The definitive guide for identifying fungi from clinical specimens

With a new team of authors,Larones Medically Important Fungi, Seventh Edition, continues the longstanding tradition of high-quality content to expand your knowledge and support your work in clinical mycology by:Providing detailed descriptions of the major mycoses as viewed in patients specimens by direct microscopic examination of stained slidesOffering a logical step-by-step process for identification of cultured organisms, utilizing detailed descriptions, images, pointers on organisms similarities and distinctions, and selected references for further informationCovering more than 150 of the fungi most commonly encountered in the clinical mycology laboratory, including new entries forEmergomyces, Metarhizium anisopliae,Rasamsonia argillacea, Rhinocladiella mackenziei, Schizophyllum commune,andThermothelomyces thermophilusPresenting details on each organisms pathogenicity, growth characteristics, relevant biochemical reactions, and microscopic morphology, illustrated with photomicrographs, unique and elegant drawings, and color photos of colony morphology and various test resultsExplaining changes in fungal taxonomy and nomenclature that are due to information acquired through molecular taxonomic studies of evolutionary fungal relationshipsProviding basic information on molecular diagnostic methods, e.g., nucleic acid amplification and sequencing, MALDI-TOF mass spectrometry, and other commercial platformsIncluding an extensive section of easy-to-follow lab protocols, a comprehensive list of media and stain procedures, guidance on collection and preparation of patient specimens, and an illustrated glossary

WithLarones Medically Important Fungi: A Guide to Identification, both novices and experienced professionals in clinical microbiology laboratories can confidently identify commonly encountered fungi.

Lars F. Westblade,PhD, D(ABMM) is the Director of the Clinical Microbiology Service at NewYork-Presbyterian/Weill Cornell Medical Center and Associate Professor at Weill Cornell Medicine with a primary appointment in the Department of Pathology and Laboratory Medicine and a secondary appointment in the Division of Infectious Diseases, Department of Medicine. He earned his doctoral degree from the University of Birmingham in the United Kingdom, and completed a fellowship in medical and public health laboratory microbiology at Washington University School of Medicine in St. Louis.

Eileen M. Burd,PhD, D(ABMM) is the Director of the Clinical Microbiology Laboratory at Emory University Hospital and Professor at Emory University School of Medicine with a primary appointment in the Department of Pathology and Laboratory Medicine and a secondary appointment in the Department of Medicine, Division of Infectious Diseases. She earned her doctoral degree from the Medical College of Wisconsin in Milwaukee and was the Division Head of Microbiology at Henry Ford Hospital in Detroit, Michigan prior to joining the faculty at Emory University in 2007.

Shawn R. Lockhart,PhD, D(ABBM) FAAM is the Senior Clinical Laboratory Advisor in the Mycotic Diseases Branch at the Centers for Disease Control and Prevention. He earned his doctoral degree from the University of Kentucky and completed his clinical microbiology fellowship at the University of Iowa Hospitals and Clinics. He directs the CDC training course in mold identification.

Gary W. Procop,MD, MS is the CEO of the American Board of Pathology and Professor of Pathology at the Cleveland Clinic Lerner School of Medicine. He remains a Consulting Staff for the Institute of Pathology and Laboratory Medicine, where he served as Medical Director for the Mycology Laboratory for more than two decades. He earned his doctoral degree from the Marshall University School of Medicine. His residency in anatomic and clinical pathology was completed at Duke University and his medical microbiology fellowship at the Mayo Clinic.

List of Tables xvii

Preface to the Seventh Edition xix

Preface to the First Edition xxi

Acknowledgments xxiii

About the Authors xxv

Basics 1

How To Use the Guide 3

Use of Reference Laboratories and Regulations for Transport 5

Safety Precautions 11

Taxonomy and Nomenclature 13

Part I Direct Microscopic Examination of Clinical Specimens 15

Introduction 17

Histological Terminology 21

Tissue Reactions to Fungal Infection 25

Stains 29

Table 1.1 Histochemical stains for fungi and/or filamentous bacteria in tissue 30

Guide to Interpretation of Direct Microscopic Examination 33

Detailed Descriptions 39

Actinomycosis 40

Mycetoma (Actinomycotic or Eumycotic) 41

Nocardiosis 43

Mucormycosis (Zygomycosis) 44

Aspergillosis 45

Miscellaneous Hyalohyphomycoses (Other than Aspergillosis) 47

Dermatophytosis (Tinea, Ringworm) 49

Tinea versicolor 50

Tinea nigra 51

Phaeohyphomycosis 52

Chromoblastomycosis 53

Sporotrichosis 54

Histoplasmosis 55

Emergomycosis 57

Talaromycosis (Penicilliosis) 58

Blastomycosis 59

Paracoccidioidomycosis 60

Lobomycosis 61

Candidiasis 62

Trichosporonosis 64

Cryptococcosis 65

Pneumocystosis 67

Protothecosis 68

Coccidioidomycosis 69

Rhinosporidiosis 70

Adiaspiromycosis 72

Part II Identification of Fungi in Culture 73

Guide to Identification of Fungi in Culture 75

Detailed Descriptions 105

Filamentous Bacteria 107

Introduction to Filamentous Bacteria 109

Table 2.1 Differentiation of filamentous aerobic actinomycetes encountered in clinical specimens 110

Nocardia spp. 111

Streptomyces spp. 114

Actinomadura spp. 116

Nocardiopsis dassonvillei 117

Yeasts and Yeastlike Organisms 119

Introduction to Yeasts and Yeastlike Organisms 121

Candida albicans 123

Table 2.2 Characteristics of the genera of clinically encountered yeasts and yeastlike organisms 124

Candida dubliniensis 125

Table 2.3 Characteristics of Candida spp. most commonly encountered in the clinical laboratory 126

Table 2.4 Characteristics that assist in differentiating Candida dubliniensis from Candida albicans 128

Candida tropicalis 129

Candida parapsilosis species complex 130

Candida lusitaniae 131

Candida krusei 132

Table 2.5 Differentiating characteristics of Magnusiomyces capitatus (formerly Blastoschizomyces capitatus) versus Candida krusei 134

Table 2.6 Differentiating characteristics of Candida krusei, Candida inconspicua, and Candida norvegensis 134

Candida kefyr 135

Candida rugosa species complex 136

Candida guilliermondii species complex 138

Table 2.7 Differentiating characteristics of Candida guilliermondii versus Candida famata 139

Candida lipolytica 140

Candida zeylanoides 141

Candida glabrata species complex 142

Candida auris 143

Candida haemulonii species complex 144

Candida pelliculosa 145

Cryptococcus neoformans species complex 147

Cryptococcus gattii species complex 149

Table 2.8 Characteristics of Cryptococcus spp. and former members of the genus 150

Table 2.9 Characteristics of yeasts and yeastlike organisms other than Candida spp. and Cryptococcus spp. 151

Rhodotorula and Cystobasidium spp. 152

Sporobolomyces salmonicolor 154

Saccharomyces cerevisiae 156

Malassezia spp. 158

Malassezia pachydermatis 160

Ustilago spp. 161

Prototheca spp. 162

Trichosporon and Cutaneotrichosporon spp. 163

Table 2.10 Key characteristics of the most common clinically encountered Trichosporon spp. and Cutaneotrichosporon spp. 165

Magnusiomyces capitatus (formerly Blastoschizomyces capitatus) 166

Geotrichum candidum 167

Thermally Dimorphic and/or Endemic Fungi 169

Introduction to Thermally Dimorphic and/or Endemic Fungi 171

Histoplasma capsulatum 172

Emergomyces spp. 175

Blastomyces dermatitidis/gilchristii 177

Coccidioides immitis/posadasii 179

Paracoccidioides brasiliensis 181

Talaromyces marneffei (formerly Penicillium marneffei) 183

Sporothrix schenckii species complex 186

Emmonsia crescens 189

Thermally Monomorphic Moulds 191

Mucormycetes 193

Introduction to Mucormycetes 195

Table 2.11 Differential characteristics of similar organisms in the class Mucormycetes 197

Table 2.12 Differential characteristics of the clinically encountered Rhizopus spp. 197

Rhizopus spp. 198

Mucor spp. 200

Rhizomucor spp. 201

Lichtheimia corymbifera species complex 202

Apophysomyces elegans species complex 204

Saksenaea vasiformis 206

Cokeromyces recurvatus 207

Cunninghamella bertholletiae 209

Syncephalastrum racemosum 211

Basidiobolus spp. 212

Conidiobolus coronatus 213

Dematiaceous Fungi 215

Introduction to Dematiaceous Fungi 217

Fonsecaea spp. 218

Myrmecridium schulzeri 221

Rhinocladiella mackenziei (formerly Ramichloridium mackenziei) 222

Phialophora verrucosa 223

Table 2.13 Characteristics of Phialophora, Pleurostoma (formerly Pleurostomophora), Phaeoacremonium, Acremonium and Sarocladium, Phialemonium, and Coniochaeta (formerly Lecythophora) 224

Pleurostoma richardsiae (formerly Pleurostomophora richardsiae) 225

Phaeoacremonium parasiticum 226

Phialemonium spp. 228

Cladosporium spp. 230

Table 2.14 Characteristics of Cladosporium spp. and Cladophialophora spp. 232

Cladophialophora carrionii 233

Cladophialophora boppii 235

Cladophialophora bantiana 236

Scedosporium apiospermum species complex 237

Table 2.15 Differentiating phenotypic characteristics of the clinically encountered members of Scedosporium spp. and Lomentospora prolificans 241

Lomentospora prolificans (formerly Scedosporium prolificans) 242

Verruconis gallopava (formerly Ochroconis gallopava) 244

Table 2.16 Characteristics of some of the black yeasts 246

Exophiala jeanselmei species complex 247

Exophiala dermatitidis 249

Hortaea werneckii 251

Madurella mycetomatis 252

Trematosphaeria grisea (formerly Madurella grisea) 253

Piedraia hortae 254

Aureobasidium pullulans 255

Table 2.17 Differential characteristics of Aureobasidium pullulans versus Hormonema dematioides 257

Hormonema dematioides 258

Neoscytalidium dimidiatum 259

Botrytis cinerea 261

Stachybotrys chartarum 262

Thermothelomyces thermophilus (formerly Myceliophthora thermophila) 264

Curvularia spp. 265

Table 2.18 Characteristics of Curvularia spp. and Exserohilum rostratum 269

Exserohilum rostratum 270

Helminthosporium spp. 272

Alternaria spp. 273

Stemphylium spp. 275

Pseudopithomyces spp. (formerly Pithomyces spp.) 276

Epicoccum spp. 277

Nigrospora spp. 279

Chaetomium spp. 280

Phoma spp. 282

Dermatophytes 285

Introduction to Dermatophytes 287

Latin Terms for Dermatophyte Infections 288

Microsporum audouinii 289

Microsporum canis 290

Paraphyton cookei species complex (formerly Microsporum cookei species complex) 292

Nannizzia gypsea species complex (formerly Microsporum gypseum species complex) 293

Lophophyton gallinae (formerly Microsporum gallinae [zoophilic form] and Microsporum vanbreuseghemii [geophilic form]) 295

Nannizzia nana (formerly Microsporum nanum) 297

Microsporum ferrugineum 299

Trichophyton mentagrophytes species complex 300

Table 2.19 Differentiation of similar conidia-producing Trichophyton spp. and Arthroderma spp. 302

Trichophyton rubrum 303

Trichophyton tonsurans 305

Arthroderma terrestre species complex (formerly Trichophyton terrestre species complex) 307

Trichophyton megninii 308

Trichophyton soudanense 309

Table 2.20 Growth patterns of Trichophyton spp. and Arthroderma spp. on nutritional test media 310

Trichophyton schoenleinii 311

Trichophyton verrucosum 312

Trichophyton violaceum 313

Arthroderma uncinatum (formerly Trichophyton ajelloi) 314

Epidermophyton floccosum 315

Hyaline Hyphomycetes 317

Introduction to Hyaline Hyphomycetes 319

Fungi in Which Arthroconidia Predominate

Table 2.21 Differential characteristics of fungi in which arthroconidia predominate 320

Malbranchea spp. 321

Pseudogymnoascus pannorum (formerly Geomyces pannorum) 323

Arthrographis kalrae 324

Hormographiella aspergillata 326

Common Species of Aspergillus

The genus Aspergillus 327

Table 2.22 Differentiating characteristics of the most common Aspergillus spp. 329

Aspergillus fumigatus species complex 331

Aspergillus niger species complex 333

Aspergillus flavus species complex 334

Aspergillus versicolor species complex 336

Aspergillus ustus species complex 338

Aspergillus tanneri 340

Aspergillus nidulans species complex 342

Aspergillus glaucus 344

Aspergillus terreus species complex 345

Aspergillus clavatus 347

Other Common Hyaline Hyphomycetes

Penicillium spp. 348

Paecilomyces variotii 350

Rasamsonia argillacea species complex (formerly Geosmithia argillacea) 351

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) 352

Table 2.23 Differential characteristics of Paecilomyces variotii, Rasamsonia argillacea, and Purpureocillium lilacinum 354

Scopulariopsis spp. 355

Table 2.24 Differential characteristics of Scopulariopsis brevicaulis versus Scopulariopsis brumptii 357

Gliocladium spp. 358

Trichoderma spp. 359

Metarhizium anisopliae species complex 361

Beauveria bassiana 363

Verticillium spp. 364

Acremonium and Sarocladium spp. 365

Fusarium spp. 367

Coniochaeta spp. (formerly Lecythophora spp.) 369

Trichothecium roseum 370

Chrysosporium spp. 371

Table 2.25 Differential characteristics of Chrysosporium versus Sporotrichum 373

Sporotrichum pruinosum 374

Sepedonium spp. 376

Chrysonilia sitophila 377

Schizophyllum commune 378

Part III Basics of Molecular Methods for Fungal Identification 379

Introduction 381

Fungal Targets 383

Table 3.1 Frequently used fungal molecular targets and primers for sequence-based species identification 385

Table 3.2 Examples of fungal molecular targets and primers for multilocus sequence-based species identification 386

Classic Molecular Identification Methods 387

Polymerase Chain Reaction 387

Non-Sequencing-Based Identification Methods 389

MALDI-TOF Mass Spectrometry 389

Signal Amplification Methods 390

PNA FISH 390

Nucleic Acid Amplification Methods 390

T2 Magnetic Resonance 390

Broad-Panel Molecular Testing and Other Emerging Sample-to-Answer Technologies 391

Sequencing-Based Identification Methods 393

Sanger Sequencing 393

Table 3.3 Lane construction for traditional bidirectional Sanger sequencing 394

Massive Parallel or Next-Generation Sequencing 394

Applications of DNA Sequencing 397

Accurate Molecular Identification 397

Table 3.4 Commonly used databases for identification of medically important fungi 398

Phylogenetic Analysis 399

Organism Typing 401

Detection of Genetic Determinants of Resistance 401

Part IV Laboratory Technique 403

Laboratory Procedures 405

Collection and Preparation of Specimens 406

Table 4.1 Common clinical sites for laboratory recovery of pathogenic fungi 409

Methods for Direct Microscopic Examination of Specimens 414

Primary Isolation 416

Table 4.2 Media for primary isolation of fungi 417

Macroscopic Examination of Cultures 419

Microscopic Examination of Growth 419

Procedure for Identification of Yeasts 421

Isolation of Yeast When Mixed with Bacteria 424

Germ Tube Test for the Presumptive Identification of Candida albicans 425

Rapid Enzyme Tests for the Presumptive Identification of Candida albicans 426

Caffeic Acid Disk Test 426

Olive Oil Disks for Culturing Malassezia spp. 426

Conversion of Thermally Dimorphic Fungi in Culture 427

Sporulation Inducement Method for Apophysomyces and Saksenaea 428

In vitro Hair Perforation Test (for Differentiation of Trichophyton  mentagrophytes and Trichophyton rubrum) 428

Temperature Tolerance Testing 429

Maintenance of Stock Fungal Cultures 429

Controlling Mites 430

Staining Methods 431

Acid-Fast Modified Kinyoun Stain for Nocardia spp. 432

Acid-Fast Stain for Ascospores 433

Ascospore Stain 433

Calcofluor White Stain 434

Giemsa Stain 435

Gomori Methenamine Silver (GMS) Stain 435

Gram Stain (Hucker Modification) 438

Lactophenol Cotton Blue 439

Lactophenol Cotton Blue with Polyvinyl Alcohol (PVA) (Hubers PVA Mounting

Medium, Modified) 439

Rehydration of Paraffin-Embedded Tissue (Deparaffination) 440

Media 441

Ascospore Media 443

Assimilation Media (for Yeasts) 444

Birdseed Agar (Niger Seed Agar; Staib Agar) 448

Brain Heart Infusion (BHI) Agar 449

Buffered Charcoal-Yeast Extract (BCYE) Agar 450

Canavanine Glycine Bromothymol Blue (CGB) Agar 450

Casein Agar 451

CHROMagar Candida Medium 452

CHROMagar Candida Plus Medium 453

CHROMID Candida Agar 453

Chromogenic Candida Agar (Brilliance Candida Agar) 454

Cornmeal Agar 454

Dermatophyte Test Medium (DTM) 455

Esculin Agar 456

Fermentation Broth for Yeasts 456

Inhibitory Mould Agar (IMA) 457

Leeming-Notman Agar (Modified) 458

Lysozyme Medium 458

Mycosel Agar 459

Potato Dextrose Agar and Potato Flake Agar 460

Rapid Assimilation of Trehalose (RAT) Broth 460

Sabouraud Brain Heart Infusion Agar (SABHI Agar) 462

Sabouraud Dextrose Agar (SDA) 463

Sabouraud Dextrose Agar with 15% NaCl 464

Sabouraud Dextrose Broth 464

Starch Hydrolysis Agar 464

Trichophyton Agars 465

Tyrosine Agar 466

Urea Agar 467

Water Agar 467

Image Appendix 469

Glossary 477

References Cited 489

Index 507