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The characterization of primary HIV infection by the analysis of serial plasma samples from newly infected persons using multiple standard viral assays.A retrospective study involving two sets of archived samples from HIV-infected plasma donors. (A) 435 samples from 51 donors detected by anti-HIV enzyme immunoassays donated during 1984–1994; (B) 145 specimens from 44 donors detected by p24 antigen screening donated during 1996–1998.Two US plasma products companies.The timepoints of appearance of HIV-1 markers and viral load concentrations during primary HIV infection.The pattern of sequential emergence of viral markers in the ‘A’ panels was highly consistent, allowing the definition and estimation of the duration of six sequential stages. From the ‘B’ panels, the viral load at p24 antigen seroconversion was estimated by regression analysis at 10 000 copies/ml (95% CI 2000–93 000) and the HIV replication rate at 0.35 log copies/ml/day, corresponding to a doubling time in the preseroconversion phase of 20.5 h (95% CI 18.2–23.4 h). Consequently, an RNA test with 50 copies/ml sensitivity would detect HIV infection approximately 7 days before a p24 antigen test, and 12 days before a sensitive anti-HIV test.The sequential emergence of assay reactivity allows the classification of primary HIV-1 infection into distinct laboratory stages, which may facilitate the diagnosis of recent infection and stratification of patients enrolled in clinical trials. Quantitative analysis of preseroconversion replication rates of HIV is useful for projecting the yield and predictive value of assays targeting primary HIV infection.