An HIV-1-infected man who experienced rapid disease progression and poor response to therapy after starting a new sexual relationship with an infected partner is known as the ‘Ottawa superinfection case'. Subsequent analysis of viral sequences of protease, reverse transcriptase, Gag p17, and Env V3 provided no evidence for the acquisition of genetically divergent viruses before disease progression or drug resistance during virological failure of combination therapy. Whether HIV-1 superinfection contributes to disease progression or the spread of drug-resistant HIV-1 remains unknown.
Subject A was first diagnosed with HIV-1 infection in 1988, and showed no evidence of disease progression over the following 9 years (Fig. 1a). In late 1997, he was found to have an abrupt increase in viral load and a decrease in the CD4 T-cell count, which prompted the initiation of combination antiretroviral therapy. Therapy for 4 months failed to suppress the plasma viral RNA level. The rapid clinical progression and poor response to therapy in this sexually active man raised concern that superinfection with a virus that was more pathogenic and drug resistant may have occurred [1,2]. He had initiated a sexual relationship, including unprotected anal intercourse, in the fall of 1997 with a man (subject B) known to have HIV infection, who had experienced virological failure despite therapy with multiple antiretroviral agents including protease inhibitors and nucleoside reverse transcriptase inhibitors. At the time of his reported sexual relationship with subject A, the partner's antiretroviral therapy consisted of nelfinavir, lamivudine and stavudine. During the 6-month period around the start of this sexual relationship with subject A, the partner's plasma RNA level ranged between 1800 and 30 000 copies/ml (Bayer branched DNA v2) and his CD4 T-cell count ranged between 94 and 166 cells/μl.
Viral sequence analysis was performed to determine whether subject A had acquired a drug-resistant virus from subject B shortly before his clinical disease progression. HIV-1 protease and reverse transcriptase sequences from subject A, subject B, and several local control individuals were determined in one laboratory (YWH) and analysed for phylogenetic relationships (data not shown). Sequences demonstrated to be identical to unrelated sequences derived in the same laboratory were deemed to have been caused by contamination and were excluded from the analysis. The sequences derived from subject A's peripheral blood mononuclear cell DNA stored in 1989 clustered separately from the sequences derived from blood plasma RNA stored in 1998. However, the sequences from these two timepoints were more similar to each other than to sequences derived from epidemiologically unrelated individuals in the same geographical area, as expected after viral genetic drift in vivo. Similarly, the majority of HIV-1 protease and reverse transcriptase sequences from subject B clustered together and were distinguishable from any other individual's viral sequences. None of the branching patterns in the phylogenetic tree of protease sequences were supported by bootstrap values greater than 70% (700 of 1000 trials), indicating that the evolutionary relationships of the protease sequences were not clearly identified. The lack of sufficient information to establish evolutionary relationships using protease sequences probably reflects the small size of the gene segment (297 bases), drug selection pressures acting at many codons in the protease gene, and extensive genetic interactions between protease and protease cleavage sites. In contrast, branching patterns of reverse transcriptase sequences were supported by significant bootstrap values, indicating that sequences from subject A and subject B represented distinct lineages.
The protease and reverse transcriptase sequences were analysed for evidence of drug resistance that might explain the poor virological responses to therapy observed in subjects A and B. Protease sequences from subject B (the partner) had the PR D30N mutation in two out of 13 clones (15%), the PR M46I mutation in an additional two out of 13 clones (15%), and the D88S mutation in three out of 13 clones (23%), indicating some genotypic evidence of protease inhibitor resistance in 53% of clones from subject B, consistent with his previous use of nelfinavir. The RT M184V mutation was detected in all 10 viral clones derived from subject B at a time when he reported treatment with a lamivudine-containing regimen. The RT M41L mutation was also detected in one out of 10 clones from subject B. In subject A, the reverse transcriptase M184V mutation (indicating lamivudine resistance) was detected in none of 16 clones from 1989, but was detected in one out of 12 clones derived from January 1998; a timepoint that was 8 months after subject A was prescribed a lamivudine-containing regimen that he did not report to subsequent clinicians. After 1997, subject A continued to be viremic during 4 months of combination antiretroviral therapy, during which time viral sequencing revealed no evidence of drug resistance at two different times.
Because final analysis of the protease sequences provided insufficient evidence to evaluate transmission linkages between subject A and subject B, sequence analysis of other viral genetic loci (gag P17 and env V3) were undertaken to evaluate further whether HIV-1 superinfection may have occurred. The confirmatory analysis of specimens from subject A was undertaken in a separate laboratory (ED) from the laboratory (RMG) analysing specimens from subject B and from epidemiologically unrelated individuals residing in the same geographical area. Phylogenetic analysis revealed evidence of an expected viral genetic drift [3–5] in subject A, and no evidence of transmission of HIV-1 from subject B to subject A (Fig. 1b and c). There was no evidence that subject A had acquired a new variant of HIV-1 before the increase in viral load and fall in CD4 T-cell count. Increases in viremia before the loss of T-cell homeostasis and clinical progression in cohorts of infected men have been described previously [6–8].
Case reports suggesting HIV-1 superinfection have recently been published [9–12] and presented at international meetings . Confirmation of superinfection by an analysis of viral sequences from source partners was not possible in these cases. Although allele-specific polymerase chain reaction indicated no evidence of dual infections at enrollment visits in the reported cases, the sensitivity of these assays is limited such that concomitant dual infection with the sequential appearance of viral variants cannot be excluded. Those reports involved individuals who were recruited into cohorts shortly after their initial HIV-1 infection, suggesting that the susceptibility to acquiring additional HIV-1 variants by superinfection may be restricted to a window period of susceptibility, as was observed in a non-human primate model of superinfection . If so, the window period in humans appears to be prolonged for at least one year for those exposed to different subtypes of HIV-1 [9,12] or longer if early antiretroviral therapy is used [11,12]. Antiretroviral therapy may increase susceptibility to HIV-1 superinfection by decreasing viral interference, increasing the density of target cells, and decreasing antiviral immune responses [15,16]. No evidence of HIV-1 superinfection could be demonstrated in the ‘Ottawa case', which involved a chronically infected individual not receiving antiretroviral treatment who was exposed to a partner with a virus of the same subtype. Whether superinfection can occur after prolonged HIV-1 infection remains to be determined. Furthermore, it remains unknown whether superinfection can accelerate disease progression or contribute to antiretroviral drug failure. Our findings suggest that special procedures are needed to control and detect laboratory contamination during the evaluation of suspected cases of superinfection.
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