Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres

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.

FIG. 1.

Template specificity for the Luminex capture probes evaluated in this study. For each candidate probe targeting each organism, templates consisted of water (white bars), the mixed vaginal panel including the target organism (gray bars), or the mixed vaginal panel excluding the target organism (black bars). The MFI of each probe for each of the target organisms was measured in triplicate, and the results are expressed as means ± standard deviations. The probes that were ultimately chosen for inclusion in the Luminex array are indicated by an asterisk. In addition to the eight targets shown here, three probes that targeted Lactobacillus vaginalis (data not shown) were evaluated, leading to the selection of probe Lvg496p (Table 1).

FIG. 2.

Nineplex detection of cloned cpn60 UT plasmid mixtures by use of the Luminex instrument. An amplicon was generated from each target organism separately and was then mixed postamplification and hybridized to the nineplex bead mix (gray bars). White bars, no-template control. Values are shown as the means of duplicate measurements ± standard deviations.

FIG. 3.

Standard curves correlating input target template amount with the postamplification MFI. Amplicons were generated by using the mixed vaginal panel (MVP; □) or equivalent copy numbers of L. iners cpn60 UT plasmid DNA (▪) and were analyzed in duplicate with a fourplex bead mixture. The results are shown as means ± standard deviations.

FIG. 4.

Paired Luminex and qPCR analysis of samples from individuals from Africa who were classified as having a normal vaginal flora (A) or as having BV (B) by the criteria of Nugent et al. (29). Samples were analyzed by a fiveplex Luminex method for the detection of G. vaginalis, A. vaginae, L. iners, and L. crispatus, as well as L. gasseri (data not shown). *, statistically significantly positive Luminex assay results (P < 0.001).

FIG. 5.

Longitudinal nineplex analysis with the Luminex instrument of vaginal swabs from selected individuals in North America. Each swab was analyzed by microscopy and was scored by using the criteria of Ison and Hay (20); a diagnosis of BV is indicated above each time point. DNA extracts were taken from the same swabs and analyzed with the Luminex instrument for the presence of each of the bacterial species targeted in this study. The MFI of each probe was measured in duplicate assays, and the results were compared to those for the no-template control. Error bars indicate standard deviations. *, statistically significant positive results (P < 0.001). The results obtained with the Luminex instrument for L. vaginae and P. anaerobius were negative for all samples and are not shown.