Identification of HIV superinfection in seroconcordant couples in Rakai, Uganda, by use of next-generation deep sequencing

Abstract

HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.

Fig. 1.

Mixture analysis of intersubtype detection by NGS. Neighbor-joining trees of p24 next-generation consensus sequences (≥10 identical reads) of control samples of A₂ (A; blue), subtypes D₁ (B; green), a mixture of A₂ and D₁ at a 50:50 ratio (C; red), and a merged tree of all three sample runs (D) are shown. The trees were constructed with a selection of subtype reference sequences and random sequences from individuals in Rakai shown in black. Brackets demonstrate the source of different clades within the merged trees. Bootstrap values higher than 80% are shown for nonmerged trees (1,000 replicates).

Fig. 2.

Inter- and intrasubtype detection of the p24 region by NGS. Neighbor-joining trees of p24 next-generation consensus sequences (≥10 identical reads) of intersubtype mixtures of A₂ and D₁ (red) at 95:5 (A) and 99:1 (B) ratios are shown. Intrasubtype mixtures of A₂ and A₁ (C; blue) and D₃ and D₄ (D; green) at the ratio of 95:5 are shown. The trees were constructed with a selection of subtype reference sequences and random sequences from individuals in Rakai shown in black. Brackets demonstrate the source of different clades within the merged trees. Bootstrap values higher than 80% are shown (1,000 replicates).

Fig. 3.

Intersubtype detection of the gp41 region by NGS. Neighbor-joining trees of gp41 next-generation consensus sequences (≥10 identical reads) of intersubtype mixtures of A₄ and D₅ (red) at 50:50 (A), 95:5 (B), 99:1 (C), and 99.9:0.1 (D) ratios are shown. The trees were constructed with a selection of subtype reference sequences and random sequences from individuals in Rakai shown in black. Brackets demonstrate the source of different clades within the merged trees. Bootstrap values higher than 80% are shown (1,000 replicates).

Fig. 4.

Detection of HIV superinfection in the p24 region. Neighbor-joining trees of HIV p24 next-generation consensus sequences (≥10 identical reads) from female_C1 (green) in 2002 (A) and 2005 (B) are shown. The trees were constructed with a selection of subtype reference sequences and random sequences from individuals in Rakai shown in black. Superinfecting strains are shown with a circle. Brackets demonstrate the individual's HIV subtypes within the trees. Bootstrap values higher than 80% are shown (1,000 replicates).

Fig. 5.

Detection of HIV superinfection in the p24 region. Neighbor-joining trees of HIV p24 next-generation consensus sequences (≥10 identical reads) from female_C3 (blue) in 2002 (A) and 2005 (B) are shown. The trees were constructed with a selection of subtype reference sequences and random sequences from individuals in Rakai shown in black. Superinfecting strains are shown with a circle. Brackets demonstrate the individual's HIV subtypes within the trees. Bootstrap values higher than 80% are shown (1,000 replicates).