SummaryThe significance of detection of human immunodeficiency virus (HIV) DNA by the polymerase chain reaction (PCR) in seronegative or sero-converting (SC) subjects remains controversial. In a previously reported study, we identified a case in which a specimen collected 12 months before serocon-version (pre-SC) was found repeatedly to be PCR positive in three experienced laboratories, while the 6-month pre-SC bleed was PCR-negative; PCR-based human leukocyte antigen (HLA)-DQA and -DRB typing of serial peripheral blood mononuclear cell (PBMC) samples from this case did not indicate a specimen mix-up or labeling error. To further investigate this case, we used HIVenvsequence and DNA heteroduplex gel-shift analyses to characterize HIV quasispecies present in serial pre- and post-SC specimens. HIVenvsequences and gel-shift pattern analyses from the 12-month pre-SC versus post-SC samples indicated that markedly distinct quasispecies were present, suggesting possible abortive infection followed by reinfection and subsequent seroconversion. However, the HIV burden of this pre-SC sample was very low (1 provirus/106PBMCs), and the quasispecies was highly heterogeneous, findings suggesting long-term rather than recent HIV infection. To test the hypothesis that the index pre-SC sample was PCR positive owing to trace blood contamination during initial processing, we analyzed the three seropositive samples collected on the same date in 1985. One of these samples was highly related to the index pre-SC sample byenvsequence and gel-shift methodologies. The source of contamination was further verified by PCR detection (using enhanced-sensitivity allele-specific primers) of HLA DR2 sequences unique to the contaminating source subject in the 12-month pre-SC PBMC sample from the index subject. This study shows that trace contamination during blood processing represents an additional basis for false-positive HIV PCR results and illustrates the usefulness of DNA heteroduplex gel-shift analysis and HLA allele-specific PCR in investigating such cases. NATIONAL GENBANK ACCESSION Numbers for these sequences: L20371 through L20380. inclusive.