M.Sc (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: Bacterial vaginosis (BV) results from a shift in normal vaginal flora and predisposes women to sexually transmitted infections (STI) including HIV. Risk factors for BV are not well understood. This analysis seeks to determine the disease frequency of BV, assess determinants of BV and quantify time to first BV episode in HIV negative women. Methods: Baseline and follow-up data from 1066 women was analysed in STATA10. Logistic regression was used to determine baseline factors associated with BV and Kaplan Meier survival analysis to estimate time to BV episode. Results: BV prevalence and incidence was estimated at 48.42% and 81 cases per 100 women years respectively. Controlling for age and education, women with Trichomonas vaginalis, Chlamydia trachomatis, Herpes Simplex Virus2 and lower socio-economic status were 67%- 380%, 31%-472%, 20%-220% and 4%-91% more likely to present with BV respectively. Consistent condom use and being a housewife or student was significantly (p<0.05) associated with lower prevalent BV, with a significant interaction between age and education (p<0.05). The median time to first BV episode was 9.7 months. Conclusion: The analysis identifies modifiable risk factors like condom use, injectable contraceptives and treatment of STIs which could potentially decrease the high BV disease burden.

Thesis (M.Sc.(Med.)(Epidemiology and Biostatistics)), Faculty of Health Sciences, University of the Witwatersrand,2009 / BACKGROUND Studies suggest an association between Bacterial Vaginosis (BV) and HIV infection; however, its temporal effect has not been greatly investigated. METHODS This is a secondary data analysis of a cohort study: set out to describe the association between BV infection and HIV acquisition. There were 750 participants enrolled in the primary cohort study. The main exposure, BV, was measured from a gram stain slide prepared from a vaginal swab. The slide was read in a laboratory qualitatively and scored by Nugents scoring. A score of 7 or above was considered positive for BV. The outcome variable (HIV) was determined by dual rapid tests and confirmed in the laboratory by a third generation ELISA. Descriptive statistics was done to describe demographic characteristics and the prevalence of BV and STIs. HIV incidence rate was calculated. Kaplan Meier survival time analysis and log rank test for significance were performed. Cox regression (univariate and multivariate) was done to determine association of BV with HIV infection. RESULTS The baseline prevalence of BV was 52 %, 95 % CI; 45 – 59. There were 21 HIV seroconversions experienced of which 7 had BV results missing and were excluded in the analysis. The remaining 14 seroconversions were followed for a mean time of 0.40 of a year and accumulated follow up time at risk of 286 person years, this represented an HIV incidence rate of 4.9 per 100 person years of follow up, 95 % CI: 2.9 – 8.27. Kaplan Meier curves revealed a higher risk of HIV-1 acquisition among women who were BV positive than the women who were BV negative. A log rank test showed that the v probability of seroconversion was different among the women depending on BV status, chi-square value 3.8, p 0.05. Controlling for confounding variables, seroconversion was high, but not significant, among BV positive women, adjusted hazard ratio 3.21; 95 % CI; 0.85-12.12, p value 0.08. CONCLUSION This study suggests that BV increases HIV seroconversion risk though statistical significance was not achieved. Vaginal cleansing education, screening and treating women with BV could maintain normal vaginal flora and reduce their susceptibility to HIV.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 41-60).

Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is of major clinical importance due to its ability to significantly affect pregnancy outcome and enhance the transmission and acquisition of HIV. BV is characterized by a dramatic shift in the vaginal microflora; in most BV cases, the predominant bacterial species is Gardnerella vaginalis. It has been demonstrated that G. vaginalis forms an adherent biofilm on the vaginal epithelium of women with BV. Furthemore, evidence suggests that the high rate of recurrence associated with BV is related to incomplete eradication of the biofilm. The overall goal of this study was to characterize G. vaginalis virulence properties, including biofilm formation, in order to better understand the pathogenesis of BV and to improve available treatment methods. In an effort to tease apart the uncertain etiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. Only G. vaginalis demonstrated all three virulence properties, including robust biofilm formation. It has been shown that the biofilm phenotype allows its constituent bacteria to be resistant to many negative environmental stimuli. Therefore, we studied the susceptibilities of biofilm vs. planktonic cultures to H2O2 and lactic acid. Biofilms tolerated higher concentrations of both chemicals; however, when the biofilm was proteolytically disrupted, sensitivity to the chemicals returned to planktonic levels. Since our data suggested a critical role for a protein in biofilm formation, a partial genome sequence of G. vaginalis was searched for sequence homology to known biofilm adhesins using the tBLASTn program. This revealed an open-reading frame encoding a hypothetical protein with significant homology to the staphylococcal Bap protein. Antibody towards a portion of the identified gene product was produced in rabbits by inoculation of a recombinant peptide to an antigenic region of the protein. Antibody inhibition assays against biofilm formation, adherence, initial adherence and aggregation were conducted. Relative expression levels of the biofilm-associated protein were analyzed under different conditions by western blot analysis. Finally, the protein was expressed in heterologous hosts and analyzed for an increase in biofilm formation.

Two vaginal phylotypes of the genus Megasphaera (phylotype 1 and phylotype 2) were recently associated with bacterial vaginosis (BV), an infection characterized by vaginal dysbiosis. Through an analysis of 16S rRNA profiles of 3,986 women enrolled in the Vaginal Human Microbiome Project, we confirmed that while both phylotypes were associated with BV, Megaspheara phylotype 1 had higher specificity for the condition. Megasphaera phylotype 2 was strongly associated with trichomoniasis. Previous studies have reported that BV-associated organisms are excluded in pregnancy. We observed that Megasphaera phylotype 1, which has been associated with adverse pregnancy outcomes, exhibited a trend of increased prevalence in the pregnant cohort. We sequenced the genomes of isolates of the two phylotypes and performed comparative analyses. We demonstrate that these two phylotypes have distinct genomic features and unique potential for metabolic processes that reveal niche specialization. These findings may provide insight into their differential associations with vaginal infections.

The human vagina is unlike that of any closely related phylogenetic species due to the abundance of a lactic acid producing bacteria, known as Lactobacillus. This microbial species is known for its direct and indirect contributions to vaginal pathogenic defense, some of which include the elicitation of host immunomodulators, release of bacteriocins and biosurfactants, and lowering of vaginal pH. While Lactobacillus is often considered the hallmark of a healthy human vagina, a significant number of women worldwide lack its abundance in their vaginal microbial communities. The lack of Lactobacillus- dominance does not necessarily equate to a disease-state but could potentially explain an increased risk of viral transmission and recurrent vaginal dysbiosis in these women. This thesis aims to investigate the numerous antimicrobial properties associated with the Lactobacillus species to better understand its significance for female reproductive and vaginal health.

Bacterial vaginosis (BV), a common female reproductive tract (FRT) condition characterized by an overgrowth of anaerobic species concurrent with the disappearance of commensal Lactobacilli species, is associated with a 60% increased risk of HIV-1 transmission. However, the role of the FRT epithelia in bacterial vaginosis-associated bacteria (BVAB)-augmented HIV- 1 transmission is unclear. To evaluate the increased risk of HIV-1 acquisition, we treated FRT epithelia with Atopobium vaginae, a prevalent BVAB, to determine the nature of the host response to BVAB exposure. Treatment of endocervical cells with A. vaginae resulted in a 1500-fold increase in the expression of the antimicrobial peptide hBD-2, an inflammatory cytokine response, and delocalization of the tight junction protein ZO-1 from cell borders. Conditioned media (CM) from the coculture of FRT epithelia and A. vaginae also generated an inflammatory immune response and lowered the transepithelial electrical resistance in polarized endocervical monolayers. Changes in HIV-1 infection were measured in TZM-bl reporter cells, which contain a luciferase gene under the control of an HIV-1 long terminal repeat (LTR) region that is activated by the binding of Tat, an HIV-1 protein that drives viral replication. NFκB is a major host-derived transcription factor that regulates the expression of many genes involved in inflammation and the innate immune response. Interestingly, NFκB has been reported to bind Tat-activated response elements within the LTR of HIV-1, driving viral transcription. TZM-bl cells were treated with CM in the absence of HIV-1, which resulted in increased luciferase production that could be suppressed by the NFκB inhibitor TPCA-1. These data suggest that epithelially derived products from the coculture of FRT cells and A. vaginae enhance HIV-1 infection by causing cervical barrier dysfunction and increasing HIV replication efficiency through NFκB.

Background: The composition of the vaginal microbiota (VMB) in the female genital tract (FGT) can impact the vaginal epithelium and protect against or increase risk of sexually transmitted viral infections. The VMB grows as a biofilm, a complex structure formed by bacteria for increased survival. When the VMB consists of a diverse bacterial community it correlates with pathogenic effects that lead to adverse health conditions and an increased risk of HIV infection. When the VMB contains Lactobacillus species, beneficial health effects and decreased susceptibility to infection are observed. The aim of this project is to optimize an in vitro model of biofilm formation for different bacteria associated with the VMB, identify the effects that biofilm has on vaginal epithelial cells and test biofilm treatment strategies. We hypothesize that a Lactobacillus biofilm will enhance barrier function and decrease cytotoxicity of vaginal epithelial cells whereas dysbiotic biofilm will decrease barrier function and induce cytotoxicity. We also hypothesize that various conditions, such as presence of estradiol and eubiotic short-chain fatty acids, will stimulate Lactobacillus biofilm growth and suppress dysbiotic biofilm growth in a vaginal epithelial cell model. Methods: For optimization of the biofilm model, VK2/E6E7 cells were grown in air-liquid interface (ALI) or liquid-liquid interface (LLI) cultures in presence or absence of L. crispatus, L. iners, G. vaginalis or P. bivia bacteria. Biofilm formation was assessed using FilmTracerTM SYPRO® Ruby biofilm matrix protein stain. Hormone effects were tested by adding estradiol (10-9 M) and progesterone (10-7 M) to culture media. Short-chain fatty acid (SCFA) effects were tested by adding lactic acid, acetic acid, succinic acid and butyric acid in varying concentrations to culture media. Enzyme effects were tested by adding sialidase to Vk2 cells before bacteria inoculation. Results: A novel in vitro model of biofilm formation on vaginal epithelial cells was created. Vk2 cells in ALI and LLI cultures remained viable in anaerobic conditions and showed mucin-1 production in aerobic and anaerobic conditions. Matrix protein staining provided a means to accurately visualize and quantify biofilm formation in this model. L. crispatus and L. iners biofilm growth maintained vaginal epithelial barrier integrity without cytotoxicity. G. vaginalis and P. bivia biofilm growth significantly reduced barrier integrity (p=0.0166, p=0.0115) and increased cytotoxicity (p=0.0024, p<0.0001). Estradiol significantly increased the growth of L. crispatus biofilm in the co-culture system (p<0.0001). Progesterone significantly increased G. vaginalis biofilm growth in the Vk2 cell co-culture (p=0.006). L. crispatus biofilm formation in the estradiol condition, G. vaginalis biofilm formation in the progesterone condition and P. bivia biofilm growth in the normal media condition were significantly decreased in the presence of sialidase (p<0.0001, p=0.0001, p=0.0380). Conclusion: A novel in vitro model of biofilm formation on a vaginal epithelial cell line that can be used to visualize and quantify biofilm growth was generated. This model was used to test various strategies for biofilm enhancement or dissociation. Estradiol enhanced beneficial Lactobacillus biofilm growth, while progesterone enhanced dysbiotic biofilm growth. Mucin- digesting enzyme sialidase was effective at dissociating all biofilms. This model can be used in the future to test different strategies of dysbiotic biofilm dissociation and enhancement of Lactobacillus biofilm in order to investigate treatments for Bacterial Vaginosis (BV) and reduce susceptibility to HIV transmission in women. / Thesis / Master of Science (MSc)

No / Objectives: High prevalence of bacterial vaginosis (BV) has been reported in lesbians but most studies were based in sexually transmitted infection clinic settings; therefore, we wished to determine the prevalence and risk factors of BV in lesbians and heterosexual women in a community setting in the UK. Methods: A cross-sectional study recruiting lesbian women volunteers from community groups, events, clubs and bars. Heterosexual women were recruited from a community family planning clinic. They self-swabbed to create a vaginal smear, which was Gram-stained and categorised as BV, intermediate or normal flora. They completed a questionnaire about age, ethnic group, smoking, genital hygiene practices and sexual history. Results: Of 189 heterosexuals and 171 lesbians recruited, 354 had gradeable flora. BV was identified in 43 (25.7%) lesbians and 27 (14.4%) heterosexuals (adjusted OR 2.45, 95% CI 1.25 to 4.82; p¿=¿0.009). Concordance of vaginal flora within lesbian partnerships was significantly greater than expected (27/31 (87%) couples, ¿¿=¿0.63; p<0.001). Smoking significantly increased the risk of BV regardless of sexuality (adjusted OR 2.65; p¿=¿0.001) and showed substantial concordance in lesbian partnerships but less than for concordance of flora. Conclusions: Women who identified as lesbians have a 2.5-fold increased likelihood of BV compared with heterosexual women. The prevalence is slightly lower than clinic-based studies and as volunteers were recruited in community settings, this figure may be more representative of lesbians who attend gay venues. Higher concordance of vaginal flora within lesbian partnerships may support the hypothesis of a sexually transmissible factor or reflect common risk factors such as smoking.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.