Knowledge of the abundance of bacterial species in vaginal communities will help us to better understand their role in health and disease. However, progress in this field has been limited because quantifying bacteria in natural specimens is an arduous process. We developed quantitative real-time PCR (qPCR) assays to facilitate assessments of bacterial abundance in vaginal specimens and evaluated the utility of these assays by measuring species abundance in patients whose vaginal floras were clinically described as normal, intermediate, or bacterial vaginosis (BV) as defined by Nugent's criteria. The qPCR measurements showed that Lactobacillus species were predominant in normal vaginal specimens and that high Lactobacillus crispatus and Lactobacillus jensenii abundance was specific to normal specimens, while Lactobacillus iners abundance was high in all categories including BV. The abundances of all non-Lactobacillus species were higher in BV specimens than in normal specimens. Prevotella species were prevalent in all specimens and represented a high percentage of total species in BV specimens. qPCR assays can be a useful tool for describing the structure of vaginal communities and elucidating their role in health and disease.Vaginal bacterial communities are composed of mixtures of diverse species, and the relative abundance of these species in part determines urogenital health and disease in women (22). It is generally acknowledged that vaginal communities predominated by Lactobacillus species are normal and healthy while communities predominated by other genera, such as Gardnerella vaginalis, are abnormal and unhealthy (36). The latter condition essentially defines a poorly understood syndrome known as bacterial vaginosis (BV). While BV can be asymptomatic and benign in some women, it is a common cause of malodorous vaginal discharge for many. Moreover, BV flora is of concern because it is associated with an increased risk of adverse sequelae, such as preterm birth (8, 24), postoperative complications in women (40), enhanced risk of acquiring sexually transmitted infections (31), and increased shedding of HIV (11). Treating BV has not proven effective for the prevention of these adverse events possibly due to the fact that standard BV treatment results in high failure and relapse rates (25,29). Furthermore, while suspected pathogens such as G. vaginalis have been implicated, no agent or factor has been identified as the cause of BV, despite experimental (10) and epidemiological (28) evidence that suggests that BV is transmissible (10). Because of all the uncertainties surrounding this syndrome, BV has been described as a microbiological and clinical enigma (16,17).Failure to understand the microbiology specific to BV is perhaps not surprising given that the basic ecology of the genitourinary microbiota, namely, the composition, relative abundance, and temporal fluctuations of vaginal species, are poorly understood. This lack of knowledge is highlighted by recent cultivation-independent broad-range PCR surveys,...

BackgroundThe epidemiology of bacterial vaginosis (BV) suggests it is sexually transmissible, yet no transmissible agent has been identified. It is probable that BV-associated bacterial communities are transferred from male to female partners during intercourse; however, the microbiota of sexual partners has not been well-studied.ResultsPyrosequencing analysis of PCR-amplified 16S rDNA was used to examine BV-associated bacteria in monogamous couples with and without BV using vaginal, male urethral, and penile skin specimens. The penile skin and urethral microbiota of male partners of women with BV was significantly more similar to the vaginal microbiota of their female partner compared to the vaginal microbiota of non-partner women with BV. This was not the case for male partners of women with normal vaginal microbiota. Specific BV-associated species were concordant in women with BV and their male partners.ConclusionsIn monogamous heterosexual couples in which the woman has BV, the significantly higher similarity between the vaginal microbiota and the penile skin and urethral microbiota of the male partner, supports the hypothesis that sexual exchange of BV-associated bacterial taxa is common.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0161-6) contains supplementary material, which is available to authorized users.

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

BackgroundSeveral methods have been reported for strain typing of Mycoplasma genitalium. The value of these methods has never been comparatively assessed. The aims of this study were: 1) to identify new potential genetic markers based on an analysis of short tandem repeat (STR) sequences in the published M. genitalium genome sequence; 2) to apply previously and newly identified markers to a panel of clinical strains in order to determine the optimal combination for an efficient multi-locus genotyping system; 3) to further confirm sexual transmission of M. genitalium using the newly developed system.ResultsWe performed a comprehensive analysis of STRs in the genome of the M. genitalium type strain G37 and identified 18 loci containing STRs. In addition to one previously studied locus, MG309, we chose two others, MG307 and MG338, for further study. Based on an analysis of 74 unrelated patient specimens from New Orleans and Scandinavia, the discriminatory indices (DIs) for these three markers were 0.9153, 0.7381 and 0.8730, respectively. Two other previously described markers, including single nucleotide polymorphisms (SNPs) in the rRNA genes (rRNA-SNPs) and SNPs in the MG191 gene (MG191-SNPs) were found to have DIs of 0.5820 and 0.9392, respectively. A combination of MG309-STRs and MG191-SNPs yielded almost perfect discrimination (DI = 0.9894). An additional finding was that the rRNA-SNPs distribution pattern differed significantly between Scandinavia and New Orleans. Finally we applied multi-locus typing to further confirm sexual transmission using specimens from 74 unrelated patients and 31 concurrently infected couples. Analysis of multi-locus genotype profiles using the five variable loci described above revealed 27 of the couples had concordant genotype profiles compared to only four examples of concordance among the 74 unrelated randomly selected patients.ConclusionWe propose that a combination of the MG309-STRs and MG191-SNPs is efficient for general epidemiological studies and addition of MG307-STRs and MG338-STRs is potentially useful for sexual network studies of M. genitalium infection. The multi-locus typing analysis of 74 unrelated M. genitalium-infected individuals and 31 infected couples adds to the evidence that M. genitalium is sexually transmitted.

Background: Trichomonias is the most common non-viral STI among women worldwide and is associated with serious reproductive morbidity, poor birth outcomes and amplified HIV transmission. Single-dose metronidazole therapy has been the treatment of choice for over three decades. There is mounting evidence, however, of high rates of repeat positives following single-dose metronidazole, and among HIVinfected women, bacterial vaginosis (BV) was found to alter treatment efficacy. The purpose of this study was to examine the effectiveness of single-dose metronidazole compared to 7 day-dose metronidazole for the treatment of trichomoniasis among HIV-uninfected, non-pregnant women and to determine if this effect was modified by BV. Methods: This was a randomized, parallel, multi-site, open-label trial of single-dose (2 g one-time) versus 7 day-dose (500 mg twice daily) for the treatment of trichomoniasis. The primary outcome was T. vaginalis infection by arm, per nucleic acid amplification test or culture, four weeks post-completion of treatment, in intentto-treat analyses. This analysis was also stratified by BV status. Findings: Of 623 women randomized, those in the 7 day-dose arm were less likely to be T. vaginalis positive at test-of-cure compared to those in the single-dose arm [34/312 (10.9%) vs. 58/311 (18.6%), p=0·001] [R.R. 0.55 (95% C.I. 0.34–0.70)]. Risk was similar by BV status (p=0·17). Self-reported adherence in both arms was > 95%. Side effects were similar by arm. Interpretation: In this sample of HIV-uninfected, non-pregnant women with trichomoniasis, compared to single-dose, 7 day-dose metronidazole treatment resulted in 45% fewer treatment failures. The 7 day-dose metronidazole should be the preferred treatment for trichomoniasis among women.

T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

This study assessed the utility of urine, vaginal, cervical, and rectal specimens for the detection of Mycoplasma genitalium in women by using our laboratory-developed PCR assay. The relative sensitivity was 85.7% for the vaginal swab specimen, 74.3% for the endocervical swab specimen, 61.4% for the urine specimen, and 24.3% for the rectal swab specimen.The diagnosis of sexually transmitted diseases (STDs) is increasingly being made using laboratory specimens that patients can collect themselves, such as urine in men and women and vaginal swabs in women. These self-collected specimens have proven useful for reliably detecting Chlamydia trachomatis and Neisseria gonorrhoeae in both men and women by using nucleic acid amplification tests (NAATs) (2, 7).Mycoplasma genitalium is strongly associated with nongonococcal urethritis in men, and its clinical significance in women and the determination of its role as a sexually transmitted agent are receiving growing attention (5, 8). Because there is currently no approved and commercially available diagnostic test for the detection of M. genitalium, clinical studies of M. genitalium infections use local laboratory-developed PCR tests to diagnose infection (11). Regardless of which NAAT is used, the determination of optimal specimen types for the detection of the organism under different circumstances is important.As part of a study of the prevalence and risk factors for M. genitalium among women, conducted in STD clinic patients in New Orleans, we assessed the utility of urine, vaginal, cervical, and rectal specimens for the detection of M. genitalium in women by using a previously described PCR assay developed in our laboratory (9, 10).Women age 18 or older who attended the New Orleans STD clinic for any reason between 28 May 2003 and 26 February 2004 were approached for inclusion in the study and were enrolled after completing the informed consent process. The study was approved by the institutional review board of the Louisiana State University Health Sciences Center. Pregnant women, women with a history of hysterectomy, and those who reported using antibiotics in the past 3 months were excluded from participation.After obtaining informed consent, a complete sexual behavior, STD, obstetric, and gynecologic history was obtained from each study participant and recorded in a standardized form, along with the patient's demographic information. To detect M. genitalium, four laboratory specimens were obtained in the following order. A first-void urine specimen was collected prior to performance of a pelvic examination and kept at 4°C before transport to the laboratory. Following insertion of a nonlubricated speculum into the vagina, a vaginal swab was obtained from the posterior fornix. After cleaning the face of the cervix, an endocervical swab was obtained. A rectal swab was obtained last. Each of the three swabs was placed in a dry transport tube and held at 4°C prior to transport to the laboratory at the end of the day.Following DNA purification, an M. genitalium-specifi...

The cobas CT/NG v2.0 test performs well using urogenital swabs, urine and cervical samples collected in PreservCyt solution.