The HIV Prevention Trials Network (HPTN) 052 trial was a phase 3, randomized, controlled trial that tested whether early initiation of antiretroviral therapy (ART) prevented sexual transmission of HIV in serodiscordant couples.1,2 Index participants in the early ART arm started ART at study enrollment with baseline CD4 cell counts of 350–550 cells per cubic millimeter. Index participants in the delayed ART arm started ART when their CD4 count fell below 250 cells per cubic millimeter or they developed an AIDS-defining illness. The primary endpoints for the trial were genetically-linked partner infections (ie, infections where the enrolled index participant was the likely source of the partner's infection). In April 2011, an interim analysis demonstrated that early ART prevented 96% of linked HIV infections and offered health benefits to the individual receiving ART.1,3 After these results were released, ART was offered to all HIV-infected index participants regardless of CD4 cell count. The trial continued until May 2015, to assess the durability of the study intervention. The final intent-to-treat analysis found that early ART prevented 93% of linked HIV infections.2 In this report, we present genetic linkage analysis and characterization of partner infections in HPTN 052, and analysis of the association of demographic, behavioral, and clinical factors with linked HIV infection.
HPTN 052 enrolled 1763 serodiscordant couples at 13 study sites in Africa, Asia, and the Americas (97% heterosexual, April 2005–May 2010).1 ART failure was defined as the first of 2 consecutive study visits where the index's viral load was >1000 copies per milliliter more than 24 weeks after ART initiation. HIV-uninfected partners were tested for HIV infection throughout the trial, as described1; partner infections were confirmed at the HPTN Laboratory Center (Baltimore, MD).
Genetic linkage of partner infections was determined using phylogenetic and statistical methods described previously.4 Linkage analysis was performed using samples collected from partners with confirmed HIV infection and the corresponding index participants (index-partner pairs); whenever possible, 2 samples from each individual were analyzed. Samples from random index participants at the same study sites were also analyzed (control samples). Three methods were used for linkage analysis: phylogenetic analysis of HIV pol sequences generated from population-sequencing; Bayesian analysis of pol sequence data; and phylogenetic analysis of env sequences obtained by next generation sequencing (Supplemental Digital Content 1, http://links.lww.com/QAI/A909).
HIV Drug Resistance Testing and HIV Subtyping
HIV genotyping was performed using the ViroSeq HIV-1 Genotyping System (Celera Diagnostics, Alameda, CA). HIV subtyping was performed using the resulting pol region sequences, as described.4
Analysis of Factors Associated With Linked vs. Unlinked Partner Infections
Associations between linkage status and categorical variables were assessed using Fishers exact test. Associations between linkage status and continuous variables were assessed using Wilcoxons rank sum test. All P-values are 2-sided. Statistical significance was defined as P < 0.05. Backwards stepwise multivariate logistic regression was performed using a cutoff of <0.05 to retain the factors considered significant.
Analysis of the Timing of Partner Infections
The timing of partner infections was analyzed in 3 cases as described5 using HIV RNA testing; serologic assays; and sequence analysis using BEAST (which estimates the time since HIV infection using phylogenetic methods for analysis of sequence data6) and Poisson Fitter (which estimates the time since HIV infection based on the accumulation of neutral mutations in the viral population7).
Ethical review committees at each participating institution approved the HPTN 052 trial (NCT00074581). Written informed consent was obtained from all study participants.
Linkage Status of Index-Partner Pairs
Seventy-eight partner infections were observed over 8295 person-years of follow-up for uninfected partners, including 6620 person-years of follow-up for partners after the corresponding index participant started ART. Forty-six infections were diagnosed before the index participant started study ART and 32 were diagnosed after the index participant started study ART (19 in the early ART arm and 13 in the delayed ART arm, Fig. 1A).
Linkage of partner infections was assessed using phylogenetic and statistical methods (Supplemental Digital Content 1, http://links.lww.com/QAI/A909). HIV sequencing and linkage analysis were successful for 72 of the 78 cases; in the other 6 cases, samples were not available for analysis or amplification failed for either the index or partner samples. Overall, 46 infections were classified as linked and 26 were classified as unlinked (Fig. 1A). Figure 1B shows the timing of linked and unlinked infections in each study arm, relative to key study milestones.
In all cases, HIV subtypes were consistent with the subtypes prevalent in the region (Supplemental Digital Content 2A, http://links.lww.com/QAI/A910). HIV drug resistance was detected in HIV from the partner in only 2 of the 46 linked cases; in both cases, the same resistance mutations were detected in the corresponding index participant, suggesting that the resistant virus was transmitted (Supplemental Digital Content 2B, http://links.lww.com/QAI/A910). Both of these cases occurred in the delayed ART arm, before the index participant started ART. HIV drug resistance was detected in the partner in only 2 of the 26 unlinked cases; in these cases, resistance mutations were not detected in HIV from the index participant (Supplemental Digital Content 2B, http://links.lww.com/QAI/A910).
Factors Associated With Linked Infections
We analyzed the association of clinical, demographic, and behavioral factors with linked partner infections (Supplemental Digital Content 3, http://links.lww.com/QAI/A911). In univariate analyses, linked partner infections were associated with the following characteristics: couple randomized to the delayed ART arm; index participant not on ART at the time of partner diagnosis; higher index viral load at the time of partner diagnosis (index viral load >400 copies per milliliter or higher log10 viral load); lower index CD4 cell count at the time of partner diagnosis; shorter time between enrollment and partner diagnosis; and fewer sexual partners in the 3 months before diagnosis. Region (Africa vs. Asia/Americas) and index sex were not associated with linked infection.
Backwards stepwise regression models were used for model building in multivariate analysis; separate analyses were used for each measure of index viral load at the time of partner infection (greater or less than 400 copies per milliliter or log10 viral load). In both models, higher index viral load was strongly associated with linked infection (P = 0.0006 and P < 0.0001). When index viral load was analyzed as a binary variable (greater or less than 400 copies per milliliter), randomization in the delayed ART arm and lower index CD4 cell count at the time of partner diagnosis were also associated with linked partner infection (P = 0.045 and P = 0.033, respectively). When index viral load was analyzed as a continuous variable (median log10 viral load), the only factor significantly associated with linked infection was index viral load at the time of partner diagnosis.
Linked Partner Infections Diagnosed After the Index Participant Started ART
Eight of the 46 linked infections occurred after the index participant started ART (3 in the early ART arm; 5 in the delayed ART arm, Fig. 1B, key cases); these 8 cases were analyzed in detail. None of the 8 partners in these key cases had HIV drug resistance. In 4 cases, the partner was diagnosed after the index participant failed ART (Supplemental Digital Content 4, http://links.lww.com/QAI/A912, Panel A). In 3 of these 4 cases, the index was viremic at the time of partner diagnosis. In the fourth case, the index was intermittently viremic before the partner diagnosis; in that case, the partner was lost to follow-up for more than one year; during that time, the partner was diagnosed with HIV infection and had started ART. In the other 4 cases, the partner was diagnosed with HIV infection shortly after the index participant started ART (Supplemental Digital Content 4, http://links.lww.com/QAI/A912, Panel B). In one of those 4 cases, the index participant did not achieve virologic suppression on ART and was viremic when the partner was diagnosed with HIV infection. In the other 3 cases (cases A–C), the index's viral load was <400 copies per milliliter when the partner was diagnosed with HIV infection.
Additional laboratory assessments were performed in cases A–C to estimate the timing of HIV transmission relative to index ART initiation (Fig. 2). Results obtained for 2 of these cases are described in a previous report5 and are updated here. In all 3 cases, the analyses indicated that the transmission event occurred either before index ART initiation (when the index was not virally suppressed), or shortly after index ART initiation (most likely before the index was virally suppressed from ART). Of note, in HPTN 052, the cumulative percentage of index participants who achieved viral suppression by 3, 6, 9, and 12 months were 76%, 87%, 90%, and 91%.
Phylogenetic methods can be used to study HIV transmission networks and hotspots within communities and populations10–12 and to assess the genetic linkage of specific transmission events.4,13 In this study, we used phylogenetic and statistical methods to identify genetically-linked partner infections in the HPTN 052 trial, which were the basis of the primary endpoint analysis for this landmark study.1,2 This report highlights the importance of genetic linkage analysis in evaluating HIV transmission in clinical trials that include serodiscordant couples. Forty-six linked partner infections were observed in the HPTN 052 trial. Only 8 of these infections were diagnosed after the index participant started ART. We identified 3 risk periods for index-to-partner (linked) transmission in couples after the index participant had started ART: (1) near the time of ART initiation, before viral suppression was achieved, (2) after ART initiation if viral suppression did not occur, and (3) after ART failure.
High HIV viral load is a major driver of sexual HIV transmission and is also associated with ART outcomes that are relevant to use of treatment as prevention. In a previous study, we showed that higher baseline viral load in index participants was associated with a longer time to viral suppression and lack of viral suppression 3 or 6 months after ART initiation was associated with a shorter time to ART failure and a higher frequency of ART failure.14 In this report, higher index viral load at study enrollment2 and higher index viral load at the time of partner seroconversion were associated with linked partner infection.
In most cases, several months of ART is required before viral suppression is achieved. More rapid viral suppression is observed with ART regimens that include integrase inhibitors15,16; however, these regimens may not be available in resource-limited settings. Previous studies have examined the risk of HIV transmission in the months after ART initiation. In a review of 6 studies of serodiscordant couples, only one linked infection was observed >6 months after ART initiation among 1672 couples with 2773 person-years of follow-up.17 In another study, no infections were observed >6 months after ART initiation (over 167 person-years follow-up).18 In HPTN 052, 4 linked infections were observed >6 months after ART initiation (over 7032 person-years of follow-up); all 4 cases occurred long after ART failure. Importantly, we did not observe any linked infections in couples where the index participant was stably suppressed on ART. These data were based on 6620 person-years of follow-up of partners after index ART initiation.
This report highlights the importance of achieving and maintaining viral suppression when ART is used to reduce the risk of HIV transmission. In this setting, special efforts should be made to minimize HIV transmission risk before the index is virally suppressed, to achieve durable viral suppression on ART, and to identify and address ART failure early.
The authors acknowledge the dedication, commitment, and efforts of the entire HPTN 052 team, and acknowledge the invaluable contributions of the participants in the HPTN 052 trial. The authors thank the laboratory staff at Johns Hopkins University, at the Rocky Mountain Laboratories, and at the HPTN 052 study sites for assistance with sample and data management.
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