Of 3408 HESN participants, 3321 had ≥1 follow-up HIV-1 test. A total of 2236 (67.3%) tested HESN participants were male, and, of these, 1336 (59.7%) were circumcised. Median ages among uninfected females and males were 31 years (interquartile range [IQR], 26–38 years) and 35 years (IQR, 30–42 years), respectively. At baseline, ≥1 partner in 1175 couples (35.4%) reported unprotected sex in the previous month, and 2261 HESN participants (68%) were HSV-2 seropositive. Furthermore, 780 HIV-1–infected partners (23%) and 344 HIV-1–uninfected partners (10%) had symptomatic genital ulcer disease. Among HIV-1–infected partners, the median baseline plasma HIV-1 RNA level was 4.1 log10 copies/mL (IQR, 3.3–4.7 log10 copies/mL).
HIV-1 Infection Incidence
Seroconversion was detected in 151 participants who were initially HESN (4.5%), of whom 24 were HIV-1 RNA RT-PCR positive at enrollment and were excluded from the analysis. Of the remaining 127 seroconverters, HIV-1 in 86 (67.7%) was genetically linked to HIV-1 in their infected partner, as revealed by viral sequencing [21
]. The overall incidence of linked infections was 1.7 cases/100 person-years and was greater in the first year of follow-up than in the second (2.0 vs 1.2 cases/100 person-years; P
HIV-1 Exposure Scores
By use of backward variable selection, the best-fitting model for HIV-1 acquisition included unprotected sex with, HIV-1 RNA load of, and symptomatic genital ulcer disease for the infected partner and HSV-2 serostatus, current pregnancy, sex, age, and male circumcision of the uninfected partner (Table ). Evaluation of Schoenfeld residuals did not suggest any time-varying effects. We used this model to calculate visit-specific exposure scores for HESN participants on the basis of the product of regression coefficients and the participant's covariates, and we normalized these to the average exposure score across the full cohort. Exposure scores ranged from −3.6 to 4.7, with a score of 0 representing the average exposure and a 1-unit increase indicating an exp(1) = 2.7-fold increased risk of infection.
Multivariable Hazard Ratios and Regression Coefficients From the Best-Fitting Cox Proportional Hazards Model Used to Estimate Human Immunodeficiency Type 1 (HIV-1) Exposure Scores
Longitudinal Changes in HIV-1 Exposure
Because of variation in time-dependent predictors, HIV-1 exposure scores varied across study visits. For instance, among 1715 (52%) participants who reported unprotected sex at ≥1 visit, 938 (47%) reported unprotected sex at ≤25% of their visits. Among 2764 HESN participants whose HIV-1–infected partner had detectable HIV-1 plasma RNA at baseline, 241 (8.5%) had undetectable plasma HIV-1 levels by the end of follow-up, and 163 HIV-1–infected partners who always had a detectable viral load experienced a decrease of ≥1 log10 copies. Of these 404 HIV-1–infected participants, 124 (31%) began using ART during the study. During follow-up, 1230 HIV-1–infected partners (37%) had genital ulcer disease by self-report or physical examination at ≥1 visit; however, ulcers were only found at 1946 (26%) of 7500 study visits attended by these participants. Finally, of 1873 visits attended by 293 uninfected women who were pregnant at any time during the study, pregnancy was documented at 699 (37%). Overall, only 51% of participants in the highest exposure score quintile at baseline remained in the highest exposure quintile at the 3-month follow-up visit.
HESN Clusters With Persistent Levels of HIV-1 Exposure
Despite variability across individual visits, participants could be divided into clusters with persistent high exposure scores (475 [14%]), stable lower risk scores (2595 [79%]), or decreasing exposure scores (214 [7%]) (Supplementary Figure 1
). Compared with participants with low exposure scores, participants in the highest exposure group exhibited riskier characteristics (Table ). Specifically, high-exposure participants were more likely to report at any time during the study that they had unprotected sex with their study partner (75% vs 46%; P
< .001), and their HIV-1–infected partners had higher mean plasma HIV-1 RNA levels (5.0 vs 3.9 log10
< .001). Furthermore, these participants were younger (mean age, 29.5 vs 34.9 years; P
< .001) and were more likely to be female (50% vs 29%; P
< .001). Among male HESN participants, the highest-exposure group was less likely to be circumcised (31% vs 59%; P
Characteristics of Longitudinal Exposure Risk Groups
Participants in the highest-exposure group had a 6.9-fold increased risk of infection than the lower exposure group on the basis of median exposure scores (1.7 [IQR, 1.5–2.1] vs −0.2 [IQR, −1.0 to 0.5]) and had similar median exposure scores as participants who acquired HIV-1 (Figure ). Furthermore, this group had the highest incidence of HIV-1 acquisition, with 49 individuals (10%) acquiring HIV-1 during follow-up, compared with only 28 (1%) in the lower-exposure group. Empirical plots showing smoothed hazards of infection among exposure clusters suggested that the risk of infection decreased over time among participants in the highest-risk group but remained constant among participants with lower exposure scores (Figure ).
Figure 1. Smoothed density curves representing human immunodeficiency virus type 1 (HIV-1) exposure score distributions for all HIV-1 seroconverters and HIV-1–exposed seronegative participants from the highest, lower, and decreasing exposure risk groups. (more ...)
Figure 2. Empirical hazard functions for human immunodeficiency virus type 1 (HIV-1) acquisition among HIV-1 exposure score risk groups, determined by clustering initially HIV-1-exposed seronegative individuals into homogenous groups on the basis of their longitudinal (more ...)
Participants in the cluster with substantial decreases in exposure over follow-up started at baseline with a median exposure risk score of 1.4 (IQR, 0.9–3.0) but had much lower scores across all subsequent visits (median, 0.4 [IQR, −0.5 to 0.9]). This drop in exposure was principally due to cessation of unprotected sex or to the HIV-1–infected partner's HIV-1 RNA levels decreasing after initiation of ART, with 39% of infected partners of participants in the decreasing exposure group reporting ART use during the study, compared with 13% and 7% in the highest- and lower-risk groups, respectively (P < .001).
Evaluation of Simplified and HESN-Only HIV-1 Exposure Score Models
To compare the best fitting longitudinal model to models that can be applied in cohorts with less data, we also evaluated a simplified model that included only baseline HIV-1 RNA levels of infected partners and longitudinal unprotected sex, along with age, sex, and male circumcision status of the HESN partner, and a HESN-only model that included predictors from the HESN partner (unprotected sex, sex, age, and male circumcision status) without behavioral or clinical data from the HIV-1–infected partner. Compared with the best-fitting model, mean individual exposure scores from the simplified model discriminated seroconverters from nonseroconverters with a high degree of sensitivity and specificity, as measured by areas under the ROC curve (AUCs) of 0.87 versus 0.85. Furthermore, 71% of participants identified in the highest-exposure cluster identified using the best-fitting model were in the highest-risk cluster identified by the simplified model. Only including baseline unprotected sex further reduced the AUC to 0.82. The discriminatory power of a model that included predictors from only the HESN participant was not as strong as either the best-fitting model or the simplified model, as indicated by an AUC of 0.71, with only 36% of highest-risk participants identified through the best-fitting model being captured as high risk in the model that was based on HESN participant data only.
To validate use of exposure scores for discriminating HIV-1 seroconverters from HESN participants, we used the simplified model described above to determine exposure scores in a second cohort of 485 HIV-1 serodiscordant couples. Although the second cohort was recruited similarly to the primary cohort, the second study only evaluated plasma HIV-1 RNA levels at baseline, hence necessitating validation with the simplified model only. Mean individual exposure scores generated by the simplified model applied to this second cohort discriminated seroconverters from nonseroconverters with a high degree of sensitivity and specificity, as measured by an AUC of 0.81, which was similar to the AUC of 0.85 achieved by the simplified model in the primary cohort (Figure ). The simplified model could also be used in the second cohort to identify a highest risk cluster composed of 48 participants (10.4%), which also showed a decreasing hazard of HIV-1 infection over time.
Figure 3. Receiver operating characteristic (ROC) curves comparing the ability of an individual's average human immunodeficiency virus type 1 (HIV-1) exposure score across all study visits to discriminate HIV-1 acquisition risk for participants in the primary and (more ...)
Simulating Effects of HIV-1 Exposure in Studies of Resistance
We used data from our primary cohort to evaluate potential effects of selecting HESN controls with low exposure when studying a hypothetical biological factor that does or does not correlate with host resistance to HIV-1. Scenario 1 assumes that increased exposure is associated with increased levels of a hypothetical host factor and that this host factor is not associated with infection (Figure A). If controls are selected at random (eg, not on the basis of HIV-1 exposure), a spurious relationship between the host factor and HIV-1 acquisition is observed (mean difference, 1.7; P < .001) that reflects confounding of HIV-1 exposure on HIV-1 acquisition (Figure B). The correct relationship between the hypothetical host factor and HIV-1 acquisition is observed once the level of HIV-1 exposure is controlled by matching HESN participants to HIV-1 seroconverters by exposure score (observed mean difference, 0; P = .6) (Figure C). Scenario 2 assumed that increased exposure is associated with increased host factor levels but that the host factor was associated with infection, with the mean level being 2 units lower among HIV-1 seroconverters (Figure D). Here, randomly selecting HESN controls results in a false-negative association (observed mean difference, 0.2; P = .4) (Figure E). Once again, this false observation is rectified by matching cases and controls by exposure levels, revealing the true association between the hypothetical host factor and HIV-1 acquisition (mean difference, –1.97; P < .001) (Figure F).
Figure 4. Simulations demonstrating potential biases when evaluating potential correlates of human immunodeficiency virus type 1 (HIV-1) resistance if HIV-1 exposure is not considered. Scenario 1 assumes that a 1-unit increase in HIV-1 exposure score is associated (more ...)