Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

Abstract

Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

Association of human papillomavirus and bacterial vaginosis with increased risk of high-grade squamous intraepithelial cervical lesions

“…Multiplex polymerase chain reaction assays were performed according to the method described by Malaguti et al19 Briefly, to optimize the detection of the 13 bacterial vaginosis agents, multiplex polymerase chain reaction was split into three different reactions: assay 1 to detect six bacteria ( G. vaginalis , M obiluncus curtisii , Bacteroides fragilis , Mycoplasma hominis , U reaplasma urealyticum , and Megasphaera type I); assay 2 to detect three bacteria ( Atopobium vaginae , Clostridia-like bacteria vaginosis-associated bacteria 1 and 2); and assay 3 to detect four bacteria ( M obiluncus mulieris mulieris , Clostridia-like bacteria vaginosis-associated bacteria 3, Sneathia sanguinegens , and Mycoplasma genitalium ). The multiplex polymerase chain reaction products were electrophoresed on 8% polyacrylamide gel stained with 1 µg/mL ethidium bromide.…”

Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women? – a position statement from the European STI Guidelines Editorial Board

“…However, both of these ureaplasmas are frequently associated with increased positivity for several traditional STIs, e.g. C. trachomatis and M. genitalium , and/or BV . The bacterial load of particularly M. hominis and to a lesser extent U. parvum and U. urealyticum can be significantly increased in the dysbiosis of BV .…”

“…The bacterial load of particularly M. hominis and to a lesser extent U. parvum and U. urealyticum can be significantly increased in the dysbiosis of BV . However, despite the association between particularly M. hominis and BV, M. hominis cannot be detected in approximately one‐third of women with BV and, accordingly, it is neither a sufficiently sensitive nor specific bacterial marker for diagnosis of BV . Despite not being susceptible to metronidazole, eradication or a decrease in the M. hominis load after BV treatment has also been reported, further indicating that M. hominis frequently belongs to the dysbiosis of BV.…”

“…Furthermore, in most studies, the strong association between bacterial vaginosis (BV) in ‘patient’ and/or BV‐associated bacteria in sexual partner of women with BV has not been adjusted for. This is particularly an issue for M. hominis but also for ureaplasmas . These and additional confounding factors make interpretation of many previous studies exceedingly difficult.…”

“…This is particularly an issue for M. hominis but also for ureaplasmas. 4,[8][9][10] These and additional confounding factors make interpretation of many previous studies exceedingly difficult.…”