1C

Blood
Cultures IV
ELLEN JO BARON, MELVIN P. WEINSTEIN,
W. MICHAEL DUNNE, JR.,
PABLO YAGUPSKY, DAVID F. WELCH, AND
DONNA M. WILSON

COORDINATING EDITOR
ELLEN JO BARON

Cumitech
CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY
Cumitech 1C Blood Cultures IV
Cumitech 2B Laboratory Diagnosis of Urinary Tract Infections
Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology
Cumitech 5A Practical Anaerobic Bacteriology
Cumitech 6A New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide
Cumitech 7B Lower Respiratory Tract Infections
Cumitech 12A Laboratory Diagnosis of Bacterial Diarrhea
Cumitech 13A Laboratory Diagnosis of Ocular Infections
Cumitech 16A Laboratory Diagnosis of the Mycobacterioses
Cumitech 18A Laboratory Diagnosis of Hepatitis Viruses
Cumitech 19A Laboratory Diagnosis of Chlamydia trachomatis Infections
Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease
Cumitech 23 Infections of the Skin and Subcutaneous Tissues
Cumitech 24 Rapid Detection of Viruses by Immunofluorescence
Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing
Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis
Cumitech 27 Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and
Laboratory Animals
Cumitech 28 Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections
Obtained from Companion and Laboratory Animals
Cumitech 29 Laboratory Safety in Clinical Microbiology
Cumitech 30A Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the
Gastrointestinal Tract
Cumitech 31 Verification and Validation of Procedures in the Clinical Microbiology Laboratory
Cumitech 32 Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained
from Food Animals and Wildlife
Cumitech 33 Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism
Cumitech 34 Laboratory Diagnosis of Mycoplasmal Infections
Cumitech 35 Postmortem Microbiology
Cumitech 36 Biosafety Considerations for Large-Scale Production of Microorganisms
Cumitech 37 Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, and Wildlife
Cumitech 38 Human Cytomegalovirus
Cumitech 39 Competency Assessment in the Clinical Microbiology Laboratory
Cumitech 40 Packing and Shipping of Diagnostic Specimens and Infectious Substances
Cumitech 41 Detection and Prevention of Clinical Microbiology Laboratory-Associated Errors
Cumitech 42 Infections in Hemopoietic Stem Cell Transplant Recipients

Cumitechs should be cited as follows, e.g.: Baron, E. J., M. P. Weinstein, W. M. Dunne, Jr., P. Yagupsky, D. F. Welch, and D. M. Wilson.
2005. Cumitech 1C, Blood Cultures IV. Coordinating ed., E. J. Baron. ASM Press, Washington, D.C.
Editorial board for ASM Cumitechs: Alice S. Weissfeld, Chair; Maria D. Appleman, Vickie Baselski, B. Kay Buchanan, Mitchell l.
Burken, Roberta Carey, Linda Cook, Lynne Garcia, Mark LaRocco, Susan L. Mottice, Michael Saubolle, David L. Sewell, Daniel Shapiro,
Susan E. Sharp, James W. Snyder, Allan Truant.
Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious
use of clinical microbiology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians,
laboratorians, and other interested stakeholders to provide a broad overview of various aspects of infectious disease testing. These
aspects include a discussion of relevant clinical considerations; collection, transport, processing, and interpretive guidelines; the clini-
cal utility of culture-based and non-culture-based methods and emerging technologies; and issues surrounding coding, medical neces-
sity, frequency limits, and reimbursement. The recommendations in Cumitechs do not represent the official views or policies of any
third-party payer.
Copyright © 2005 ASM Press
American Society for Microbiology
1752 N Street NW
Washington, DC 20036-2904
All Rights Reserved
10 9 8 7 6 5 4 3 2 1
 Blood Cultures IV

Ellen Jo Baron
Stanford University Medical Center, Stanford, CA 94305
Melvin P. Weinstein
Robert Wood Johnson University Hospital, New Brunswick, NJ 08903
W. Michael Dunne, Jr.
Washington University School of Medicine, St. Louis, MO 63110
Pablo Yagupsky
Soroka University Medical Center, Ben Gurion University of the Negev,
Beer-Sheva 84101, Israel
David F. Welch
Medical City Dallas and North Texas Children’s Hospital, Dallas, TX 75230
Donna M. Wilson
California Department of Health Services, Medi-Cal Benefits Branch, Medical
Policy Section, Sacramento, CA 94234
COORDINATING EDITOR: Ellen Jo Baron
Stanford University Medical Center, Stanford, CA 94305

Introduction and Rationale for Performing Blood Cultures . . . . . . . . . . . . . . 2

Characteristics of Bacteremia and Fungemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Clinical Use of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Recommended Volume of Blood To Be Cultured . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Recommended Number of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Timing of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Importance of Separate Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Limitations of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Collection of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Skin Antisepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Methods of Obtaining Blood for Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Transport and Initial Processing of Blood Culture Bottles . . . . . . . . . . . . . . . 8
Checklist for Blood Cultures before Leaving the Patient’s Bedside . . . . . . . . . . . . . . . . . . . 8
Transport to the Laboratory and Handling and Moving within the Laboratory . . . . . . . . . . . 8
Bottle Examination, Processing Protocols, and Rejection Criteria . . . . . . . . . . . . . . . . . . . . 8
Safe Handling of Blood Cultures in the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Media and Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Blood-to-Broth Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Atmosphere of Incubation and Use of Anaerobic Blood Culture Vials . . . . . . . . . . . . . . . . 12
Length of Incubation of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Commercially Available Manual Blood Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Automated Blood Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Incubation and Examination of Blood Cultures . . . . . . . . . . . . . . . . . . . . . . 13
General Concepts for Detecting and Initial Handling of Positive Blood Cultures . . . . . . . . 13
Incubation Durations with Automated Blood Culture Systems . . . . . . . . . . . . . . . . . . . . . 13
Incubation Duration and Detecting Positive Blood Cultures in Nonautomated Systems . . . 13
Visual Examination of Smears from Positive Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . 13
Initial Subcultures of Positive Blood Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Direct Identification and Susceptibility Testing from Blood Culture Broth . . . . . . . . . . . . . . 14
False-Positive Blood Cultures (“Contaminants”) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Polymicrobic Bacteremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Saving Blood Culture Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
CPT-4 Coding and Billing Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1
2 Baron et al. CUMITECH 1C

Laboratory Diagnosis of Sepsis Caused by a Colonized Indwelling Vascular

Catheter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Conventional Methods for Detecting Pathogens That Fail To Grow in Stan-
dard Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Bartonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Brucella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Campylobacter and Helicobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Leptospira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Mycobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Molecular Methods for Detecting BSIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Specimen Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Numbers of Cultures Drawn per Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Positive Culture Rate and Number of Cultures per 1,000 Patient Days . . . . . . . . . . . . . . . 25
Technologist Competency and Result Reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ordering, Billing, and Reimbursement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

INTRODUCTION AND RATIONALE FOR A sudden influx of bacteria ordinarily is cleared

PERFORMING BLOOD CULTURES from the bloodstream in minutes to hours, except in
the case of overwhelming infection or an intravascu-

T
he presence of living microorganisms in the
blood of a patient is an event of major diag-
nostic and prognostic importance (16, 163,
165). When microorganisms multiply at a rate that
exceeds the capacity of the reticuloendothelial system
to remove them, bloodstream infection (BSI) is the
result (13). If there is failure of the host’s defenses to
localize infection at its primary focus or failure of a
physician’s attempts to remove, drain, or adequately
treat localized infection, persistent BSI may result.
When blood cultures yield a clinically important
pathogen, not only is an infectious cause established
for the patient’s illness, but the etiologic agent also
becomes available for antimicrobial susceptibility
testing and optimization of therapy. Identification of
an etiologic agent of septicemia results in more
appropriate reimbursement for the resources needed
to maintain quality laboratory services, an important
issue for health care institutions.
Characteristics of Bacteremia and Fungemia
Microorganisms usually enter the blood from extra-
vascular sites via lymphatic vessels. Direct entry of
bacteria and fungi into the bloodstream occurs with
intravascular infections such as infective endocardi-
tis, infected arteriovenous fistulas, mycotic aneurysms,
suppurative thrombophlebitis, and colonized intra-
vascular devices (e.g., intravenous catheters, arterial
lines, subcutaneous ports, and vascular grafts).
lar focus (41). The fixed macrophages in the liver and
spleen play major roles in clearing bacteria from the
blood. Bacterial capsules and other virulence factors
delay clearance; specific antibodies enhance removal.
Although polymorphonuclear leukocytes (PMNs) are
crucial for localizing infections in extravascular sites,
intravascular PMNs play a minor role, if any, in
clearance. When the body responds to the presence
of an infectious agent in the bloodstream with sys-
temic signs and symptoms of illness (e.g., a systemic
inflammatory response syndrome), the condition is
called septicemia.
An understanding of the circumstances in which
different types of bacteremia and fungemia are likely
to occur is helpful in planning diagnostic studies and
interpreting results of blood cultures. The common
sources of BSIs are intravascular devices (19%), the
genitourinary tract (17%), the respiratory tract (12%),
the bowel and peritoneum (5%), skin (5%), the bil-
iary tract (4%), intra-abdominal abscesses (3%),
other known sites (8%), and unknown sites (27%)
(166).
The clinical pattern of BSIs can be transient, inter-
mittent, or continuous. Transient bacteremia is most
common and occurs after manipulation of infected
tissues (e.g., abscesses, furuncles, and cellulitis),
instrumentation of contaminated mucosal surfaces
(dental procedures; urologic manipulations such as
cystoscopy, urethral dilatation, and catheterization;
CUMITECH 1C
suction abortion; and upper and lower gastrointesti-
nal endoscopic procedures), and surgery involving
contaminated areas (transurethral resection of the
prostate, vaginal hysterectomy, and debridement of
infected burns). Bacteremia also occurs early in the
course of many systemic and localized infections and
has been reported in various studies in 50 to 80% of
patients with meningitis, 5 to 30% of patients with
pneumonia, 20 to 70% of patients with pyogenic
arthritis, 30 to 50% of patients with osteomyelitis,
and 5 to 90% of patients with gonococcal and
meningococcal infections (121).
Intermittent bacteremia most often is associated
with undrained intra-abdominal, pelvic, perinephric,
hepatic, prostatic, and other abscesses. Such abscess-
es are a common cause of fever of unknown origin.
Continuous bacteremia is a cardinal feature of
endovascular infections, most notably acute and sub-
acute infective endocarditis (119, 168). This pattern
may also be seen during the first few weeks of
typhoid fever and brucellosis.
“Breakthrough” bacteremia occurs while a patient
is receiving systemic therapy with antimicrobial agents
to which the infecting microorganism is susceptible
(4, 164). Breakthrough bacteremia that occurs early
in therapy is usually due to inadequate concentra-
tions of the antimicrobial agent, whereas break-
through episodes that occur later usually are due to
inadequate drainage of a focus of infection or impair-
ment of host defenses (4).
CLINICAL USE OF BLOOD CULTURES
Based on the discussion above, recommendations for
the clinical use of blood cultures can be made. Ideal-
ly, blood should be obtained for culture prior to the
administration of systemic antimicrobial therapy
from any patient who is sufficiently ill to be hospi-
talized and who has fever (
38°C) or hypothermia
(36°C); leukocytosis (total peripheral leukocyte
count of more than 10,000 leukocytes per liter),
especially with a left shift toward immature or band
forms; absolute granulocytopenia (less than 1,000
mature PMNs per liter); or a combination of the
above (153). Blood cultures complement urine and
cerebrospinal fluid cultures in the evaluation of
neonates with suspected sepsis whose only clinical
findings, in addition to fever or hypothermia, may be
poor feeding and failure to thrive (46). Young chil-
dren, especially those 2 years old or less, with pneu-
mococcal and Haemophilus influenzae bacteremia
may present as outpatients, with a marked fever
(
39.4°C) and leukocytosis (total peripheral leuko-
cyte count often
20,000 leukocytes per liter) (39).
Nondescript complaints, such as myalgia, malaise, or
fatigue, may result from bacteremia in elderly patients
Blood Cultures IV 3
who may remain afebrile (53, 119). A low-grade
fever in elderly patients may signal infective endo-
carditis, especially when accompanied by malaise,
myalgia, or stroke (119, 167).
Finally, a critically important and fundamental
concept is that a blood culture, often referred to as a
culture set, is defined as a volume of blood obtained
under aseptic conditions (preferably by venipunc-
ture) that is inoculated to one or more bottles or vials
(usually containing broth culture medium). A cul-
ture, whether it consists of only one bottle or several
bottles inoculated from the same venipuncture or line
draw, is considered to be positive if one or more than
one of the bottles demonstrates growth. The individ-
ual bottles from a single venipuncture or line draw
are not considered separately. However, for organ-
isms other than putative “contaminants,” discussed
in more detail later, higher numbers of circulating
organisms per ml of blood result in more positive
blood culture bottles among those obtained.
Recommended Volume of Blood To Be Cultured
The volume of blood that is obtained for each blood
culture (culture set) is the single most important vari-
able in recovering microorganisms from patients
with BSIs (7, 27, 55, 60, 77, 93, 110, 129, 146, 158).
Adult Patients
Several studies using conventional manual blood cul-
ture methods and one using an early-generation semi-
automated blood culture system demonstrated a
direct relationship between the diagnostic yield from
a blood culture and the volume of blood cultured
from adults (55, 60, 77, 146). When the volume of
blood was increased from 2 to 20 ml, the yield of
positive cultures increased by 30 to 50%. More
recently, Cockerill and colleagues reassessed this rela-
tionship using the BACTEC 9240 (Becton Dickinson
Diagnostic Instrument Systems, Sparks, Md.) blood
culture system (27). These authors used an uncon-
ventional definition of a blood culture to assess the
volume versus yield relationship. In adult patients,
separate 20-ml blood samples obtained within a 30-
min period, each divided equally between an aerobic
and anaerobic blood culture vial in 10-ml aliquots,
were considered to represent a single culture. Using
this definition, the investigators then assessed the
incremental yield over a volume range from 10 to 40
ml. In patients without infective endocarditis, vol-
umes of 20 ml increased the yield by 30% compared
with volumes of 10 ml, and volumes of 30 ml increased
the yield 47% compared with volumes of 10 ml.
Although there was an additional increase in yield
when 40 ml of blood was cultured, the increment
compared with 30 ml was only 7%. Based on all
of the currently available data, the recommended
4 Baron et al.
volume of blood to obtain from adults per culture is
20 to 30 ml.
Pediatric Patients
The optimal volume of blood that should be
obtained from infants and children has not been
defined with certainty, but the available data suggest
that a direct relationship between the volume of
blood cultured and detection of BSIs also exists in
this patient population (67, 145). An early study doc-
umented that in infants and young children, blood
samples of
1 ml detected more bacteremias than
samples of 1 ml (145). More recently, Kellogg et al.
documented that low-level bacteremia (10 CFU/ml)
occurred in 68% of infants up to 2 months of age
(66) and 60% of children from birth to 15 years of
age (67). Twenty-three percent of episodes had 1
CFU/ml of blood (67). Although the authors did not
quantify the incremental yield as a function of blood
volume, they concluded logically that volumes larger
than the 1 to 5 ml recommended previously will
improve detection of BSIs in pediatric patients and
improve patient care (38, 161). Based on the premise
that it is safe to obtain as much as 4 to 4.5% of a
patient’s total blood volume for culture and on the
known relationship between total blood volume and
patient weight, the recommendations of Kellogg et
al. (65, 67), which we have modified slightly, are
shown in Table 1. As a practical matter, however, it
may be impossible to obtain the recommended vol-
umes from tiny, premature infants.
Recommended Number of Blood Cultures
Several studies have addressed the number of blood
cultures needed to detect BSIs in adults. In 1975,
using a manual system with basal broth culture
media and 20 ml of blood per culture, Washington
reported sequential results from 80 patients with
bacteremia at the Mayo Clinic (157). The first blood
culture detected 80% of episodes, two blood cultures
detected 88%, and three blood cultures detected
99% (79 of 80 bacteremias studied). Subsequently,
Weinstein et al. reported results from 282 patients
Table 1. Blood volumes suggested for cultures from infants and childrena
Wt of patient Total blood
vol (ml)
kg lb
1 2.2 50–99
1.1–2 2.2–4.4 100–200
2.1–12.7 4.5–27 200
12.8–36.3 28–80 800
36.3 80 2,200
a
Modified from reference 67.
CUMITECH 1C
with bacteremia and fungemia from the University of
Colorado (165); these results were based on use of a
manual blood culture system with basal broth cul-
ture media and culture volumes of 15 ml. In this
study, 91% of episodes were detected with the first
culture, and 99% (281 of 282 bacteremias) were
detected with two blood cultures. Cockerill et al.
recently reported results from the mid-1990s using
the BACTEC 9240 continuous monitoring blood cul-
ture instrument with aerobic resin and anaerobic lyt-
ic broth culture media, 20 ml per culture (27). In 163
BSIs without endocarditis, 65% were detected with
the first blood culture, 80% with two blood cultures,
96% with three blood cultures, and the remainder
with four blood cultures. In patients with endocardi-
tis, the first blood culture was positive in approxi-
mately 90% of episodes (27). Cockerill et al. specu-
lated as to the reason why more rather than fewer
blood cultures were needed despite using a newer
culture system with enhanced culture media. One
possibility was that lower levels of bacteremia and
fungemia were being detected but that detection of
low numbers of organisms required more blood cul-
tures. Another possibility was that more patients
were receiving broad-spectrum antimicrobial therapy
(an unmeasured variable in their study), with a con-
comitant impairment of bacterial growth that increased
the number of blood cultures required for optimal
diagnostic sensitivity. It can be concluded from the
available data that two to four blood cultures are
necessary to optimize detection of bacteremia and
fungemia. Although the traditional recommendation
in routine circumstances has been two or three blood
cultures of at least 20 ml, the recent report by Cock-
erill et al. (27), if corroborated for other systems and
media, may establish four blood cultures (80-ml total
volume) as the optimum, although this volume of
blood may lead to nosocomial anemia and may not
be safe to remove from some severely ill and septic
patients. Given that a substantial number of bac-
teremias would be missed if only one blood culture
were obtained, laboratory policy with appropriate
medical board approval should facilitate including a
Recommended vol of Total % of total
blood for culture (ml) vol for blood
Culture no. 1 Culture no. 2 culture (ml) vol
2 2 4
2 2 4 4
4 2 6 3
10 10 20 2.5
20–30 20–30 40–60 1.8–2.7
CUMITECH 1C Blood Cultures IV 5

second blood culture as a reflexive test, even if the Table 2. Recommendations for the timing of blood
cultures in different clinical conditions and syndromes
physician has specified only one blood culture. A
mechanism for physicians to opt out of this standard Condition or syndrome Recommendations
protocol must, of course, be available. Documenta-
Suspected acute primary Obtain two or three blood
tion of the medical necessity of the second blood cul- bacteremia or fungemia, cultures, one right after the
ture requires that the billing system recognizes that it meningitis, osteomyelitis, other, from different sites
is not a duplicate order, but a distinct procedural arthritis, or pneumonia following the clinical events
service. This is usually done by adding a modifier that precipitated the blood
culture
(-59) to the Current Procedural Terminology 4
Fever of uncertain origin (e.g., Obtain two or three blood
(CPT-4) code (i.e., 87040-59). occult abscess, typhoid cultures, one right after the
fever, brucellosis or other other, from different sites
Timing of Blood Cultures undiagnosed febrile initially. If these are nega-
Few studies have systematically evaluated the timing syndrome) tive after 24–48 h of incuba-
tion, obtain two more blood
of blood cultures and the optimum interval between
cultures, one right after the
successive blood cultures. Experimental studies have other, from different sites
shown that after an influx of bacteria into the blood- Suspected bacteremia or Consider alternative blood
stream, there is a lag time of approximately 1 h, after fungemia with persistently culture methods designed
which chills (rigors) occur (13). Fever then follows. negative blood cultures to enhance recovery of
mycobacteria, fungi, and
However, Thomson et al. observed no significant dif-
rare or fastidious micro-
ferences in positivity rates of blood cultures obtained organisms (see text)
in relation to the fever spikes of patients (147). As a
practical matter, blood cultures are usually obtained
after the onset of fever; however, by this time the tant, the number of blood culture sets that grow
blood may be sterile due to the efficiency of clearance microorganisms, especially when measured as a func-
mechanisms. Thus, blood cultures should be obtained tion of the total number of sets obtained, has proved
as soon as possible after the onset of fever or chills. to be a useful aid in interpreting the clinical signifi-
Some authorities have recommended arbitrary cance of positive blood cultures (83, 165, 166). In
intervals of 30 to 60 min between blood cultures, contrast to patients with infective endocarditis (con-
except in critically ill septic patients, from whom tinuous bacteremia or fungemia) or other true posi-
specimens should be obtained rapidly, prior to initia- tive BSIs (transient or intermittent bacteremia or
tion of antibiotic therapy (142). However, Li et al. fungemia), patients whose blood cultures grow con-
demonstrated no difference in yield whether blood taminants usually have only a single blood culture
samples for cultures performed within a 24-h period (when two or more are obtained) that is positive
were obtained simultaneously or at spaced intervals (Fig. 1). This information has great practical value
(77). For these reasons, the clinical status of the for both clinicians and microbiologists.
patient should be the primary guide to the timing of
Limitations of Blood Cultures
blood cultures. In urgent situations where prompt
administration of antimicrobial therapy is mandated, Blood culture methods and techniques, as described
blood cultures should be obtained simultaneously or in this Cumitech, represent the current state of the art
over a short time frame (e.g., less than 1 h). In less and optimal practice. However, a true gold standard
urgent situations in which the patient is relatively sta- for the diagnosis of BSIs does not exist. Current sys-
ble, drawing blood at spaced intervals, such as 1 to 2 tems require hours to days of incubation until micro-
h apart, may be indicated. This may be especially bial growth is detected. No single commercially
helpful if the clinician wishes to document continu- available system or culture medium has been shown
ous bacteremia in patients with suspected endovas- to be best suited for the detection of all potential
cular infections. Recommendations for the timing of blood pathogens. Some microorganisms grow poor-
blood cultures are shown in Table 2. ly, if at all, using conventional methods and reagents
and require special diagnostic techniques as detailed
Importance of Separate Blood Cultures elsewhere in this document. In the future, it is possi-
Obtaining more than a single blood culture is impor- ble that current culture-based systems will be
tant both for diagnostic sensitivity and for interpre- replaced or supplemented with molecular techniques
tation of the clinical significance of a positive result. that are not only more sensitive clinically and
As indicated previously, studies have shown that two analytically, but also faster. Whether any new system
to four blood cultures may be needed for optimum will be more cost-effective than cultures remains to
diagnostic sensitivity (27, 157, 165). Equally impor- be determined.
6 Baron et al.
Figure 1. Patterns of positivity in successive blood cultures: evidence of the diagnostic importance of separate cultures. Reprinted from
reference 165 with permission from the University of Chicago Press.
COLLECTION OF BLOOD CULTURES
Skin Antisepsis
The likelihood that a positive blood culture repre-
sents infection rather than contamination is, at least
in part, a function of skin antisepsis at the time blood
is obtained. Failure to adequately cleanse the skin
using meticulous technique and an appropriate anti-
septic agent increases the risk that microbial flora of
the skin, such as coagulase-negative staphylococci or
Corynebacterium spp., will contaminate the blood
culture. Initial cleansing with 70% isopropyl alcohol,
followed by use of 1 to 2% iodine tincture or an
iodophor has been recommended as standard prac-
tice (38, 121). Iodophors (aqueous iodine solutions)
require 1.5 to 2 min of contact time for maximum
antiseptic effect, whereas iodine tincture (iodine in
alcohol) exerts its effect after 30 s (71). It is the
change of state from wet to dry of the agent that
causes bacterial cell wall disruption, and alcohol-
based antiseptics dry more quickly than water-based
products. Health care workers who obtain blood cul-
tures (many of whom have little or no formal med-
ical education) are often in a hurry, do not under-
stand the importance of antiseptic preparation
contact time, and are less likely to wait 1.5 to 2 min
as opposed to half a minute before obtaining blood.
At least two studies have documented lower contam-
ination rates using iodine tincture rather than an
iodophor (80, 143). Another report compared the
use of 0.2% chlorine peroxide and 10% povidone-
iodine and demonstrated lower contamination rates
when chlorine peroxide was used (139). A study
CUMITECH 1C
comparing an alcoholic solution of 0.5% chlorhexi-
dine gluconate versus povidone-iodine for skin anti-
sepsis prior to blood culture demonstrated reduced
contamination rates with chlorhexidine (94). Finally,
a recent report assessed contamination rates when
skin was prepared with iodine tincture versus a com-
mercial product containing 2% chlorhexidine glu-
conate and 70% isopropyl alcohol (11); in this study,
there was no significant difference in the contamina-
tion rates associated with the two preparation meth-
ods. Thus, the available data suggest that iodine tinc-
ture and chlorhexidine products are likely to be
equivalent and that both may reduce contamination
rates to a greater degree than products containing
povidone-iodine preparations. Chlorhexidine prepa-
rations have the advantage of being both colorless
and less irritating to skin, so that their use may allow
one to abandon the additional step necessary with
iodine preparations of removing the iodophor using
a final alcohol scrub after the venipuncture is com-
pleted. Both iodophors and chlorhexidine may have
toxicity for neonates; further studies are needed (47).
One study found skin preparation for catheter inser-
tion using 0.5% chlorhexidine in 70% isopropyl
alcohol was safe and more effective than 10% povi-
done-iodine for neonates at least 7 days old (49).
Until further studies are available, it is important to
wash the area with alcohol when these disinfectants
are used on the skin of neonates. Some institutions
substitute two separate alcohol cleansings, allowing
the alcohol to dry thoroughly after each use, for the
more active disinfectants if neonates are known to be
sensitive to the other agents. A 2002 guideline for
CUMITECH 1C
catheter insertion suggests 2% chlorhexidine as the
agent of first choice for skin antisepsis, but no rec-
ommendations were made for children 2 years old
(45).
The venipuncture site should be prepared by first
vigorously cleansing for 30 s back and forth across
the site with 70% isopropyl alcohol followed by
either 1 to 2% tincture of iodine, which is allowed to
dry for at least 30 s, or 2% chlorhexidine, which is
allowed to dry for at least 30 s, before inserting the
needle. If aqueous iodophors are used after the alco-
hol step, they are applied in increasing concentric cir-
cles from the actual venipuncture spot because the
disinfectant takes so long to act (to dry) that viable
contaminants might be reintroduced to the area that
had been prepared if the swab is allowed to recontact
the initial site. There are no data supporting applying
alcoholic disinfectants in an outward concentric cir-
cle, but vigorous friction is important. If palpation of
the vein is necessary after skin disinfection, the
gloved finger should be cleansed with the antiseptic
agent and allowed to dry before touching the site.
Some commercial preparations incorporate alcohol
and disinfectant into a sponge to allow a one-step
process.
Methods of Obtaining Blood for Culture
Venipuncture remains the technique of choice for
obtaining blood for culture (6, 161). Arterial blood
cultures are not associated with higher diagnostic
yields than venous blood cultures and are not recom-
mended (121). Blood cultures obtained from indwelling
intravascular access devices are associated with
greater contamination rates than are blood cultures
obtained by venipuncture (17, 31, 42). Although
physicians and nurses may think they are saving
patients the pain of an extra needle stick when blood
cultures are obtained from catheters as opposed to
venipuncture, they may actually be doing their patients
and the health care system a disservice if contami-
nants are grown, resulting in the need for even more
blood cultures and costly additional diagnostic stud-
ies or the immediate institution of long-term intra-
venous antibiotic therapy. Blood obtained through
an indwelling line is twice as likely to yield a con-
taminant than blood obtained through a properly
prepared skin site (17). So although blood occasion-
ally 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 inter-
pretation in the event of a positive result.
In the era before human immunodeficiency virus
(HIV) infection, blood cultures were obtained with a
“two-needle” technique, using a sterile needle and
syringe to obtain blood, then changing to a second
Blood Cultures IV 7
needle before inoculating the culture vial. This tech-
nique was used to reduce potential contamination in
case any skin microorganisms might be present on
the first needle following the venipuncture. With
increased concern about the risk of HIV transmission
to health care workers associated with needle-stick
injuries, the two-needle technique was abandoned
when several studies showed that contamination
rates were not significantly increased when a single
needle was used for both venipuncture and inocula-
tion of blood culture vials (61, 73, 76). Although a
subsequent meta-analysis demonstrated somewhat
higher contamination rates with the single-needle
method (3.7%) compared with the two-needle
method (2.0%), the higher rates are tolerated in
order to reduce the risk of occupational needle-stick
injuries (118).
Blood may also be inoculated directly to evacuat-
ed culture vials containing broth media through a
transfer set or a double-ended needle, or into an
evacuated blood collection tube containing sodium
polyanetholsulfonate (SPS) used for transfer to the
laboratory where the specimen is then inoculated to
culture vials. If the latter method is used, SPS is the
preferred anticoagulant, since citrate, heparin,
EDTA, and oxalate may be toxic for some bacteria.
However, SPS is also toxic to some bacteria, such as
Neisseria meningitidis (140). If a direct inoculation
method is used, collection bottles or tubes must be
held below the level of the venipuncture needle to
minimize the risk of reflux. In general, use of inter-
mediate collection tubes is discouraged, since (i) SPS
present in collection tubes will be added to that pres-
ent in blood culture bottles, thereby increasing the
final concentration of SPS in the blood-broth mix-
ture; (ii) the extra step of transferring blood to ulti-
mate bottles, tubes, or plates provides additional
opportunity for contamination; (iii) transferring
blood increases the risk of exposure of laboratory
personnel to blood-borne pathogens; and (iv) use of
collection tubes instead of direct inoculation into
vials containing media may compromise cultures that
are delayed or require transport to a distant labora-
tory. Use of direct-draw methods does not allow the
amount of backpressure to be controlled as is the
case when a needle and syringe method is used; the
arbitrary vacuum may result in collapse of veins and
inability to obtain the correct volume of blood from
some patients, such as the frail elderly or those on
long-term chemotherapy. In addition, the vacuum
present within culture vials may not be precise, and
the volume of blood obtained may not be optimal.
After the appropriate volume of blood is obtained
and inoculated to culture vials, the mixture should be
gently agitated or the vials inverted to prevent clot-
ting. If the vein is missed initially, a new needle (or
8 Baron et al.
transfer set) should be used for each repeat venipunc-
ture.
As with all specimens submitted to the laboratory,
blood culture vials should be labeled with the appro-
priate identification information and accompanied
by an electronic or written requisition showing date
and time of collection, the identification of the per-
son collecting the specimen, and any other informa-
tion required by institutional policy.
TRANSPORT AND INITIAL PROCESSING
OF BLOOD CULTURE BOTTLES
Checklist for Blood Cultures before
Leaving the Patient’s Bedside
1. All bottles or tubes are labeled with two unique
patient identifiers, such as name and birthdate or
name and Social Security number.
2. All bottles or tubes and requisitions are labeled
with collector’s employee name, number, or code.
3. The specific site of collection (which vein, which
arm, etc.) is recorded for each set of bottles or
tubes obtained. Denoting individual lumens has
no additional value, and there is no literature to
support the clinical relevance of results from an
individual lumen. One method of obtaining this
information is to supply test codes for order entry
that represent specific sites. Examples of such a
code are as follows: AL, arterial line; DIALL, dial-
ysis catheter left; and MEDL, mediport left.
4. If the volume in any bottle is less than the total
amount desired, the actual volume of blood inject-
ed into the bottle should be documented on infor-
mation accompanying the bottle to the laboratory.
The volume of blood in collection tubes can be
measured when the blood is transferred into blood
culture broth.
Transport to the Laboratory and Handling and
Moving within the Laboratory
1. Timing. Blood should be transported as quickly as
possible to the laboratory, preferably within 2 h,
and should be placed into the incubator as quick-
ly as possible. Although most modern blood cul-
ture instruments will detect growth at any stage,
delay beyond 2 h in incubating cultures usually
results in delay in detection of positives. Bottles
from systems that depend on colorimetric detec-
tion of positives, such as BacT/ALERT (Table 3),
should be examined visually for color change of
the indicator before being placed into the instru-
ment. Those with positive color changes can be
managed immediately. For systems that depend on
other detection methods, the manufacturer’s rec-
ommendations for incubating bottles delayed in
CUMITECH 1C
transit should be followed. For example, BACTEC
(Table 3) bottle handling is based on media type.
For Aerobic PLUS, Anaerobic PLUS, Peds PLUS,
and lytic media, bottles may still be placed into the
instrument after delays of 20 h at 35°C or 48 h at
room temperature (20 to 25°C). Standard aerobic
and anaerobic bottles, however, can only be
placed into the instrument within 12 h at 35°C or
48 h at room temperature. Those samples not
received within these time frames must be stained
and subcultured and incubated manually off-line.
2. Temperature. Blood for culture should never be
refrigerated or allowed to cool. Keeping the bot-
tles warm (no warmer than 37°C) is preferable to
leaving them at room temperature, although
organisms in blood culture broths should remain
viable at room temperature for several days. Blood
collected in tubes should remain at room temper-
ature until it is injected into culture bottles.
Because of their relatively small headspace and
thin walls, tubes could be stressed by gas forma-
tion from growing organisms enough that they
may explode if they are incubated.
3. Safety for transport. Blood culture bottles should
be carried in some sort of container that will pro-
tect them from dropping and from knocking
against each other. Carrying tubes or bottles in the
hands is dangerous and should not be done, even
for short distances within the laboratory. If tubes
or bottles must travel through a pneumatic tube
system, they should be checked in advance for
ability to withstand the harshest conditions of
transport. Many people feel that if the container is
intact after being dropped to the floor from a 4-
foot height, it will be strong enough for transport
in a pneumatic tube. There are no published stud-
ies on container strength.
Bottle Examination, Processing Protocols, and
Rejection Criteria
Staff who receive blood cultures in the laboratory
should check the bottles and requisitions carefully to
detect a number of problems or errors, listed here.
1. Depending on laboratory and hospital guidelines,
specimens with improper labeling may need to be
rejected. Bottles with no labels are usually rejected
at all times. Patient identification data on the cul-
ture bottles and requisition must match, and mis-
matched specimens may have to be rejected,
depending on laboratory policy. Whenever rejec-
tion is being considered, the physician and/or
nursing unit must be notified immediately so they
can recollect the samples or discuss the options
with a laboratory director or supervisor.
Table 3. Characteristics of the three major continuously monitored automated blood culture systems currently used in the United States
Characteristic
Manufacturer
Growth indicator
Detection mechanism
Positive culture signal
Bottle capacity/unit
Unit capacity/system
Maximum bottle capacity/system
Bottle monitor frequency
Aerobic bottle agitation
Blood volume
Additional indications
Available media formulations
Miscellaneous
BacT/ALERT 3D
bioMerieux, Inc., Marcy I’Etoile, France, and
Durham, N.C.
CO2 production
Colorimetric CO2 sensor in base of bottle, LED
illuminator and photodiode detector in incu-
bator module
Visual and audible alerts with remote alarm
capability
120 or 240
6 5
1,440
Once every 10 min
Rocking, 70 cycles/min
10 ml standard; 4 ml for pediatric bottles;
requires external volume control
Mycobacterial cultures; monitoring bacterial
contamination of platelets
Standard aerobic (SA) and anaerobic (SN), FAN
aerobic (FA) and anaerobic (FN), pediatric
FAN (PF), mycobacteria process (MP) and
blood (MB). FAN media formulations contain
activated charcoal particles for nonspecific
removal of toxic inhibitors of bacterial
growth.
Media now available in shatter-resistant, plastic
bottles that retain transparency and thermal
resistance of glass. Bottles do not require
venting and contain a liquid emulsion CO2
sensor and larger headspace with perform-
ance equal to traditional glass bottles. Con-
troller module has touch-activated panel for
text-free random loading and unloading of
samples. Bar code scanner recognizes bottle
type and patient accession data.
BACTEC 9000
BD Diagnostic Systems, Sparks, Md.
CO2 production
Fluorimetric CO2 sensor in base of bottle, LED
illuminator and photodiode detector in incu-
bator module
Visual and audible alerts with remote alarm
capability
50, 120, or 240 depending on system choice
1,200
Once every 10 min
Rocking, 30 cycles/min
10 ml standard; 3 ml for pediatric bottles;
requires external volume control
Mycobacterial cultures, possibly sterile fluid
cultures
BACTEC Standard/10 AEROBIC/F and Standard
10 ANAEROBIC/F (aerobic and anaerobic
media without resins), BACTEC PLUS AERO-
BIC/F and PLUS ANAEROBIC/F (aerobic and
anaerobic media with antimicrobial removal
resins), BACTEC PEDS PLUS/F medium
(small sample volume aerobic medium with
resins), BACTEC Lytic/10 ANAEROBIC/F
medium (contains a lysing agent to release
intracellular organisms—supports the growth
of obligate anaerobic and facultative organ-
isms), BACTEC Myco/F Lytic for growth of
mycobacteria and fungi
A new system called the BACTEC LX is cur-
rently in clinical trials. This system detects
CO2 production directly in the headspace of
the bottle through the use of infrared laser
spectroscopy.
VersaTREK
Trek Diagnostic Systems, Cleveland, Ohio
CUMITECH 1C
CO2, H2, and N2 production, O2 consumption
Pressure transducer detects increase/decrease
in bottle headspace pressure
Visual and audible alerts with remote alarm
capability
240 or 528
6
3,168
Once every 12 min for stirred bottles; once
every 24 min for stationary bottles
Vortexing with a magnetic stir bar at 3,100 rpm
5 ml maximum (40-ml bottle) or 10 ml maxi-
mum (80-ml bottle), 0.1 ml minimum; accu-
rate vacuum draw
Culture of sterile body fluids, mycobacterial cul-
tures and susceptibility testing
REDOX 1 (aerobic) and REDOX 2 (anaerobic) in
80-ml and 40-ml bottles, the latter available
as direct measured draw bottles, MYCO
medium with compressed cellulose sponges
for culture of mycobacteria
The VersaTREK system represents a total
redesign of the ESP blood culture system
originally developed by Difco Laboratories
and provides numerous enhancements in
ergonomic and mechanical design as well as
software upgrades. The system LCD screen
provides one-touch access to bottle informa-
Blood Cultures IV
tion and allows new bottles to be scanned
and loaded into any available culture station.
9
10 Baron et al.
2. Bottles must be checked for leaking or blood on
the outside of the bottle. All blood products
should be handled with gloves to avoid skin con-
tact with blood. If the bottle is intact but blood is
found on the outside, use a disinfectant solution
(10% bleach is best) to wipe off the bottle and
then proceed to accession and process it. The col-
lecting person should be notified immediately by
telephone of the potential for causing risk to lab-
oratory workers, and a note should be placed on
the report stating that the sample was received
contaminated, e.g., “container received leaking;
specimen was accepted and nurse manager (name)
was called by (name) at (time, date).” Lack of
observation of laboratory rules for specimen sub-
mission by an individual or patient care unit
should be monitored and appropriate educational
activities or sanctions should be undertaken for
continual violations.
3. In some rare instances, bottles may crack before
use or in transit to the laboratory. If the crack does
not destroy the integrity of the bottle, i.e., there is
no detectable fluid leaking out, place a note exter-
nally to warn handlers to be careful and continue
to process the bottle.
4. Specimens submitted in wrong containers or tubes,
such as a purple-top EDTA Vacutainer tube, can-
not be processed as blood cultures. Notify the
physician or collector immediately that the speci-
men is being rejected and explain the proper
method. Request resubmission of a sample using
correct protocols.
5. Blood cultures submitted in expired tubes or bot-
tles should be processed, but the collecting site
should be notified immediately to discard all
expired bottles and obtain current supplies. The
report should note that media were expired when
specimens were received and that results may not
be reliable. Part of ongoing quality assurance is to
monitor all media for expiration dates and prevent
such failures from recurring.
6. Incorrect volume of blood in the bottle, either too
low or too high, should be noted on the report,
but the blood cultures should be processed. Annu-
al monitoring of volumes should be used for an
ongoing educational process to reinforce the
importance of correct volumes for the system.
7. Too few bottles, or a single blood culture set when
two sets are recommended, should be noted on the
report, but the blood cultures should be processed.
Continual monitoring of adherence to protocols
and recommendations by units or clinics is part
of annual Quality Assurance. Repeat violators
should be counseled and sanctioned as needed.
CUMITECH 1C
8. More than the recommended number of blood
cultures per 24-h period should be processed, but
the physician should be called to discuss the lack
of improved detection after a threshold volume of
blood is in progress. Infectious diseases specialists
can be enlisted to discuss these issues with the
ordering physicians.
9. If blood cultures were inoculated more than 2 h
before receipt in the laboratory, it is prudent to
examine the bottles for evidence of growth (bub-
bles, sediment, lysis of red cells, change in color
indicator, as indicated above in the Timing sec-
tion) before incubating the bottles. Some rapidly
growing bacteria or large numbers of organisms in
the blood could be recovered at this time without
further incubation. Lysis-centrifugation tubes (Iso-
lator; Wampole Laboratories, Princeton, N. J.; see
description below), because of the lysing compo-
nent and other additives in the tubes, may show
delayed or even decreased recovery of organisms if
processing is performed more than 8 h after col-
lection.
Practically speaking, the laboratory should
attempt to process all correctly labeled blood cul-
tures, despite problems with transport or delayed
delivery. Results should then include a disclaimer
statement documenting the extent of the delay
between collection and incubation, with a comment
that results may not be as reliable as those from
properly handled cultures.
Safe Handling of Blood Cultures
in the Laboratory
1. Blood culture bottles should be transported in a
container that prevents them from falling, knock-
ing into each other, or rolling off the surface. They
should never be carried from one area of the lab-
oratory to another in the hands of laboratorians,
but should be placed in a carrier device.
2. Blood and body fluids require standard precau-
tions for handling (48). That entails wearing gloves
and working with the bottle behind a shield, or
wearing goggles during performance of any manip-
ulations that may generate droplets or aerosols.
3. Withdrawing blood from positive blood culture
bottles can be dangerous. Especially if the septum
is seen to be bulging or if gas is visible in the
broth, the bottle can explode or pressure can
cause aerosols when the septum is pierced. Such
bottles should be handled in a shielded safety cab-
inet, and the person inserting a needle into the bot-
tle should wear a full face shield to guard against
splashes that might breach the laminar flow barri-
er. It is advisable to handle all positive blood cul-
CUMITECH 1C
ture bottles in a biological safety cabinet, if pos-
sible. Bottles with mycelial phase mold growth
should always be handled in a safety cabinet. If
the mold is white and potentially Coccidioides, the
bottle should not be opened at all unless it is the
only way to make the diagnosis.
4. Special blunt-ended needles should be used to
insert into the septa for withdrawing samples from
positive blood culture bottles, to decrease the pos-
sibility of a needle-stick injury. The bottles should
be held firmly, since these needles require addi-
tional pressure to penetrate the septum. Careful
attention to technique is important to avoid acci-
dents.
5. Bottles should be inspected for leaks and cracks at
the time of accessioning and again before placing
them into the incubator system. Bottles that leak
or those with cracks all the way through the bot-
tle must be rejected and redrawn.
6. Used blood culture bottles, both positive and neg-
ative, should be disposed of in hard-sided “sharps”
biohazard containers or other puncture-resistant
infectious waste containers, rather than plastic
bags, which are prone to rip and spill the bottles
out. The containers should not be filled so full that
disposal workers have difficulty lifting them.
7. Most laboratories either terminally decontaminate
(e.g., autoclave) all disposed blood culture bottles
and tubes before sending them to the landfill, or
make arrangements with a licensed contractor to
properly handle them as biohazardous infectious
waste.
MEDIA AND INCUBATION
Culture Media
Multiple nutritionally enriched broth-based culture
media have been used successfully; no one medium
or system will detect all potential pathogens optimal-
ly. The most widely used medium is soybean-casein
digest broth (118). Other media include brain heart
infusion, supplemented peptone, Columbia, and bru-
cella broths. Most manufacturers supplement their
base media with proprietary additives designed to
enhance microbial growth, so it cannot be assumed
that common generic media (e.g., soybean-casein
digest broth) from different manufacturers will per-
form in an equivalent manner. Decisions as to the
choice of medium formulation in individual labora-
tories should be based on data from well-controlled
field trials in which large numbers of cultures were
assessed. In general, most commercially marketed
blood culture media perform well for common blood
pathogens. Medium formulations designed to enhance
Blood Cultures IV 11
the detection of anaerobes, fungi, and mycobacteria
are also marketed commercially. A more detailed dis-
cussion of detection of fungemia and mycobac-
teremia can be found elsewhere in this Cumitech.
Most commercially available blood culture media are
able to support the growth of yeasts such as Candida
spp.
Although blood should be obtained for culture
before the administration of antibiotics, many
patients with suspected BSIs are already receiving
empiric antimicrobial therapy at the time blood cul-
tures are obtained (166), potentially reducing the
sensitivity of the test. With dilution of the blood in
the culture broth, the concentration of the antimicro-
bial agents will be reduced and the inhibitory effects
minimized. Moreover, the amount of SPS in most
commercial blood culture media will inactivate some
aminoglycosides and polymyxins. Nevertheless,
because of concerns about microbial recovery from
blood cultures in patients receiving systemic antimi-
crobials, some manufacturers have marketed prod-
ucts to enhance detection in this common clinical
situation. These include media containing antibiotic-
binding resins (Plus/F, BD Diagnostic Systems,
Sparks, Md.) and activated charcoal (FAN, FA, FN;
bioMerieux, Durham, N.C.). Data from large, multi-
center studies comparing media with activated char-
coal versus basal media formulations suggested that
the medium formulations with activated charcoal
(i) had improved yield of microorganisms, including
staphylococci, enteric gram-negative rods, and yeasts
(162, 170); (ii) had improved yields in patients
receiving theoretically effective antimicrobial therapy
(90); and (iii) detected more contaminants, especially
coagulase-negative staphylococci (162). Compara-
tive studies of resin media versus charcoal-containing
media have suggested generally equivalent perfor-
mance overall (64, 111). Interpretation of Gram-stain
results from positive cultures may require more tech-
nical expertise and experience when the charcoal-
containing medium formulations are used (144).
Blood-to-Broth Ratio
Human blood contains substances (e.g., complement,
lysozyme, and phagocytic leukocytes) capable of
inhibiting microbial growth. Diluting blood in cul-
ture broth reduces the concentrations of these
inhibitory substances, as well as the concentrations
of any antimicrobial agents that may have already
been administered to patients. Studies that have
addressed the blood-to-broth ratio have recommend-
ed 5- to 10-fold dilution (8, 120, 128). Dilutions less
than 1:5 may result in reduced yield and may
increase the chances that blood will clot, thereby
trapping potential pathogens within the clot; thus,
12 Baron et al. CUMITECH 1C

filling blood culture bottles with more than the rec- of the widely used continuous monitoring blood cul-
ommended volume of blood should be avoided. ture systems was used, demonstrated that 99.5% of
nonendocarditis BSIs and 100% of endocarditis
Atmosphere of Incubation and Use of episodes were detected within 5 days of incubation
Anaerobic Blood Culture Vials (27). These data suggest that the extended incubation
periods recommended in the past for detection of fas-
Traditional two-vial blood culture systems have
tidious microorganisms that sometimes cause endo-
included one aerobic and one anaerobic vial, with
carditis, including Brucella, Capnocytophaga, and
blood distributed equally to the two vials. Since the
Campylobacter spp. (see the section on “difficult to
1970s, the proportion of BSIs due to anaerobes has
grow” organisms later in this document) and the
decreased, whereas the proportion due to fungi has
HACEK group (Haemophilus, Actinobacillus, Car-
increased (35, 54, 81, 99). Several studies in the
diobacterium, Eikenella, and Kingella spp.), are usu-
1990s of adults (95, 99, 107, 135) and children (178)
ally not necessary for microbiology laboratories that
concluded that routine use of anaerobic blood cul-
use modern continuous monitoring blood culture
ture vials was not necessary and recommended that
systems.
they be used only selectively for patients who are at
high risk for anaerobic bacteremia. By contrast, a
recent study comparing two FAN aerobic bottles Commercially Available Manual
with activated charcoal versus one FAN aerobic and Blood Culture Systems
one FAN anaerobic bottle, both with activated char-
The Isolator System
coal, demonstrated increased yield of staphylococci,
The Isolator system (Wampole Laboratories), both
Enterobacteriaceae, and anaerobes when the aerobic/
adult- (Isolator 10) and pediatric-size tubes, contains
anaerobic combination culture set was used (124).
a lysing agent that lyses red and white blood cells,
As very few fungemias were present in this study, the
thus releasing intracellular bacteria. For the adult
potential reduction in detection of these obligate aer-
Isolator, blood is drawn directly into a vacuum-
obes with use of the combination set could not be
suction tube containing a lytic compound. The well-
assessed.
mixed adult-size 10-ml maximum volume tube of
It is difficult to make conclusive recommendations
blood is centrifuged in a fixed-angle rotor centrifuge,
regarding routine versus selective use of anaerobic
and a specially designed pipette is used to withdraw
blood culture vials for several reasons. Patient popu-
the supernatant containing the plasma, human cellu-
lations differ according to the type of institution or
lar debris, and any antimicrobial materials (such as
laboratory, and not all blood culture systems and
antibiotics or complement) that had been in the
medium combinations have been, or likely ever will
blood. A second pipette is used to mix the sediment
be, studied. Moreover, selective use of anaerobic vials
containing lysed white blood cells, red cell mem-
requires that certain logistical issues be addressed.
branes, and microbes and withdraw the inoculum.
These include adequate notification of physicians and
The sediment containing the etiologic agent can then
staff of a change in laboratory procedure and the rea-
be inoculated to media and handled in a process that
sons the change was implemented; determining
maximizes recovery of the organism being sought.
which, if any, patient care locations (e.g., gynecologic
The blood drawn into a Pediatric Isolator 1.5-ml
or colon and rectal surgery units) should be stocked
tube is lysed in the tube and the entire volume is sub-
routinely with anaerobic vials; and developing algo-
cultured directly. A direct colony count of growth on
rithms for the physicians ordering blood cultures
the media can be extrapolated to yield a quantitative
and/or the individuals who obtain blood for culture
blood culture result. The bacteria-containing portion
(i.e., phlebotomists, nurses, clinical care technicians,
can be inoculated onto any medium, including media
house officers, and medical students). A sequential
specific for the pathogen being sought, such as
plan that includes in-service education and quality
buffered charcoal-yeast extract agar (BCYE) for
assurance after implementation should be undertaken
Legionella, appropriate media for mycobacteria, or
to confirm that the protocols are being followed.
fungal media for isolation of molds. Limitations
include increased detection of some contaminants
Length of Incubation of Blood Cultures due to manipulation of the sample, and inhibition or
In routine circumstances, using automated continu- decreased recovery of some pathogens, such as Strep-
ous monitoring systems, blood cultures need not be tococcus pneumoniae (26, 59, 75, 156). It is, how-
incubated for longer than 5 days (27, 40, 58, 87, ever, a useful and perhaps essential method for recov-
169). For laboratories using manual blood culture ery of some molds and fastidious organisms that fail
systems, 7 days should suffice in most circumstances to grow in routine blood culture broths, as detailed
(38). A recent study at the Mayo Clinic, in which one in the fastidious organism section below.
CUMITECH 1C
The Septi-Chek System
The Septi-Chek system (BBL; BD Diagnostic Systems,
Sparks, Md.) consists of either an adult bottle con-
taining 70 ml of broth (tryptic soy broth, Columbia
broth, brain heart infusion broth, and thioglycolate
broth are available) for inoculation of 8 to 10 ml of
blood, or a pediatric bottle containing 20 ml of broth
(tryptic soy or brain heart infusion broth) for inocu-
lation of 1 to 3 ml of blood. After inoculation, the
cap is removed and replaced with a device containing
an agar-coated slide (three agars are included: choco-
late, MacConkey, and malt). Inverting the bottle
allows inoculation of the agar, so that the system
becomes a closed biphasic blood culture (26). Aero-
bic media only are available.
Automated Blood Culture Systems
There are three common continuously monitored
blood culture systems used in the United States at this
time. Summaries of key features are seen in Table 3.
INCUBATION AND EXAMINATION OF
BLOOD CULTURES
General Concepts for Detecting and Initial
Handling of Positive Blood Cultures
In an ideal world, blood cultures (either visually or
automatically monitored) would be inspected several
times on all three shifts to ensure the rapid identifi-
cation of a potentially life-threatening condition. In
light of personnel limitations and budget reductions,
however, this goal is not often achieved. Blood cul-
tures represent one of the two most important diag-
nostic culture procedures performed in the clinical
microbiology laboratory (the other being cere-
brospinal fluid cultures) and, therefore, they deserve
a higher degree of attention than other analyses.
Automated blood culture instruments should be
inspected for signal-positive cultures at least once per
8-h shift. Ideally, bottles should be removed and eval-
uated as soon as the instrument indicates growth. A
recent study has shown that a telephone report of a
positive blood culture with Gram stain results has a
greater influence on antimicrobial therapy than does
the final issue of antimicrobial susceptibility results
(97). If sufficient microbiology staff are lacking, tech-
nical staff in other round-the-clock laboratories (e.g.,
a rapid response laboratory) could be cross-trained
to perform this task. All signal-positive bottles that
occur during the primary daily working shift should
be subcultured, smears prepared for Gram stain
analysis, and the results of the Gram stain reported
by telephone to the patient’s health care team. The
amount of blood culture monitoring and processing
of positives on additional shifts must be determined
based on staffing availability and the priorities of the
Blood Cultures IV 13
institution. If an automated blood culture system
breaks down or fails, all bottles must be manually
subcultured.
Incubation Durations with Automated
Blood Culture Systems
All manufacturers of continuously monitored blood
culture systems received Food and Drug Administra-
tion clearance based on performance that included a
5-day incubation cycle. However, two studies using
the ESP blood culture system, which is no longer
marketed, indicated that either a 4-day or a 3-day
incubation cycle did not reduce the detection of clin-
ically significant isolates (32, 56). Laboratories wish-
ing to deviate from the manufacturer’s product insert
must validate their modified procedure in their own
setting with sufficient data to justify a change, and
must notify their physicians of their protocol. For all
blood culture systems, the final report for a negative
culture should state the number of days in which the
bottle itself was incubated, and not the total number
of incubation days of the bottle plus subcultures,
if any.
Incubation Duration and Detecting Positive
Blood Cultures in Nonautomated Systems
In the absence of an automated detection system,
bottles should be visually examined for signs of bac-
terial growth initially after 6 to 12 h of incubation
and daily thereafter for 7 days (12, 37). Growth is
enhanced by shaking the bottle, but visual inspection
is then precluded and blind subcultures are essential.
An initial blind subculture should be performed after
12 to 18 h. Gram or acridine orange fluorescent
staining can be performed during the initial exami-
nation to aid in the early detection of microbial
growth (37, 89, 148), but blind or terminal subcul-
tures are of little use thereafter, particularly if bottles
are allowed to settle and can be visually inspected
(18, 50). Nonautomated cultures in anaerobic media
are incubated without shaking, and anaerobic bacte-
ria growing in anaerobic blood culture media are
almost always detectable by visual signs, either tur-
bidity, lysis of red blood cells, visible microcolonies,
or gas in the broth. Blind subcultures from anaerobic
bottles are not necessary (98, 108).
Visual Examination of Smears from Positive
Blood Cultures
The single most important test to perform on any
visual- or signal-positive blood culture is the Gram
stain. It is highly likely that the information provid-
ed by this test when coupled with pertinent patient
information will dictate the choice of primary
antimicrobial therapy. Terminology used to report
Gram stain results should be as descriptive as possi-
14 Baron et al.
ble without being misleading or ambiguous (37). For
example, a report of “gram-positive cocci” could
indicate a variety of microorganisms including
staphylococci, streptococci, and enterococci, but a
report of “gram-positive cocci in pairs” narrows the
field considerably. This information can be detailed
further by denoting whether cocci are present in pairs
and short chains (suggestive of pneumococci, entero-
cocci, or group B streptococci) or long chains (sug-
gestive of other beta-hemolytic streptococci and viri-
dans streptococci) for better differentiation. The use
of a term like “diphtheroid” should be avoided
because it could be misinterpreted by the clinician to
mean a contaminant, when many potentially devas-
tating microorganisms such as Rhodococcus or
Mycobacterium species can share the morphological
characteristics of coryneform gram-positive bacteria.
These organisms could be described as “coryneform
gram-positive bacilli” or “pleomorphic gram-posi-
tive rods not resembling Bacillus or Clostridium.”
It is often useful to examine Gram-stain-negative,
signal-positive blood cultures microscopically using
an acridine orange stain visualized with an ultravio-
let microscope using a fluorescein isothiocyanate fil-
ter. While this method does not distinguish between
gram-positive and gram-negative organisms (it is a
nonspecific nucleic acid fluorescent stain), it is often
useful to identify bacteria with poor counterstaining
qualities such as Mycoplasma, Campylobacter, and
Brucella spp. (22, 23) or to reduce the need to exten-
sively subculture truly false-positive cultures (1).
Initial Subcultures of Positive Blood Cultures
Independent of the microscopic results, all signal or
visually positive blood cultures require an initial sub-
culture to an appropriate selection of agar-based
growth media. Positive blood cultures may appear
cloudy, show bubbles of gas formation, or change
color from red to brownish. Other positive signs are
the presence of granular or spherical structures (tiny
floating colonies or colonies at the edge of the menis-
cus), fluffy structures (potential mold colonies), or
lines of turbidity resembling comet tails. The subcul-
ture media should consist, at the very least, of Tryp-
ticase soy agar with 5% sheep blood as an all-pur-
pose medium and a chocolate agar plate for the
isolation of nutritionally fastidious organisms. These
two media are inoculated with a few drops of well-
mixed blood culture broth which is streaked for iso-
lation, and they are incubated at 35 to 37°C in 3 to
5% carbon dioxide (CO2) for a minimum of 48 h
and examined daily for the appearance of distinct
colonies. If growth occurs in the anaerobic bottle
only, or if organisms seen in both bottles morpholog-
ically suggest anaerobes, a supplemented (vitamin K
and hemin) anaerobic blood agar plate should be
CUMITECH 1C
inoculated and incubated in an anaerobic atmos-
phere for 48 to 72 h. Selective or specific media such
as MacConkey agar (for gram-negative bacilli), co-
listin-nalidixic acid agar (for mixed infections or
streptococci), or mycologic agar (for yeasts and
molds) can be added based on Gram-stain results.
The second edition of the Clinical Microbiology Pro-
cedures Handbook contains suggested media based
on Gram stain criteria (177). Certain yeasts (Malas-
sezia species) may be visible as small budding yeasts
that resemble bowling pins in blood culture smears,
but the yeasts will not grow on routine fungal media
without the addition of a thin film of sterile olive oil
as a growth factor. If the Gram stain of the blood cul-
ture bottle indicates the possibility of staphylococci
or enterococci, vancomycin and methicillin screening
agar can be added to the regimen of subculture media
when resistance is a consideration, a form of direct
susceptibility testing further described in the next
section. Supplemental media such as BCYE or rabbit
blood agar for the isolation of opportunistic
pathogens can be used for subculture when dealing
with immunocompromised individuals or when
infections caused by fastidious organisms (e.g., Bar-
tonella spp.) are considered and this information is
conveyed to the laboratory (36). Recovery of fastidi-
ous organisms is discussed more extensively in the
next section. Some rapidly growing mycobacteria
grow well on routine media within a few days and
can be mistaken for coryneform rods. The organisms
may stain as gram-positive, slightly pointed rods, or
they may not take the stain well at all and appear as
clear “ghosts.” Mycobacteria should be ruled out by
acid-fast stain for any small colonies of gram-positive
or variable rods that cannot be otherwise identified.
Direct Identification and Susceptibility
Testing from Blood Culture Broth
For Gram-stain-positive blood culture bottles, a
number of protocols have been designed to facilitate
rapid identification and susceptibility testing of bac-
teria directly from the blood culture medium. These
protocols have included direct identification using
commercial biochemical panels (57, 79, 155), detec-
tion of bacterial-specific enzymes such as tube coag-
ulase (20, 152), immunologic detection using anti-
body assays (62, 112), probe hybridization (20),
protein-nucleic acid fluorescence in situ hybridiza-
tion (Food and Drug Administration cleared for
Staphylococcus aureus, Enterococcus faecalis, and
Candida albicans) (20, 106), and PCR (115), to
name a few. In a similar fashion, protocols have been
designed for susceptibility testing of microorganisms
directly from positive blood culture bottles (21, 29,
57, 74, 114) or PCR detection of resistance genes
(82). One method involves centrifugation of 5 ml of
CUMITECH 1C
the broth at 160  g for 5 min to pellet the blood
cells, followed by centrifugation of the supernatant
at 650  g for 10 min to pellet bacteria. The bacte-
rial pellet is adjusted to a McFarland 0.5 turbidity and
handled as for standard susceptibility testing (79). To
facilitate inoculation into growth-requiring systems,
the organism may also be enriched from the blood
culture broth by adding 1 drop of the broth to 0.5 ml
of brain heart infusion broth, incubating (with shak-
ing if possible) for 3 to 4 h to achieve a McFarland
0.5 turbidity, and adding the entire volume to the
inoculation liquid for microtiter MIC trays (177). A
modification of this protocol uses a serum separator
tube (BD Vacutainer Systems, Franklin Lakes, N.J.)
to perform a single centrifugation at 1,300 to 1,400
 g for 10 min (152). A number of derivations of this
process have been used (21, 57, 74, 79, 114). Because
this is not a standardized means of susceptibility test-
ing, a confirmatory antimicrobial susceptibility test is
generally recommended once the organism has been
recovered in pure culture; however, a recent article
reported 97.6% correlation with very few major
errors when a direct susceptibility method was com-
pared to standard susceptibility testing of the isolate
(79). From an economic standpoint, rapid identifica-
tion and susceptibility testing has been shown to
reduce the length of hospitalization of patients with
sepsis, which translates into reductions in cost of
patient care (10, 12). However, the laboratory can-
not bill for two susceptibility tests, so if the direct
and standard susceptibilities are both performed,
only one of them will receive reimbursement. The
logistics, cost, and performance of an assay must be
carefully weighed when the laboratory is considering
the feasibility of incorporating identification and sus-
ceptibility testing of microorganisms directly from
positive blood cultures. Further, it should be noted
that with certain organism-drug combinations, false
susceptibility or resistance results can be generated
using direct testing (21, 82, 114). Laboratories
should validate their method against the standard
results.
False-Positive Blood Cultures (“Contaminants”)
One of the most perplexing problems associated with
blood cultures is establishing a means of avoiding or
dealing with microorganisms that enter the bottle
during procurement but were not actually circulating
in the patient’s bloodstream. Differentiating “con-
taminants” from true pathogens is extremely diffi-
cult, especially because the same species can easily be
found in either situation. Policies for processing and
reporting possible/likely blood culture contaminants
should be in place to standardize the minimal labo-
ratory evaluation and to avoid unnecessary therapy
for the patient. Obviously, avoidance is the best pol-
Blood Cultures IV 15
icy and this can be accomplished most effectively by
paying strict attention to the process of skin antisep-
sis, venipuncture, and specimen transfer to blood cul-
ture bottles, as described previously (37). Beyond
preemptive techniques, however, it is considered dif-
ficult to reduce the overall contamination rate below
2% (38). Further, the organisms commonly associ-
ated with contaminated blood cultures (Bacillus [not
B. anthracis] spp., Corynebacterium spp., Propioni-
bacterium spp., coagulase-negative staphylococci,
viridans group streptococci, Aerococcus spp., Micro-
coccus spp., and many others) are perfectly capable
of causing serious infection in the appropriate set-
ting. Coagulase-negative staphylococci, because of
their ability to colonize and form a biofilm on
indwelling and prosthetic devices and their ubiqui-
tous presence on the human skin, are the primary
agents of both catheter-associated septicemia and
false-positive blood cultures (122). In many cases
when only one blood culture is received from a given
patient, a potential contaminant is recovered from
one or both bottles and without a second blood cul-
ture for comparison, interpretation of the clinical rel-
evance of that positive culture is impossible. Physi-
cians may be forced to initiate treatment for practical
and legal reasons if susceptibilities are reported by
the laboratory.
Therefore, the evaluation of an isolate with low
virulence potential recovered from a single blood cul-
ture set (one or both bottles) should be limited to the
extent to which phenotypically similar but medically
important organisms can be safely excluded from the
identification. Some examples include the perfor-
mance of a bile solubility test to differentiate S. pneu-
moniae from viridans group streptococci; an aggluti-
nation assay or coagulase test to distinguish S. aureus
from coagulase-negative staphylococci; and a moti-
lity assay or demonstration of hemolysis to separate
non-anthracis Bacillus spp. from B. anthracis. Rou-
tine susceptibility testing is not necessary for suspect-
ed contaminants, but all isolates should be saved so
that additional studies can be performed if an identi-
cal organism is recovered from a subsequent blood
culture from the same patient. At that point, full
identification of both isolates along with susceptibil-
ity testing should be initiated. More than one publi-
cation provides a detailed description of a laboratory-
based algorithm for minimizing the extent of
evaluation of blood culture contamination (122,
150, 160).
Polymicrobic Bacteremia
The incidence of true polymicrobic bacteremia in the
absence of contamination is currently unknown,
since most blood cultures are not reincubated after
the initial positive subculture. A recent report on the
16 Baron et al.
epidemiology of bloodstream infections in the United
States over a 20-year period indicates that polymi-
crobic bacteremia is relatively rare, accounting for
only 4.7% of all septic episodes (86). However, in
select patient populations, blood cultures positive for
two or more organisms can range from 10% in chil-
dren (171) to nearly 30% in immunocompromised
patients (70, 84). Dental extractions represent one
known risk factor for polymicrobic bacteremia (5).
Polymicrobic bacteremia in association with
Pseudomonas aeruginosa carries a higher risk for
mortality and is more frequently seen among older
patients (2). Polymicrobic bacteremia can be an indi-
cator of underlying disease and represent transloca-
tion of microorganisms from the gut, skin, and
mucosal surfaces (84). Even so, the laboratory
approach toward the evaluation of blood cultures
positive for multiple organisms should include a fair
degree of common sense. Whether isolated singly or
in conjunction with other organisms, bacteria with
high pathogenic potential such as P. aeruginosa, S.
aureus, or Escherichia coli require complete identifi-
cation and susceptibility testing. When potential con-
taminating bacteria such as coagulase-negative
staphylococci, Bacillus species, coryneform gram-
positive aerobic bacilli, or Propionibacterium acnes
accompany the mix of recovered bacteria, it throws
the validity of a positive blood culture in doubt, and
clinical relevance of the presence of true pathogens is
difficult to interpret. In this instance, the rules
described above under the heading of “contami-
nants” come into play. All potential contaminants
are identified to a limited extent and saved pending
future isolation of the same organism from a subse-
quent blood culture.
Saving Blood Culture Isolates
All blood culture isolates should be maintained as
frozen stock cultures for a minimum of 6 months
pending the possibility of additional studies. Centers
with sufficient resources should attempt to save iso-
lates much longer, for as long as storage space is
available. Possible/probable contaminants are saved
for comparison purposes in case a similar organism
is recovered from the same patient in subsequent
blood cultures, albeit for a shorter time frame (10
days to 2 weeks). If freezer space is limited, labora-
tories should negotiate with their infection control
practitioners to arrive at a mutually acceptable list of
isolates to save for specified time periods longer than
that used for isolates from other sources. A variety of
techniques are available for the preparation of frozen
stock cultures of bacteria, including the use of 10%
skim milk or Trypticase soy broth supplemented with
10% glycerol as freezer media (117). One convenient
method is to place approximately 1 ml of freezer
CUMITECH 1C
medium in a sterile freezer vial. A sterile swab is used
to obtain growth from a fresh subculture of the
blood culture isolate. The swab is immersed into the
freezer medium and broken off below the cap of the
vial. If subcultures are required, the vial needs only to
be thawed and the swab removed with a flamed for-
ceps and transferred to solid growth medium for iso-
lation. Commercial products containing plastic beads
with linear holes for preserving frozen suspensions of
bacteria are also available (Cryocare; Key Scientific
Products, Round Rock, Tex.). After making a sus-
pension of the organism in the broth, excess liquid is
removed and the beads are frozen. To reconstitute
the culture, a single bead is removed and rolled on an
agar plate or dropped into a broth and incubated.
Reporting Results
Unlike many other laboratory results, positive blood
cultures can have an immediate impact on patient
care decisions, and clinically relevant results must be
reported to caregivers as quickly as they are avail-
able. It is often helpful to review other cultures, such
as urine or sputum, received previously from the
same patient for possible clues to the identity of the
isolate before making the call. The number of posi-
tive sets (all bottles from a single phlebotomy) and
the total number of sets received, the time from col-
lection until positivity, and a description of the organ-
ism seen in the smear should be reported. Several
studies have shown broadly improved outcomes and
efficiencies when such reports are delivered quickly
(10, 12, 33, 97). As much information as possible
should be conveyed to the clinician, although the
microbiologist may have to explain the nuances of
these interpretations to non–infectious diseases spe-
cialist physicians. For example, “tiny gram-negative
coccobacilli” is more useful than “gram-negative
bacilli.” A report such as “gram-positive, boxcar-
shaped rods, no visible spores, and abundant gas in
the blood culture bottle” can alert a physician to pos-
sible clostridial sepsis, whereas “gram-positive rods”
raises no alarms. Using cellular arrangement to
report “gram-positive cocci resembling staphylococ-
ci” is more meaningful than reporting “gram-posi-
tive cocci in clusters.” In some circumstances, not all
results must be called in to physicians, but the algo-
rithms of which results require a phone call and the
person to whom positive blood culture results can be
delivered are unique to each health care institution.
For example, in some institutions, a single coagu-
lase-negative staphylococcal isolate from one blood
culture set is not reported to the caregiver, but if a
second blood culture becomes positive for coagulase-
negative staphylococci, the result is reported and fur-
ther workup is initiated. Conversely, any coagulase-
negative staphylococci isolated from the blood of a
CUMITECH 1C
pediatric patient, even when only one bottle has been
sent to the laboratory, is called in immediately.
Whether positive results can be delivered to a nurse,
a unit clerk, or to a physician only should be dic-
tated by the laboratory’s institutional policy. How
such information is conveyed can also vary. Some
busy clinical services, such as the emergency depart-
ment, may prefer faxed results, or they may monitor
the hospital information system closely enough to
preclude any additional report of positive culture
results. Clinically relevant positive blood cultures are
considered “critical values” and reported according
to guidelines developed to satisfy the Joint Commis-
sion on Accreditation of Healthcare Organizations
2003 Patient Safety Goals (see http://www.jcaho.org/
accreditedorganizations/patient+safety/npsg.htm).
The receiver of a critical value is expected to write
down the information and read it back to the caller;
the laboratorian making the call must ask the receiv-
ing person to “please read back the results.” Regard-
less of who receives the report, the microbiologist
must document to whom the report was given and
read back by, and the date and time of report
delivery.
Reports should be updated on a regular basis,
preferably daily, as to whether there is growth in the
blood culture or not. This is to document that the
specimen was received in the laboratory. A report
such as “no growth detected so far” is acceptable.
Once an organism has been detected, a preliminary
report should be issued, either on paper or electroni-
cally into the laboratory information system. Reports
should include the total number of days that the bot-
tle was incubated before being detected as positive,
the type of bottle(s) such as aerobic or anaerobic, and
the source of the sample (such as “PICC line” or
“right hand”). As new information is determined, the
report should be updated. At the end of the pre-
scribed incubation period, negative reports should
state the number of days the bottle was incubated.
Any comments about the specimen or its handling
that could compromise results, such as extended
delays before the bottle was incubated in the instru-
ment, or suboptimal volume fills, should be added to
the report. Interpretive comments, such as the prob-
able clinical significance of the isolate, if added at all,
should be carefully worded and developed in part-
nership with the infectious disease specialists or
other interested medical specialists at the institution.
One example of a comment that was developed with
infectious diseases physician input is used when
Enterococcus species are isolated from blood cul-
tures: “For treatment of endocarditis, the combina-
tion of penicillin or ampicillin and an appropriate
aminoglycoside (streptomycin or gentamicin) is indi-
cated. Vancomycin is usually reserved for penicillin-
Blood Cultures IV 17
allergic patients.” It is prudent to avoid use of the
word contaminant because of the ease with which it
can be misinterpreted. An alternative comment for
reporting a potential contaminant is “This isolate is
possibly a collection-associated skin organism; notify
the microbiology laboratory if further studies are
indicated.”
As with all laboratory reports, the format should
be clear and easy to interpret, and the physician
should find all relevant information easily. It is best
to work with physician groups to jointly agree on a
reporting format. Part of an ongoing quality assur-
ance program includes auditing reports to ensure
that what the physician sees at the user end is what
the laboratory intended and that no information is
missing or confusing.
CPT-4 CODING AND BILLING ISSUES
One bacterial blood culture billed with CPT-4 code
87040 (aerobic and anaerobic bottles filled from a
single phlebotomy) includes the incubation, initial
smear of a positive bottle, and subculture to appro-
priate media. A second blood culture obtained on the
same day, which is clearly the standard of practice,
should be billed in a manner that allows recognition
of the fact that it represents a distinct procedural
service (e.g., by attaching a modifier, 87040–59).
However, some payers may prefer use of modifier -91
(repeat service on the same date). It is best to check
with your specific local reimbursement organization
about appropriate CPT-4 coding if you have been
denied reimbursement with what you consider to be
correct coding, especially since some payers have
developed inappropriate payment policies limiting
blood cultures to one unit of service per day. Blood
cultures for isolation of yeasts or molds are billed
with CPT-4 code 87103, and blood cultures for iso-
lation of mycobacteria are billed with CPT-4 code
87116.
When isolates are identified using a combination
of cellular and colonial morphology and up to three
tests (e.g., “spot” biochemicals), the identifications
are considered presumptive for reimbursement pur-
poses and are included in the primary culture code
described above. However, when organisms are iden-
tified using more than three biochemical tests or by
other complex processes, an additional definitive
identification code may be added (e.g., 87077 for
aerobic isolates, 87076 for anaerobic isolates, 87106
for yeasts, 87107 for molds, and 87118 for mycobac-
teria). In addition, identifications using molecular
methods, immunofluorescent stains, or other im-
munologic methods such as agglutination assays are
also billed with the appropriate CPT-4 codes. Each
antiserum used for an agglutination grouping, for
18 Baron et al.
example, is billed separately with CPT-4 code 87147.
Susceptibilities are billed separately. If an organism is
tested for beta-lactamase using a cefinase disk, that
test can be billed with CPT-4 code 87185 in addition
to the routine method, which is billed with CPT-4
code 87186 for each microdilution plate or 87184
for each disk diffusion plate (12 or fewer disks). If
the antibiotic gradient strip system Etest (AB Biodisk
N. A., Inc., Piscataway, N.J.) is used for some antibi-
otics, such as when testing is requested by a physician
but the agents are not included in the laboratory’s
standard automated panel, each antibiotic strip can
be billed separately with CPT-4 code 87181.
The lysis-centrifugation system (Isolator; Wam-
pole Laboratories, Princeton, N.J.) calls for different
billing. An adult-size Isolator tube is billed for con-
centration with CPT-4 code 87015 in addition to the
blood culture code 87040, and positive plates can
also be billed for identification and susceptibilities as
above. There is no CPT-4 code for the quantitative
aspect of an Isolator blood culture. However, if the
Isolator blood culture is ordered for isolation of
fungi only, the CPT-4 code is 87103 (plus 87015 if it
is centrifuged). In all matters pertaining to correct
coding and billing for blood cultures, clinical micro-
biologists should be aware of reimbursement policies
issued by payers for which the laboratory is an
approved provider of services.
LABORATORY DIAGNOSIS OF SEPSIS
CAUSED BY A COLONIZED INDWELLING
VASCULAR CATHETER
Indwelling vascular catheters are a cornerstone of
modern medical care, enabling administration of flu-
ids, parenteral nutrition and drugs, easy blood sam-
pling, and monitoring of a patient’s physiological
parameters. However, even when careful antisepsis is
followed during the catheter insertion and mainte-
nance, these devices tend to become colonized by
commensal indigenous and pathological skin flora
over time, and catheter-related infections (CRI) are
now among the most common sources of nosoco-
mial bacteremia. Microorganisms attach to the inner
and outer surfaces of the intravenous device, and
bacteria such as Staphylococcus epidermidis and
other species of coagulase-negative staphylococci
secrete an excess of extracellular matrix (“slime”) and
form a tenacious biofilm that protects the organism
from the host immunological response. Flushing of
the colonized catheter by intravenous fluid solutions
provides nutritional support to residing organisms
and contributes to dispersal of the infection through
the bloodstream. Studies performed in patients as
well as animal models have demonstrated that the
CUMITECH 1C
risk for CRI, either local infection or bacteremia,
correlates with the number of organisms colonizing
the catheter (137).
Although purulence or frank cellulitis at the
catheter exit site makes the diagnosis of CRI obvious
in some cases, local signs of inflammation are absent
in more than 70% of catheter-related bloodstream
infections, and the disease usually manifests as onset
of a new febrile episode in a hospitalized patient
(138). The ideal laboratory method for diagnosing
CRI should meet all of the following criteria.
1. It should be rapid enough to enable timely thera-
peutic decisions such as catheter removal and/or
institution of antimicrobial therapy.
2. It should be technically simple.
3. It should be able to detect intraluminal organisms
as well as those attached to the external catheter
surface.
4. It should be highly sensitive.
5. It should be highly specific (i.e., able to differenti-
ate between colonization and true infection).
6. It should be able to confirm or rule out the diag-
nosis of CRI without the need to remove the
indwelling vascular device.
7. It should enable isolation of the infecting bacteria
or fungi to enable complete identification, antibi-
otic susceptibility testing, and eventual typing.
8. It should be safe for the patient.
Although a long list of different laboratory meth-
ods has been used over the years to confirm or rule
out CRI, unfortunately none of them meet all the
aforementioned requirements. In addition, most eval-
uations of methods to diagnose indwelling vascular
catheter infections have been hampered by lack of a
precise “gold standard” for defining CRI, enrollment
of a small number of patients with culture-proven
infection, different case mixes (immunocompetent
and immunocompromised patients, children and
adults), multiple catheter types, lack of uniformity of
the culture techniques, and use of different criteria
for positivity, precluding valid comparisons of results
and making the laboratory diagnosis of CRI a much-
debated issue.
The traditional and simple methods of performing
microscopy and nonquantitative culture of the exit
site or the catheter tip are clearly unsatisfactory
because they do not allow quantification of the num-
ber of microbes and thus are not able to differentiate
between true CRI, skin colonization around the
catheter, and contamination of the external catheter
CUMITECH 1C
surface by a small number of organisms during with-
drawal of the intravascular device through the skin
tunnel (78).
Accepted laboratory methods for diagnosing CRI
can be divided, for practical purposes, into two
broad categories according to the need to remove the
indwelling catheter in order to make the diagnosis.
This issue is of clinical importance for patients with
suspected CRI and no signs of life-threatening infec-
tion because, if the diagnosis of CRI is not confirmed,
an alternative source of infection should be sought,
and there is no indication for removing the catheter.
However, in a patient with a central line and signs of
overwhelming infection such as septic shock but no
apparent clinical focus, removal of the catheter under
suspicion of infection without waiting for laboratory
confirmation of CRI is probably indicated.
Diagnostic methods that leave the catheter in
place are based on the rationale that if the central
line is the focus of bacteremia, because of the in situ
replication of organisms, a high microbial concentra-
tion should be present in blood samples taken from
the infected catheter. This high microbial load can be
readily detected by examining an acridine orange-
stained or Gram-stained sample of the blood cell
layer obtained by the cytocentrifugation of 5
l of
blood drawn from the catheter (127). In one experi-
mental method, the sensitivity was enhanced by
pushing a sterile endoluminal brush through the
catheter and then sending the brush for culture (72).
Although there were no adverse events in this study
of 230 catheters, the safety of this method has not
been evaluated in a large trial and it remains contro-
versial. The high concentration of organisms present
in intravascular catheters of patients with CRI can
also be compared to that found in blood specimens
obtained from distant peripheral veins. The differ-
ence in the microbial concentration between blood
specimens obtained from the infected central line and
from a peripheral site can be measured using a quan-
titative blood culture system such as the pour plate or
the lysis-centrifugation method (43). A colony count
ratio greater than 4 to 10:1 between the central
venous blood and a peripheral vein blood specimen
has been found to have 78 to 94% sensitivity and 99
to 100% specificity for diagnosing CRI (19). When
only a quantitative central venous blood sample but
no peripheral vein culture is obtained, the diagnosis
of CRI can still be confirmed by finding more than
100 CFU of bacteria/ml of blood or 25 CFU of
fungi/ml, because bacteremia and fungemia caused
by infections other than CRI in adult patients tend to
yield lower CFU counts (19).
Because the lysis-centrifugation method is not
readily available in all clinical microbiology labora-
Blood Cultures IV 19
tories and has the disadvantage of being manual,
time-consuming, and prone to contamination, an
alternative approach for diagnosing CRI based on
the use of broth-based blood culture systems has
been recently developed. Continuous monitoring of
blood culture vials by modern automated instru-
ments enables determining and recording the precise
time-to-positivity in each vial. Finding of a differen-
tial time to positivity greater than 2 h between vials
inoculated with blood drawn through the catheter
and those obtained from a peripheral vein has been
found to be reliable for the diagnosis of CRI by more
than one series of investigations (14, 116), although
some others have not found such positive results
(123).
Diagnostic laboratory methods that require
removal of the central catheter have the obvious dis-
advantage that the diagnosis is always made retro-
spectively. In cases where the diagnosis of CRI is
ruled out, insertion of a new blood vessel access
device would still be required, adding hospital costs
and potential risks to the patient. Methods that
require catheter removal for making the diagnosis of
CRI include (alone or in combination) (i) use of an
endoluminal brush (72); (ii) sonication or flushing of
the catheter lumen to remove adherent organisms
(136), followed by a staining procedure and/or a
quantitative culture; and (iii) a semiquantitative cul-
ture of the catheter tip (85) and/or hub (44). Of all
these procedures, the semiquantitative culture of the
catheter tip (Maki method) is the most widely used
and evaluated (138). This method involves culturing
the external surface of the removed catheter tip by
rolling a 3- to 4-cm segment four times over the sur-
face of a blood-agar plate with the aid of a sterile for-
ceps. After incubation in aerobic conditions, the
number of growing colonies is counted. The original
cutoff value to diagnose CRI recommended by Maki
(15 CFU of bacteria) has been lowered by some
investigators to 5 to improve sensitivity (28). Num-
bers for yeast have not been as well studied. It should
be noted, however, that the method does not detect
indwelling vascular catheters with exclusive endolu-
minal colonization. This problem has been addressed
by flushing the catheter as described by Cleri et al.
(25) or by sonication (68). The Cleri method consists
of cutting and separating the first 1-cm-long intra-
dermal segment of the removed catheter from the
intravascular portion. A needle is inserted into the
proximal end of the intravascular segment, which is
then immersed in 2 or 10 ml of Trypticase soy broth,
depending on the length of the segment to be cul-
tured, and flushed three times. The broth is serially
diluted 100-fold, and 0.1 ml of each dilution is
streaked onto blood agar plates. In the sonication
20 Baron et al.
procedure, the catheter is placed into a tube contain-
ing 4 ml of brain heart infusion (68). The tube is then
sonicated for 1 min followed by vortexing for 15 s.
Aliquots of 0.1 and 0.001 ml of the sonicated broth
are then plated onto solid media and incubated.
When sonication methods are evaluated, cutoff val-
ues of 102 CFU (136) or 103 CFU (15, 68) have been
shown to yield 94 to 97% sensitivity and 75 to 88%
specificity for diagnosing CRI.
The recently introduced brush technique appears to
improve the diagnosis of CRI by mechanically remov-
ing organisms attached to the endoluminal biofilm,
which are then suitable for detection by microscopy
and culture (72). Although the early experience with
this novel method appears promising, concerns
regarding its safety when used while the catheter is still
in place have been raised because of the theoretical
possibility of blood-borne dissemination of dislodged
organisms and distal embolization (72). More wide-
spread studies using this technique are needed.
The question of which is the most appropriate lab-
oratory method for diagnosing CRI remains unset-
tled. Despite the lack of consensus, the following rec-
ommendations are made.
1. If septic shock of undetermined source develops in
a patient with an indwelling vascular catheter, or
when local signs of infection such as purulence or
cellulitis are present, the catheter should be
removed and a semiquantitative culture (by the
Maki method) or quantitative culture (by the Cleri
or sonication methods) of the catheter tip should
be performed in addition to at least two blood cul-
ture sets obtained peripherally.
2. If the patient with suspected CRI is in a stable clin-
ical condition, confirmation of the diagnosis by
ruling out other sources, for example, prior to
administration of antibiotics and removal of the
catheter, should be attempted.
3. When quantitative blood cultures are available,
blood specimens should be drawn through the
catheter and from a peripheral vein and concen-
tration of microorganisms should be compared. A
catheter-blood to peripheral-blood ratio 4 is
indicative of CRI (19).
4. When an automated broth-based blood culture
system is used, a differential time to positivity of
2 h between hub blood and peripheral blood can
be used to confirm CRI (116).
5. Complementary methods such as the Gram stain
or acridine orange cytospin tests and use of the
endoluminal brush seem promising, but addition-
al experience with these techniques is needed
prior to their recommendation.
6. Only aerobic cultures of the intravascular device
should be performed.
CUMITECH 1C
CONVENTIONAL METHODS FOR
DETECTING PATHOGENS THAT
FAIL TO GROW IN STANDARD MEDIA
A number of microorganisms present in blood, either
transiently or as agents of intravascular infections
including endocarditis, cannot be recovered with
standard routine or automated blood culture proto-
cols. Some bacteria traditionally thought to require
longer incubation times for recovery, including the
HACEK agents and Brucella (see specifics in this sec-
tion), are now routinely recovered by the usual auto-
mated blood culture procedures and incubation
times. Nutritionally deficient streptococci, Abio-
trophia species, and Granulicatella species grow well
in blood culture media and exhibit positive signals in
the bottles. They are readily visualized by Gram
staining but fail to grow on blood agar plates,
although they should be viable on chocolate agar.
Subculturing to blood agar with a Staphylococcus
aureus cross-streak also reveals the satelliting
colonies typical for these species. Each new lot of
chocolate agar should be tested with a nutritionally
variant streptococcus to ensure that blood culture
subcultures will reveal the agent. If such an added
quality assurance activity is not feasible, S. aureus
should be cross-streaked on blood agar subcultures
for gram-positive cocci that fail to grow on initial
subcultures. Francisella tularensis grows sufficiently
in commercial systems to be routinely recovered
from blood. Not only is this organism a potential
bioterrorism agent, but it is considered a biosafety
level 3 organism and should be handled minimally in
routine clinical laboratories. For example, if an iso-
late exhibits extremely tiny, pale-staining gram-nega-
tive or gram-variable coccobacilli on the smear, all
plates should be sealed immediately and all handling
should be performed in a biological safety cabinet by
using Sentinel Laboratory Response Network proto-
cols (see http://www.bt.cdc.gov/labissues/#testing). If
the isolate is found to be catalase negative or weak
positive, urease negative, oxidase negative, and
nitrate negative, it should be referred to the appro-
priate Response Network Reference Level Labora-
tory to rule out Francisella.
Some agents, including Coxiella, Chlamydia,
Rickettsia, and Tropheryma spp., do not grow on
artificial media and are best diagnosed using serolog-
ical tests (slow) or molecular amplification methods
(available at limited reference laboratories). Although
serology is virtually the only method for diagnosis of
Chlamydia psittaci endocarditis, cross-reactivity with
antibodies directed against Bartonella has been prob-
lematic (88). Blood for molecular amplification test-
ing may require EDTA tubes, plasma preparation
tubes, or acid-citrate tubes, and reference laborato-
CUMITECH 1C
ries often request that the sample be frozen at 70°C
and shipped on dry ice. The performing laboratory
should be consulted before obtaining the blood to
learn which type of tube, preservative, and tempera-
ture should be used for handling.
Musso and Raoult have isolated Coxiella burnetii
from the blood of 53% of untreated patients with Q
fever by inoculation of human fibroblasts grown in
shell vials (100). Immunofluorescent staining of the
monolayer is required to detect infection. This
method has not been validated in other laboratories
and is generally not available elsewhere, but labora-
tories should again follow Sentinel Laboratory
Response Network protocols for referral to a net-
work confirmatory laboratory for testing.
Another group of organisms may be present in
blood and viability is maintained in standard blood
culture media, but either the organisms do not grow
sufficiently to produce the metabolic end products
that serve as growth indicators in the system or their
growth is too scant to be visible in the broth. Cryp-
tococcus neoformans, Legionella species, some Heli-
cobacter species, and molds, including the systemic
dimorphic species, fall into this category.
Fungi
Although systemic cryptococcal disease is most rap-
idly diagnosed with the serum cryptococcal antigen
test (either agglutination or ELISA format), the organ-
ism may be flagged by automated systems and can
certainly be recovered from blood culture broths by
performing blind subcultures to fungal media at the
end of a standard 5- or 7-day incubation protocol.
Other yeasts do grow well in commercially available
blood culture broths and do not need special han-
dling, with the exception of Malassezia furfur, which
requires the addition of olive oil to the subculture
agar. The best recovery of M. furfur is achieved with
a very thin coating of olive oil (extra virgin, sterilized
before use) applied to the fungal plate medium (brain
heart infusion, Sabouraud dextrose, or potato flake
agar) with a swab before inoculation. Infants under-
going lipid emulsion therapy and neonates in the
intensive care unit are most at risk for M. furfur sep-
sis, and special handling should be requested if this
organism is suspected. Because the pH, nutrients,
and oxygen partial pressure of standard blood cul-
ture broths have been optimized for bacteria, molds
are rarely recovered. If circulating molds, such as
Fusarium or Histoplasma, are suspected, either a
special fungal liquid medium, such as MYCO/F
Lytic medium (BACTEC), BacT/ALERT MB, or the
Isolator lysis-centrifugation system should be used
(96). For Cryptococcus and molds, inoculation onto
media suited for culture of systemic fungi such as
Blood Cultures IV 21
brain heart infusion, Sabouraud glucose and brain
heart infusion, or potato dextrose agar affords the
best recovery (26).
Bartonella
Antibody testing is the most reliable method for diag-
nosis of Bartonella bacteremia (45). Failing this,
nucleic acid amplification methods are also quite sen-
sitive (179). Blood cultures may be attempted using
lysis centrifugation, with the sediment plated onto
freshly prepared Columbia agar base blood, choco-
late, or BCYE agar plates incubated at 35°C in a
moist 5 to 7% CO2 atmosphere for 14 to 21 days.
Bartonella bacilliformis, unlike B. henselae and B.
quintana, grows best at 25 to 30°C. Tierno and col-
leagues have isolated Bartonella species from
BacT/ALERT blood culture bottles that signaled pos-
itive by injecting 7.5 ml of the “positive” broth into
lysis-centrifugation tubes and processing the sedi-
ment as above (149).
Brucella
Depending on the automated system, Brucella can be
isolated using standard or slightly prolonged incuba-
tion protocols. A review of methods reported that
continuous monitoring systems were faster than
lysis-centrifugation, although all isolates detected by
the Isolator grew within 7 days (173). However, the
Isolator system failed to detect a substantial number
of positives (6 of 28) detected by BACTEC (174).
Some studies reported 100% recovery within 4 days
for either BACTEC or BacT/ALERT (125, 174).
Although variable results were reported for different
commercial systems, only 1 of 41 true-positive cul-
tures was detected by a blind subculture at 7 days
versus automated detection in the BACTEC (175).
One study reported detection of 3 of 97 positive cul-
tures by the BACTEC between days 8 and 9 (9); thus,
a 10-day total incubation in an automated blood cul-
ture system should approach 100% sensitivity for
detection of Brucella. However, with sufficient vol-
ume of blood, it is reasonable to expect almost all
Brucella to be detected within the standard 5-day
protocol.
This genus is among the most common labora-
tory-acquired infectious agents as well as a potential
bioterrorism agent, and all manipulations should be
performed in a biosafety level 2 laboratory setting
within a biological safety cabinet. Plates should be
taped shut, and as soon as preliminary tests suggest
Brucella (including small gram-negative coccobacilli,
rapid positive urease, positive oxidase, and nitrate),
the isolate should be referred to the appropriate Con-
firmatory Laboratory Response Network laboratory.
Two recent articles reported Brucella appearing as
22 Baron et al.
gram-positive or gram-variable coccobacilli in the
initial Gram stain from broth, and that has been seen
in other laboratories as well, so all small gram-vari-
able coccobacilli should be handled as if they were
bioterrorism agents until proven otherwise (91, 104).
Campylobacter and Helicobacter
These two genera consist of small, thin, curved rods
that may require acridine orange for visualization in
instrument-flagged positive bottles. Several species of
Campylobacter, including C. jejuni, C. lari, and C.
fetus, are isolated from blood occasionally, and they
usually grow within a 5-day protocol. Subculture
plates should be incubated to Columbia blood-agar
base, specific Campy media (without antibiotics, if
possible), and incubated in a microaerophilic atmos-
phere at 35 to 37°C for up to 3 days. Helicobacter
cinaedi and other species (H. westmeadii, for exam-
ple) have been recovered from blood, usually from
immunocompromised patients (69, 151). These iso-
lates were detected after a 7-day incubation, but
standard Gram stains failed to reveal organisms and
acridine orange staining was required.
Legionella
Although legionellae survive fairly well in commer-
cial blood culture broth, they do not multiply (24). In
fact, bacteremia does occur during systemic illness.
Detection requires either specialized BCYE broth (no
longer available commercially) or lysis-centrifugation
and plating onto BCYE followed by incubation in a
moist atmosphere for up to 5 days. In a seeded blood
culture study, recovery of the organism decreased
within 30 min of inoculation of the Isolator tube, and
only 10% of the original inoculum was recovered if
Isolator tubes were processed after 15 h (24).
Mycoplasma
Mycoplasma hominis has been implicated as a cause
of postpartum sepsis, but its clinical significance is
still in doubt. It is recovered in blood cultures occa-
sionally and may be subcultured on standard sheep
blood-agar plates. M. pneumoniae may also cause
septicemia. Standard blood culture systems rarely
support growth of these cell-wall-absent organisms
without the addition of gelatin to neutralize the
inhibitory effect of SPS and arginine for enhancement
of the growth of M. pneumoniae, but the organisms
can be visualized in “positive” bottles if acridine
orange stain is used. Subculture to specialized
Mycoplasma medium such as Shepard’s A-7 or SP4
biphasic medium and incubation for up to 7 days at
35°C in 5% CO2 or anaerobically (best) is essential,
although occasional colonies may grow on Columbia
CNA agar plates. Physicians should explain how
results of cultures positive for Mycoplasma will alter
CUMITECH 1C
patient treatment as a prerequisite for the laboratory
attempting to isolate this difficult genus.
Leptospira
Blood cultures should be obtained during the first
week of illness, as the bacteremia is no longer pres-
ent beyond 7 days. The organism does not survive at
35°C, so cultures must be held and incubated at
30°C. Ideally, two drops of blood should be inocu-
lated directly into 10 ml of semisolid oleic acid albu-
min medium such as Ellinghausen-McCullough-
Johnson-Harris broth or PLM-5 broth (Intergen Co.,
Purchase, N.Y.) at the bedside. These media or oth-
ers that contain bovine serum albumin and Tween 80
are recommended (176). This medium is incubated at
28 to 30°C for up to 13 weeks and examined under
darkfield microscopy weekly for the presence of the
tightly coiled, hooked spirochetes. Alternatively,
blood may be collected into heparin, oxalate, or cit-
rate tubes and maintained at ambient temperature
for shipment to a reference laboratory for culture.
Mycobacterium
In most cases, mycobacterial blood cultures are
requested for detection of disseminated Mycobacte-
rium avium-M. intracellulare complex in patients
with AIDS. Commercial broth systems for mycobac-
terial cultures have specialized broths for isolating
mycobacteria from blood. In a study reported in
2001, the MYCO/F Lytic medium for the BACTEC
instrument performed better than other media and
worked well in combination with an Isolator tube
(154). A more recent controlled study found that
mycobacteria (predominantly M. avium complex)
were recovered equally well in both MYCO/F Lytic
and BacT/ ALERT MB media in their respective con-
tinuously monitored systems with comparable sensi-
tivity, approximately 81 to 85%, but faster time to
positivity than the previous standard Isolator 10 tube
(30). When commercial mycobacterial blood culture
media are used, the manufacturer’s recommendations
for handling and processing should be followed. In
some cases, such as for the BacT/ALERT MB system,
blood is inoculated directly into the bottle at the bed-
side and a special enrichment fluid is added to those
bottles in the laboratory before incubation. Blood
can also be collected in a heparin tube (not EDTA or
acid-citrate tubes) and submitted to the laboratory
for inoculation directly onto plate media (7H-11)
and automated instrument broths. If blood collected
in Isolator tubes is then inoculated into BACTEC
12B bottles, only 0.2 ml of the bacterial pellet should
be used, as a component in the Isolator was found to
be inhibitory (34, 159). Inhibitory effects of Isolator
samples should be checked before using with other
systems. Blood may be obtained in Isolator tubes,
CUMITECH 1C
processed, and plated directly onto mycobacterial
media for colony count determination. Several earli-
er studies showed the Isolator to be effective for
recovery of mycobacteria in blood (51, 52, 134,
154). Although high numbers of circulating M. avi-
um-M. intracellulare complex in patients with AIDS
is rarely seen today, the success of treatment can be
monitored by performing quantitative cultures with
the Pediatric 1.5-ml Isolator plated directly onto
Middlebrook 7H11 or 7H12 agar plates (172).
MOLECULAR METHODS FOR
DETECTING BSIs
For certain organisms (more commonly viruses),
molecular amplification methods including PCR,
nucleic acid sequence-based amplification assays,
and occasionally other protocols are the state of the
art. This is true today for cytomegalovirus, where the
presence and quantity of circulating virus are used to
aid diagnosis of exacerbating disease, particularly in
patients on immunosuppressive therapies or who are
otherwise immunocompromised, or neonates with
congenital disease (92). Quantitative tests for Epstein-
Barr virus are used to monitor transplant recipients
for posttransplantation lymphoproliferative disease
(126). And for several years, quantitative molecular
tests have been the standard for monitoring viral
loads in staging and therapy of hepatitis C and HIV
(109, 132, 141).
As discussed above with reference to fastidious
agents of septicemia, molecular amplification may be
the only method for detecting some bacterial agents,
such as Rickettsia, Bartonella, Chlamydia, Borrelia
burgdorferi, Ehrlichia, and Anaplasma, as well
as blood-borne parasites, including Plasmodium,
Babesia, Leishmania, and Toxoplasma spp. If the
agents cause endocarditis, blood may not be the best
specimen; the valve tissue removed during surgery
would be the preferred sample for detection of the
organism (e.g., Coxiella). When whole blood or
white blood cell components only are tested, there is
a concentration or extraction step followed by the
amplification step, and finally a detection step to
identify and in some cases quantify the amplified
products. However, for infectious agent molecular
testing, a single analyte- and method-specific code is
used to represent the entire procedure. The most spe-
cific CPT-4 codes available should be used, and non-
specific codes should be used only when an analyte-
specific code does not yet exist.
Several manufacturers are currently evaluating
broad-spectrum universal primer amplification for
detection of groups of organisms known to cause
septicemia, followed by more specific tests when a
positive result is obtained. A few leading-edge labo-
Blood Cultures IV 23
ratories are also evaluating their own internally
developed molecular amplification methods (102,
115). Unfortunately, the new molecular methods will
likely not preclude use of conventional blood cul-
tures for a long time to come, as the organisms must
be isolated for susceptibility testing and epidemiolog-
ical typing studies. In addition, there are too many
chances of false-negative or false-positive molecular
tests unless a traditional method is used to corrobo-
rate molecular results (103). However, as a supple-
mental procedure, nucleic acid amplification may
allow more rapid recognition of septicemia and the
earlier use of appropriate antimicrobials, thus
enhancing patient outcomes, but it will certainly not
decrease overall laboratory costs in the short term.
QUALITY CONTROL
Meeting standards for the performance of media and
reagents, monitoring temperatures and instrument
performance parameters, susceptibility results with
organisms of known susceptibility, and many other
activities are part of the routine quality control (QC)
performed by all laboratories. National regulations
promulgated by the Clinical Laboratory Improve-
ment Amendments (CLIA) and published in the
Federal Register (42a; also available to download at
http://www.asm.org/ASM/files/LeftMarginHeader
List/DOWNLOADFILENAME/0000000809/cliafin
reg[1].pdf) should be followed unless state guidelines
supersede the national guidelines. The College of
American Pathologists (CAP) has deemed status
within CLIA and publishes checklists available to all
laboratories, even those not accredited by CAP, that
contain excellent criteria for quality control (http://
www.cap.org/apps/docs/laboratory_accreditation/
checklists/checklistftp.html). Another excellent re-
source is the chapters on quality assurance and QC
in the Clinical Microbiology Procedures Handbook,
second edition (63, 130). A future Cumitech on qual-
ity systems will also discuss monitoring activities for
the blood culture process (D. L. Sewell et al., unpub-
lished work).
CLIA and the Clinical and Laboratory Standards
Institute (CLSI; formerly NCCLS) both state that
commercially produced blood culture media do not
require additional in-laboratory QC testing beyond
visual inspection if the manufacturer follows CLSI
guidelines (101). Users should, however, keep accu-
rate records of lot numbers, dates received and
expired, and the product inserts certifying the proper
QC by the manufacturer. Isolator tubes do require
some in-laboratory QC, as outlined by the manufac-
turer, including monitoring of vacuum draw volume,
breakage in the centrifuge, lysis of blood cells, and
appropriate organism recovery.
24 Baron et al. CUMITECH 1C

QUALITY ASSURANCE of blood based on weight of patient (for infants and

children), and how to handle a low-volume speci-
Blood culture accessioning, processing, incubating,
men. Criteria for rejection are essential. To monitor
and all the activities that occur within the laboratory
that all workers who collect blood are following
for identification of isolates, susceptibility testing,
proper procedures, the laboratory should monitor
and results formatting are activities for which QC
monthly the contamination rate using parameters
parameters should be measured and monitored.
such as those developed for the CAP Q-Probe series
Quality assurance activities include attention to pre-
(131). One suggested definition of contamination is
analytical stages, which include venipuncture, filling
“a single culture positive for coagulase-negative
and labeling the bottles, and transport to the labora-
staphylococci or coryneform gram-positive rods or
tory; and postanalytical activities, which include
Micrococcus or Propionibacterium, or a single bottle
results reporting to physicians and patient outcomes.
with Bacillus species, not anthracis.”
The laboratory’s responsibilities include all aspects of
Because some hospital care units (typically the
testing, and for those functions performed outside
emergency department or a pediatric oncology ward)
the laboratory, communication and cooperation with
have higher contamination rates, units should be
other caregivers are essential. Some suggested quality
monitored separately. Contamination rates for blood
assurance activities are summarized in Table 4.
cultures obtained through lines should be monitored
Specimen Collection separately from those obtained percutaneously. If
rates do not fall below national standards, which are
The laboratory must provide protocols for proper
currently placed at 2 to 3% (131, 139), monitoring
collection of blood cultures from all sites, including
of an individual phlebotomist may be instituted with
those drawn through indwelling lines, and specific
counseling and retraining where necessary.
guidelines on numbers of cultures to obtain, volume
Neonatal blood cultures pose a special problem in
monitoring for contamination, as it is common prac-
Table 4. Quality assurance parameters to monitor tice to obtain only a single blood culture. It has been
suggested that the C-reactive protein value may be
Factors to monitor Timing used to distinguish contamination from infection for
“Contamination” rate based Monthly, with timely report- blood cultures yielding coagulase-negative staphylo-
on laboratory-specific ing to units that exceed cocci (113). However there is no current consensus
parameters. Separate standards and the relative clinical relevance of coagulase
audits for patient care
staphylococci in such cultures must be determined by
units, different phleboto-
mists (nurses vs laboratory, clinicians and infection control practitioners.
for example), and line- The volume of blood obtained should also be
drawn vs. peripheral monitored, at least annually, for a period of time suf-
cultures. ficient to get information on the areas where the
Volume of blood obtained by Annually, for a limited time
majority of blood cultures are drawn. One method is
unit. If problems are found,
individual phlebotomist to weigh bottles before they are sent to the wards,
monitoring may be and to reweigh them once they have been inoculated.
necessary. One milliliter of blood weighs approximately 1 g.
Single blood cultures Monthly at first, longer Different patient parameters, such as pediatric versus
increments if this is not a
adult, must be taken into consideration when aver-
problem
Too many blood cultures Constantly ages are computed. The percentage of blood samples
Percent of positive blood Monthly, per unit and patient 2 ml over or below the recommended volume for
cultures type the vial type should be determined and appropriate
Number of blood cultures/ Annually inservice activities presented to those phlebotomy
1,000 patient days
groups with problems. False-negative results can be
Correlation between smear Annually, per technologist
result and culture results attributed to both too little blood and too much
Time to calling results to care- For some limited time period, blood, which may allow clotting and trapping of
giver from time of detec- annually or more often if organisms inside the clot.
tion of positive blood necessary (physician
culture complaints) Numbers of Cultures Drawn per Patient
Direct susceptibility results Constantly
compared with definitive Given that a single blood culture is never appropri-
susceptibility results from ate, if a laboratory does not have an automatic reflex
pure isolates policy for a second blood culture, the number of sin-
Paperwork, clerical errors, Periodically, such as quarterly gle blood cultures should be monitored. Some patient
billing, reimbursement
care areas, such as the neonatal intensive care nurs-
CUMITECH 1C
ery, may need to be exempt from the “two-bottle
minimum” rule due to the difficulty of collecting
multiple cultures, although in truth, coagulase-nega-
tive staphylococci are both the most common
pathogen and most common contaminant in that set-
ting as well (65). Physicians or patient care areas
where single-bottle cultures are more commonly
obtained should be counseled. A CAP Q-Probe study
evaluated this parameter (105). The authors found
that in the 10% lowest-performing hospitals, solitary
blood cultures comprised 42.5% of all blood cul-
tures, whereas the top 10% of hospitals had only
3.4% solitary blood cultures. Another quality assur-
ance monitor is for the occasional occurrence of too
many blood cultures ordered. Given that increased
detection of additional positive cultures with vol-
umes of 80 ml is extremely unlikely (27), blood cul-
tures beyond this total volume are probably unneces-
sary and the physician should be informed of other
potential testing methods. This audit should be ongo-
ing, so the laboratory director, infectious diseases
specialist, or clinical pathologist can discuss diagnos-
tic options with the ordering physicians in real time.
Positive Culture Rate and Number of Cultures
per 1,000 Patient Days
If the positive culture rate is too high or too low,
physicians may not be ordering blood cultures
appropriately. A national audit comprising 649 labo-
ratories found an average of 7.7% positives by labo-
ratory criteria and 8.2% by clinical criteria (131).
Depending on the type of hospital (primary care ver-
sus tertiary, academic versus community), the rate
could be higher or lower. But if positivity drops
below 5% or rises above 15%, an investigation
should be initiated into whether physicians are order-
ing blood cultures appropriately. Another monitor of
appropriate ordering practices is to audit the number
of blood cultures ordered per 1,000 patient days. A
sampling of a number of hospitals found values
between 103 and 188 (L. Peterson and J. M. Miller,
ClinMicroNet Microbiology Laboratory Directors
World Wide Web-based forum, 1999). A number
between these two extremes is recommended.
Technologist Competency and Result Reporting
Correlation of smear results (which should be report-
ed immediately to the caregiver) and culture results is
one measure of the technologist’s ability to properly
interpret smear results. Lack of correlation has
patient care consequences, as physicians may begin
inappropriate antibiotics if the smear interpretation
is incorrect. This should be monitored individually
for the technologist and overall, and the results can
become part of each technologist’s annual compe-
tency assessment. Another aspect of the communica-
Blood Cultures IV 25
tion between laboratory and physician is turnaround
time from first detection of positive to informing the
caregiver. This should be part of ongoing quality
assurance monitoring. Both Doern and Barenfanger
have shown major improvement in patient outcomes
with faster results reporting (10, 12, 33, 97). For
those laboratories that perform immediate suscepti-
bility tests from the blood culture broth directly, the
results of the preliminary tests should be compared
with those of the definitive test performed on the pure
culture isolates. Cumitech 41 has additional sugges-
tions for quality assurance monitoring and other
activities to prevent laboratory-associated errors (3).
Ordering, Billing, and Reimbursement
Finally, laboratories deserve to be fairly compensated
for the effort and materials expended on the care of
patients. If possible, laboratories should periodically
audit a series of patient results to determine whether
the CPT-4 coding was correct, the billing was correct,
and whether the payment was actually received. The
information about specific reimbursement may not be
available without special requests, but billing errors
and incorrect coding can result in huge fines, as well
as lost revenues, so these functions merit scrutiny.
One strategy is to pick randomly five patient results
to audit quarterly, including the transfer of orders to
the computer system, the correct translation of labo-
ratory results to the hospital information system, and
the appropriate billing. Another strategy is to request
the rate of reimbursement for a particular CPT code.
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