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,...

PCR was used to survey bacterial vaginosis flora before and after metronidazole treatment. The species composition for pretreatment patients was variable. Lactobacillus iners was prominent in all patients posttreatment. Atopobium vaginae concentrations were highest for patients who failed or responded incompletely to treatment and lowest for patients who were cured.Bacterial vaginosis (BV) is the most common cause of vaginal irritation and is associated with adverse pregnancy outcomes (4, 13) and an increased risk of human immunodeficiency virus infection (14,15). BV results when the normal, predominantly Lactobacillus vaginal flora shifts to one dominated by Gardnerella vaginalis, Mycoplasma hominis, and a variety of anaerobic organisms. However, no specific pathogen has been identified, and the cause of BV is unknown (6). Metronidazole is the most commonly prescribed antibiotic for treatment of BV, but failure and recurrence rates are high (7). Recent cultivation-independent analyses of PCR-amplified 16S rRNA gene sequences reveal that there are bacterial genera associated with BV that were not previously recognized, including a metronidozole-resistant anaerobe, Atopobium vaginae (8,9,17,19). We examined the species composition of the vaginal flora of BV patients before and 1 month after metronidazole treatment by using PCR assays directed toward a broad range of bacterial genera and a quantitative PCR assay targeting A. vaginae.Clinical assessments of BV were made just prior to treatment and at 4 weeks posttreatment. All six BV patients in the study met all Amsel criteria (2) and had Nugent scores of Ն4 (12). Treatment was a 0.75% topical metronidazole gel applied once daily for 5 days. The study was approved by the Louisiana State University Health Sciences Center institutional review board, and informed consent was obtained from each participant. Vaginal swabs were collected prior to treatment and 30 days posttreatment by using a standard protocol and were stored frozen. To isolate DNA, swabs were agitated in 0.5 ml of molecular-biology-grade water, suspensions were centrifuged, and nucleic acids were isolated from pellets by using an AquaPure genomic DNA kit (Bio-Rad, Hercules, CA). Primers for quantitative PCR assays of A. vaginae 16S rRNA genes were designed using Primrose (3); primers were 5Ј-GTTAGG TCAGGAGTTAAATCTG-3Ј and 5Ј-TCATGGCCCAGAC C-3Ј. Real-time amplifications were performed on an iCycler (Bio-Rad) using iQ-SYBR Green Supermix (Bio-Rad). Thermal cycling consisted of 95°C for 2.5 min, followed by 40 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s; 10 ng of template DNA was used in each amplification. PCR amplification of vaginal DNA using primers targeting conserved regions of the16S rRNA gene were used to generate clone libraries with a TOPO

Cultivation-independent analysis of 16S rRNA gene sequences in vaginal samples revealed two previously unrecognized, uncultivated Megasphaera-like phylotypes. Phylogenetic analysis and environmental distribution suggest that these Megasphaera types may be unique to the vaginal environment. Quantitative PCR suggests that both phylotypes are present in higher concentrations in women with bacterial vaginosis.

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methods with relatively poor sensitivity compared to nucleic acid amplification methods. Our aim was to determine TV prevalence using the APTIMA TV Assay (ATV, Gen-Probe Incorporated) and the frequency of co-infections with Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) in the USA among women being screened. Methods Samples from 7593 women aged 18e89 years undergoing routine CT and NG screening at obstetrics/gynaecology, emergency room, hospital in-patient, family practice, family planning, internal medicine, jail, and STD clinic populations in 21 states were collected. Consecutive samples previously tested for CT and NG by the APTIMA COMBO 2 Assay (Gen-Probe Incorporated) were retrospectively tested with the ATV assay. Endocervical, urine, vaginal swab and PreservCyt liquid Pap samples (Hologic Inc.) diluted into APTIMA specimen transport buffer were tested. Results Overall prevalences of TV, CT and NG in surveyed women were 8.7%, 6.7%, and 1.7%, respectively. TV prevalence ranged from 7.5 to 8.6% in women age 18 to 39 yr, and increased to 9.8% in women age 40e44 yr. Highest observed TV prevalences were in women ages 45e49 yr (13.4%) and over 50 yr (13.0%). CT and NG prevalences were less than 2% in the 40+ age group and highest in women less than 30 years of age ranging from 5.2% to 14.3% for CT and 1.3%e3.3% for NG. TV was the more prevalent STD than either CT or NG in all age groups, except the 18-19 yr group (CT: 14.3%; TV: 8.5%). TV prevalence differed by race/ethnicity (20.2% blacks; 5.7% whites; 5.0% Hispanics; 3.8% Asians). TV prevalence was 14.4% in the Southeast, 9.5% in the Southwest and Midwest, and 4.3% in the Northeast and ranged from 5.4% in Family Planning clinics to 22.3% in jails. Co-infections in most age groups were <1%, and were highest in the 18e19 yr group (TV/CT: 2.1%; TV/NG: 0.88%). Conclusions TV prevalence was highest in women over 40 years of age, in contrast to CT and NG prevalences which were highest in women under 30 years of age. Co-infection of TV with CT or NG was relatively low. The high TV prevalence in all age groups suggests that all women being screened for CT/NG should also be screened for TV. Routine TV screening should also be considered for at-risk sexually active women of any age.