Culture-Based Method with Performance Comparable to That of PCR-Based Methods for Detection of Group B Streptococcus in Screening Samples from Pregnant Women

Abstract

We compared five approaches for group B streptococcus (GBS) detection: three culture-based methods and two methods using broth-enhanced real-time PCR. Carrot broth-enhanced subculture to GBS Detect (Hardy Diagnostics, Santa Maria, CA) exhibited sensitivity and specificity comparable to carrot broth- and LIM broth-enhanced real-time PCRs.

TEXT

Group B streptococcus (GBS) colonization during pregnancy has been implicated as a major risk factor for severe neonatal infections, including sepsis, pneumonia, and meningitis (1, 2). In the United States, the Centers for Disease Control and Prevention recommend screening women between 35 and 37 weeks of pregnancy to determine GBS carrier status and prophylactic antibiotic treatment during labor for women at risk of transmitting this organism (2).

Several published studies have demonstrated the usefulness of culture-based (3, 4) and PCR-based (5–10) methods for detecting GBS. We compared five approaches for GBS detection: chromogenic culture detection by Northeast Laboratory GBS agar (NEL; Northeast Laboratory Services, Winslow, ME) and StrepB carrot broth (carrot broth; Hardy Diagnostics, Santa Maria, CA), carrot broth-enhanced subculture to GBS Detect (Detect; Hardy Diagnostics, Santa Maria, CA), and carrot broth-enhanced and LIM broth-enhanced (Remel, Lenexa, KS) real-time PCRs.

A total of 211 vaginal/rectal swabs submitted for routine prenatal GBS screenings were evaluated, including 104 BactiSwab double swab transports (Remel, Lenexa, KS) and 107 Eswabs (Copan Diagnostics, Inc., Murrieta, CA). All specimens were directly inoculated on NEL plates using the specific transport swab (Fig. 1). When a BactiSwab double-swab transport was received, both swabs were used to inoculate NEL plates. One swab was placed in the carrot broth (according to the manufacturer's instructions), and the second swab was placed in the LIM broth. When an Eswab was received, both the carrot and LIM broths were each inoculated with approximately 500 μl of the Eswab modified Liquid Amies transport solution and the swab was placed in the carrot broth (according to the manufacturer's instructions). NEL plates were incubated anaerobically at 35°C and examined at 18 to 24 h and 48 h for orange colonies (indicative of GBS). Carrot and LIM broths were incubated aerobically at 35°C for 18 to 24 h. Prior to PCR, all carrot broths were examined for any orange color in the broth, which is indicative of GBS, and were subcultured to Detect plates. Detect plates were incubated aerobically at 35°C for 18 to 24 h and examined for any beta-hemolytic colonies, which is a specific characteristic of GBS on Detect regardless of hemolysis exhibited on sheep blood agar. Real-time PCR was performed on the Cepheid SmartCycler system (Cepheid, Sunnyvale, CA) using the BD GeneOhm StrepB assay (Becton, Dickinson, Franklin Lakes, NJ) as described previously for 50 μl aliquots of carrot and LIM broths (5). All NEL, carrot broth, and Detect isolates were confirmed GBS positive by performing CAMP tests. CAMP negative isolates were confirmed negative by GBS latex agglutination (Slidex Strepto Plus; bioMérieux, Durham, NC).

Fig 1.

Study design for the evaluation of culture- and PCR-based methods for the detection of GBS. (a) NEL, Northeast Laboratory GBS agar. (b) Confirmed by CAMP/latex agglutination. (c) Detect, GBS Detect agar.

Performance characteristics were calculated using the consensus results from carrot broth-enhanced subculture to Detect, carrot broth-enhanced PCR, and LIM broth-enhanced PCR as reference standards. Samples were considered true positives if 2 of 3 broth-enhanced methods yielded positive results on initial testing. Samples with discordant results from these three methods were further analyzed by repeat PCR testing from the broth, but the results of repeat testing were not used in the calculation of performance characteristics. Additionally, samples with positive discrepant PCR results were subcultured from broth to Detect. All broths were stored at 4°C up to 72 h prior to discrepant analysis.

Forty specimens were positive for GBS (40/211, 19.0%) as determined by the consensus standard. Performance characteristics of culture- and PCR-based methods are shown in Table 1. Carrot broth-enhanced subculture to Detect had a sensitivity of 100% and specificity of 100%. The sensitivities and specificities were 100% and 98.8%, respectively, for carrot broth-enhanced PCR, and 95.0% and 99.4%, respectively, for LIM broth-enhanced PCR. Although their specificities were 100%, the sensitivities of NEL agar and carrot broth, when used alone, were lower than the other methods (Table 1). There were no differences in the performance of any method based on the swab type used for specimen collection (see Table S1 in the supplemental material). However, there were eight inhibited (i.e., PCR failed to amplify and no result was generated) carrot broth-enhanced PCR samples (6/104 BactiSwab samples and 2/107 Eswab samples) and two inhibited LIM broth-enhanced PCR samples (2/107 Eswab samples). All PCR-inhibited samples tested negative by NEL, carrot broth, carrot broth-enhanced subculture to Detect, and the alternative PCR-based method. Inhibited samples were not included in the calculation of the performance of the individual PCR-based method that demonstrated inhibition.

Table 1.

Performance of culture- and PCR-based methods for detection of GBS^a

Method	No. tested positive/total no. positive	Sensitivity (%) (95% CI)	Specificity (%) (95% CI)	PPV (%) (95% CI)	NPV (%) (95% CI)
NEL	32/40	80.0 (63.9–90.4)	100 (97.3–100)	100 (86.7–100)	95.5 (91.1–97.9)
Carrot broth	33/40	82.5 (66.6–92.1)	100 (97.3–100)	100 (87.0–100)	96.1 (91.7–98.3)
Carrot broth-enhanced subculture to Detect	40/40	100 (89.1–100)	100 (97.3–100)	100 (89.1–100)	100 (97.3–100)
LIM broth-enhanced PCR	38/40	95.0 (81.8–99.1)	99.4 (96.2–99.9)	97.4 (84.9–99.9)	98.8 (95.4–99.8)
Carrot broth-enhanced PCR	40/40	100 (89.1–100)	98.8 (95.2–99.8)	95.2 (82.6–99.2)	100 (97.1–100)

^aPPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; NEL, Northeast Laboratory GBS agar; Detect, GBS Detect agar.

Fourteen of 211 samples (6.6%) evaluated had discordant results (Table 2). There were 5 samples with discordant results between LIM broth-enhanced PCR, carrot broth-enhanced PCR, and carrot broth-enhanced subculture to Detect (samples A to E). Two false-negative LIM broth-enhanced PCRs (samples A and B) were positive by carrot broth-enhanced PCRs, and each had GBS isolated when subcultured from carrot broth to Detect. In these two samples, repeat PCRs from LIM broths yielded repeat negative results. These false-negatives could have occurred from a sampling error during the processing of specimens with low levels of GBS, as bacteria may not have been inoculated in sufficient amounts on all media. There was a false-positive LIM broth-enhanced PCR (sample C) and a false-positive carrot broth-enhanced PCR (sample D)—both samples yielded negative results upon repeat PCR testing. Additionally, both of these broth-enhanced PCR false positives were negative by all culture methods and subcultures of broths to Detect were negative. Finally, there was a false-positive carrot broth-enhanced PCR (sample E) that was negative by LIM broth-enhanced PCR and negative by all culture methods. Repeat carrot broth-enhanced PCR of this false-positive sample yielded a repeat positive result. From this sample, a beta-hemolytic streptococcus was cultured and identified as S. porcinus by Vitek 2 (bioMérieux, Durham, NC). The BD GeneOhm StrepB package insert provides no information regarding the species of Streptococcus included in their specificity analysis, but it has been shown that some S. porcinus can be CAMP positive (cfb is the target of this PCR assay) (11). As we did not directly test this isolate in the PCR assay, further study is required to assess whether this false positive represents analytical cross-reactivity with S. porcinus.

Table 2.

Samples with discordant results between culture- and PCR-based methods for detection of GBS

Sample	NELa		Carrot broth	Carrot broth-enhanced subculture to Detectb	Carrot broth-enhanced PCR	LIM broth-enhanced PCR	Final interpretation
	18–24 h	48 h
A	−	−	−	+	+	−c	False-negative NEL, carrot broth, and LIM broth-enhanced PCR
B	−	−	−	+	+	−c	False-negative NEL, carrot broth, and LIM broth-enhanced PCR
C	−	−	−	−	−	+d,e	False-positive LIM broth-enhanced PCR
D	−	−	−	−	+d,e	−	False-positive carrot broth-enhanced PCR
E	−	−	−	−e	+c,e,f	−c	False-positive carrot broth-enhanced PCR
F	−	−	+	+	+	+	False-negative NEL
G	−	−	+	+	+	+	False-negative NEL
H	−	−	+	+	+	+	False-negative NEL
I	−	−	+	+	+	+	False-negative NEL
J	−	+	−	+	+	+	False-negative carrot broth
K	−	+	−	+	+	+	False-negative carrot broth
L	+	+	−	+	+	+	False-negative carrot broth
M	−	−	−	+	+	+	False-negative NEL and carrot broth
N	−	−	−	+	+	+	False-negative NEL and carrot broth

^aNEL, Northeast Laboratory GBS agar.

^bDetect, GBS Detect agar.

^cRepeat PCR testing from broth yielded identical result.

^dRepeat PCR testing from broth yielded negative result.

^eSubculture of broth to Detect was GBS negative.

^fStreptococcus porcinus identified from culture of carrot broth.

There were 8 false-negative NEL cultures (Table 2, samples A, B, F to I, M, and N) and 7 false-negative carrot broth cultures (samples A, B, and J to N), and each had GBS isolated from the same sample when subcultured from carrot broth to Detect (samples A, B, and F to N). This is consistent with previously published studies demonstrating increased performance of broth-enhanced GBS detection methods compared to direct culture (12–14).

Although the carrot broth- and LIM broth-enhanced PCR methods have the ability to be reported more quickly, our data show they did not improve GBS detection compared to carrot broth-enhanced subculture to Detect. Additionally, the cost and complexity of performing PCR may limit its use in some laboratories. In our laboratory, the estimated cost of reagents and labor for carrot broth- or LIM broth-enhanced PCR was 13 times more expensive than carrot broth-enhanced subculture to Detect (data not shown). Carrot broth-enhanced subculture to Detect also allowed for isolation of GBS, including strains typically nonhemolytic on sheep blood agar, for antimicrobial susceptibility testing as necessary. Our study indicates that a nonmolecular testing approach utilizing carrot broth-enhanced subculture to Detect has acceptable performance characteristics for the detection and isolation of GBS.