Following the establishment of the mean plasma viral setpoint, subsequent plasma viral loads increased only slightly (+0.005 log10 HIV-1 copies/ml per month; P = 0.24) through 24 months.
We also found a significant difference in the time to mean plasma viral setpoint by HIV-1 subtype. Time to setpoint was fastest for subtype D (100 days; 95% CI 67–109 days), followed by subtype A (139 days; 95% CI 109–157 days), and was slowest for subtype C infections (183 days; 95% CI 152–200 days).
We observed a direct correlation between HIV-1 cervical and plasma viral RNA levels during early infection (Spearman's r = 0.47; P < 0.0001) (Fig. 1). We found an equilibrium level or ‘setpoint’ among genital secretions similar to that in the peripheral blood. The mean cervical setpoint was 1.64 log10 HIV-1 copies/swab (95% CI 1.46–1.82) and occurred at 174 days (95% CI 145–194) from the estimated infection date. Similar to plasma viral loads, cervical viral loads were higher during acute infection (mean of 3.01 log10 copies/swab) than during periods 1–2, 2–4, and 4–6 months after infection (means of 2.30, 2.00, and 1.92 log10 HIV-1 copies/swab; P = 0.03, P < 0.01, and P < 0.01, respectively) (Table 3). Cervical specimens taken 1–2 months after HIV-1 infection had higher mean viral loads than specimens taken 2–4 and 4–6 months from time of infection (P < 0.01). The comparisons were similar when each country was considered individually. Following the establishment of a setpoint at 174 days after infection, cervical viral loads did not change significantly (+0.001 log10 HIV-1 copies/swab per month; P = 0.85) through 24 months.
In multivariable analysis, having a nonviral STI (chlamydia, gonorrhea, or trichomoniasis) (+0.29 log10 copies/swab; P = 0.03), a partner spending nights away from home (+ 0.22 log10 copies/swab; P < 0.01), unprotected sex within 3 days (+0.21 log10 copies/swab; P = 0.06), and Zimbabwe-subtype C infection (+0.26 log10 copies/swab; P = 0.05) were associated with increased cervical viral loads (Table 4). The effect of subtype D infection on mean cervical viral load (+0.30 log10 copies/swab) was of similar magnitude as subtype C infection but was not statistically significant (P = 0.09). Greater duration since HIV infection (−0.11 log10 copies/swab per month; P < 0.01) was associated with decreased cervical viral loads. There was no association between DMPA (+0.12 log10 copies/swab; P = 0.35) or COC use (+0.08 log10 copies/swab; P = 0.50) and cervical HIV-1 viral loads. Age, pregnancy, breastfeeding, and genital ulcer disease were also not significantly associated with cervical HIV-1 levels.
We also considered our final multivariate model predicting cervical viral loads adjusted for plasma viral load. Higher plasma viral loads were strongly associated with higher mean cervical loads (+0.30 log10 copies/swab; P = <0.001) and time since HIV infection remained strongly associated with decreased mean cervical loads (−0.09 log10 copies/swab per month; P < 0.001). Having a partner who spent nights away from home also remained associated with higher cervical loads (+0.20 log10 copies/swab; P < 0.01). However, HIV-1 subtype, nonviral STIs, and having unprotected sex within the last 3 days were no longer significantly associated with mean cervical loads. Instead, breastfeeding (+0.25 log10 copies/swab; P = 0.04) and the number of coital acts per month (15–29 acts: +0.17 log10 copies/swab; P = 0.04; >30 acts: +0.35 log10 copies/swab; P = 0.18) were associated with higher cervical viral loads.
We found that women in Uganda and Zimbabwe established a plasma viral setpoint of 4.20 log10 HIV-1 copies/ml at 121 days and an analogous cervical viral ‘setpoint’ of 1.64 log10 HIV-1 copies/swab at 174 days from estimated date of HIV-1 infection suggesting that setpoint is achieved later in the genital compartment than in the plasma. Cervical viral loads were strongly correlated with plasma viral loads during the first 6 months of HIV-1 infection (P < 0.0001) and were significantly higher (0.7–1.1 log10 copies/ml higher) during acute infection than subsequently during the early infection period.
Our findings concerning the level and timing of the plasma viral setpoint are similar to those reported by other studies. For example, a study of 161 sex workers in Mombassa, Kenya, reported a median viral setpoint of 4.46 log10 copies/ml attained at 4 months after infection . A study of high-risk Kenyan men and women found a virus setpoint of 4.60 log10 copies/ml at 209 days after infection . Similarly, a study among newly HIV-infected adults in the United States estimated the viral setpoint at 4.56 log10 copies/ml at 117 day after infection .
We found that subtype D infection, pregnancy, and breastfeeding at the time of HIV infection were associated with a higher plasma viral setpoint, whereas young age was associated with a decreased plasma setpoint. These findings concerning predictors of plasma viral setpoint contrast with a previous study conducted among Kenyan sex workers. In that study, DMPA use was associated with a higher viral setpoint (compared with no use of hormonal contraception) but no association was reported between older age, pregnancy, breastfeeding, or subtype D infection and plasma viral setpoint . Although no other analyses of predictors of viral setpoint exist, several studies have reported on predictors of HIV-1 disease progression. A Zambian study found an increased risk of disease progression (CD4 cell count <200 cells/μl or death) among women using hormonal contraception compared with women randomized to copper intrauterine devices . Conversely, a study of postpartum Kenyan women found no differences in change in plasma viral load or CD4 cell counts among women initiating COCs or DMPA . Additionally, several studies suggest that older age  and subtype D HIV-1 infection [31–34] are associated with more rapid HIV-1 disease progression. On the contrary, most studies conclude that pregnancy, although causing transient CD4 cell count decline, is not associated with more rapid disease progression [35–38].
We found a dynamic in the female genital compartment similar to the plasma viral setpoint – high levels of HIV-1 genital viremia during acute infection falling to a steady-state level at about 6 months. Following the establishment of this ‘setpoint’, genital viral loads remained constant up to 2 years after infection. We are not aware of previous reports of a ‘setpoint’ in the genital compartment. Most previous studies have not had substantial genital viral load data from the acute and early infection periods. However, although it is well documented that the plasma viral setpoint is predictive of subsequent disease progression [2-4], the utility of a genital ‘setpoint’ as a predictor of potential infectivity to a sex partner remains to be established.
Our findings corroborate recent reports of high levels of HIV-1 genital shedding early in infection in both women and men with declining levels thereafter [5,6]. Genital and plasma viral loads have also been strongly correlated in other studies (r = 0.4–0.7) [20,39–41]; plasma RNA load is often the factor most strongly associated with genital RNA load in multivariable models [40,42]. However, the strong correlation between genital and plasma viral loads has not previously been clearly documented during early infection.
Subtype C infection, nonviral STIs, having a partner who spends nights away from home, and recent unprotected sex were associated with higher cervical HIV-1 loads, whereas time since infection was associated with decreased cervical loads. Hormonal contraceptive (COC and DMPA) use was not associated with cervical viral loads during early HIV-1 infection. Our results corroborate the findings of much previous research. For example, previous studies have identified nonviral STIs [6,9,40,43], recent unprotected sex, and subtype C HIV-1 infection  as associated with higher genital viral loads. Our finding that hormonal contraception is not associated with HIV RNA genital shedding also agrees with those of most (but not all) previous studies suggesting that hormonal contraception appears to be associated with shedding of HIV-infected cells (measured by HIV-1 DNA) but not cell-free virus (measured by HIV-1 RNA) in the female genital tract [8–10,12,15,20]. However, we are unaware of previous research assessing correlates of HIV-1 genital shedding among women during early infection.
Our study has a number of important strengths. The study was prospective with samples for both plasma and cervical viral loads being collected every 12 weeks beginning before HIV infection. We measured HIV infection timing with precision by conducting HIV PCR testing on serial samples that were serologically negative. We accurately measured many variables that were potentially associated with both HIV viral setpoint and genital shedding, including hormonal contraceptive use and reproductive tract infections. We also measured viral subtype from women with a variety of non-B HIV-1 clades. Finally, we enrolled women seeking family planning services in two sub-Saharan countries. This allows for greater generalizability of study results than a study population drawn from a selected high-risk group (e.g., sex workers).
Our study also had limitations. We used RNAlater as storage media for cervical specimens. This resulted in lower cervical viral load levels compared with specimens collected in dimethylsulfoxide (compared at later study visits). We only sequenced the C2–V3 region of env and thus cannot fully explore the issue of recombinant viruses. Also, some women had unprotected sex during the 3 days prior to their study visit and thus measured genital viral loads at these visits could have been a combination of a participant's and her partner's viral load. However, we measured unprotected sex acts in the last 3 days and adjusted for this in our model of cervical viral loads and believe that this improves the accuracy of our estimates of predictors of cervical viral loads (Table 4). Finally, we are unable to address whether a genital viral ‘setpoint’ is meaningful in terms of long-term transmission risk.
In summary, we found that cervical HIV-1 viral loads were highest during acute infection and then declined up to 6 months after infection when they appeared to reach a setpoint. Factors associated with a higher plasma viral setpoint included older age, subtype D infection, pregnancy, and breastfeeding. Factors associated with higher HIV-1 cervical loads during early infection included nonviral STIs, recent unprotected sex, subtype C infection, and shorter duration since infection. Modification of these factors could result in slower disease progression (pregnancy, breastfeeding) or HIV-1 transmission risk (prevention of STI and unprotected sex). However, the prognostic value of a cervical viral setpoint on future transmission risk remains to be established.
This project has been funded with federal funds from the National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Department of Health and Human Services through a contract with Family Health International (FHI) (Contract Number N01-HD-0-3310).
C.S.M. is the study principal investigator and directed the design and analysis of the study and wrote the manuscript draft; K.D., S.B., M.M., and B.V.D.P. planned, supervised, conducted (S.B.) and did quality assurance (B.V.D.P.) for the laboratory work including managing the laboratory data in Uganda and Zimbabwe; C.K. conducted the data analysis; A.R. monitored the study sites and performed data management; M.D., J.B., and T.C. are site principal investigators and supervised the study teams in Zimbabwe and Uganda; E.A. is the laboratory co-investigator and designed, tested, and supervised the virology assays; R.A.S. is the study co-principal investigator and study clinical consultant; all authors contributed to drafts of the manuscript and approved the final manuscript.
We would like to thank Pai-Lien Chen, PhD, for designing the statistical analysis plan and supervising data analysis as well as Immaculate Nankya, MBCHB, PhD, for assuming directorship of the Uganda laboratory in K.D.'s absence. We would also like to thank the GS Study participants in Uganda and Zimbabwe for their participation in the Study.
B.V.D.P. consults for Roche Diagnostics.
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