| Abstract: | ![]()
Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.Bacterial vaginosis (BV), which is defined by a reduction in the vaginal Lactobacillus populations and an increase in the number of microbial species present, including Gardnerella spp., Atopobium spp., and others, is increasingly recognized as an important risk factor for adverse reproductive health outcomes, such as miscarriage and premature birth, and sexually transmitted diseases, including human immunodeficiency virus infection (25). Despite intense investigation, the etiology and clinical course of BV have not been well defined, probably because the normal variation of the vaginal microbial communities between individuals and their dynamics over time are complex and poorly understood. Consequently, the accurate diagnosis of BV, as well as the elaboration of effective prevention and treatment strategies, remains a major challenge (26, 44).The current “gold standard” for the diagnosis of BV in the laboratory setting relies on microscopic profiling of microbial types in Gram-stained vaginal swab smears by using the criteria of Nugent et al. (29) or Ison and Hay (20). These scoring systems are based on the relative abundance of Lactobacillus morphotypes (large gram-positive rods) compared to the abundance of the bacterial morphotypes suggestive of BV (gram-negative or gram-variable coccobacilli and curved rods and gram-positive cocci). Although this method provides reliable information and is particularly well suited for use in resource-poor settings, it requires well-trained, highly experienced individuals to interpret the results and is simplistic in its reflection of the variety of organisms present in the microbial communities present in healthy individuals with a normal vaginal flora and patients with BV. Potential problems with the subjective interpretation of the Gram stain result, along with an increasing appreciation of the fact that the complexity of the vaginal microbiota can be missed by these classical methods, have resulted in a search for other methods for the diagnosis of BV that can also identify the different species present (5, 25).In-depth studies of the makeup of vaginal microbial communities are quite common, including studies that use the older culture-based approaches (31) and more recent molecular studies that involve the construction of a clone library on the basis of the 16S rRNA gene (30) or the chaperonin-60 (cpn60) (16) target. While the information generated by these studies is critical to obtaining a greater understanding of the variability and ecological dynamics of vaginal microbial communities, they remain far too labor-intensive at present for the screening of large numbers of samples or for the routine laboratory diagnosis of BV.Most recently described molecular approaches for the diagnosis of BV are based on quantitative PCR (qPCR) of the 16S rRNA gene or other targets in bacteria extracted from vaginal samples. For example, one recent study found that the simultaneous quantification of the Gardnerella cpn60 target and the Atopobium 16S rRNA gene target resulted in the sensitive and specific diagnosis of BV in comparison to the sensitivity and specificity of the diagnosis by use of the scoring system of Nugent et al. (29). The advantages of the cpn60 universal target (UT) as an alternative to the 16S rRNA gene for the identification of bacterial species are increasingly recognized and include the increased resolution available at the species level and its presence at a single copy per genome, improving the reliability of quantitation (7).Multiplexed, bead-based flow cytometric detection and quantification of bacterial targets by use of the Luminex platform have been described previously (3, 9, 14, 38). The main advantage of this approach is its ability to detect a large number of molecular targets simultaneously after a single PCR, making high-throughput analysis of multiple samples from large study groups or longitudinal studies technically feasible. In the context of BV, the use of this multiplex approach, coupled with highly discriminatory cpn60-specific probes, facilitates screening for multiple species of Lactobacillus and a number of BV-associated bacteria simultaneously, providing an unprecedented level of information about the variability of vaginal bacterial communities, defined as those associated with a normal vaginal flora and those associated with BV, in a single, rapid assay.The goal of the present study was the design of a flow cytometric bead array for the rapid profiling of microbial communities extracted from vaginal samples and its evaluation as a potential technique for the rapid diagnosis of BV in the laboratory setting. |
