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
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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:
[email protected]; Website: www.clsi.org.
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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
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 [email protected].
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)
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Volume 34 M56-A
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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
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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
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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
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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
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©
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,
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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
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EP05
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EP09 EP18
EP23
EP29
M02
M11
M35
M40 MM09
MM18
MM19
QMS01
MM19
QMS01
MM19
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EP18
MM19
QMS01
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MM19
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MM19
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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
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Sam
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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
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