Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

Abstract

c Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

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

“…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].…”

“…According to the literature, discrimination of Lactobacillus species by qPCR may inform about a shift on the vaginal population of this genus. 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].…”

“…This method is considered a reliable diagnostic approach from the viewpoint of reproducibility [27] and nowadays, it is considered the gold standard for BV diagnosis [10]. However, this system is a qualitative method, requires specialized training and does not provide information of some BV-associated bacteria, like A. vaginae and M. hominis [5,23].…”