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ObjectivesAs pelvic inflammatory disease (PID) aetiology is not completely understood, we examined the relationship between select novel bacteria, PID and long-term sequelae.MethodsFastidious bacterial vaginosis (BV)-associated bacteria (Sneathia (Leptotrichia) sanguinegens, Sneathia amnionii, Atopobium vaginae and BV-associated bacteria 1 (BVAB1)), as well as Ureaplasma urealyticum and Ureaplasma parvum were identified in cervical and endometrial specimens using organism-specific PCR assays among 545 women enrolled in the PID Evaluation and Clinical Health study. Risk ratios and 95% CIs were constructed to determine associations between bacteria, histologically confirmed endometritis, recurrent PID and infertility, adjusting for age, race, gonorrhoea and chlamydia. Infertility models were additionally adjusted for baseline infertility.ResultsPersistent detection of BV-associated bacteria was common (range 58% for A. vaginae to 82% for BVAB1) and elevated the risk for persistent endometritis (RRadj 8.5, 95% CI 1.6 to 44.6) 30 days post-cefoxitin/doxycycline treatment, independent of gonorrhoea and chlamydia. In models adjusted for gonorrhoea and chlamydia, endometrial BV-associated bacteria were associated with recurrent PID (RRadj 4.7, 95% CI 1.7 to 12.8), and women who tested positive in the cervix and/or endometrium were more likely to develop infertility (RRadj 3.4, 95% CI 1.1 to 10.4). Associations between ureaplasmas and PID sequelae were modest.ConclusionsTo our knowledge, this is the first prospective study to demonstrate that S. sanguinegens, S. amnionii, BVAB1 and A. vaginae are associated with PID, failure of the Centers for Disease Control and Prevention-recommended treatment to eliminate short-term endometritis, recurrent PID and infertility. Optimal antibiotic regimens for PID may require coverage of novel BV-associated microbes.

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

Although not designed to look specifically at the effect of H2-blockers on the incidence of NEC, our study suggests that their use lowers fecal microbial diversity and shifts the microfloral pattern toward Proteobacteria. These alterations in fecal microbiota may predispose the vulnerable immature gut to necrotizing enterocolitis and suggest prudence in the use of H2-blockers in the premature infant.

Objectives:As the aetiology of bacterial vaginosis (BV) is not well understood, this study sought to determine the relationships between several fastidious microbes, BV and selected clinical characteristics of BV.Methods:Endometrial and cervical specimens from 50 women with non-gonococcal, non-chlamydial endometritis were tested for Leptotrichia sanguinegens/amnionii, Atopobium vaginae, bacterial vaginosis-associated bacteria 1 (BVAB1), Ureaplasma urealyticum biovar 2 (UU-2) and Ureaplasma parvum using PCR. BV was categorised using Nugent’s and Amsel’s criteria. Odds ratios (OR) adjusted for age and race were estimated using multivariable logistic regression.Results:Although elevated pH was a universal feature, other BV characteristics differed by pathogen, suggesting variable clinical presentation. Only UU-2 was strongly associated with vaginal discharge, but a positive whiff test and a 20% or greater classification of epithelial cells as clue cells were more common among women with L sanguinegens/amnionii, A vaginae and BVAB1. For each of these bacteria, there were trends towards associations with BV defined by Amsel’s criteria (L sanguinegens/amnionii OR 2.9, 95% CI 0.5 to 15.7; A vaginae OR 2.6, 95% CI 0.6 to 11.4; BVAB1 OR 5.7, 95% CI 1.0 to 31.1) and significant associations with BV defined by Gram stain (L sanguinegens/amnionii OR 17.7, 95% CI 2.8 to 113.0; A vaginae OR 19.2, 95% CI 3.7 to 98.7; BVAB1 OR 21.1, 95% CI 2.2 to 198.5).Conclusions:L sanguinegens/amnionii, A vaginae and BVAB1 are associated with clinical characteristics consistent with BV and BV defined by Nugent’s and Amsel’s criteria. These fastidious bacteria may cause unrecognised infection, as none was associated with abnormal vaginal discharge.