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Genital HIV-1 RNA Predicts Risk of Heterosexual
HIV-1 Transmission - pdf attached
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"Our large sample size of heterosexual African HIV-1 serodiscordant couples and prospective follow-up with collection of genital samples before HIV-1 transmission permitted analyses demonstrating that the concentration of HIV-1 RNA in endocervical and seminal samples from HIV-1-infected individuals strongly correlated with risk of HIV-1 transmission to their HIV-1-susceptible sexual partners.....We found a stepwise association between genital HIV-1 levels and HIV-1 incidence, with an about twofold increased risk for each one log10 increase in genital HIV-1.....We found that acyclovir reduced genital HIV-1 levels by ~0.3 log10"
Sci Transl Med 6 April 2011
Jared M. Baeten,1,2,3* Erin Kahle,1,3 Jairam R. Lingappa,1,2,4 Robert W. Coombs,2,5 Sinead Delany-Moretlwe,6 Edith Nakku-Joloba,7 Nelly R. Mugo,1,8 Anna Wald,2,3,5,9 Lawrence Corey,2,3,5,9 Deborah Donnell,1,10 Mary S. Campbell,2 James I. Mullins,2,5 Connie Celum1,2,3
1Department of Global Health, University of Washington, Seattle, WA 98195, USA. 2Department of Medicine, University of Washington, Seattle, WA 98195, USA. 3Department of Epidemiology, University of Washington, Seattle, WA 98195, USA. 4Department of Pediatrics, University of Washington, Seattle, WA 98195, USA. 5Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA. 6Wits Institute for Reproductive Health and HIV, University of the Witwatersrand, Johannesburg 2001, South Africa. 7Department of Epidemiology and Biostatistics, Makerere University, Kampala, Uganda. 8Department of Obstetrics and Gynaecology, University of Nairobi, Nairobi, Kenya. 9Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 10Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Abstract
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. There was a correlation between genital and plasma HIV-1 RNA concentrations: For endocervical swabs, Spearman's rank correlation coefficient ρ was 0.56, and for semen, ρ was 0.55. Each 1.0 log10 increase in genital HIV-1 RNA was associated with a 2.20-fold (for endocervical swabs: 95% confidence interval, 1.60 to 3.04) and a 1.79-fold (for semen: 95% confidence interval, 1.30 to 2.47) 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 11 cases, 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.
Introduction
Many studies have measured HIV-1 concentrations in semen, cervicovaginal, and anorectal secretions to assess the infectiousness of HIV-1 transmitted sexually (1-3). Higher genital HIV-1 concentrations are thought to 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 (7-13), 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 have raised questions regarding whether genital HIV-1 concentrations can predict the risk of HIV-1 sexual transmission (1, 2, 16-18).
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.
Results
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 to 38] for HIV-1-infected partners and 34 years (IQR, 28 to 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 to 8), and 28.6% of couples reported unprotected sex during the month before enrollment. Among HIV-1-infected participants, the median CD4+ T cell count was 469 cells/mm3 (IQR, 350 to 638) and the median plasma HIV-1 RNA concentration was 4.0 log10 copies/ml (IQR, 3.3 to 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.
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 to 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 drug acyclovir [which suppresses herpes simplex virus type 2 (HSV-2)] versus placebo: median, 2.98 versus 3.29 log10 copies/swab for endocervical swabs (P < 0.001) and 2.38 versus 2.76 log10 copies/ml for semen (P = 0.008).
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 (ρ) was 0.56 (P < 0.001) among women and 0.55 (P < 0.001) among men; the correlation was the same (ρ = 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 to 0.56; P < 0.001] and a 0.46 log10 copies/ml increase in semen HIV-1 RNA (95% CI, 0.40 to 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 to 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 to 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 stepwise relationship between genital HIV-1 quantity and HIV-1 transmission incidence was observed (Fig. 1); a similar stepwise 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 about twofold greater risk of HIV-1 transmission (Table 3). The hazard ratio (HR) was 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 the HR was 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 by 1.67-fold (P = 0.02) and the risk of male-to-female HIV-1 transmission by 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.
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 because genital samples were not collected at study enrollment. We obtained data that were similar to the overall results: multivariate HR 2.38 (95% CI, 1.13 to 4.78) per log10 copies/swab increase in endocervical HIV-1 RNA and risk of female-to-male HIV-1 transmission, and multivariate HR 2.89 (95% CI, 1.03 to 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. Again, the data were similar to the overall results: multivariate HR 1.88 (95% CI, 1.11 to 3.19) per log10 copies/swab increase in endocervical HIV-1 RNA and risk of female-to-male HIV-1 transmission, and multivariate HR 1.81 (95% CI, 1.04 to 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, 0.3 to 1.3). 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 to 2.1). For these 11 transmissions, the median time between collection of the genital sample and HIV-1 seroconversion was 4.6 months (range, 0 to 16.3) 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 to 5.9).
Discussion
Our data provide empirical evidence that differences in genital tract concentrations of HIV-1 RNA influence the transmission risk of HIV-1 infection, and we found that this relationship was independent of plasma HIV-1 concentrations. Our large sample size of heterosexual African HIV-1 serodiscordant couples and prospective follow-up with collection of genital samples before HIV-1 transmission permitted analyses demonstrating that the concentration of HIV-1 RNA in endocervical and seminal samples from HIV-1-infected individuals 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 posttreatment genital tract samples, cure of sexually transmitted infections and initiation of ART significantly reduced genital HIV-1 RNA concentrations (7-9, 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 nontransmitting partners (23).
We found a stepwise association between genital HIV-1 levels and HIV-1 incidence, with an about twofold 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 stepwise manner. This linear risk relationship between log10 HIV-1 RNA concentrations and HIV-1 outcomes has been previously reported for systemic HIV-1 concentrations and both sexual and perinatal HIV-1 transmission (4, 24). The Ugandan study first demonstrated that higher blood HIV-1 concentrations resulted in increased heterosexual infectiousness (4) and that plasma HIV-1 levels are 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 biological mechanism is not possible with the samples we tested in 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. 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 (that is, the point estimate of risk of HIV-1 transmission versus log10 genital HIV-1 levels) but would contribute to the precision of the estimate (that is, the width of the CIs). 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 because of genital tract infections or local immunological factors (1, 18, 26-29). 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 previous 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 because the variability in HIV-1 concentrations is greater in genital samples than in plasma samples (2). However, despite 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 sexually transmitted infections (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 6 months, and for most 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 to 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.
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