Clsi bloodculture

M47-AVol. 27 No. 17

Replaces M47-P Vol. 26 No. 31

Principles and Procedures for Blood Cultures; Approved Guideline

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

(Formerly NCCLS)Licensed to: Giancarlo Scoppettuolo, MD UCSC

This document is protected by copyright. CLSI order # 52644, id # 453704, Downloaded on 4/27/2008.

Clinical and Laboratory Standards Institute Advancing Quality in Healthcare Testing Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the healthcare community. It is recognized worldwide for the application of its unique consensus process in the development of standards and guidelines for patient testing and related healthcare issues. Our process is based on the principle that consensus is an effective and cost-effective way to improve patient testing and healthcare services.

In addition to developing and promoting the use of voluntary consensus standards and guidelines, we provide an open and unbiased forum to address critical issues affecting the quality of patient testing and health care.

PUBLICATIONS

A document is published as a standard, guideline, or committee report.

Standard A document developed through the consensus process that clearly identifies specific, essential requirements for materials, methods, or practices for use in an unmodified form. A standard may, in addition, contain discretionary elements, which are clearly identified.

Guideline A document developed through the consensus process describing criteria for a general operating practice, procedure, or material for voluntary use. A guideline may be used as written or modified by the user to fit specific needs.

Report A document that has not been subjected to consensus review and is released by the Board of Directors.

CONSENSUS PROCESS

The CLSI voluntary consensus process is a protocol establishing formal criteria for:

• the authorization of a project

• the development and open review of documents

• the revision of documents in response to comments by users

• the acceptance of a document as a consensus standard or guideline.

Most documents are subject to two levels of consensus—“proposed” and “approved.” Depending on the need for field evaluation or data collection, documents may also be made available for review at an intermediate consensus level.

Proposed A consensus document undergoes the first stage of review by the healthcare community as a proposed standard or guideline. The document should receive a wide and thorough technical review, including an overall review of its scope, approach, and utility, and a line-by-line review of its technical and editorial content.

Approved An approved standard or guideline has achieved consensus within the healthcare community. It should be reviewed to assess the utility of the final document, to ensure attainment of consensus (i.e., that comments on earlier versions have been satisfactorily addressed), and to identify the need for additional consensus documents.

Our standards and guidelines represent a consensus opinion on good practices and reflect the substantial agreement by materially affected, competent, and interested parties obtained by following CLSI’s established consensus procedures. Provisions in CLSI standards and guidelines may be more or less stringent than applicable regulations. Consequently, conformance to this voluntary consensus document does not relieve the user of responsibility for compliance with applicable regulations.

COMMENTS

The comments of users are essential to the consensus process. Anyone may submit a comment, and all comments are addressed, according to the consensus process, by the committee that wrote the document. All comments, including those that result in a change to the document when published at the next consensus level and those that do not result in a change, are responded to by the committee in an appendix to the document. Readers are strongly encouraged to comment in any form and at any time on any document. Address comments to Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA 19087, USA.

VOLUNTEER PARTICIPATION

Healthcare professionals in all specialties are urged to volunteer for participation in CLSI projects. Please contact us at [email protected] or +610.688.0100 for additional information on committee participation.

Licensed to: Giancarlo Scoppettuolo, MD UCSCThis document is protected by copyright. CLSI order # 52644, id # 453704, Downloaded on 4/27/2008.

M47-A ISBN 1-56238-641-7

Volume 27 Number 17 ISSN 0273-3099

Principles and Procedures for Blood Cultures; Approved Guideline Michael L. Wilson, MD Michael Mitchell, MD Arthur J. Morris, MD, FRCPA Patrick R. Murray, PhD Larry G. Reimer, MD L. Barth Reller, MD Michael Towns, MD Melvin P. Weinstein, MD Sybil A. Wellstood, PhD W. Michael Dunne, Jr, PhD Robert C. Jerris, PhD David F. Welch, Ph, D(ABMM) Abstract Clinical and Laboratory Standards Institute document M47-A—Principles and Procedures for Blood Cultures; Approved Guideline addresses the laboratory detection of bacteremia and fungemia by use of blood cultures. Included in this guideline are recommendations for the: 1) clinical importance of blood cultures; 2) specimen collection and transportation; 3) critical factors in the recovery of pathogens from blood specimens; 4) special topics, including pediatric blood cultures, catheter-related bloodstream infections, infective endocarditis, patients receiving antimicrobial therapy, rare and fastidious pathogens, and test of cure; 5) reporting results; 6) interpreting blood culture results; 7) safety issues; and 8) quality assurance. Clinical and Laboratory Standards Institute (CLSI). Principles and Procedures for Blood Cultures; Approved Guideline. CLSI document M47-A (ISBN 1-56238-641-7). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007.

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 healthcare 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/NCCLS documents. Current editions are listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. 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: [email protected]; Website: www.clsi.org

(Formerly NCCLS)


Number 17 M47-A

ii

Copyright ©2007 Clinical and Laboratory Standards Institute. Except as stated below, neither this publication nor any portion thereof may be adapted, copied or otherwise reproduced, by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from Clinical and Laboratory Standards Institute (“CLSI”). 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, contact the Executive Vice President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Suggested Citation (CLSI. Principles and Procedures for Blood Cultures; Approved Guideline. CLSI document M47-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2007.) Proposed Guideline October 2006 Approved Guideline May 2007 ISBN 1-56238-641-7 ISSN 0273-3099


Volume 27 M47-A

iii

Committee Membership Area Committee on Microbiology Mary Jane Ferraro, PhD, MPH Chairholder Massachusetts General Hospital Boston, Massachusetts James H. Jorgensen, PhD Vice-Chairholder University of Texas Health Science Center San Antonio, Texas Donald R. Callihan, PhD BD Diagnostic Systems Sparks, Maryland Freddie Mae Poole FDA Center for Devices and Radiological Health Rockville, Maryland John H. Rex, MD, FACP AstraZeneca Cheshire, United Kingdom Daniel F. Sahm, PhD Eurofins Medinet Herndon, Virginia Fred C. Tenover, PhD, ABMM Centers for Disease Control and Prevention

John D. Turnidge, MD Women’s and Children’s Hospital North Adelaide, Australia Michael L. Wilson, MD Denver Health Medical Center Denver, Colorado Advisors Ellen Jo Baron, PhD Stanford Univ. Hospital & Medical School Stanford, California Lynne S. Garcia, MS LSG & Associates Santa Monica, California Richard L. Hodinka, PhD Children’s Hospital of Philadelphia Philadelphia, Pennsylvania Michael A. Pfaller, MD University of Iowa College of Medicine Iowa City, Iowa

Robert P. Rennie, PhD University of Alberta Hospital Edmonton, Alberta, Canada Thomas R. Shryock, PhD Elanco Animal Health Greenfield, Indiana Jana M. Swenson, MMSc Centers for Disease Control and Prevention Atlanta, Georgia Melvin P. Weinstein, MD Robert Wood Johnson Medical School New Brunswick, New Jersey Matthew A. Wikler, MD, MBA, FIDSA Pacific Beach BioSciences San Diego, California Gail L. Woods, MD University of Arkansas for Medical Sciences Little Rock, Arkansas

Atlanta, Georgia Subcommittee on Principles and Procedures for Blood Cultures Michael L. Wilson, MD Chairholder Denver Health Medical Center Denver, Colorado Michael Mitchell, MD U Mass Memorial Medical Center Worcester, Massachusetts Arthur J. Morris, MD Diagnostic Medlab Auckland, New Zealand Patrick R. Murray, PhD NIH Bethesda, Maryland Larry G. Reimer, MD University of Utah School of Medicine Salt Lake City, Utah L. Barth Reller, MD DUHS Clinical Laboratories Durham, North Carolina

Michael Towns, MD BD Diagnostic Systems Sparks, Maryland Melvin P. Weinstein, MD Robert Wood Johnson University Hospital New Brunswick, New Jersey Sybil A. Wellstood, PhD US Department of Agriculture Riverdale, Maryland Advisors W. Michael Dunne, Jr., PhD Washington University School of Medicine St. Louis, Missouri Robert C. Jerris, PhD Children’s Healthcare of Atlanta Atlanta, Georgia

David F. Welch, PhD, D(ABMM) University of Texas Southwestern Medical Center Dallas, Texas Staff Clinical and Laboratory Standards Institute Wayne, Pennsylvania John J. Zlockie, MBA Vice President, Standards Tracy A. Dooley, BS, MLT(ASCP) Staff Liaison Donna M. Wilhelm Editor Melissa A. Lewis Assistant Editor


Number 17 M47-A

iv


Volume 27 M47-A

v

Contents

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

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

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

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

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

3 Definitions .................................................................................................................................1 3.1 Acronyms......................................................................................................................3

4 Clinical Importance of Blood Cultures ......................................................................................4 4.1 Diagnostic Importance ..................................................................................................4 4.2 Prognostic Importance ..................................................................................................4

5 Specimen Collection and Transportation...................................................................................4 5.1 Timing of Blood Cultures .............................................................................................4 5.2 Number of Blood Cultures............................................................................................4 5.3 Volume of Blood for Culture........................................................................................5 5.4 Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles ...........5 5.5 Disinfection of Skin and Prevention of Blood Culture Contamination ........................6 5.6 Blood Culture Collection ..............................................................................................6 5.7 Specimen Rejection Criteria for Blood Culture Specimens..........................................7

6 Methods and Procedures ............................................................................................................7 6.1 Critical Factors in the Recovery of Pathogens From Blood Specimens .......................7 6.2 Blood Culture Methods...............................................................................................10 6.3 Fungal Blood Cultures ................................................................................................13 6.4 Mycobacterial Blood Cultures ....................................................................................14 6.5 Special Topics.............................................................................................................15

7 Reporting Results.....................................................................................................................23 7.1 Written and Electronic Reports...................................................................................23

8 Contaminants ...........................................................................................................................26

9 Safety Issues ............................................................................................................................26 9.1 Agents Associated With Laboratory-Acquired Infections..........................................27 9.2 Protective Measures ....................................................................................................27

10 Quality Assurance....................................................................................................................31 10.1 Preexamination Process ..............................................................................................31 10.2 Examination Process...................................................................................................34 10.3 Postexamination Process.............................................................................................35

References.............................................................................................................................................36

Additional References...........................................................................................................................45

Summary of Delegate Comments and Subcommittee Responses.........................................................46


Number 17 M47-A

vi

Contents (Continued)

The Quality Management System Approach ........................................................................................50

Related CLSI/NCCLS Publications ......................................................................................................51


Volume 27 M47-A

vii

Foreword The incidence of sepsis continues to increase in the United States: the most recent published data indicate that as many as 660,000 cases occur annually.1 Because the morbidity and mortality attributable to sepsis is high, the prompt and accurate detection of bacteremia and fungemia is important for improving patient care. The laboratory test that is used to detect the presence of bacteria (bacteremia) or fungi (fungemia) in the blood is the blood culture. During the past 30 years, a number of studies have been conducted to: 1) define the clinical significance of blood cultures; 2) define the critical factors in the recovery of pathogens from the blood; 3) establish the best medium formulations and other laboratory practices; 4) evaluate and compare commercial blood culture systems; and 5) develop interpretive criteria. Because of the clinical importance of bacteremia and fungemia, and therefore the importance of blood cultures, guidelines are needed so that laboratories and providers use optimal laboratory methods and interpret the results correctly. To date there has not been a single document that incorporates these data into consensus guidelines. Such guidelines are also needed to help control healthcare costs, as the costs attributable to the recovery of contaminants from blood cultures are high. Key Words Bacteremia, bacteria, blood culture, bloodstream infection, fungemia, fungi, mycobacteria, sepsis


Number 17 M47-A

viii


Volume 27 M47-A

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

Principles and Procedures for Blood Cultures; Approved Guideline 1 Scope The laboratory detection of bacteremia and fungemia remains one of the most important functions of clinical microbiology laboratories. During the past 30 years, a number of studies have defined the critical factors in the recovery of pathogens from blood and the optimal laboratory methods for recovering specific pathogens, and have established the performance characteristics of blood culture systems. Despite this information, there remains a need for guidelines for the collection, processing, and interpretation of blood cultures. Several in vitro blood culture devices are cleared by the United States Food and Drug Administration (FDA) for use in the United States. These devices typically are available for use in other countries. This guideline is intended to provide guidance to clinical microbiologists and other laboratorians (e.g., pathologists, laboratory supervisors, laboratory managers) for the recovery of pathogens from blood specimens taken from patients who are suspected of having bacteremia or fungemia. Specific recommendations will be offered for the collection, transport, and processing of blood cultures. The existing blood culture technology will be reviewed and the relative benefits of these technologies will be compared. Procedures for the identification of pathogens will not be addressed. Antimicrobial susceptibility testing of bacteria is addressed in CLSI documents M2—Performance Standards for Antimicrobial Disk Susceptibility Tests,2 M7—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically,3 and M11—Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria.4 Antimicrobial susceptibility testing of fungi is covered in CLSI/NCCLS documents M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts5 and M38—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi.6 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 infectious agents and thus are more comprehensive than universal precautions which are intended to apply only to transmission of blood-borne pathogens. Standard and universal precaution guidelines are available from the US Centers for Disease Control and Prevention.7 For specific precautions for preventing the laboratory transmission of all infectious agents from laboratory instruments and materials and for recommendations for the management of exposure to all infectious disease, refer to the most current edition of CLSI document M29—Protection of Laboratory Workers From Occupationally Acquired Infections.8 3 Definitions antiseptic - a substance that inhibits the growth and development of microorganisms without necessarily killing them.9 automated blood culture system - a blood culture system that uses mechanical systems to incubate, agitate, and/or monitor blood culture bottles for microbial growth. bacteremia – the presence of bacteria in the bloodstream; NOTE: Bacteria isolated from blood may be the cause of sepsis, indeterminate as a cause of sepsis, or contaminants.10


Number 17 M47-A


biphasic blood culture system - a blood culture system in which a single container (vial) has separate chambers for solid- and liquid-based media; NOTE: Most biphasic systems are designed so that the solid medium can be irrigated with the liquid medium. blind subcultures – subcultures performed as a routine laboratory procedure irrespective of any objective evidence of microbial growth.10 blood culture – a specimen of blood that is submitted for bacterial or fungal culture; NOTE: This is irrespective of the number of bottles or tubes into which the specimen is divided or distributed.10 blood culture series – a group of temporally related blood cultures that are collected to determine whether a patient has bacteremia or fungemia.10

blood culture set – the combination of blood culture bottles or tubes into which a single blood specimen is inoculated.10 bloodstream infection – an infection associated with bacteremia or fungemia. breakthrough bacteremia – bacteremia that persists while a patient is receiving antimicrobial therapy for an episode of bacteremia; NOTE 1: Breakthrough bacteremia that occurs early usually is the result of inappropriate or inadequate antimicrobial chemotherapy; NOTE 2: Breakthrough bacteremia that occurs late usually is the result of a focus of infection (e.g., an abscess) that has not been drained adequately.10 chlorhexidine gluconate – the digluconate salt of chlorhexidine9; NOTE: It is used as a topical agent for cleansing and disinfecting the skin. contaminant – a microorganism isolated from a blood culture that was introduced into the culture during specimen collection or processing and that was not pathogenic for the patient from whom blood was collected10 (i.e., the isolates were not present in the patient’s blood when the blood was sampled for culture). conventional (manual) blood culture system – a blood culture system that processes bottles without the use of mechanical systems (i.e., manually). culture – 1) the intentional growing of microorganisms, such as bacteria or fungi, in a controlled environment, for purposes of identification or other scientific study, or for commercial and/or medicinal use; 2) the product resulting from the intentional growth of microorganisms. culture medium – a substance or preparation used for the cultivation and growth of microorganisms. disinfectant – a substance used to reduce the concentration of bacteria, fungi, or viruses on a surface. false positive – a positive test result for a disease or condition when the disease or condition is not present; NOTE: For blood cultures, 1) a culture that yields a microbial isolate(s) that is determined not to be the cause of sepsis, or 2) a culture with objective evidence of microbial growth (i.e., an instrument signal that indicates microbial growth) but for which subcultures and stains are negative. fungemia – the presence of fungi (yeasts or molds) in the bloodstream.10 inadequate blood volume – a blood culture bottle containing less than 80% of the recommended minimum volume indicated on the bottle label. indeterminate isolates – a microorganism of undetermined clinical importance.10


Volume 27 M47-A


lysis-centrifugation blood culture systems - blood culture systems that utilize chemicals to lyse the blood cells, thereby releasing microorganisms into the fluid, followed by centrifugation to concentrate the microorganisms in the pellet; NOTE: These systems are most often used for fungal and mycobacterial blood cultures.

manual blood culture - see conventional blood culture system. povidone iodine – a water-soluble complex of iodine with polyvinylpyrrolidone11; NOTE 1: Applied as an antiseptic in the form of solutions or ointments, it releases iodine; NOTE 2: Used as a topical agent for disinfecting the skin. sepsis – systemic inflammatory response syndrome (SIRS) plus infection.12,13 septic episode – an episode of sepsis, severe sepsis, or septic shock for which a blood culture or blood culture series is drawn; NOTE: The blood cultures may or may not yield microorganisms that are subsequently determined to be the cause of sepsis, severe sepsis, or septic shock.10

septic shock – sepsis with arterial hypotension despite adequate fluid resuscitation.12,13 severe sepsis – sepsis associated with organ dysfunction, hypoperfusion, or hypotension.12,13 subculture – a bacterial or fungal culture made from material derived from another culture, such as the blood-broth mixture in blood culture bottles or a culture made by transferring microorganisms to a fresh medium from a previous culture; a method used to prolong the life of a particular strain where there is a tendency to degeneration in older cultures.11,14 systemic inflammatory response syndrome (SIRS) – a physiologic state believed to be triggered by systemic activation of the innate immune response; NOTE: Under the current definition, SIRS is considered to be present when patients have more than one of the following clinical and/or laboratory findings: body temperature >38 °C or <36 °C; heart rate >90/minute; hyperventilation evidence by respiratory rate >20/minute or PaCO2 <32 mm Hg; and white blood cell count >12,000 cells/µL or <4000 cells/µL.12,13 terminal subculture – a subculture taken from the blood-broth mixture in blood culture bottles at the end of the routine incubation and testing period; NOTE: Because subcultures are done on blood culture when there is objective evidence of microbial growth, terminal subcultures may be done as a matter of routine on blood culture bottles lacking evidence of microbial growth (i.e., “negative” blood culture bottles). tincture of iodine – an alcoholic solution of iodine and potassium iodide9; NOTE: It is used as a topical agent for disinfecting the skin. true positive – a positive test result for a disease or condition when the disease or condition is present; NOTE: For blood cultures, a culture that yields a microbial isolate(s) that is determined to be the cause of sepsis. venipuncture – puncture of a vein 9; NOTE: A method used to collect blood specimens for culture. 3.1 Acronyms CMBCSs continuous monitoring blood culture systems CRBSI catheter-related bloodstream infection HACEK group Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella HBV hepatitis B virus


Number 17 M47-A


HCV hepatitis C virus HIV human immunodeficiency virus LIS laboratory information system VAD venous access device 4 Clinical Importance of Blood Cultures 4.1 Diagnostic Importance The presence of living microorganisms circulating in the bloodstream of a patient has substantial diagnostic and prognostic importance.15 The positive blood culture either establishes or confirms that there is an infectious etiology for the patient’s illness. Moreover, it also provides the etiologic agent for antimicrobial susceptibility testing which, in turn, allows optimization of antibiotic therapy. 4.2 Prognostic Importance From a prognostic standpoint, a blood culture that grows a clinically important pathogen indicates failure of the host’s defenses to contain the infection at its primary location or failure of the physician to remove, drain, or otherwise eradicate that focus of infection. The type of pathogen recovered from blood also provides important prognostic information.15 5 Specimen Collection and Transportation 5.1 Timing of Blood Cultures Only a few studies have tried to establish the optimal timing of blood cultures to maximize the recovery of pathogens from blood. Experimental data show that an influx of bacteria into the bloodstream is followed by a lag of approximately one hour before chills and fever develop.16 Although it has been common practice to obtain blood cultures at arbitrary intervals of 30 to 60 minutes,17 Li et al18 showed no difference in microbial recovery when blood specimens were drawn for culture simultaneously or at spaced intervals for up to 24 hours. Thompson et al observed no significant differences in positivity rates of blood cultures obtained in relation to the fever spikes of patients.19 As a practical matter, blood cultures should be obtained simultaneously (or over a short timeframe). Drawing blood at intervals is only indicated when it is necessary to document continuous bacteremia in patients with suspected infective endocarditis or other endovascular (e.g., catheter-related) infections. 5.2 Number of Blood Cultures Several studies have been published addressing the optimal number of blood cultures that are needed to detect bacteremia or fungemia. This variable is reviewed in detail by Aronson and Bor,20 who present the theoretical basis for this issue. The first publication was in 1975, when Washington reported results from 80 patients using 20-mL blood specimens.21 In that study, the cumulative yield of pathogens from three blood cultures was 80% for the first culture, 88% from two cultures, and 99% from three cultures.21 In 1983, Weinstein et al reported results from 282 patients using 15-mL blood specimens. The results from that study were similar to those reported by Washington; the cumulative yield of pathogens from these cultures was 91% from the first culture and >99% (281/282) from two cultures.22 In both of these studies, blood cultures were performed using manual blood culture systems; in 2004 Cockerill reported the results of a similar study from 163 patients when blood cultures were performed using a continuous-monitoring blood culture system (CMBCS). In that study, the cumulative yield of pathogens from three blood cultures, with a blood


Volume 27 M47-A


volume of 20 mL each (excluding patients with infective endocarditis), was 65% from the first culture, 80% from two cultures, and 96% with three cultures.23 For patients with infective endocarditis, the yield was 90% from first blood culture.23 The lower cumulative yield from the cultures performed on a CMBCS, as compared with the yields from manual blood culture systems, may be the result of a number of variables: differences in the broth media that were used, the detection mechanisms, use of different antimicrobial agents over the 20 years that these studies were performed, the volume of blood cultured, and, most importantly, different definitions of true-positive and contaminated blood cultures.21-23 The present guideline is to collect two to three sets per episode. Single blood cultures should never be drawn from adult patients; this practice results in an inadequate volume of blood cultured, and the results of single blood cultures are more difficult to interpret. Blood cultures should not be repeated for two to five days, because the blood does not become sterile immediately following the start of antimicrobial therapy. The use of so-called surveillance blood cultures has been advocated as a means to allow earlier detection of sepsis in certain patient populations—such as those in intensive care, undergoing transplantation, or with vascular catheters—or as a "test of cure." Blood cultures obtained for the prediction of septic episodes are of limited value and should not be performed routinely, as these cultures do not improve patient management but add substantial costs.24-26

Similarly, most patients with bacteremia or fungemia can be followed clinically and do not need follow-up blood cultures to document that the bacteremia or fungemia has cleared. There are, however, two exceptions to this: the first is for patients with infective endocarditis, where documenting that bacteremia or fungemia has cleared may be used to assess and guide therapy; the second is for patients with Staphylococcus aureus bacteremia not related to infective endocarditis, where positive follow-up blood cultures drawn at 48 to 96 hours were the strongest predictors of complicated S. aureus bacteremia.27 5.3 Volume of Blood for Culture The volume of blood drawn for culture is the most important variable in detecting bacteremia or fungemia.18,23,28-35 This observation is based on data published from many studies of adult patients with bacteremia and fungemia; only limited data have been published from studies of infants and young children. For adult patients, the yield of pathogens increases in direct proportion to the volume of blood that is cultured from 2 to 30 mL.18,23,28,29,33 The yield still increases when 40-mL (or even higher) volumes of blood are cultured, although the increase may no longer be in direct proportion to the volume of blood cultured.18 For pediatric patients, the limited data that have been published also indicate that the yield of pathogens increases in direct proportion to the volume of blood that is cultured.34,36 Some of these data are based on observations of the numbers of bacteria that are present in blood, with the implication that culturing higher volumes of blood containing small numbers of bacteria will improve recovery of bacteria by culture.34,36-45 For adults, the recommended volumes for blood cultures are 20 to 30 mL per culture (i.e., per venipuncture). For infants and younger children, the volume of blood drawn should be no more than 1% of the patient’s total blood volume. There are no published data for determining when volumes considered to be appropriate for adults can be used for older children. 5.4 Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles The historic practice has been to divide the blood drawn for culture equally between aerobic and anaerobic blood culture bottles. This practice was questioned in the late 1980s and early 1990s after several studies reported that the incidence of anaerobic bacteremia began to decrease, starting in the 1970s.42-47 Several studies performed at about the same time reported data to support the concept of routinely inoculating blood drawn for culture into aerobic bottles only, using anaerobic bottles only for select patients. Based on these data, a number of authors have recommended use of aerobic bottles only for routine cultures.39,45-50 A recent study reported data that raise doubts about those recommendations.49 In this study, use of paired aerobic/anaerobic blood culture bottles yielded more staphylococci, members of the family Enterobacteriaceae, and anaerobes when compared to paired aerobic blood culture bottles.50


Number 17 M47-A


Because the data are conflicting and inconclusive, and because the recommendation that anaerobic blood culture bottles be limited to use in select patient populations has never been validated by controlled clinical studies to define these patient populations, it is recommended that routine blood cultures include paired aerobic/anaerobic blood culture bottles.50-52 When less than the recommended volume of blood is drawn for culture, the blood should be inoculated into the aerobic vial first; any remaining blood should then be inoculated into the anaerobic vial. Inoculating blood culture bottles in this manner is important because most bacteremias are caused by aerobic and facultative bacteria, which will be recovered better from aerobic bottles. In addition, pathogenic yeasts are recovered almost exclusively from aerobic bottles, as are strict aerobes, such as Pseudomonas and Stenotrophomonas. For those laboratories opting to use aerobic bottles only, it is important that two bottles be used for each blood culture to help ensure that adequate volumes of blood are cultured. 5.5 Disinfection of Skin and Prevention of Blood Culture Contamination Most blood cultures are drawn by venipuncture. In order to minimize the risk of contamination with skin flora, the venipuncture site requires disinfection. A number of disinfectants have been used clinically during the past 50 years, including rubbing alcohol (70% isopropyl), tincture of iodine, povidone-iodine, iodophors, chlorine-peroxide, and chlorhexidine gluconate.39,53-59 Several studies comparing these disinfectants have been published, from which the following conclusions can be made: • Tincture of iodine, chloride peroxide, and chlorhexidine gluconate are superior to povidone-iodine

preparations. • Tincture of iodine and chlorhexidine gluconate are probably equivalent. Iodine-containing preparations require sufficient time to disinfect surfaces (30 seconds for tincture of iodine and 1.5 to 2 minutes for iodophors). Chlorhexidine gluconate requires the same amount of time as tincture of iodine, but is not associated with allergic reactions and does not need to be cleaned off the skin after the venipuncture is completed. The primary disadvantage to chlorhexidine gluconate is that it cannot be used to disinfect skin of infants less than two months of age; however, it is the recommended skin disinfectant for older infants, children, and adults. 5.6 Blood Culture Collection Blood cultures should be collected using standard precautions. Strict aseptic technique should be used throughout the procedure. Blood for culture should be drawn from veins, not arteries.20,40,53 Arterial blood cultures are not associated with higher diagnostic yields than venous blood cultures and are not recommended.53 Blood cultures obtained from indwelling intravascular access devices, such as intravenous catheters and ports, are associated with greater contamination rates than are blood cultures obtained by venipuncture.60-62 Although blood occasionally may need to be obtained from intravenous lines and similar access devices, a culture of blood from such a device should be paired with another culture of blood obtained by venipuncture to assist in interpretation in the event of a positive result. If blood cultures for bacteria or fungi are collected through an intravenous line, it is not necessary to discard the initial volume of blood or flush the line with saline to eliminate residual heparin or other anticoagulants.63 Moreover, the antimicrobial activity of heparin is effectively eliminated in protein-rich culture media.64,65

After the venipuncture site is identified, the rubber septum on the blood culture bottle(s) or tube(s) should be disinfected with 70% isopropyl alcohol and allowed to dry. The site of the venipuncture should then be disinfected; typically this means cleansing the site first with 70% isopropyl alcohol, allowing it to air dry, followed by application of the main disinfectant, then allowing that substance to sit for the recommended amount of time. The person drawing the culture should not palpate the vein after skin disinfection unless a


Volume 27 M47-A


sterile glove is worn. It is recommended that blood be drawn into a sterile syringe and then transferred to the blood culture tube or bottle. Blood can be drawn directly into collection tubes containing sodium polyanetholsulfonate (SPS), but should never be drawn into tubes containing other anticoagulants. The blood from an SPS tube can then be transferred to blood culture medium. Drawing blood directly into blood culture vials (e.g., using a needle holder designed for collecting blood into tubes) is not recommended because of the risk of reflux of the broth media back into the vein and also because the volume of blood draw into the bottle or tube cannot be controlled. Collection devices are available from some manufacturers; these should be used according to the manufacturers’ recommendations. Blood culture bottles should be kept upright when these devices are used. Blood culture bottles/tubes should be inverted gently several times to prevent clotting. For many years it was standard practice to change needles before inoculating blood culture bottles/tubes. Although several studies showed that using the same needle to both draw blood as well as to inoculate blood culture bottles did not significantly increase contamination rates,66-68 a meta-analysis did show slightly higher contamination rates when needles were not changed.69 Because of the risk of sustaining an accidental needlestick, many facilities activate the safety feature of the needle, remove and discard the contaminated sharp, and apply a safety transfer device to the syringe before filling the bottles. Regardless of the method used to collect blood cultures, laboratories should validate that their process is effective in minimizing contamination rates to an acceptable range, typically ≤ 3%. 5.6.1 Transporting Specimens to the Laboratory Blood culture bottles/tubes should be sent to the laboratory within two hours; delays in entering blood culture bottles into continuous-monitoring blood culture instruments (particularly if the bottles are incubated at 35 to 37 °C) may delay or impede detection of growth. Holding bottles at room temperature is not recommended for anything longer than a few hours. Blood culture bottles/tubes should never be refrigerated or frozen after they have been inoculated; refrigeration or freezing can kill some microorganisms, and freezing fluid-filled containers may cause the container to break. Blood culture specimens collected for lysis-centrifugation also should not be held for more than eight hours; holding cultures longer than this may decrease the yield or delay the growth of certain bacterial pathogens.70-73 5.7 Specimen Rejection Criteria for Blood Culture Specimens Blood culture specimens that meet the following criteria should be rejected and another specimen collected: • incorrectly labeled or unlabeled vials; • broken, damaged, or leaking bottles/tubes; • clotted tubes; or • tubes containing anticoagulants other than SPS (refer to Section 6.1, Critical Factors in the Recovery

of Pathogens From Blood Specimens). 6 Methods and Procedures 6.1 Critical Factors in the Recovery of Pathogens From Blood Specimens VOLUME The optimal recovery of bacteria and fungi from blood depends on culturing an adequate volume of blood. The direct correlation between the volume of blood cultured and yield relates to the low number of colony forming units (CFU) in a milliliter of adult blood. For each additional milliliter of blood cultured,


Number 17 M47-A


the yield of microorganisms recovered from adult blood increases.30,33 Pediatric patients often have higher numbers of microorganisms in their blood, and satisfactory results are achieved with smaller blood culture volumes. However, low-level bacteremia may also occur in children, and the recommended volume of blood to collect is based on the patient’s total blood volume and age.38 Section 5, Specimen Collection and Transportation, discusses the recommended blood volumes to collect safely from adult and pediatric patients in order to maximize yield. Laboratories must also follow manufacturers’ instructions and collect the recommended blood volumes for the system. Surveys74 indicate that laboratories often receive less than the recommended culture amounts; laboratories should process these cultures and note on the report that the volume collected was less than optimal to achieve adequate sensitivity. Monitoring blood volume and providing feedback to staff should be part of an institution’s quality assurance program to improve patient care and resource utilization.75 BLOOD-TO-BROTH RATIO Normal human blood contains substances that inhibit microbial growth. Among these are complement, lysozyme, phagocytes, antibodies, and antimicrobial agents (if the patient is receiving treatment with antimicrobial agents prior to collection of blood for culture). To reduce the concentration of these inhibitory factors, and thereby minimize their inhibitory activity, blood should be diluted in broth media at a blood-to-broth ratio of 1:5 to 1:10.76 Failure to maintain this ratio may result in false-negative cultures from underfilling bottles. Some commercial blood culture systems use a blood-to-broth ratio that is less than 1:5—a ratio that is acceptable because of the addition of proprietary substances that bind to and inactivate the inhibitory substances present in blood. Pediatric blood specimens can be inoculated into pediatric vials designed to maintain the blood-to-broth ratio with smaller blood volumes, but there is minimal data to indicate that use of these bottles improves microbial recovery. MEDIA (TYPES, INDICATIONS/FORMULATIONS) Numerous broth medium formulations are available for conventional and automated blood culture methods. Soybean-casein digest broth is the most widely used basal medium, and brain-heart infusion (BHI), Columbia, Brucella, thiol, thioglycolate, and supplemented peptone broths have also been used to recover aerobic and anaerobic microorganisms. Several Middlebrook formulations are available for automated and commercial blood culture systems to enhance recovery of mycobacteria. Each automated blood culture system has its own specific medium formulations to culture aerobic and anaerobic microorganisms. Several well-controlled, large clinical studies comparing the systems and media indicate that most perform acceptably.39,77-79 However, comparisons are complicated by the variations in the same basal medium formulations marketed by different manufacturers. Manufacturers enrich their media with a variety of proprietary supplements, varying concentrations of SPS, and different headspace atmospheres to enhance microbial growth.79 Regardless of the method used, a combination of complementary medium formulations may be necessary to optimize recovery rates of all potential pathogens, because no single medium or system recovers all microorganisms optimally. Recommendations for quality control requirements for media, including procedures for receiving media from the manufacturer (e.g., visual inspection and contamination checks) and quality assurance guidance, are outlined in CLSI document M22—Quality Control for Commercially Prepared Microbiological Culture Media.80 Other blood culture methods include biphasic systems containing both broth medium and agar or a solid surface, and lysis-centrifugation.39,77,78 Details about these systems and how to select a system are discussed in Section 6.2, Blood Culture Methods.


Volume 27 M47-A


ADDITIVES (ANTICOAGULANTS, RESINS, CHARCOAL) All broth blood culture media contain anticoagulants to inhibit blood clot formation. The most effective anticoagulant, SPS, neutralizes lysozyme, inhibits phagocytosis, inactivates some aminoglycosides, and inhibits part of the complement cascade.79 The typical concentration of SPS ranges from 0.025 to 0.05%, although some commercial systems have concentrations as low as 0.006%. Despite inhibiting the growth of several bacteria, including Neisseria species, Peptostreptococcus anaerobius, Moraxella catarrhalis, and Gardnerella vaginalis,81-83 SPS remains the most common anticoagulant and has been shown to increase the rate and speed of recovery of both gram-positive and gram-negative microorganisms. Heparin, ethylenediamine tetraacetic acid (EDTA), and citrate are toxic to microorganisms; blood should not be inoculated into blood collection tubes containing these anticoagulants. In addition to diluting blood in broth, and adding SPS to reduce the inhibitory effects of antimicrobials in blood cultures, some manufacturers have added antimicrobial-binding or adsorbing agents to their systems to enhance the recovery of microorganisms from the blood of patients receiving antimicrobial therapy. Compared to media without additives, more microorganisms, particularly staphylococci and yeasts, are recovered from formulations with additives.21,73,84,85 However, these media are more expensive, and there are increased costs to the laboratory to work up the increased numbers of contaminants that also are recovered. INCUBATION CONDITIONS Temperature Bacterial and fungal blood cultures should be incubated at 35 °C after collection and delivery to the laboratory. Although delays in incubating cultures after collection do not affect yield, delays should be minimized to avoid prolonging the time to detection of microbial recovery. Because there are conflicting reports about the effects of delays on microbial recovery in lysis-centrifugation tubes, these tubes should be processed within eight hours after blood collection.73 Atmosphere To accommodate the volume of blood to be inoculated into a bottle, a portion of the headspace atmosphere is evacuated to create a partial vacuum; thus, blood culture bottles contain an atmosphere in the bottle headspace that has a lower pressure than the atmosphere. With some manual systems, one bottle must be vented with a needle to create an aerobic atmosphere. Automated blood cultures are also manufactured with varying amounts of carbon dioxide added to the ambient atmosphere in the headspace of the aerobic vial. The atmosphere in the anaerobic bottle headspace contains carbon dioxide and nitrogen. See Section 5.4, Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles, for additional discussion of this issue. Length of Incubation For conventional manual methods, incubation for seven days is recommended; some slower-growing, fastidious bacterial pathogens (Bartonella, Legionella, Brucella, Nocardia) and dimorphic fungi may require longer incubation. The standard incubation period for routine blood cultures performed by automated systems is five days,39,86 including cultures for Brucella spp., Haemophilus spp., Actinobacillus spp., Cardiobacterium spp., Eikenella corrodens, Kingella spp., and the nutritionally variant streptococci (e.g., Abiotrophia and Granulicatella spp.). Prolonged incubation and testing periods to recover bacteria


Number 17 M47-A


from patients suspected of having infective endocarditis also appear unnecessary, as discussed in Section 6.5.3, Special Considerations: Infective Endocarditis.23,87

Published data88,89 suggest that four or even as few as three days90,91 may be adequate to recover 95 to 97% of clinically significant bacteria and yeasts from some systems. However, the current recommendation remains five days of incubation for automated systems. Adequate data from studies validating a shorter incubation time may satisfy accrediting bodies and permit laboratories to adapt shorter blood culture monitoring protocols. AGITATION Studies indicate that bottle agitation, particularly during the first 24 hours of incubation, increases yields and improves the speed of detecting microorganisms in aerobic bottles.92,93 This presumably is due to increased oxygenation of the broth medium. The current CMBCSs agitate bottles continuously. Agitation of anaerobic bottles does not adversely affect growth of bacteria. MONITORING FREQUENCY/SUBCULTURES Conventional blood culture methods require daily or more frequent visual examination for evidence of macroscopic growth. Cultures should be examined with bright fluorescent bulbs or with incandescent, transmitted light for turbidity, hemolysis, gas production, surface colony formation, or change in blood color. Subcultures to blood and chocolate agar and/or Gram or acridine orange stains of aerobic vials after 24 to 48 hours of incubation facilitate early detection of microorganisms. Blind/terminal subcultures of negative cultures are considered unnecessary by many and may increase contamination rates and needlestick injuries.39 Repeat subcultures of previously positive blood cultures are ineffective for detecting polymicrobial bacteremias. Some manual blood culture systems, particularly those with agar media that are an integral part of the bottle, also require daily visual inspection to detect evidence of microbial growth. CMBCSs monitor aerobic and anaerobic vials at regular 10- to 24-minute intervals for evidence of growth. Subcultures are performed when a positive signal indicates growth. Routine, blind, or terminal subcultures from negative blood cultures performed on automated systems are unnecessary when cultures have been monitored for at least five days.94 Subcultures should be held for a period determined by the type of organism(s) being considered. 6.2 Blood Culture Methods Blood cultures can be processed in the laboratory either by manual or by automated methods. For the sake of discussion, the term manual blood culture techniques will refer to methods that do not employ instrumentation to monitor the growth of microorganisms. Manual blood cultures can be further subdivided into visually monitored (‘conventional’) broth-based cultures, cultures using biphasic media, and the lysis-centrifugation technique. Automated CMBCSs use instrumentation to detect microbial growth in broth media by monitoring byproducts of bacterial or fungal metabolism. 6.2.1 Manual Blood Cultures 6.2.1.1 Conventional Broth Cultures Conventional blood cultures typically consist of paired aerobic and anaerobic bottles. Standard medium choices include brain-heart infusion, Columbia, or trypticase soy broths for aerobic to microaerophilic microorganisms, and thiol or thioglycolate formulations for microaerophilic to anaerobic bacteria. Each medium may be supplemented with additional factors (e.g., hemin, vitamin K1, L-cysteine) to support the


Volume 27 M47-A


growth of fastidious microorganisms. The volume of broth selected for conventional cultures should reflect the anticipated draw volume so that a blood-to-broth ratio ranging from 1:5 to 1:10 is achieved.53 Commercially prepared blood culture bottles have broth volumes ranging from 18 to 100 mL. The atmosphere in the headspace of commercially prepared blood culture bottles is evacuated to remove O2 and replaced with CO2 and N2 at pressures lower than atmosphere pressure. Following inoculation, the bottle designated for aerobic microorganisms may require venting to reintroduce O2 for aerobic metabolism (refer to the manufacturer’s recommendations). After inoculation with blood, bottles are incubated at 35 °C and examined visually for evidence of bacterial growth. Changes that suggest microbial growth include turbidity of the blood-broth mixture, growth of microcolonies, hemolysis, color changes, and gas production. Initial inspection occurs after 12 to 24 hours of incubation with twice daily inspection for two days followed by daily inspection for days 3 to 7.95 Gram staining of the broth along with subculture during the initial examination can hasten early detection of growth in conventional cultures.96,97 However, this practice does little to improve detection of growth thereafter, and terminal subcultures of negative bottles should be discouraged.98,99 Bottles should be incubated for seven days. 6.2.1.2 Biphasic Cultures The use of a blood culture bottle containing both agar and broth medium was initially designed for the recovery of Brucella species from blood.100 These “biphasic” blood culture systems have also been shown to be effective for the recovery of fungi and bacteria from blood. The popularity of this method has been hindered by the complexity of bottle preparation and the relative lack of commercial sources for the bottles. Currently, a commercially prepared system is available that consists of a broth culture bottle and an attachable cylinder housing a paddle coated with an agar-based medium on either side. Bottles are available with a variety of broth and volume combinations to support use for pediatric or adult patients and a range of aerobic to anaerobic microorganisms. Formulations are also available for the recovery of mycobacteria. To initiate a culture, blood is introduced into the broth bottle, and the agar-containing cylinder is attached to the top of the bottle. Subcultures are accomplished simply by inverting the bottle and allowing the blood/broth mixture to wash over the agar surface. The assembly is then incubated in the upright position and observed for the growth of microorganisms either in the broth (as described for conventional blood culture systems) or on the agar surface. Compared to conventional manual blood culture bottles, biphasic blood culture systems provide improved recovery of aerobic and facultative anaerobic bacteria and fungi; biphasic systems also have decreased time to detection and allow for recovery of isolated colonies.101-110 However, the commercial biphasic systems yield higher contamination rates and do not perform as well for the recovery of anaerobic bacteria.105,106,108,110 Similar to manual blood cultures, biphasic media should be monitored visually for a total of seven days. Biphasic bottles should be incubated for seven days. 6.2.1.3 Lysis-Centrifugation Lysis-centrifugation refers to a blood culture method in which microorganisms are released from lysed blood and are then separated from blood components by centrifugation into a density layer at the bottom of the collection tube. Microorganisms concentrated into this layer are then transferred from the lysis-centrifugation tube to solid media. The performance of the lysis-centrifugation method is equivalent to that of biphasic blood culture systems in terms of time to recover isolated colonies and the overall recovery rate of filamentous fungi and yeasts.


Number 17 M47-A


6.2.2 Continuous-Monitoring Blood Culture Systems79 Two of the three commercially available CMBCSs rely on detection of increased CO2 production by microorganisms. Both of these systems are modular in design to support low or high bottle capacity, depending on the number of blood cultures that need to be processed. A variety of media formulations are available for use with each system, including aerobic and anaerobic formulations with or without additives designed to enhance recovery of microorganisms from patients receiving antimicrobial therapy. Media for the recovery of mycobacteria from blood have also been developed for both systems. Aerobic bottles are mechanically aerated during the course of incubation to increase the pO2 in the broth medium. The growth detection apparatus of both systems is similar. A sensor located on the bottom of the blood culture bottle is separated from the liquid medium by a membrane that is selectively permeable to CO2. As CO2 is produced by actively metabolizing microorganisms, it diffuses across the membrane and displaces hydrogen ions, causing acidification of the sensor with a concomitant shift in the emission spectrum of the detection dye (either colorimetric or fluorometric, depending on the system). A light-emitting diode illuminates the sensor every ten minutes, and reflected light is captured by a photo detector. The change in signal intensity is proportionate to the emission shift of the sensor dye, which is related to the amount of dissolved CO2 in the culture medium. Data is collected and transferred to a computer, where software algorithms analyze the data and recognize either increasing rates or sustained production of CO2. Bottles matching criteria for significant CO2 increases are signaled for removal and subculture. The third CMBCS detects microbial growth by measuring the headspace gas pressure within the blood culture bottle. This pressure change is attributable to microbial O2 consumption and/or N2, H2, or CO2 production. Aerobic and anaerobic bottles are monitored every 12 and 24 minutes, respectively, and pressure readings are translated into growth curves over time after correction for atmospheric pressure changes. A software algorithm translates pressure changes into likely microbial growth patterns and signals a positive bottle for removal and subculture. At present, two medium formulations in two bottle sizes are approved for general use with this system. The advantages to CMBCSs include decreased staff needs for laboratories processing large numbers of blood cultures, a reduction in contamination rates because monitoring is noninvasive, and decreased time to detection of positive cultures secondary to the increased rate of monitoring. However, the latter is dependent on the frequency of instrument inspection and subculture of flagged bottles. On shifts when microbiologists are not available, central laboratory personnel should be scheduled to inspect the instrument(s), remove positive bottles for subculture and prepare Gram stains for evaluation at a later time, or be trained to examine and report Gram-stained smears. The ability to prepare and interpret Gram stains and smears on all shifts can affect information delivery time and changes in therapeutic options.111 6.2.3 Preservation of Isolates for Further Testing Blood culture isolates are of unique value in patients with infectious processes. Microorganisms may be used for research, clinical, epidemiological, educational, microbiological, therapeutic, and commercial reasons. During the period of active testing, preserving isolates is most conveniently accomplished by serial subculture to appropriate media on a periodic basis. The frequency of subculture varies for different organisms because of intrinsic viability, as well as phenotypic and genotypic stability. Subculture every two to three days is likely to preserve the organism, at least through several passages. Laboratories should develop procedures for short- and long-term storage of blood culture isolates to ensure access to viable organisms in case additional testing is needed. All blood culture isolates, except known contaminants, should be maintained for seven to ten days. This is most important for microorganisms whose clinical significance cannot be determined by initial blood culture results. The simplest approach to the storage of bacteria and yeasts for short periods of time is to keep them on agar media. These cultures may be stored at room temperature, but storage at 5 to 8 °C may enhance the


Volume 27 M47-A


duration of viability. Cultures maintained in this fashion are subject to drying, but most clinically important bacteria will survive for days to weeks. Sealing the cultures to prevent water loss from the medium may allow preservation of viability during intermediate-term storage. There are no detailed descriptions of the adequacy of these approaches, and individual laboratories will have variable success, depending on internal environmental conditions. If possible, laboratories should have procedures for both short- and long-term storage of pathogenic blood culture isolates in case additional testing is needed. This is particularly important for isolates that may be associated with repeated/relapsing infections in an individual, changing resistance patterns caused by antimicrobial therapy, and confusing interpretation (e.g., patients who may also have endogenous infection with the same species). Ultra-low temperature (e.g., -70 oC) storage or lyophilization is recommended for long-term storage. Sterile cryoprotective agents, such as 20% skim milk, 10% glycerol, and 5% DMSO, improve the viability of organisms preserved at ultra-low temperatures, but the optimal agent varies by organism group. To prepare isolates for long-term storage, an aliquot of late log-phase growth of a fresh broth culture is centrifuged and the pellet resuspended in several milliliters of fresh broth medium to which the cryoprotective agent has been added at the appropriate concentration. Aliquots, typically ~0.5 mL, of this suspension are transferred into cryopreservation vials. Such vials must be able to withstand ultra-low temperatures, be securely sealed, and provide a suitable surface for labeling the contents of the vial. A record of the location of each preserved isolate should be prepared to facilitate retrieval of isolates as needed. When needed, cryopreserved cultures should be rapidly thawed by use of a 35 °C waterbath, followed by subculture appropriate for the organism. Lyophilization may provide the most dependable method for long-term storage of many bacteria, but is not appropriate for microorganisms damaged by removal of intracellular water, such as molds and some bacteria. Specialized equipment, storage vials, and procedures are required for lyophilization. Additional detailed methods for microorganism storage are available elsewhere.112-114

6.3 Fungal Blood Cultures 6.3.1 Population at Risk The increased incidence of infections with documented fungemia corresponds to improvements in blood culture techniques and changes in the medical management of patients.15,45 Fungemia with yeasts is well-documented in patients with traumatic injuries or ulcerations of the gastrointestinal mucosa, recipients of broad-spectrum antibacterial antimicrobial agents or hyperalimentation, patients with intravenous access devices, and those with immunosuppressive diseases. In recent years there has been an increase in the recovery of dimorphic fungi (e.g., Histoplasma, Blastomyces, Coccidioides) and filamentous fungi (e.g., Fusarium, Scedosporium) in blood cultures of patients with AIDS, hematologic malignancies, bone marrow and solid organ transplantations, and other causes of severe immune compromise. Although infections with Aspergillus and Zygomycetes are common in severely immunocompromised patients, documentation of fungemia in these patients is infrequent. The yeasts most commonly isolated in blood cultures include Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Cryptococcus neoformans. Other Candida species (e.g., Candida krusei, Candida lusitaniae, Candida guilliermondii), Malassezia furfur, Rhodotorula species, and Trichosporon species are recovered less frequently.115,116 Histoplasma capsulatum is the most commonly isolated dimorphic fungus; Blastomyces dermatitidis and Coccidioides immitis are recovered less frequently.101,117,118 Fusarium and Scedosporium are the most commonly isolated filamentous fungi, with Exophiala, Rhinocladiella, and Aspergillus recovered less frequently.119-121


Number 17 M47-A


6.3.2 Critical Factors Numerous studies have documented better recovery of yeasts in aerobic broths compared with anaerobic broths.45,46,115 Although improved recovery of yeasts was obtained with biphasic blood culture systems using both agar and broth media, this is generally unnecessary with current commercial blood culture systems. Agitation of broth-based systems increases aeration and improves recovery of yeasts.122 This can be accomplished either through the use of automated commercial blood culture systems or mechanical agitation of manual broth systems for the first 24 hours of incubation. Most yeasts and yeast-like fungi will be detected within two to five days of incubation, although some yeasts (e.g., C. glabrata, C. neoformans) may require extended incubation. Optimal growth of M. furfur requires supplementation of media with lipids (e.g., olive oil). Although dimorphic fungi can grow in most commercially prepared aerobic blood culture broth media, detection may require up to two to four weeks of incubation. For this reason, use of broth-based automated blood culture systems is unreliable. If infection with a dimorphic or filamentous fungus is suspected, blood should be processed by lysis-centrifugation and plated onto agar media.70,118 Although some fungi (e.g., Fusarium and Scedosporium) may grow within three to five days, cultures should be incubated for a minimum of four weeks. 6.3.3 Methods—Manual Three types of manual blood culture systems are used for the detection of fungemia: 1) bottles of nutrient broth, 2) biphasic systems, and 3) lysis-centrifugation systems. Yeasts can be satisfactorily recovered in any of the three systems; however, dimorphic and filamentous fungi can only be recovered reliably in biphasic broths and in the lysis-centrifugation system. A variety of aerobic media have been used with equivalent success for the detection of yeast infections. Anaerobic broths should not be used. Biphasic systems are acceptable for the recovery of yeasts and dimorphic and filamentous fungi, although incubation of the cultures for four weeks is required for reliable detection of dimorphic fungi. For optimum performance of the biphasic systems, gentle continuous mixing of the bottles for the first 24 hours of incubation is required.122 Poor recovery of dimorphic fungi in biphasic systems can be attributed to an insufficient incubation period. Dimorphic and filamentous fungi are detected earlier in lysis-centrifugation systems than with other manual blood culture systems. For optimal recovery of fungi in lysis-centrifugation systems, the concentrated blood sediment should be inoculated onto multiple agar-based media and incubated at both 27 to 30 °C and 35 to 37 °C.123 6.3.4 Methods—Automated With the current CMBCSs, recovery of yeasts is best in aerobic broth formulations. In some studies, the use of factors that inactivate antimicrobial agents has improved recovery and time to detection of yeasts.85 In general, special media formulated for the recovery of yeasts are unnecessary.124,125 6.4 Mycobacterial Blood Cultures 6.4.1 Population at Risk The incidence of positive blood cultures with mycobacteria was relatively uncommon until the advent of the AIDS epidemic. Mycobacteremia is also documented in patients with other immunosuppressive diseases (e.g., leukemia, severe combined immunodeficiency syndrome, multiple myeloma, and other solid tumor malignancies), recipients of high-dose steroid therapy or cytotoxic chemotherapy, and patients with long-term vascular access devices. Documented mycobacteremia with Mycobacterium tuberculosis is relatively uncommon in developed countries. However, in developing countries recovery of this organism in blood cultures from immunocompromised patients is commonplace.124 In contrast, Mycobacterium avium complex (MAC) is


Volume 27 M47-A


the most common mycobacterium isolated in the blood of immunocompromised patients in developed countries, although in recent years the incidence of mycobacteremia has decreased.126-128 Other slowly growing mycobacteria, including Mycobacterium kansasii, Mycobacterium simiae, Mycobacterium xenopi, and Mycobacterium genavense, have been recovered from immunocompromised patients with disseminated disease.127,128 Bacteremia with rapidly growing mycobacteria (e.g., M. fortuitum, M. chelonae, M. abscessus, and M. mucogenicum) is more commonly associated with contamination of long-term intravascular catheters and prosthetic devices.129 6.4.2 Critical Factors Mycobacteria are not particularly fastidious microorganisms and can grow in conventional blood culture broths if the incubation period is extended.130 Despite this observation, optimal recovery of mycobacteria from blood specimens requires supplementation of broth cultures with fatty acids (e.g., oleic acid), albumin, and carbon dioxide. Some species (e.g., M. genavense, M. haemophilum) require supplements such as mycobactin and hemin, hemoglobin, or ferric ammonium citrate. Whereas some mycobacterial species have a temperature preference of 25 to 30 °C, strains associated with mycobacteremia can be recovered at 35 to 37 °C. Because mycobacteria have a slow generation time, all cultures should be incubated for a minimum of four weeks. 6.4.3 Methods—Manual Blood specimens must be processed with a lytic agent to release intracellular bacteria before culture media are inoculated. Blood specimens should be collected in either a sterile tube with an anticoagulant (e.g., SPS, heparin, or citrate but not EDTA) or a lysis-centrifugation tube. Whole blood should not be directly inoculated onto solid media or into broths without a lytic agent. Blood specimens collected in a lysis-centrifugation tube can be inoculated onto a variety of solid, broth, or biphasic media; however, recovery of mycobacteria in specimens processed on solid media is inferior to recovery in broth and biphasic systems.131,132 Detection of positive cultures in the biphasic systems is slower than in broth-based automated systems.133,134 6.4.4 Methods—Automated A variety of automated commercial systems have been developed for the recovery of mycobacteria. Each manufacturer has developed broth-based media supplemented with various growth factors and antimicrobial agents that are used specifically with their culture systems. Some systems require the initial lysis of blood before inoculation into the broth cultures; whereas in other systems anticoagulated, whole blood can be processed. Some differences have been reported in the overall detection and time to detection in these systems135-137; however, extensive comparisons have not been performed. 6.5 Special Topics 6.5.1 Pediatric Blood Cultures As for adult patients, two to three blood cultures should be collected within a 24-hour period. Because anaerobic bacteria are rarely recovered in pediatric patients, some investigators have recommended the use of aerobic bottles only.138,139 Anaerobic bottles may be considered in high-risk groups that include neonates from mothers that have had prolonged rupture of membranes during childbirth or maternal chorioamnionitis; chronic oral or sinus infection; cellulitis (especially perianal and sacral); abdominal signs and symptoms; bite wounds; septic phlebitis; and neutropenic patients receiving steroids.139 The same methods of skin antisepsis for adults apply to pediatrics, with the exception in neonates with the potential to develop subclinical hypothyroidism due to iodine.140 For all patients, topical iodine


Number 17 M47-A


compounds must be completely removed after phlebotomy. Chlorhexidine gluconate as a skin antiseptic is approved for use in pediatric patients two months of age and older. For patients who are younger than two months of age, use of 70% isopropyl alcohol is an acceptable alternative for skin disinfection. As with recommendations in adults, blood drawn from an intravenous catheter should be accompanied by a peripheral draw. Pediatric blood cultures differ from adult blood cultures primarily in the amount of blood obtained for culture. Many of the studies in adults indicating blood volume, ratio of blood to broth, number and timing of cultures, and the need to use blood culture bottles specifically designed for adult blood cultures are lacking for pediatrics. Caution must be used in extrapolating these data to pediatric patients. It has been common practice to draw the minimum amounts of blood because of the: 1) smaller volumes of blood in pediatric patients; 2) difficulty in phlebotomy; 3) potential for increased transfusions due to the amounts of blood drawn for all laboratory procedures; and 4) presumed high levels of bacteria in the blood in pediatric patients with bacteremia.141 While many pediatric infections are characterized by high numbers of microorganisms in the blood, a small but significant number of infections have relatively low numbers of microorganisms.36-38,142-145 Processing larger blood volumes increases the pathogen recovery yield,138,142,146-148 and decreases the time to detection.36,146,149 As stated previously, for infants and younger children, the volume of blood to be drawn for culture should not exceed 1% of the patient's blood volume. On an individual basis, suggested blood culture volumes to be drawn have been based on other clinical parameters, such as patient weight and hematocrit.38,147,150

6.5.2 Catheter-Related Bloodstream Infections Catheter-related bloodstream infections (CRBSIs) are common causes of healthcare-associated infections. It is estimated that there are more than 250,000 cases of CRBSI annually in the United States,151 with an attributable mortality of 12 to 35%.152 Important risk factors for CRBSI include: type of catheter (non-tunneled central venous catheters long-term, tunneled central venous catheters short-term, peripheral catheters), duration of catheter placement, and site of insertion.153 Despite their frequent occurrence, CRBSIs are difficult to diagnose. Clinically, there may be absence of inflammation around the catheter exit site and the presence of only nonspecific clinical signs suggestive of sepsis. Documentation of these infections in the clinical microbiology laboratory is also problematic due to the lack of a well-established gold standard for laboratory diagnosis. Diagnosis of catheter exit site and tunnel tract infections will not be covered in these guidelines. There have been a number of proposed methods for diagnosing these infections in the clinical microbiology laboratory. These methods include: semiquantitative and quantitative cultures of catheter segments; paired peripheral and catheter blood cultures; quantitative peripheral and catheter blood cultures; differential time-to-positivity for peripheral versus catheter blood cultures; and endoluminal brush staining with acridine orange, among others. A meta-analysis of the various methods used to diagnose CRBSI was published in 1997.154 Although that review failed to show conclusively which of these methods was best for diagnosis of CRBSI, it did conclude that a quantitative culture was the most accurate method for catheter-segment culture, while unpaired quantitative catheter blood culture was the single most cost-effective test, especially for long-term catheters. Diagnostic methods that rely on catheter-segment cultures, however, require removal or exchange of the catheter, and should not be performed in the absence of simultaneous peripheral (i.e., obtained by venipuncture) blood cultures. It has been estimated that 75 to 85% of catheters are removed unnecessarily during evaluation of new fever.155 In order to avoid the unnecessary removal of a central venous catheter, methods that permit the diagnosis with the catheter in place may be attempted. Furthermore, because removal of a surgically implanted catheter is frequently a management challenge, it is important to distinguish CRBSI from skin contamination, catheter colonization, or infection from a source other than the catheter.


Volume 27 M47-A


6.5.2.1 Recommendations for Short-term Peripheral Catheters Obtain two sets of peripheral blood cultures from the patient via venipuncture. Remove the catheter aseptically and culture by the semiquantitative method of Maki156 (with these catheters it is usually the external surface of the catheter that is colonized, leading to infection). • Interpretation of culture results:

— If one or more blood culture sets are positive AND the catheter-segment culture is positive (>15 colonies for semiquantitative) for the same organism: suggestive of CRBSI.

— If one or more blood culture sets are positive AND the catheter-segment culture is negative:

inconclusive; however, suggestive of CRBSI if positive for Staphylococcus aureus or Candida spp. AND in the absence of any other identifiable source of infection.

— If both blood culture sets are negative AND the catheter-segment culture is positive, despite the

colony count: suggestive of catheter colonization, not CRBSI. — If both blood culture sets are negative AND the catheter-segment culture is negative: CRBSI is

unlikely. 6.5.2.2 Recommendations for Nontunneled and Tunneled Central Venous Catheters and Venous Access Ports (VAP) Obtain at least two sets of blood cultures from the patient suspected of having a CRBSI, with at least one set drawn from a peripheral venipuncture and labeled as such. The other set should be drawn aseptically from the catheter hub or through the VAP septum, close to the same time of collection of the peripheral set and labeled as such. • Interpretation of culture results (Method 1):

— If both sets recover the same organism (as determined by identification and antimicrobial susceptibility profiles): suggestive of CRBSI, in absence of any other identifiable source of infection.

— If both sets are positive for the same organism AND the set drawn through the catheter becomes

positive >120 minutes earlier: suggestive of CRBSI, in absence of any other identifiable source of infection. (If the differential time to positivity is <120 minutes, a CRBSI is still possible, if both sets are positive for an organism with the same identification and antibiotic susceptibility profile.)

— If both sets are positive AND the set drawn through the catheter has at least a fivefold greater

number of CFUs/mL: suggestive of CRBSI, in absence of any other identifiable source of infection. This method requires use of a manual quantitative blood culture system, such as lysis/centrifugation method.

— If only the blood culture set drawn from the catheter becomes positive: inconclusive for CRSBI,

and suggests either colonization of the catheter or contamination during the collection of the culture.

— If only the blood culture set drawn peripherally becomes positive: inconclusive; however,

suggestive of CRBSI if positive for Staphylococcus aureus or Candida spp. AND in the absence of any other identifiable source of infection. Documentation of CRSBI would require a positive semiquantitative or quantitative culture of the catheter segment with the same organism, or


Number 17 M47-A


additional positive catheter or peripheral blood cultures with the same organism, in the absence of any other identifiable source of infection.

— If both sets are negative: CRBSI is unlikely.

Or, • Obtain two sets of blood cultures aseptically through independent peripheral venipunctures. • Remove the suspected catheter and aseptically cut the distal 5-cm segment of the catheter and submit

it to the laboratory for culture by either Maki’s semiquantitative roll-plate method, or quantitative culture following vortexing or sonication.

• Interpretation of culture results (Method 2):

— If one or more of the blood culture sets AND the catheter-segment culture are positive with the same organism, as determined by identification phenotype and antimicrobial susceptibility profiles, then a CRSBI is likely.

— If one or more of the blood culture sets are positive AND the catheter-segment culture is

negative, this may represent a CRSBI if positive for Staphylococcus aureus or Candida spp. AND in the absence of any other identifiable source of infection. Documentation of CRSBI would require additional positive peripheral cultures with the same organism, in the absence of any other identifiable source of infection.

— If the blood culture sets are negative AND the catheter-segment culture is positive: suggests

colonization of the catheter, as opposed to a CRBSI. — If both blood culture sets and the catheter-segment culture are negative: a CRBSI is unlikely.

Method 1 might be appropriate in those instances in which an attempt is being made to save the catheter from having to be removed from the patient. If the decision has been made to remove the suspected catheter from the patient, then Method 2 might be more appropriate. 6.5.3 Special Considerations: Infective Endocarditis Blood culture results are critical to the diagnosis and management of patients with infective endocarditis. It therefore is imperative that optimal procedures are used, such as those stated in the earlier sections of this guideline. If optimal culture techniques are used, positive blood cultures will be obtained in over 90% of infective endocarditis cases.87 The following recommendations are those that specifically apply to the diagnosis of infective endocarditis. When to Obtain Cultures The first issue when evaluating a patient with suspected infective endocarditis is to determine when to obtain the blood cultures. The continuous nature of most bacteremias associated with infective endocarditis, however, renders timing less important. • Acute Infective Endocarditis In cases of suspected or known infective endocarditis that may be caused by highly virulent pathogens such as Staphylococcus aureus, blood cultures should be drawn immediately to avoid unnecessary delays in treatment.


Volume 27 M47-A


Recommendation: Obtain blood culture sets within a 30-minute period before administration of empiric antimicrobial agents. • Subacute Infective Endocarditis If the patient presents with complaints that are suggestive of subacute disease, there is no urgent need to obtain the cultures before starting initiation of empiric therapy. For these infections, it is far more important to attempt to establish the microbiological diagnosis. Recommendation: Obtain blood culture sets with the sets spaced 30 minutes to one hour apart. This may help document a continuous bacteremia, which may provide additional clinical value, especially if the echocardiogram is negative or equivocal. Skin Antisepsis Adequate skin disinfection is particularly important when evaluating patients with suspected infective endocarditis. In patients who present with subacute infective endocarditis and have a prosthetic heart valve(s), the principal pathogens are skin flora such as coagulase-negative staphylococci. Therefore, it is especially important that blood cultures should be obtained by venipuncture and not from indwelling intravascular devices. Catheter-drawn blood cultures are associated with an increased risk of contamination, which might lead to a false conclusion of infective endocarditis. Recommendation: Following adequate skin antisepsis, obtain blood for culture from separate, peripheral venipuncture sites. Do not obtain blood for culture from indwelling catheters. Number of Blood Cultures The optimal number of blood cultures to be drawn varies, but a single blood culture set clearly is inadequate. It has been demonstrated that positive blood cultures due to skin contaminants usually result in a single-positive blood culture set when multiple sets were obtained.15 Therefore, multiple sets would help a clinician to distinguish a “false-positive” blood culture, due to skin contaminants, from “true-positive” blood cultures. The other reason that multiple blood culture sets should be obtained is due to the increased volume of blood cultured, which is the single most important factor in the recovery of microorganisms from blood. A single blood culture, therefore, would not enable determination of possible continuous bacteremia, would not enable distinction between contamination and true bacteremia, and would not provide the appropriate volume. Because subacute infective endocarditis has the highest pretest probability of a positive blood culture, a total of three to five blood culture sets should suffice. If initial blood cultures are negative, alternative diagnostic strategies should be considered. Recommendation: Initially obtain three blood culture sets from patients presenting with possible infective endocarditis. If those sets are negative at 24 hours, obtain two more sets of cultures, for a total of five sets overall. Blood Culture Media No single blood culture system, nor any single culture medium, can detect all microorganisms that might be present in the bloodstream of patients with suspected infective endocarditis. Use of an anaerobic culture complements aerobic media in the recovery of facultative anaerobes, such as streptococci, especially the nutritionally variant streptococci, and as such should still be included with every blood culture set. Frequently patients who are undergoing investigation for possible infective endocarditis have already been placed on antimicrobial agents; this is the single most common reason for “culture-negative”


Number 17 M47-A


infective endocarditis. In order to overcome the potential inhibitory activity of those antimicrobial agents on bacterial growth, it is important to utilize a blood culture medium that has been designed to counteract or neutralize this inhibitory effect. In earlier studies of infective endocarditis, it was reported that another potential cause of “culture-negative” endocarditis is the presence of the so-called “nutritionally variant streptococci” (NVS). These streptococcal species, now called Abiotrophia defectiva, Granulicatella adiacens, and Abiotrophia elegans, require supplemental vitamin B6 or cysteine for growth. Commercially available blood culture media today either already contain these supplements, or the human blood added to the culture medium provides them. Subcultures of blood culture bottles showing chains of gram-positive cocci consistent with streptococci but without growth on sheep blood agar with a tryptic soy base are clues to the presence of NVS. It is best practice to employ techniques to recover these microorganisms, which account for an estimated 5 to 6% of viridans streptococcal endocarditis cases.157 Duration of Incubation of Blood Culture Commercial blood culture systems recover most clinically important pathogens from blood within five days, which is now the recommended duration of incubation for routine blood cultures. Previously it was held that at least two weeks of incubation were required158 for optimal recovery of the more fastidious microorganisms that may cause infective endocarditis. The data to support this recommendation were based on older culture systems and media, and do not reflect the situation today. Recently reported data23 from the Mayo Clinic documented that all episodes of infective endocarditis were detected within a five- day period of incubation of blood cultures using a CMBCS. In addition, a recent study in which an extensive blood culture protocol was used with extended incubation showed only three clinically relevant results after five days of incubation in 215 patients with suspected infective endocarditis159; these were two Mycobacterium avium complexes and one Legionella pneumophila. All HACEK microorganisms (24 isolates) were recovered within five days.159,160

Recommendation: Incubate blood culture bottles for five days. If all blood culture sets are negative at five days and the diagnosis of infectious endocarditis is still under consideration, subculture all bottles from the blood culture sets to chocolate agar. 6.5.3.1 Special Considerations: Diagnosis of Fungal Infective Endocarditis Fungal infective endocarditis includes cases caused by both yeasts and molds. This disease entity, once considered rare, is now being reported more frequently.161 It has been stated that the increase in this disease can be attributed to “medical progress,” because many of the risk factors for this disease are associated with technologic advances: long-term intravenous catheterization; hyperalimentation; immunosuppression; implantation of prosthetic devices, including heart valves and pacemakers; prolonged use of antimicrobial agents; and cardiac surgery.161 Use of drugs of addiction is another important risk factor for the development of fungal endocarditis.162 Typically a fungal etiology for infective endocarditis is not under initial consideration until the routine blood culture results are reported as negative. In a recent worldwide review of 270 cases of fungal endocarditis, this diagnosis was included in the differential diagnosis in only 48 (18%) of the cases at the time of presentation.162 The most common fungal pathogens implicated in infective endocarditis are Candida albicans and non-albicans Candida spp.161,162 Many of the same technical factors for diagnosis of bacterial endocarditis apply to the diagnosis of fungal endocarditis. Most routine manual and automated blood culture system media are able to support the growth of yeasts such as Candida spp. If routine blood cultures are negative with these media, then it may be warranted to specifically request a “fungal blood culture,” in which media are used that optimally support the growth of most yeasts. This is rarely indicated, however, and only if the routine methods used


Volume 27 M47-A


are known to be suboptimal for yeasts. Overall, if optimal methods are utilized, the percentage of positive blood cultures in fungal endocarditis for Candida spp., is 83 to 95%.163 6.5.4 Patients Receiving Antimicrobial Therapy Patients are frequently receiving antimicrobial therapy at the time of blood culture collection. The possibility that these antimicrobials inhibit recovery of bacterial pathogens has long been a concern. Recent research corroborates this concept by showing that when blood cultures are obtained before and after initiation of antimicrobial agents, the postantibiotic cultures are less likely to yield microorganisms.164 Medium formulations have been developed for continuous-monitoring blood culture systems that add resins or proprietary activated charcoal substances intended to remove antimicrobial agents. In laboratory experiments, the effectiveness of these substances in adsorbing or neutralizing antimicrobials varies from one drug class to another, and the interval required for removal may be hours to days.165 Comparisons of each of these products against each other show that they perform similarly in overall recovery rates of microorganisms.166,167 In multiple trials, these media perform better than standard media.85,168-172 In some studies, recovery is enhanced exclusively for samples obtained from patients who are receiving antimicrobials at the time of blood culture collection, but in others these supplemented media perform better for overall recovery of microorganisms, even in patients who have not been treated. Hence, it is not clear that the advantage these formulations have is specifically the result of antimicrobial removal. Among individual microorganisms, staphylococci consistently grow more often from one of these media, and in selected studies, streptococci, Enterobacteriaceae, nonfermenters, and yeasts are also recovered more often. Microorganisms judged to be contaminants may also grow more frequently in these media.173 Moreover, resin- and charcoal-containing products are more expensive than standard blood culture media. In one study, these media only enhanced recovery of microorganisms that were already being effectively treated with antimicrobials, and their recovery had little impact on patient management.174 In contrast, another study showed important clinical benefits from the improved recovery in a tertiary care setting.174 6.5.5 Rare and Fastidious Pathogens These microorganisms are infrequently recovered from blood, but when encountered may represent serious infection. The recovery of a member of the HACEK group of bacteria in blood is among the major criteria for diagnosis of infective endocarditis.175 In the past, the recovery of these microorganisms required special procedures,98,176 but the current automated technology provides generally reliable detection of fastidious isolates (e.g., Haemophilus spp., Abiotrophia spp., Cardiobacterium spp., Actinobacillus spp.) within the same timeframe of routine blood cultures.159,160 In contrast to the majority of blood cultures yielding nonfastidious pathogens, which become signal-positive in the first 24 to 36 hours of incubation, bacteremia caused by fastidious microorganisms may require up to five days of incubation and testing before becoming signal-positive. Microorganisms such as Legionella, Bartonella, or Mycoplasma are more optimally diagnosed by means of immunodiagnostic or molecular techniques, as are the uniformly uncultivable (by usual bacterial culture systems) agents such as Coxiella, Chlamydophila, Rickettsia, and Tropheryma.177,178 With the exception of Bartonella spp., most rare or fastidious bacteria are still cultivable by usual bacterial culture systems and can be recovered using blood culture protocols currently in use by most clinical laboratories. The usual five-day cycle practiced in many laboratories with continuous-monitoring instruments will permit detection of even Abiotrophia/Granulicatella and Francisella. A phenomenon sometimes associated with highly fastidious bacteria in blood cultures is that of signal-positive/Gram stain-negative cultures. The bacterium may not be revealed by Gram stain of the contents of a positive bottle for a number of reasons. Failure to observe the bacterium usually derives from an


Number 17 M47-A


atypical morphology or unusually small size (e.g., Abiotrophia spp. may produce bizarre shapes or sizes, while Francisella may be so small and indistinct that they blend in with background on the slide). In some cases, as with the mycoplasmas, they may be unable to be Gram stained. An acridine orange stain may, in these cases, reveal the bacterium.179 For example, use of either acridine orange or a Gram's stain using carbol fuchsin as the counterstain may be necessary to see Campylobacter, Helicobacter, or Brucella on smears. Detection of fastidious gram-negative bacteria in blood cultures should always elicit safety precautions because of the highly infectious nature of some of these bacteria (e.g., Francisella or Brucella). These can be safely handled and presumptively identified in microbiology laboratories that use Biological Safety Level-2 (BSL-2) practices; use of the biological safety cabinet is recommended at the earliest possible stage of blood culture examination. Abiotrophia and Granulicatella: These bacteria can be detected in the automated blood culture systems, usually after the second day of incubation.180 Isolation in subculture depends on the presence of pyridoxal or cysteine in the medium. This requirement can be met by supplemented media, by cocultivation with staphylococci around which satellite colonies will grow, or by use of enriched chocolate and some anaerobe agars. Consequently, these bacteria may be isolated on chocolate or anaerobe subcultures but not on blood agar if a base such as TSA is used. Bartonella: Bartonella spp. can be recovered from blood by culture but the yield is low; in most cases special handling is necessary. The persistent and recurring nature of Bartonella bacteremia elicits strong antibody responses, making serology a diagnostically useful approach.181 Numerous PCR assays have been developed for direct detection of Bartonella spp. in clinical specimens, including those from patients with endocarditis.182 If blood cultures are performed, lysis-centrifugation in combination with plating on freshly prepared, enriched, blood or chocolate media is optimal.183 Plates should be incubated at 35 °C in a humid atmosphere containing elevated CO2 for 14 to 21 days. Alternatively, a lytic broth system may be used, followed by subculture to solid media upon termination of a seven-day incubation period. Brucella: In the past, isolation of Brucella from blood was done using biphasic media and prolonged incubation. With the advent of CMBCSs, recovery of Brucella spp. differs little from that of other bacteria.184,185 While a commercially available biphasic system would still be suitable, the sensitivity and efficiency of detection is best (and also when compared to lysis-centrifugation) with the CMBCSs.186 Studies from geographic areas endemic for brucellosis suggest that most isolates can be recovered within three days. The rate of detection by the seventh day is at least 95%, and rarely an isolate may require longer than seven days for detection.187,188 When one of the CMBCSs is used, most isolates are recovered from aerobic bottles only. Campylobacter: Systemic disease due to various species of Campylobacter can occur.189 Though usually associated with acute gastroenteritis, C. jejuni may also be recovered from blood. Infrequently encountered Campylobacter species, such as C. fetus, C. lari, or C. upsaliensis, can be recovered using the CMBCS typically after two to three days of incubation. Growth of C. jejuni is slower at 35 to 36 °C than at 42 °C, so subcultures may appear negative after the first 24 hours. Colonies should be visible after 48 hours of incubation, provided the plates are incubated in a microaerobic atmosphere. Francisella: F. tularensis grows in standard blood culture media. Although these bacteria typically require cysteine supplementation for optimal recovery, this is usually not necessary. F. tularensis varies in its growth rate; hence, the length of incubation required before detection is unpredictable.190,191 There are relatively rapidly growing strains that behave as practically nonfastidious, while other strains require more than ten days of incubation. Due to its tiny, pleomorphic characteristics, F. tularensis is easily missed upon examination of Gram-stained contents of blood culture bottles. Subcultures of F. tularensis may at first grow on standard sheep blood agar (SBA), but upon subsequent passage will fail to grow on SBA. Some isolates may initially grow only on chocolate agar plates.


Volume 27 M47-A


HACEK Group: Identification of a bacterium from the group of HACEK microorganisms is highly suggestive of infective endocarditis, even in the absence of an obvious focus of infection and of typical physical findings. The group includes Haemophilus spp. (other than H. influenzae), Actinobacillus actinomycetemcomitans, Cardiobacterium spp., Eikenella corrodens, and Kingella spp. They cause a variety of infections, in addition to endocarditis, which also may be accompanied by bacteremia. Regardless of the focus of infection, most blood cultures that yield these microorganisms are positive within the first week.192 In a study focused on HACEK endocarditis employing biphasic and lysis-centrifugation methods, blood cultures became positive in a mean of 3.4 days.193 If a high index of suspicion for HACEK endocarditis exists, and cultures are negative after five days, a longer incubation period or terminal subculture may be necessary. Helicobacter: There are several newly described or recently reclassified species of Helicobacter. They can occur as bacteremic pathogens, usually in immunocompromised patients, with H. cinaedi as the most frequently isolated species. These bacteria can be detected as early as the third day of incubation by automated blood culture systems, but they often require more than five days to signal positive.194-196 Optimal recovery of these bacteria requires extending the incubation period to seven days and adding a terminal subculture onto enriched blood agar followed by incubation in a hydrogen-enriched microaerobic atmosphere. Legionella: Legionella spp. are common causes of community-acquired pneumonia in many geographic regions, but Legionella bacteremia secondary to pneumonia is unusual.197 Legionella bacteremia can also occur following renal transplantation, and cases of prosthetic valve endocarditis caused by Legionella spp. have been reported.198 Culture of Legionella requires use of buffered charcoal yeast extract (BCYE), and subculture onto BCYE after a five-day incubation period of routine blood cultures is acceptable, as is use of BCYE agar in conjunction with the lysis-centrifugation system. Leptospira: Although methods for recovering leptospires from blood have been described, these bacteria are unlikely to be recovered from blood cultures.199 Other laboratory methods for diagnosis should be used in cases of suspected leptospirosis. Mycoplasma: Current automated blood culture systems designed to recover common pathogenic bacteria are not reliable for recovery of Mycoplasma spp. from blood. M. hominis may be fortuitously isolated from conventional blood cultures, and addition of supplements such as gelatin or arginine may enhance recovery, but slow growth and specialized medium requirements and mycoplasma-specific cultivation techniques should be employed when this organism is suspected on clinical grounds.200 For optimal recovery, blood should be inoculated onto specific media (e.g., SP4 glucose at pH 4.5) that can be used for both Mycoplasma pneumoniae and Mycoplasma hominis (provided that arginine is added for the latter). 7 Reporting Results The results of blood cultures, whether positive or negative, are critical to patient care. Therefore, the reporting of blood culture results must be effective and consistent throughout the culture process. The report must allow the healthcare provider to quickly and accurately identify the status and any test results for an ordered blood culture. Though a fairly wide range of reporting mechanisms may exist for different types of laboratories and healthcare systems, several generic guidelines pertain.

7.1 Written and Electronic Reports 7.1.1 Specimen Status It is important that the healthcare provider be able to determine the status of a specimen quickly and efficiently. Specific information includes the answers to the following questions:


Number 17 M47-A


• Has the specimen been ordered? • Has the specimen been collected? • What testing was ordered on the specimen? • Has the specimen been received by the laboratory? • Is the specimen acceptable for the testing requested? • Is the requested testing in progress? A record should be created in the laboratory information system (LIS) as soon as possible for each blood culture ordered. Data concerning change in the specimen status, as described above, should be entered into the record as soon as possible.

7.1.2 Culture Data Each record should include a result, as described below. Culture data and other information should be verified as quickly as possible. Critical value results, as described below, must be communicated as soon as possible (within 60 minutes). Noncritical culture information should be entered into the culture record within four hours of verification. 7.1.3 Preliminary Written Results The following may serve as examples of specimen status reports: • blood culture ordered, specimen not received; • testing in progress, no results to date; • no growth at 24 hours; • no growth at 48 hours; and • positive blood culture.

At the first detection of a positive blood culture, written and verbal reports, including the Gram stain results, should be issued to the healthcare provider responsible for the patient as described in Section 7.1.5, Critical Value (Verbal) Reports. Preliminary written reports include, as available:

— final Gram stain result; — preliminary identification (based on colonial and microscopic morphology, preliminary test

results, etc., according to laboratory protocol); and — final antimicrobial susceptibility data.

7.1.4 Final Written Results The final blood culture result should include one of the following: • Test Canceled • No Growth – The total incubation time, according the laboratory protocol, should be indicated in

the final report. • Positive Blood Culture – A verbal report, as described in Section 7.1.5, Critical Value (Verbal)

Reports, should be issued for any final positive blood cultures if one was not issued during the preliminary status of the testing.


Volume 27 M47-A


The final written report includes, as available:

— final Gram stain result; — final identification; and — final antimicrobial susceptibility data.

• Other Information—Any additional information that may impact the interpretation of the final test result should be included in the written report. Examples of such information include inadequate blood volume collected, prolonged transport time, or other factors as described in Section 10, Quality Assurance.

7.1.5 Critical Value (Verbal) Reports Any blood culture test result that may have an important impact on patient care (i.e., critical values) should be reported according to laboratory policy. Such policies must be designed, in consultation with the healthcare provider served by the laboratory, to ensure compliance with standards of medical care. Critical values should be communicated verbally, as described below, unless alternative communication strategies have been established and validated to ensure timely and accurate communication of results; also, document that the critical value results were received by the healthcare provider. The policy regarding communication of critical values should ensure that information is communicated quickly and accurately to the healthcare provider. A mechanism for identifying an alternative licensed caregiver must be provided for cases in which the requesting healthcare provider is unavailable. The first laboratory result (whether stain or culture) documenting a positive blood culture should be communicated as a critical value for every positive blood culture. Critical value reports should be issued as soon as possible (within 60 minutes) after laboratory verification of the abnormal result. Communication of information, generated by subsequent testing, may not be required unless the information is significantly different from the information communicated in the original critical value communication.

Several other specimen reports should be communicated as critical values. A critical value report should be issued when unacceptable specimens are received for blood culture testing and a new blood culture is needed (e.g., a broken blood culture bottle).

A critical value result should be issued for any correction to previously issued preliminary or final results, whether the changed results were issued as critical values or by routine reporting. The written report should clearly indicate that the results represent a corrected report, and provide details regarding the change to the previously issued report.

Each critical value report should include the following components, which must be documented in the laboratory record:

• full name of the person issuing the report; • date and time of unsuccessful and successful attempts to contact a healthcare provider responsible

for the patient; • full name of the person to whom the report is issued; • the abnormal value reported, with emphasis that the result is a critical value; and • documentation that the person receiving the report “read-back” the result.


Number 17 M47-A


Written and/or electronic reporting, according to the laboratory protocol, must follow all critical value verbal reports.

8 Contaminants In order to minimize blood culture contamination, each laboratory should have policies describing: 1) blood culture collection techniques that minimize contamination; and 2) a standardized process for the evaluation of blood culture isolates to determine the contamination rate. Even when the best blood collection protocols are used it may not be possible to reduce the contamination rate below 2%.39

Microorganisms commonly associated with contaminated blood cultures (e.g., Bacillus spp., Corynebacterium spp., Propionibacterium spp., coagulase-negative staphylococci, Aerococcus spp., Micrococcus spp.) are also capable of causing systemic, blood-borne infection in the appropriate setting. In many instances, a potential contaminant is recovered from one or both bottles of a single blood culture set. Without a second blood culture for comparison, it is virtually impossible to assign significance to a questionable isolate. Therefore, the evaluation of an isolate with low virulence potential recovered from a single blood culture set (one or both bottles) should be limited to the extent at which medically important microorganisms can be excluded from the identification. Susceptibility testing should not be done on suspected contaminants. All isolates should be saved for a few days so that additional studies can be performed if an identical organism is recovered from subsequent blood cultures of the same patient. Recovery of multiple isolates of the same organism(s) from independent blood cultures warrants full identification and susceptibility testing of the initial isolate(s). The publication of Richter et al. provides a detailed description of a laboratory-based algorithm for minimizing the extent of evaluation of blood culture contamination.201 9 Safety Issues The risk of injury or laboratory-acquired infection must be minimized for laboratory personnel. Publications such as CLSI/NCCLS document GP17—Clinical Laboratory Safety202 and ISO 15190, Medical laboratories—Requirements for safety203 provide information on the implementation of an effective safety program for laboratory activities. These documents address important issues, including program organization, facility design, maintenance and inspection of equipment, personal safety, signage, and labeling. Specific areas addressed include fire prevention; chemical, electrical, microbiological, and radiation safety; waste disposal; evacuation and emergency response; program evaluation; and other laboratory safety issues. CLSI/NCCLS documents M298 and H3—Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture204 provide additional information regarding safety issues directly related to blood culture examination. Pathogens present in samples collected for blood culture examination have the potential for causing infection in laboratory workers through percutaneous injury or by direct contact with the worker’s skin, eyes, or mucus membranes. In addition, indirect exposure of personnel, by contaminated surfaces or to pathogens isolated in culture, provides another potential mechanism for laboratory-acquired infection. The risk of infection is decreased by implementation of procedures that minimize the chance of exposure of personnel to infectious materials. Such procedures include use of standard precautions, use of personal protective equipment and barriers, use of safety devices and equipment, and proper handling of specimens and biohazardous waste. For certain pathogens, like hepatitis B virus and Neisseria meningitidis, immunization of laboratory personnel provides an effective adjunct to engineering controls. Institution policies must comply with federal and local regulations.


Volume 27 M47-A


9.1 Agents Associated With Laboratory-Acquired Infections 9.1.1 Transmission of Hepatitis Viruses and Retroviruses The importance of preventing percutaneous injury cannot be overemphasized, especially during the collection of samples by venipuncture. The average risk of infection after needlestick injury is ~18% for hepatitis B virus (HBV), 1.8% for hepatitis C virus (HCV), and ~0.3% for human immunodeficiency virus (HIV). See the most current edition of CLSI document M29.8 Though transmission of HBV, HCV, and HIV through direct cutaneous and mucus membrane exposure have been well documented, precise incidence studies defining the risk of transmission after such exposures have not been well defined. Safety strategies to prevent transmission of HBV are considered sufficient for prevention of HCV and HIV transmission. 9.1.2 Transmission of Agents Other Than Hepatitis Viruses and Retroviruses While the hepatitis viruses and retroviruses present the greatest risk of transmitting infection from blood samples collected for blood culture examination, the handling and manipulation of patient samples and cultures in the laboratory present an increased risk of infection due to other pathogens. Though percutaneous exposure represents the most common mode of transmission of the hepatitis viruses and retroviruses, transmission of other infectious agents within the laboratory is most commonly due to aerosol or small-droplet formation. Therefore, engineering and work practice controls that minimize the formation and dissemination of infectious droplets and aerosols must be employed for activities that might produce them. Some naturally occurring agents that are associated with high risk for laboratory-acquired infection may be isolated by blood culture procedures. Because the isolation of such isolates is not predictable, all blood culture isolates should be handled according to protocols based on Biosafety in Microbiological and Biomedical Laboratories (BMBL).205 9.2 Protective Measures Prevention of infection transmitted by samples submitted for blood culture examination, or pathogens isolated from these samples, depends on compliance with an effective laboratory-wide exposure control plan. Protocols for activities related to blood culture examination should provide detailed instructions for the following protective measures: Hand Washing Frequent hand washing is a critical component for preventing transmission of laboratory-acquired infection. Hands, or other skin surfaces, must be washed immediately after direct contact with blood or any potentially infectious material. Hands should also be washed before donning gloves and after removal of gloves, after completion of work, and when leaving the laboratory or moving into a clean area within the laboratory. MMWR Guideline for Hand Hygiene in Health-Care Settings206 provides information for establishing a hand hygiene program for a variety of healthcare settings. Barrier Protection OSHA regulations mandate that institutions provide employees with appropriate personal protective equipment. Gloves must be worn during the collection of samples for blood culture examination and changed between each patient contact. Gloves must be changed immediately if they become contaminated by blood or show any sign of breakage or loss of barrier function. Gloves should also be worn in the laboratory specimen receiving and processing areas, in the mycobacteriology laboratory, and other areas


Number 17 M47-A


of the laboratory where hands may come into contact with infectious materials or contaminated surfaces. Gloves should be worn when handling and disposing of biohazardous waste. Facial protection should be used whenever there is a possibility of splashing of blood or other infectious material. A face shield, fluid-resistant mask with eye protection, or splashguard can provide this protection. Fluid-resistant, long-sleeved, closed-front protective clothing should be worn for activities related to blood culture processing and examination. Protective clothing must be removed immediately if visibly contaminated. Contaminated protective clothing should be discarded as biohazardous waste or laundered according to the institution’s policy. Protective clothing must be removed before leaving the laboratory or moving into a clean area of the laboratory. Biological Safety Cabinets Class IIA or IIB biological safety cabinets should be used for laboratory activities that have a significant risk for formation of small droplets or aerosols. Sterilization, Disinfection, and Decontamination The collection and manipulation of samples for blood culture examination presents risks for contamination of equipment and environmental surfaces with infectious material. Detailed instructions for the prevention and management of infectious material spills must be provided by general laboratory procedures. Decontamination of laboratory equipment should follow instructions provided by the manufacturer as well as standard laboratory practice. Individuals responsible for transporting diagnostic specimens must be trained in packaging that is compliant with applicable local and federal regulations. Transport procedures must provide containment for potential spills and provide instructions for response to spills recognized during transport. Samples for blood culture examination may be transported through a pneumatic tube system if care is taken to prevent breakage. In the event of breakage during transport, detailed instructions must be provided for response to different types of potential contamination. If potentially infectious material may have leaked out of the transport carrier, the entire system must be shut down while potentially contaminated routes and carriers are identified from the system’s traffic records. Detailed instructions for decontamination of the stations, tube system, and carrier components (carrier, liner, containment pouches, etc.) must be prepared according to the manufacturer’s instructions. If a spill involves agents that are transmissible via inhalation, the room should be closed for a minimum of 30 minutes to allow droplets to settle. Appropriate personal protective equipment must be worn during clean-up procedures; gown, gloves, and facial protection are a minimum. Use of puncture-resistant gloves is required if broken glass or other sharp object is involved in the spill. Use of respirators is required if an infectious aerosol may have been formed, or for agents with high risk for respiratory transmission, such as M. tuberculosis. Water impermeable shoe covers must be worn for large spills on the floor. The exact procedures employed by laboratory protocol should take into account the volume and type of spilled material, infectious agent potentially present and its concentration, and type of contaminated surface. In general, the response to spills includes the following components: • containment and absorption; • removal of residual material using an aqueous detergent;


Volume 27 M47-A


• decontamination by flooding the surface using an intermediate hospital disinfectant, such as diluted household bleach. Concentration and time of exposure will depend on the type of surface contaminated;

• removal of disinfectant and rinse with water; • drying surface to prevent slipping; and • disposal of materials used for decontamination and all contaminated materials that could not be

effectively decontaminated. Standard Precautions Standard precautions provide engineering and work practice controls that will minimize the risk of contact with infectious materials or, in the case of accidental contact, minimize the duration of exposure. All employees must receive initial training in the basic principles and practices related to standard precautions. In addition, retraining related to the concepts of standard precautions should be incorporated into the laboratory’s continuing education program. A critical component of standard precautions is the prevention of percutaneous exposure. CLSI/NCCLS document X3—Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory207 provides guidelines for implementing an appropriate program for activities related to blood culture examination. Several strategies should be considered: • Minimize the use of needles and other sharp instruments (e.g., use safety devices for venting bottles

and subculturing). • When needles must be used, as in the collection of samples for blood culture examination, use

safety devices and practices to minimize the chance of needlestick injury. Recapping needles, when necessary, and other manipulation of exposed needles, like those that may be required to vent aerobic blood culture bottles, should be performed using techniques that prevent directing the point of the needle to any part of the body.

• If blood is collected by syringe, the needle used for collection should not be replaced before

inoculation of blood culture bottles. The use of intermediate collection tubes or bottles increases the risk of blood culture contamination, as well as the risk of injury during subsequent transfer; their use, therefore, is discouraged.

• Needles should be equipped with a mechanism to minimize the risk of injury. The safety

mechanism should be used, according to the manufacturer’s instructions. • Needles must be placed in an approved sharps container after use. The container should be close to

the site of use with the opening clearly visible. Sharp containers should not be overfilled. • A sharps container must be used if exposed needles or other sharps must be transported to or within

the laboratory. • The use of glass should be avoided for laboratory supplies that come in contact with patient

samples or infected materials.


Number 17 M47-A


Management of Laboratory Accidents Laboratory personnel must be trained and maintain competence in risks, prevention, and management of accidents related to job performance. The components of postexposure management should include acute exposure management, exposure reporting, evaluation of risk associated with exposure, and postexposure prophylaxis. Detailed protocols, in compliance with OSHA and other federal regulations as well as the institution’s infection control program, should be established to address all aspects of personnel management after exposure to potentially infectious materials. Engineering controls and processes that will minimize exposure of laboratory personnel to infectious material should be provided for activities performed in dirty areas. Specifically, activities that increase the risk of exposure of laboratory personnel to infectious material in dirty areas should be clearly prohibited by protocol. Such high-risk activities include: mouth pipetting; nail biting; smoking; eating; contact lens manipulation; contact of eye, nose, or mouth with hands or other environmental surfaces; etc. Laboratory personnel should avoid use of contact lenses in dirty areas. If absolutely required, goggles or other eye protection should be used. Procedures associated with a high risk for splashing should be performed in a biological safety cabinet or behind a splashguard. Centrifuges must be operated according to the manufacturer’s instructions. Centrifuges should be equipped with sealed rotors or safety cups. Plastic tubes with sealing screw-top tubes should be used whenever possible. Before use, tubes must be examined carefully for signs of damage. The contents should be examined for signs of breakage before opening the sealed rotor or safety cups. Rotors or safety cups and centrifuged samples should be opened within a biological safety cabinet for samples likely to contain agents spread by airborne transmission. Respiratory Protection Respiratory protection must be provided when laboratory personnel may be exposed to agents associated with a high risk for airborne transmission, through contact with infected patients, specimens obtained from infected patients, or laboratory isolates. Laboratory personnel performing activities related to blood culture examination for mycobacteria may be exposed to such risks. Performance of personal respirators must comply with current CDC, NIOSH, and OSHA recommendations with regard to filter efficiency for 1-µm particles; fit testing to document face-seal integrity; ability to fit various facial sizes and characteristics; the ability of healthcare workers to evaluate face piece fit each time they put on a respirator; and other applicable criteria. Laboratory Instruments and Equipment Instruments and equipment should only be operated according to the manufacturer’s instructions and with sufficient engineering and work practice controls to minimize any type of injury to laboratory personnel. Parts of any device that contact patient samples or infectious material as part of normal use should be decontaminated regularly during routine maintenance and before disposal or repair. Equipment that has been contaminated, or operated in a “dirty” area of the laboratory, must be decontaminated immediately according to manufacturer’s instructions. NOTE: Dried blood must be considered as infectious as liquid blood. Uncontaminated supplies, reagents, and materials must be stored separately from patient samples and stored cultures. Subcultures of patient or other isolates must be stored in an organized and secure manner that ensures easy access and identification at the time of retrieval or disposal. Containers (ideally plastic) designed for low-temperature storage must be used for samples or isolates that require freezer storage.


Volume 27 M47-A


Safety Training Initial safety training and evaluation of ongoing competence must be provided to laboratory personnel, in an effective format, for all activities they perform in the laboratory. The training, monitoring activities, incidence response, and documentation related to safety must comply with federal and local regulations and should be a core component of the laboratory’s quality assurance program. Special Precautions Related to Blood Culture Examination Unless there is increased suspicion for the isolation of a highly infectious agent requiring higher-level biosafety practices, BSL2 practices are adequate for routine activities related to blood culture examination. CLSI/NCCLS document H3204 provides guidance for preparing protocols for blood sample collection and blood culture examination. The phlebotomist must follow procedures to minimize the risk of transmitting infection from contact with the patient or specimens taken from the patient. An important aspect of patient safety related to blood culture examination is the procedure for identification of the patient. Two reliable identifiers must be obtained and compared to the requisition form. The protocol must be reliable for patients who are conscious and cooperative as well as for those patients who are not. Examples of reliable information include: full name, current address, hospital identification number, social security number, or birth date. The patient’s location is not reliable for patient identification. Samples collected for blood culture examination should be placed in a leakproof primary container with a secure closure. The requisition slip should not be placed in the primary container because of the possibility of contamination. Primary containers with separate pockets, like plastic bags, provide a convenient system to minimize contamination of requisition forms. The samples should be transported to the laboratory in secondary containers that are able to contain the specimen in the case of a break with leakage from the primary container. For blood culture specimens transported by pneumatic tube, plastic bags that have been shown to be leakproof within the system should be used for transport. Place bottles in a primary plastic transport bag and close securely. Secure the bottles together using tape or a rubber band around both bottles. Place the secured bottles into a secondary plastic transport bag and close securely. The sample and test requisition slip are placed in the transport carrier that is then closed, locked, and transported to the laboratory by standard protocol. No other specimens should be transported with a specimen for blood culture examination. Waste generated by activities related to blood culture examination must be managed according to biohazardous waste disposal procedures of the institution, in compliance with relevant federal and local regulations. CLSI/NCCLS document GP5—Clinical Laboratory Waste Management208 provides information concerning key elements for waste management and guidance for establishing an effective waste management system within the laboratory. 10 Quality Assurance 10.1 Preexamination Process The preexamination process for blood culture examination includes the following components: patient evaluation; test selection and ordering; sample collection; sample transport; sample receipt; and sample processing. Each of these components includes multiple procedures or processes for successful completion.


Number 17 M47-A


10.1.1 Patient Evaluation

Blood culture examination may provide critical information that impacts therapy and/or prognosis for selected patient groups; therefore, it is imperative that every organization develops guidelines to identify appropriate patients for blood culture analysis. Guidelines should also identify clinical scenarios with a low prior probability of bacteremia or fungemia for which blood culture examination is not recommended. False-positive blood culture examination for such low-risk patients is likely to have a negative impact on the patient’s outcome and/or the institution’s cost of caring for the patient. Example QA Indicator: Proportion of patients admitted with presumed bacterial pneumonia, from whom blood cultures were collected as part of the initial diagnostic work-up. 10.1.2 Test Selection and Ordering The laboratory should work with the organization’s healthcare practitioners to ensure that clinical practice standards result in selection of the most appropriate use of blood cultures. Protocols for when to draw or not to draw blood cultures should be available for all practitioners who use a laboratory. Requests for blood culture examination must be submitted using a standardized process (paper or electronic) established by the organization in compliance with relevant local and federal regulations. The laboratory must ensure that healthcare providers have been trained in the accurate completion of relevant paper or electronic requisition forms. All critical information must be included in the requisition in a legible form, including, but not limited to: unequivocal patient identifiers; unequivocal identification of the authorized healthcare provider requesting the examination; specimen type and detail; specific examination requested; patient diagnosis (ICD-9-CM code); and pertinent clinical information. Example QA Indicator 1: Proportion of patients with blood cultures who have the recommended number of blood culture sets submitted. Collection of two or three blood culture sets is recommended per episode. Example QA Indicator 2: Proportion of patients with more than the recommended number of blood cultures submitted. Collection of two or three blood culture sets is recommended per episode for the initial patient evaluation. Collection of another two or three blood culture sets may be indicated after 48 to 72 hours if the initial culture sets were noninformative. “Surveillance” cultures are not recommended. 10.1.3 Sample Collection Upper extremity venipuncture is recommended for the collection of samples for blood culture examination under most circumstances. CLSI/NCCLS document H3204 provides guidance to laboratories for the preparation of procedures related to sample collection for blood culture. Collection of samples by arterial puncture or lower extremity venipuncture may increase the risk of patient injury and of culture contamination. Specimens collected through lines also have a greater risk for contamination. Detailed protocols for collection of samples will minimize the medical errors that can occur with venipuncture (including misidentification of samples or patients, incorrect collection vessel, incorrect timing of collection, formation of hematomas, nosocomial anemia, and hemoconcentration). A training program with documentation of trainee competency is essential for healthcare providers who will draw blood cultures. Such protocols should be designed with the goals of minimizing the risks for both the patient and phlebotomist, as well as ensuring the collection of a sample that will produce informative results from the blood culture examination. The use of a dedicated team for collecting blood culture samples should be considered.

The following information must be provided for all blood culture specimens:

• patient’s first and last name;


Volume 27 M47-A


• a unique identification number; • date and time of collection; • other information or label required by institution policy; and • identification of the person collecting the specimen.

Compared to blood samples collected for most other types of laboratory examination, samples for blood culture examination require additional procedures to maximize the detection of microbial pathogens and to minimize the risk of contaminating the blood culture. Protocols for blood culture examination must follow the manufacturer’s instructions for specific blood sample requirements. When possible, samples should be collected before administering antimicrobial agents. Example QA Indicator 1: Blood culture contamination rate. The goal for blood culture contamination rate, whether analyzed overall or stratified by location, phlebotomist, etc., should be less than 3%. Example QA Indicator 2: Proportion of blood culture bottles inoculated with more or less than the recommended volume of blood. For adults, each blood culture bottle should be inoculated with 10 mL of blood. Example QA Indicator 3: Proportion of blood cultures submitted that include only a single inoculated bottle. Example QA Indicator 4: Proportion of blood cultures submitted that must be rejected for any cause. 10.1.4 Sample Transport Samples for blood culture should be transported to the laboratory as quickly as possible in a manner to ensure maintenance of sample integrity and compliance with applicable safety regulations. It is recommended that samples for blood culture examination be delivered to the laboratory within two hours of collection and transport. Example QA Indicator: Proportion of blood cultures submitted with prolonged (>2 hour) transport time. 10.1.5 Sample Receipt and Processing Samples for blood culture examination must be promptly received after delivery to the laboratory, assessed for acceptability (with respect to collection, specimen volume, transport time and condition, paperwork, labeling, etc.), accessioned into laboratory records, processed with media inoculation, as required (refer to CLSI document M2280 for general QC requirements for media), and transferred to the site of blood culture examination. Special instructions for the handling or incubation of samples should be provided for times when processing within the laboratory will be delayed. The status of samples for blood culture examination that are determined to be unacceptable at arrival in the laboratory must be communicated immediately to the ordering physician or patient location according to the laboratory’s policy for reporting critical test results. Blood culture specimens that meet the following criteria should be processed but the provider notified that the specimen is not optimal: • inadequately filled bottles/tubes; • insufficient numbers of cultures; • single blood cultures; and • blood inoculated into only aerobic or anaerobic bottles.


Number 17 M47-A


When more than the recommended number of blood cultures is submitted, the specimens should be processed but the provider notified that this will increase the risk of phlebotomy-induced anemia and is more likely to result in the recovery of contaminants. There are two common approaches to determine the adequacy of fill of bottles: visual comparison with a volume standard and weighing bottles. Weighing bottles is more accurate, whereas visual comparison can be done more quickly. Example QA Indicator: Documentation of communication for rejected blood culture specimens. 10.2 Examination Process The examination process for blood cultures includes the following components: • procedures for the detection of microorganisms; • identification of isolates; • susceptibility testing of relevant isolates; • verifying the reliability of test results; and • interpretation of test results. Each of these components includes multiple procedures or processes for successful completion. Detailed laboratory protocols must be prepared for each process. These protocols must comply with relevant regulations of the institution, government, and accreditation agencies. The specific procedures must also comply with manufacturers’ instructions for equipment or kits, as described in package inserts or operator’s manuals. CLSI document GP2—Laboratory Documents: Development and Control209 provides guidelines for the preparation of effective procedures. Rules for submitting isolates from blood culture samples for additional identification and susceptibility testing must be included in the procedure for blood culture examination. The extent of testing may be limited for likely contaminants or subsequent isolates of previously worked-up pathogens; however, the procedure for limiting work-up of blood culture isolates must be validated and periodically reevaluated to ensure that clinically relevant information is not being missed. Activities performed during the examination process may produce information that could be important for patient care. Such information, like results of staining or “preliminary” identification procedures, must be communicated to the healthcare providers, but in a manner that ensures that the preliminary nature of the report is clearly indicated. Verification of the final results should include a review of preliminary results. Significant inconsistencies (e.g., preliminary report shows gram-negative diplococci, but final report shows Streptococcus pneumoniae) should be reviewed by the laboratory director (or designee) and communicated immediately to the healthcare practitioner according to the laboratory’s policy for critical result reporting. Preliminary results should be included in the final blood culture examination report. Objective guidelines for the interpretation of blood culture examinations should be available to ordering clinicians (e.g., interpretive criteria and clinical significance for both positive and negative results). The procedure manual for blood culture examination should include guidelines for the identification of potential false-negative and false-positive results, as well as causes for such. Documentation should identify other clinical or laboratory information that might be necessary to accurately interpret a blood culture examination and/or avoid false-positive or false-negative results (e.g., number of samples collected for blood culture examination; antimicrobial therapy before blood culture collection; other laboratory signs, such as increased WBC; positive cultures from other infected sites; type and severity of immune compromise; pretest probability of infection caused by a fastidious organism not reliably detected by the routine blood culture examination; etc.).


Volume 27 M47-A


Example QA Indicator 1: Effectiveness of critical value results and documentation of communication. Critical results from blood cultures should be communicated to an appropriate healthcare provider within one hour of validation, and details of the communication should be clearly recorded in the culture record.

Example QA Indicator 2: Proportion of blood cultures that require correction to transmitted results.

10.3 Postexamination Process The postexamination process for blood culture examination includes the following components: • result reporting and archiving; and • sample management. Each of these components includes multiple procedures or processes for successful completion. 10.3.1 Reporting Blood culture results must be verified as accurate before being reported. Methods and guidelines for manual validation must be validated before use. After verification, the results must be communicated to clinicians in accordance with well-documented, verified protocols. The laboratory reports must be legible and organized so that results are clearly and unequivocally accessible to the healthcare providers reviewing the report. Clear, standardized terminology should be used and abbreviations should be avoided. When necessary, use of only institutionally defined abbreviations will minimize the chance of misinterpreting the results. The use of “standard report” result comments may also minimize transcription errors in preparation of examination reports. The laboratory director or designee should review corrected reports on a daily basis. Cumulative summaries of examinations with corrected reports should be reviewed periodically as part of the laboratory QA process. Example QA Indicator: The results of QA activities should be communicated with healthcare providers in order to identify opportunities for improvement. The effectiveness of interventions designed to improve quality related to blood cultures should be documented by continued monitoring of the relevant QA indicator. 10.3.2 Record Management Laboratory records for activities related to blood culture examination, including final reports, must be archived in accordance with federal, local, and accreditation or certification service standards. Protocols for record storage must clearly define the records to be stored, storage medium, methods for efficient retrieval, duration of storage, and destruction of records after the end of storage. CLSI/NCCLS document GP26—Application of a Quality Management System Model for Laboratory Services,210 Appendix D, provides guidance concerning the retention schedule for various laboratory records. 10.3.3 Consultation The laboratory medical director should be available to healthcare providers to discuss questions related to the results or interpretation of blood culture examinations in the context of the patient’s specific clinical condition. The laboratory director should recommend any appropriate additional clinical information or follow-up laboratory examinations that could contribute to the interpretation of blood culture examination reports.


Number 17 M47-A


References 1 Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med.

2003;348:1546-1554. 2 CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Ninth Edition. CLSI document M2-A9.

Wayne, PA: Clinical and Laboratory Standards Institute; 2006. 3 CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Seventh Edition.

CLSI document M7-A7. Wayne, PA: Clinical and Laboratory Standards Institute; 2006. 4 CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Seventh Edition. CLSI document M11-

A7. Wayne, PA: Clinical and Laboratory Standards Institute; 2007. 5 CLSI/NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second Edition.

CLSI/NCCLS document M27-A2. Wayne, PA: NCCLS; 2002. 6 CLSI/NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard.

CLSI/NCCLS document M38-A. Wayne, PA: NCCLS; 2002. 7 Garner JS, Hospital Infection Control Practices Advisory Committee. Guideline for isolation precautions in hospitals. Infect Control Hosp

Epidemiol. 1996;17(1):53-80. 8 CLSI. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—Third Edition. CLSI document

M29-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. 9 Dorland’s Medical Dictionary, 27thedition. Philadelphia: W.B. Saunders; 1998. 10 Wilson ML. Blood cultures: introduction. Clin Lab Med. 1994;14:1-7. 11 Online Medical Dictionary: A Medical Dictionary of Medical Related Terminology. Available at: http://medical-

dictionary.com/dictionaryresults.php. Accessed 21 September 2006. 12 Levy MM, Fink MP, Marshall JC, et al. International Sepsis Definitions Conference. 2001SCCM/ESICM/ACCP/ATS/SIS International

Sepsis Definitions Conference. Intensive Care Med. 2003;29:530-538. 13 Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee. Definitions

for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992;20:864-874. 14 Taber Medical Dictionary, 18th edition. Philadelphia: F.A. Davis; 1997. 15 Weinstein MP, Towns ML, Quartey SM, et al. The clinical significance of positive blood cultures in the 1990s: a prospective

comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis. 1997;24:584-602.

16 Bennett IL Jr., Beeson PB. Bacteremia: a consideration of some experimental and clinical aspects. Yale J Biol Med. 1954;26:241-262. 17 Strand CL. Blood cultures: consensus recommendations in 1988. Microbiology no. MB 88-1 (MB-172). American Society for Clinical

Pathologists Check Sample Continuing Education Program. Chicago: American Society for Clinical Pathologists; 1988. 18 Li J, Plorde J, Carlson L. Effects of volume and periodicity on blood cultures. J Clin Microbiol. 1994;32:2829-2831. 19 Thompson RB Jr., Evans BL, Sourtherland JL. Collecting blood for culture. Generalist Microbiology Tech Sample No. G-1. American

Society of Clinical Pathologists; 1991. 20 Aronson MD, Bor DH. Blood cultures. Ann Intern Med. 1987;106:246-253. 21 Washington JA. Blood cultures: principles and techniques. Mayo Clin Proc. 1975;50:91-95. 22 Weinstein MP, Reller LB, Murphy JR, Lichtenstein KA. The clinical significance of positive blood cultures; a comprehensive analysis of

500 episodes of bactermia and fungemia in adults. I. Laboratory and epidemiologic observations. Rev Infect Dis. 1983;5:35-53. 23 Cockerill FR III, Wilson JW, Vetter EA, et al. Optimal testing parameters for blood cultures. Clin Infect Dis. 2004;38:1724-1730. 24 Levin PD, Hersch M, Rudensky B, Yinnon AM. Routine surveillance blood cultures: their place in the management of critically ill patients.

J Infect. 1997;35:125-128. 25 Czirok E, Prinz GY, Denes R, Remenyi P, Herendi A. Value of surveillance cultures in a bone marrow transplantation unit. J Med

Microbiol. 1997;46:785-791. 26 Nielsen J, Kolmos HJ, Rosdahl VT. Poor value of surveillance cultures for prediction of septicaemia caused by coagulase-negative

staphylococci in patients undergoing haemodialysis with central venous catheters. Scand J Infect Dis. 1998;30:569-572.


Volume 27 M47-A


27 Fowler VG Jr., Olsen MK, Corey GR, et al. Clinical identifiers of complicated Staphylococcus aureus bacteremia. Arch Intern Med.

2003;163:2066-2072. 28 Hall MM, Ilstrup DM, Washington JA II. Effect of volume of blood cultured on detection of bacteremia. J Clin Microbiol. 1976;3:643-645. 29 Ilstrup DM, Washington JA II. The importance of volume of blood cultured in the detection of bacteremia and fungemia. Diagn Microbiol

Infect Dis. 1983;1:107-110. 30 Mermel LA, Maki DG. Detection of bacteremia in adults: consequences of culturing an inadequate volume of blood. Ann Intern Med.

1993;119:270-272. 31 Plorde JJ, Tenover FC, Carlson LG. Specimen volume versus yield in the BACTEC blood culture system. J Clin Microbiol. 1985;22:292-

295. 32 Sandven P, Hoiby EA. The importance of blood volume cultured on detection of bacteraemia. Acta Pathol Microbiol Scand. 1981;89:149-

152. 33 Tenney JH, Reller B, Mirrett S, Wang WLL, Weinstein MP. Controlled evaluation of the volume of blood cultured in detection of

bacteremia and fungemia. J Clin Microbiol. 1982;15:558-561. 34 Washington JA II. Conventional approaches to blood culture. In: JA Washington II, ed. The Detection of Septicemia. West Palm Beach, FL:

CRC Press; 1978:41-88. 35 Arpli M, Bentzon MW, Jensen J, Fredriksen W. Importance of blood volume cultured in the detection of bacteremia. Eur J Clin Microbiol

Infect Dis. 1989;8:838-842. 36 Szymczak EG, Barr JT, Durbin WA, Goldman DA. Evaluation of blood culture procedures in a pediatric hospital. J Clin Microbiol.

1979;9:88-92. 37 Kellogg JA, Ferrentino FL, Goodstein MH, Liss J, Shapiro SI, Bankert DA. Frequency of low-level bacteremia in infants from birth to two

months of age. Pediatr Infect Dis J. 1997;16:381-385. 38 Kellogg JA, Manzella JP, Bankert DA. Frequency of low-level bacteremia in children from birth to fifteen years of age. J Clin Microbiol.

2000;38:2181-2185. 39 Dunne WM Jr., Nolte FS, Wilson ML. Blood Cultures III. Hindler JA, coordinating ed. Washington, DC: American Society for

Microbiology; 1997. 40 Weinstein MP. Current blood culture methods and systems: clinical concepts, technology, and interpretation of results. Clin Infect Dis.

1996;23:40-46. 41 Kellogg JA, Ferrentino FL, Liss J, Shapiro SI, Bankert DA. Justification and implementation of a policy requiring two blood cultures when

one is ordered. Lab Med. 1994;25:323-330. 42 Dorsher CW, Rosenblatt JE, Wilson WR, Ilstrup DM. Anaerobic bacteremia: decreasing rate over a 15-year period. Rev Infect Dis.

1991;13:633-636. 43 Gomez J, Banos V, Ruiz J, et al. Clinical significance of anaerobic bacteremias in a general hospital. A prospective study from 1988 to

1992. Clin Invest. 1993;71:595-599. 44 Lombardi DP, Engleberg N. Anaerobic bacteremia: incidence, patient characteristics, and clinical significance. Am J Med. 1992;92:53-60. 45 Murray PR, Traynor P, Hopson D. Critical assessment of blood culture techniques: analysis of recovery of obligate and facultative

anaerobes, strict aerobic bacteria, and fungi in aerobic and anaerobic blood culture bottles. J Clin Microbiol. 1992;30:1462-1468. 46 Ortiz E, Sande MA. Routine use of anaerobic blood cultures: Are they still indicated? Am J Med. 2000;108:445-447. 47 Zaidi AKM, Knaut AL, Mirrett S, Reller LB. Value of routine anaerobic blood cultures in pediatric patients. J Pediatr. 1995;127:263-268. 48 Morris AJ, Wilson ML, Mirrett S, Reller LB. Rationale for selective use of anaerobic blood cultures. J Clin Microbiol. 1993;31:2110-2113. 49 Sharp SE, McLaughlin JC, Goodman JM, et al. Clinical assessment of anaerobic isolates from blood cultures. Diagn Microbiol Infect Dis.

1993;17:19-22. 50 Riley JA, Heiter BJ, Bourbeau PP. Comparison of recovery of blood culture isolates from two BacT/ALERT FAN aerobic blood culture

bottles with recovery from one FAN aerobic bottle and one FAN anaerobic bottle. J Clin Microbiol. 2003;41:213-217. 51 Kellogg JA. Selection of a clinically satisfactory blood culture system: the utility of anaerobic media. Clin Microbiol Newslett.

1995;17:121-124. 52 Bartlett JG, Dick J. The controversy regarding routine anaerobic blood cultures. Am J Med. 2000;108:505-506.


Number 17 M47-A


53 Reller LB, Murray PR, MacLowry JD. Cumitech 1A, Blood Cultures II. Washington JA. II, coordinating ed. Washington DC: American

Society for Microbiology; 1982. 54 King TC, Price PB. An evaluation of iodophors as skin antiseptics. Surg Gynecol Obstet. 1963;116:361-365. 55 Little JR, Murray PR, Traynor PS, Spitznagel E. A randomized trial of povidone-iodine compared with iodine tincture for venipuncture site

disinfection: effects on rates of blood culture contamination. Am J Med. 1999;107:119-125. 56 Strand CL, Wajsbort RR, Sturmann K. Effect of iodophor vs. iodine tincture skin preparation on blood culture contamination rate. JAMA.

1993;269:1004-1006. 57 Souvenir D, Anderson DE Jr., Palpant S, et al. Blood cultures positive for coagulase-negative staphylococci: antisepsis, pseudobacteremia,

and therapy of patients. J Clin Microbiol. 1998;36:1923-1926. 58 Mimoz O, Karim A, Mercat A, et al. Chlorhexidine compared with povidone-iodine as skin preparation before blood culture: a randomized

controlled trial. Ann Intern Med. 1999;131:834-837. 59 Barenfanger J, Drake C, Lawhorn J, Verhuist SJ. Comparison of chlorhexidine and tincture of iodine for skin antisepsis in preparation for

blood sample collection. J Clin Microbiol. 2004;42:2216-2217. 60 Bryant JK, Strand CL. Reliability of blood cultures collected from intravascular catheter versus venipuncture. Am J Clin Pathol.

1987;88:113-116. 61 DesJardin JA, Falagas MA, Ruthazer R, et al. Clinical utility of blood cultures drawn from indwelling central venous catheters in

hospitalized patients with cancer. Ann Intern Med. 1999;131:631-647. 62 Everts RJ, Vinson EN, Adholla PO, Reller LB. Contamination of catheter-drawn blood cultures. J Clin Microbiol. 2001;39:3393-3394. 63 Everts RJ, Harding H. Catheter drawn blood cultures: Is withdrawing the heparin lock beneficial? Pathology. 2004; 36:170-173. 64 Warren JR, Graham F. The effect of heparin on the growth of bacteria and yeast. J Bacteriol. 1950;60:171-174. 65 Christman JF, Doherty DG. The antimicrobial action of heparin. J Bacteriol. 1956;72:433-435. 66 Krumholz H, Cummings S, York M. Blood culture phlebotomy: Switching needles does not prevent contamination. Arch Intern Med.

1990;113:290-292. 67 Isaacman DJ, Karasic RB. Lack of effect of changing needles on contamination of blood cultures. Pediatr Infect Dis J. 1990;9:274-278. 68 Leisure MK, Moore DM, Schwartzman JD, Hayden GF, Donowitz LG. Changing the needle when inoculating blood cultures: a no-benefit

and high-risk procedure. JAMA. 1990;264:2111-2112. 69 Spitalnic SJ, Wollard RH, Mermel LA. The significance of changing needles when inoculating blood cultures: a meta-analysis. Clin Infect

Dis. 1995;21:1003-1006. 70 Bille J, Stockman L, Roberts GD, Horstmeier CD, Ilstrup DM. Evaluation of a lysis-centrifugation system for recovery of yeasts and

filamentous fungi from blood. J Clin Microbiol. 1983;18:469-471. 71 Hamilton DJ, Amos D, Schwartz RW, Dent CM, Counts GW. Effect of delay in processing on lysis-centrifugation blood culture results

from marrow transplant patients. J Clin Microbiol. 1989;27:1588-1593. 72 Stockman L, Roberts GD, Ilstrup DM. Effect of storage of the DuPont lysis-centrifugation system on recovery of bacteria and fungi in a

prospective clinical trial. J Clin Microbiol. 1984;19:283-285. 73 Washington JA. Collection, transport and processing of blood cultures. Clin Lab Med. 1994;14:59-68. 74 Schifman RB, Strand CL. Blood culture contamination data analysis and critique. In: Q-Probes. Chicago, IL: College of American

Pathologists; 1995:1-19. 75 Barenfanger J. Improving the utility of microbiology data: an update. Clin Microbiol Newsletter. 2003;25:1-7. 76 Aukenthaler R, Ilstrup DM, Washington JA II. Comparison of recovery of microorganisms from blood cultures diluted 10%

(volume/volume) and 20% (volume/volume). J Clin Microbiol. 1982;15:860-864. 77 Magadia RR, Weinstein MP. Laboratory diagnosis of bacteremia and fungemia. Infect Dis Clin N Am. 2001;15:1009-1024. 78 Mirrett S, Reller LB, Petti CA, et al. Controlled clinical comparison of BacT/ALERT standard aerobic medium with Bactec standard

aerobic medium for culturing blood. J Clin Microbiol. 2003;41:2391-2394. 79 Reimer LG, Wilson ML, Weinstein MP. Update on detection of bacteremia and fungemia. Clin Microbiol Rev. 1997;10:444-465.


Volume 27 M47-A


80 CLSI/NCCLS. Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard—Third Edition.

CLSI/NCCLS document M22-A3. Wayne, PA: NCCLS; 2004. 81 Eng J. Effect of sodium polyanetholsulfonate in blood cultures. J Clin Microbiol. 1975;1:119-123. 82 Reimer LG, Reller LB. Effect of sodium polyanetholsulfonate on the recovery of Gardnerella vaginalis from blood culture media. J Clin

Microbiol. 1985;21:686-688. 83 Staneck JL, Vincent S. Inhibition of Neisseria gonorrhoeae by sodium polyanetholsulfonate. J Clin Microbiol. 1981;13:463-467. 84 Wilson ML, Weinstein, MP, Mirrett S, et al. Controlled evaluation of BacT/ALERT standard anaerobic and FAN anaerobic blood culture

bottles for the detection of bacteremia and fungemia. J Clin Microbiol. 1995;33:2265-2270. 85 Weinstein MP, Mirrett S, Reimer LG, et al. Controlled evaluation of BacT/ALERT standard aerobic and FAN aerobic blood culture bottles

for detection of bacteremia and fungemia. J Clin Microbiol. 1995;33:978-981. 86 Wilson ML, Mirrett S, Reller LB et al. Recovery of clinically important microorganisms from the BacT/Alert blood culture system does not

require testing for 7 days. Diagn Microbiol Infect Dis. 1993;16:31-34. 87 Towns ML, Reller LB. Diagnostic methods: current best practices and guidelines for isolation of bacteria and fungi in infective

endocarditis. Infect Dis Clin N Am. 2002;16:363-376. 88 Doern GV, Brueggemann AB, Dunne WM, et al. Four-day incubation period for blood culture bottles processed with the Difco ESP blood

culture system. J Clin Microbiol. 1997;35:1290-1292. 89 Cornish N, Kirkley BA, Easley KA, Washington JA. Reassessment of the incubation time in a controlled clinical comparison of the

BacT/Alert aerobic FAN bottle and standard anaerobic bottle used aerobically for the detection of bloodstream infections. Diagn Microbiol Infect Dis. 1998;32:1-7.

90 Bourbeau PP, Pohlman JK. Three days of incubation may be sufficient for routine blood cultures with BacT/ALERT FAN blood culture

bottles. J Clin Microbiol. 2001;39:2079-2082. 91 Han XY, Truant AL. The detection of positive blood cultures by the AccuMed ESP-384 system: the clinical significance of three-day

testing. Diagn Microbiol Infect Dis. 1999;33:1-6. 92 Hawkins BL, Peterson EM, de la Maza LM. Improvement of positive blood culture detection by agitation. Diagn Microbiol Infect Dis.

1986;5:207-213. 93 Weinstein MP, Mirrett S, Reimer LG, Reller LB. Effect of agitation and terminal subcultures on yield and speed of detection of the Oxoid

Signal blood culture system versus the BACTEC radiometric system. J Clin Microbiol. 1989;27:427-430. 94 Hardy DJ, Hulbert BB, Migneault PC. Time to detection of positive BacT/Alert blood cultures and lack of need for routine subculture of 5-

to 7-day negative cultures. J Clin Microbiol. 1992;30:2743-2745. 95 Blazevic DJ, Stemper JE, Matsen JM. Comparison of macroscopic examination, routine Gram stains, and routine subcultures in the initial

detection of positive blood cultures. Appl Microbiol. 1974;27:537-539. 96 McCarthy LR, Senne JE. Evaluation of the acridine orange stain for detection of microorganisms in blood cultures. J Clin Microbiol.

1980;2:107-111. 97 Tierney BM, Henry NK, Washington II JA. Early detection of positive blood cultures by the acridine orange staining technique. J Clin

Microbiol. 1983;18:830-833. 98 Campbell J, Washington JA II. Evaluation of the necessity for routine terminal subcultures of previously negative blood cultures. J Clin

Microbiol. 1980;12:576-587. 99 Gill VJ. Lack of clinical relevance in routine terminal subculturing of blood cultures. J Clin Microbiol. 1981;14:116-118. 100 Casteneda MR. A practical method for routine blood cultures in brucellosis. Proc Soc Exp Biol Med. 1947;64:114-115. 101 Roberts GD, Washington JA II. Detection of fungi in blood cultures. J Clin Microbiol. 1975;1:309-310. 102 Caplan LM, Merz WG. Evaluation of two commercially prepared biphasic media for recovery of fungi from blood. J Clin Microbiol. 1978;

8:469-470. 103 Hall MM, Mueske CA, Ilstrup DM, Washington JA II. Evaluation of biphasic medium for blood cultures. J Clin Microbiol. 1979;10:673-

676. 104 Bille J, Roberts GD, Washington JA II. Retrospective comparison of three blood culture media for the recovery of yeasts from clinical

specimens. Eur J Clin Microbiol. 1983;2:22-25.


Number 17 M47-A


105 Bryan LE. Comparison of a slide blood culture system with a supplemented peptone broth culture method. J Clin Microbiol. 1981;14:389-

392. 106 Pfaller MA, Sibley TK, Westfall LM, et al. Clinical laboratory comparison of a slide blood culture system with a conventional broth system.

J Clin Microbiol. 1982;16:525-530. 107 Kiehn TE, Capitolo C, Mayo JB, Armstrong D. Comparative recovery of fungi from biphasic and conventional blood culture media. J Clin

Microbiol. 1981;14:681-683. 108 Henry NK, Grewell CM, McLimans CA, Washington JA II. Comparison of the Roche Septi-Chek blood culture bottle with a brain heart

infusion biphasic medium bottle and with a tryptic soy broth bottle. J Clin Microbiol. 1984;19:315-317. 109 Weckbach LS, Staneck JL. Performance characteristics of a commercially prepared biphasic blood culture bottle. J Clin Microbiol.

1986;23:700-703. 110 Weinstein MP, Reller LB, Mirrett S, et al. Controlled evaluation of trypticase soy broth in agar slide and conventional blood culture

systems. J Clin Microbiol. 1985;21:626-629. 111 Savinelli et al. What happens when automated blood culture instruments detect growth but there is no technologist in the microbiology

laboratory. DMID. 2004;48:173-174. 112 Alexander M, Daggett PM, Gherna R, et al. Laboratory manual on preservation, freezing, and freeze-drying as applied to algae, bacteria,

fungi and protozoa. In: American Type Culture Collection Methods I. Rockville, MD: American Type Culture Collection; 1980:1-46. 113 Heckly RJ. Preservation of microorganisms. Adv Appl Microbiol. 1978;24:1-53. 114 Reimer LG, Carroll KC. Procedures for the storage of microorganisms. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH,

eds. Manual of Clinical Microbiology, 8th ed. Washington, DC: ASM Press; 2003:67-73. 115 Geha DJ, Roberts GE. Laboratory detection of fungemia. Clin Lab Med. 1994;14:83-97. 116 Guerra-Romero L, Edson RS, Cockerill FR, Hoorstmeier CD, Roberts GD. Comparison of DuPont Isolator and Roche Septi-Chek for

detection of fungemia. J Clin Microbiol. 1987;25:1623-1625. 117 Graybill JR. Histoplasmosis and AIDS. J Infect Dis.1988;158:623-626. 118 Paya CV, Roberts GD, Cockerill FR. Laboratory methods for the diagnosis of disseminated histoplasmosis: clinical importance of the lysis-

centrifugation blood culture technique. Mayo Clin Proc. 1987;62:480-485. 119 Husain S, Munoz P, Forrest G, et al. Infections due to Scedosporium apiospermum and Scedosporium prolificans in transplant recipients:

clinical characteristics and impact of antifungal agent therapy on outcome. Clin Infect Dis. 2005;40:89-99. 120 Jensen TG, Gahrn-Hansen BG, Arendrup M, Bruun B. Fusarium fungaemia in immunocompromised patients. Clin Microbiol Infect.

2004;10:499-501. 121 Nucci M, Akita T, Barreiros G, et al. Nosocomial fungemia due to Exophiala jeanselmei and a Rhinocladiella species: newly described

causes of bloodstream infection. J Clin Microbiol. 2001;39:514-518. 122 Murray PR. Comparison of the lysis-centrifugation and agitated biphasic blood culture systems for detection of fungemia. J Clin Microbiol.

1991;29:96-98. 123 Procop GW, Cockerill FR, Vetter EA, Harmsen WS, Hushes JG, Roberts GD. Performance of five agar media for recovery of fungi from

Isolator blood cultures. J Clin Microbiol. 2000;38:3827-3829. 124 Fuller DD, Davis TE, Denys GA, York MK. Evaluation of BACTEC Myco/F Lytic medium for recovery of mycobacteria, fungi, and

bacteria from blood. J Clin Microbiol. 2001;39:2933-2936. 125 Wilson ML, Davis TE, Mirrett S, et al. Controlled comparison of the BACTEC high-blood-volume fungal medium, BACTEC Plus 26

aerobic blood culture bottle, and 10-milliliter Isolator blood culture system for detection of fungemia and bacteremia. J Clin Microbiol. 1993;31:865-871.

126 Inderlied CB, Kemper CA, Bermudez LEM. The Mycobacterium avium complex. Clin Microbiol Rev. 1993;6:266-310. 127 Falkinham JO. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev. 1996;9:177-215. 128 Doucette K, Fishman JA. Nontuberculous mycobacterial infection in hematopoietic stem cell and solid organ transplant recipients. Clin

Infect Dis. 2004;38:1428-1444. 129 Brown-Elliott BA, Wallace RJ. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing

mycobacteria. Clin Microbiol Rev. 2002;15:716-746.


Volume 27 M47-A


130 Hanscheid T, Monteiro C, Cristino JM, Lito LM, Salgado MJ. Growth of Mycobacterium tuberculosis in conventional BacT/Alert FA blood

culture bottles allows reliable diagnosis of mycobacteremia. J Clin Microbiol. 2005;43:890-891. 131 Archibald LK, McDonald LC, Addison RM, et al. Comparison of BACTEC Myco/F Lytic and Wampole Isolator 10 (Lysis-centrifugation)

systems for detection of bacteremia, mycobacteremia, and fungemia in a developing country. J Clin Microbiol. 2000;38:2994-2997. 132 Kiehn TE, Cammarata R. Laboratory diagnosis of mycobacterial infections in patients with acquired immunodeficiency syndrome. J Clin

Microbiol. 1986;24:708-711. 133 Sewell DL, Rashad AL, Rourke WJ, Poor SL, McCarthy JA, Pfaller MA. Comparison of the Septi-Chek AFB and BACTEC systems and

conventional culture for recovery of mycobacteria. J Clin Microbiol. 1993;31:2689-2691. 134 van Griethuysen AJ, Jansz AR, Buiting AG. Comparison of fluorescent BACTEC 9000 MB system, Septi-Chek AFB system, and

Lowenstein-Jensen medium for detection of mycobacteria. J Clin Microbiol. 1996;34:2391-2394. 135 Witebsky FG, Keiser JF, Conville PS, et al. Comparison of BACTEC 13A medium and DuPont Isolator for detection of mycobacteremia. J

Clin Microbiol. 1988;26:1501-1505. 136 Waite RT, Woods GL. Evaluation of BACTEC Myco/F Lytic medium for recovery of mycobacteria and fungi from blood. J Clin

Microbiol. 1998;36:1176-1179. 137 Crump JA, Tanner DC, Mirrett S, McKnight CM, Reller LB. Controlled comparison of BACTEC 13A, MYCO/F LYTIC, BacT/ALERT

MB, and Isolator 10 systems for detection of mycobacteremia. J Clin Microbiol. 2003;41:1987-1990. 138 Dunne WM Jr, Tillman J, Havens PL. Assessing the need for anaerobic medium for the recovery of clinically significant blood culture

isolates in children. Pediatr Infect Dis J. 1994;13:203-206. 139 Lee CS, Tang RB, Chung RL, Chen SJ. Evaluation of different blood culture media in neonatal sepsis. J Microbiol Immunol Infect.

2000;33:165-168. 140 Linder N, Davidovitch N, Reichman B, et al. Topical iodine-containing antiseptics and subclinical hypothyroidism in preterm infants. J

Pediatr. 1997;131(3):434-439. 141 Dietzman DE, Fischer GW, Schoenknecht FD. Neonatal Escherichia coli septicemia—bacterial counts in blood. J Pediatr. 1974;85:128-

130. 142 Durbin WA, Szymczak EG, Goldmann DA. Quantitative blood cultures in childhood bacteremia. J Pediatr. 1978;92:778-780. 143 Krisher KK, Whyburn DR, Koepnick FE. Comparison of the BacT/Alert pediatric blood culture system, Pedi-BacT, with conventional

culture using the 20-milliliter Becton-Dickinson supplemented peptone broth tube. J Clin Microbiol. 1993;31:793-797. 144 Weisse ME, Bass JW, Young LM. Pediatric blood culture: comparison of yields using aerobic, anaerobic and hypertonic media. Pediatr

Infect Dis J. 1992;11:123-125. 145 Wiswell TE, Hachey WE. Multiple site blood cultures in the initial evaluation for neonatal sepsis during the first week of life. Pediatr Infect

Dis J. 1991;10:365-369. 146 Isaacman DJ, Karasic RB, Reynolds EA, Kost SI. Effect of number of blood cultures and volume of blood on detection of bacteremia in

children. J Pediatr. 1996;128:190-195. 147 Kaditis AG, O'Marcaigh AS, Rhodes KH, et al. Yield of positive blood cultures in pediatric oncology patients by a new method of blood

culture collection. Pediatr Infect Dis J. 1996;15:615-620. 148 Schelonka RL, Chai MK, Yoder BA, et al. Volume of blood required to detect common neonatal pathogens. J Pediatr. 1996;129:275-278. 149 McGowan KL, Foster JA, Coffin SE. Outpatient pediatric blood cultures: time to positivity. Pediatrics. 2000;106:251-255. 150 Gaur AH, Giannini MA, Flynn PM, et al. Optimizing blood culture practices in pediatric immunocompromised patients: evaluation of

media types and blood culture volume. Pediatr Infect Dis J. 2003;22:545-552. 151 Karchmer AW. Nosocomial bloodstream infections: microorganisms, risk factors, and implications. Clin Infect Dis. 2000;31(Suppl

4):S139-S143. 152 Pittet D, Tarara D, Wenzel RP. Nosocomial bloodstream infection in critically ill patients: excel length of stay, extra costs, and attributable

mortality. JAMA. 1994;271:1598-1601. 153 O’Grady NP, Alexander M, Dellinger EP, et al. Guidelines for the prevention of intravascular catheter-related infections. MMWR

Recommendations and Reports. 2002;51(RR10):1-26. 154 Siegman-Igra Y, Anglin AM, Shapiro DE, et al. Diagnosis of vascular catheter-related bloodstream infection: a meta-analysis. JCM.

1997;35(4):928-936.


Number 17 M47-A


155 Bouza E, Burillo A, Munoz P. Catheter-related infections: diagnosis and intravascular treatment. Clin Microbiol Infect. 2002;8(5):265-274. 156 Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous catheter-related infection. NEJM.

1997;296:1305-1309. 157 Roberts RB, Krieger AG, Schiller NL, Gross KC. Viridans streptococcal endocarditis: role of various species, including pyridoxal-

dependent streptococci. Rev Infect Dis. 1979;1:955-966. 158 Washington JA II. The role of the microbiology laboratory in the diagnosis and antimicrobial treatment of infective endocarditis. Mayo Clin

Pro. 1982;57:22-32. 159 Baron EJ, Scott JD, Tompkins LS. Prolonged incubation and extensive subculturing do not increase recovery of clinically significant

microorganisms from standard automated blood cultures. Clin Infect Dis. 2005;41:1677-1680. 160 Petti CA, Bhally HS, Weinstein MP. Utility of extended blood culture incubation for isolation of Haemophilus, Actinobacillus,

Cardiobacterium, Eikenella, and Kingella organisms: a retrospective multicenter evaluation. J Clin Microbiol. 2006;44:257-259. 161 Rubenstein E, Lang R. Fungal endocarditis. Eur Heart J. 1995;16(Suppl B):84-89. 162 Ellis ME, Al-Abdely H, Standridge A, Greer W, Ventura W. Fungal endocarditis: evidence in the world literature, 1965-1995. Clin Infect

Dis. 2001;32:50-62. 163 McLeod R, Remington JS. Fungal endocarditis. In: Rahimtoola SH, et al., eds. Infective Endocarditis. New York, NY: Grune & Stratton;

1978:211-290. 164 Grace CJ, Leiberman J, Pierce K, Littenberg B. Usefulness of blood culture for hospitalized patients who are receiving antibiotic therapy.

Clin Infect Dis. 2001;32:1651-1655. 165 Crepin O, Roussel-Delvallez M, Martin GR, Courcol RJ. Effectiveness of resins in removing antimicrobial agents from blood cultures. J

Clin Microbiol. 1993;31:734-735. 166 Jorgensen JH, Mirrett S, McDonald LC, et al. Controlled clinical laboratory comparison of BACTEC Plus Aerobic/F resin medium with

BacT/Alert aerobic FAN medium for detection of bacteremia and fungemia. J Clin Microbiol. 1997;35:53-58. 167 Pohlman JK, Kirkley BA, Easley KA, Basille BA, Washington JA. Controlled clinical evaluation of BACTEC Plus Aerobic/F and

BacT/Alert aerobic FAN bottles for detection of bloodstream infections. J Clin Microbiol. 1995;33:2856-2858. 168 Doern GV, Barton A, Rao S. Controlled comparative evaluation of BacT/Alert FAN and ESP 80A aerobic media as means for detecting

bacteremia and fungemia. J Clin Microbiol. 1998;36:2686-2689. 169 Frank U, Malkotsis D, Mlangeni D, Daschner FD. Controlled clinical comparison of three commercial blood culture systems. Eur J Clin

Microbiol. 1999;18:248-255. 170 Koontz FP, Flint KK, Reynolds JK, Allen SD. Multicenter comparison of the high volume (10 mL) NR BACTEC PLUS system and the

standard (5 mL) NR BACTEC system. Diagn Microbiol Infect Dis. 1991;14:111-118. 171 Krisher KK, Gibb P, Corbett S, Church D. Comparison of the BacT/Alert PF pediatric FAN blood culture bottle with the standard pediatric

blood culture bottle, the Pedi-BacT. J Clin Microbiol. 2001;39:2880-2883. 172 Wilson ML, Mirrett S, Meredith FT, et al. Controlled clinical comparison of BACTEC Plus Anaerobic/F to standard Anaerobic/F as the

anaerobic companion bottle to Plus Aerobic/F medium for culturing blood from adults. J Clin Microbiol. 2001;39:983-989. 173 Jessamine PG, Hoban DJ, Forward KR. Positive BACTEC resin cultures do not influence antimicrobial selection. Diagn Microbiol Infect

Dis. 1990;13:281-284. 174 McDonald LC, Fune J, Gaido LB, et al. Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture

bottles. J Clin Microbiol. 1996;34:2180-2184. 175 Durack DT, Lukes AS, Bright DK, Duke Endocarditis Service. New criteria for diagnosis of infective endocarditis: utilization of specific

echocardiographic findings. Am J Med. 1994;96:200-209. 176 Geraci JE, Greip PR, Wilkowske CJ, Wilson WR, Washington JA II. Cardiobacterium hominis endocarditis: four cases with clinical and

laboratory observations. Mayo Clin Proc. 1978;53:49-53. 177 Houpikian P, Raoult D. Diagnostic methods: current best practices and guidelines for identification of difficult-to-culture pathogens in

infective endocarditis. Infect Dis Clin North Am. 2002;16:377-392. 178 Lisby G. Molecular methods for diagnosis of infective endocarditis. Infect Dis Clin North Am. 2002;16:393-412. 179 Larson AM, Dougherty MJ, Nowowiejski DJ, et al. Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of

broth blood cultures. J Clin Microbiol. 1994;32:1492-1496.


Volume 27 M47-A


180 Murray CK, Walter EA, Crawford S, McElmeel ML, Jorgensen JH. Abiotrophia bacteremia in a patient with neutropenic fever and

antimicrobial susceptibility testing of Abiotrophia isolates. Clin Infect Dis. 2001;32:e140-e142. 181 Foucault C, Barrau K, Brouqui P, Raoult D. Bartonella quintana bacteremia among homeless people. Clin Infect Dis. 2002;35:684-689. 182 Zeaiter Z, Fournier PE, Greub G, Raoult D. Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum. J Clin

Microbiol. 2003;41:919-925. 183 Slater LN, Welch D, Hensel D, Coody DW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N

Engl J Med. 1990;323:1587-1593. 184 Bannatyne RM, Jackson MC, Memish Z. Rapid detection of Brucella bacteremia by using the BACTEC 9240 system. J Clin Microbiol.

1997;35:2673-2674. 185 Ruiz J, Lorente I, Perez J, Simarro E, Martinez-Campos L. Diagnosis of brucellosis by using blood cultures. J Clin Microbiol. 1997;

35:2417-2418. 186 Yagupsky P, Peled N, Press J, Abramson O, Abu-Rashid M. Comparison of BACTEC 9240 Peds Plus medium and isolator 1.5 microbial

tube for detection of Brucella melitensis from blood cultures. J Clin Microbiol. 1997;35:1382-1384. 187 Yagupsky P. Detection of Brucellae in blood cultures. J Clin Microbiol. 1999;37:3437-3442. 188 Ozturk R, Mert A, Kocak F, et al. The diagnosis of brucellosis by use of BACTEC 9240 blood culture system. Diagn Microbiol Infect Dis.

2002;44:133-135. 189 Krause R, Ramschak-Schwarzer S, Gorkiewicz G, et al. Recurrent septicemia due to Campylobacter fetus and Campylobacter lari in an

immunocompetent patient. Infection. 2002;30:171-174. 190 Haristoy X, Lozniewski A, Tram C, Simeon D, Bevanger L, Lion C. Francisella tularensis bacteremia. J Clin Microbiol. 2003;41:2774-

2776. 191 Clarridge JE III, Raich TJ, Sjosted A, et al. Characterization of two unusual clinically significant Francisella strains. J Clin Microbiol.

1996;34:1995-2000. 192 Doern GV, Davaro R, George M, Campognone P. Lack of requirement for prolonged incubation of Septi-Chek blood culture bottles in

patients with bacteremia due to fastidious bacteria. Diagn Microbiol Infect Dis. 1996;24:141-143. 193 Das M, Badley AD, Cockerill FR, Steckelberg JM, Wilson WR. Infective endocarditis caused by HACEK microorganisms. Ann Rev Med.

1997;48:25-33. 194 Orlicek SL, Welch DF, Kuhls TL. Septicemia and meningitis caused by Helicobacter cinaedi in a neonate. J Clin Microbiol. 1993;31:569-

571. 195 Trivett-Moore NL, Rawlinson WD, Yuen M, Gilbert GL. Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of

two AIDS patients. J Clin Microbiol. 1997;35:1144-1150. 196 Kiehlbauch JA, Tauxe RV, Baker CN, Wachsmuth IK. Helicobacter cinaedi-associated bacteremia and cellulitis in immunocompromised

patients. Ann Intern Med. 1994;121:90-93. 197 Rihs JD, Yu VL, Zuravleff JL, Goetz A, Muder RR. Isolation of Legionella pneumophila from blood with the BACTEC system: a

prospective study yielding positive results. J Clin Microbiol. 1985;22:422-424. 198 Chen TT, Schapiro JM, Loutit J. Prosthetic valve endocarditis due to Legionella pneumophila. J Cardiovasc Surg. 1996;37;631-633. 199 Palmer MF, Zochowski WJ. Survival of leptospires in commercial blood culture systems revisited. J Clin Pathol. 2000;53:713-714. 200 Waites KB, Canupp KC. Evaluation of BacT/ALERT system for detection of Mycoplasma hominis in simulated blood cultures. J Clin

Microbiol. 2001;39:4328-4331. 201 Richter SS, Beekmann SE, Croco JL, et al. Minimizing the workup of blood culture contaminants: implementation and evaluation of a

laboratory-based algorithm. J Clin Microbiol. 2002;40:2437-2444. 202 CLSI/NCCLS. Clinical Laboratory Safety; Approved Guideline—Second Edition. CLSI/NCCLS document GP17-A2. Wayne, PA:

NCCLS; 2004. 203 ISO. Medical laboratories—Requirements for safety. ISO 15190. Geneva: International Organization for Standardization; 2003. 204 CLSI/NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—Fifth Edition.

CLSI/NCCLS document H3-A5. Wayne, PA: NCCLS; 2003.


Number 17 M47-A


205 Biosafety in Microbiological and Biomedical Laboratories (BMBL). 5th edition. US Department of Health and Human Services; Public

Health Service, Centers for Disease Control and Prevention; and National Institutes of Health. February 2007. 206 Guideline for hand hygiene in health-care settings. MMWR. Vol. 51/No. RR-16; October 25, 2002. 207 CLSI/NCCLS. Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory; A Report. CLSI/NCCLS

document X3-R. Wayne, PA: NCCLS; 2002. 208 CLSI/NCCLS. Clinical Laboratory Waste Management; Approved Guideline—Second Edition. CLSI/NCCLS document GP5-A2. Wayne,

PA: NCCLS; 2002. 209 CLSI/NCCLS. Clinical Laboratory Technical Procedure Manuals; Approved Guideline—Fourth Edition. CLSI/NCCLS document GP2-

A4. Wayne, PA: NCCLS; 2002. 210 CLSI/NCCLS. Application of a Quality Management System Model for Laboratory Services; Approved Guideline—Third Edition.

CLSI/NCCLS document GP26-A3. Wayne, PA: NCCLS; 2004.


Volume 27 M47-A


Additional References McDonald LC, Weinstein MP, Fune J, Mirrett S, Reimer LG, Reller LB. Controlled comparison of BacT/Alert FAN aerobic medium and BACTEC fungal blood culture medium for detection of fungemia. J Clin Microbiol. 2001;39:622-624. Mirrett S, Joyce M, Reller LB. Validation of performance of plastic versus glass bottles for culturing anaerobes from blood in BacT/ALERT SN medium. J Clin Microbiol. 2005;12:6150-6151. Reimer LG. Laboratory detection of mycobacteremia. Clin Lab Med. 1994;14:99-105. Salfinger M, Stool EW, Piot D, Heifets L. Comparison of three methods for recovery of Mycobacterium avium complex from blood specimens. J Clin Microbiol. 1988;26:1225-1226. Weinstein MP. Emerging data indicating that extended incubation of blood cultures has little clinical value. Clin Infect Dis. 2005;41:1681-1682.


Number 17 M47-A


Summary of Delegate Comments and Subcommittee Responses M47-P, Principles and Procedures for Blood Cultures; Proposed Guideline General 1. I suggest the following sections and text be reviewed to make the document less USA-centric and more global: - Section 6.2.2—delete “cleared by US FDA” in two locations - Section 6.3.4—delete “available in United States” - Section 6.5.2—the reference to a US study is OK in the context, but information about the rest of the world

should also be provided - Section 9—delete the last sentence that refers to specific OSHA and CFR requirements - Section 9.1.2—delete the specific US regulation or at least cite comparable regulations from other countries. • Sections 6.2.2 and 6.3.4: The wording has been changed as suggested.

Section 6.5.2: This is intended as a supporting citation only. No change is made. Section 9: This sentence has been deleted as suggested. Section 9.1.2: This section has been revised substantially in response to comment #15. The cited reference is now available online (without cost) and thus should be considered a global resource. No other changes are made.

2. Excellent document. Recommendations for culturing blood in order to specifically isolate brucella should focus

on optimal use of media for the CMBCSs or use Isolator Tubes with subculture to appropriate agar media. For the CMBCS the current formulations of the anaerobic media do not support the growth of brucella as documented in some of the studies reporting on isolation of brucella from the automated systems. Therefore, the recommendation for brucella culture with a CMBCS should be the use of a set of aerobic media and not use the anaerobic bottle. The number of sets to collect should probably be at least two.

• This section (6.5.5) has been revised to note that most isolates are recovered only from aerobic bottles

when a CMBCS is used. Section 5.2, Number of Blood Cultures 3. The brief paragraph on page 5 regarding surveillance blood cultures remains unclear with regard to the value of

these cultures in certain patient populations. • This section has been revised to address this. Section 5.6, Blood Culture Collection 4. I am writing to submit a comment pertaining to an acceptable contamination rate for blood cultures. Last

paragraph: “...minimizing contamination rates to an acceptable range, typically < 3%.” Compare that to page 33 of the document, “The goal for blood culture contamination rate...should be below 2%.” Was it the intent that the second statement would specify a different percentage because it is a QA Indicator? This is a bit confusing. Our lab, as well as many others, monitors blood culture contamination rates on a monthly basis and reports that rate to Infection Control, using the 3% rate as acceptable rate. We would like to ask the committee to consider 3% as the rate in both sections of the document.

• The second percentage found in Section 10.1.3 has been changed to 3% to make the text internally

consistent.

Clinical and Laboratory Standards Institute consensus procedures include an appeals process that is described in detail in Section 8 of the Administrative Procedures. For further information, contact CLSI or visit our website at www.clsi.org.


Volume 27 M47-A


5. Third paragraph, eighth sentence “....because of the risk of reflux of the broth media back into the vein...”: this “reflux” experience was related to the old practice of directly inserting the blood culture bottle into the venipuncture needle holder attached to the needle in the vein. Newer and safer blood culture needle devices were introduced about five to ten years ago; these are typically butterfly needles attached to long tubing attached to “domes” fitted over the blood culture bottles, with blood flow controlled by the vacuum in the blood culture bottles and a septum over the protected needle contained within the “dome.” These devices have eliminated the possibility of “reflux” of broth media into the vein. These devices are the preferred (and predominant) method of collecting blood in the various hospitals I’ve worked. I rarely hear anymore of blood cultures being collected by syringes or directly from a vacutainer holder attached to a needle inserted in the arm.

From my limited perspective, these devices are preferred for use in most settings because of the improved safety (fewer needlesticks, more controlled venipuncture/blood collection). I don't know about the rest of the world, but perhaps these devices should be mentioned in this section.

The device we use at my current lab is the Saf-TWing Blood Collection Set (Smiths Medical International Ltd., Hythe Kent, UK). This product is also latex-free and sterile.

• It is the policy and practice of CLSI not to use brand names or to make recommendations regarding the

use of specific commercial products. No changes are made. 6. Fourth paragraph – Changing needles before culture inoculation created a major safety hazard and numerous

needlesticks occurred, as correctly stated. But, and related to comment #5 above, the newer blood collection devices (dome assembly set) allow the same needle to draw directly into both bottles of a single set of blood cultures. And use of these devices has dramatically reduced needlestick injuries from this procedure.

I’d recommend an addition to this paragraph discussing the advantage of these blood collection “dome” devices, especially relative to the safety considerations (i.e., needlestick injuries).

• It is the policy and practice of CLSI not to use brand names or to make recommendations regarding the

use of specific commercial products. No changes are made. 7. Fifth paragraph: This paragraph states that the acceptable blood culture contamination rate is < 3%. Yet, two

other sections (p. 26, Section 8, Contaminants; p. 33, Example QA Indicator 1) state “below 2%.” Please be consistent.

• Please see the response to comment #4. Section 6.2.1.1, Conventional Broth Cultures 8. Third paragraph – “…incubated at 35 degrees,” yet on page 9 under “Temperature” it is 35 - 37 degrees. • The text has been changed to 35 degrees to make the document internally consistent. Section 6.2.3, Preservation of Isolates for Further Testing 9. Third paragraph - What is the expected temperature for the ultra-low temperature storage? • The text has been changed to include: (e.g., -70 oC). Section 6.5.2, Catheter-Related Bloodstream Infections 10. Typo on page 16, 4th paragraph: “250 000.” • This has been changed to "250,000."


Number 17 M47-A


Section 6.5.2.1, Recommendations for Short-Term Peripheral Catheters 11. Interpretation of results should be Interpretation of Culture results (the same as in Section 6.5.2.2). • This change has been made as suggested. Section 6.5.3, Special Considerations: Infective Endocarditis 12. Under “Blood Culture Media,” first paragraph, second sentence—“recovery of facultative anaerobes.” • This change has been made as suggested. Section 8, Contaminants 13. First sentence: What does the point mean? An example would help explain the second point. • The first part of this paragraph has been revised. Section 9, Safety Issues 14. This section might benefit from a discussion of the blood culture collection devices (“dome” assembly, or

whatever this thing is called if it has a generic name). • Please see response to comment #5. Section 9.1.2, Transmission of Agents Other Than Hepatitis Viruses and Retroviruses 15. Second paragraph, last sentence: List below is missing. • This section has been revised and the reference has been updated to the February 2007 edition. Because

this information already is available from a number of other sources, and therefore would be redundant here, it has been deleted.

Section 10.1.4, Sample Transport 16. Sample transport has a two-hour timeframe included here. This information was not included in Section 5.6.1,

Transport of Specimens to the Laboratory. • The text on page 7 has been changed to be consistent with the wording in Section 10.1.4. Section 10.3.2, Specimen Management 17. This section is Record Management. • The title of the section has been changed.


Volume 27 M47-A


NOTES


Number 17 M47-A


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 approach is based on the model presented in the most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any healthcare service’s path of workflow (i.e., 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 quality system essentials (QSEs) are: Documents & Records Equipment Information Management Process Improvement Organization Purchasing & Inventory Occurrence Management Customer Service Personnel Process Control Assessments―External and

Internal Facilities & Safety

M47-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.

Doc

umen

ts

& R

ecor

ds

Org

aniz

atio

n

Pers

onne

l

Equi

pmen

t

Purc

hasi

ng &

In

vent

ory

Proc

ess

Con

trol

Info

rmat

ion

Man

agem

ent

Occ

urre

nce

Man

agem

ent

Ass

essm

ent

Proc

ess

Impr

ovem

ent

Serv

ice

&

Satis

fact

ion

Faci

litie

s &

Safe

ty

X

Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. 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. For example, CLSI/NCCLS document GP26⎯Application of a Quality Management System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow these processes to deliver the laboratory’s services, namely quality laboratory information. M47-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/NCCLS Publications section on the following page.

Preexamination Examination Postexamination

Exam

inat

ion

orde

ring

Sam

ple

colle

ctio

n

Sam

ple

trans

port

Sam

ple

rece

ipt/p

roce

ssin

g

Exam

inat

ion

Res

ults

revi

ew

and

follo

w-u

p

Inte

rpre

tatio

n

Res

ults

repo

rting

an

d ar

chiv

ing

Sam

ple

man

agem

ent

X X X X X X

Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.


Volume 27 M47-A


Related CLSI/NCCLS Publications∗ GP2-A5 Laboratory Documents: Development and Control; Approved Guideline— Fifth Edition (2006). This

document provides guidance on development, review, approval, management, and use of policy, process, and procedure documents in the medical laboratory community.

GP17-A2 Clinical Laboratory Safety; Approved Guideline—Second Edition (2004). This document contains general

guidelines for implementing a high-quality laboratory safety program. The framework is adaptable to any laboratory. An NCCLS-CAP joint project.

H3-A5 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—

Fifth Edition (2003). This document provides procedures for the collection of diagnostic specimens by venipuncture, including line draws, blood culture collection, and venipuncture in children. It also includes recommendations on order of draw.

HS1-A2 A Quality Management System Model for Health Care; Approved Guideline—Second Edition (2004).

This document provides a model for healthcare service providers that will assist with implementation and maintenance of effective quality systems.

M22-A3 M29-A3

Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard—Third Edition (2004). This standard contains quality assurance procedures for manufacturers and users of prepared, ready-to-use microbiological culture media. 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.

X3-R Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory; A

Report (2002). This report presents a step-by-step approach for implementing safer medical devices that reduce or eliminate sharps injuries to laboratory personnel. X3-R is written in an expanded checklist format, outlines a process that goes beyond general recommendations, and specifically addresses the needs of professionals performing specimen collection and clinical laboratory procedures.

∗ Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process; therefore, readers should refer to the most recent editions.


Number 17 M47-A


NOTES


Volume 27 M47-A


NOTES


Active Membership (as of 1 April 2007)

Sustaining Members Abbott American Association for Clinical Chemistry AstraZeneca Pharmaceuticals Bayer Corporation BD Beckman Coulter, Inc. bioMérieux, Inc. CLMA College of American Pathologists GlaxoSmithKline Ortho-Clinical Diagnostics, Inc. Pfizer Inc Roche Diagnostics, Inc. Professional Members AABB American Academy of Family Physicians American Association for Clinical Chemistry American Association for Laboratory Accreditation American Association for Respiratory Care American Chemical Society American College of Medical Genetics American Medical Technologists American Society for Clinical Laboratory Science American Society for Microbiology American Type Culture Collection, Inc. ASCP Associazione Microbiologi Clinici Italiani (AMCLI) British Society for Antimicrobial Chemotherapy Canadian Society for Medical Laboratory Science - Société Canadienne de Science de Laboratoire Médical COLA College of American Pathologists College of Medical Laboratory Technologists of Ontario College of Physicians and Surgeons of Saskatchewan Elkin Simson Consulting Services ESCMID Family Health International Hong Kong Accreditation Service Innovation and Technology Commission Italian Society of Clinical Biochemistry and Clinical Molecular Biology International Federation of Biomedical Laboratory Science International Federation of Clinical Chemistry JCCLS Joint Commission on Accreditation of Healthcare Organizations National Society for Histotechnology, Inc. Ontario Medical Association Quality Management Program-Laboratory Service RCPA Quality Assurance Programs PTY Limited SDS Pathology SIMeL Sociedad Espanola de Bioquimica Clinica y Patologia Molecular Sociedade Brasileira de Analises Clinicas Sociedade Brasileira de Patologia Clinica Turkish Society of Microbiology Washington G2 Reports World Health Organization Government Members Association of Public Health Laboratories BC Centre for Disease Control Caribbean Epidemiology Centre Centers for Disease Control and Prevention Centers for Disease Control and Prevention – Tanzania Centers for Medicare & Medicaid Services Centers for Medicare & Medicaid Services/CLIA Program Chinese Committee for Clinical Laboratory Standards

Department of Veterans Affairs FDA Center for Devices and Radiological Health FDA Center for Veterinary Medicine Health Canada Massachusetts Department of Public Health Laboratories Ministry of Health and Social Welfare - Tanzania National Center of Infectious and Parasitic Diseases (Bulgaria) National Health Laboratory Service (South Africa) National Institute of Standards and Technology National Pathology Accreditation Advisory Council (Australia) New York State Department of Health Ontario Ministry of Health Pennsylvania Dept. of Health Saskatchewan Health-Provincial Laboratory Scientific Institute of Public Health; Belgium Ministry of Social Affairs, Public Health and the Environment University of Iowa, Hygienic Lab Industry Members AB Biodisk Abbott Abbott Diabetes Care Abbott Molecular Inc. Abbott Point of Care Inc. Access Genetics ACM Medical Technologies, Inc. Acupath AdvaMed Advanced Liquid Logic Advancis Pharmaceutical Corporation Affymetrix, Inc. Agilent Technologies Ammirati Regulatory Consulting Anapharm, Inc. Anna Longwell, PC Aptium Oncology ARK Diagnostics, Inc. Arpida Ltd A/S ROSCO AstraZeneca Pharmaceuticals Aviir, Inc. Axis-Shield POC AS Bayer Corporation – Tarrytown, NY Bayer Corporation – West Haven, CT Bayer HealthCare, LLC, Diagnostics Div. – Elkhart, IN BD BD Biosciences – San Jose, CA BD Diabetes Care BD Diagnostic Systems BD Vacutainer Systems Beckman Coulter, Inc. Beth Goldstein Consultant (PA) Bioanalyse, Ltd. Bio-Development S.r.l. Bio-Inova Life Sciences International Biomedia Laboratories SDN BHD bioMérieux (NC) bioMérieux, Inc. (MO) Bio-Rad Laboratories, Inc. Bio-Rad Laboratories, Inc. – France Bio-Rad Laboratories, Inc. – Irvine, CA Bio-Rad Laboratories, Inc. – Plano, TX Black Coast Corporation – Health Care Systems Consulting Blaine Healthcare Associates, Inc. Center for Measurement Standards/ITRI Cepheid Chen & Chen, LLC Comprehensive Cytometric Consulting Control Lab Copan Diagnostics Inc. Cosmetic Ingredient Review Cubist Pharmaceuticals Cumbre Inc. Dade Behring Inc. – Cupertino, CA Dade Behring Inc. – Deerfield, IL Dade Behring Inc. – Glasgow, DE Dade Behring Inc. – Marburg, Germany Dade Behring Inc. – Sacramento, CA David G. Rhoads Associates, Inc. Decode Genetics, Inc. Diagnostic Products Corporation Diagnostica Stago Digene Corporation Eiken Chemical Company, Ltd.

Elanco Animal Health Electa Lab s.r.l. Enterprise Analysis Corporation Eomix, Inc. Eurofins Medinet FasTraQ Inc. (NV) Future Diagnostics B.V. Gavron Group, Inc. Gen-Probe Genaco Biomedical Products, Inc. Genomic Health, Inc. Gentris Corporation Genzyme Clinical Specialty Laboratory Genzyme Diagnostics GlaxoSmithKline Gluco Tec, Inc. GluMetrics, Inc. Greiner Bio-One Inc. HistoGenex N.V. Immunicon Corporation Instrumentation Laboratory IT for Small Business Janssen Ortho-McNeil Pharmaceutical Japan Assn. of Clinical Reagents Industries Johnson & Johnson Pharmaceutical Research and Development, L.L.C. K.C.J. Enterprises LabNow, Inc. Laboratory Specialists, Inc. LifeScan, Inc. (a Johnson & Johnson Company) Maine Standards Company, LLC Medical Device Consultants, Inc. Merck & Company, Inc. Micromyx, LLC MicroPhage MultiPhase Solutions, Inc. Mygene International, Inc. Nanogen, Point-of-Care Diagnostics Div. NeED Pharmaceuticals Srl Nippon Becton Dickinson Co., Ltd. Nissui Pharmaceutical Co., Ltd. NovaBiotics (Aberdeen, UK) Novartis Institutes for Biomedical Research Nucryst Pharmaceuticals Olympus America, Inc. Optimer Pharmaceuticals, Inc. Orion Genomics, LLC Ortho-Clinical Diagnostics, Inc. (Rochester, NY) Oxonica (UK) Panaceapharma Pharmaceuticals Paratek Pharmaceuticals Pathology Services Inc. PathWork Informatics Pfizer Animal Health Pfizer Inc Pfizer Italia Srl Phadia AB Powers Consulting Services PPD, Inc. Primera Biosystems, Inc. QSE Consulting Radiometer America, Inc. Radiometer Medical A/S Reliance Life Sciences Replidyne Rib-X Pharmaceuticals Roche Diagnostics GmbH Roche Diagnostics, Inc. Roche Laboratories Roche Molecular Systems Sanofi Pasteur Sarstedt, Inc. Schering Corporation Seneca Medical, Inc. Sequenom, Inc. SFBC Anapharm Sphere Medical Holding Streck Laboratories, Inc. Sysmex America, Inc. (Long Grove, IL) Sysmex Corporation (Japan) Tethys Bioscience, Inc. The Clinical Microbiology Institute TheraDoc Therapeutic Monitoring Services, LLC Theravance Inc. Third Wave Technologies, Inc. Thrombodyne, Inc. Transasia Bio-Medicals Limited Trek Diagnostic Systems, Inc. TrimGen Corporation Watin-Biolife Diagnostics and Medicals Wyeth Research XDX, Inc. YD Consultant Trade Associations AdvaMed

Japan Association of Clinical Reagents Industries (Tokyo, Japan) Associate Active Members 3rd Medical Group (AK) 48th Medical Group/MDSS (APO, AE) 59th MDW/859th MDTS/MTL (TX) Aberdeen Royal Infirmary (Scotland) Academisch Ziekenhuis -VUB (Belgium) Acibadem Labmed Clinical Laboratory (Turkey) ACL Laboratories (IL) ACL Laboratories (WI) Akron’s Children’s Hospital (OH) Alameda County Medical Center (CA) Albany Medical Center Hospital (NY) Albemarle Hospital (NC) Alfred I. du Pont Hospital for Children (DE) All Children’s Hospital (FL) Allegheny General Hospital (PA) Allina Labs (MN and WI) Alton Memorial Hospital (MN) American Hospital Dubai (UAE) American University of Beirut Medical Center (NY) Arkansas Methodist Medical Center (AR) Arnett Clinic, LLC (IN) Asante Health System (OR) Aspirus Wausau Hospital (WI) Associated Regional & University Pathologists (UT) Atlantic Health System (NJ) Augusta Medical Center (VA) AZ Sint-Jan (Belgium) Azienda Ospedale Di Lecco (Italy) Balfour Hospital (Scotland) Baptist Hospital for Women (TN) Barbados Reference Laboratory (Barbados) Bassett Army Community Hospital (AK) BayCare Health System (FL) Baystate Medical Center (MA) BC Biomedical Laboratories (Surrey, BC, Canada) Borders General Hospital (Scotland) Boulder Community Hospital (CO) British Columbia Cancer Agency – Vancouver Cancer Center (BC, Canada) Broward General Medical Center (FL) Cadham Provincial Laboratory – MB Health (Canada) Calgary Laboratory Services (Calgary, AB, Canada) Canterbury Health Laboratories (New Zealand) Cape Breton Healthcare Complex (Canada) Cape Fear Valley Medical Center Laboratory (NC) Capital Health - Regional Laboratory Services (Canada) Capital Health System Fuld Campus (NJ) Capital Health System Mercer Campus (NJ) Catholic Health Initiatives (KY) CDC/HIV (APO, AP) CDPH (CO) Central Baptist Hospital (KY) Central Kansas Medical Center (KS) Central Texas Veterans Health Care System Centralized Laboratory Services (NY) Centura Laboratory (CO) Chang Gung Memorial Hospital (Taiwan) Chesapeake General Hospital (VA) Chester County Hospital (PA) Children’s Healthcare of Atlanta (GA) Childrens Hospital of Wisconsin (WI) Christiana Care Health Services (DE) CHUM Hopital Saint-Luc (Canada) City of Hope National Medical Center (CA) Clarian Health – Clarian Pathology Laboratory (IN) Cleveland Clinic Health System Eastern Region (OH) Clovis Community Hospital (CA) CLSI Laboratories (PA) Commonwealth of Kentucky Community Care 5 (OH) Community Hospital (IN) Community Hospital (OH) Connolly Hospital (Ireland) Covance CLS (IN) Creighton Medical Laboratories (NE) Creighton University Medical Center (NE) Crosshouse Hospital (Scotland) Cumberland Regional Health Care Centre (Canada) Danish Institute for Food and Veterinary Research (Denmark) Licensed to: Giancarlo Scoppettuolo, MD UCSC

This document is protected by copyright. CLSI order # 52644, id # 453704, Downloaded on 4/27/2008.

Darwin Library NT Territory Health Services (Australia) David Grant Medical Center (CA) Daviess Community Hospital (IN) Dekalb Memorial Hospital (IN) DeWitt Healthcare Network (USA Meddac) (VA) DHHS NC State Lab of Public Health (NC) Diagnofirm Med Labs Diagnostic Laboratory Services, Inc. (HI) Diagnostic Services of Manitoba (Canada) Dianon Systems/Lab Corp. (OK) Dr. Everette Chalmers Regional Hospital (NB) Driscoll Children’s Hospital (TX) DSI of Bucks County (PA) DUHS Clinical Laboratories (NC) Dumfries and Galloway Royal Infirmary (Scotland) Edinburgh Royal Infirmary (Scotland) EMH Regional Medical Center (OH) Emory University Hospital (GA) Evangelical Community Hospital (PA) Evanston Hospital (IL) Exeter Hospital (NH) Federal Medical Center (MN) Firelands Regional Medical Center (OH) First Health of the Carolinas Moore Regional Hospital (NC) Fisher-Titus Memorial Hospital (OH) Flaget Memorial Hospital (KY) Fleury S.A. (Brazil) Forum Health Northside Medical Center (OH) Fox Chase Cancer Center (PA) Gamma Dynacare Medical Laboratories (Ontario, Canada) Garden City Hospital (MI) Geisinger Medical Center (Danville, PA) Geisinger South Wilkes Barre Laboratory (PA) Geisinger Wyoming Valley Medical Center (Wilkes-Barre, PA) Genesis Healthcare System (OH) Gilbert Bain Hospital (Scotland) Glasgow Royal Infirmary (Scotland) Good Samaritan Hospital (NE) Hagerstown Medical Laboratory (MD) Hamad Medical Corporation (Qatar) Harris Methodist Fort Worth (TX) Hartford Hospital (CT) Health Network Lab (PA) Health Partners Laboratories (VA) Health Waikato (New Zealand) Heidelberg Army Hospital (APO, AE) High Desert Health System (CA) Hoag Memorial Hospital Presbyterian (CA) Holy Cross Hospital (MD) Holy Family Medical Center (WI) Holy Spirit Hospital (PA) Holzer Medical Center (Gallipolis, OH) Holzer Medical Center (Jackson, OH) Hopital Cite de La Sante De Laval (Canada) Hôpital Maisonneuve - Rosemont (Montreal, Canada) Hôpital Sacré-Coeur de Montreal (Quebec, Canada) Hôpital Sainte - Justine (Quebec) Hopital Santa Cabrini Ospedale (Canada) Hospital Albert Einstein (Brazil) Hospital de Dirino Espirito Santa (Portugal) Hospital De Sousa Martins (Guarda) (Portugal) Hospital for Sick Children (Toronto, ON, Canada) Hôtel Dieu Grace Hospital Library (Windsor, ON, Canada) Humility of Mary Health Partners (OH) Hunterdon Medical Center (NJ) Icon Laboratories (Ireland) IGate Clinical Research Intl., Pvt., LTD (India) Indiana University - Chlamydia Laboratory (IN) Inova Fairfax Hospital (VA) Institut fur Stand. und Dok. im Med. Lab. (Germany) Institut National de Santé Publique du Quebec Centre de Doc. – INSPQ (Canada) Integrated Regional Laboratories South Florida (FL)

International Health Management Associates, Inc. (IL) Island Hospital (WA) Jackson Hospital & Clinic, Inc. (AL) Jackson Purchase Medical Center (KY) Jacobi Medical Center (NY) John C. Lincoln Hospital (AZ) John F. Kennedy Medical Center (NJ) John H. Stroger, Jr. Hospital of Cook County (IL) John Muir Medical Center (CA) Johns Hopkins Medical Institutions (MD) Johns Hopkins University (MD) Kadlec Medical Center (WA) Kaiser Permanente (CA) Kaiser Permanente (MD) Kangnam St. Mary’s Hospital (Korea) Kenora-Rainy River Reg. Lab. Program (Canada) King Fahad Medical City (Saudi Arabia) King Faisal Specialist Hospital (MD) Kosciusko Laboratory (IN) LabCorp (NC) Laboratory Alliance of Central New York (NY) LabPlus Auckland Healthcare Services Limited (New Zealand) Lakeland Regional Medical Center (FL) Landstuhl Regional Medical Center (APO, AE) Langlade Memorial Hospital (WI) Legacy Laboratory Services (OR) Lewis-Gale Medical Center (VA) L’Hotel-Dieu de Quebec (Quebec, Canada) Licking Memorial Hospital (OH) LifeBridge Health Sinai Hospital (MD) Lions Gate Hospital (BC, Canada) Long Beach Memorial Medical Center (CA) Long Island Jewish Medical Center (NY) Los Angeles County Public Health Lab. (CA) Louis A Johnson Medical Center (WV) Madison Parish Hospital (LA) Magruder Memorial Hospital (OH) Main Line Clinical Laboratories, Inc. (PA) Malmo University Hospital (Sweden) Manipal Acunova (India) Marshfield Clinic (WI) Martin Luther King/Drew Medical Center (CA) Martin Memorial Health Systems (FL) Marymount Medical Center (KY) Massachusetts General Hospital (Microbiology Laboratory) Maxwell Air Force Base (AL) MDS Metro Laboratory Services (Burnaby, BC, Canada) Mease Countryside Hospital (FL) Mease Dunedin Hospital (FL) Medical Centre Ljubljana (Slovenia) Medical College of Virginia Hospital (VA) Medical Univ. of South Carolina (SC) Memorial Health Center, Inc. (WI) Memorial Hospital (OH) Memorial Hospital Miramar (FL) Memorial Hospital Pembroke (FL) Memorial Hospital West (FL) Memorial Regional Hospital (FL) Mercy Medical Center (CO) Mercy Medical Center (ID) Mercy Medical Center (OR) Methodist Hospital (MN) Methodist Hospital (TX) Methodist Hospital Pathology (NE) Monklands Hospital (Scotland) Montreal General Hospital (Canada) Morgan Hospital & Medical Center (IN) Morton Plant Hospital (FL) Mount Sinai Hospital (NY) Mountainside Hospital (NJ) National Serology Reference Laboratory, Australia Naval Hospital Cherry Point Laboratory (NC) Naval Hospital Oak Harbor (WA) NB Department of Health & Wellness (New Brunswick, Canada) The Nebraska Medical Center New England Fertility Institute (CT) New Lexington Clinic (KY) New York City Department of Health and Mental Hygiene (NY) The New York Hospital Medical Center of Queens (NY) New York-Presbyterian Hospital (NY) New York University Medical Center (NY)

Ninewells Hospital (Scotland) North Bay Hospital (FL) North Mississippi Medical Center (MS) North Shore Hospital Laboratory (Auckland, New Zealand) North Shore-Long Island Jewish Health System Laboratory (NY) Northern Plains Laboratory (ND) Northridge Hospital Medical Center (CA) Northwest Texas Hospital (TX) Northwestern Memorial Hospital (IL) Norton Healthcare (KY) Ochsner Clinic Foundation (LA) Oklahoma Heart Hospital, LLC (OK) Onze Lieve Vrouw Ziekenhuis (Belgium) Orlando Regional Healthcare System (FL) Our Lady of the Way Hospital (KY) Overlook Hospital (NJ) Palisades Medical Center (NJ) Pathologists Associated (IN) Pathology and Cytology Laboratories, Inc. (KY) Pathology Associates Medical Laboratories (WA) PathWest (Australia) PCA Southeast (TN) Pediatrix Screening Inc. (PA) Pennsylvania Hospital (PA) Penrose St. Francis Health Services (CO) Perry County Memorial Hospital (IN) Physicians Reference Laboratory (KS) Pitt County Memorial Hospital (NC) Powell River General Hospital (BC, Canada) PPD (KY) Prince George Medical Lab (Prince George, BC) Providence Health Care (Canada) Provincial Health Services Authority (Vancouver, BC, Canada) Provincial Laboratory for Public Health (Edmonton, AB, Canada) Queen Elizabeth Hospital (Canada) Queensland Health Pathology Services (Australia) Quest Diagnostics, Inc Quest Diagnostics, Inc (San Juan Capistrano, CA) Quintiles Laboratories, Ltd. (GA) Raigmore Hospital (UK) Redington-Fairview General Hospital (ME) Régie Régionale Dela Santé Beaséjour (Canada) Regional Health Authority - Central Manitoba Inc (Canada) Regional Health Authority Four (RHA4) (Canada) Regions Hospital (MN) Research Medical Center (MO) Richmond General Hospital (BC, Canada) Riverside Methodist Hospital (OH) Riverview Hospital (WI) Riyadh Armed Forces Hospital (Riyadh, Saudi Arabia) Robert Wood Johnson University Hospital (NJ) Roxborough Memorial Hospital (PA) Royal Alexandra Hospital (Scotland) Sahlgrenska Universitetssjukhuset (Sweden) Saint Elizabeth Regional Medical Center (NE) Saint Francis Hospital & Medical Center (CT) St. Agnes Healthcare (MD) St. Anthony Hospital Central Laboratory (CO) St. Anthony’s Hospital (FL) St. Barnabas Medical Center (NJ) St. Christopher’s Hospital for Children (PA) St. Eustache Hospital (Quebec, Canada) St. Francis Medical Center (MN) St. John Hospital and Medical Center (MI) St. John’s Hospital & Health Ctr. (CA) St. Joseph Medical Center (MD) St. Joseph Mercy (WI) St. Joseph Mercy Hospital (MI) St. Joseph’s Hospital (FL) St. Joseph's Medical Center (CA) St. Joseph's Regional Medical Center (NJ) St. Jude Children’s Research Hospital (TN) St. Louis Children’s Hospital (MO) St. Luke’s Hospital (PA) St. Margaret Memorial Hospital (PA) St. Mary Corwin Regional Medical Center Laboratory (CO) St. Mary Medical Center (CA) St. Mary’s Health Center (MO) St Mary’s Healthcare (SD) St. Mary’s Hospital (BC, Canada) St. Mary’s Medical Center (IN) St. Rose Dominican Hospitals (NV) St. Thomas More Hospital (CO)

San Antonio Community Hospital (TX) San Francisco General Hospital-University of California San Francisco (CA) Santa Monica Hospital Med. Ctr. (CA) Seoul Clinical Laboratories (Korea) Shands at the University of Florida Shape Healthcare Clinic (APO, AE) Sheik Kalifa Medical City (UAE) Shore Memorial Hospital (NJ) SJRMC Plymouth Laboratory (IN) Sonora Quest JV (AZ) South Bend Medical Foundation (IN) South Dakota State Health Laboratory (SD) South Florida Baptist Hospital (FL) South Texas Laboratory (TX) Southern Health Care Network (Australia) Southwest Nova District Health Authority (Canada) Specialty Laboratories, Inc. (CA) Squamish General Hospital (BC, Canada) Starke Memorial Hospital Laboratory (IN) State of Connecticut Department of Public Health (CT) State of Washington Public Health Labs Steele Memorial Hospital (ID) Stirling Royal Infirmary (Scotland) Stony Brook University Hospital (NY) Stormont-Vail Regional Medical Center (KS) Stratford General Hospital (Canada) Sunnybrook & Women’s College Health Sciences Centre (Toronto, Ontario) Sunnybrook Health Science Center (ON, Canada) Swedish Medical Center (CO) Sydney South West Pathology Service (Australia) Taipei Veterans General Hospital (Taiwan) Taiwan Society of Laboratory Medicine Tampa General Hospital (FL) Temple Univ. Hospital - Parkinson Pav. (PA) Texas Department of State Health Services (TX) The Bermuda Hospitals (Bermuda) The Community Hospital (OH) The Hospital for Sick Children (Canada) The Nebraska Medical Center (NB) The New York Hospital Medical Center of Queens (NY) The Ottawa Hospital (Canada) The Permanente Medical Group (CA) The Public Health Laboratory, H47 (AR) The Toledo Hospital (OH) The University of Texas Medical Branch (TX) The Wisconsin Heart Hospital (WI) Thomason Hospital (TX) Timmins and District Hospital (Canada) Touro Infirmary (LA) TPMG Inc. (CA) Tri-Cities Laboratory (WA) Tripler Army Medical Center (HI) Tuen Mun Hospital (Hong Kong) Tufts New England Medical Center Hospital (MA) Tuttle Army Health Clinic (GA) UBC Hospital (BC, Canada) UCLA Immunogenetics Lab (CA) UCLA Medical Center (CA) UCSD Medical Center (CA) UCSF Medical Center China Basin (CA) UMC of Southern Nevada (NV) UNC Hospitals (NC) Union Clinical Laboratory (Taiwan) United Clinical Laboratories (IA) Unity HealthCare (IA) Universita Campus Bio-Medico (Italy) Universitair Ziekenhuis Antwerpen (Belgium) University of Colorado Health Sciences Center (CO) University of Colorado Hospital University of Illinois Medical Center (IL) University of Maryland Medical System University of Medicine & Dentistry, NJ University Hosp. (NJ) University of MN Medical Center - Fairview University of Missouri Hospital (MO) University of MS Medical Center (MS) University of the Ryukyus (Japan) University of Virginia Medical Center University of Washington U.S. Army Health Clinic – Vicenza (APO) US LABS, Inc. (CA) U.S.A. Meddac (Pathology Division) (MO) U.T. Health Center (TX) UZ-KUL Medical Center (Belgium) VA (Asheville) Medical Center (NC) VA (Bay Pines) Medical Center (FL) VA (Cincinnati) Medical Center (OH) VA (Colmery-O’Neil) Medical Center (KS) VA (Des Moines) Central Iowa Healthcare Systems (IA)


VA (Fargo) Medical Center (ND) VA (Fayetteville) Medical Center (AR) VA (Iowa City) Medical Center (IA) VA (Lincoln) Nebraska Western Iowa Healthcare System (NE) VA New Jersey Health Care System (NJ) VA (Phoenix) Medical Center (AZ) VA (San Diego) Medical Center (CA) VA (Tucson) Medical Center (AZ)

Valley Health (VA) Vancouver Hospital and Health Sciences Center (BC, Canada) Virga Jessezieukenhuis (Belgium) Virginia Regional Medical Center (MN) ViroMed Laboratories (LabCorp) (MN) Waianae Coast Comprehensive Health Center (HI) Walter Reed Army Medical Center (DC) Warren Hospital (NJ) Washington Hospital Center (DC)

Waterbury Hospital (CT) Waterford Regional Hospital (Ireland) Wellstar Health Systems (GA) West China Second University Hospital, Sichuan University (P.R. China) West Valley Medical Center Laboratory (ID) Westchester Medical Center (NY) Western Isles Hospital (Scotland) Wheaton Franciscan & Midwest Clinical Laboratories (WI)

Wheeling Hospital (WV) Whistler Health Care Centre (BC, Canada) Whitehorse General Hospital (Canada) William Beaumont Hospital (MI) Winchester Hospital (MA) Winn Army Community Hospital (GA) Womack Army Medical Center (NC) Women’s Health Laboratory (TX) Woodlawn Hospital (IN) York Hospital (PA) Yorkshire Hospital (Scotland)

OFFICERS

BOARD OF DIRECTORS

Robert L. Habig, PhD, President Abbott Gerald A. Hoeltge, MD, President-Elect The Cleveland Clinic Foundation Wayne Brinster, Secretary BD W. Gregory Miller, PhD, Treasurer Virginia Commonwealth University Thomas L. Hearn, PhD, Immediate Past President Centers for Disease Control and Prevention Glen Fine, MS, MBA, Executive Vice President

Susan Blonshine, RRT, RPFT, FAARC TechEd Maria Carballo Health Canada Russel K. Enns, PhD Cepheid Mary Lou Gantzer, PhD Dade Behring Inc. Lillian J. Gill, DPA FDA Center for Devices and Radiological Health Prof. Naotaka Hamasaki, MD, PhD Nagasaki International University

Valerie Ng, PhD, MD Alameda County Medical Center/ Highland General Hospital Janet K.A. Nicholson, PhD Centers for Disease Control and Prevention Timothy J. O’Leary, MD, PhD Department of Veteran Affairs Klaus E. Stinshoff, Dr.rer.nat. Digene (Switzerland) Sàrl Michael Thein, PhD Roche Diagnostics GmbH James A. Thomas ASTM International


940 West Valley Road Suite 1400 Wayne, PA 19087 USA PHONE 610.688.0100 FAX 610.688.0700 E-MAIL: [email protected] WEBSITE: www.clsi.org ISBN 1-56238-641-7
