Analyses for detection and quantification of HIV in sperm samples
S.L. Castillo, E. Sturba, M.S. Ojeda, M.I. Gutierrez
Laboratory Stamboulian, Molecular Biology, Buenos Aires, Argentina
Background: HAART therapy allowed discordant couples have their own children. Since assisted fertilization protocols require detection of HIV in sperm samples, our goal was to evaluate commercial qualitative and quantitative tests.
Methods: COBAS Ampliprep/COBAS AMPLICOR HIV-1 MONITOR Test version 1.5 (Roche) for viral load in seminal plasma and Amplicor HIV-1 DNA test version 1.5 (Roche) for HIV proviral DNA in sperm cells.
Results: We first tested 31 sperm samples for amplification of the internal control (QS) to discard potential inhibition. Seminal plasmas were analyzed directly and diluted in normal human plasma and all yielded valid QS. We also compared results from 12 samples with NASBA (Nuclisens HIV-1 QT, Biomerieux) and confirmed lack of false negative results in the new system. Finally, we spiked 22 of these seminal samples with human plasma from HIV-positive patients and confirmed that HIV RNA was also detectable and the range of viral loads were similar. The limit of detection tested was 100 copies/ml.We analyzed the cellular fraction of the sperm (swim up) for the presence of integrated HIV. All 25 samples tested from sperm donors, were negative for HIV, but the amplification of the QS was successful. To ensure amplification of HIV, we spiked 13 swim up samples with white blood cells from HIV-patients with low levels of CD4 lymphocytes (surrogate marker of infected cells) and performed the Amplicor test. All were positive for HIV, with CD4 counts as little as 4/mm3 (1%).
Conclusions: We conclude that the new assays are adequate methodologies to analyze HIV in sperm samples, testing the viral load in seminal plasma and the presence of the virus in the sperm cells. We did not observed inhibition of the internal controls, indicating that these different sample matrix can be analyzed. None of the samples demonstrated a false negative result regardless of the CD4 counts.
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