A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

Abstract

A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus crispatus and Lactobacillus iners. Together with a short questionnaire, a vaginal swab for PCR and vaginal smear for microscopy were taken by their general practitioner or gynaecologist. Data from 151 women (median age 32) were available. Nugent Score (NS) was used to classify the samples and 83 samples were classified as normal (NS 0–3), 13 as intermediate (NS 4–6), and 55 as bacterial vaginosis (NS 7–10). In women with a NS of 7–10, PCR detected Gardnerella vaginalis, Atopobium vaginae and Megasphaera phylotype 1 in respectively, 96 %, 87 % and 60 %, whereas in women with a NS of 1–3 these species were detected in 27 %, 6 % and 2 % (P <0.001). A ratio of Lactobacillus crispatus over Lactobacillus iners of <1 (as calculated from the quantification cycle value (Cq)) was present in women with a NS of 7–10 in 66 % versus 33 % in women with a NS of 1–3 (P <0.001). The BV-PCR displayed a sensitivity of 92 % and specificity of 96 % with a positive predictive value of 94 % and a negative predictive value of 95 %. The Lactobacillus-index improved the correct classification of samples where only one of the other bacterial species was detected. Compared to the Nugent Score this multiplex qPCR offers a convenient tool for performing observer independent diagnosis of BV.

Assessment of vaginal microbiota in Brazilian women with and without bacterial vaginosis and comparison with Nugent score

“…G. vaginalis, A. vaginae, M. hominis and Mobiluncus spp. were detected at higher concentrations in vaginal microbiota of BV compared to non-BV women, corroborating the findings of recent studies [3,24,30,33,38]. In contrast, Lactobacillus spp.…”

“…A recent study has investigated the community structure of healthy and BV vaginal microbial ecosystems and detected A. vaginae only in BV group [25]. However, other studies that also investigated microbial communities associated with BV showed results that corroborate our findings [23,24].…”

“…Lactobacillus iners is a species associated with both healthy and abnormal vaginal microbiota [17,23,33]. Unlike Lactobacillus crispatus L. gasseri and L. jensenii, L. iners may be common and abundant in abnormal microbiota, sharing space with BV-marker organisms [3,24,33].…”

“…Recent advances have been achieved by culture-independent techniques, allowing association of bacteria species with BV for the first time, such as A. vaginae [17,[20][21][22]. Polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) have been used to investigate BV-associated bacteria composition, while real-time PCR (qPCR) stands out as a useful tool for bacteria quantification [23][24][25]. In addition, qPCR schemes have recently been developed as an alternative for BV diagnosis [23,24].…”

“…Polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) have been used to investigate BV-associated bacteria composition, while real-time PCR (qPCR) stands out as a useful tool for bacteria quantification [23][24][25]. In addition, qPCR schemes have recently been developed as an alternative for BV diagnosis [23,24].…”