Diagnostic Microbiology and Infectious Disease 36 (2000) 159 –162

Comparison of Gen-Probe AccuProbe Group B streptococcus culture

identification test with conventional culture for the detection of Group
B streptococci in broth cultures of vaginal-anorectal specimens from
pregnant women夞
Natalie Williams-Bouyer*, Barbara S. Reisner, Gail L. Woods
University of Texas Medical Branch at Galveston, Department of Pathology, 301 University Boulevard, Galveston, TX 77555-0740, USA

Received 3 August 1999; accepted 9 November 1999

Abstract

The performance of the AccuProbe Group B Streptococcus Culture Identification Test (Gen-Probe Incorporated, San Diego, CA, USA)
for the detection of group B streptococci (GBS) directly from LIM broth cultures of vaginal-anorectal swab specimens from pregnant women
(two swabs per patient in most cases) was evaluated by comparing results to those of conventional GBS culture. Of 411 specimens analyzed,
82 were positive and 312 were negative for GBS by both methods. After initial testing, the percent agreement was 95.9%. The initial
sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 90.1%, 97.5%, 91.1%, and 97.2%,
respectively. Results were discrepant for 17 specimens: eight were GBS positive by probe and negative by culture; nine were negative by
probe and positive by culture. To resolve discrepancies, culture plates were re-examined for GBS colonies, AccuProbe testing was repeated
on the initial LIM broth cultures, and the second swab (if received) was inoculated to LIM broth for AccuProbe testing after overnight
incubation. After discrepant resolution testing, the percent agreement between the two test methods was 97.8%. The final sensitivity,
specificity, and positive and negative predictive values for the AccuProbe test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively. These
data suggest that the AccuProbe test is a reliable method for detecting GBS in vaginal-anorectal specimens, providing results more rapidly
than conventional culture. However, strict adherence to the manufacturer’s test protocol is necessary to limit technical errors. © 2000
Elsevier Science Inc. All rights reserved.

Keywords: Group B streptococcus; Broth cultures; Vaginal-anorectal specimens; Intrapartum antibiotics

1. Introduction
Streptococcus agalactiae, or group B streptococcus
(GBS), is an important cause of neonatal meningitis, sepsis,
and pneumonia. Disease can occur as early-onset (⬍7 days
of age), late-onset (7 days-3 months of age), or late-late-
onset (⬎90 days of age) (Baker, 1997). Infants born to
women colonized with GBS in the genital or rectal area are
at an increased risk for developing GBS disease. Prevention
of early-onset GBS infection through the use of intrapartum
antibiotics has been demonstrated (Allen et al., 1993; Boyer
夞 Data from this article were presented in part at the 99th General
Meeting of the American Society for Microbiology, Chicago, Illinois, May
30 –June 3 [poster c-24]
* Corresponding author. Tel.: 1-409-772-2173; fax: ⫹1-409-772-5683.
E-mail address: nmwillia@utmb.edu (N. Williams-Bouyer).
and Gotoff, 1986). However, the use of this type of therapy
has not been shown to prevent late- or late-late-onset of
disease. The Centers for Disease Control and Prevention
(CDC) recommends two approaches to identify mothers
who should receive intrapartum antibiotics to prevent early-
onset GBS disease (Centers for Disease Control, 1996): 1)
identification of important risk factors, including previous
delivery of an infant with invasive GBS disease, GBS bac-
teriuria during the pregnancy, delivery at ⬍37 weeks’ ges-
tation, duration of ruptured membranes ⱖ18 hours, and
intrapartum temperature ⱖ100.4°F; and 2) performance of
routine cultures of vaginal-rectal swab specimens of women
at 35–37 weeks gestation to detect GBS colonization. To
enhance the recovery of GBS, it is recommended that spec-
imens be inoculated to a selective broth medium that is then
subcultured overnight to blood agar plates. Solid media are
then incubated an additional 24 to 48 hours. There are

0732-8893/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 7 3 2 - 8 8 9 3 ( 9 9 ) 0 0 1 4 6 - 7
160 N. Williams-Bouyer et al. / Diagnostic Microbiology and Infectious Disease 21 (2000) 159 –162
several acceptable methods to identify suspicious colonies
of GBS. One of these, the Accuprobe nucleic acid probe,
can be used both to test colonies growing on a plate or an
aliquot of broth from a liquid culture medium. Bourbeau et
al. (1997) found that the sensitivity and specificity of the
AccuProbe assay for direct detection of GBS in broth cul-
tures of 502 specimens were 94.7% and 99.5%, respec-
tively, suggesting that the test could eliminate the need to
subculture the broth and incubate plates an additional 2
days. However, to our knowledge, the findings of Bourbeau
et al. (1997) have not been confirmed by other investigators.
To evaluate the use of the probe at our institution, we
compared the results of the traditional GBS culture per-
formed essentially according to the CDC guidelines to those
of the Accuprobe assay tested on 24-hour broth cultures.
2. Materials and Methods
2.1. Specimens
For the majority of patients included in this study, two
vaginal-anorectal swab specimens (Culterette II brand
swabs, BBL, Cockeysville, MD, USA) were obtained from
pregnant women receiving care at the University of Texas
Medical Branch. Occasionally, only one swab was submit-
ted for testing. Specimens were transported to the laboratory
at ambient temperature. One swab was used to inoculate one
Columbia agar plate with 5% sheep blood supplemented
with nalidixic acid and colistin (CNA), and then immersed
for at least 1 min. in a 3-ml tube of LIM broth (Todd Hewitt
broth supplemented with 10 ␮g/ml nalidixic acid, 15 ␮g/ml
colistin, and 10 mg/ml yeast extract). The tube was vor-
texed, and the swab expressed on the inside of the tube and
discarded. Plated and tubed media (all obtained from BBL)
were incubated at 35° C in 5% CO2. When available, the
second swab was stored at refrigerator temperature for up to
3 days for additional testing to resolve discrepancies be-
tween Accuprobe Group B streptococcus culture identifica-
tion test and culture results.
2.2. Culture and identification
After 18- to 24-h incubation at 35° C in 5% CO2, primary
CNA plates were examined for beta- and gamma-hemolytic
colonies resembling GBS, and LIM broth cultures were
subcultured to a blood agar plate (BAP). BAPs were incu-
bated overnight and examined for suspect colonies with the
morphologic appearance of GBS. Primary CNA and sub-
culture plates that were negative for GBS were incubated
for an additional 24 h and re-examined. Suspect colonies
were gram-stained, and gram-positive cocci resembling
streptococci were tested with Strep B grouping latex reagent
(PathoDx Strep grouping kit, Diagnostic Products Corpora-
tion, Los Angeles, CA, USA).
2.3. Nucleic acid probe
LIM broth cultures were vortexed, and 50 ␮l of the
well-mixed suspensions were transferred into Probe Re-
agent Tubes. When used for culture confirmation, three to
four suspect GBS colonies were transferred to a Probe
Reagent Tube, suspended in 50 ␮l of lysis reagent, and
thoroughly mixed. Testing was performed according to
manufacturer’s directions. Results were interpreted as fol-
lows: ⬍40,000 relative light units (RLU), negative;
ⱖ50,000 RLUs, positive; 40,000 – 49,999 RLUs, indetermi-
nate. Testing was repeated for specimens with results in the
indeterminate range, as specified by the manufacturer. S.
agalactiae ATCC 13813 (positive control) and S. bovis,
ATCC 33317 (negative control) were included in each test
run.
2.4. Discrepant analysis
For those specimens with discrepant results, the follow-
ing were performed: 1) culture plates were re-examined for
possible GBS colonies, and if present, these colonies were
subcultured to a blood agar plate and then tested by Accu-
Probe, and 2) the AccuProbe test was repeated on LIM broth
cultures from initial swab specimens that had been main-
tained at room temperature for up to 48 h. If the discrepancy
remained unresolved, the second swab specimen, if avail-
able, was inoculated to LIM broth, which was tested by
probe after overnight incubation.
2.5. Statistical methods
Percent agreement, sensitivity, specificity, and positive
and negative predictive values were calculated according to
standard formulas (Dawson-Saunders and Trapp, 1994).
3. Results
A total of 414 specimens from 407 patients were evalu-
ated. Upon testing LIM broth cultures by AccuProbe, 90
specimens were positive for GBS, 321 were negative, and
three gave indeterminate results. Only one of the latter three
specimens (48,671 RLU) was available for repeat testing
(the other two were inadvertently discarded). The repeat
result for this specimen, which was negative for GBS by
culture, also was indeterminate (49,553 RLU). Because the
second AccuProbe result for this specimen was indetermi-
nate and the other two with indeterminate results could not
be re-tested, these three specimens were excluded from the
final analysis.
Of the 411 specimens (404 patients) which were evalu-
ated, 91 were culture-positive for GBS. For 64 of the 91
(70.3%), GBS was detected on inspection of primary CNA
plates after overnight incubation, whereas 27 (29.7%)
showed growth of GBS only upon subculture of the LIM
 N. Williams-Bouyer et al. / Diagnostic Microbiology and Infectious Disease 21 (2000) 159 –162 161

Table 1
Summary of probe and culture results for samples with discrepancies after initial testing

Parameter Specimen RLU value (interpretation) for:

no.
1st LIM broth Repeat of 1st Probe result of suspect 2nd LIM brothb
LIM broth GBS coloniesa

Probe (⫹)/Culture (⫺) 1 80772 (⫹) 107790 (⫹) NC 42920 (I)

2 402302 (⫹) 14810 (⫺) 10351 (⫺) —
3 79674 (⫹) 38794 (⫺) NC —
4 400826 (⫹) 14549 (⫺) 12162 (⫺) —
5 62896 (⫹) 338029 (⫹) 10747 (⫺) 8101 (⫺)
6 666341 (⫹) 506566 (⫹) 19463 (⫺) NA
7 72259 (⫹) 50938 (⫹) 12983 (⫺) NA
8 52620 (⫹) 54758 (⫹) NC 13555 (⫺)
Probe (⫺)/Culture (⫹)
9 26517 (⫺) 66170 (⫹) NC —
10 38063 (⫺) 45550 (I) 340436 (⫹) 395150 (⫹)
11 10110 (⫺) 11172 (⫺) 9010 (⫺) 500 (⫺)
12 8056 (⫺) 14867 (⫺) 8983 (⫺) 3322 (⫺)
13 24163 (⫺) 27554 (⫺) NC 435855 (⫹)
14 33336 (⫺) 228628 (⫹) NC —
15 35758 (⫺) 6825 (⫺) NC 14629 (⫺)
16 23830 (⫺) 40390 (I) NC 14036 (⫺)
17 19578 (⫺) 16346 (⫺) NC 643770 (⫹)

RLU ⫽ relative light units (⬍40,000 RLUs, negative [⫺]; ⱖ50,000 RLUs, positive [⫹]; 40,000 – 49,999 RLUs, indeterminate [I]); NA ⫽ only one swab
received; NC ⫽ no colonies resembling GBS.
a
Primary CNA and subculture plates were examined for suspect colonies with the morphologic appearance of GBS, to confirm culture results.
b
A second swab specimen was inoculated to a LIM broth only if a discrepancy between conventional culture and AccuProbe testing of the initial LIM
broth remained.

broth. Culture and AccuProbe results agreed for 394 spec-
imens: 82 were positive and 312 were negative for GBS by
both methods (95.9% agreement). Thus, the initial sensitiv-
ity, specificity, and positive and negative predictive values
for the AccuProbe test were 90.1%, 97.5%, 91.1%, and
97.2%, respectively.
Discrepant analysis of the 17 specimens with discordant
Gen-Probe and culture results (eight positive for GBS by
probe and negative by culture; nine negative by probe and
positive by culture) is summarized in Table 1. Table 2
describes the assessment of initial AccuProbe test results
after discrepant analysis. Re-examination of culture plates
and AccuProbe testing of suspect colonies did not change
initial culture results. For three of the eight specimens that
were GBS positive by probe and negative by culture, repeat
testing of the initial LIM broth by AccuProbe gave a neg-
ative result, thus agreeing with culture. Two of the nine
specimens that were originally GBS negative by probe and
positive by culture were positive by Accuprobe retesting of
the initial LIM broth (AccuProbe sensitivity, specificity, and
positive and negative predictive value were 92.3%, 98.4%,
94.4% and 97.8%, respectively). If results were not resolved

Table 2
Assessment of initial AccuProbe testing

Specimen Interpretation of initial Rationale

no. test result

1 Probable false-positive probe Second LIM broth RLU is indeterminate and no suspect GBS colonies present.
2 False-positive probe Repeat of first LIM broth (⫺) and result of probe of suspect GBS colonies (⫺).
3 False-positive probe Repeat of first LIM broth (⫺) and result of second LIM broth (⫺).
4 Probable false-positive probe Probe of suspect colonies (⫺).
5 False-positive probe Second LIM broth (⫺) and probe of suspect colonies (⫺).
6/7 Probable false-positive probe Probe of suspect colonies (⫺), although repeat of first LIM (⫹).
8 Probable false-positive probe Second LIM broth (⫺) and no suspect colonies present.
9 False-negative probe Repeat of 1st LIM broth.
10 False-negative probe Second LIM broth (⫹) and probe of suspect GBS colonies (⫹).
11/12 True-negative probe Probe of suspect colonies (⫺).
13/17 False-negative probe Second LIM broth (⫹).
14 Probable false-negative probe Second LIM broth (⫹).
15/16 Probable false-negative probe Second LIM broth (⫺); no suspect colonies were available to confirm initial culture result.
162 N. Williams-Bouyer et al. / Diagnostic Microbiology and Infectious Disease 21 (2000) 159 –162
by re-testing the initial LIM broth, a second swab specimen
(available for 10 of 12 patients) was inoculated to LIM
broth and tested by probe after overnight incubation. For
three of these 10 that were initially GBS negative by probe
and positive by culture, the AccuProbe result for the second
LIM broth culture was positive. Based on the resolution of
these discrepancies, 402 test results were in agreement; 87
(21.6%) specimens were positive for GBS, and 315 (78.4%)
were negative for GBS. The percent agreement between test
methods was 97.8%. The final sensitivity, specificity, and
positive and negative predictive values for the AccuProbe
test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively.
4. Discussion
Results of our evaluation of using AccuProbe for detec-
tion of GBS in LIM broth cultures differ somewhat from
those of Bourbeau et al. (1997), which to our knowledge is
the only other published study in which the probe was used
for this purpose. After initial testing in our hands, the
performance of the AccuProbe GBS test (sensitivity, 90.1%;
specificity, 97.5%) appeared to be lower than it had been in
the previous evaluation [sensitivity, 94.7%; specificity,
99.5%]. Results of our discrepant analysis, however, sug-
gest that many of the discordant probe results most likely
were due to technical error. The fact that most of the
discrepancies occurred early in the study tends to support
this hypothesis. For example, insufficient mixing of the
initial broth specimen could cause a false negative result.
Inadequate vortexing of the tested aliquot before the selec-
tion step, on the other hand, may be responsible for false
positive probe results. For two of our false positive speci-
mens, however, the RLU values were quite high, which is
puzzling. Suppression of growth of GBS by Enterococcus
faecalis has been described (Dunne and Holland-Staley,
1998) and theoretically, is possible for these specimens.
Unfortunately, the presence or absence of E. faecalis on the
corresponding culture plates was not recorded. In addition
to technical error, a few of our initially false negative results
apparently were due to sampling error (i.e., GBS present
only on one swab), based on a positive probe result on the
broth culture of the second swab. False-negative probe re-
sults also could have been due to the presence of low
numbers of GBS in the specimen, as previously shown by
Bourbeau et al. (1997); however, we cannot document this,
because quantitation was not performed.
To determine the potential impact of our false negative
and false positive probe results, the medical records of the
involved patients were reviewed. For all patients, the
screening was performed at delivery, and culture results
were not available until after the patient was discharged
from the hospital. In no case was review of the culture result
acknowledged. For those patients who had positive cultures,
the information available was insufficient to assess outcome
of the infant.
With regard to cost, use of the AccuProbe for direct
detection of GBS in broth cultures is most cost effective for
laboratories that do a relatively large volume of GBS
screening tests. In 1996, Bourbeau et al. (1997) determined
that the cost of the AccuProbe ($5.16) was $0.25 less than
a positive GBS culture and about $1.50 more than a nega-
tive GBS culture. Because the costs are dependent on vol-
ume (of both screens for GBS and number of other Gen-
Probe products used by the laboratory [which will influence
the ultimate cost of the AccuProbe kit]), method used to
identify GBS, and percent positive cultures, performing a
cost analysis that would be relevant to all laboratories is not
possible. Each laboratory must determine cost based on
their data.
In summary, AccuProbe is a reliable method for direct
detection of GBS in broth cultures if testing personnel are
adequately trained and pay close attention to detail when
performing the test. In our opinion, use of the test in this
manner is most likely to be implemented for the purpose of
increasing labor efficiency in laboratories with a high vol-
ume of screens for GBS. Our data suggest that for optimal
sensitivity, if two swab specimens are received, both should
be inoculated to the selective broth. To optimize specificity,
if the initial RLU value is between 50,000 and 100,000,
reporting indeterminate or a preliminary result of positive
and subculturing the broth to a solid medium for confirma-
tion should be considered.
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