October 2012

M54-APrinciples and Procedures for Detection of Fungi in Clinical Specimens—Direct Examination and Culture; Approved Guideline

This guideline provides protocols, quality control parameters, and interpretive criteria for performing fungal cultures and for the detection and identification of fungi in direct examinations.

A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.

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Clinical and Laboratory Standards Institute Setting the standard for quality in clinical laboratory testing around the world.

The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a common cause: to foster excellence in laboratory medicine by developing and implementing clinical laboratory standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability. Consensus Process

Consensus—the substantial agreement by materially affected, competent, and interested parties—is core to the development of all CLSI documents. It does not always connote unanimous agreement, but does mean that the participants in the development of a consensus document have considered and resolved all relevant objections and accept the resulting agreement. Commenting on Documents

CLSI documents undergo periodic evaluation and modification to keep pace with advancements in technologies, procedures, methods, and protocols affecting the laboratory or health care.

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Comments on published CLSI documents are equally essential, and may be submitted by anyone, at any time, on any document. All comments are addressed according to the consensus process by a committee of experts. Appeals Process

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Clinical and Laboratory Standards Institute950 West Valley Road, Suite 2500 Wayne, PA 19087 USA P: 610.688.0100F: 610.688.0700www.clsi.orgstandard@clsi.org

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ISBN 1-56238-857-6 (Print) ISBN 1-56238-858-4 (Electronic) ISSN 1558-6502 (Print) M54-A ISSN 2162-2914 (Electronic) Vol. 32 No. 14

Principles and Procedures for Detection of Fungi in Clinical Specimens—Direct Examination and Culture; Approved Guideline

Volume 32 Number 14 Nancy L. Wengenack, PhD, D(ABMM), FIDSA Gary W. Procop, MD Gail L. Woods, MD Glenn D. Roberts, PhD Lynette Y. Berkeley, PhD Michael A. Saubolle, PhD, D(ABMM) Vishnu Chaturvedi, PhD Wiley A. Schell, MS Michael J. Dykstra, PhD Audrey N. Schuetz, MD, MPH Annette W. Fothergill, MA, MBA, MT(ASCP) Guy St-Germain, BS Bharat Gandhi, M(ASCP), S(CCM), BSc Deanna A. Sutton, PhD, MT, SM(ASCP) Brian J. Harrington, PhD, MPH Paula H. Vance Patricia L. Kammeyer, MT(ASCP) Paula Williams, MT(ASCP)SM Kathi LK Krankoski, MT(ASCP) Jianlong Jim Zhou, PhD A. Leonel Mendoza, PhD

Abstract Clinical and Laboratory Standards Institute document M54-A—Principles and Procedures for Detection of Fungi in Clinical Specimens—Direct Examination and Culture; Approved Guideline provides recommended processes for the plating and examination of fungal cultures and principles and procedures for the detection of fungi in clinical specimens, including criteria for the performance and interpretation of direct microscopic examinations. Safety considerations unique to mycology laboratories and a discussion of appropriate levels of laboratory service (eg, when to refer samples to more experienced laboratories) are highlighted. Specimen collection, transport, and processing guidelines, including rejection criteria, are provided to guide the reader in the collection of high-quality specimens for direct examinations and fungal cultures. Fungal stains and interpretive criteria appropriate for the detection and characterization of fungal elements in direct microscopic examinations are emphasized as critical components for rapid, cost-effective detection of fungi. Media selection, incubation conditions, and other growth requirements for fungal cultures are provided with suggested culture examination schedules, interpretations for growth on positive cultures, and reporting criteria. Clinical and Laboratory Standards Institute (CLSI). Principles and Procedures for Detection of Fungi in Clinical Specimens—Direct Examination and Culture; Approved Guideline. CLSI document M54-A (ISBN 1-56238-857-6 [Print]; ISBN 1-56238-858-4 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2012.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org.

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Copyright ©2012 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to permissions@clsi.org. CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, e-mail permissions@clsi.org. Suggested Citation CLSI. Principles and Procedures for Detection of Fungi in Clinical Specimens—Direct Examination and Culture; Approved Guideline. CLSI document M54-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. Approved Guideline October 2012 ISBN 1-56238-857-6 (Print) ISBN 1-56238-858-4 (Electronic) ISSN 1558-6502 (Print) ISSN 2162-2914 (Electronic)

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Volume 32 M54-A

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Contents

Abstract .................................................................................................................................................... i

Committee Membership ........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

1 Scope .......................................................................................................................................... 1

2 Standard Precautions .................................................................................................................. 1

3 Terminology ............................................................................................................................... 1

3.1 A Note on Terminology ................................................................................................ 1 3.2 Definitions .................................................................................................................... 2 3.3 Additional Descriptive Terms and Synonyms .............................................................. 3 3.4 Abbreviations and Acronyms ....................................................................................... 3

4 Biosafety .................................................................................................................................... 4

4.1 Risk Assessment ........................................................................................................... 5 4.2 Facilities ........................................................................................................................ 7 4.3 Other Biosafety Considerations .................................................................................... 7 4.4 Personnel ....................................................................................................................... 8

5 Levels of Laboratory and Referral Services .............................................................................. 8

6 Quality Control and Maintenance of Stock Cultures ................................................................. 9

6.1 Quality Control of Media, Reagents, and Other Supplies ............................................. 9 6.2 Quality Control Reference Strains ................................................................................ 9 6.3 Corrective Action ........................................................................................................ 12

7 Specimen Collection and Transport ......................................................................................... 12

7.1 Rejection Criteria ........................................................................................................ 13 7.2 Specimen Types .......................................................................................................... 14

8 Specimen Processing ............................................................................................................... 18

8.1 Concentration .............................................................................................................. 18 8.2 Direct Inoculation ....................................................................................................... 18 8.3 Mincing and Homogenization..................................................................................... 18 8.4 Specimen Retention and Storage ................................................................................ 19

9 Direct Examination of Clinical Specimens .............................................................................. 19

9.1 Detection and Differentiation of Fungi in Direct Examination .................................. 19 9.2 Microscopy Procedures ............................................................................................... 37 9.3 Molecular Methods ..................................................................................................... 44

10 Specimen Plating and Incubation ............................................................................................. 44

10.1 Inoculation of Specimens ............................................................................................ 45 10.2 Media .......................................................................................................................... 46 10.3 Media Selection .......................................................................................................... 47 10.4 Incubation Requirements and Conditions ................................................................... 48

11 Culture Assessment .................................................................................................................. 49

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Contents (Continued)

11.1 Reading Schedule ....................................................................................................... 49 11.2 Interpretation of Growth ............................................................................................. 50

References ............................................................................................................................................. 54

Appendix A. Specimen Collection, Transport, and Processing Chart .................................................. 59

Appendix B. Principles of Fluorescent Microscopy ............................................................................. 63

Appendix C. Histopathological Stains .................................................................................................. 69

Appendix D. Control of Mite Infestations in Fungal Cultures .............................................................. 74

Appendix E. Additional Isolation Media for Culturing Sputum and Other Respiratory Tract

Samples From Cystic Fibrosis Patients ................................................................................................. 76

The Quality Management System Approach ........................................................................................ 80

Related CLSI Reference Materials ....................................................................................................... 81

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Foreword

CLSI document M54, Principles and Procedures for Detection of Fungi in Clinical Specimens—Direct

Examination and Culture, addresses the principles and procedures for the detection of fungi in clinical

specimens with an emphasis on direct microscopic examinations and fungal culturing methods. In

addition, unique safety risks associated with mycology laboratories are discussed, and guidance on the

performance of a risk assessment, the facilities necessary for fungal culturing, and other relevant safety

considerations such as personnel training and education are provided. A discussion of when it may be

prudent to refer samples to more experienced laboratories is also provided.

QC processes unique to the mycology laboratory, including methods for the maintenance and storage of

stock strains, are included as well as the performance standards and expected results for the QC of

nonexempt mycological media. Specimen collection and transport criteria are detailed, including

acceptable specimen types and specimen rejection criteria. Specimen processing procedures including

concentration, direct inoculation, and mincing/homogenization of tissues are discussed, with emphasis on

the recovery of fungi from tissues by either increasing surface area exposure of the organism to the

medium or by preventing loss of viability for mucoraceous moulds.

The ability of the laboratory scientist to detect and characterize fungal elements directly from patient

specimens provides substantial information about possible pathogens in a rapid fashion. Therefore, the

characterization of yeast and yeast-like fungi based on size and other microscopic characteristics is

emphasized together with, where possible, differentiation of moulds in direct examinations. Stains

commonly used for direct examinations in the mycology laboratory are presented along with information

on the utility of histopathology stains that may come to the attention of the mycologist.

In addition to direct examination, fungal cultures play an integral role in the diagnosis of fungal

infections. M54 provides information on fungal media, including applications, selection, inoculation,

incubation, and schedules for reading.

The creation of this document required significant effort and valuable input from a number of experts in

the field. I would like to personally thank them for their time and contributions to the document.

Nancy L. Wengenack, PhD

Chairholder of the Document Development Committee on Principles and Procedures for Fungal Cultures

Key Words

Culture, direct examination, fungi, mould, mycology, safety, specimen collection, stock cultures, yeast

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Volume 32 M54-A

©Clinical and Laboratory Standards Institute. All rights reserved. 1

Principles and Procedures for Detection of Fungi in Clinical Specimens—

Direct Examination and Culture; Approved Guideline

1 Scope

Mycologists have recognized the need for a consensus guideline that serves as a concise reference for the

performance of fungal culturing and the preparation and reading of direct microscopic examinations. M54

provides principles and procedures for biosafety in the mycology laboratory, QC of fungal media,

maintenance of stock strains, specimen collection and transport, specimen processing, direct microscopic

examinations, fungal culturing methods, and assessment of cultures.

This guideline is intended for laboratory scientists who process specimens for fungal culture and/or

perform fungal direct microscopic examinations. Although they can be encountered growing on

mycology media, Nocardia spp. and other aerobic actinomycetes can also be found growing on media in

other areas of the laboratory (eg, bacteriology, mycobacteriology). Therefore, culture and direct

examinations for this group of organisms will not be discussed in this document. Furthermore, the

identification of fungi is outside of the scope of this document.

2 Standard Precautions

Because it is often impossible to know what isolates or specimens might be infectious, all patient and

laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard

precautions are guidelines that combine the major features of “universal precautions and body substance

isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus

are more comprehensive than universal precautions, which are intended to apply only to transmission of

blood-borne pathogens. The Centers for Disease Control and Prevention address this topic in published

guidelines that focus on the daily operations of diagnostic medicine in human and animal medicine while

encouraging a culture of safety in the laboratory.1 For specific precautions for preventing the laboratory

transmission of all known infectious agents from laboratory instruments and materials and for

recommendations for the management of exposure to all known infectious diseases, refer to CLSI

document M29.2

3 Terminology

3.1 A Note on Terminology

CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization

wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences

while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the

global metrological community have evolved differently in the United States, Europe, and elsewhere; that

these differences are reflected in CLSI, International Organization for Standardization (ISO), and

European Committee for Standardization (CEN) documents; and that legally required use of terms,

regional usage, and different consensus timelines are all important considerations in the harmonization

process. In light of this, CLSI’s consensus process for development and revision of standards and

guidelines focuses on harmonization of terms to facilitate the global application of standards and

guidelines.

In Europe, the term “clinical” often refers to clinical studies of drugs under stringent conditions;

therefore, the term medical laboratory is used in place of the term clinical laboratory.

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Clinical and Laboratory Standards Institute. All rights reserved. 2

3.2 Definitions

biosafety level (BSL) – laboratory designation determined by risk assessment of the pathogenicity of the

agent, mode of transmission, amount of the agent manipulated, and the nature of the work performed3,4

;

NOTE: In the United States and Europe, this is subdivided into four levels (BSL-1, BSL-2, BSL-3, BSL-

4) for microbiological and biomedical laboratories.

blastoconidium (pl. blastoconidia) – a conidium that is formed by budding from a hyphal,

pseudohyphal, or yeast cell.

capsule – a mucopolysaccharide material surrounding a cell.

chancre – a sore or ulcer located at the initial point of entry of a pathogen.

chromoblastomycosis – a cutaneous or subcutaneous infection caused by darkly pigmented fungi (eg,

Phialophora, Fonsecaea) characterized by the presence of dematiaceous (phaeoid) sclerotic cells.

coenocytic – aseptate with no cross-walls separating the cells.

coin lesion – a well-circumscribed, calcified lung lesion resembling the shadow of a coin on an X-ray.

conidium (pl. conidia) – an asexual reproductive structure that forms on the side or the end of a hypha or

conidiophore.5

dematiaceous (phaeoid) – having conidia, spores, or hyphae that are brown to black in color due to the

presence of melanin.6

dermatophyte – a fungus that obtains nutrients from keratin and infects skin, hair, and nails; consists of

species within the genera Microsporum, Trichophyton, and Epidermophyton.

dichotomous – acute angle branching into two equal branches.

dimorphic – having two morphological forms.

endemic – restricted to a particular geographic region.

endospore – a spore produced within a spherule.

exudate – the accumulation of a fluid having a high concentration of protein in a body cavity caused by

increased capillary permeability usually secondary to inflammation.

fastidious – requiring specialized conditions for growth.

granuloma – a chronic inflammatory tissue response characterized by activated histiocytes and possibly

giant cells.

grind – to homogenize by friction (eg, with a sterile mortar and pestle or tissue grinder).

homogenize – to reduce a patient sample to a small, uniform size dispersed in a liquid.

hypha (pl. hyphae) – a septate or aseptate filament of a fungus.

kerion – a severe pustular inflammation involving the hair follicles.6

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80 ©Clinical and Laboratory Standards Institute. All rights reserved.

The Quality Management System Approach Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the

development of standards and guidelines, which facilitates project management; defines a document structure via a

template; and provides a process to identify needed documents. The quality management system approach applies a

core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care

service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The

QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs

are as follows:

Organization Personnel Process Management Nonconforming Event Management

Customer Focus Purchasing and Inventory Documents and Records Assessments

Facilities and Safety Equipment Information Management Continual Improvement

M54-A addresses the QSEs indicated by an “X.” For a description of the other documents listed in the grid, please

refer to the Related CLSI Reference Materials section on the following page.

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Path of Workflow

A path of workflow is the description of the necessary steps to deliver the particular product or service that the

organization or entity provides. A laboratory path of workflow consists of three sequential processes:

preexamination, examination, and postexamination. All laboratories follow these processes to deliver the

laboratory’s services, namely quality laboratory information.

M54-A addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other

documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.

Preexamination Examination Postexamination

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Volume 32 M54-A

©Clinical and Laboratory Standards Institute. All rights reserved. 81

Related CLSI Reference Materials

GP26-A4 Quality Management System: A Model for Laboratory Services; Approved Guideline—Fourth Edition

(2011). This document provides a model for medical laboratories that will assist with implementation and

maintenance of an effective quality management system.

M22-A3 Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard—

Third Edition (2004). This document contains quality assurance procedures for manufacturers and users of

prepared, ready-to-use microbiological culture media.

M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—

Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission

of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific

precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and

materials; and recommendations for the management of exposure to infectious agents.

M47-A Principles and Procedures for Blood Cultures; Approved Guideline (2007). This document provides

recommendations for the collection, transport, and processing of blood cultures as well as guidance for the

recovery of pathogens from blood specimens taken from patients who are suspected of having bacteremia or

fungemia.

CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to

the most current editions.

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950 West Valley Road, Suite 2500, Wayne, PA 19087 USA

P: 610.688.0100 Toll Free (US): 877.447.1888 F: 610.688.0700

E: customerservice@clsi.org www.clsi.org

PRINT ISBN 1-56238-857-6

ELECTRONIC ISBN 1-56238-858-4

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