Understanding the correlates of HIV transmission is important to determining effective preventative strategies. The pandemic spread of HIV-1, in contrast to the limited spread of HIV-2, primarily in West Africa, is likely due to different rates of transmission of these two viruses [1–4]. Nonetheless, both HIV-1 and HIV-2 are primarily spread by heterosexual transmission [5,6]. Similar to other sexually transmitted infections (STIs), risk of HIV transmission is in part a function of the quantity of HIV in genital tract secretions [7–17]. A greater understanding of patterns and risk factors associated with genital tract HIV-1 and HIV-2 shedding will permit the development of more efficient strategies to control HIV transmission . Only three studies have compared risk factors for detecting HIV-1 versus HIV-2 in the genital tract; all were performed in women [19–22]. Potential differences in HIV shedding rates among subjects infected with HIV-1 versus HIV-2 have been hypothesized to result from lower HIV plasma loads among HIV-2 infected persons [23–25]. Indeed, HIV-1 plasma viral load has been the only factor directly linked to transmission [26–30]. Factors shown to be associated with shedding in semen have been less well studied but plasma viral load appears to be a strong correlate [13–15] as do concurrent STIs [31–34].
In this study we examined semen shedding in 22 HIV-1 and 10 HIV-2-infected men from Senegal, West Africa. These data may help explain the different transmission rates between HIV-1 and HIV-2 and may provide new insights regarding prevention.
Materials and methods
Study population and study design
Men aged 18 years and older presenting to the University of Dakar, Fann Hospital, Infectious Disease Clinic were offered HIV-1 and HIV-2 serologic testing. Sera were tested using a microwell plate HIV-1/HIV-2 enzyme immunoassay (Genetic Systems, Redmond, Washington, USA), with positive sera confirmed using a rapid synthetic peptide-based membrane immunoassay (Multispot; Sanofi Diagnostic Pasteur, Redmond, Washington, USA) which classifies subjects as HIV-1, HIV-2, or dually seropositive. Semen and blood samples for HIV viral load assays were obtained from 22 men infected with HIV-1 and 10 with HIV-2. All subjects were antiretroviral therapy naive. This study was conducted according to procedures approved by the institutional review boards of the Universities of Washington and Dakar, and the Senegalese National AIDS Committee. All subjects provided informed consent for study participation.
Collection of specimens and clinical assessment
Subjects completed an interview with questions concerning demographic characteristics, and sexual and other behaviors. A routine medical history was taken and recorded on a standardized form by a clinician who also performed a general physical examination and a genital examination and clinical assessment for sexually transmitted infections (STI). Five milliliters of peripheral blood was collected into tubes containing ethylenediamine tetra-acetic acid (EDTA) or acid citrate dextrose (ACD) and analyzed using the FACSCount analyzer (Becton Dickinson Biosciences, San Jose, California, USA) to determine the number of CD4, CD8 and CD3 cells/μl of blood; 10 ml of peripheral blood was collected for HIV-1 and HIV-2 quantitative RNA viral load assays. Semen was obtained by masturbation into a sterile container.
Quantitative detection of HIV-1 and HIV-2 RNA
Quantitative viral load assays for HIV-1 and HIV-2 plasma and semen RNA were performed using polymerase chain reaction-based assays developed at Roche Molecular Systems (Pleasanton, California, USA) as previously described [25,35].
Fisher's exact tests were performed to compare detection of semen and plasma HIV RNA among men with HIV-1 versus HIV-2. Continuous variables (e.g., CD4 cell count, log10 plasma/semen RNA) were compared using the Wilcoxon rank-sum test. Undetectable levels of plasma or semen HIV-1 and HIV-2 RNA were set to the lower limit of detection minus one for statistical analyses. To evaluate the sensitivity and robustness of our study findings to samples that had negative results on quantitative assay for HIV RNA we performed a series of analyses, setting the values for those samples to a range of values between 0 and the limit of detection. None of these analyses gave qualitatively different results. The level of statistical significance used in all analyses was P < 0.05. Data analyses were conducted using SAS 8.2 for Windows and JMP for Macintosh (SAS Institute, Cary, North Carolina, USA).
The demographic, clinical, immunologic and virologic characteristics of the 22 HIV-1 and 10 HIV-2 infected men in this study are shown in Table 1. The mean age of the HIV-1 and HIV-2 subjects was 37 and 40, respectively. Experiencing greater than 10 life-time sexual partners and having sex with female sex workers was common in these men, whereas consistent condom usage was less frequent. Symptomatic STI were infrequent: none of the men had penile discharge or inguinal lymphadenopathy, genital ulcer disease (GUD) was also uncommon (Table 1). The majority of subjects had WHO stage 3 disease and the mean CD4 cell count was 222 and 276/μl for HIV-1 and HIV-2-infected subjects, respectively (P = 0.8 for CD4 cell count difference by Wilcoxon rank sum test). The mean plasma viral load was 4.7 and 3.0 log10 copies/ml for HIV-1 and HIV-2, respectively (P = 0.002 for difference by Wilcoxon rank sum test). The presence of HIV RNA in semen was marginally higher in those with HIV-1 compared to those with HIV-2, (being detected in 21 of 22 (95%) of HIV-1 and seven of 10 (70%) of HIV-2 infected subjects; P = 0.07 by Fisher's exact test), However, the levels of HIV RNA present in semen were markedly different between those with HIV-1 compared to HIV-2, with a mean of 4.4 log10 copies/ml among those with HIV-1 and a mean of 2.6 log10 copies/ml among those with HIV-2 (P < 0.001 for difference by Wilcoxon rank sum test). Semen HIV RNA viral load correlated with plasma viral load (adjusted R2 = 0.53, P < 0.0001) irrespective of HIV type (P = 0.007 for HIV-1 and P = 0.05 for HIV-2) (Fig. 1). However, HIV-2 infected subjects had 0.7 log10 lower HIV semen load than those with HIV-1 after adjusting for log10 plasma level (P = 0.07). In addition, semen HIV RNA viral load was inversely correlated with CD4 cell count (adjusted R2 = 0.16; P = 0.02) irrespective of HIV type (P = 0.03 for HIV-1 and P = 0.18 for HIV-2) (Fig. 2). However, HIV-2 infection was associated with a 1.5 log10 lower HIV semen load than that in HIV-1 after adjusting for CD4 cell count (P < 0.001). In multivariate analysis, plasma viral load and HIV type, but not CD4 cell count, were independently predictive of semen viral load (P-values = 0.03, 0.05, 0.48, respectively).
To our knowledge this is the first study to examine levels of HIV-2 in the male genital tract or compare them to those of HIV-1. We examined semen HIV shedding in 22 HIV-1 and 10 HIV-2-infected men from Senegal, West Africa. We found significantly lower levels of HIV in semen of those with HIV-2 compared to HIV-1, and shedding generally correlated with plasma viral load irrespective of virus type. This study confirms previous reports of a strong correlation between HIV-1 male genital shedding and plasma viral load [13–15] and expands the data on male genital tract HIV-1 levels in individuals from sub-Saharan Africa [36–39]. In addition, we have previously shown that equal plasma viral loads predict a similar rate of CD4+ T cell decline in HIV-1 and HIV-2-infected individuals from our Senegalese cohort and that viral load but not HIV type is the most important factor . Our current data suggest that while levels of semen viral loads were lower in HIV-2 compared to HIV-1, plasma viral load is stronger predictor than HIV type in determining the level of semen shedding. Furthermore, our current study of HIV-1 and HIV-2 in the male genital tract shows similar findings to previous studies comparing HIV-1 and HIV-2 levels in the female genital tract; namely that HIV-2 levels are typically lower than HIV-1 in both plasma and the genital tract [19–22]. Taken together, these data help explain the different transmission rates between HIV-1 and HIV-2 and may provide new insights regarding prevention.
Limitations of this study include the small sample size, especially the number of HIV-2 infected subjects, and the lack of longitudinal follow-up. The lack of longitudinal follow-up leaves unanswered the question of whether the usually low plasma HIV-2 RNA viral load throughout the course of HIV-2 infection  leads to persistently reduced seminal shedding. Moreover, the question of whether the similar levels of peripheral blood mononuclear cell HIV DNA found in HIV-1 and HIV-2 infection  are also found in HIV-infected seminal cells also remains unanswered. Given the small sample size of HIV-2 subjects, other potential correlates, such as factors previously found to be associated with HIV-1 shedding, could not be assessed. Specifically, it is unclear whether concurrent STI increase HIV-2 seminal shedding as they do in HIV-1 infection. Symptomatic STI in this study were rare. None of the men had penile discharge or inguinal adenopathy suggesting that gonorrhea and/or chlamydia were unlikely and consequently swabs for gonorrhea/chlamydia were not taken in asymptomatic men. GUD was present in 11% of the HIV-1 and 13% of the HIV-2 infected men. It was not further assessed for specific pathogens (e.g. syphilis, herpes simplex virus, chancroid). There was no association between presence of GUD and semen or plasma loads (P = 0.46 and 0.23, respectively) although power to detect an effect was low given the small sample size. However, given the GUD data and the low prevalence of symptomatic STI we believe semen viral loads were unlikely to be deferentially biased in the HIV-1 versus HIV-2 groups. Unfortunately this study was prematurely ended due to community concerns regarding semen donation; however, we do not believe this premature termination systematically biased our results. Despite these important constraints, we found a significant correlation of HIV-2 semen viral load with plasma viral load.
Despite the correlation between plasma and semen viral load, evidence suggests that the genital tract can be a persistent reservoir for HIV-1 replication in men and that treatment with antiretroviral drugs only suppresses semen HIV-1 shedding to varying degrees [40–44]. In addition, antiretroviral drug-resistant HIV-1 can be detected in semen of men on antiretroviral therapy [45,46]. Whether these observations will be seen in HIV-2-infected men treated with antiretroviral therapy will require further studies. Ultimately, a thorough understanding of the correlates of HIV shedding in the male genital tract will help in defining strategies to prevent HIV transmission and curtail its epidemic spread.
We thank Deana Rich, Elise Reay-Ellers, Macoumba Touré, Dr Mame B. Diouf, Dr Mame A. Faye Niang and Dr Awa M. Coll-Seck for their invaluable co-ordination and supervision of study procedures in Senegal; Dr Aissatou Diop, Dr Pierre Ndiaye, Marie Pierre Sy and Mame Dieumbe Mbengue-Ly for patient care; and Alison Starling for forms development and data management. In addition, we would like to thank Shirley Kwok, Rich Respess, Kelly Lagassic, Cindy Christopherson and Jane Kuypers for their work in the development of assays and testing of study samples for HIV-1 and HIV-2 RNA and David Nickle and Jim Mullins for helpful discussions. We would like to thank the study participants without whom these studies would not be possible.
Sponsorship: These studies were supported by grants from the NIH/NIAID and the University of Washington CFAR.
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