This prospective study was carried out between March and September 2017. Pregnant women who presented for medical appointments at the outpatient, antenatal, and labor and delivery units of the HCPA Department of Obstetrics and Gynecology were recruited. The study (number: 2016-0560) was approved by the institutional Research Ethics Committee of Gynecology and Obstetrics Research and Postgraduate Group (GPPG-GO) and conducted in accordance with the provisions of the Declaration of Helsinki. All patients provided written informed consent prior to enrollment. In case of participants under 18 years of age, the responsible also signed the consent form. The inclusion criterion was gestational age ≥24 weeks, while the exclusion criterion was any use of antibiotics in the 30 days preceding enrollment.
Among 300 enrolled women, 30 were excluded: 5 refused to participate, 10 had already undergone GBS screening and received their results at the time of study inclusion, and 15 had used antibiotics in the last 30 days (Figure 1). Thus, the final sample comprised 270 women.
Three vaginal and rectal swabs (one sample for each screening method – Xpert GBS, qPCR, and culture) – were collected from each patient and immediately stored in Stuart transport medium, according to CDC recommendations. The swabs collected for qPCR were sent to the HCPA microbiology and molecular biology laboratories; the culture swabs were sent to an outside laboratory (Endocrimeta®); and the Xpert GBS samples were analyzed on specific equipment provided by Cepheid. All samples were sent for evaluation within 24 hours of collection.
Xpert® GBS (Cepheid)
The collected swab was transferred to the designated chamber of the Xpert GBS cartridge, which was loaded into a Cepheid GeneXpert device, as recommended by the manufacturer. A trained physician performed all Xpert GBS assays. The result could be negative or positive based on the detection of the target gene sequence adjacent to the GBS cfb gene, as defined by the GeneXpert software. Xpert GBS performs automation and integration of sample lysis, amplification and purification of nucleic acids, and detection of the target sequence using qPCR. The total assay runtime was around 50 minutes.
The collected swab was inoculated on blood agar plates and incubated at 37°C for 48 hours in a microbiological incubator. After incubation, the plates were inspected for the presence of beta-hemolytic colonies. If there was any suspicion of beta-hemolytic plaque growth after 48 hours, plaques were reincubated for another 24 hours and inspected again. Beta-hemolytic colonies whose morphology were consistent with GBS were subcultured and CAMP-tested . CAMP-positive colonies were deemed presumably positive for GBS.
Real-time polymerase chain reaction (qPCR)
Sample preparation and DNA (deoxyribonucleic acid) extraction
The swabs were incubated for 18 to 24 hours into Todd Hewitt selective medium containing gentamicin and nalidixic acid. After centrifugation of the broth, the precipitate was washed with 1X PBS (phosphate-buffered saline) solution (pH = 7.2) and centrifuged again. Then, the precipitate was washed with 1X Tris-EDTA (TE) buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH = 7.5), and DNA extracted by thermal lysis. The thermal lysis protocol was performed using TE solution for 15 min at 100°C followed by 15 min at −80°C to lyse bacterial cell walls. The quality and quantity of DNA extracted from samples were estimated spectrophotometrically in a Nanodrop ND-1000 system (Thermo Fisher Scientific, USA), at 260 nm (A260) and 280 nm (A280) absorbance, with the sample diluted to 5 ng/µL.
Real-time polymerase chain reaction
For the qPCR, we used the cfb gene region that encodes the CAMP factor present in GBS. The primers used were 5′-TTT CAC CAG CTG TAT TAG AAG TA-3′ and 5′-GTT CCC TGA ACA TTA TCT TTG AT-3′. For internal control, a synthetic single-chain DNA (5′-ATC GCT GAT CCG GCC ACA TAT CGC GTT TAT GCG AGG TCG GGT GGG CGG GTC GTT AGT TTC GTT TTG GGC CTA CGT GGC CTT TGT CAC CGA-3′) was used to detect amplification inhibition in all samples using the primers 5′-ATC GCT GAT CCG GCC ACA-3′ and 5′-TCG GTG ACA AAG GCC ACG TA-3′.
The amplification reagents were prepared as follows: Platinum® SYBR® Green (Invitrogen) concentrated mix 6.25 μL, SBG primers 1.25 μL, ROX 1:50 0.25 μL, and DNAse- and RNAse-free water 2.5 μL; 0.5 μL of internal control (IC) solution and 0.75 μL of primers were added to the IC tube.
The extracted DNA solution was added to 10 μL of amplification reagents. Amplification and fluorescent detection were measured by qPCR in a 7500 Real-Time PCR System (Applied Biosystems). The amplification was performed with one cycle at 50°C for 2 minutes for DNA polymerase activation, followed by one cycle at 95°C for 10 minutes for initial denaturation, than 40 cycles at 95°C for 15 sec and 60°C for 1 min for amplification, followed by two cycles at 95°C for 15 sec and 60°C for 15 sec for fluorescence detection and melting temperature (Tm) measurement. Samples are considered positive when the amplification curve is detected and the Tm is in the acceptable range (GBS Tm 76-78°C and IC Tm 82-84°C). The negative control should not have an amplification curve for the GBS target, while the positive control should be positive for the two targets tested. To ensure high sensitivity in the PCR, the cutoff point was set at a threshold cycle value of Ct = 40 [10, 11].
Statistical analysis and sample size
Sample size was calculated in WinPEPI Version 11.63, based on the findings of a previous study . Considering a 16% prevalence of GBS positivity with the gold-standard method (culture) and 95% power to detect a 5% difference in prevalence of a positive result, with an estimated 10% attrition rate, the final sample size required was 230 participants.
Regarding the data processing, we used a database double entry, and review were performed using the SPSS, version 18.0. [SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc.]. Symmetric data was expressed as mean and standard error of mean (±SEM), or by median and interquartile range [Percentiles 25th–75th, P25–P75]. The Shapiro-Wilk test was used to determine the normality of data distribution. Categorical variables were described as absolute (n) and relative (n%) frequencies.
Agreement between assays was determined using the kappa and Cronbach’s alpha coefficients. The sensitivity, specificity, negative predictive value, and positive predictive value of the tests were evaluated in accordance with STARD (Standards for Reporting of Diagnostic Accuracy) initiative recommendations.
Spearman’s ρ coefficients were estimated for obstetric characteristics and GBS positivity.
The level of significance was set at 5% for all analysis.