PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Sci Transl Med. Author manuscript; available in PMC Oct 6, 2011.
Published in final edited form as:
PMCID: PMC3087186
NIHMSID: NIHMS283752
Genital HIV-1 RNA Quantity Predicts Risk of Heterosexual HIV-1 Transmission
Jared M. Baeten, MD, PhD,1,2,3 Erin Kahle, MPH,1,3 Jairam R. Lingappa, MD, PhD,1,2,4 Robert W. Coombs, MD, PhD,2,5 Sinead Delany-Moretlwe, MD, PhD,6 Edith Nakku-Joloba, MBChB, PhD,7 Nelly R. Mugo, MBChB, MPH,1,8 Anna Wald, MD, MPH,2,3,5,9 Lawrence Corey, MD,2,3,5,9 Deborah Donnell, PhD,1,10 Mary S. Campbell, MD,2 James I. Mullins, PhD,2,5 and Connie Celum, MD, MPH1,2,3, for the Partners in Prevention HSV/HIV Transmission Study Team*
1 Department of Global Health, University of Washington, Seattle, USA
2 Department of Medicine, University of Washington, Seattle, USA
3 Department of Epidemiology, University of Washington, Seattle, USA
4 Department of Pediatrics, University of Washington, Seattle, USA
5 Department of Laboratory Medicine, University of Washington, Seattle, USA
6 Wits Institute for Reproductive Health and HIV, University of the Witwatersrand, Johannesburg, South Africa
7 Department of Epidemiology and Biostatistics, Makerere University, Kampala, Uganda
8 Department of Obstetrics and Gynaecology, University of Nairobi, Nairobi, Kenya
9 Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, USA
10 Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, USA
Correspondence to: Jared Baeten, University of Washington, Box 359927, 325 Ninth Ave, Seattle, WA 98104, Tel – 206-520-3808, Fax – 206-520-3831, jbaeten/at/u.washington.edu
*The Partners in Prevention HSV/HIV Transmission Study team members are listed under Acknowledgements
High plasma HIV-1 RNA concentrations are associated with an increased risk of HIV-1 transmission. Although plasma and genital HIV-1 RNA concentrations are correlated, no study has evaluated the relationship between genital HIV-1 RNA and the risk of heterosexual HIV-1 transmission. In a prospective study of 2521 African HIV-1 serodiscordant couples, we assessed genital HIV-1 RNA quantity and HIV-1 transmission risk. HIV-1 transmission linkage was established within the partnership by viral sequence analysis. We tested endocervical samples from 1805 women, including 46 who transmitted HIV-1 to their partner, and semen samples from 716 men, including 32 who transmitted HIV-1 to their partner. Genital and plasma HIV-1 concentrations were correlated: For endocervical swabs, Spearman’s rank correlation coefficient rho was 0.56 (p<0.001), and for semen rho was 0.55 (p<0.001). Each 1 log10 increase in genital HIV-1 RNA was associated with a 2.20-fold (for endocervical swabs, 95% confidence interval 1.60–3.04, p<0.001) and a 1.79-fold (for semen, 95% confidence interval 1.30–2.47, p<0.001) increased risk of HIV-1 transmission. Genital HIV-1 RNA independently predicted HIV-1 transmission risk after adjusting for plasma HIV-1 quantity (hazard ratio 1.67 for endocervical swabs and 1.68 for semen). Seven female-to-male and four male-to-female HIV-1 transmissions (incidence <1% per year) occurred from persons with undetectable genital HIV-1 RNA, but in all eleven plasma HIV-1 RNA was detected. Thus, higher genital HIV-1 RNA concentrations are associated with greater risk of heterosexual HIV-1 transmission, and this effect was independent of plasma HIV-1 concentrations. These data suggest that HIV-1 RNA in genital secretions could be used as a marker of HIV-1 sexual transmission risk.
Many studies have measured HIV-1 concentrations in semen, cervicovaginal, and anorectal secretions to assess the infectiousness of HIV-1 transmitted sexually [13]. It is predicted that higher genital HIV-1 concentrations reflect greater HIV-1 infectivity. First, plasma HIV-1 levels predict sexual and perinatal HIV-1 transmission risk [1, 4]. Second, higher cervicovaginal HIV-1 concentrations among HIV-1 infected pregnant women have been associated with increased risk of perinatal HIV-1 transmission [5, 6]. Third, factors that heighten sexual HIV-1 transmission risk in epidemiological studies, such as genital tract infections resulting in inflammation of the genital mucosa, increase genital HIV-1 [713], and factors that decrease HIV-1 transmission risk, such as antiretroviral therapy (ART) [14], decrease genital HIV-1 [15]. However, although plasma and genital HIV-1 concentrations are correlated, higher variability in mucosal versus blood plasma HIV-1 and discordance between mucosal and plasma HIV-1 in some individuals has raised questions regarding whether genital HIV-1 concentrations can predict the risk of HIV-1 sexual transmission [1, 2, 1618].
To date, no prospective study has assessed whether genital HIV-1 concentrations correlate with HIV-1 sexual transmission risk. Ninety percent of new HIV-1 infections worldwide are transmitted sexually, and a greater understanding of the biological mechanisms underlying HIV-1 infectiousness is needed. In a prospective cohort of heterosexual African HIV-1 serodiscordant couples, we evaluated the relationship between genital HIV-1 quantity in the HIV-1 infected partner and HIV-1 transmission risk.
Of 3408 HIV-1 serodiscordant couples enrolled in the trial, 2521 (74.0%), including 1805 of 2299 couples with HIV-1 seropositive women (78.5%) and 716 of 1109 couples with HIV-1 seropositive men (64.6%), provided genital samples for HIV-1 RNA quantification and were included in this analysis. The median age was 32 years (interquartile range [IQR] 27–38) for HIV-1 infected partners and 34 years (IQR 28–41) for HIV-1 uninfected partners (Table 1). Most couples were married and cohabiting. The median monthly frequency of sex was four times (IQR 2–8), and 28.6% of couples reported unprotected sex during the month prior to enrollment. Among HIV-1 infected participants, the median CD4+ T cell count was 469 cells/mm3 (IQR 350–638) and the median plasma HIV-1 RNA concentration was 4.0 log10 copies/mL (IQR 3.3–4.6). There was no statistically significant difference in plasma HIV-1 RNA concentrations or CD4+ T cell counts for those who provided versus those who did not provide a genital sample.
Table 1
Table 1
Enrollment characteristics
Follow-up and HIV-1 seroincidence
During 3509 person-years of follow-up for assessment of HIV-1 seroincidence among the 2521 HIV-1 seronegative partners included in this analysis, 113 partners (73 men and 40 women) seroconverted to HIV-1 (incidence 3.2 per 100 person-years). Median follow-up was 18 months (IQR 12–24 months). Of the 113 incident HIV-1 infections, 78 (69.0%), including 46 among men (63.0%) and 32 among women (80.0%), were determined by viral sequencing to be linked within the partnership; these frequencies are similar to the study population as a whole (68.9% linked in the overall cohort) [19].
Detection and quantity of genital HIV-1 RNA
HIV-1 RNA was detected in 59.9% of endocervical swab samples and 56.5% of semen samples (Table 2); median HIV-1 concentrations were 3.20 log10 copies/swab for endocervical samples and 2.57 log10 copies/mL in semen samples. Genital HIV-1 concentrations were significantly lower among those randomized to receive the HSV-2 suppressive drug acyclovir versus placebo: median 2.98 vs. 3.29 log10 copies/swab for endocervical swabs (p<0.001) and 2.38 vs. 2.76 log10 copies/mL for semen (p=0.008).
Table 2
Table 2
Genital and plasma HIV-1 RNA concentrations
Genital HIV-1 RNA concentrations were correlated with plasma HIV-1 levels measured at the closest visit. For 99.6% of endocervical and 63.7% of semen samples, a concurrent plasma sample was collected for HIV-1 RNA quantification; for most of the remainder, a plasma sample for HIV-1 RNA quantification was available within 6 months of collection of the genital sample. Spearman’s rank correlation coefficient (Spearman’s rho) was 0.56 (p<0.001) among women and 0.55 (p<0.001) among men; the correlation was the same (Spearman’s rho 0.55, p<0.001) when restricted to those semen samples that had concurrent plasma HIV-1 RNA results. By linear regression, each 1 log10 copies/mL increase in plasma HIV-1 RNA was associated with a 0.52 log10 copies/swab increase in endocervical HIV-1 RNA (95% confidence interval [CI] 0.48–0.56, p<0.001) and a 0.46 log10 copies/mL increase in semen HIV-1 RNA (95% CI 0.40–0.52, p<0.001).
For the 46 genetically-linked female-to-male HIV-1 transmission events, the median time from endocervical swab collection to the visit at which HIV-1 seroconversion was detected was 5.7 (IQR 0–8.9) months, with 11 (23.9%) samples collected at the same study visit as seroconversion was detected and an additional 20 (43.5%) samples collected within 3 months of the seroconversion visit. Twenty (43.5%) seroconversions occurred after collection of the swab sample. For the 32 male-to-female linked HIV-1 transmission events, the median time from semen sample collection to HIV-1 seroconversion was 3.0 (IQR 0–6.1) months, with 4 (12.5%) samples collected at the same study visit as seroconversion and an additional 14 (43.8%) samples collected within 3 months of the seroconversion visit. Thirteen (40.6%) seroconversions occurred after collection of the semen sample.
Genital HIV-1 concentrations and HIV-1 transmission risk
Genital HIV-1 levels were significantly higher among those who did versus those who did not transmit HIV-1: median 3.89 versus 3.18 log10 copies/swab for endocervical swabs (p<0.001) and 3.44 versus 2.54 log10 copies/mL for semen (p<0.001). A strong step-wise relationship between genital HIV-1 quantity and HIV-1 transmission incidence was observed (Figure 1); a similar step-wise effect was seen for the relationship between plasma HIV-1 RNA and HIV-1 transmission incidence. In a Cox proportional hazards model, each 1.0 log10 increase in genital HIV-1 RNA was associated with an approximately 2-fold greater risk of HIV-1 transmission (Table 3): hazard ratio [HR] 2.20, p<0.001 per log10 copies/swab increase in endocervical HIV-1 RNA and risk of female-to-male HIV-1 transmission and HR 1.79, p<0.001 per log10 copies/mL increase in semen HIV-1 RNA and risk of male-to-female HIV-1 transmission. This effect of genital HIV-1 RNA concentration remained statistically significant after adjustment for plasma HIV-1 RNA levels and for demographic and clinical characteristics in multivariate analysis. In the final multivariate models, each 1.0 log10 increase in genital HIV-1 RNA increased the risk of female-to-male HIV-1 transmission 1.67-fold (p=0.02) and the risk of male-to-female HIV-1 transmission 1.68-fold (p=0.02). Higher plasma HIV-1 RNA concentrations were associated with increased HIV-1 transmission risk, although only the effect on female-to-male transmission was statistically significant (HR 2.16 per log10 copies/mL increase, p=0.001), whereas the male-to-female HIV-1 transmission effect was not statistically significant (HR 1.38 per log10 copies/mL increase, p=0.2) in multivariate analysis. Thus, plasma and genital HIV-1 RNA concentrations independently predicted female-to-male HIV-1 transmission risk but plasma HIV-1 RNA was not significantly associated with male-to-female transmission risk after adjustment for seminal HIV-1 RNA quantity.
Figure 1
Figure 1
Step-wise association between genital and plasma HIV-1 RNAquantity and HIV-1 transmission risk
Table 3
Table 3
Genital HIV-1 RNA predicts HIV-1 transmission risk independent of plasma HIV-1 RNA.
We performed two sensitivity analyses to assess the contribution of timing of genital sample collection to our findings. First, we considered only follow-up time after collection of genital specimens for HIV-1 RNA quantification as genital samples were not collected at study enrollment. We found similar findings to the overall results: multivariate HR 2.38 (95% CI 1.13–4.78) per log10 copies/swab increase in endocervical HIV-1 RNA and the risk of female-to-male HIV-1 transmission, and multivariate HR 2.89 (95% CI 1.03–8.11) per log10 copies/mL increase in semen HIV-1 RNA and risk of male-to-female HIV-1 transmission. Second, we analyzed only those transmitting couples who had a genital sample collected within 3 months of HIV-1 seroconversion, and the results were again similar to those overall: multivariate HR 1.88 (95% CI 1.11–3.19) per log10 copies/swab increase in endocervical HIV-1 RNA and the risk of female-to-male HIV-1 transmission, and multivariate HR 1.81 (95% CI 1.04–3.14) per log10 copies/mL increase in semen HIV-1 RNA and risk of male-to-female HIV-1 transmission.
Seven of 46 (15.2%) female-to-male HIV-1 transmissions occurred from women with undetectable endocervical HIV-1 RNA concentrations. HIV-1 incidence among the 724 couples in which the women had undetectable HIV-1 RNA concentrations was 0.6 per 100 person-years (95% CI 0.2–1.2). Four of 32 (12.5%) male-to-female HIV-1 transmissions occurred from men with undetectable semen HIV-1 RNA concentrations; HIV-1 transmission from the 311 men who had undetectable semen HIV-1 RNA concentrations was 0.8 per 100 person-years (95% CI 0.2–1.8). For these 11 transmissions, the median time between collection of the genital sample and HIV-1 seroconversion was 4.6 months (range 0–16.3) months and all had detectable plasma HIV-1 RNA at the visit closest to collection of the genital sample (median 4.4 log10 copies/mL, range 2.4–5.9).
Our data provide empirical evidence that differences in genital tract concentrations of HIV-1 influence the transmission risk of HIV-1 infection, and we found that this relationship was independent of plasma HIV-1 concentration. Our large sample size of heterosexual African HIV-1 serodiscordant couples and prospective follow-up with collection of genital samples prior to HIV-1 transmission permitted analyses demonstrating that the concentration of HIV-1 RNA in endocervical and seminal samples from HIV-1 infected individuals was strongly correlated with risk of HIV-1 transmission to their HIV-1 susceptible sexual partners. Genomic analysis of HIV-1 isolates to confirm HIV-1 transmission within the study partnerships further strengthens our findings. These data support the concentration of HIV-1 RNA in genital secretions as a marker of HIV-1 sexual transmission risk.
The first studies of genital HIV-1 using viral culture provided qualitative evidence for infectious virus in genital secretions as a mechanism for HIV-1 transmission [20, 21]. Subsequent studies have used nucleic acid amplification to quantify genital HIV-1, with results suggesting that higher genital HIV-1 levels are likely to be a measure of increased HIV-1 infectiousness [1]. Higher plasma HIV-1 levels, genital tract infections, and advanced HIV-1 disease have been associated with increased genital HIV-1 levels [2]. In prospective interventional studies with pre- and post-treatment genital tract samples, cure of sexually transmitted infections and initiation of ART significantly reduced genital HIV-1 RNA concentrations [79, 15, 22]. However, to demonstrate that genital HIV-1 levels predict risk of HIV-1 sexual transmission required longitudinal studies of HIV-1 infected persons and their initially uninfected partners. The establishment of such cohorts has been logistically challenging [1]. Only one previous case-control study, among men who have sex with men, assessed the relationship between genital HIV-1 RNA concentrations and risk of HIV-1 sexual transmission. This study reported that plasma and seminal fluid HIV-1 RNA concentrations in 15 transmitting partners were significantly higher than in 32 non-transmitting partners [23].
We found a step-wise association between genital HIV-1 levels and HIV-1 incidence, with an approximately two-fold increased risk for each one log10 increase in genital HIV-1. This was comparable to the association between endocervical HIV-1 RNA and female-to-male HIV-1 transmission, and seminal HIV-1 RNA and male-to-female transmission. We also found that plasma HIV-1 RNA quantity predicted HIV-1 transmission risk in a similar step-wise manner. This linear risk relationship between log10 HIV-1 RNA concentrations and HIV-1 outcomes has been previously reported for systemic HIV-1 concentrations versus both sexual and perinatal HIV-1 transmission [4, 24], including in the Ugandan study that first demonstrated that higher blood HIV-1 concentrations resulted in increased heterosexual infectiousness [4], as well as for plasma HIV-1 levels as a predictor of the risk of HIV-1 clinical progression to AIDS. The consistency of this relationship raises the question of whether the log10 quantity is a fundamental pathogenic property of the virus, although discerning the precise biologic mechanisms is not possible with the samples we tested for this study.
We observed a small number of HIV-1 transmission events (annual incidence <1%) among couples in which the HIV-1 infected partner had genital HIV-1 levels below the limit of quantification. Importantly, plasma HIV-1 was detectable for all 11 persons with undetectable genital HIV-1 concentrations who transmitted HIV-1 to their partners. The reason for this could be that a single assessment of genital HIV-1 burden may miss intermittent shedding of genital virus [16, 25].
In our study, as in multiple previous studies, plasma and genital HIV-1 concentrations were only modestly correlated [1]. We found that genital HIV-1 concentrations remained independently associated with HIV-1 transmission risk after adjustment for plasma HIV-1 levels, as well as other clinical and behavioral factors. Genital HIV-1 levels display greater variability than do plasma HIV-1 levels [13]; greater variability in the measurement of genital versus plasma HIV-1 would not alter the accuracy of our findings (i.e., the point estimate of risk of HIV-1 transmission versus log10 genital HIV-1 levels) but would contribute to the precision of the estimate (i.e., the width of the confidence intervals). Recent work suggests that genital HIV-1 levels, like those in plasma, establish a relatively stable set point after acute infection [18]. Thus, a single measurement, as done in this study, may provide a useful biomarker of HIV-1 infectiousness, particularly given the challenges of obtaining repeat genital HIV-1 measurements in large studies. HIV-1 replication may be different at genital mucosal sites compared to other sites that contribute virus to the blood, potentially due to genital tract infections or local immunological factors [1, 18, 2629]. Thus, genital HIV-1 levels, as potentially the most relevant and proximate marker of HIV-1 exposure for sexual HIV-1 transmission, may predict HIV-1 risk as well as or better than plasma HIV-1 concentrations alone. We found that only genital HIV-1 levels in men were statistically related to HIV-1 transmission risk in a model that included both genital and plasma HIV-1 RNA concentrations, whereas for women both blood plasma and genital HIV-1 RNA were independently predictive. These findings could reflect the biology of menses and the contribution of blood HIV-1 to the female genital tract, which is not a consideration for men. More limited statistical power for our analysis of male-to-female HIV-1 transmission (given a smaller number of HIV-1 infected men compared to women in our study population) may also explain these findings. Future studies of genital HIV-1 should explore characteristics of those variants that are transmitted, including genetic sequence differences, viral fitness, and whether the source of transmitted virus is cell-free or cell-associated HIV-1 [1, 30].
We found that acyclovir reduced genital HIV-1 levels by ~0.3 log10, a result that was statistically significant and similar to prior studies of HSV-2 suppressive therapy [13]; in our trial, acyclovir reduced plasma HIV-1 levels by 0.25 log10 copies/mL but did not reduce HIV-1 transmission [19]. We recently estimated that a nearly 0.75 log10 copies/mL reduction in plasma HIV-1 RNA would be necessary to decrease HIV-1 transmission by 50% [24]. Thus, interventions that greatly reduce HIV-1 levels, like ART, are likely to have more substantial effects on HIV-1 transmission risk than interventions that reduce HIV-1 concentrations minimally.
We only collected one sample per study participant for genital HIV-1 quantification. Repeat measurements might have increased precision in our regression estimates as the variability in HIV-1 concentrations is greater in genital samples than in plasma samples [2]. However, in spite of this potential for improvement in analytical precision, we still observed a strong relationship between genital HIV-1 levels and HIV-1 transmission risk. Previous studies of genital HIV-1 have collected a single or a small number of genital samples per individual to measure the effect on genital HIV-1 shedding of interventions aimed at decreasing HIV-1 infectiousness, including HSV-2 suppressive therapy [9, 11], treatment of curable STIs [7, 8, 22], and initiation of ART [15]. Our results confirm that a single measurement of genital HIV-1 quantity is a strong surrogate marker of HIV-1 transmission risk, and suggest that the potential impact of new interventions aimed at reducing HIV-1 transmission can be assessed through studies of genital HIV-1 RNA. With >2500 participants, this is the largest study of genital HIV-1 in African persons.
A limitation of this study is that some HIV-1 transmission events occurred before or several months after collection of the genital sample. However, the median time from acquiring the genital sample to HIV-1 seroconversion was less than six months and for the majority of HIV-1 transmission events the genital sample was collected before or at the time of seroconversion. Sensitivity analyses assessing the timing of genital sample collection relative to HIV-1 transmission and the collection of a plasma sample for HIV-1 RNA quantification generated results similar to those from the analysis of all participants. Etiological screening for sexually transmitted infections was done at study enrollment and not when genital HIV-1 RNA samples were collected. Finally, HIV-1 infected partners were also HSV-2 seropositive. HSV-2 is common among persons with HIV-1 (seroprevalence 50–90%) [31], and thus this is unlikely to limit the generality of our findings.
Understanding the relationship between genital HIV-1 replication and the risk of HIV-1 transmission is central to describing the fundamental biological mechanisms underlying HIV-1 transmission. ART and other potential new interventions (such as HIV-1 vaccines) that reduce systemic and genital HIV-1 replication, and interventions that reduce genital HIV-1 concentrations alone (such as treatment of genital tract infections and antiretroviral-based microbicides) should continue to be evaluated for their potential to reduce HIV-1 transmission. Genital sampling should be used to quantify the potential reduction in HIV-1 transmission risk of interventions that are directed at reducing the infectiousness of persons with HIV-1.
Population and Procedures
Between November 2004 and April 2007, heterosexual HIV-1 serodiscordant couples were enrolled from 14 sites in 7 African countries (Botswana, Kenya, Rwanda, South Africa, Tanzania, Uganda, and Zambia) in a randomized, placebo-controlled, clinical trial of herpes simplex virus type 2 (HSV-2) daily suppressive acyclovir therapy for prevention of HIV-1 transmission (Clinicaltrials.gov number NCT00194519) [32]. Follow-up was for up to 24 months per couple; some couples were followed for less than 24 months due to scheduled study closure. All study follow-up was completed by October 2008. HSV-2 suppressive therapy, provided to the HIV-1 infected members of the couples, failed to reduce HIV-1 transmission, in spite of an average 0.25 log10 copies/mL reduction in plasma HIV-1 levels [19].
Couples were eligible if both members were ≥18 years of age and if they reported ≥3 episodes of vaginal intercourse during the three months prior to enrollment. HIV-1 infected partners were HIV-1 and HSV-2 seropositive, not using ART, and had a CD4 T cell count of ≥250 cells/mm3 and no history of AIDS-defining conditions.
HIV-1 infected partners were seen monthly. At the time the study was conducted, national guidelines generally recommended ART initiation at CD4 T cell counts of less than 200–250 cells/mm3 or in persons with clinical AIDS. HIV-1 infected persons who met national guidelines for initiation of ART during follow-up, as a result of CD4 T cell decline or change in clinical status, were referred to local HIV-1 care clinics to start ART. HIV-1 infected men provided one semen sample for HIV-1 RNA quantification at any visit ≥3 months after enrollment. HIV-1 infected women underwent a speculum pelvic examination at a visit six months after enrollment during which an endocervical Dacron swab for HIV-1 RNA quantification was obtained; swabs were not collected at a defined time in the menstrual cycle, although women usually deferred sampling when they were menstruating. HIV-1 uninfected partners were seen quarterly for risk assessment and tests for HIV-1 antibodies.
All participants received pre- and post-test HIV-1 counseling, risk reduction counseling (individually and as a couple), free condoms, and treatment of sexually transmitted infections according to WHO guidelines. The study protocol was approved by the University of Washington Human Subjects Review Committee and ethical review committees at each collaborating organization. All participants provided written informed consent.
Laboratory Analyses
HIV-1 serological testing was by dual rapid HIV-1 antibody tests, with positive results confirmed by HIV-1 enzyme immunoassay (EIA) and Western blot. For HIV-1 seroconverters, HIV-1 transmission endpoints were classified as either “genetically linked” within the partnership or “unable to be linked” (i.e., likely acquired outside of the study partnership), based on sequencing of HIV-1 C2-V3-C3 regions of env and p17/p24 regions of gag amplified from plasma from the seroconverting partner and the HIV-1 infected partner with whom they enrolled in the study. Phylogenetic analysis and posterior probability of linkage using pair-wise nucleotide distances between sequences was performed as previously detailed [19].
HSV-2 serostatus was determined by HSV Western blot [33]. At study conclusion, batched testing of enrollment samples included nucleic acid amplification for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis of endocervical swab or urine samples (Gen-Probe, San Diego, CA) [32].
CD4 T cell quantification was performed for HIV-1 infected participants every six months using flow cytometry. HIV-1 RNA was quantified from plasma at baseline, at months 3, 6, 12, and at study exit. HIV-1 RNA was quantified from seminal plasma and endocervical swabs using the COBAS Ampliprep/COBAS TaqMan real-time HIV-1 RNA assay, version 1.0 (Roche Diagnostics, Indianapolis, IN) [19]. The assay was validated for seminal plasma and fluid eluted from endocervical swabs using HIV-1 spiked Virology Quality Assurance Program standards and published specimen-processing procedures [11, 13]. Endocervical swabs were eluted in 1000 μL of GUSCN lysis buffer, eluted for 15 minutes, vortexed briefly, and microfuged for 5 seconds at 14,000 g to pellet debris prior to removal of fluid for testing. A final dilution step with 10x PBS was used to achieve sufficient volume for the COBAS AP/TM assay, with a lower limit of quantification of 240 copies (per mL for blood plasma and seminal plasma and per swab for endocervical samples). Validation of the COBAS assay against an independently validated quantitative HIV-1 real-time PCR assay showed assay precision of <0.24 log10 copies/mL, which was not significantly different between the two assay platforms [11, 13]. Assay inhibitors were removed by the COBAS Ampliprep procedure.
Data Analysis
Plasma and genital HIV-1 RNA concentrations were log10-transformed to approximate normality. Samples below the limit of quantification were assigned values at half that limit. Genital HIV-1 RNA concentrations and male-to-female and female-to-male transmission were analyzed separately.
Couples in which the HIV-1 infected participant contributed a genital sample for HIV-1 RNA quantification were included in this analysis [19]. The primary outcome measure was detection of HIV-1 seroconversion, and we restricted analyses to genetically linked HIV-1 transmissions. Participants with genetically unlinked HIV-1 transmissions contributed follow-up time until HIV-1 seroconversion; data from subsequent visits were censored. Couples in which the HIV-1 infected partners initiated ART were censored at ART initiation and genital samples collected subsequently were not analyzed (n=43). Twenty couples were excluded because semen samples had insufficient volume for testing.
We used Cox proportional hazards analysis to assess the relationship between genital HIV-1 concentrations and risk of genetically-linked HIV-1 seroconversion among initially HIV-1 seronegative partners. Acyclovir did not reduce the risk of HIV-1 transmission, but significantly reduced plasma HIV-1 RNA levels, by an average of 0.25 log10 copies/mL [19]; thus, analyses were stratified by randomization arm. Analyses were also stratified by study site, to account for potential unmeasured differences across the 14 sites. Analyses were adjusted for plasma HIV-1 RNA levels as a time-dependent variable and for other potential correlates of HIV-1 transmission, including unprotected sex, sexually transmitted infections, HSV-2 serostatus of the HIV-1 uninfected partner, CD4 T cell count of the HIV-1 infected partner, circumcision status of the male partner, and age. Given that genital samples were collected only once during the study, genital HIV-1 RNA quantity was analyzed as a time-independent variable, with follow-up time beginning at enrollment; sensitivity analyses were performed to assess the effect of timing of genital sample collection. Chi-square and Mann-Whitney U tests were used to compare categorical and continuous characteristics, respectively. Spearman’s correlation coefficient and linear regression were used to assess the relationship between genital and plasma HIV-1 concentrations. Data were analyzed using SAS version 9.20.
Acknowledgments
We gratefully acknowledge the invaluable contributions of the HIV-1 serodiscordant couples who participated in this study. We thank the teams at the study sites and at the University of Washington for work on data and sample collection and management. We acknowledge Joan Dragavon and the University of Washington Retrovirology Laboratory for measurement of genital HIV-1 RNA and Dr. Renee Ridzon from the Bill & Melinda Gates Foundation for study oversight.
Funding: The Bill and Melinda Gates Foundation (grant ID #26469) and the United States National Institutes of Health (grant AI038858 and a developmental grant from the University of Washington Center for AIDS Research [AI027757]).
Role of the funding source: The authors designed and executed the study, had full access to the raw data, performed all analyses, wrote the manuscript, and had final responsibility for the decision to submit for publication.
Partners in Prevention HSV/HIV Transmission Study Team
University of Washington Coordinating Center and Central Laboratories, Seattle, USA: Connie Celum (principal investigator), Anna Wald (protocol co-chair), Jairam Lingappa (medical director), Jared M. Baeten, Mary Campbell, Lawrence Corey, Robert W. Coombs, James P. Hughes, Amalia Magaret, M. Juliana McElrath, Rhoda Morrow, James I. Mullins
Study sites and site principal investigators
Cape Town, South Africa (University of Cape Town): David Coetzee; Eldoret, Kenya (Moi University, Indiana University): Kenneth Fife, Edwin Were; Gaborone, Botswana (Botswana Harvard Partnership): Max Essex, Joseph Makhema; Kampala, Uganda (Infectious Disease Institute, Makerere University): Elly Katabira, Allan Ronald; Kigali, Rwanda (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Kayitesi Kayitenkore, Etienne Karita; Kisumu, Kenya (Kenya Medical Research Institute, University of California San Francisco): Elizabeth Bukusi, Craig Cohen; Kitwe, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, William Kanweka; Lusaka, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Bellington Vwalika; Moshi, Tanzania (Kilimanjaro Christian Medical College, Harvard University): Saidi Kapiga, Rachel Manongi; Nairobi, Kenya (University of Nairobi, University of Washington): Carey Farquhar, Grace John-Stewart, James Kiarie; Ndola, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Mubiana Inambao; Orange Farm, South Africa (Reproductive Health Research Unit, University of the Witwatersrand): Sinead Delany-Moretlwe, Helen Rees; Soweto, South Africa (Perinatal HIV Research Unit, University of the Witwatersrand): Guy de Bruyn, Glenda Gray, James McIntyre; Thika, Kenya (University of Nairobi, University of Washington): Nelly Rwamba Mugo
Data management was provided by DF/Net Research, Inc. (Seattle, USA) and site laboratory oversight was provided by Contract Lab Services (University of the Witwatersrand, Johannesburg, South Africa).
Footnotes
Author contributions: Data and specimens were collected by JMB, JRL, RWC, SDM, ENJ, NRM, AW, LC, MSC, JIM, and CC. JMB, EK, and DD performed the statistical analysis. JMB completed the first draft of the manuscript. All authors contributed to the study design, analysis, interpretation of the data, and the writing of the final draft of the article.
Competing interests: JRL is a member of the scientific advisory board of Prosetta Bioconformatics, Inc; CC has served as a paid scientific advisor to the research advisory boards of Merck and Gilead. The other authors report no competing interests.
1. Baeten JM, Overbaugh J. Measuring the infectiousness of persons with HIV-1: opportunities for preventing sexual HIV-1 transmission. Curr HIV Res. 2003;1:69–86. [PubMed]
2. Coombs RW, Reichelderfer PS, Landay AL. Recent observations on HIV type-1 infection in the genital tract of men and women. AIDS. 2003;17:455–480. [PubMed]
3. Anderson BL, Cu-Uvin S. Determinants of HIV shedding in the lower genital tract of women. Curr Infect Dis Rep. 2008;10:505–511. [PubMed]
4. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F, Meehan MO, Lutalo T, Gray RH. Viral load and heterosexual transmission of human immunodeficiency virus type 1 Rakai Project Study Group. N Engl J Med. 2000;342:921–929. [PubMed]
5. Chuachoowong R, Shaffer N, Siriwasin W, Chaisilwattana P, Young NL, Mock PA, Chearskul S, Waranawat N, Chaowanachan T, Karon J, Simonds RJ, Mastro TD. Short-course antenatal zidovudine reduces both cervicovaginal human immunodeficiency virus type 1 RNA levels and risk of perinatal transmission Bangkok Collaborative Perinatal HIV Transmission Study Group. J Infect Dis. 2000;181:99–106. [PubMed]
6. Gaillard P, Verhofstede C, Mwanyumba F, Claeys P, Chohan V, Mandaliya K, Bwayo J, Plum J, Temmerman M. Exposure to HIV-1 during delivery and mother-to-child transmission. AIDS. 2000;14:2341–2348. [PubMed]
7. Cohen MS, Hoffman IF, Royce RA, Kazembe P, Dyer JR, Daly CC, Zimba D, Vernazza PL, Maida M, Fiscus SA, Eron JJ., Jr Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1 AIDSCAP Malawi Research Group. Lancet. 1997;349:1868–1873. [PubMed]
8. McClelland RS, Wang CC, Mandaliya K, Overbaugh J, Reiner MT, Panteleeff DD, Lavreys L, Ndinya-Achola J, Bwayo JJ, Kreiss JK. Treatment of cervicitis is associated with decreased cervical shedding of HIV-1. AIDS. 2001;15:105–110. [PubMed]
9. Nagot N, Ouedraogo A, Foulongne V, Konate I, Weiss HA, Vergne L, Defer MC, Djagbare D, Sanon A, Andonaba JB, Becquart P, Segondy M, Vallo R, Sawadogo A, Van de Perre P, Mayaud P. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N Engl J Med. 2007;356:790–799. [PubMed]
10. Anderson BL, Wang CC, Delong AK, Liu T, Kojic EM, Kurpewski J, Ingersoll J, Mayer K, Caliendo AM, Cu-Uvin S. Genital tract leukocytes and shedding of genital HIV type 1 RNA. Clin Infect Dis. 2008;47:1216–1221. [PMC free article] [PubMed]
11. Zuckerman RA, Lucchetti A, Whittington WL, Sanchez J, Coombs RW, Magaret A, Wald A, Corey L, Celum C. HSV suppression reduces seminal HIV-1 levels in HIV-1/HSV-2 co-infected men who have sex with men. AIDS. 2009;23:479–483. [PMC free article] [PubMed]
12. Zuckerman RA, Lucchetti A, Whittington WL, Sanchez J, Coombs RW, Zuniga R, Magaret AS, Wald A, Corey L, Celum C. Herpes simplex virus (HSV) suppression with valacyclovir reduces rectal and blood plasma HIV-1 levels in HIV-1/HSV-2-seropositive men: a randomized, double-blind, placebo-controlled crossover trial. J Infect Dis. 2007;196:1500–1508. [PubMed]
13. Baeten JM, Strick LB, Lucchetti A, Whittington WL, Sanchez J, Coombs RW, Magaret A, Wald A, Corey L, Celum C. Herpes simplex virus (HSV)-suppressive therapy decreases plasma and genital HIV-1 levels in HSV-2/HIV-1 coinfected women: a randomized, placebo-controlled, cross-over trial. J Infect Dis. 2008;198:1804–1808. [PMC free article] [PubMed]
14. Attia S, Egger M, Muller M, Zwahlen M, Low N. Sexual transmission of HIV according to viral load and antiretroviral therapy: systematic review and meta-analysis. AIDS. 2009;23:1397–1404. [PubMed]
15. Graham SM, Holte SE, Peshu NM, Richardson BA, Panteleeff DD, Jaoko WG, Ndinya-Achola JO, Mandaliya KN, Overbaugh JM, McClelland RS. Initiation of antiretroviral therapy leads to a rapid decline in cervical and vaginal HIV-1 shedding. AIDS. 2007;21:501–507. [PubMed]
16. Sheth PM, Kovacs C, Kemal KS, Jones RB, Raboud JM, Pilon R, la Porte C, Ostrowski M, Loutfy M, Burger H, Weiser B, Kaul R. Persistent HIV RNA shedding in semen despite effective antiretroviral therapy. AIDS. 2009;23:2050–2054. [PubMed]
17. Pilcher CD, Joaki G, Hoffman IF, Martinson FE, Mapanje C, Stewart PW, Powers KA, Galvin S, Chilongozi D, Gama S, Price MA, Fiscus SA, Cohen MS. Amplified transmission of HIV-1: comparison of HIV-1 concentrations in semen and blood during acute and chronic infection. AIDS. 2007;21:1723–1730. [PMC free article] [PubMed]
18. Morrison CS, Demers K, Kwok C, Bulime S, Rinaldi A, Munjoma M, Dunbar M, Chipato T, Byamugisha J, Van Der Pol B, Arts E, Salata RA. Plasma and cervical viral loads among Ugandan and Zimbabwean women during acute and early HIV-1 infection. AIDS. 2010;24:573–582. [PMC free article] [PubMed]
19. Celum C, Wald A, Lingappa JR, Magaret AS, Wang RS, Mugo N, Mujugira A, Baeten JM, Mullins JI, Hughes JP, Bukusi EA, Cohen CR, Katabira E, Ronald A, Kiarie J, Farquhar C, Stewart GJ, Makhema J, Essex M, Were E, Fife KH, de Bruyn G, Gray GE, McIntyre JA, Manongi R, Kapiga S, Coetzee D, Allen S, Inambao M, Kayitenkore K, Karita E, Kanweka W, Delany S, Rees H, Vwalika B, Stevens W, Campbell MS, Thomas KK, Coombs RW, Morrow R, Whittington WL, McElrath MJ, Barnes L, Ridzon R, Corey L. Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2. N Engl J Med. 2010;362:427–439. [PMC free article] [PubMed]
20. Zagury D, Bernard J, Leibowitch J, Safai B, Groopman JE, Feldman M, Sarngadharan MG, Gallo RC. HTLV-III in cells cultured from semen of two patients with AIDS. Science. 1984;226:449–451. [PubMed]
21. Vogt MW, Witt DJ, Craven DE, Byington R, Crawford DF, Schooley RT, Hirsch MS. Isolation of HTLV-III/LAV from cervical secretions of women at risk for AIDS. Lancet. 1986;1:525–527. [PubMed]
22. Wang CC, McClelland RS, Reilly M, Overbaugh J, Emery SR, Mandaliya K, Chohan B, Ndinya-Achola J, Bwayo J, Kreiss JK. The effect of treatment of vaginal infections on shedding of human immunodeficiency virus type 1. J Infect Dis. 2001;183:1017–1022. [PubMed]
23. Butler DM, Smith DM, Cachay ER, Hightower GK, Nugent CT, Richman DD, Little SJ. Herpes simplex virus 2 serostatus and viral loads of HIV-1 in blood and semen as risk factors for HIV transmission among men who have sex with men. AIDS. 2008;22:1667–1671. [PMC free article] [PubMed]
24. Lingappa JR, Hughes JP, Wang RS, Baeten JM, Celum C, Gray GE, Stevens WS, Donnell D, Campbell MS, Farquhar C, Essex M, Mullins JI, Coombs RW, Rees H, Corey L, Wald A. Estimating the impact of plasma HIV-1 RNA reductions on heterosexual HIV-1 transmission risk. PLoS ONE. 2010;5:e12598. [PMC free article] [PubMed]
25. Krieger JN, Coombs RW, Collier AC, Ho DD, Ross SO, Zeh JE, Corey L. Intermittent shedding of human immunodeficiency virus in semen: implications for sexual transmission. J Urol. 1995;154:1035–1040. [PubMed]
26. Ellerbrock TV, Lennox JL, Clancy KA, Schinazi RF, Wright TC, Pratt-Palmore M, Evans-Strickfaden T, Schnell C, Pai R, Conley LJ, Parrish-Kohler EE, Bush TJ, Tatti K, Hart CE. Cellular replication of human immunodeficiency virus type 1 occurs in vaginal secretions. J Infect Dis. 2001;184:28–36. [PubMed]
27. Zhu T, Wang N, Carr A, Nam DS, Moor-Jankowski R, Cooper DA, Ho DD. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol. 1996;70:3098–3107. [PMC free article] [PubMed]
28. Sheth PM, Danesh A, Sheung A, Rebbapragada A, Shahabi K, Kovacs C, Halpenny R, Tilley D, Mazzulli T, MacDonald K, Kelvin D, Kaul R. Disproportionately high semen shedding of HIV is associated with compartmentalized cytomegalovirus reactivation. J Infect Dis. 2006;193:45–48. [PubMed]
29. Kaul R, Pettengell C, Sheth PM, Sunderji S, Biringer A, MacDonald K, Walmsley S, Rebbapragada A. The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission. J Reprod Immunol. 2008;77:32–40. [PubMed]
30. Butler DM, Delport W, Kosakovsky Pond SL, Lakdawala MK, Cheng PM, Little SJ, Richman DD, Smith DM. The origins of sexually transmitted HIV among men who have sex with men. Sci Transl Med. 2010;2:18re11. [PMC free article] [PubMed]
31. Strick LB, Wald A, Celum C. Management of herpes simplex virus type 2 infection in HIV type 1-infected persons. Clin Infect Dis. 2006;43:347–356. [PubMed]
32. Lingappa JR, Kahle E, Mugo N, Mujugira A, Magaret A, Baeten J, Bukusi EA, Cohen CR, Katabira E, Ronald A, Kiarie J, Farquhar C, Stewart GJ, Makhema J, Essex M, Were E, Fife K, Debruyn G, Gray G, McIntyre J, Manongi R, Kapiga S, Coetzee D, Allen S, Inambao M, Kayitenkore K, Karita E, Kanweka W, Delany S, Rees H, Vwalika B, Coombs RW, Morrow R, Whittington W, Corey L, Wald A, Celum C. Characteristics of HIV-1 discordant couples enrolled in a trial of HSV-2 suppression to reduce HIV-1 transmission: the Partners Study. PLoS ONE. 2009;4:e5272. [PMC free article] [PubMed]
33. Ashley RL, Militoni J, Lee F, Nahmias A, Corey L. Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera. J Clin Microbiol. 1988;26:662–667. [PMC free article] [PubMed]