1. Detection of HIV-1 RNA in seminal plasma samples from treated patients with undetectable HIV-1 RNA in blood plasma Marcelin AG 1, Tubiana R 1, Lambert-Niclot S 1, Lefebvre G 1, Dominguez S 1, Bonmarchand M 1, Vauthier-Brouzes D 1, Marguet F 1, Peytavin G 2, Poirot C 1 and the Pitié-Salpêtrière AMP à risque viral Study Group 1 Hôpital Pité-Salpêtrière, Université Pierre et Marie Curie, Paris, France 2 Hôpital Bichat-Claude-Bernard, Université Denis Diderot, Paris, France

2. Background (I)  Recently, the Swiss Federal Commission for HIV/AIDS stated that a seropositive individual, with no other sexual transmitted disease (STD), under antiretroviral treatment and with an undetectable HIV-1 plasma viral load (< 40 copies/ml) for at least 6 months, does not sexually transmit HIV.  However, using a model-based analysis, it has been shown that the risk of HIV transmission in heterosexual partnerships in the presence of effective treatment is low but non-zero and that the transmission risk in male homosexual partnerships is higher over repeated exposures 1.  Indeed, studies have shown that there is a strong relation between HIV plasma viral load and heterosexual transmission rates 2. 1- Wilson et al. Lancet 2008. 2- Quinn et al. NEJM 2000.

3. Background (II)  However, HIV plasma viral load might not always reflect HIV replication levels in semen and some factors could increase the risk of transmission, such as:  Incomplete adherence  Variable drug penetration in compartments?  Other STDs  The aim of this study was to evaluate the residual HIV-1 RNA shedding in semen from patients who were enrolled in an assisted reproductive technology program (ART).

4. Methods (I)  Since 2002, our clinical centre has managed HIV-1 serodiscordant couples within the male partner is infected and not the female to allow pregnancies with assisted reproduction techniques (ART) in order to:  Avoid the risk of HIV sexual transmission  In some cases, to treat infertility  For this purpose, before ART, sperm is “washed” and frozen until virological testing has shown the absence of HIV RNA detection.

5. Sperm Density gradient centrifugation 90% fraction Spermatozoa Seminal plasma 90% 10% Washing centrifugation HIV-1 ARN detection Washed Spermatozoa HIV-1 ARN detection Freezing Until ART

6. Methods (II)  145 HIV-1 infected men attending the Pitié-Salpêtrière Hospital in the multidisciplinary ART program provided 264 paired blood and semen samples between January 2002 and January 2008.  The Cobas Taqman HIV-1 Assay was used to quantify HIV-1 RNA in blood and in seminal plasma as previously described with a limit of quantification of 40 copies/ml in blood and 200 copies/ml in seminal plasma.  ARV pharmacological measurements were performed in blood and seminal plasma.

7. Results (I)  Among the total 264 paired samples:  32 blood samples had HIV-1 RNA > 40 copies/ml Median HIV RNA = 6325 copies/ml (range = 222 – 28300 cp/ml)  16 seminal samples had HIV-1 RNA > 200 copies/ml Median HIV RNA = 1770 copies/ml (range = 255 – 25100 cp/ml)  234/264 paired samples were concordant:  225 samples with undetectable HIV-1 RNA both in blood and semen (85.3 %)  9 samples with detectable HIV-1 RNA in blood and semen (3.4%)

8. Results (II)  30/264 paired samples were discordant:  23 with detectable HIV-1 RNA in blood although the seminal viral load was undetectable  7 with detectable HIV-1 RNA in seminal plasma although the blood viral load was undetectable

9. Results (III)  These 7 discordant paired samples corresponded to 7 distinct patients.  All these patients were under stable HAART with an undetectable HIV-1 RNA in blood plasma for at least 6 months and had no other STDs that are systematically screened in the program.  Among these 7 patients, 6 had an undetectable concordant result in blood and semen in at least one other time point during follow up.

10. Blood plasma Seminal plasma

11. Patient Age (ys) CD4 (cell/mm 3 ) HIV-1 RNA (cp/mL) Circular cells (10 6 /mL) ARV Drug concentration (ng/mL) bloodSeminalBloodSeminal 139368<409400.4ZDV+3TC+IDV/rNA 239529<402573.43TC EFV LPV/r 1594 3486 9656 726 <10 <30 355360<4012306.6ZDV 3TC LPV/r <10 <30 <10 388 <30 443779<402551.5TDF FTC ATV 81 <10 <30 87 <10 <30 547526<4080211ZDV 3TC IDV/r <10 626 2726 <10 3516 1756 640500<4026712FTC+ATV/rNA 743692<406203.7TDF FTC EFV 15 <10 1219 95 163 <10 When antiretroviral drugs such as lamivudine, tenofovir and indinavir were present in blood they were also detected in semen

12. Conclusions (I)  These results show that 5% of patients had detectable HIV-1 RNA in seminal plasma although they had concomitantly undetectable HIV-1 RNA in blood while they were under effective HAART and with no other STDs.  This does not seem to be related to a specific type of ARV treatment.  In these cases, HIV-1 RNA in seminal plasma is comprised between 255 to 1230 copies/ml.  This study confirms that HIV RNA shedding is intermittent in semen over time and that the rate of 5% should be considered as a low estimation of this phenomenon.

13. Conclusions (II)  Although, the presence of HIV-1 RNA does not mean necessarily infectious viruses, this result suggests that cell free virus can be still present in semen despite fully active ARV treatment.  These results should be taken into account in public health messages.  Indeed, while effective antiretroviral therapy is likely to substantially reduce HIV transmission at a population level, residual HIV RNA shedding can occur.

14. Conclusions (III)  Longitudinal studies should be conducted to measure more precisely the frequency and quantity of genital HIV shedding like those previously conducted for HSV.  Search for any relationship between the type of ARV use and the occurrence of viral shedding in a larger cohort of patients.

15. Acknowledgments  ART multidisciplinary group  Infectious Disease : R. Tubiana, S. Dominguez, C. Katlama  Internal Medicine: M. Bonmarchand, A. Simon  Gynecology : G. Lefebvre, D. Vauthier, M. Dommergues  Reproductive biology : C. Poirot  Virology : V. Thibault, V. Calvez  Hepatology: P. Lebray  Pediatrics : I. de Montgolfier  Psychiatry : O. Rosenblum  This study was supported in part by ANRS