IDENTIFICATION OF PATHOGENIC FUNGI

IDENTIFICATION OF PATHOGENIC FUNGIColin K. Campbell PhDHealth Protection Agency Mycology Reference LaboratoryBristol, UK (retired)

Elizabeth M. Johnson PhDHealth Protection Agency Mycology Reference LaboratoryBristol, UK

David W. Warnock PhD, FAAM, FRCPathNational Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and PreventionAtlanta, Georgia, USA

SECOND EDITION

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DisclaimerThis text was co-authored by David W. Warnock in his private capacity. No official support or endorsement by the U.S. Centers for Disease Control and Prevention or by the U.S. Department of Health and Human Services is intended or should be inferred.

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Library of Congress Cataloging-in-Publication DataCampbell, Colin K. Identification of pathogenic fungi / Colin Campbell, Elizabeth Johnson, David W. Warnock. – 2nd ed. p. ; cm. Rev. ed. of: Identification of pathogenic fungi / Colin K. Campbell ... [et al.]. London : Public Health Laboratory Service, c1996. Includes bibliographical references and index. ISBN 978-1-4443-3070-0 (hardback) I. Johnson, Elizabeth M., Ph. D. II. Warnock, D. W. III. Identification of pathogenic fungi. IV. Title. [DNLM: 1. Fungi–isolation & purification. 2. Fungi–pathogenicity. 3. Mycoses–diagnosis. QW 180]

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v

Preface, ixAcknowledgements, xi

  1  Introduction, 1

  2  Identification of Moulds, 11Media for Mould Identification, 14Mounting Fluids, 16

  3  Moulds with Arthrospores, 18Neoscytalidium dimidiatum, 20Coccidioides species, 24Onychocola canadensis, 28

  4  Moulds with Aleuriospores: I. The Dermatophytes, 31Microsporum gypseum, 38Microsporum canis, 40Microsporum equinum, 42Epidermophyton floccosum, 44Trichophyton terrestre, 46Trichophyton rubrum, 48Trichophyton interdigitale, 52Trichophyton mentagrophytes, 54Trichophyton erinacei, 56Trichophyton equinum, 58Trichophyton soudanense, 60Microsporum persicolor, 62Trichophyton tonsurans, 64Microsporum audouinii, 66Trichophyton violaceum, 68Trichophyton verrucosum, 70Trichophyton schoenleinii, 72Trichophyton concentricum, 74Other Microsporum and Trichophyton species, 76

  5  Moulds with Aleuriospores: II. Others, 80Geomyces pannorum, 82Chrysosporium keratinophilum, 84Myceliophthora thermophila, 86

CONTENTS

vi

CONTENTS

Histoplasma capsulatum, 88Blastomyces dermatitidis, 92Paracoccidiodes brasiliensis, 96

  6  Moulds with Holoblastic Conidia, 99Aureobasidium pullulans, 102Sporothrix schenckii, 104Cladophialophora bantiana, 106Cladosporium sphaerospermum, 108Fonsecaea pedrosoi, 110Rhinocladiella atrovirens, 112Rhinocladiella mackenziei, 114Ochroconis gallopava, 116Alternaria alternata, 118Ulocladium chartarum, 120Curvularia lunata, 122Bipolaris hawaiiensis, 124Exserohilum rostratum, 126

  7  Moulds with Enteroblastic Conidia Adhering in Chains, 129Aspergillus flavus species complex, 134Aspergillus fumigatus species complex, 136Aspergillus glaucus, 138Aspergillus nidulans species complex, 140Aspergillus versicolor species complex, 142Aspergillus ustus species complex, 144Aspergillus niger species complex, 146Aspergillus terreus species complex, 148Aspergillus candidus species complex, 150Penicillium marneffei, 152Scopulariopsis brevicaulis, 154Purpureocillium lilacinum, 156Paecilomyces variotii, 158

  8  Moulds with Enteroblastic Conidia Adhering in Wet Masses, 161Fusarium lichenicola, 166Fusarium dimerum species complex, 168Fusarium semitectum, 170Fusarium proliferatum, 172

vii

CONTENTS

Fusarium oxysporum species complex, 174Fusarium solani species complex, 176Acremonium strictum, 178Acremonium kiliense, 180Lecythophora mutabilis, 182Scedosporium prolificans, 184Scedosporium apiospermum, 186Phaeoacremonium parasiticum, 188Pleurostomophora richardsiae, 190Phialophora verrucosa, 192Hortaea werneckii, 194Exophiala spinifera, 196Exophiala dermatitidis, 198Exophiala jeanselmei, 200

  9  Mucoraceous Moulds and Their Relatives, 203Cunninghamella bertholletiae, 208Lichtheimia corymbifera, 210Rhizomucor pusillus, 212Mucor circinelloides, 214Rhizopus microsporus, 216Rhizopus arrhizus, 218Mucor hiemalis, 220Basidiobolus ranarum, 222Conidiobolus coronatus, 224Pythium insidiosum, 226Apophysomyces elegans, 228Saksenaea vasiformis, 230Mortierella wolfii, 232

10  Miscellaneous Moulds, 235Aphanoascus fulvescens, 238Monascus ruber, 240Chaetomium species, 242Phoma herbarum, 244Myxotrichum deflexum, 246Schizophyllum commune, 248Leptosphaeria senegalensis, 250Neotestudina rosatii, 252

viii

CONTENTS

Piedraia hortae, 254Lasiodiplodia theobromae, 256Pyrenochaeta romeroi, 258Madurella mycetomatis, 260

11  Identification of Yeasts, 263Media for Yeast Identification, 272Candida albicans, 274Candida tropicalis, 276Candida krusei, 278Candida lipolytica, 280Candida kefyr, 281Candida lusitaniae, 282Candida parapsilosis, 284Candida pelliculosa, 286Candida guilliermondii, 287Candida glabrata, 288Cryptococcus neoformans and Cryptococcus gattii, 290Rhodotorula glutinis, 292Saccharomyces cerevisiae, 294Geotrichum candidum, 296Saprochaete capitata, 298Trichosporon species, 300Malassezia furfur species complex, 302Malassezia pachydermatis, 304

12  Identification of Fungi in Sections, Smears and Body Fluids, 305

Appendix 1:  Common Mycological Terms, 321Appendix 2:  Further Reading, 325

Species Index, 327Subject Index, 333

ix

In the seventeen years that have elapsed since the first edition of this manual was pub-lished, much progress has been made in the diagnosis, treatment and prevention of fungal diseases. Nonetheless, these infections continue to be a leading cause of serious illness and death in many different patient populations. New methods of diagnosis have been intro-duced and new antifungal agents have been licensed for use, but these developments have been offset by the emergence of resistance to several classes of drugs and an increase in infections caused by fungi with innate resistance to one or more classes.

Many of the moulds that are now recognized as being capable of producing serious disease in immunocompromised or debilitated individuals are environmental organisms whose natural habitat is in the soil or on plants, wood, compost heaps, or rotting food. Most are familiar to mycologists, plant pathologists and food microbiologists, but they present problems for the clinical microbiologist who often has had no formal training in the identification of fungi. This process can be challenging and sometimes frustrating because of the importance placed on the morphological characteristics of the organisms, and the need to become familiar with a wide range of different structures and terms.

As before, this manual has been designed for use by medical, scientific and technical staff in hospital laboratories in the UK and abroad, but we hope it will also be of interest to other groups of scientists. The organisms described have been grouped in chapters accord-ing to spore-bearing structures produced in culture, rather than simply being ordered on an alphabetical basis. Each chapter has been arranged so that the descriptions for similar organisms may be found on adjacent pages. In addition, we have attempted to provide differential diagnoses on the basis of both colonial appearance and microscopic character-istics for the organisms described. Although molecular methods are assuming an ever greater importance, routine identification of moulds still rests, for the most part, on mor-phological examination. To assist with this, we have added colour illustrations of cultures and microscopic structures to the line drawings that are found throughout the manual. Lack of space has precluded the inclusion of every rare organism that might be isolated from a clinical specimen. In some cases a single representative member of a genus is described, and isolates that appear similar to the description provided may need to be referred to a specialist for confirmation of the identification.

For this edition, we have added a new chapter on the identification of fungi in his-topathogical sections and smears. As before, we have included two appendices: the first giving definitions of many mycological terms in common use and the second listing some useful monographs and more comprehensive texts that the reader may wish to consult.

Colin K. CampbellElizabeth M. Johnson

David W. Warnock

PREFACE

xi

We are very grateful to our colleagues, Dr Andrew Borman, Mrs Adrien Szekely and Dr Christopher Linton from the Mycology Reference Laboratory, Bristol, for their input during the development of this new edition, and for their many helpful suggestions for its improve-ment. We are also grateful to our late colleague Dr Christine Philpot for her involvement in the first edition. We also wish to thank Kate Newell of Wiley-Blackwell and project manager Kathy Syplywczak for their invaluable help in the design and production of this monograph.

Colin K. CampbellElizabeth M. Johnson

David W. Warnock

ACKNOWLEDGEMENTS

1

The kingdom Fungi consists of a distinct group of eukaryotic organisms that absorb their nourishment from living or dead organisms or organic matter. Fungi are found throughout nature, performing an essential service in returning to the soil nutrients removed by plants. There is, however, a large group of species that are parasitic on plants and a smaller group that are parasitic on animals, as well as on man. Fungi show considerable variation in size and form, but can be divided into three main groups: multicellular filamentous fungi (moulds); unicellular fungi (yeasts); and dimorphic fungi which are capable of changing their growth to either a multicellular or unicellular form, depending on the growth conditions.

In most multicellular fungi, the vegetative stage consists of a system of tubular, branch-ing filaments, or mycelium. Each individual filament, or hypha, has a rigid cell wall and increases in length as a result of apical growth. In the more primitive fungi, the hyphae remain aseptate (without cross-walls). In the more advanced groups however, the hyphae are divided into compartments or cells by the development of more or less frequent cross-walls, termed septa. Such hyphae are termed septate.

Yeasts are unicellular fungi consisting of separate, round, oval or elongated cells or blastospores that propagate by an asexual process called budding in which the cell develops a protuberance from its surface. The bud enlarges and may become detached from the parent cell, or it may remain attached and itself produce another bud. In this way a chain of cells may be produced. Under certain conditions, continued elongation of the parent cell before it buds results in a chain of elongated cells, termed a pseudohypha, which resembles the hypha of moulds. Unlike a true hypha, however, the connection between adjacent pseudohyphal cells shows a marked constriction. Some yeasts can also produce true hyphae, with cross-walls. A small number of yeasts reproduce by fission. Yeasts are neither a natural nor a formal taxonomic group, but are a growth form shown by a wide range of unrelated fungi.

Some medically important fungi change their growth form during the process of tissue invasion. These dimorphic pathogens usually change from a multicellular hyphal form in the natural environment to a budding, single-celled yeast form in tissue.

Fungi reproduce by means of microscopic propagules, termed spores, that consist of a single cell or several cells contained within a rigid wall. Spores may be produced by an asexual process (involving mitosis only) or by sexual reproduction (involving meiosis). Some species of fungi are homothallic and able to form sexual structures within individual colonies. Most, however are heterothallic and do not form their sexual structures unless

1  INTRODUCTION

Identification of Pathogenic Fungi, Second Edition. Colin K. Campbell, Elizabeth M. Johnson, and David W. Warnock.© 2013 Health Protection Agency. Published 2013 by Blackwell Publishing Ltd.

INTRODUCTION

2

two different mating strains come into contact. Thus, sexual reproduction is often difficult to obtain in culture. The sexual spores and the structures in which they are produced form the traditional basis for fungal classification. Most recently the kingdom Fungi has been divided into a number of lesser groups, termed phyla, based on differences in their sexual structures. Two of these phyla (the Ascomycota and the Basidiomycota) and two sub-phyla (the Mucoromycotina and Entomophthoromycotina) contain species that are pathogenic to humans and animals.

Sexual Reproduction

Of the kingdom Fungi, the majority of species belong to the sub-kingdom Dikarya (literally ‘two nuclei’ as their sexual reproduction involves a cell containing two fusing nuclei). This group is made up of two phyla (the Basidiomycota and the Ascomycota). Outside the Dikarya there are many other smaller groups, with sexual reproduction often involving the fusion of multiple nuclear pairs in a single cell. Examples of the latter are seen in the sub-phyla Mucoromycotina and Entomophthoromycotina. These two sub-phyla have replaced the phy-lum Zygomycota, a grouping now abandoned as misrepresenting phylogenetic relation-ships. Both show fusion of the multinucleate tips of two hyphae leading to the formation of a single, large zygospore, lying between them. This is a multinucleate thick-walled struc-ture that has evolved to endure adverse environmental conditions. Meiosis occurs on germination and the vegetative haploid mycelium develops.

In contrast, in the Ascomycota and Basidiomycota, sexual reproduction has evolved into a means of rapid dispersal to new habitats, unlike the resting nature of the zygospore. In both these groups the diploid stage is transient, with meiosis resulting in the production of enormous numbers of short-lived haploid spores. In the Ascomycota, the sexual spores or ascospores are produced in sacs, or asci. Each ascus usually contains eight ascospores. The group shows a gradual transition from primitive forms that produce single asci to species that produce large structures, or ascocarps, containing large numbers of asci. Three main

zygospore ascocarp basidiocarp

INTRODUCTION

3

forms of ascocarp are common: the perithecium which releases its spores through an apical opening; the cleistothecium, which splits open to liberate its contents; and the gymnothecium, which is an open loose network of protective hyphae.

In most of the Basidiomycota the sexual spores or basidiospores are borne on projections at the tip of club-shaped basidia. These are produced in macroscopic structures or basidiocarps.

Whilst the reproductive structures associated with sexual life cycles are important to a full understanding of the fungi, most of the organisms described in this manual may be identified on the basis of their asexual reproductive structures and spores.

Asexual Reproduction

Fungi may also produce asexual spores by simple haploid nuclear division. Again, short lived propagules are produced in enormous numbers to ensure spread to new habitats. In many fungi this asexual (anamorph or imperfect) stage has proved so successful that the sexual (teleomorph or perfect) stage has diminished or even disappeared. These species have long been known as the Fungi Imperfecti or Deuteromycetes. This by convention con-tained all the asexual relatives of the Ascomycota and Basidiomycota, but not those of the former Zygomycota. With advances in molecular phylogenetic analysis, the concept of Fungi Imperfecti is becoming increasingly redundant as a useful taxonomic grouping, since most asexually-reproducing species can now be placed with their sexually reproducing relatives.

Conidia

In the Ascomycota and Basidiomycota the asexual spores are termed conidia, and are produced from a conidiogenous cell. In some species the conidiogenous cell is not different from the rest of the mycelium. In others the conidiogenous cell is contained in a specialised hyphal structure or conidiophore. There are two basic methods of asexual spore production: thallic in which an existing hyphal cell is converted into a conidium; and blastic, in which the conidium is produced as a result of some form of budding process.

Thallic Conidiogenesis

In thallic conidiogenesis the conidium is produced from an existing hyphal cell. This occurs when a hypha breaks up into sections to form individual cells, or arthrospores, or when one cell develops a thick wall to form a resting spore or chlamydospore.

Arthrospores are derived from the fragmentation of an existing hypha and represent the simplest form of asexual sporulation. In most species the septum separating two cells splits

INTRODUCTION

4

down the middle, leaving a trace of the resulting torn wall on the end of the spore. In a few instances the arthospores are intercalated with separating cells and are liberated after these cells have dissolved. This leaves a marked annular frill at the ends of the detached arthrospores. Moulds which produce arthrospores as their principal reproductive spores are described in detail in Chapter 3.

Aleuriospores represent an intermediate state between thallic and blastic conidiogenesis. These spores are formed from the side or tip of a hypha and during the initial stage before a septum is laid down, can resemble short, hyphal branches. As in all genuine cases of thallic conidiogenesis, it is not possible for a second spore to be formed at the same point. This form of conidium is characteristic of the dermatophytes (described in Chapter 4) but is also found in a number of other fungi of medical importance (described in Chapter 5).

Blastic Conidiogenesis

Many fungi evolved some form of repeated budding that permits them to produce large numbers of asexual spores from a single conidiogenous cell. Two forms of blastic conidio-genesis are now recognised: holoblastic development in which both the inner and the outer wall of the conidiogenous cell swell out to form the conidium, and enteroblastic development, in which the conidium is produced from within the conidiogenous cell, the outer layer of the hyphal wall being ruptured and an inner layer extending through to become the new spore wall. These two forms of blastic conidiogenesis can be further subdivided according to the details of spore development.

Holoblastic Conidiogenesis

In some fungi, the conidiogenous cells each produce a single holoblastic conidium. In others, however, the first-formed conidium produces a second conidium and the second

arthrospores chlamydospores aleuriospores

INTRODUCTION

5

produces a third, and so on, until a chain of spores is produced with the youngest at its tip. As each conidium can produce more than one bud, a branching chain becomes possible. Examples of moulds that produce holoblastic branching chains of spores include species of Cladosporium. In other species, the conidiogenous cell that produced the first-formed spore then grows past it to produce a second (sympodial spore production). If this process is repeated, it will result in an elongated conidiogenous cell, known as a geniculate conidi-ophore, with numerous lateral single spores along its sides. This happens, for example, in species of Alternaria and Bipolaris. Moulds which produce holoblastic conidia are described in detail in Chapter 6.

Enteroblastic Conidiogenesis

In fungi that produce enteroblastic spores, the wall of the conidia is derived from the inner layer of the wall of the conidiogenous cell and the conidia are produced from an opening in the outer wall of the conidiogenous cell. This permits a succession of spores to be pro-duced at the same point. The specialised conidiogenous cell from which the conidia are produced is termed a phialide. In some fungi, such as species of Aspergillus and Penicillium, continuous replenishment of the inner wall of the tip of the phialide results in the forma-tion of an unbranched chain of connected spores, with the youngest at the base. Moulds which produce enteroblastic conidia in chains are described in detail in Chaper 7.

In other fungi, such as species of Fusarium and Acremonium, a new inner layer of wall material is produced for each successive spore. Repeated conidiogenesis results in an accu-mulation of the unused remains of these layers within the tip of the phialide. The spores are not firmly attached to each other and often move aside to accumulate in a wet mass around the phialide. Unlike the spores of species of Aspergillus and Penicillium, these spores do not spread on air currents, but are coated with a wettable slime which appears to be an

Holoblastic conidia

INTRODUCTION

6

adaptation to water dispersal. Moulds which produce enteroblastic conidia in wet masses are described in detail in Chapter 8.

Annellides, like phialides, are cells which produce conidia at their tips in unbranched chains (as in the genus Scopulariopsis) or in wet masses (as in the genus Scedosporium). Unlike phialides, annellides increase in length each time a new spore is produced. An old annellide that has produced many spores will have a number of apical scars or annellations at its tip. These scars, which are left as successive spores break off, are often difficult to see under the optical microscope.

phialides

annellide

Enteroblastic conidia in dry chains

annellides

phialides

Enteroblastic conidia in wet masses

INTRODUCTION

7

Sporangiospores

In the former Zygomycota, one major group, the order Mucorales often produces the asexual spores, or sporangiospores, inside a closed sac, or sporangium the wall of which ruptures to liberate them. The sporangium is held above the substratum on an unbranched or branched sporangiophore. The different species of fungi in this group are distinguished from one another by the sporangiophores, sporangia and sporangiospores, as well as by the presence or absence of rhizoids that anchor the sporangiophores to the substratum. In addition these organisms have large, aseptate or almost aseptate hyphae. These are described in detail in Chapter 9.

sporangia

rhizoids

Other Forms of Sexual and Asexual Spore Production

Most of the moulds described in this manual are identified on the basis of their asexual reproductive structures and spores. However, there are a number of pathogenic moulds that produce sexual spores in ascocarps or basidiocarps in culture, rather than asexual spores, and these are described in detail in Chapter 10. This chapter also includes descrip-tions of several moulds that produce macroscopic fruiting bodies (pycnidia) containing conidia (pycnidiospores). In addition, several non-sporing pathogenic moulds have been included.

Yeasts

Yeasts are neither a natural nor a formal taxonomic group, but are a growth form found in a wide range of unrelated ascomycetous and basidiomycetous fungi. Their identification, unlike that of moulds, relies on a combination of morphological, physiological and bio-chemical characteristics. Chapter 11 deals with the organisms that are most frequently

INTRODUCTION

8

encountered in clinical laboratories and describes the tests that are most commonly employed for their identification.

The ‘black yeasts’ are not a formal taxonomic group, but the term is applied to a wide range of unrelated ascomycetous and basidiomycetous fungi that have darkly pigmented cell walls and are able to produce budding cells at some stage in their life cycle. Many of these fungi form both holoblastic and enteroblastic conidia, sometimes even in the same mycelium.

Nomenclature of Fungi

The names of fungi are subject to the International Code of Botanical Nomenclature which must be followed when proposing the name for a new fungal species. Many common and widely distributed species of fungi have been described as new many times and thus have come to have more than one name. In general, the correct name for any species is the earli-est name published in line with the requirements of the Code. To avoid confusion, however, the Code permits certain exceptions. The most significant of these is when an earlier generic name has been overlooked, a later name is in common use, and a reversion to the earlier name would cause problems.

Another reason for changing the name of a fungus is when new research necessitates the transfer of a species from one genus to another, or establishes it as the type of a new genus. Such changes are quite in order, but with the provision that the specific epithet should remain unchanged, except for inflection according to the rules of Latin grammar. However often a species is transferred to a new genus, the correct species epithet is always the first one that was applied to that particular organism.

If there is one complication of fungal nomenclature that is confusing, it is the fact that a large number of fungi bear more than one name. This is an apparent departure from the basic principle of biological classification, in which an organism can only have one correct name. In many cases where fungi bear two names, one designates their sexual stage (or teleomorph) and the other their asexual stage (or anamorph). Often this situation has arisen because the anamorphic and teleomorphic stages were described and named at dif-ferent times without the connection between them being recognized. Both names are valid under the International Code of Botanical Nomenclature, but that of the teleomorph should take precedence over that of the anamorph. In practice, however, it is more common to refer to a fungus by its asexual name because this is the stage which is usually obtained in culture.

The nomenclature of fungi that have both asexual and sexual stages is challenging. Some teleomorphic fungi can produce more than one asexual form of propagation, the term synanamorph being used to describe each of these different anamorphs. These may bear separate names. However, in each organism only a single teleomorph can be produced.

INTRODUCTION

9

Identification of Fungi

Most of the chapters in this manual include one or more dichotomous keys to the species described. Below we present an overall key to direct the reader to the particular groups covered in individual chapters. As with all the keys, the information required for identifica-tion is arranged as pairs (or triplets) of contrasted characteristics, the pairs being consecu-tively numbered on the left. Each member of the pair leads, on the right side of the page, either to the name of a group of fungi or to another higher number, i.e. to a further pair of contrasted characteristics. To use the key, start at point number 1 and follow through in the sequence indicated. Except for Chapter 11 (the yeasts), the arrangement of the descriptions follows the order given by the keys, so placing similar species close together for easy comparison.

INTRODUCTION

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Key to the main groups of fungi described

1a Mucoid colonies consist of budding cells with few or no hyphae

2

1b Colonies consist of hyphae 3

2a Colonies black Chapter 6 (Aureobasidium pullulans) and Chapter 8 (in part)

2b Colonies white, cream, pink or red Chapter 11 (yeasts)

3a Hyphae mostly aseptate Chapter 9 (mucoraceous moulds)3b Hyphae septate 4

4a Hyphae breaking into arthrospores; other types of spore absent

Chapter 3 (moulds with arthrospores)

4b Arthrospores absent or other types of spore present in addition

5

5a Spores formed in chains 65b Spores not formed in chains 7

6a Spores chains branching Chapter 6 (moulds with holoblastic conidia)

6b Spores chains unbranched Chapter 7 (moulds with enteroblastic conidia in chains)

7a Spores formed singly on sides of hyphae, or on short branches

Chapters 4 and 5 (moulds with aleuriospores), Chapter 6 (in part)

7b Spores formed repeatedly at the same point, aggregating in wet masses

Chapter 8 (moulds with annellides or phialides)

7c Fruiting bodies visible Chapter 10 (miscellaneous moulds)

11

The identification of filamentous fungi is based on the examination of their macroscopic (colonial) and microscopic characteristics. In the seventeen years since the first edition of this manual, there has been an explosion of new species names, many based more heavily on nucleic acid sequencing methodology than on the traditional morphological approach. It is thus important to recognise that definitive identification of, for example, atypical, unusual or non-sporing moulds, will often require molecular analysis to fully support morphological identification.

One of the consequences of molecular analysis of moulds has been the recognition that many medically important species are in fact complexes that are composed of a number of genetically distinct, but morphologically identical species. Nonetheless, as an introduction to the major groups of pathogens for clinical diagnosis, microscopic morphology remains the standard approach. Wherever possible we have indicated where a species complex has been deliberately simplified for primary identification.

Macroscopic features such as colonial form, surface colour and production of pigments are often helpful in identification. The growth rate of mould colonies depends on the culture medium and temperature of incubation but provided conditions are standardised, these characteristics can be taken into consideration in the process of identification. Morphological examination of microscopic structures such as spores and spore-bearing cells is an essential part of mould identification. Moulds that fail to sporulate are often impossible to speciate and it is therefore important to select culture conditions which favour sporulation.

Many clinical laboratories today employ DNA sequencing as part of their routine proto-col for fungal identification. In circumstances where morphology-based identification is not helpful, an isolate may be a candidate for DNA-based identification. This approach may be useful when an isolate displays atypical morphology, fails to sporulate, requires lengthy incubation or incubation on specialized media in order to sporulate, or if the phe-notypic results are nonspecific or confusing.

Media

The texture and colour of mould colonies often depend on the age of the culture and agar medium on which the organism is grown. Nevertheless, these characteristics are useful in identification. Owing to the almost universal use of Sabouraud’s glucose peptone agar, the descriptions in this manual are based on cultures prepared on it. However, there are

2  IDENTIFICATION  OF  MOULDS

Identification of Pathogenic Fungi, Second Edition. Colin K. Campbell, Elizabeth M. Johnson, and David W. Warnock.© 2013 Health Protection Agency. Published 2013 by Blackwell Publishing Ltd.

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numerous formulations of that medium, both with and without antibiotics, and it is advis-able to confine supplies to one manufacturer as the morphological appearance of moulds, and pigmentation in particular can differ from one formulation to another. Moulds often grow best on rich media, such as glucose peptone agar, but over-production of mycelium often results in loss of sporulation. If a mould isolate fails to produce spores or other rec-ognisable structures after two weeks, it should be subcultured to a less-rich medium to encourage sporulation and permit identification. The composition of a number of useful media is given at the end of this chapter.

Methods of Slide Preparation

Microscopic examination of slide preparations is the most important part of the identifica-tion of a mould culture. If well prepared, these will often give sufficient information on the form and arrangement of spores and other structures for an identification of the fungus to be made. The usual method is to remove some of the surface growth from a culture plate with a sharp rigid needle and place it in a drop of mounting fluid (such as lacto-fuchsin or lactophenol cotton blue) on a clean microscope slide. The material is then teased apart with two sharp needles and a cover slip applied. Gentle pressure is used to spread out the preparation before it is examined under a microscope using ×10 and ×40 objective lenses.

There are several other methods for the preparation of slides for microscopic examination of fungi. One of the most helpful is to use clear adhesive tape. A small flag of tape (about 20mm long) is cut with scissors and placed on the end of a rigid needle. The tape is pressed, adhesive side downwards, on to the surface of the culture using a second needle applied to the back of the tape. The coated tape is then placed, adhesive side upwards, in a small drop of mounting fluid on a microscope slide. A second small drop of mounting fluid is placed on the preparation and a cover slip is applied.

If a slide preparation shows no spores, it is often helpful to try nearer the centre of the colonies, where the mould is older and has had more time to sporulate. If there are too many spores and the sporing structures cannot be discerned, it is useful to try nearer the edge of the colonies. If no spores are found in slide preparations, it is sometimes worthwhile to remove the lid from the culture plate and examine the colonies for evidence of sporula-tion under the low-power objective of a microscope.

Both the ‘needle’ and the ‘tape’ methods give suitable preparations for microscopic examination but each has its drawbacks with certain forms of fungal growth. Needle preparations dislodge chains and wet masses of spores, and these features are best seen with tape preparations. On the other hand, structures such as pycnidia and spores hidden deep in the mycelium are not picked up on tape and require dissection with a needle. The

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needle also allows sub-agar growth to be studied. Mounting fluids such as lactophenol and lactofuchsin attack some types of adhesive tape and render it unsuitable for preparation of permanent mounts. Needle preparations can be sealed around the edge with DPX for long-term storage.

Slide Culture

The slide culture technique is useful for observing the intact arrangement of spores or spore-bearing structures. A thin, square block of a suitable nutrient agar (smaller than a cover slip) is placed on a sterile microscope slide supported on a bent glass rod in a petri dish. The four sides of the agar block are then inoculated with portions of mycelium of the fungus to be identified. The block is then covered with a sterile cover slip, sterile distilled water added to the base of the petri dish, the lid replaced and the plate incubated at 30oC. Once adequate sporulation has occurred, the cover slip is removed from the agar and placed on a drop of mounting fluid on a clean glass slide with the adherent mycelium downwards. The agar block is then removed and discarded, leaving adherent mycelium on the slide. Mounting fluid is added and a clean cover slip applied. The preparations can be sealed for long term preservation.

Conversion of Dimorphic Pathogens from Mould to Yeast Phase

Commercial kits are available for the rapid identification of the dimorphic fungi Histoplas­ma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Sporothrix schenckii. These include the AccuProbe test (Gen-Probe Inc., San Diego, California, USA) and the exoantigen test (ImmunoMycologics Inc., Norman, Oklahoma, USA). In addition, the iden-tification may be further supported by inducing conversion from the mycelial to the yeast phase. Conversion of Coccidioides spp. to the pathogenic (spherule) phase is problematic and less commonly attempted. Note that all these fungi, with the exception of S. schenckii are hazardous pathogens and should only be handled under safe laboratory conditions (UK Hazard Group 3 or equivalent).

Conversion to yeast phase usually requires special media and a temperature above 35°C. An extensive body of literature has been devoted to conversion conditions as they apply to the various species, and specialist laboratories will have their favourite procedures. Bacteriological blood agar, heat-treated blood (‘chocolate’) agar, brain-heart infusion agar or even Sabouraud’s glucose peptone agar may achieve conversion. Whatever medium is used, conversion often leads to a colony of yeast cells mixed with hyphae, and serial sub-cultures may be needed to fully convert to the yeast phase. It is also helpful to keep the agar surface from becoming too dry.

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MEDIA FOR MOULD IDENTIFICATION

Cornmeal Agar

This medium is useful for stimulating ascocarp and pycnidium production in some moulds.

cornmeal extract 2 g

agar 15 g

distilled water 1 L

Heat to dissolve. Autoclave at 121°C for 15 min.

Czapek-Dox Agar

This defined medium is recommended for the identification of Aspergillus and Penicillium spp. It is also useful for stimulating sporangium production in mucoraceous moulds.

sucrose 30 g

sodium nitrate 2 g

potassium chloride 0.5 g

magnesium glycerophosphate 0.5 g

potassium sulphate 0.35 g

ferrous sulphate 0.01 g

agar 12 g

distilled water 1 L

Heat to dissolve. Autoclave at 121°C for 15 min.

Dermatophyte Test Agar

This medium turns red in colour with dermatophytes and is useful for distinguishing those species from other moulds. It is important to remember that some non-dermatophyte moulds can also produce that colour change.

glucose 40 g

mycological peptone 10 g

phenol red 0.2 g

agar 12 g

distilled water 1 L

Heat to dissolve. Autoclave at 121°C for 15 min.

Malt Extract Agar

This rich medium is recommended as an alternative to Sabouraud’s glucose peptone agar for stimulating sporulation in a wide range of moulds, including the dermatophytes.

malt extract 30 g

mycological peptone 5 g

agar 15 g

distilled water 1 L

Heat to dissolve. Autoclave at 115°C for 10 min.

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Philpot’s Urea Agar

This medium turns red in colour if the fungus produces urease enzyme. It is used to distinguish Trichophyton rubrum (usually urease negative) from T. interdigitale (urease positive). It is important to remember that the granular form of T. rubrum gives a positive result, as will most dermatophytes.

glucose 5 g

mycological peptone 1 g

sodium chloride 5 g

potassium-dihydrogen orthophosphate

2 g

phenol red 0.012 g

agar 15 g

distilled water 1 L

Heat to dissolve. Autoclave at 115°C for 20 min. Cool to 50°C and add 50 ml of sterile 40% urea solution.

Potato Dextrose Agar

This is a good general purpose medium which stimulates sporulation in many moulds. It stimulates pigment production in some dermatophytes.

glucose 20 g

potato extract 4 g

agar 15 g

distilled water 1 L

Heat to dissolve. Autoclave at 121°C for 15 min.

Sabouraud’s Glucose Peptone Agar

This medium is recommended for the isolation and cultivation of dermatophytes and other moulds requiring a rich substrate with a high content of organic nitrogen. Antibacterial antibiotics (in particular chloramphenicol) can be added to control bacterial contamination.

glucose 40 g

mycological peptone 10 g

agar 15 g

distilled water 1 L

Heat to dissolve. Autoclave at 121°C for 15 min.

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Lactophenol

phenol crystals 20 g

lactic acid 20 mL

glycerol 40 mL

distilled water 20 mL

Heat gently to dissolve. Store away from direct sunlight.

MOUNTING FLUIDS

Lactophenol cotton blue

cotton blue 0.075 g

lactophenol 100 mL

Store away from direct sunlight.

Lactofuchsin

acid fuchsin 0.1 g

lactic acid 100 mL

Store away from direct sunlight.