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Incidence rates of human immunodeficiency virus type 1 (HIV-1) superinfection differ among cohorts and, as yet, only 2 cohorts of homosexual men have been screened. Here, we investigated the incidence of HIV-1 superinfection during the first year after infection among homosexual participants in the Amsterdam Cohort Studies on HIV infection and AIDS who seroconverted between 1985 and 1997.We analyzed env C2-C4 diversity in the serum of therapy-naive participants, using a heteroduplex mobility assay; heteroduplexes were considered to be indicators of potential dual infections, in which case env C2-C4 polymerase chain reaction (PCR) products were cloned and sequenced. Sequences were subjected to phylogenetic analysis. Data on the sexual behavior of participants were collected from 1 year before seroconversion until the end of the investigated period.For 89 seroconverters with a detectable viral load (>1000 copies/mL), env PCR products were generated from serum samples obtained at seroconversion and 1 year later. Heteroduplexes were observed in 68 of the 89 patients; among these 68 patients, a median of 9 molecular clones per time point was sequenced. Phylogenetic analysis did not reveal evidence for superinfection; 1 patient was HIV-1 coinfected. Shortly after diagnosis of HIV infection, the number of sex partners decreased, the frequency of anal intercourse declined, and condom use increased.The incidence of HIV-1 superinfection soon after seroconversion in this cohort is low. Risk reduction shortly after HIV-1 diagnosis early during the HIV-1 epidemic in the Netherlands may have contributed to the absence of HIV-1 superinfection observed in this study.