Sexually transmitted infections (STIs) increase the risk of acquiring HIV, and STI control is recommended for HIV prevention, particularly in areas of generalized HIV epidemic where the dominant mode of HIV transmission is sexual, such as southern Africa.1–3 Trichomonas vaginalis (T. vaginalis) is the most common nonviral STI in the world, with roughly 170 million cases annually.4 There are an estimated 30 million new cases of T. vaginalis each year in sub-Saharan Africa.4 Though up to 50% of cases of T. vaginalis may be asymptomatic, infection with T. vaginalis may produce an inflammatory response, irritation, discharge, pruritus, and hemorrhagic lesions.4,5 T. vaginalis is an etiological agent of vaginitis in women, urethritis in men,4,5 and had been associated with pelvic inflammatory disease, endometritis, and adverse pregnancy outcomes.6–8
There is an increasing body of epidemiologic evidence indicating that T. vaginalis may increase susceptibility to HIV infection.3,9–13 Most recently, a case–control study among general populations in Zimbabwe and Uganda demonstrated that women infected with T. vaginalis had a nearly 3-fold increased risk of HIV acquisition (odds ratio, 2.74; 95% confidence interval [CI], 1.25–6.00).12 This study was compelling because it established a conclusive temporal relationship between the 2 infections. Likewise, longitudinal studies in Kenya10 and Zaire9 found increased risk of HIV among women infected with T. vaginalis, and 2 well-conducted meta-analyses reached similar conclusions.11,14
Epidemiologic synergy between HIV and STIs has been examined in a number of reviews;2,3,11,15–19 however, epidemiologic studies that have looked at the effect of HIV on STI acquisition are limited. A small number of cross-sectional20,21 and longitudinal studies 22–27 suggest that HIV infection may increase susceptibility to some STIs. If confirmed, these results would be the cause for concern, given that STIs causing an inflammatory response generally result in increased genital shedding of HIV and thus increased likelihood of transmission to HIV-uninfected sexual partners.9,28–30
Despite the coexisting global epidemics of T. vaginalis and HIV, little attention has focused on emerging evidence that T. vaginalis increases susceptibility to, and potentially transmission of, HIV. In this study, we evaluated T. vaginalis infection in the context of a multisite, randomized controlled trial, among women in South Africa and Zimbabwe, to determine if risk of HIV acquisition was increased among women recently infected with T. vaginalis, and if risk of T. vaginalis acquisition was increased among women infected with HIV.
MATERIALS AND METHODS
A general population of women were recruited from September 2003 to September 2005 from 3 trial sites in Harare, Zimbabwe, and Durban and Johannesburg, South Africa to participate in the Methods for Improvement of Reproductive Health study, a randomized controlled trial to assess the effectiveness of the diaphragm and lubricant gel for the prevention of HIV/STI acquisition (ClinicalTrials.gov, NCT00121459). The study protocol was reviewed and approved by ethics committees at the University of California, San Francisco, and at all local collaborating organizations. All women provided written informed consent before initiating study procedures. Details of this study are described elsewhere.31 Briefly, HIV-negative women were recruited and randomized to receive either a diaphragm, lubricant gel and condoms, or condoms only. A total of 4948 women provided data for the HIV endpoint: 2455 in Harare, 1485 in Durban, and 1008 in Johannesburg. A total of 4968 women provided data for the T. vaginalis end point: 2468 in Harare, 1492 in Durban, and 1008 in Johannesburg. Women were between 18 and 49 years, sexually active (≥4 sex acts per month), and not pregnant or planning to become pregnant during trial participation. Women were followed up quarterly between 12 and 24 months, with a median of 21-months follow-up. At each visit, women received product adherence and risk-reduction counselling, treatment of curable STIs, condoms, and resupply of Replens gel (for intervention arm). All women were asked to use condoms at every sex act.
HIV testing was conducted at scheduled quarterly visits using 2 rapid tests, Determine HIV-1/2 (Abbott Laboratories, Tokyo, Japan) and Oraquick (OraSure Technologies, Bethlehem, PA) on samples obtained by finger prick or venipuncture. Discordant and concordant positive results were confirmed using ELISA (Vironostika, Biomerieux, Durham, NC; BioRad, Redmond, WA; or AxSYM HIV Ag/Ab Combo, Abbott Laboratories, Abbott Park, IL).
Women were tested for a range of STIs. Herpes Simplex Virus Type 2 (HSV-2) testing was conducted using ELISA (FOCUS Diagnostics, Cypress, CA). If negative at baseline, HSV-2 testing was conducted at the closing visit and for positive participants retrospective testing was conducted on stored sera to determine timing of infection. A urine sample was provided for Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) testing using DNA PCR (Roche Pharmaceuticals, Branchburg, NJ) at screening and quarterly visits. T. vaginalis DNA was detected from the same urine specimen using an adaptation of this commercial assay as described elsewhere.32 Women positive for any treatable STIs, including C. trachomatis, N. gonorrhoeae, and T. vaginalis, were contacted and scheduled for an interim visit for treatment or were treated at their next scheduled visit. Pelvic examination, urinalysis, and wet mount were conducted at quarterly visits when clinically indicated.
Demographic data were collected at baseline, and data on medical and sexual history, and sexual behavior were collected at baseline and all quarterly follow-up visits in face-to-face interviews, and by Audio Computer-Assisted Self-Interviewing.
A composite variable was created to assess high versus low participant risk behavior, with high-risk behavior defined ≥1 of the following indicators: ever exchanged sex for money, food, drugs, or shelter in the past 3 months; ≥2 sexual partners in the past 3 months; having sex under the influence of drugs/alcohol in the past 3 months; ever injecting drugs; ever having anal sex. Low risk was defined as the absence of all of these indicators. A composite variable was also created to assess high versus low partner risk behavior. High partner risk behavior was defined as the participant reporting ≥1 of the following indicators: ever having had a sexual partner test positive for HIV; suspecting or knowing that a regular partner had other sex partners in past 3 months; having had vaginal sex when the regular partner was under the influence of alcohol/drugs in past 3 months; regular partner was away from home for ≥1 mo/yr. Low risk was defined as the absence of all of these indicators.
Age at baseline was divided into 3 categories based on distribution of data (≤24 years, 25–34 years, ≥35). Education was divided into 2 categories (less than high school education, high school education or more). Living with partner, and circumcision status of regular partner (based on participant report) were also assessed at baseline. Partner change was assessed at each visit, and participants were categorized as having the same primary sexual partner versus several primary partners during the course of study participation.
Timing of incident HIV infection was defined as the first visit with a positive HIV ELISA test, following a negative test. All participants who tested positive for T. vaginalis were treated after the screening visit, and were considered T. vaginalis-negative at study entry. Timing of incident T. vaginalis infection was defined as the first visit with a positive T. vaginalis PCR test after time of enrollment. Current hormonal contraception use was assessed at quarterly visits and was dichotomized (yes/no). To address potential recall bias, we assessed male condom use at last sex (yes/no). Infection with N. gonorrhoeae or C. trachomatis was combined to form a composite cervical STI status variable (positive for either infection or both, vs. no infection). HSV-2 status was also assessed as a time-dependent risk factor.
To establish the risk of HIV acquisition associated with T. vaginalis infection, we used a lagging procedure, looking at T. vaginalis at the visit immediately before the HIV detection visit. If T. vaginalis results were not available from the prior visit, the participant was dropped from the risk set at that event time. The primary analysis compared HIV incidence in the 2 groups (T. vaginalis-positive vs. T. vaginalis-negative at previous visit) using Cox proportional hazard regression. Results were summarized by the estimated hazard ratio (HR) comparing HIV incidence in the T. vaginalis-positive and T. vaginalis-negative groups. There was no difference in either HIV (HR = 1.06, P = 0.64) or T. vaginalis infection (HR = 0.89, P = 0.18) by study arm, and we therefore conducted a pooled analysis combining study arms. To remove the possible effects of confounding, we controlled for study site, study arm, and for the measures discussed above (age, education, living with partner, partner circumcised, partner change, condom use at last sex, participant behavioral risk, primary partner behavioral risk, HSV-2, N. gonorrhea/C. trachomatis, hormonal contraception) in all analyses. Tied event times were handled using the Efron approximation.33 The assumption of proportionality was checked by plotting the negative log of the survival function against log time. Other STIs (HSV-2, N. gonorrhoeae/C. trachomatis) were handled using a similar lagging procedure, looking at infection at the visit immediately before the HIV detection visit. Behavioral time-dependent variables were not lagged.
An identical procedure was used to examine whether HIV infection increased risk of T. vaginalis acquisition. T. vaginalis incidence was defined as the first infection from the time of enrollment. Subsequent T. vaginalis infections were not considered in this analysis due to the small number of recurrent infections (2.0%) in this sample. All statistical analysis was conducted using SAS version 9.1 (SAS Institute, Cary, NC). We considered a P < 0.05 to be significant.
Baseline characteristics are shown in Table 1: 62% of women were ≥25 years, 68% were living with their partner, and 44% had completed high school. Median age at first sex was 18 years (range, 10–31) and the median number of lifetime partners was 1 (range, 1–30). Only 22% of women reported that their partner was circumcised and 20% reported that they did not know if he was circumcised. Overall 11.4% of women were infected with a nonviral STI at baseline, with 3.4% of women infected with T. vaginalis at baseline, and 4.8% infected with N. gonorrhoeae/C. trachomatis.
T. vaginalis infection was common, with 500 women (10%) experiencing at least one incident case over the course of follow-up. Of these, 1.6% had 1 recurrent infection, and 0.4% had >1 recurrent infection during follow-up. The overall incidence rate of T. vaginalis was 6.5/100 women-years. We saw the highest incidence in Durban (8.6/100 women-years). The Harare and Johannesburg sites had rates of 5.4 (P < 0.0001) and 6.6 (P = 0.04)/100 women-years, respectively.
The HIV end point analysis was based on 4948 women, who contributed a total of 7655 years of follow-up. There were 309 HIV seroconversions during the follow-up period, for an overall HIV incidence rate of 4.0/100 women-years. Of these, 252 (82%) women had a T. vaginalis test result available from their previous study visit. Participants with an available T. vaginalis test result did not differ from those without an available T. vaginalis test result with regard to our selected baseline characteristics (data not shown). Participants infected with T. vaginalis were more likely to test positive for HIV at the following visit, compared to participants uninfected with T. vaginalis (HR = 2.60; 95% CI, 1.38–4.91) (Table 2). HSV-2, C. trachomatis/N. gonorrhoea, and young age (≤24 years) were also strongly associated with increased risk of HIV. Primary partner risk behavior was moderately associated with risk of HIV, and living with a partner showed a strong protective effect against HIV. Participant risk behavior was not associated with increased risk of HIV. After controlling for these potentially confounding factors, the elevated risk of HIV associated with T. vaginalis infection remained significant (adjusted HR = 2.05; 95% CI, 1.05–4.02).
The T. vaginalis end point analysis was based on 4968 women, who contributed a total of 7636 years of follow-up. For all 500 participants with an incident T. vaginalis infection, there was confirmation of HIV status at the previous visit. HIV-positive women were significantly more likely to acquire T. vaginalis infection at the following visit, compared to HIV-negative women (HR = 2.57; 95% CI, 1.79–3.68) (Table 3). Other risk factors for incident T. vaginalis infection included HSV-2, C. trachomatis/N. gonorrhoea, and participant high-risk behavior. Having at least a high school education showed a significant protective effect against T. vaginalis. After controlling for potential confounding factors, the increased risk of T. vaginalis infection among HIV-positive women remained significant (adjusted HR = 2.12; 95% CI, 1.35–3.32).
This is the largest study to date to prospectively examine the association between T. vaginalis and HIV infection. This study represents one of the few longitudinal examinations of the risk of HIV acquisition associated with T. vaginalis in a general population of women, and is the first to examine the risk of T. vaginalis acquisition associated with HIV infection among a general population of women. We found that risk of HIV acquisition was increased with previous T. vaginalis infection, and risk of T. vaginalis acquisition was increased with recent HIV infection. These data suggest that control of T. vaginalis may be an important HIV control strategy.
T. vaginalis was a common infection in this population, with 10% of women experiencing at least one incident case over the course of follow-up. We detected >2-fold increased risk of HIV acquisition among women infected with T. vaginalis, after adjusting for potential confounding. The risk of T. vaginalis acquisition was also >2-fold among HIV-positive women, as compared to their HIV-negative counterparts. While other STI infection (HSV2, C. trachomatis/N. gonorrhoea) were risk factors for both T. vaginalis and HIV acquisition, other variables differed between each outcome. Specifically, while more education was protective and participant behavioral risk was a predictor of T. vaginalis infection, these were not significantly associated with HIV acquisition. Young age was a risk factor for HIV while living with a partner had a protective effect against HIV, but not T. vaginalis, acquisition. These differences highlight the importance of disentangling risk associated with sexual behavior from other risk factors that may have a different impact on transmission of HIV compared to other STIs. Additionally, while other STIs were significant risk factors for HIV acquisition, T. vaginalis remained independently associated with HIV risk. This is an important finding given the high prevalence of T. vaginalis compared to other STIs. The combination of (1) high prevalence, (2) independent effect on HIV risk, (3) simplicity of diagnosis, and (4) availability of inexpensive, effective treatment suggest that infection with T. vaginalis is a risk factor that can be effectively reduced in populations at high risk for HIV.
We used highly sensitive methods for measurement of biologic predictors and outcomes, which likely resulted in good precision of our estimate. The large sample size provides ample statistical power to assess the relationship between T. vaginalis and HIV. Furthermore, we conducted our study among a general population of women in 3 cities in southern Africa, with 12 to 24 months of follow up per woman. Thus our findings were robust and are likely to be generalizable to other female populations, although stringent criteria for study entry were applied.
Results of our analysis of T. vaginalis on risk of HIV acquisition are consistent with that of other studies and meta-analyses of this association.9–12,14 Epidemiologic synergy between T. vaginalis and HIV is biologically plausible. STIs including T. vaginalis may opportunistically infect HIV-positive women, and may increase susceptibility and/or persistence of other STIs due to a compromised immune system,19,20 which may explain the observed increased risk of T. vaginalis among HIV-positive women. Infection with T. vaginalis is associated with vaginal inflammation, which elicits a cellular immune response, inducing recruitment of leucocytes including HIV target cells.34–37 It can also cause microscopic mucosal hemorrhaging, compromising the vaginal mucosal barrier, and increasing the quantity of HIV target cells.38,39 Studies have also shown a degradation of secretory leukocyte protease inhibitors associated with T. vaginalis infection, which block HIV cellular attachment.40 This may explain the observed increased risk of HIV acquisition among T. vaginalis-infected women.
We found an adjusted 2.11-fold increased risk of incident T. vaginalis in HIV-positive women. This finding is noteworthy, as this relationship has rarely been examined.21,27 To the best of our knowledge, only one previous study among sex workers in Kenya has identified a trend toward increased risk of T. vaginalis acquisition among HIV-positive women (HR = 1.3; 95% CI, 1.0–1.7).27 In the same study, risk of genital ulcer disease and candidiasis were associated with level of immune suppression, but T. vaginalis was not. We did not collect data on CD4 count or HIV plasma viral load in our study, and therefore cannot examine the relationship with T. vaginalis acquisition.
There are inherent weaknesses in any study of STIs and HIV acquisition. Because both are transmitted by sexual behavior, observational studies to determine the association between T. vaginalis and HIV acquisition will be susceptible to behavioral confounding.11,41 We used a lagging procedure for T. vaginalis and other sexually transmitted confounding factors, looking at the visit immediately before the HIV detection visit, to address the temporal nature of the association with HIV. Syphilis infection was measured only at baseline, where women who showed positive results were then treated, and at the closing visit. As such, we cannot be sure of the timing of infection for those who acquired syphilis during study follow-up and could not accurately control for this; however, just 2.2% of women showed positive results for syphilis at the closing visit. Though self-report and recall bias associated with behavioral measures are always of concern, the fact that different behavioral factors impacted incident T. vaginalis and incident HIV to a different extent gives us greater confidence that we achieved adequate control for sexual behavior. Infectivity per sex act is much higher for bacterial and protozoan STIs than for HIV, so T. vaginalis incidence will be a better indicator of recent individual risk behavior than HIV, which may take months or even years of exposure before infection occurs. In our study, we found high participant behavioral risk to be highly associated with T. vaginalis acquisition. We found that high partner behavioral risk increased risk of HIV acquisition, and living with the partner had a highly protective effect against HIV. These findings underscore what has been repeatedly shown in other studies, that for HIV (which has low infectivity per sex act), and in a general population of African women where individual risk behavior is low, most risk for HIV acquisition comes from women's partner's risk behavior, and not from their own.42–44 In previous studies, BV has been shown to be associated with T. vaginalis infection,12 therefore concomitant BV may have contributed to the risk of HIV acquisition or to T. vaginalis infection among HIV-positive women in our study. However, data on BV infection were not collected systematically, and therefore we could not control for this in our analysis.
STI coinfection in HIV-positive women is associated with increased genital HIV shedding, and hence infectivity,9,29,45–47 therefore women infected with T. vaginalis may be both at increased risk of acquisition and transmission of HIV. This bidirectional relationship between T. vaginalis and HIV represents a potentially important factor in sustaining the HIV epidemic in populations where T. vaginalis is endemic. In addition, T. vaginalis infection itself is associated with a range of reproductive health issues. T. vaginalis is easily treated and drugs are inexpensive and generally available. Effective control of this common infection is therefore recommended as part of a comprehensive HIV prevention package.
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