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Preejaculatory fluid, a viscous, lubricating secretion expressed by penile urethral glands during sexual excitement, may play a role in the sexual transmission of HIV-1. The urethra has been shown to be an important site of HIV infection in men and male macaques, and preejaculatory fluid and urethral swabs from HIV-1-infected men often contain HIV. Recent studies have shown that HAART reduces but does not eliminate seminal HIV shedding in infected men, and that the penile urethra remains a site of persistent simian immunodeficiency virus infection in HAART-treated macaques. The objective of this study was to determine whether HIV-infected men on stable HAART continue to shed HIV into preejaculatory secretions.Single-center prospective study.Sixty HIV-infected men on HAART were recruited to provide preejaculatory fluid, semen, and blood for HIV RNA quantification by reverse transcription-PCR.Eight men had detectable HIV in blood; of these four had HIV in semen (range: 40–96 000 copies/ml), and one had HIV in preejaculate (2400 copies/sample). Fifty-two men had undetectable HIV RNA in blood; of these 10 (19.2%) had HIV RNA in semen (range: 59–800 copies/ml) whereas none (0%) had HIV RNA in preejaculate (P = 0.004).This study documents for the first time high levels of HIV RNA in preejaculate fluid. However, none of the men on stable HAART with undetectable blood viral load had HIV RNA in preejaculate, even though many had detectable HIV in semen. The urethral glands do not appear to be a principal source of HIV in men on suppressive HAART.