Amalia Meier

Influence of the Normal Menstrual Cycle on Vaginal Tissue, Discharge, and Microflora

The objective of this study was to examine genital tissue, vaginal fluid, and vaginal microbial flora at 3 phases of the menstrual cycle in asymptomatic women. Vaginal examinations were performed 3 times in 74 women: at the menstrual phase (days 1-5), the preovulatory phase (days 7-12), and the postovulatory phase (days 19-24). Flora of 50 women without bacterial vaginosis (BV) was analyzed separately from flora of 24 women with BV. The volume of vaginal discharge increased and the amount of cervical mucus decreased over the menstrual cycle. Among subjects without BV, the rate of recovery of any Lactobacillus changed little (range, 82% to 98%; P = .2); however, a small increase occurred in the rate of recovery of heavy (3+ to 4+ semiquantitative) growth of Lactobacillus over the menstrual cycle (P = .04). A linear decrease occurred in the rate of recovery of heavy growth of any non-Lactobacillus species, from 72% at days 1-5 to 40% at days 19-24 (P = .002). A linear decrease also occurred in the rate of recovery of Prevotella species, from 56% on days 1-5 to 28% on days 19-24 (P =. 007), while a small linear increase occurred in the rate of recovery of Bacteroides fragilis (P=.05). Among subjects with BV, the only significant change was an increase in the rate of recovery of Lactobacillus, from 33% at days 1-5 to 54% at days 19-24 (P = .008). Among all subjects, the rate of recovery of heavy growth of Lactobacillus increased over the menstrual cycle and, in contrast, the concentration of non-Lactobacillus species tended to be higher at menses, which is evidence that the vaginal flora becomes less stable at this time.

Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR

ABSTRACT The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P < 0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The MasterPure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Depomedroxyprogesterone-induced hypoestrogenism and changes in vaginal flora and epithelium ☆

Objective To identify the effects of depomedroxyprogesterone acetate (DMPA) on vaginal microbial flora and epithelium. Methods Women who desired DMPA for contraception were evaluated before and at 3 and 6 months after initiation of 150-mg DMPA injections every 3 months. At each visit, we assessed genital symptoms, vaginal signs, vaginal micro-flora, and histopathology by vaginal biopsies. Results Among 38 women observed for 6 months, there was significant reduction in mean serum estradiol level (99.9 ± 9.3 pg/mL to 26.6 ± 1.6 pg/mL, P < .001). The number of subjects with any Lactobacillus did not change, but the number with hydrogen peroxide (H2O2)–positive Lactobacillus decreased from 20% before to 12% after 6 months of DMPA (P = .005). The log concentration in colony-forming units per milliliter of vaginal fluid of H2O2–positive Lactobacillus decreased in a linear manner from 4.0 ± 0.6 at baseline to 2.5 ± 0.6 after 6 months of DMPA use (P = .006). The mean number of cell layers in the epithelium was reduced slightly from 28.1 ± 0.7 to 25.9 ± 0.9 (P = .05), epithelial thickness decreased from 1.02 ± 0.04 mm to 0.89 ± 0.05 mm (P = .005), and the glycogen-positive thickness decreased from 0.81 ± 0.04 mm at baseline to 0.66 ± 0.05 after 6 months of DMPA use (P = .005). Conclusion Depomedroxyprogesterone acetate produced a systemic hypoestrogenic state associated with decreased H2O2–positive Lactobacillus colonization and slight thinning of the glycogen vaginal epithelial layer. Such changes possibly compromise the vaginal barrier to infection.

Immunoepidemiologic Profile of Chlamydia trachomatis Infection: Importance of Heat-Shock Protein 60 and Interferon-γ

Epidemiological, animal, and in vitro investigations suggest that Chlamydia trachomatis infection engenders acquired immunity, the basis for which is incompletely defined, especially in humans. In a prospective cohort study of women at high risk for C. trachomatis infection, we found that, at baseline and after adjustment for age and other potential confounding variables, production of interferon- gamma by peripheral-blood mononuclear cells (PBMCs) stimulated with chlamydia heat-shock protein 60 strongly correlated with protection against incident C. trachomatis infection. This investigation supports a direct role for C. trachomatis-specific immune responses in altering the risk of infection and suggests immune correlates of protection that are potentially useful in vaccine development.

Diversity of the CD8 + T-cell response to herpes simplex virus type 2 proteins among persons with genital herpes

ABSTRACT Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8+ T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/106 CD8+ T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8+ T-cell response within individuals is associated with the frequency of viral reactivation warrants further study.

Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya.

Objective: The objective of this study was to assess the risk factors for and persistence of Mycoplasma genitalium (MG) in a highly exposed female population in Kenya. Study Design: Two hundred fifty-eight sex workers in Nairobi, Kenya, 18 to 35 years of age, were enrolled. Every 2 months, cervical samples were collected for MG, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC) testing by polymerase chain reaction. Results: At enrollment, 16% were infected with MG. Seventy-seven subjects acquired 107 MG infections, giving an incidence of 22.7 per 100 women-years. Incident CT (adjusted hazard ratio [HR] = 2.4; 95% confidence interval [CI] = 1.5–4.0), GC (HR = 2.0; 95% CI = 1.2–3.5), and HIV infection (adjusted HR = 2.2; 95% CI = 1.3–3.7) were associated with an increased risk of MG. Seventeen percent, 9%, and 21% of MG infections persisted 3, 5, and ≥7 months, respectively. Conclusion: The high incidence of MG, greater than that for both CT (14.0%) and GC (8%), association with common sexually transmitted infection risk factors, and persistence in the female genital tract supports its role as a common sexually transmitted infection in Kenyan women.

Effects of Vaginal Intercourse with and without a Condom on Vaginal Flora and Vaginal Epithelium

Effects of a single episode of intercourse on vaginal flora and epithelium were examined in subjects randomly assigned to groups that used no condom or lubricated nonspermicide condoms. Subjects were evaluated at visits before (1 month and 1-2 days) and after (8-12 h, 2-3 days, and 6-8 days) an index episode of sexual intercourse. The 22 subjects who used no condoms had significantly more Escherichia coli and a high concentration (> or =10(5) cfu/mL) of E. coli in the vagina (both, P<.001) and urine (all <10(5) cfu/mL; P=.004) at visit 3 than at visits 1 and 2. The 20 subjects who used condoms had a trend toward more vaginal E. coli (P=.06) and a significant increase in other enteric gram-negative rods (P=.001) after intercourse. Intercourse was not associated with gross, colposcopic, or histologic vaginal epithelial abnormalities.

Bacterial Vaginosis: Risk Factors Among Kenyan Women and Their Male Partners

Objectives: To simultaneously examine associations of bacterial vaginosis (BV) with potential risk factors in both the female and her male partner. Study Design: We recruited women 18–45 years of age and their male partners from clinics in Nairobi, Kenya. All underwent face-to-face standardized interview physical examination, human immunodeficiency virus (HIV)-1 and syphilis serologic testing, endocervical cultures for Neisseria gonorrhoeae, and vaginal swabs for diagnosis of BV by Gram stain and trichomoniasis by culture. Results: Of 219 women, 97 (44%) had BV. BV was significantly associated by univariate analyses with women’s own risk factors (young age, being unmarried, early sexual debut, more than 1 sexual partner, lifetime, rectal sex, trichomoniasis, HIV infection, and by principal components analysis, with low socioeconomic status [SES]) and also with male partners’ characteristics (HIV infection, and by principal components analysis, low SES, and poor hygiene). In multivariate analysis including risk factors from both genders, the odds of having BV was 5.7 times higher if either partner was HIV seropositive, 13.2 times higher if the female had trichomoniasis, 2.5 times higher if the female had more than 1 sex partner ever, and decreased with increasing age of the female. Conclusion: In this population, characteristics of males and of females were independently associated with BV. Close association of male hygiene and male HIV status precluded distinguishing the influence of male hygiene on partner’s BV.

Effects of oral contraceptive pill use on vaginal flora and vaginal epithelium.

The objective of this study was to examine the effect of oral contraceptive (OC) use on vaginal discharge, epithelium, and flora. Thirty women who planned to use OC for contraception were evaluated before and 2 months after the start of OC use. At both visits, genital symptoms and exposures were assessed by questionnaire; vaginal signs were assessed by speculum examination and colposcopy; vaginal microflora was evaluated by quantitative culture; and a vaginal biopsy was obtained for histopathologic evaluation. Variables were compared between the initial visit and after 2 months of OC use. It was found that OC use did not change the gross, colposcopic, or histologic appearance of the vaginal epithelium or characteristics of vaginal or cervical discharge. Vaginal flora essentially remained unchanged after 2 months of OC use, except that a small decrease occurred in the number of subjects with > or =10(5) colony forming units/mL of H(2)O(2) producing Lactobacillus from 16 at baseline to 9 (p = 0.04) and in the total number of subjects with Ureaplasma urealyticum from 17 at baseline to 10 of 29 (p = 0.04). The results indicate minimal effect of OC use on the vaginal epithelium and vaginal and cervical discharge, and a small effect on vaginal flora.

Oral herpes simplex virus type 2 reactivation in HIV-positive and -negative men.

BACKGROUND Previous studies using viral cultures rarely reported herpes simplex virus type 2 (HSV-2) isolation from the mouth. We sought to characterize oral HSV-2 shedding as detected by HSV DNA polymerase chain reaction among HSV-2-seropositive men. METHODS Participants collected daily swabs from oral and anogenital areas for HSV detection with a quantitative polymerase chain reaction assay. RESULTS A total of 109 HSV-2-seropositive men (59 of whom were human immunodeficiency virus [HIV] negative, and 50 of whom were HIV positive) were sampled for a median of 64 consecutive days. Forty-four (40.4%) had HSV-2 detected from oral swabs on at least 1 day. Oral HSV-2 was detected on 148 (2.3%) of 6,422 days, genital HSV-2 was detected on 1,110 (17%) of 6,505 days, oral HSV-1 was detected on 220 (5.5%) of 4,018 days, and genital HSV-1 was detected on 88 (2.2%) of 4,073 days. Oral HSV-2 shedding was never associated with an oral lesion, but it was often concurrent with genital HSV-2 shedding. Both oral and genital HSV-2 were detected on 90 (61%) of 148 days with oral HSV-2 shedding. Oral HSV-2 shedding occurred on 90 (8.2%) of 1,110 days with genital HSV-2 shedding, versus 58 (1.1%) of 5,316 days without genital HSV-2 shedding (P<.001). The HIV-positive men shed HSV-2 orally more frequently than did the HIV-negative men (odds ratio, 2.7 [95% confidence interval, 1.1-7.1]). CONCLUSIONS Oral HSV-2 reactivation was common (especially among HIV-positive men), was always asymptomatic, and often occurred on days of genital HSV-2 reactivation.