Lower Genitourinary Tract Sources of Seminal HIV Robert W. Coombs, MD, PhD,*† David Lockhart, MSc,‡ Susan O. Ross, RN,§ Leslie Deutsch, RN,§ Joan Dragavon, BSc, MLM,* Kurt Diem, BSc,* Thomas M. Hooton, MD,† Ann C. Collier, MD,† Lawrence Corey, MD,*† and John N. Krieger, MD§ Objective: To investigate genital tract sources of HIV-1, we conducted extensive genitourinary sampling of 23 seropositive men without urethritis who shed HIV in their seminal plasma. Design: Semen was collected, then samples were obtained for HIV RNA in blood plasma, urethral fluid, preYprostate massage fluid/urine (PMF/U) and post-PMF/U, and expressed prostatic secretions. System- atic transrectal ultrasound-guided prostate biopsies obtained from multiple prostate areas were evaluated for HIV RNA and DNA. Results: Seminal HIV RNA levels correlated with HIV RNA levels in urethral fluid and post-PMF/U and with prostate biopsies HIV DNA, but not with expressed prostatic secretions HIV RNA. However, only the HIV RNA level in post-PMF/U independently predicted that in semen (2.77-fold change in semen for each 10-fold change in post-PMF/U; 95% confidence interval, 1.0Y7.7) accounting for one third of the seminal HIV RNA level variation, irrespective of adjustment for antiretroviral therapy. Conclusions: These data indicate that distal genitourinary sources other than the prostate appear to be the major source of seminal HIV in men without clinical urethritis or prostatitis. Because the HIV RNA level in blood plasma is not reliable as an independent clinical predictor of virus levels in seminal plasma, these findings also extend the concept that the male genital tract is a distinct virological compartment from blood. Key Words: AIDS, HIV, RNA, DNA, semen, prostate gland, compartmentalization, urethra, sexual transmission, male (J Acquir Immune Defic Syndr 2006;41:430Y438) S emen is well established as a primary vehicle for sexual transmission of HIV-1. Surprisingly, the anatomic sites and sources of seminal HIV are largely unknown. Based on earlier studies showing that seminal shedding of HIV RNA and HIV DNA continued after vasectomy, we and others deduced that distal genitourinary anatomic sites are likely important sources of seminal HIV. 1Y4 We report a pilot study to test the hypotheses that the urethra and prostate are critical viral sources and that these sites remain reservoirs of genital virus in men who continue to shed seminal HIV while on antiretroviral therapy (ART). We localized and quantified HIV viral burdens in specimens systematically obtained from various genitourinary sites by using systematic anatomic sampling of genital fluids and prostate biopsies (Pbx). METHODS Study Design Our clinical population of HIV-seropositive men was screened between April 1999 and January 2003 to identify men either on no ART or on stable regimens (for at least 3 months) and who had more than 200 HIV RNA copies/mL of blood and seminal plasma. Following written informed consent, each participant agreed to provide 3 separate samples of semen and blood before collecting urethral fluid (UrF; both a urethral swab and/or a less invasive wick), ex- pressed prostate secretions (EPSs), and 6 Pbx. All nonsemi- nal genitourinary specimens were collected during a single clinic visit. The initial study design included only UrF and the prostate biopsy procedure (since we obtained multiple biopsies). However, we modified the protocol to also include preYprostate massage fluid collected in urine (PMF/U) and post-PMF/U when we noted a low rate of HIV detection in biopsies from the initial participants. Each subject had a standardized history and physical examination by the same examiner to exclude potential sub- jects with symptoms or signs of active urethritis, prostatitis, or other active genitourinary tract problems. Semen samples were collected at each subject’s home and transported by taxi to the urology laboratory. 5 After semen analysis and staining, the specimen was sent to the retrovirus laboratory by our courier service. The semen analysis studies were accom- plished within 2 hours, and viral studies were initiated within 4 hours of sample collection. 6 Blood Specimens Peripheral blood was collected into EDTA-containing tubes (Becton Dickinson, Franklin Lakes, NJ), separated within 8 hours of collection into plasma and peripheral blood mononuclear cells by Ficoll-Hypaque gradient centrifugation, then frozen at j 70-C. All specimens were tested in batch for CLINICAL SCIENCE 430 J Acquir Immune Defic Syndr & Volume 41, Number 4, April 1, 2006 Received for publication August 8, 2005; accepted November 16, 2005. From the Departments of *Laboratory Medicine, †Medicine, ‡Biostatistics, and §Urology, University of Washington, Seattle, WA. Informed consent was obtained from all subjects following human experimen- tation guidelines of the United States Department of Health and Human Services and the University of Washington. The authors have no commercial associations that might pose a conflict of interest. Presented in part at the 11th Conference on Retroviruses and Opportunistic Infections; February 8 to 11, 2004; San Francisco, CA (abstract 420). Funding sources: DK-49477; Centers for AIDS Research AI-27757; AIDS Clinical Trials Group AI-27664 and AI-38858, AI-41535, and HD-40540. Reprints: Robert W. Coombs, MD, PhD, Harborview Medical Center, Research & Training Building, Room 706, 325-9th Avenue, Seattle, WA 98104-2499 (e-mail: bcoombs@u.washington.edu). Copyright * 2006 by Lippincott Williams & Wilkins Copyr ight © Lippincott Williams & Wilkins. Unauthor iz ed reproduction of this article is prohibited.