Sequence divergence between PR or RT sequences obtained at different times from HIV-1—infected individuals receiving antiretroviral treatment may result from the acquisition or loss of mutations at positions associated with drug resistance [7
], from genetic bottlenecks in which a new therapy selects for preexisting rare variants with differences at positions not associated with drug resistance [11
], from mix-ups with a sample from a different person [13
], and from superinfection with a virus from a different person [14
]. Of these possibilities, superinfection is the most interesting to clinicians and researchers because of its implications for protective immunity, viral interference, and the development of recombinant virus strains [17
]. We have, therefore, focused our discussion on the ability of our data to detect superinfection. However, our study also provides new data on the extent to which PR and RT sequences of HIV-1 isolates from treated individuals may change over time. An understanding of these data is essential for maintaining the quality of PR and RT sequencing in laboratories performing genotypic resistance testing.
The present study shows that, although HIV-1 PR and/or RT genes from treated persons may become highly divergent, this divergence almost always results from intrahost sequence evolution, rather than from superinfection. We arrived at this conclusion by identifying 37 (5%) individuals with the most-divergent isolates from our sample of 718 individuals, reviewing their treatment histories, and then sequencing tat and/or gag—genes that are not under selective drug pressure—from these individuals. For 16 of these 37 individuals, the sequence divergence was consistent with a documented treatment interruption or with the resumption of therapy after a treatment interruption. For another 15 individuals, paired tat and/or gag genes were monophyletic, which argues against superinfection. Stored samples were not available for tat and gag sequencing for the remaining 7 individuals.
Although numerous cases of simultaneous infection with 2 distinct HIV-1 strains have been reported [17
], there have been only 4 well-documented cases of superinfection, in which a second virus infected a person well after an initial infection [14
]. All 4 cases occurred in persons who were monitored prospectively after the identification of primary infection. In 3 of the 4 cases, superinfection occurred with a virus belonging to a different subtype than the primary strain [14
Approximately 99% of isolates in our cohort belong to subtype B [21
], which makes it more difficult to detect superinfection and requires the use of 2 new methods for assessing sequence divergence: the mixture-weighted distance allows the calculation of nucleotide distances between sequences containing nucleotide mixtures [4
], whereas the GPS score allows the detection of signature polymorphisms within an infected individual that may be present even in the absence of phylogenetic clustering. The GPS method bears some similarity to the signature pattern analysis method of Korber et al. [22
] but differs in that it evaluates 2 sequences at a time from a large set of sequences for which the amino acid profile (distribution of variants) at each position is known.
Each of the previous published cases of superinfection was initially identified by population-based sequencing. We cannot, however, exclude the possibility that superinfection occurred in our cohort but was undetected, because the superinfection virus remained a minor variant. To detect these cases, it would be necessary to sequence multiple clones from each of the plasma samples, rather than restricting the analysis to those samples with the most genetic divergence. Such a strategy would be optimally suited to a small cohort of persons at high risk of superinfection, rather than a large cohort like ours in which exposure history was not available.
Because HIV-1 is likely to undergo recombination when 2 viruses infect the same cell, we also examined sequences for the possibility that a second virus may have recombined with virus present in the first plasma sample. We divided each of the PR sequences into 2 segments of ~150 nt and each of the RT sequences into 5 segments of 150 nt and calculated the divergence between matched gene segments from the same person. This analysis identified 61 additional persons with virus isolates having PR or RT segments with a mixture-weighted distance >4.5% (~7 nt) between at least 1 pair of matched 150-nt segments. In nearly all these additional cases, divergence appeared to be caused by a major change in treatment (usually an interruption) or by a few unexplained nucleotide changes (data not shown), which suggests that, even if recombination with a superinfecting strain had occurred, it was extremely rare.
Surveillance data from the San Francisco Department of Health suggest that, among persons with the most common risk factors for HIV-1 in Northern California (e.g., male homosexuality and intravenous drug use), there is an ~1%–2% annual incidence of new HIV-1 infection [23
]. Prospective studies in cohorts of individuals for whom risk behavior is documented have the potential to better define the incidence of superinfection. However, such studies are difficult to perform. Although we do not know the risk profile of our cohort, it is likely that ~10–20 new cases of HIV-1 would have been expected during 1072 person-years of follow-up had the individuals in the cohort not already been infected.
Superinfection may be prevented as a result of partial immunity, the effect of antiretroviral drugs on superinfection with a drug-susceptible strain, or viral interference from the original virus strain. However, we cannot quantify the risk of superinfection because we do not know the extent of HIV-1 exposure within our cohort and because we cannot completely exclude the possibility that some cases of superinfection escaped detection. Therefore, infected individuals, even those receiving anti-retroviral therapy, should continue to avoid activities that could transmit HIV-1 to others or increase their risk of a second infection.