo National Institute of Infectology
Department of Pharmaceutical Sciences Fundação Oswaldo Cruz
Center for Health Sciences, UFPE Brazil
Brazil
Glaucia Morgana de Melo Guedes
Carolina Maria da Silva Specialized Medical Mycology Center
Department of Mycology Department of Pathology and Legal
Center for Biosciences Medicine
Federal University of Pernambuco (UFPE) Federal University of Ceará
Cidade Universitária Brazil
Recife, Brazil

Marcos de Abreu Almeida Débora de Souza Colares Maia

National Institute of Infectology Castelo-Branco
Fundação Oswaldo Cruz Department of Pathology and Legal
Brazil Medicine
Federal University of Ceará
Rossana de Aguiar Cordeiro Brazil
Specialized Medical Mycology Center
Department of Pathology and Legal Medicine Márcia dos Santos Lazéra
Federal University of Ceará National Institute of Infectology
Brazil Fundação Oswaldo Cruz
Brazil
João Nobrega de Almeida Júnior
Central Laboratory Division
Reginaldo Gonçalves de Lima-Neto
Hospital das Clínicas
Department of Tropical Medicine
Faculty of Medicine
Center for Health Sciences, UFPE
University of São Paulo
and
Brazil
Department of Mycology
Rodrigo de Almeida Paes Biosciences Center
National Institute of Infectology Federal University of Pernambuco (UFPE)
Fundação Oswaldo Cruz Brazil
Brazil
Rejane Pereira Neves
André Luiz Cabral Monteiro de Azevedo Department of Mycology
Santiago Center for Biosciences
Department of Tropical Medicine Federal University of Pernambuco (UFPE)
Center for Health Sciences, UFPE Cidade Universitária
Brazil Recife, Brazil

xi
xii    Contributors

Rosane Orofino-Costa Anderson Messias Rodrigues

Dermatology Department Laboratory of Emerging Fungal Pathogens,
School of Medical Sciences Cell Biology Division
Rio de Janeiro State University Department of Microbiology, Immunology
Brazil and Parasitology
Federal University of São Paulo
Germana Costa Paixão Brazil
Center of Health Sciences
State University of Ceará José Júlio Costa Sidrim
Brazil Specialized Medical Mycology Center
Department of Pathology and Legal
Patrice Le Pape Medicine
IICiMed – EA 1155 Cibles et médicaments Federal University of Ceará
des infections de l’immunité et du cancer Brazil
University of Nantes
France Luciana Trilles
National Institute of Infectology
Claudia Vera Pizzini Fundação Oswaldo Cruz
National Institute of Infectology Brazil
Fundação Oswaldo Cruz
Brazil Bodo Wanke
National Institute of Infectology
Marcos Fábio Gadelha Rocha Fundação Oswaldo Cruz
Specialized Medical Mycology Center Brazil
Department of Pathology and Legal Medicine
Federal University of Ceará Rosely Maria Zancopé-Oliveira
and National Institute of Infectology
School of Veterinary Fundação Oswaldo Cruz
State University of Ceará Brazil
Brazil
List of Abbreviations

AD atopic dermatitis
AdoMet/SAM S-adenosylmethionine
AFLP amplified fragment length polymorphism
ARF ADP-ribosylation factor
ART antiretroviral therapy
ASR analyte-specific reagent
AST Antimicrobial susceptibility testing
BAL Bronchoalveolar lavage
BDG 1–3-β-D-glucan
BHI brain heart infusion agar
BSL biosafety level
CDC Centers for Disease Control and Prevention
CF complement fixation
CFW calcofluor white fluorescent stain
CGB l-canavanine glycine bromothymol blue
CLSI Clinical Laboratory Standards Institute
CNS central nervous system
CrAg cryptococcal capsular polysaccharide antigen
CSF cerebrospinal fluid
CVC central venous catheters
DGGE denaturing gradient gel electrophoresis
ECV or ECOFF epidemiological cut-off value
EDTA ethylenediamine tetraacetic acid
EIA enzyme immunoassay
ELISA enzyme-linked immunosorbent assay
EUCAST European Committee on Antimicrobial Susceptibility Testing
FFPE formalin-fixed paraffin-embedded tissue
FISH fluorescence in situ hybridization
FITC fluorescein isothiocyanate
FL folliculitis
FTD fast track diagnostics
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GMS Gomori methenamine-silver nitrate stain
gp43 glycoprotein of 43,000 daltons (Paracoccidioides antigen)
GXM capsular antigen glucuronoxylomannan from Cryptococcus
HE hematoxylin and eosin
HIV human immunodeficiency virus
HPA H. capsulatum polysaccharide antigen
ID immunodiffusion
IF immunofluorescence
IGS intergenic spacer-1
IRIS immune reconstitution inflammatory syndrome
ISHAM International Society of Human and Animal Mycology
ITS internal transcribed spacer
KL-6 Krebs von den Lungen-6 antigen
KOH potassium hydroxide
LA latex agglutination test
LAMP loop-mediated isothermal amplification
LDH lactate dehydrogenase
LFA lateral flow immunoassay
LSU large subunit
MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
MEC minimum effective concentration
MGG May-Grünwald Giemsa
MIC minimum inhibitory concentration
ML maximum likelihood
MLST multilocus sequence typing

xiii
xiv    List of Abbreviations

Msg major surface glycoprotein (Pneumocystis antigen)

NAALADase N-acetylated α-linked acidic dipeptidase (H. capsulatum antigen)
NJ Neighbor-joining
NSA niger seed agar
NWT non-wild type
PAS periodic acid-Schiff
PCM paracoccidioidomycosis
PCP pneumonia by Pneumocystis carinii
PCR polymerase chain reaction
PCR-RFLP PCR-based restriction fragment length polymorphism
PFGE pulsed field gel electrophoresis
PJP Pneumocystis pneumonia
PMF peptide mass fingerprint
PS psoriasis
PV pityriasis versicolor
RAPD random amplification of polymorphic DNA
RCA rolling circle amplification
rDNA ribosomal DNA genes
RIA radioimmunoassay
RSA recombinant synthetic antigen
SADH secondary alcohol dehydrogenase
SD seborrheic dermatitis
SDA Sabouraud dextrose agar
SHP hypersensitivity pneumonitis
SPS sodium polyanethol sulfonate
SSKI saturated solution of potassium iodide
TP tube precipitin Coccidioides antigen
WB Western blot
WT wild type
1
Mycological Diagnosis
General Principles
Rossana de Aguiar Cordeiro

Contents

1.1 Introduction 1
1.2 Collection of clinical specimens 1
1.3 Mycological processing (conventional tests) 5
1.3.1 Direct microscopic examination 5
1.3.2 Culture 5
1.4 Identification 5
1.4.1 Standard culture-based methods: Phenotypical analysis 5
1.4.2 Culture-independent methods 11
1.4.2.1 Molecular diagnosis 11
1.4.2.2 Immunological diagnosis 15
1.4.2.3 Matrix-assisted laser desorption ionization-time of flight
mass spectrometry 15
1.5 Conclusions 18
Bibliography 18

1.1 Introduction

Mycological laboratories have an important role in the complex scenario of understanding the
etiology of fungal infections. They provide information not only related to diagnosis, but also to
the treatment, prevention, and control of mycosis. Through such technical data, it is also possible
to gain insight into the epidemiology of fungal infections, which brings great responsibilities to
the lab staff. Therefore, standardized and internationally validated protocols and routines must
be followed.

In order to achieve these goals, mycological labs need to be equipped with biosafety items:
individual and community barriers must be available (Table 1.1), and personnel must be
engaged in continuous biosafety training. Labs pertaining only to dermatological routines
need to be designed to reach level 2 biological safety criteria. On the other hand, labs with
complex attendance routines, that is, oncological patients, post-transplant patients, and
patients suspected of deep-seated infections, need risk 3 biological safety criteria. Additionally,
mycologists need continued education to become familiar with new fungal species and modern
diagnostic procedures. A general scheme of mycological diagnosis stages is shown in Figure 1.1.

1.2 Collection of clinical specimens

After clinical examination of the patient by physicians, the first step of mycological diagnosis is
to collect clinical specimens. Mycologists should be aware that: (a) specimens must always be
collected from the representative site of infection, (b) collection should preferably be performed
prior to the start of antifungal therapy, (c) the amount of material collected should be suitable
for performing all the required tests, (d) specimens should be transported to the lab and
processed as soon as possible, and (e) all clinical material can be infected with other hazardous