+++
Collection and Processing
of Clinical Specimens
++
No degree of laboratory expertise can correct the error of inappropriately
collected and transported specimens. The proper collection and handling
of clinical specimens is as important as selecting the correct medication
for treatment.1,2 Common problems with clinical specimen
collection and handling include insufficient quantity, contamination,
inappropriate transport conditions, and delay in transport to the
laboratory.1,2 The most optimal specimen for the
isolation of bacteria fluid scrapings, biopsy, or tissue samples
increase the chance of recovery of the pathogen and reduce the isolation
of contaminants that may mislead or result in misdiagnosis. Swabs,
although commonly sent are less likely to yield useful results. Swabs
are commonly sent, but they are not the most optimal specimen for
the isolation of bacteria. Fluid, scrapings, biopsy, or tissue samples
increase the chance of recovery of the pathogen and reduce the isolation
of contaminants that may mislead or result in misdiagnosis.
++
Transport media are necessary to maximize pathogen survival. They
are designed to prevent drying, provide minimal nutrients, and maintain
a balanced physiochemical environment that prevents the oxidation
and enzymatic destruction of the pathogen. Some transport media
may contain antimicrobial agents that suppress normal or contaminating flora
to enhance the isolation of a specific pathogen. Ideally, specimens
for bacterial culture should not be stored for longer than 24 hours
before processing. When specimens cannot be transported or processed
immediately, appropriate alternatives are available. Holding conditions
are specimen or pathogen specific. Urine can be refrigerated at
2°C to 8°C for up to 24 hours. Inoculated blood culture bottles
can be held at room temperature for up to 24 hours. Specimens for
the isolation of Neisseria gonorrhoeae should be
inoculated onto specific media (warmed to room temperature), preferably
transported to the laboratory expeditiously in a CO2-enriched transport
pack (Gono-Pack, Jemec) and immediately incubated at 35°C in 5% CO2.
Cerebrospinal fluid (CSF) should be transported rapidly at room
temperature or at 35°C to 37°C and never refrigerated. Stool for
cytotoxin assay for Clostridium difficile must
be refrigerated or frozen at –70°C if not processed within 1
hour because toxin activity decays rapidly at ambient temperature.
A dry swab is preferred for detection of Streptococcus pyogenes antigen
from pharyngeal specimens. Dacron swabs can be used for viral and S
pyogenes isolation. Culturette swab is a commonly used transport
system. Frequently, transport kits are assembled and supplied by
local laboratories to ensure the optimal recovery of microorganisms. Specimens
from infected sites where anaerobic bacteria may be causative agents,
such as brain or lung abscesses or peritoneal fluid, require special
anaerobic transport medium. Transporting the fluid to the laboratory
for an anaerobic culture in a syringe is not adequate. Blood culture
bottles should be inoculated by the bedside with an appropriate
amount of blood. Synovial joint and peritoneal fluids can also be
inoculated into blood culture bottles to enhance recovery yield.3-6 Sterile
screw-cap containers can be used to transport urine, feces, CSF,
and other body fluids.
++
Various types of culture media are used within the clinical microbiology
laboratory. Enriched media support the growth of
fastidious bacteria. Examples include chocolate agar, sheep blood
agar, and thioglycolate broth. Selective media
permit the selective growth of certain groups of bacteria, while suppressing
others. Example include colistin-nalidixic agar (CNA) for the isolation
of gram-positive organisms. Differential media assist
in distinguishing among similar groups of bacteria. MacConkey agar
is both selective (permitting the growth of aerobic gram-negative
bacteria only) and differential (distinguishing gram-negative bacilli
by their ability to ferment lactose). A lactose-fermenter organism
(purple-pink colonies) on MacConkey agar may represent Escherichia
coli, whereas a nonlactose-fermenter organism (clear colonies)
may represent Pseudomonas aeruginosa or Shigella.
The use of these media increases the yield of recovery of certain
fastidious organisms and hastens the time to identification and
availability of susceptibility data.
+++
Specimen-Specific Isolation
Methods
++
Venipuncture sites are cleansed with 70% isopropyl or
ethyl alcohol, followed by disinfection with 1% to 2% tincture
of iodine, 10% povidone-iodine, or 2% chlorhexidine
gluconate with 70% isopropyl alcohol (allowing it to dry
for at least 1 minute, with no wiping).7 The rubber
stopper of the blood culture bottle or tube is also disinfected
using 70% isopropyl or ethyl alcohol. Iodine is not recommended
because its use may lead to cracks. Blood culture specimens from
venipuncture sites are preferred as they have a lower contamination
rate than specimens obtained through a newly inserted intravenous
catheter.8 The proportion of blood to broth is
important. A blood-to-broth ratio > 1:5 is desirable.9 Inadequate
volume is the most important cause of a false-negative blood culture,
and it appears to be a common problem among pediatric patients.10 Bacteremia
in children is usually quantitatively higher than in adults, but
low-level bacteremia does occur. An appropriate volume of blood
per bottle and number of blood cultures being submitted is critical.11,12 One
to 5 mL may be an adequate sample from children, whereas 20 to 30
mL is required from adults.9 Appropriate volumes would
be 1 to 2 mL in neonates, 2 to 3 mL in infants, 3 to 5 mL in children,
and 10 to 20 mL in adolescents.13
++
Most broth culture systems contain an anticoagulant, sodium polyanetholesulfonate (SPS),
0.025% to 0.05%, which inhibits phagocytosis and
serum bactericidal activity, inactivates complement, and neutralizes lysozymes
and some antibiotics such as aminoglycosides. Although it may enhance
the recovery of certain pathogens, SPS can inhibit the growth of
others such as Neisseria, Streptobacillus
moniliformis (one of the causes of rat-bite fever), and Francisella
tularensis (cause of tularemia). An appropriate ratio of blood-to-broth
volume dilutes SPS, thereby decreasing its natural inhibitory factors,
and also dilutes any antimicrobial agent that may be present, without
affecting the growth of important pathogens.
++
Although a single sampling may be sufficient for most patients
with bacteremia, especially with Staphylococcus aureus,
multiple samples are needed under certain circumstances (eg, in
patients with suspected endocarditis in whom three or more samples
are desirable to obtain a sensitivity of 96%, especially
if the patient has received antimicrobial agents). In a more recent
study in adults, as many as four blood cultures in a 24-hour period
were needed to achieve a detection rate of >99%.14 The
total volume of blood submitted for culture is also critical. Similar
studies in children are lacking at this time. For children with
indwelling intravascular catheters, at least two sets of cultures
are desirable: one from the vascular catheter and another from a venipuncture
site.15-17
++
Once blood is inoculated into a broth bottle, rapid incubation
is essential. In one study, a delay of 3 hours resulted in a 25% reduction
in recovery of Streptococcus pneumoniae.18
++
Current blood culture systems were developed to increase yield,
reduce the time to recovery of bacteria, and diminish technologist time.
Newer systems also maximize the recovery of fastidious organisms.
Commonly used systems are lysis centrifugation–direct plating or
the Isolator system (Wampole Laboratories), the VersaTREK (Trek
Diagnostic Systems), the BacT/Alert (bioMérieux,
Inc), and the BACTEC system (Becton Dickinson Division Instrument
Systems). Comparisons between systems are detailed in additional
text on DVD. For the latter three systems, depending on the type
of suspected pathogen, several broth formulas are available. Unfortunately,
no single system is optimal for all microorganisms.
++
The lysis centrifugation–direct plating system consists
of a sterile tube of two different sizes (1.5 and 10 mL) that contains
saponin and SPS, which lyse red and white blood cells in the blood
specimen and inactivate complement and immunoglobulins. Hemoglobin binds
to SPS and prevents the inhibition of bacteria.19 The
larger tubes are centrifuged at 300 g for 30 minutes
in a fixed-angle rotor. With the use of an Isostat press, the lysate
is removed and inoculated directly onto agar medium, which is incubated
and examined daily for growth. Media can be selected to maximize
the recovery of suspected pathogens. Many facilities use this system
for the isolation of non-Candida fungi and to perform quantitative
blood cultures.20,21 Unfortunately, contamination
has been a problem with this system. Contamination can be decreased
by careful handling and inoculation of agar under laminar flow.
++
BACTEC has demonstrated increased recovery of organisms and decreased
time to detection, particularly for Mycobacterium. BACTEC
9240 is a continuous monitoring, “noninvasive” blood
culture system that uses internal fluorescent CO2 sensors
to detect the metabolic activity of bacteria. Various types of media
have been developed for this system. BACTEC Plus Aerobic/F,
Plus Anaerobic/F, and Peds Plus/F bottles contain
resins that may decrease the effect on bacterial growth of antibiotics.
By using a differential time to positivity between a positive culture
result from a central venous catheter site and a peripheral venipuncture
site (Å 2 hours), the diagnosis of catheter-related bloodstream
infection can be confirmed.15,22 The BacT/Alert
and VersaTREK systems are two additional continuous monitoring blood
culture systems with demonstrable usefulness in the detection of
bacteremia and fungemia, both having a higher yield of recovery
and a reduction in time to detection.23
++
Older “conventional” broth bottles were incubated
at 35°C for 7 days. Optimally, two bottles were inoculated; one
was vented with a needle to yield aerobic conditions, and an unvented
one was used to maintain anaerobic conditions. Bottles were inspected
daily for macroscopic growth and subcultured blindly onto chocolate
agar after 72 hours of incubation. Acridine orange staining was
recommended after 18 to 24 hours’ incubation. If biphasic
bottles were used, they were inverted at least twice daily for the
first 2 days and then daily for 5 days. Most pediatric pathogens
were isolated within 72 hours of incubation. These systems are labor
intensive and continuous monitoring is not feasible, so most institutions
have replaced them with newer automated systems.
++
When using the continuously monitored blood culture systems,
most bacterial pathogens are detected within 48 hours.12,23-29 Rapid
detection of pathogens leads to the prompt initiation of effective
therapies; shortens hospital stays, and diminishes health care–related
expenses.30 Unfortunately, contaminants such as coagulase-negative
staphylococci (CONS) may also grow within this time frame but generally
at a slower rate; true infections with CONS are usually detected
within 15 hours, whereas contaminants usually take longer than 22
hours.31 In another study, a culture that was positive
within 18 hours was 13 times more likely to contain a pathogen than
a contaminant. The mean time to positive cultures for contaminants
was 31.1 hours.32 Incubation for more than 5 days
is not usually warranted, except in certain situations (eg, suspected
fungi, Bartonella henselae,Corynebacterium,
Actinomyces) as detailed further in DVD text.33
++
In the era of the automated blood culture system, there is no
need for prolonged incubations for Brucella species. For
the isolation of cell wall–deficient bacteria, hypertonic
medium (containing 10% sucrose or mannitol) is required.
BACTEC NR-8A and BacT/Alert could also be used. Nutritionally
deficient organisms such as Abiotrophia and Granulicatella should
be suspected when organisms are evident on Gram stain but fail to
grow when subcultured. Broth can be subcultured onto a blood agar
plate streaked with Staphylococcus aureus, or media
supplemented with 0.001% pyridoxal hydrochloride and 0.05% to
0.1% l-cysteine. A pyridoxal-impregnated
disk can also be used. Plates are usually incubated overnight at
35°C to 37°C in 5% CO2. The HACEK group of microorganisms
(Haemophilus, Actinobacillus, Cardiobacterium, Eikenella,
and Kingella), which are rare causes of bacteremia
and endocarditis, will grow within the routine incubation period for
automated blood culture systems.33,34
++
Special techniques are needed for the isolation of Bartonella species
and should only be performed by laboratories familiar with culture
and identification of the organism. Bartonella henselae,
a rickettsialike coccobacilli, is responsible for most cases of
cat-scratch disease in children. The Isolator system is used, with
subsequent inoculation onto media enriched with fresh 5% rabbit
blood (fresh Columbia blood agar, fresh chocolate agar, and buffered
charcoal yeast extract agar), and incubated in 5–7% CO2 at
35°C to 37°C for 3 weeks.
++
To ensure the prompt initiation of pathogen-specific antimicrobial
therapy, the rapid detection and identification of pathogens, along
with susceptibility data, are critical. Direct inoculation of automated
systems such as VITEK from blood culture subcultures was 22 hours
faster than controls in providing susceptibility data, and 13 hours
faster in providing identification of the pathogen.35
++
The direct detection of Staphylococcus aureus and
methicillin resistance from positive blood cultures can be a valuable
diagnostic and therapeutic advance. The StaphSR assay (BD GeneOhm,
San Diego, CA), a real-time polymerase chain reaction (PCR) assay,
was found to be 100% sensitive and 98.4% specific when
detecting methicillin-resistant S aureus.36 More
recently, the US Food and Drug Administration approved the use of
GeneXpert, a self-contained real-time PCR system for the detection
of enterovirus,37 for group B Streptococcus colonization,38 and
the use of methicillin-resistant S aureus directly
from blood cultures.
++
Germ tube tests directly from blood culture bottles to identify Candida
albicans may facilitate the transition of therapy to potentially less
toxic agents.39
++
Although clean-voided midstream urine is an acceptable specimen
for culture in older children and adults, this technique is difficult
in young children. The collection of urine by a bag fixed to the
perineum is a poor substitute (because of contamination), especially
when antibiotic therapy will be initiated after collection. In young
infants, urine obtained by bladder catheterization or by suprapubic
aspiration is preferred. A quantitative culture is required to differentiate
significant isolates from contaminants because the distal part of
the urethra is normally colonized with bacteria. A known volume
of urine (0.01–0.001 mL) is inoculated by means of a calibrated
loop or pipette onto agar media to permit quantification of isolated
colonies. Detection of Å 104 colony-forming
units (CFU)/mL of a single bacterial isolate from a clean-voided
midstream urine specimen (Å 102/mL
from a specimen obtained by catheter) correlates well with a probable urinary
tract infection. A prolonged time in ambient temperature from collection
to inoculation (>2 hours) is associated with an increase in colony
counts leading to false-positive culture results. Interpretation
of quantitation parameters in urine specimens from uncircumcised boys
when obtained by bladder catheterization or clean void should be
considered identical because both collection methods are prone to contamination.40 Any
bacterial growth from urine obtained by suprapubic aspiration is
considered clinically relevant. Isolation of multiple organisms
with low colony counts usually indicates contamination, except when
a person is unable to concentrate or retain urine or when an obstructive
uropathy is present.
++
Gram stain or acridine orange stain of unspun urine in which
at least two bacteria per high-power field of the same type are
demonstrated indicates significant bacteriuria (ie, Å 105 CFU/mL).
+++
Specimen Collection
and Processing
++
Because CSF glucose, protein, and cell counts or the isolation
of bacteria from blood culture cannot reliably exclude the presence
of meningitis, CSF must be transported to the laboratory without
delay because fluid is hypotonic and cells can lyse, thereby affecting
the cell count and contributing to a falsely abnormal biochemical
analysis (low glucose). At room temperature, cell counts (especially
neutrophils) decrease approximately 32% by 1 hour and 50% by
2 hours after collection.42 Refrigeration can render
fastidious bacteria such as Neisseria nonviable.
If a delay is expected, samples should be stored at room temperature or
incubated at 37°C. The first tube collected should be the one sent
for culture because contamination is less likely. The minimal volume acceptable
for culture of fungi and Mycobacterium is 2 mL;
10 to 15 mL is preferred.
++
Routinely, CSF should be inoculated onto sheep blood agar and
enriched chocolate agar and incubated for 4 days at 35°C to 37˚C
in 5% CO2. Some laboratories have limited the use
of enriched broth (thioglycolate) to patients with CSF shunts. The
isolation of bacteria such as coagulase-negative staphylococci from
the broth tube only (with no growth on agar plates) in patients
with no prior antimicrobial therapy almost always represents a contaminant.
Frequently, Propionibacterium acnes grows only
from the broth tube in patients with shunt infections caused by
the organism. To avoid the prolonged use of antibiotics or unnecessary
changes in antimicrobial therapy based on broth growth only, routine
use of broth tubes for all CSF specimens is not recommended.43,44
++
Centrifugation by cytospinning (2000 rpm; 350 g)
maximizes pellet formation of bacteria, producing a yield on direct
examination of CSF by Gram stain superior to that of unconcentrated
samples.45Routinely, CSF should be inoculated
onto sheep blood agar and enriched chocolate agar and incubated
for 4 days at 35°C to 37°C in 5% CO2. Centrifugation,
with Gram stain demonstrates organisms in 75% to 90% of
untreated patients with meningitis; the yield decreases to 40% to
60% in patients who have received an oral antibiotic. An
acridine orange stain, or 3,6-bis(dimethylamino)acridine, is a more
sensitive technique than Gram stain for detecting bacteria, especially
in patients who have received antimicrobial therapy.47,48 Patients
with Streptococcus pneumoniae infection are more
likely to have a positive Gram stain (90%) than are those with Neisseria
meningitidis (75%).46If
the Gram stain is positive but the culture demonstrates no growth
at 72 hours, the culture should be held for an additional 4 days.
An acridine orange stain, or 3,6-bis(dimethylamino)acridine, is
a more sensitive technique than Gram stain for detecting bacteria,
especially in patients who have received antimicrobial therapy.47,48
++
Real-time polymerase chain reaction of 16S ribosomal DNA appears
to be an ideal tool for the diagnosis of bacterial meningitis, especially in
those children pretreated with antibiotics. Multiple studies have
demonstrated its clinical usefulness, with sensitivities similar
to routine culture technology but a shorter turnaround time.49,50 The
technology is highly sensitive and rapid in the detection of pathogens
associated with CSF shunt infections such as Propionibacterium
acnes and Staphylococcus.51
++
Bacterial antigen detection (BAD) is usually not useful in the
diagnosis and management of meningitis. Its value appears to be
limited to patients with negative Gram stains, CSF, or blood cultures;
pretreated patients (antibiotics for more than 24 hours); or patients
in whom the lumbar puncture is postponed because of the severity
of illness.46,52
++
Although not yet routinely recommended, in one study the inoculation
of BACTEC blood culture systems with CSF led to the rapid isolation
of pathogens such as Streptococcus pneumoniae.53
++
Throat swabs for the detection of Streptococcus pyogenes (group
A streptococcus) is the most common respiratory tract specimen sent
for culture. Proper collection of the sample by swabbing the tonsillar surface,
posterior pharyngeal wall, and opposite tonsillar surface, while
avoiding the tongue and saliva, affects the yield of the culture. The
isolation and reporting of “all” oropharyngeal
organisms from a throat swab is of limited clinical value and may
lead to the treatment of nonpathogenic bacterial in persons with
nonbacterial causes of pharyngitis. An exception would be the young
child with cystic fibrosis in whom the isolation of Pseudomonas
aeruginosa from a deep pharyngeal culture may be clinically
relevant.
+++
Rapid Tests
for Group a, Beta-Hemolytic Streptococci
++
Routine culture on agar plates requires 24 to 48 hours to detect
group A streptococci and may result in a delay in therapy for some patients.
Rapid detection assays for streptococcal antigen may yield results
in 10 to 70 minutes. The specificity of various tests is 62% to
100%, but the sensitivity is lower (52–99%).54 Results
of rapid antigen tests are influenced by the skill, experience,
and expertise of the person obtaining the throat swab and performing
the assay. Although initial studies performed in microbiology laboratories
demonstrated a higher sensitivity for most antigen detection assays,
performance in other settings has shown variable results. Rapid
antigen tests using optical immunoassay (OIA) have consistently
demonstrated sensitivities and specificities higher than other assays.
++
A culture should be obtained from all individuals with a negative
rapid antigen test.55 Although the sensitivities
of rapid antigen assay increase with severity of illness, the sensitivity
of many assays is low enough to merit culture.56 Positive
tests do not require confirmation or verification by culture.57 In
addition, the isolation of Streptococcus pyogenes on
an agar plate allows for the antimicrobial susceptibility testing
for macrolide resistance.
++
New technology using chemiluminescent single-stranded DNA probes
and one-rapid-cycle real-time polymerase chain reaction have demonstrated
high specificity and good sensitivity. Unfortunately, the tests
are difficult to perform, costly, and not suitable as point-of-care
tests.58-60
++
Antigen detection assays have been used to detect the presence
of group A streptococci, such as in pyogenic arthritis, cellulitis,
and parapneumonic effusions, at extrapharyngeal sites. Abbott TestPack
Plus Signify Strep Atest (Abbott Laboratories) and Directigen 1-2-3
(BD Biosciences) have shown high sensitivity when testing synovial
fluid, soft tissue aspirate, and pleural fluid.61 Specificity
rates were also high (99–100%).62
++
Tympanocentesis and sinus aspiration for culture are extremely
useful in special situations (eg, immunocompromised patients, patients
with intracranial complications, and those who fail to respond to
antimicrobial therapy). Data are conflicting regarding the validity
of culture of the nasopharynx in predicting the pathogens of sinusitis
and otitis media, and routine use is not indicated.
++
Collection of sputum from children with lower respiratory tract
infections is technically difficult. Aspiration of deep pharyngeal/tracheal
secretions (with a Luken trap) is used by many. In older children,
sputum can be a valuable specimen. The presence of 10 or more squamous
epithelial cells per low-power field is highly suggestive of oropharyngeal
contamination, and the specimen should not be processed. Conversely,
the presence of more than 25 white blood cells per low-power field
denotes an adequate specimen. Gram stain should be used to aid in
the interpretation of isolates from culture.
++
Transtracheal aspirates are technically difficult to obtain in
young children and are seldom performed. Bronchoscopy, especially
a quantitativebronchoalveolar lavage specimen or protected brush,
is extremely useful in the diagnosis of Pneumocystis jiroveci and
mycobacterial, fungal, and bacterial infections. Isolation of Å 104 CFU/mL
is predictive of the pathogen of infection, as proved by biopsy.63-65 More
studies in children are needed.
++
Isolation of organisms belonging to the Burkholderia
cepacia complex from the sputum of patients with cystic
fibrosis requires the use of selective media. Either B cepacia selective
agar (BCSA) or oxidative-fermentative base–polymyxin B–bacitracin–lactose
(OFPBL) medium can be used.66,67 BCSA appears to
be superior to OFPBL in its ability to support the growth of B
cepacia complex. Genovar identification and classification
require PCR-based assays performed in laboratories specializing
in this type of molecular typing.
++
Currently available diagnostic tests for Bordetella pertussis have
variable sensitivity, depending on the stage of the disease, level
of immunization, adequacy of collection and transport, and diagnostic
method.68 When properly performed, culture is superior
to direct immunofluorescence assay (DFA) on nasopharyngeal secretions. Serologic
tests by enzyme immunoassay (EIA) (B pertussis–specific
immunoglobulin IgA, IgG, and IgM) are sensitive but difficult to
interpret and require acute and convalescent sera. PCR assays may provide
higher sensitivity with a quicker turnaround than culture.69,70 A
combination of culture, serology, and PCR provide a greater sensitivity
when performed on individuals with cough illness.68,71,72
+++
Stool and Other Gastrointestinal Secretions
+++
Bacterial Enteric
Pathogens
++
Most children with diarrhea have an uncomplicated course that
resolves within days without antimicrobial therapy. These children
rarely require laboratory testing.Infants and children with fever and/or
bloody diarrhea may require specific antibacterial therapy or need
close monitoring for complications such as hemolytic uremic syndrome.
Stool specimens are submitted to laboratories for the routine isolation
of Salmonella, Shigella, Campylobacter,
and Escherichia coli O157:H7. When clinically and epidemiologically
indicated, the isolation of other bacterial causes such as Plesiomonas, Aeromonas, Yersinia,
and enteroaggregative E coli can be attempted.
Stool specimens can also be examined for the presence of shigalike toxin
from non-O157:H7 enterohemorrhagic E coli isolates.
++
Stool
is inoculated onto various selective and differential agar media
to facilitate the recovery and identification of enteric pathogens.
++
The
routine identification of all aerobic organisms isolated from stool
specimens is of no clinical utility and results in wasted technologist
time and hospital charges. At times, clinicians may inappropriately
treat Pseudomonas aeruginosa or Candida
albicans because it was isolated as a predominant organism,
but the isolation of either organism usually reflects prior antibiotic
use.
++
The yield of stool cultures (and examinations for ova and
parasites) in persons with onset of diarrhea more than 3 days after
hospitalization is extremely low (< 1%). In these patients, nosocomial
pathogens such as rotavirus, Clostridium difficile,
and norovirus are more likely. Institutions should educate clinicians not
to order “routine” stool cultures on these patients,
or the laboratory should implement rejection criteria where the
test would not be performed if ordered. These strategies result in
significant financial savings to the institution and shortened hospital
stays, as well as eliminating the wait for laboratory results of limited
clinical value.44,73
+++
Clostridium
Difficile
++
In most laboratories, testing for Clostridium difficile toxin
consists of a two-step algorithm for toxin detection. Stool is initially
screened by an immunoassay for C difficile glutamate
dehydrogenase antigen (C. DIFF CHEK-60, TechLab). Results are usually
available within 1 hour. If this test is negative, no further testing
is merited. Screen-positive specimens undergo toxin detection using
cell culture cytotoxicity neutralization assay or a rapid toxin
A/B assay.74,75 Culture, with detection
of toxin production by C difficile isolates, is
another technique for identification of C difficile disease.
Recovery of the organism has the added advantage of allowing further
characterization of the isolate for pathogenic mechanisms such as
binary toxin production. The use of this algorithm results in a
decrease in the empiric use of antibiotics such as metronidazole
and oral vancomycin by several days while waiting for test results.
Colonization of the gastrointestinal tract of young infants with C
difficile is common and disease is unlikely because enterocytes
in young children are not susceptible to toxin. Stool specimens
from young infants, especially those younger than 6 months, need not
be routinely tested for C difficile.76
++
Multiple tests are available for the diagnosis
of Helicobacter pylori. Histologic examination
of tissue stained with Warthin-Starry silver and hematoxylin-eosin stains is
still considered one of the gold standards.77In
patients with suspected treatment failure for whom antimicrobial
susceptibility testing is desirable, Isolation of the organism can
also be attempted. For culture, commercially available Brucella
agar plates with 5% horse blood or brain heart infusion
(BHI) agar supplemented with 7% horse blood is the preferred
medium. Biopsy samples can be tested for the presence of H
pylori by testing for urease. The 13C-labeled
urea breath test and serology have been advocated as noninvasive methods
of diagnosing H pylori disease in children because
neither test requires endoscopy. The breath test requires some degree
of cooperation for 60 minutes. This assay is highly sensitive and
specific. In addition, it appears to be less costly than other tests.78-80 An antigen
assay performed on stool has now replaced the urea breath test at
many institutions.81 Detection of specific antibodies
may not differentiate between acute or past infection, and detection
of specific IgM antibody is not sufficiently sensitive to be useful.82 Although
PCR may be a promising diagnostic tool for H pylori infection,
the rapid urease testing and histologic examination provide reliable
reproducible results. PCR may be too expensive for routine clinical
use. Gastric aspiration provides a useful specimen and avoids endoscopy.83
+++
Synovial and Peritoneal
Fluid
++
Inoculation of synovial fluid and peritoneal fluid into BACTEC
blood culture bottles results in a higher recovery rate of bacteria
than with conventional media. In one study, Kingella kingae,
a cause of septic arthritis, was exclusively detected in specimens
inoculated into BACTEC bottles.3 Investigators
from France found K kingae to be the leading cause of
osteoarticular infections in children at their institution by using
a PCR for 16S rDNA directly on osteoarticular specimens.84 In
patients with spontaneous bacterial peritonitis, inoculation of
peritoneal fluid into Bactec bottles more than doubled the recovery
of gram-negative bacteria.85 Although none of the
fluids inoculated onto conventional media yielded streptococci and
enterococci, 33% of those inoculated into Bactec bottles
grew the organisms. Similar findings have been demonstrated in patients
with peritonitis complicating continuous ambulatory peritoneal dialysis.86,87
+++
Antimicrobial Susceptibility Testing
++
A microorganism is considered to be susceptible to an antimicrobial
agent if in vitro growth is inhibited at a concentration
one fourth to one eighth of that achievable in the patient’s blood,
given a usual dose of the agent. In vitro resistance
is highly predictive of clinical treatment failure. However, in
vitro susceptibility does not ensure clinical efficacy.
The site of infection and accessibility by the antimicrobial agent,
protein binding, route of administration, immune status of the host,
and presence of an abscess requiring drainage also influence outcomes. Organisms
warrant testing if susceptibility cannot be reliably predicted.
Susceptibility testing of Streptococus pyogenes is seldom
necessary because of its predictable antibiogram, whereas testing
for Pseudomonas aeruginosa is always indicated.
The test results of certain antimicrobial agents are reported routinely,
but other results are reported selectively to gain information about
individual and community susceptibility patterns, as well as to
control inappropriate use of certain agents, limit cost, and prevent
the emergence of resistant bacteria.
+++
Standard Media
and Test Conditions
++
A variety of methods and media have been developed for susceptibility
testing and standards have been set for testing and interpretation
of results by the Clinical and Laboratory Standards Institute (CLSI).
Methods and interpretation are pathogen specific.
++
Disk diffusion, or the Kirby-Bauer test, is a standardized technique
for testing rapidly growing pathogens.88 An inoculum
is prepared by direct suspension of colonies to yield a standardized
inoculum that is swabbed onto the surface of a Mueller-Hinton agar
plate. Reproducibility depends on the log-growth phase of organisms;
therefore, fresh subcultures are always required. Filter paper disks
impregnated with a standardized concentration of an antimicrobial agent
are placed on the surface, and the size of the zone of inhibition
around the disk is measured after overnight incubation. Classification
of zone diameters into susceptible, intermediate, or resistant categories
is accomplished by following CLSI interpretive guideline tables.
++
Another commonly employed susceptibility method is broth microdilution
(MIC).93 This system allows a quantitative measurement
of in vitro activity. The use of concentrations
around the breakpoint MICs for resistance allows testing of more
agents with fewer wells. Usually, five to eight concentrations representing
therapeutically achievable ranges are tested against each organism,
or one to three concentrations are used to determine activity at
the breakpoint MIC. Because the exact MIC is not known, only qualitative
assessment of susceptibility is determined. One must be careful
with the interpretation of these values because they apply to a
specific organism, the likely site of infection, the concentration
of the organism at the site, and the type of antimicrobial agent
being used.
++
The E-test (AB Biodisk) is a method that integrates disk diffusion
to determine an actual MIC value and that provides accurate, reproducible
results. It also allows a simple methodology for testing of anaerobes99 and
fastidious bacteria.100-102 An impervious inert
strip carries a marked, continuous concentration gradient of a predefined
antibiotic consisting of more than 15 twofold dilutions. After incubation
on seeded agar, the MIC is read at the edge of the zone of inhibition
as it intersects the strip. There is good agreement between disk diffusion,
agar dilution, and broth microdilution.103 This
technique is especially useful for testing the susceptibility of Streptococcus pneumoniae to
penicillin.104-107
++
When interpreting MIC results, lower MIC values for a specific
antimicrobial agent do not imply that the agent is more active than
one with a higher value when both are considered susceptible for
that organism. Because these MIC values will differ between organisms
and antimicrobial agents, comparisons deserve caution.
++
Automated antimicrobial susceptibility testing can provide results
in 2 to 18 hours.114 Systems commonly used in the
United States are the BioMerieux VITEK2 System, the Dade MicroScan
WalkAway, and the BD Phoenix system. The VITEK2 system may provide identification
as early as 2 hours (susceptibility as early as 6 hours) after inoculation.
Plastic reagent cards containing small wells or microcuvettes allow
the simultaneous testing for many different antimicrobial agents.
Growth is detected by means of a densitometer. It uses turbidimetrically
determined kinetic measurements of growth to compute MIC values
by regression analysis. The Dade MicroScan WalkAway system can provide
results between 4.5 and 18 hours of incubation. The BD Phoenix System
can accommodate up to 100 simultaneous tests. An indicator is added
to the broth of each isolate, and redox indicator measures bacterial
growth. Reliable results can be available after approximately 10
hours of incubation.115,116Advantages of automated systems
are that they can be connected to the laboratory computer system,
provide rapid test results, allow intralaboratory and interlaboratory
standardization, are less labor intensive, and have the potential
of artificial intelligence for data review. Systems can also be
too restrictive for some laboratories. Panels and cards are formatted
by the manufacturer with predetermined antimicrobial agents. These
may not match the hospital pharmacy formulary. Systems are not appropriate
for all organisms; unacceptable results (usually falsely susceptible
test results) can occur for fastidious bacteria such as P
aeruginosa, S pneumoniae, enterococci, Stenotrophomonas
maltophilia, and coagulase-negative staphylococci.114 The
initial capital investment may be prohibitive for some facilities,
and a backup system is always needed in case of system failure.117,118
+++
Specific Testing Methods
++
Testing of staphylococci for methicillin (oxacillin) resistance
is best achieved by detection of the mecA gene
by real-time polymerase chain reaction (PCR) or its product, penicillin-binding
protein (PBP) 2a by latex agglutination (LA). Both tests demonstrate
a 100% correlation.89,90 The LA test requires approximately
15 to 20 minutes to perform. Real-time PCR for mecA
gene can rapidly detect methicillin-resistant Staphylococcus
aureus directly from positive blood culture bottles with
a high degree of sensitivity and specificity.36,91 For
smaller laboratories, a Kirby-Bauer test using a cefoxitin disk
is a simple and inexpensive method for detecting methicillin resistance.
Disk diffusion is still useful in testing staphylococcal isolates.
Testing can be performed on TMP-SMX and other antimicrobials commonly
used for treatment of MRSA infections that may not be available
on automated system panels. Detection of inducible macrolide-lincosamide-streptogramin
B resistance in S aureus isolates (D-test) is easily
performed using disk diffusion.92
++
Inhibitory zones < 20 mm require confirmation using minimal
inhibitory concentration (MIC) methodology. Because penicillin-intermediate
and penicillin-resistant strains are common, blood and CSF isolates
should be tested directly by MIC methods.
++
An oxacillin disk (1-μg disk on Mueller-Hinton
blood agar) can be used as a screening test for penicillin resistance
of Streptococcus pneumoniae. An inhibition zone
of Å 20 mm is suggestive of β-lactam susceptibility. β-lactamase
production is the most frequent mechanism of resistance with Haemophilus species, whereas
ampicillin resistance mediated by an alteration in protein binding
is relatively uncommon and requires MIC or disk diffusion testing
for detection.
++
The extended-spectrum β-lactamases (ESBLs) produced
by certain strains of Klebsiella species and Escherichia
coli have been responsible for treatment failures with
extended-spectrum cephalosporins. ESBL-producing isolates are resistant
to all penicillins and cephalosporins, as well as aztreonam. ESBLs
have also been detected in isolates of Salmonella, Pseudomonas
aeruginosa, Enterobacter, and Citrobacter. Currently,
no reliable test is available for detecting ESBLs in organisms other than Klebsiella species, E
coli, and Proteus mirabilis.
ESBL-producing isolates can be detected by testing specific ESBL
screening drugs and confirmatory testing using cefotaxime and ceftazidime
with and without clavulanic acid.94
++
Routine testing of Neisseria gonorrhoeae isolates
is limited to detection of β-lactamase. Any other
testing should be referred to public health or other specialty laboratories.95
++
Testing of β-lactam agents requires a strictly
standardized inoculum (5 × 105 CFU/mL for
MIC and 108 CFU/mL for disk diffusion testing);
an increased inoculum falsely increases the MIC.96
++
Carbapenemases in Klebsiella pneumoniae (KPC)
are now being reported more frequently in parts of the United States.
These are difficult to detect, and automated systems may fail to
consistently detect their presence. Clinical failures with imipenem,
meropenem, and ertapenem may suggest the presence of KPC.97
++
Testing of enterococci for high-level resistance to gentamicin
and streptomycin is useful to predict synergistic bacterial killing
when combined with a β-lactam or vancomycin. Gentamicin-
and streptomycin-containing agar or MIC wells (500 µg/mL
gentamicin, 2000 μg/mL streptomycin) is
used to detect high-level resistance.98 If high-level
resistance to gentamicin is noted, resistance to other aminoglycosides
is predictable. High-level resistance to streptomycin is specific
only to this agent. Incubation for a full 24 hours is necessary
(48 hours for streptomycin).
++
Testing for drug resistance among isolates of Streptococcus
pneumoniae, enterococci, and Staphylococcus aureus deserves
special mention. Most laboratories use a 1-μg oxacillin
disk screening test to detect penicillin resistance among pneumococci
as described previously. Penicillin MIC tests are then performed
on any zone diameter of ⩽19 mm to determine if the isolate is indeed
resistant. All CSF and blood S pneumoniae isolates
should bypass the disk screening test and have MIC tests performed
initially. Penicillin, cefotaxime or ceftriaxone, meropenem, and
vancomycin should be routinely tested on these isolates. Interpretive
criteria differ for cefotaxime, ceftriaxone, and penicillin for
patients with meningitis and nonmeningitis. The laboratory rarely
knows the clinical condition of the patient, so they will report
both interpretations for each drug for non-CSF isolates.
++
Many susceptibility methods have problems detecting low-level
vancomycin resistance among enterococci. Disk diffusion testing
of vancomycin with incubation for at least 24 hours, using an agar
dilution plate with brain heart infusion (BHI) agar with 6 μg of
vancomycin per mL (vancomycin agar screen plate).108Vancomycin
agar screen plate can also be used to detect vancomycin resistance
in S aureus, which is commonly missed by disk diffusion
and automated systems.
++
Despite the standardization of susceptibility testing by the
Clinical and Laboratory Standards Institute and other professional
organizations for common pathogens, there are many organisms that
can cause serious infections in which methods are still not standardized
and interpretative guidelines are not available. The choice of antimicrobial
therapy for these infections is based on clinical experience published
in the medical literature rather than on in vitro susceptibility
testing.
++
Susceptibility testing of anaerobic bacteria should be limited
to laboratories with special qualifications.109 Testing
is indicated when failure of the usual therapeutic regimens has occurred
or when the severe infection requiring long-term therapy is anticipated
(eg, for brain abscesses, osteomyelitis, endocarditis, or refractory
bacteremia). Agar dilution testing is the referenced standard for
determination of MICs, but E-test methodology is more widely used
by clinical laboratories.99
++
Testing of fastidious bacteria should be limited to a few specialized
laboratories. For mycobacteria, an agar dilution test has been standard
for testing. A standardized concentration of test organism is inoculated
onto agar (ie, Middlebrook 7H10) containing the antimicrobial agent
to be tested. After incubation for up to 3 weeks (35°C, CO2),
susceptibility is determined by comparing growth on the antibiotic-containing
medium with that on antibiotic-free medium.
++
The BACTEC 460TB radiometric system provided a major advance
in the susceptibility testing of slow-growing Mycobacterium species.
BACTEC 12B or Middlebrook 7H12 broth containing an antimicrobial
agent was inoculated with the test organism and incubated for 5
days at 37°C. The growth index was compared with that of a control
bottle containing no antimicrobial agent.110An
automated nonradiometric system, BACTEC MGIT 960, appears to be
as reliable as older methods.111-113