TABLE 1 Stains for direct microscopic observation of fungi and/or filamentous bacteria in tissue

TABLE 2 Differentiation of filamentous aerobic actinomycetes encountered in clinical specimens

TABLE 3 Phenotypic characteristics of most common clinically encountered Nocardia spp.

TABLE 4 Characteristics of the genera of clinically encountered yeasts and yeastlike organisms

TABLE 5 Characteristics of Candida spp. most commonly encountered in the clinical laboratory

TABLE 6 Characteristics that assist in differentiating Candida dubliniensis from Candida albicans

TABLE 7 Differentiating characteristics of Blastoschizomyces capitatus versus Candida krusei

TABLE 8 Differentiating characteristics of Candida krusei, Candida inconspicua, and Candida norvegensis

TABLE 9 Differentiating characteristics of Candida guilliermondii versus Candida famata

TABLE 10 Characteristics of Cryptococcus spp.

TABLE 11 Characteristics of yeasts and yeastlike organisms other than Candida spp. and Cryptococcus spp.

TABLE 12 Key characteristics of the most common clinically encountered Trichosporon spp.

TABLE 13 Characteristics for differentiating species of the Sporothrix schenckii complex

TABLE 14 Differential characteristics of similar organisms in the class Mucormycetes

TABLE 15 Differential characteristics of the clinically encountered Rhizopus spp.

TABLE 16 Characteristics of Phialophora, Pleurostomophora, Phaeoacremonium, Acremonium, Phialemonium, and Lecythophora

TABLE 17 Characteristics of Cladosporium spp. and Cladophialophora spp.

TABLE 18 Differentiating phenotypic characteristics of the clinically encountered members of the Scedosporium spp. complex and Lomentospora prolificans

TABLE 19 Differentiation of the clinically encountered Verruconis species

TABLE 20 Characteristics of some of the “black yeasts”

TABLE 21 Differential characteristics of Aureobasidium pullulans versus Hormonema dematioides

TABLE 22 Characteristics of Bipolaris, Drechslera, and Exserohilum spp.

TABLE 23 Differentiation of similar conidia-producing Trichophyton spp.

TABLE 24 Growth patterns of Trichophyton species on nutritional test media

TABLE 25 Differential characteristics of fungi in which arthroconidia predominate

TABLE 26 Differentiating characteristics of the most common species of Aspergillus

TABLE 27 Differential characteristics of Paecilomyces variotii versus Purpureocillium lilacinum

TABLE 28 Differential characteristics of Scopulariopsis brevicaulis versus Scopulariopsis brumptii

TABLE 29 Differential characteristics of Chrysosporium versus Sporotrichum

TABLE 30 Media for primary isolation of fungi

TABLE 31 Inhibitory mould agar versus Sabouraud dextrose agar as a primary medium for isolation of fungi

With the writing of this new edition, we are grateful for the willingness of so many in our field to help in numerous ways. Our everlasting gratitude is also extended to the many colleagues who assisted in the preparation of previous editions; most of their contributions are now substantive and integral parts of the ongoing Guide.

Dr. Sanchita Das from North Shore University Hospital, Evanston, IL, generously contributed her time and expertise to update the section entitled “Basics of Molecular Methods for Fungal Identification” (Part III) which she had originally written for the 5th edition.

Our colleagues in Dr. Francis Barany’s molecular microbiology research laboratory at Weill Cornell Medicine have been extremely helpful and supportive during the writing of this edition as well as the previous edition.

Dr. Stephen Jenkins, Director of the Clinical Microbiology Laboratory of the NewYork–Presbyterian Hospital/Weill Cornell Medicine (NYPH/WCM) has always readily offered valuable advice and been very supportive. Dr. Lars Westblade, Associate Director of the Clinical Microbiology Laboratory, contributed numerous helpful suggestions for additions to this edition. As noted in previous editions, almost all the organisms shown microscopically and/or as cultured colonies were prepared in the Mycology Laboratory of NYPH/ WCM. We will forever be indebted to the staff of the mycology lab for their enormously significant contributions over the years.

Pat Kuharic of the Photography Department of Weill Cornell Medicine has given us the benefit of her outstanding expertise in preparing the excellent color photographs of fungal colonies as well as the black and white photomicrographs. With her talent and professional passion to get it “just right,” she is a great asset to the book.

Aleina Zehra, administrative assistant to Dr. Walsh, meticulously reviewed and updated the many Internet websites and suppliers’ locations mentioned throughout the book, as well as assisting in the preparation of working manuscript drafts of various sections.

We thank the staff of the St. Jude Children’s Research Hospital, Department of Environmental Health and Safety, for their assistance in reviewing the section related to packaging and shipping of hazardous materials.

Dr. Sybren de Hoog kindly provided a 2-year subscription to the Atlas of Clinical Fungi. Drs. David Warnock and Michael Pfaller discussed perspectives with us on fungal taxonomy and nomenclature.

Last, but definitely not least, we extend our deep appreciation to the members of ASM Press. Christine Charlip, Director, has been highly supportive of this 6th edition as an important contribution to ASM Press’s library of clinical microbiological resources. Kudos also goes to Larry Klein, Production Manager; Ellie Tupper, Senior Production Editor; and the marvelous associates who contributed to the production of this edition: Mark Via, copyeditor; Susan Schmidler, interior design; and Debra Naylor, cover design. They have been extremely helpful, creative, flexible, and patient and are much appreciated.

• How To Use the Guide
• Use of Reference Laboratories and Regulations for Transport
• Safety Precautions
• Taxonomy and Nomenclature

Before beginning to use the guide, the reader should understand several points.

Fungi often appear different in living hosts than they do in cultures. Part I (pp. 17–72) is designed as a guide for preliminary identification of fungi seen on direct microscopic examination of clinical specimens.

In Part II (pp. 73–331), the descriptions of the macroscopic and microscopic morphologies of the cultured fungi pertain to those on Sabouraud dextrose agar (SDA) unless otherwise specified. SDA may not be as regularly used for primary isolation of fungi directly from specimens as it was in the past, due to evidence that it is not as supportive as once believed (Scognamiglio et al., 2010). Fortunately, the descriptions can also be applied to growth on alternative media.

Many moulds begin as white mycelial growths, and coloration occurs at the time of conidiation or sporulation. Hence, organisms are listed under their most likely color(s) at maturity, when the typical microscopic reproductive formations are more readily observed.

In Parts I and II, when feasible, organisms are arranged in an order based on morphologic similarities (rather than alphabetical order) to facilitate convenient comparison.

This book is a guide to identification. Standard texts and our suggested references should be used for additional information concerning clinical disease, history, ecology, immunology, and therapy.

As molecular assays are increasingly being employed for identification of fungi, the basics of these methods and their utility in the clinical mycology laboratory are discussed in Part III (pp. 333–357).

Instructions for general laboratory procedures, i.e., collection of specimens, direct microscopic preparations, primary isolation, slide cultures, special tests, maintenance of stock cultures, and the like, are given in Part IV (pp. 359–384). Staining methods are described on pp. 385–394; the preparation and use of media are on pp. 395–425.

Any terms used that may not be familiar to the reader can most likely be found in the Glossary on pp. 457–468.

Once the organism has been properly collected, cultured, isolated, and observed microscopically, use of the guide is quite simple.

1. 1. Note the morphology of the unknown fungus.
   1. a. Is it a filamentous bacterium, yeastlike, thermally dimorphic, or a thermally monomorphic mould?
   2. b. Record color of surface and reverse (underside) of colony.
2. 2. Using the initial “Guide to the Identification of Fungi in Culture” on pp. 75–107, refer to a page that shows drawings of the microscopic morphologies of organisms having the appropriate macroscopic appearance. Here one may see either the exact organism under examination or several possibilities.
3. 3. Proceed to the page given in parentheses under the likely organism(s) to find more detailed information, including pathogenicity, rate of growth, colony morphology, an enlarged drawing of the microscopic appearance, a photomicrograph, and references for additional information. Where applicable, there will be reference to tables and color plates* and discussions of tests or characteristics that may help to differentiate extremely similar organisms.
4. 4. Ordinarily, the identification will be quite certain. If, however, any doubt remains, the organism should be sent to a reference laboratory for confirmation of identification as discussed in the following section.

Rare or atypical fungi can be difficult to identify even for a very experienced microbiologist or medical technologist. After a possible identification of an isolated organism is reached, confirmation is often necessary.

When the identification of an isolated fungus is dubious or when the fungus appears to be one that the laboratory worker has never before encountered, a reliable reference laboratory should be asked to confirm the identification. A reference laboratory should also be utilized if identification to species level is needed and requires molecular methods that are not available in the source laboratory. Because of the toxicity and known spectrum of activity of various antifungal medications, it is especially important to confirm the identification of organisms suspected of causing mycoses. Ordinarily, the state health department acts as a reference laboratory; otherwise, a reputable commercial laboratory of proven competency should be chosen to provide the service.

Cultures sent to reference laboratories should be pure, young, and actively growing on agar slants. Petri plates should not be transported. For details on the labeling, mailing, and delivering of potentially pathogenic isolates, one should consult the reference laboratory for specific requirements and comply with the shipping codes. Personnel involved in packing and shipping infectious substances must receive training in the proper procedures, and the training must be maintained and documented.

The regulations for transport of infectious substances and biological substances are developed and issued by three major authorities: (i) the International Civil Aviation Organization (ICAO), a United Nations agency; (ii) the International Air Transport Association (IATA); and (iii) the U.S. Department of Transportation (DOT). Thanks to a concerted effort made during the past few years, their regulations are now in substantial agreement with one another. All of the required supplies are commercially available.

Infectious substances are now classified as either Category A or Category B. The definition of each category and the instructions for packing and shipping isolates from humans follow.

A Category A Infectious Substance is an infectious substance in a form capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. If there is doubt as to whether or not an isolate meets the criteria for Category A, it must be handled as a Category A organism. At the time of this writing, the lone fungus expressly listed in Category A is Coccidioides immitis (includes Coccidioides posadasii), in cultured form only. The list is not all-inclusive, so other fungi may, with careful consideration, qualify as Category A.

The instructions for packing Category A organisms are as follows.

1. 1. The tube containing the slanted culture must be made of glass, metal, or plastic and should be labeled with the organism identification (in general terms if the identification is unknown). It must be securely closed and rendered leakproof by positive means (such as heat seal or metal crimp); if a screw cap is used, it must be secured with adhesive tape, paraffin sealing tape, or manufactured locking closure. The maximum allowable volume per tube is 50 ml (50 g). The culture tube is considered the “primary receptacle.”
2. 2. Position enough packing and absorbent material at the top, bottom, and sides of the culture tube to prevent breakage and to absorb the entire volume of the culture in case of leakage. If more than one tube is being transported, each one must be wrapped individually to ensure that contact between them, and consequent breakage, is prevented.
3. 3. Insert the wrapped culture tube(s) into durable, watertight “secondary packaging” (it must be made of material that is certified for ability to withstand an increased amount of pressure if it is to be shipped by air). Although several culture tubes may be placed in a single secondary container, the total contents cannot exceed 50 ml (50 g).
4. 4. Attach an itemized list of the contents and quantities of the primary receptacle(s) to the outside of the secondary packaging.
5. 5. Place the secondary package(s) in an outer shipping container that measures at least 4 × 4 inches on at least one surface and is constructed of sturdy material that meets the strict U.N. specifications for shipping Category A substances.

The outer shipping container must show (i) a “Class 6 Infectious Substance—In Case of Leakage” label; (ii) a label showing the proper shipping name, “Infectious Substance Affecting Humans,” and the UN2814 marking; (iii) the U.N. package certification mark; and (iv) shipper (or consignee) identification.

A Shipper’s Declaration must accompany shipment of a Category A substance. The necessary form has vertical red “candy stripes” along the left and right edges. Most carriers require that the form be typed (not handwritten). FedEx provides online software to generate the form (http://images.fedex.com/us/services/pdf/DiscColumnsColor_PDF.pdf). The form can also be found at http://www.econotech.com/shipping/index.php. Note that a color printer is required. A 24/7 emergency response telephone number must be supplied; it must be monitored by a person (not an answering machine or message service) who has access to information concerning the hazards, and actions required, in case of human exposure to the contents of the shipment. This role can be filled by an agency or commercial company that provides this service.

A Category B Infectious Substance is an infectious substance that does not meet Category A criteria. The proper, complete designation for this type of organism is “Biological Substance, Category B.”

The instructions for packing Category B organisms are very similar to those for Category A, but differ in the following ways.

• The primary receptacle does not require sealing by a positive means.
• The maximum allowable volume per primary receptacle is 1 liter (4 kg).
• The secondary packaging must be leakproof and sift-proof, but may be a sealed plastic bag or other intermediate packaging.
• Either the primary or secondary container must be pressure resistant for transport by air.
• There are no U.N. manufacturing specifications for the outer packaging, but it must be rigid and strong enough for its intended use and able to pass a 3.9-foot drop test.
• The outer shipping container must show a label showing the proper shipping name, “Biological Substance, Category B,” and the UN3373 marking. The name and telephone number of a responsible person may be placed on the outer packaging or on a document such as an air waybill.
• The Shipper’s Declaration for Dangerous Goods is not required.
• An emergency response telephone number is not required.

For updates on the regulations, consult the Federal Motor Carrier Safety Administration site https://www.fmcsa.dot.gov/regulations/hazardous-materials/how-comply-federal-hazardous-materials-regulations.

Following are suppliers of packaging materials.

• Saf-T-Pak (http://www.saftpak.com/; the only one the author has used; can highly recommend)
• CARGOpak (http://www.cargopak.com/)
• EXAKT-PAK (http://www.exaktpak.com/)

Since many fungi produce conidia or spores that easily become airborne, precautions are essential to prevent contamination of the laboratory environment and infection of personnel.

• If at all possible, laboratories culturing filamentous fungi should ideally be in a separate room with negative air pressure; the minimum alternative is a designated separate space in the open microbiology laboratory.
• A suitable biological safety cabinet (Class II BSC) must be used when dealing with moulds. Yeast cultures can be handled on the bench in the same manner that bacterial cultures are routinely handled.
• Care must be taken not to spatter infectious material by careless flaming of wire needles or loops. Use of a benchtop microbiology incinerator is one way to avoid this hazard. Sterile disposable implements provide the best solution by also eliminating the need to flame.
• All used implements and any supernatants should be placed in a vessel containing disinfectant.
• Screw-cap tubes of slanted media are safer to handle than are petri plates. If plates are used, shrink seals should be employed. Petri plates should NEVER be used if Coccidioides immitis is suspected or if a culture of any fungus is to be mailed or otherwise transported to another laboratory.
• Plates and slants should be observed for growth before opening.
• A wet preparation should be made of all moulds before setting up a slide culture; DO NOT make slide cultures of isolates that may be Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Coccidioides sposadasii, Paracoccidioides brasiliensis, Talaromyces marneffei, or Cladophialophora bantiana. These organisms can usually be identified on well-made wet preps. DNA probes are commercially available for confirmation of Histoplasma, Blastomyces, and Coccidioides.
• All contaminated materials must be autoclaved before being discarded.
• Laboratory coats must be worn at all times in the work area.
• Gloves should be worn when manipulating a mould culture that is possibly a dermatophyte; when the task is completed, hands and wrists must be washed well (removing watches and bracelets).
• Personnel must wash their hands thoroughly with a disinfectant soap after handling any mycology cultures.
• The work area must be cleaned with disinfectant at least daily.
• Mouth pipetting must NOT be done.
• There can be no smoking, drinking, eating, gum chewing, application of cosmetics, or insertion of contact lenses in the laboratory.
• A culture must NEVER be sniffed to determine whether it has an odor; the typical Streptomyces sp. can be smelled from afar.

For further information, see

• Centers for Disease Control and Prevention/National Institutes of Health, 2009, Biosafety in Microbiology and Biomedical Laboratories, 5th ed.
• Miller, J. M. (ed.), 2011, Guidelines for Safe Work Practices in Human and Animal Diagnostic Laboratories. MMWR Supplement.