An overview of the methods used to assess the genetic linkage of seroconversion events in HPTN 052 is shown in . HIV pol
region sequences were obtained for 2 samples from different study visits for 70 of the 76 participants (92.1%) studied (38 index-partner pairs); 3 index participants and 3 partners had only 1 pol
sequence result. The median time between collection of the 2 partner samples was 27 days (range, 1–126 days), the median time between collection of the 2 index samples was 356 days (range, 27–1211 days), and the median time between collection of the earliest index sample and the latest partner sample was 380 days (range, 1–1211 days); in some cases, the second index sample was collected after the seroconversion event. A single phylogenetic tree was generated using all available index and partner sequences, 80 local control sequences, subtype reference sequences, and an outgroup sequence (simian immunodeficiency virus). In all cases, paired sequences from the same individual grouped closely together on monophyletic branches; the median genetic similarity of the paired sequences was 99.5% (range, 94.7%–100%). For all 38 index-partner pairs, the HIV subtype of the index and partner was the same and was consistent with the prevalent subtype(s) in the region (). Antiretroviral drug resistance mutations were detected in samples from 4 index-partner pairs. In all 4 cases, the pattern of resistance mutations detected was consistent with the final linkage assessment (Supplementary File 2
). Phylogenetic trees were also generated for each geographic region (Africa, Asia, and North and South America). The phylogenetic clustering of sequences from index-partner pairs was the same in the large composite tree and the region-specific trees (not shown). A representative tree that includes 5 seroconversion events is shown ().
Outline of approach used for linkage analysis (see text).
In 30 of the 38 couples analyzed, the sequence(s) obtained for the index grouped with the sequence(s) obtained for the corresponding partner on a unique, monophyletic branch. The bootstrap values for the grouped sequences were 100% in all but 1 case (for 1 event, the bootstrap value was 99%). Those 30 seroconversion events were provisionally characterized as linked. In the remaining 8 couples, sequences from the index and partner did not group together. Those 8 seroconversion events were provisionally characterized as unlinked. There was no difference in the linkage assessments using other models for determining genetic distance (see Materials and Methods).
The genetic linkage of HIV from index-partner pairs was also assessed by comparing genetic similarity of paired HIV pol sequences using Bayesian analysis. A posterior probability of linkage was computed for each index-partner sequence pair (up to 4 sequences per couple) using pooled and unpooled training data (). Pooled results were used for the primary determination of linkage. Using this approach, 27 of the 38 (71%) seroconversion events were classified as linked and 11 as unlinked.
Figure 3. Densities of similarities from linked and unlinked sequence pairs. A, Densities of similarities from linked and unlinked sequence pairs from 32 subtype C couples, along with subtype C training data. B, Densities of similarities from linked and unlinked (more ...)
In 35 of the 38 (92.1%) seroconversion events analyzed, the linkage assessments based on phylogenetic and Bayesian analyses were concordant (). In 27 cases, both were linked; in 8 cases, both were unlinked. In the remaining 3 cases, phylogenetic analysis suggested that the seroconversion events were linked, whereas statistical analysis suggested that they were not linked; those events were provisionally characterized as “to be determined” (TBD). Examples of results from phylogenetic and Bayesian analyses are shown in .
Representative Results Obtained From Linkage Analysisa
Samples from 12 of the 38 (31.6%) seroconversion events were further analyzed using NGS (env region [gp41]). This included 1 event provisionally characterized as linked, 8 events provisionally characterized as unlinked, and 3 events provisionally characterized as TBD. NGS analysis confirmed that 7 of the 8 provisionally unlinked events were unlinked. However, for 1 event that was provisionally classified as unlinked, NGS analysis revealed that the event was linked (event 052-2989; ). NGS results for 1 event provisionally characterized as TBD indicated that the event was linked. NGS results for the other 2 TBD events did not sufficiently meet the criteria established for linkage; the status for those events was not changed. One sample provisionally characterized as linked was confirmed to be linked by NGS.
Figure 4. Next-generation sequencing results for the env region (gp41). The figure shows phylogenetic trees generated using data from next-generation sequencing (NGS, env region [gp41]). Each panel shows results obtained for 1 seroconversion event where linkage (more ...)
Final linkage status was determined for 36 of the 38 events analyzed (). In all but 1 case, the final linkage assessment was the same as the assessment made based on data available at the time of the April 2011 DSMB meeting. In 1 case, a seroconversion event in the delayed ART study arm that was previously characterized as TBD was subsequently characterized as linked (event 052-1168; ).
The median minimum time between collection of index and partner samples was 28 days (interquartile range [IQR], 0–84 days; range, 0–1083 days) and was longer for the 7 unlinked events (median, 266 days; IQR, 180–537 days; range, 77–696 days) than for the 29 linked events (median, 4.0 days; IQR, 0–66 days; range, 0–1083 days). Nonetheless, the range of time between paired index specimens (median, 355 days; IQR, 178–503 days), for which 29 of 30 similarities were >97%, was similar to the range of time between unlinked index-partner samples, all of which had similarities <95%. This suggests that the timing of specimen collection did not significantly influence the final linkage assessment.
We analyzed the association of demographic, behavioral, and clinical factors with the linkage status of seroconversion events (). Linked HIV transmission was more frequent when the index participant was in the delayed ART study arm and was not receiving ART at the time of the partner’s seroconversion. There was also a significant association between unlinked transmission and the partner’s report of a higher number of sexual partners during the 3 months before seroconversion. We did not find an association between linked transmission and geographic region (Africa, Asia, and America), index sex, index CD4 cell count at the time of the partner’s seroconversion, or the length of time between enrollment and seroconversion. Linked transmission was less frequent in male–male couples. However, there were only 2 male–male couples in the analysis.
Factors Associated With Linkage of Seroconversion Events