July 2014

M56-APrinciples and Procedures for Detection of Anaerobes in Clinical Specimens; Approved Guideline

This document presents standardized, cost-effective, and efficient best practice processes for anaerobe bacteriology to assist clinical laboratories in selecting those methods that lead to improved patient care.

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

SAMPLE

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.

CLSI’s consensus process depends on experts who volunteer to serve as contributing authors and/or as participants in the reviewing and commenting process. At the end of each comment period, the committee that developed the document is obligated to review all comments, respond in writing to all substantive comments, and revise the draft document as appropriate.

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

If it is believed that an objection has not been adequately addressed, the process for appeals is documented in the CLSI Standards Development Policies and Process document.

All comments and responses submitted on draft and published documents are retained on file at CLSI and are available upon request.

Get Involved—Volunteer!Do you use CLSI documents in your workplace? Do you see room for improvement? Would you like to get involved in the revision process? Or maybe you see a need to develop a new document for an emerging technology? CLSI wants to hear from you. We are always looking for volunteers. By donating your time and talents to improve the standards that affect your own work, you will play an active role in improving public health across the globe.

For further information on committee participation or to submit comments, contact CLSI.

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

SAMPLE

ISBN 1-56238-969-6 (Print)

ISBN 1-56238-970-X (Electronic)

ISSN 1558-6502 (Print) M56-A

ISSN 2162-2914 (Electronic) Vol. 34 No. 10

Principles and Procedures for Detection of Anaerobes in Clinical

Specimens; Approved Guideline

Volume 34 Number 10

Maria D. Appleman, PhD

Diane M. Citron, BS, M(ASCP)

Jeffrey O‘Kelly, MS, SM(ASCP)CM

Kathy Bernard, MSc, ARM(CCM)

James C. Copeland, PhD

Mike E. Cox, BS

Jennifer Dien Bard, PhD, D(ABMM), FCCM

Peter Heseltine, MD, FACP, FACB, FIDSA

Nilda V. Jacobus

Susan M. Kircher, MS, MT(ASCP)

Brian Mochon, PhD, D(ABMM)

Andrew B. Onderdonk, PhD, DLM (ASCP)CM

Linda G. Otterson, BS, MT(ASCP)

Pravin H. Patel, PhD, D(ABMM)

Ribhi M. Shawar, PhD, D(ABMM)

Angela Thompson, MS

Abstract Clinical and Laboratory Standards Institute document M56-A—Principles and Procedures for Detection of Anaerobes in Clinical

Specimens; Approved Guideline provides procedures for performing testing and providing accurate, reliable, and useful results to

laboratories with differing levels of expertise in anaerobe bacteriology. Preexamination requirements for specimen selection,

collection, transport, timely processing, and examination procedures are discussed. Rapid and complex methods are compared for

their ability to provide definitive identifications. Because the delivery of preliminary reports is vital to patient care when complex

final reports are delayed, interpretations of direct smears and culture results are presented to help laboratorians confidently issue

preliminary reports. Descriptions of anaerobes involved in human disease and a discussion of diagnostic methods for Clostridium

difficile disease are presented. Guidelines for establishing competency testing to laboratories at their various levels of expertise

and complexity are included. Because failures in good practices for preexamination, examination, and postexamination

techniques can put patients at risk, a discussion of risk assessment during the design and implementation of anaerobe

bacteriology protocols is included.

Clinical and Laboratory Standards Institute (CLSI). Principles and Procedures for Detection of Anaerobes in Clinical Specimens;

Approved Guideline. CLSI document M56-A (ISBN 1-56238-969-6 [Print] ISBN 1-56238-970-X [Electronic]). Clinical and

Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2014.

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 you or your organization are 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.

SAMPLE

Number 10 M56-A

ii

Copyright ©2014 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 Anaerobes in Clinical Specimens; Approved Guideline.

CLSI document M56-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.

Approved Standard July 2014

ISBN 1-56238-969-6 (Print)

ISBN 1-56238-970-X (Electronic)

ISSN 1558-6502 (Print)

ISSN 2162-2914 (Electronic)

SAMPLE

Volume 34 M56-A

v

Contents

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

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

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

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

2 Introduction ................................................................................................................................ 1

3 Standard Precautions .................................................................................................................. 2

4 Terminology ............................................................................................................................... 2

4.1 A Note on Terminology ................................................................................................ 2 4.2 Definitions .................................................................................................................... 3 4.3 Abbreviations and Acronyms ....................................................................................... 3

5 Introduction to Anaerobe Bacteriology...................................................................................... 5

5.1 Basic Concepts of Clinical Anaerobe Bacteriology ...................................................... 5 5.2 Indigenous Flora ........................................................................................................... 5 5.3 Common Anaerobe Infections ...................................................................................... 9

6 Preexamination Considerations ............................................................................................... 11

6.1 When Is an Anaerobic Specimen Indicated? .............................................................. 11 6.2 Specimen Collection, Transport, and Rejection.......................................................... 11

7 Examination Methods .............................................................................................................. 15

7.1 Direct Microscopic Examination of the Specimen ..................................................... 15 7.2 Processing Specimens for Culture of Anaerobes ........................................................ 18 7.3 Culture Requirements ................................................................................................. 18 7.4 Media Selection .......................................................................................................... 20 7.5 Identification Procedures ............................................................................................ 25

8 Description of Clinically Significant Anaerobes ..................................................................... 54

8.1 Gram-Negative Rods .................................................................................................. 55 8.2 Anaerobic Cocci ......................................................................................................... 65 8.3 Gram-Positive Rods .................................................................................................... 72 8.4 Diagnosis of Clostridial Diseases (Excluding Clostridium difficile) .......................... 82 8.5 Diagnosing Clostridium difficile Infection ................................................................. 92

9 Antimicrobial Susceptibility Testing ....................................................................................... 99

9.1 General Considerations ............................................................................................... 99 9.2 Selection of Microorganisms to Test .......................................................................... 99 9.3 Selection of Antimicrobial Agents for Testing ........................................................... 99 9.4 β-Lactamase Testing ................................................................................................. 100 9.5 Methods for Antimicrobial Susceptibility Testing of Anaerobes ............................. 100 9.6 Interpretative Criteria/Reporting of Results .............................................................. 100

10 Postexamination Information ................................................................................................. 100

10.1 Reporting: Preliminary and Final Results ................................................................. 100 10.2 Interpretation of Results ............................................................................................ 101

SAMPLE

Number 10 M56-A

vi

Contents (Continued)

11 Additional Information .......................................................................................................... 101

11.1 Risk Management ..................................................................................................... 101 11.2 Determination of Competency .................................................................................. 104 11.3 Stocking and Shipping of Isolates............................................................................. 106

References ........................................................................................................................................... 107

Appendix A. Gram Stain Procedure ................................................................................................... 126

Appendix B. Taxonomy of Common Clinical Isolates ....................................................................... 128

The Quality Management System Approach ...................................................................................... 132

Related CLSI Reference Materials ..................................................................................................... 134

SAMPLE

Volume 34 M56-A

vii

Foreword

This document provides information on timely, standardized, and cost-effective methods that constitute

best practices for anaerobe bacteriology and has been created to assist clinical laboratories in the selection

and use of high-quality, efficient methods to improve patient care.

Anaerobes can be involved in infections of the head and neck, central nervous system, pleuropulmonary

sites, intra-abdominal sites, female genital tract, blood, skin, soft tissues, and bones. These infections are

known to contribute significantly to patient morbidity and mortality and adequate therapy plays an

important role in the outcome. Previously, sites of anaerobe infection and the anaerobic microorganism‘s

antimicrobial susceptibilities were thought to be predictable, but this is no longer true. Clinicians are

seeing more anaerobe infections in immunocompromised patients and in those patients with complex

diseases. Anaerobic isolates that are resistant to antimicrobial agents are encountered more frequently.

This changing clinical picture requires an adequate laboratory response.

Anaerobe bacteriology can be challenging. The microorganisms are sensitive to oxygen exposure; thus

requiring specialized supplies, equipment, and methods. They are part of the normal human flora often

causing infections by penetration of the deeper tissues when physical damage to tissue or structure has

occurred. Therefore, the diagnostic process must differentiate between the infecting and noninvolved

microorganisms. The isolation and ID of those specific anaerobes involved in infections depend on

appropriate methods beginning with specimen selection, collection, transportation, and, finally, ID

determinations. New technical microbiology tools now allow IDs to be reported rapidly and to be more

extensive and precise. Additional training and collaboration with clinicians will be required to interpret

the extensive reports but this can improve patients‘ outcomes, which is the ultimate goal of testing,

diagnosis, and treatment.

Key Words

Anaerobe identification, anaerobe taxonomy, anaerobes, anaerobic culture, antimicrobial susceptibility

testing, Clostridium difficile detection

SAMPLE

Volume 34 M56-A

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

Principles and Procedures for Detection of Anaerobes in Clinical Specimens;

Approved Guideline

1 Scope

This document provides guidance for preexamination, examination, and postexamination procedures

associated with the culture of anaerobic bacteria. Because anaerobic bacteria are part of human normal

flora and are sensitive to oxygen exposure, good preexamination methods are essential. These

recommendations include methods for collecting proper specimens from appropriate clinical sites and for

transport procedures that protect anaerobes from oxygen exposure so that all pathogens involved in

infections can be detected. The optimal methods needed to provide accurate, timely, and sufficient

information for appropriate medical decisions are included, along with a discussion of the use and value

of partial and full isolate IDs. Also included in this guideline are recommendations for interpreting

results, assistance in understanding the value of rapid preliminary results, and guidance on issues of QC,

QA, and competency.

The intended audience includes medical technologists, infectious disease physicians, microbiology

laboratory directors, pathologists, and researchers.

Because anaerobe antimicrobial susceptibility testing (AST) methods are presented in CLSI documents

M111 and M100,

2 this document limits its discussion to the need and indications for AST.

2 Introduction

Institutions allocate varying resources to their laboratories for the development of the expertise and

processes needed to work with anaerobic bacteria. The goal of this document is to present procedures that

result in accurate and timely anaerobe bacteriology reports at different categories of expertise (refer to

Table 1). Users of each laboratory should be aware of the level of expertise and extent of testing provided

by that laboratory.

When an institution determines the extent of anaerobe bacteriology that will be provided, it is performing

a risk assessment. An extreme risk for potential patient harm is introduced when a hospital decides not to

provide resources for detection of anaerobes because of a perception that its patients do not have anaerobe

infections or that those types of infections are not important. Other laboratories limit IDs of polymicrobial

infections to less than four isolates. Some laboratories even recommend limited workup of specimens

such as abdominal abscesses. This recommendation is based on the belief that empiric therapy does not

require culture results; but it overlooks the fact that all organisms grown from an abscess were involved in

its formation and that some may be antibiotic resistant. Because many anaerobic infections are

polymicrobic and the bacterial constituents may act synergistically, ID of all organisms present may

provide both prognostic and therapeutic guidance.

Newer techniques may allow the rapid ID of many microorganisms. If a decision is made to conserve

resources by limiting the number of isolates identified, each case must be communicated to the clinician

so that the risk of incomplete but potentially available information is known, and additional studies can be

done if clinically indicated. Without this collaboration between the laboratory and clinician, the risks of

arbitrary censure of the data may pose an unacceptable risk. The additional information provided by new

technical tools such as 16s ribosomal RNA (rRNA) gene sequencing and matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) adds valuable clinical insight

regarding the disease process and role of the pathogens. Although it is tempting to restrict the use of

these new advances in laboratory medicine because of supposed users‘ unfamiliarity with new taxonomy

SAMPLE

Number 10 M56-A

©

Clinical and Laboratory Standards Institute. All rights reserved. 2

or financial costs, withholding or delaying reports from the clinician on the grounds that the report is not

perceived by the laboratory to be clinically important can add to patient risk.

Risk for patients can also be introduced by a lack of rigor in specimen collection and transport or by a

laboratory‘s failure to understand and use basic concepts of anaerobe bacteriology. Every delay and

inaccuracy introduces potential harm. Every appropriate decision on specimen collection, transport,

acceptance for processing, method of processing, and extent of testing can minimize risk and improve

patient care.

Table 1. Categories of Anaerobe Bacteriology Expertise Category Extent of Expertise

1 Laboratory accepts specimens for anaerobic culture, but only detects the presence of anaerobes.

Anaerobes are referred to another laboratory for ID and AST.

2 Laboratory screens for the major anaerobic groups. Definitive ID and AST are performed in a

reference laboratory.

3 Laboratory can identify anaerobes to genus and species level using phenotypic and enzymatic

tests. It may perform some AST.

4 Laboratory provides final definitive IDs using 16s rRNA gene sequencing or MALDI-TOF MS

technology and may perform quantitative AST. Abbreviations: AST, antimicrobial susceptibility testing; ID, identification; MALDI-TOF MS, matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry; rRNA, ribosomal ribonucleic acid.

3 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 (CDC) address this topic in

published guidelines that address the daily operations of diagnostic medicine in human and animal

medicine while encouraging a culture of safety in the laboratory.3 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 M29.4

4 Terminology

4.1 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 order to align the usage of terminology in this document with that of ISO and CLSI document

QMS01,5 the terms preexamination, examination, and postexamination have replaced preanalytical,

SAMPLE

Number 10 M56-A

©

Clinical and Laboratory Standards Institute. All rights reserved. 132

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

M56-A addresses the QSE 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.

Org

aniz

atio

n

Cu

stom

er F

ocu

s

Fac

ilit

ies

and

Saf

ety

Per

son

nel

Pu

rchas

ing

and

Inven

tory

Equ

ipm

ent

Pro

cess

Man

agem

ent

Do

cum

ents

an

d

Rec

ord

s

Info

rmat

ion

Man

agem

ent

No

nco

nfo

rmin

g

Ev

ent

Man

agem

ent

Ass

essm

ents

Con

tinual

Imp

rov

emen

t

MM19

QMS01

MM19

QMS01

M29

MM19

QMS01

MM19

QMS01

QMS03

MM19

QMS01

MM19

QMS01

X

EP05

EP06

EP09 EP18

EP23

EP29

M02

M11

M35

M40 MM09

MM18

MM19

QMS01

MM19

QMS01

MM19

QMS01

EP18

MM19

QMS01

EP18

MM19

QMS01

EP18

MM19

QMS01

SAMPLE

Volume 34 M56-A

©Clinical and Laboratory Standards Institute. All rights reserved. 133

Path of Workflow

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

organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination,

examination, and postexamination and their respective sequential subprocesses. All laboratories follow these

processes to deliver the laboratory‘s services, namely quality laboratory information.

M56-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

Ex

amin

atio

n o

rder

ing

Sam

ple

coll

ecti

on

Sam

ple

tra

nsp

ort

Sam

ple

rece

ipt/

pro

cess

ing

Ex

amin

atio

n

Res

ult

s re

vie

w a

nd

foll

ow

-up

Inte

rpre

tati

on

Res

ult

s re

po

rtin

g a

nd

arch

ivin

g

Sam

ple

man

agem

ent

QMS01

X

MM09 MM19

QMS01

X

M40

MM09 MM19

QMS01

X

MM09 MM19

QMS01

X

EP23 M02

M11

M35

MM09 MM19

QMS01

X

EP23 M02

M11

M35

M100

MM09 MM19

QMS01

X

EP23 M02

M11

M35

M100

MM09 MM19

QMS01

X

M02

M11

M35

M100

MM09

QMS01

MM09

QMS01

SAMPLE

For more information, visit www.clsi.org today.

Explore the Latest Offerings from CLSI!

Where we provide the convenient and cost-effective education resources that laboratories need to put CLSI standards into practice, including webinars, workshops, and more.

Visit the CLSI U Education Center

See the options that make it even easier for your organization to take full advantage of CLSI benefits and our unique membership value.

Find Membership Opportunities

About CLSIThe 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 standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability.

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: membership@clsi.org

Introducing CLSI’s New Individual Membership! CLSI is offering a new membership opportunity for individuals

who support or volunteer for CLSI but whose organizations are not currently members.

Individuals

Student Member ($25)—Full-time students enrolled in an academic program

Associate Member ($75)—Professionals associated with the health care profession and/or clinical and laboratory services

Full Member ($250)—Professionals associated with the health care profession and/or clinical and laboratory services

Benefits include:

Participation on document development committees

Discount on educational products

Benefits include:

Participation on document development committees Discount on educational products A 15% discount on products and services

Benefits include:

Participation on document development committees Voting on all documents (concurrent with delegate voting) Participation in governance activities (vote for the Board of Directors, be nominated for the Board, and

be eligible to be selected for Board committee service) Discount on educational products A 25% discount on products and services

Effective January 1, 2013, all CLSI volunteers are required to be members at one of the above levels if their organization is not a CLSI member. For current volunteers (those who are still actively on committees as of January 1), we have waived the requirement of membership until the end of their current volunteer assignment, and they may continue participating without incurring any membership fees. Please feel free to contact CLSI’s Membership department with any questions at membership@clsi.org.

Apply Today! Visit www.clsi.org/membership for an application.

Introducing CLSI’s New Membership OpportunitiesMore Options. More Benefits. More Value.

We’ve made it even easier for your organization to take full advantage of the standards resources and networking opportunities available through membership with CLSI.

As we continue to set the global standard for quality in laboratory testing, we’re adding initiatives to bring even more value to our members and customers.

Including eM100, the interactive searchable database for drug selection, interpretation, and quality control procedures within M100.

Shop Our Online Products

Including eCLIPSE Ultimate Access™, CLSI’s cloud-based, online portal that makes it easy to access our standards and guidelines—anytime, anywhere.

Shop Our Online Products

CLIPSEUltimate Access

eTM

SAMPLE

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 1-56238-969-6

ELECTRONIC ISBN 1-56238-970-X

SAMPLE