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The presence of detectable HIV-1 prior to the appearance of HIV-1-specific antibody was assessed in 41 incident infections that occurred during a 6-year prospective cohort study. All available antibody-negative samples (n = 138) and the first antibody positive sample (n = 41) were tested, under code, by the polymerase chain reaction (PCR) in two laboratories and by HIV-1 isolation in a third laboratory. Samples were available as long as 66 months and at least 18 months before seroconversion for 24/41 subjects. An equal number of time-matched control specimens from persistently seronegative homosexual men and 103 samples from normal blood donors were also tested under code. Samples with discordant results were subjected to coded repeat analysis along with appropriate controls. All but one of the 41 first antibody-positive specimens (97.6%) were PCR positive and 65% were isolation positive. Two control specimens from seronegative homosexual men were PCR positive and one was culture positive, but HLA typing provided clear evidence of specimen mix-up in the specimen archive. For 37/41 seroconvertors, all available antibody negative specimens were negative by both PCR and virus isolation. In three cases, the specimen obtained 6 months before seroconversion was PCR and isolation positive. One specimen, obtained 12 months before SC, was PCR positive and isolation negative but was determined to be the result of sample contamination. Both PCR and isolation were negative in this subject 6 months before SC. In conclusion, we were unable to detect immunosilent infection > 6 months before seroconversion in high-risk homosexual men.

Concordance between two primer pairs and the clinical sensitivity of a single primer pair-probe system were evaluated for a human immunodeficiency virus type 1 (HIV-1) polymerase chain reaction (PCR) assay. Six-hundred sixty-three clinically defined HIV-1 specimens were analyzed in a blind fashion for HIV-1 DNA using an optimized, well-characterized PCR assay. All samples were amplified in duplicate with each of two primer pairs targeting distinct, highly conserved regions within the HIV-1 gag genome. Amplified product was detected by oligomer hybridization using 32P-labeled probes. Correlation between PCR, culture, and serology data was achieved in 661 of 663 (99.7%) specimens. After initial analysis, discordant PCR results due to discrepancies between primer pairs were observed in 9 of 663 (1.4%) specimens. All nine specimens were resolved as negative on reevaluation. After retesting of discordant PCR results, concordance between the two primer pairs was achieved in 100% of samples. These data indicate that the two primer pair systems performed comparably and that good clinical sensitivity (100%) would be achieved using a single primer-probe system. Factors and procedures that influence the reproducibility and accuracy of this HIV-1 PCR assay are discussed.

The sensitivity and specificity of the polymerase chain reaction (PCR) for the detection of HIV-1 proviral DNA was determined in five laboratories with extensive experience in PCR testing. Five panels consisting of 105 HIV-1-seronegative specimens from regularly repeating blood donors with no risk factors for HIV infection and 99 HIV-1-seropositive and culture-positive specimens from a cohort of homosexual/bisexual men were sent under code to each laboratory. Amplification procedures and testing algorithms by which specimens were judged positive, negative, or indeterminate varied between laboratories. The average sensitivity for the five laboratories was 99.0%, with two laboratories achieving 100%. The average specificity was 94.7%, varying between 90.5 and 100%. The overall false-positive rate was 1.8%, the false-negative rate was 0.8%, and the indeterminate rate was 1.9%. Of 1,005 determinations made by the five laboratories, 32 (3.2%) were misclassifications. Most of the classification errors occurred in specimens from uninfected individuals and were distributed among the laboratories in such a way as to indicate laboratory error rather than the inherent reactivity of some samples. This emphasizes the need for standardization of PCR testing and caution in interpreting positive PCR reactions in HIV-1-seronegative persons.