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Shedding of HIV and human herpesviruses in the semen of effectively treated HIV-1 infected men who have sex with men
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Clinical Infectious Diseases Advance Access published April 17, 2013
Sara Gianella1, Davey M. Smith1,2, Milenka V. Vargas1, Susan J. Little1, Douglas D. Richman1,2, Eric S. Daar3, Michael P. Dube4, Fan Zhang5, Christina C. Ginocchio5, Richard H. Haubrich1, Sheldon R. Morris1 and the CCTG 592 Team
"In summary, the present study evaluated a large number of seminal samples (n=114) from HIV-infected sexually active MSM treated with ART with an extensive battery of tests for viral and bacterial co-infections. The association between isolated seminal HIV shedding and high-level CMV replication and EBV replication in the genital tract suggests that the presence of these viruses could play a role in HIV transmission not only in ART-naïve subjects as previously described [19, 21] but also in individuals on ART. These finding have important implications for the development of strategies to reduce HIV transmission."
"We previously described that seminal shedding of different herpesviruses (i.e. high-level CMV, EBV, HHV-8) was associated with increased levels of HIV within the genital tract of ART-naïve MSM. The present study aimed to investigate if this remains true as well in subjects receiving ART with suppressed HIV levels in blood plasma.....We previously described that seminal CMV replication is positively associated with levels of immune activation in the male genital tract [18], and our present study on a larger cohort, suggests that high-level CMV replication likely plays a role in HIV seminal shedding also in successfully treated HIV infected individuals."
"Since HIV in male genital secretions accounts for the majority of HIV transmission in both women and MSM [28], it is crucial to understand the factors associated with increased seminal HIV replication in order to inform strategies to reduce sexual HIV transmission and the development of HIV drug resistance. Effective ART substantially reduces the HIV levels in blood and semen [1-4]; however, a significant number of subjects intermittently shed HIV in semen despite having suppressed levels in blood plasma [9-12]. In our study of sexually active, ART-treated MSM with blood plasma HIV levels <500 copies/ml, 11 (9.6%) had detectable HIV in seminal plasma at very low levels between 50 and 230 copies/ml. The prevalence of seminal HIV shedding falls within the range previously described in the literature (2%-48%) [9-12], depending on the characteristics of the observed population. Detectable levels of seminal HIV were present significantly more often in persons with plasma HIV between 50 and 500 copies/ml compared to those <50 copies/ml.
Subjects with evidence of HIV replication in plasma while on ART (i.e. blood plasma HIV levels 50-500 copies/ml) had detectable levels of HIV in semen more frequently than subjects with <50 HIV copies/ml (35.7% versus 6.0%, FDR-adjusted p-value 0.08)."
"In a sub-analysis of the 100 subjects with HIV in blood of <50 copies/ml, of whom six had detectable HIV seminal levels, high CMV levels remained the only factor associated with HIV shedding with a relative risk of 5.7 (95% CI 1.1-29.3). Specifically, 15.4% of semen samples with high-level CMV also shed HIV compared to 2.7% in those without high levels of CMV. There was also a trend for an increased frequency of HIV shedding in semen being associated with EBV shedding (13.8% versus 2.8%) with a relative risk of 4.9 (95% CI 0.9-25.3). The presence of bacterial STI (rectal, urethral, and/or pharyngeal) and nonspecific urethritis was not associated with HIV seminal shedding. Only the subject with rectal C. trachomatis and N. gonorrhoea co-infection shed detectable HIV in the semen (table 3).....
High-level CMV seminal shedding was positively associated with presence of pharyngeal STIs, with seminal EBV and seminal HHV-8. Moreover, seminal EBV was associated with detectable HHV-7 in semen. Seminal CMV and EBV were both associated with lower CD4 counts....."
"Consistent with previous studies [9], we found a high prevalence of herpesviruses shedding in our treated cohort. Compared to our previous results on a similar cohort of ART-naïve HIV-infected MSM, we observed a lower frequency of seminal shedding for most herpesviruses, except for CMV and HHV-6. Specifically, in semen samples of ART-treated compared to untreated HIV-infected subjects we found: 49.1% versus 51.3% seminal samples with detectable CMV DNA, 30.7% versus 40.9% with detectable EBV DNA. 2.6% versus 10.4% for with detectable HSV DNA, 7.0% versus 7.0% with HHV-6 DNA, 8.8% versus 14.8% with HHV-7 DNA, and 3.5% versus 11.3% with HHV-8 DNA."
"These observations suggest that ART initiation may reduce seminal shedding of many herpesviruses, but CMV DNA seminal shedding, in particular, remains highly prevalent in treated HIV-infected subjects. This finding is in contrast with other reports, showing decreased frequency of CMV replication after ART initiation [29, 30], but these previous studies included subjects with lower CD4 counts and evaluated CMV replication in blood and not in semen."
Abstract
Background. Current antiretroviral therapy (ART) suppresses HIV in blood to undetectable levels in most infected individuals; however, some men shed HIV in semen despite suppressed levels in blood.
Methods. This study included 114 chronically HIV-1 infected men who have sex with men, who were receiving ART with blood plasma HIV <500 copies/ml.
Asymptomatic participants were screened for bacterial sexually transmitted infections (STIs) and non-specific genital inflammation. Levels of HIV and seven human herpesviruses were measured by real-time (RT)-PCR in seminal plasma. Predictors of HIV seminal shedding were determined on the entire cohort, and on the subset of 100 subjects with blood plasma HIV <50 copies/ml.
Results. Eleven subjects (9.6%) had detectable levels of seminal HIV (median: 2.1 log10copies/ml), and 72 (63.2%) had at least one herpesvirus detected in their seminal plasma. Detectable levels of seminal HIV were present more often in persons with plasma HIV between 50-500 copies/ml compared to those <50 copies/ml (p-values adjusted for false discovery rate (FDR)=0.08). There was a trend for high-level CMV (>4 log10 DNA copies/ml) (FDR-adjusted p=0.08), and presence of EBV (FDR-adjusted p=0.06) in semen to be associated with detectable seminal HIV levels. In a sub-analysis of 100 subjects with blood plasma HIV <50 copies/ml, high-levels of CMV in semen was the only significant predictor for seminal HIV shedding.
Introduction
Current antiretroviral therapy (ART) suppresses human immunodeficiency virus type 1 (HIV) RNA in blood and semen to below the level of detection in most infected men, substantially decreasing the risk of sexual HIV transmission [1-4]. Recent studies reported a >90% reduction in HIV transmission among serodiscordant heterosexual couples after starting ART [2, 3], and a similar reduction in HIV transmission associated with anal intercourse was noted in men who have sex with men (MSM) receiving ART [5]. Nevertheless, sexual HIV transmission [6, 7], and intermittent seminal HIV shedding [8] can occur despite ART. The frequency of seminal HIV shedding in treated individual range from 2 to 48% [9-12]. HIV shedding in the semen of ART-treated individuals might be a consequence of viral compartmentalization [13] with poor drug penetration within the genital tract [8, 14], or stimulation of virus replication by concurrent sexually transmitted infections (STIs) and genital inflammation [9, 10, 15-17].
Seminal cytomegalovirus (CMV) was previously associated with increased activation of CD4+ T-cells in the genital tract [18], and several groups have described an association between CMV and Epstein-Barr virus (EBV) with HIV seminal shedding in individuals with detectable HIV in blood plasma [19-23]. From a public health perspective, it is crucial to understand if concurrent CMV and EBV shedding are associated with HIV shedding in semen of immunologically reconstituted and virologically controlled individuals.
Results
Study participants' demographics and clinical data HIV-infected participants (n=114) were high-risk MSM without clinical evidence of STIs who had a mean age of 44 years, receiving ART and with HIV <500 copies/ml in blood plasma within three month from semen collection (median time between semen collection and blood HIV level determination: 33 days, interquartile range (IQR): 14-70). One hundred subjects (88%) had HIV levels <50 copies/ml. The median time on ART at the time of semen collection was 2.5 years (IQR: 13.5 months- 4.8 years), and the median CD4 count was 580 cells/uL (IQR: 532-628). For 112 subjects with available specific ART information, 76.8% were on a regimen including tenofovir; 39.3% were on a regimen including a non nucleoside reverse transcriptase inhibitor (NNRTI); 55.4% were on a regimen including a protease inhibitor (PI); and 18.8% were on a regimen including an integrase inhibitor. Of the 21 subjects who were taking a regimen including an integrase inhibitor, 10 also included a PI, and 4 included a NNRTI. Self-reported levels of ART adherence during the last month were >90% for 99 (87%) of the subjects. Self-reported substance abuse (including marijuana, cocaine, amphetamines, club drugs, and opiates) was high at 34.2%, and methamphetamine use within one month from sample collection was reported by 13.6% of participants. Characteristics and demographics of the subjects are summarized in table 1.
Bacterial and viral genital infections
Eleven subjects (9.6% of the entire cohort) had detectable levels of HIV in semen, with a median of 2.1 log10 RNA copies/ml among detectable samples (range 1.7-2.5), and 72 (63.2%) had at least one herpesvirus detected in their seminal plasma. These herpesviruses included CMV (frequency 49.1%, median detectable viral load of 4.3 log10 DNA copies/ml, IQR 3.2-4.9), Epstein-Barr virus (EBV) (30.7%, 2.9 log10 copies/ml, IQR 2.5-3.4), herpes simplex virus type 2 (HSV-2) (2.6% 5.1 log10 copies/ml), human herpes virus type 6 (HHV-6) (7.0%, 3.3 log10 copies/ml), human herpes virus type 7 (HHV-7) (8.8%, 3.2 log10 copies/ml) and human herpes virus type 8 (HHV-8) (3.5%, 3.4 log10 copies/ml) (figure 1). In contrast to our previous results on ART naïve subjects [19], no HSV type 1 shedding was found in the semen of this cohort. Urethral, rectal or pharyngeal bacterial STIs were detected in 17 (14.9%) of the individuals, and non-specific urethritis in an additional 8 (7.0%) (Table 1). Specifically, one subject had syphilis, five subjects had urethral STIs (three with M. genitalium and two with C. trachomatis), six patients had rectal STIs (two with C. trachomatis, three with N. gonorrhoea, and one subject co-infected with both C. trachomatis and N. gonorrhoea), and six patients had pharyngeal STIs (two with C. trachomatis, three with N. gonorrhea and one with trichomonas). All subjects were included in the study and treated for these infections after specimen collections. Subjects were all asymptomatic at the time of screening.
Key Predictors of isolated HIV seminal shedding on ART
Subjects with evidence of HIV replication in plasma while on ART (i.e. blood plasma HIV levels 50-500 copies/ml) had detectable levels of HIV in semen more frequently than subjects with <50 HIV copies/ml (35.7% versus 6.0%, FDR-adjusted p-value 0.08). Neither the class of ART, the time on ART nor the self-reported pill-adherence were significantly associated with HIV shedding in semen. Also self-reported substance abuse was not associated with HIV seminal shedding.
Similar to our previous findings in ART-naïve HIV-infected MSM [19], there was a trend for high-level CMV (>4 log10 DNA copies/ ml) and presence of EBV to be associated with detectable HIV seminal levels (tables 2 and 3). Specifically, 7 subjects (21.9%) with high-level CMV replication in semen had concomitant HIV seminal shedding compared to 4 of those (4.9%) with no or low CMV shedding in semen (FDR-adjusted p-value=0.08), with a relative risk of 4.5 (95% CI, 1.4-14.2). A trend towards an increased frequency of detectable HIV seminal shedding was observed when comparing any level of CMV shedding to no CMV (14.3% versus 5.2%, p=0.12). For those subjects with both CMV and HIV detectable in semen, the mean CMV DNA level was 1 log10 higher in semen compared to non-shedders, 3.9 versus 2.9 log10 DNA copies/ml, but this difference was not statistically significant. Any detectable EBV shedding was also associated with HIV shedding in semen (22.9% HIV seminal shedding in subjects with concomitant EBV replication compared to 3.8% in those without EBV, FDR-adjusted p-value=0.06), but there was no evidence that higher EBV levels were associated with greater HIV shedding. In fact, for patients with HIV and EBV co-shedding, the levels of EBV DNA copies was lower in those shedding HIV in semen compared to those with no HIV shedding, 2.6 versus 3.1 log10 DNA copies/ml (p=0.06). High-level CMV shedding in semen was also significantly associated with EBV shedding.
In a sub-analysis of the 100 subjects with HIV in blood of <50 copies/ml, of whom six had detectable HIV seminal levels, high CMV levels remained the only factor associated with HIV shedding with a relative risk of 5.7 (95% CI 1.1-29.3). Specifically, 15.4% of semen samples with high-level CMV also shed HIV compared to 2.7% in those without high levels of CMV. There was also a trend for an increased frequency of HIV shedding in semen being associated with EBV shedding (13.8% versus 2.8%) with a relative risk of 4.9 (95% CI 0.9-25.3). The presence of bacterial STI (rectal, urethral, and/or pharyngeal) and nonspecific urethritis was not associated with HIV seminal shedding. Only the subject with rectal C. trachomatis and N. gonorrhoea co-infection shed detectable HIV in the semen (table 3).
Factors associated with seminal CMV and EBV shedding on ART
High-level CMV seminal shedding was positively associated with presence of pharyngeal STIs, with seminal EBV and seminal HHV-8. Moreover, seminal EBV was associated with detectable HHV-7 in semen. Seminal CMV and EBV were both associated with lower CD4 counts. There was a trend for individuals receiving PI-based ART regimens to have higher CMV shedding as compared to those receiving NNRTI-based regimens (33.8% versus
18.2%). There was also a trend for patients with seminal EBV shedding to have a shorter time on ART compared to people without seminal EBV shedding (974 versus 1357 days of ART). Time on ART, however, was not associated with presence of CMV seminal shedding.
Discussion
Since HIV in male genital secretions accounts for the majority of HIV
transmission in both women and MSM [28], it is crucial to understand the factors associated with increased seminal HIV replication in order to inform strategies to reduce sexual HIV transmission and the development of HIV drug resistance. Effective ART substantially reduces the HIV levels in blood and semen [1-4]; however, a significant number of subjects intermittently shed HIV in semen despite having suppressed levels in blood plasma [9-12]. In our study of sexually active, ART-treated MSM with blood plasma HIV levels <500 copies/ml, 11 (9.6%) had detectable HIV in seminal plasma at very low levels between 50 and 230 copies/ml. The prevalence of seminal HIV shedding falls within the range previously described in the literature (2%-48%) [9-12], depending on the characteristics of the observed population. Detectable levels of seminal HIV were present significantly more often in persons with plasma HIV between 50 and 500 copies/ml compared to those <50 copies/ml.
We previously described that seminal shedding of different herpesviruses (i.e. high-level CMV, EBV, HHV-8) was associated with increased levels of HIV within the genital tract of ART-naïve MSM. The present study aimed to investigate if this remains true as well in subjects receiving ART with suppressed HIV levels in blood plasma.
Consistent with previous studies [9], we found a high prevalence of herpesviruses shedding in our treated cohort. Compared to our previous results on a similar cohort of ART-naïve HIV-infected MSM, we observed a lower frequency of seminal shedding for most herpesviruses, except for CMV and HHV-6. Specifically, in semen samples of ART-treated compared to untreated HIV-infected subjects we found: 49.1% versus 51.3% seminal samples with detectable CMV DNA, 30.7% versus 40.9% with detectable EBV DNA. 2.6% versus 10.4% for with detectable HSV DNA, 7.0% versus 7.0% with HHV-6 DNA, 8.8% versus 14.8% with HHV-7 DNA, and 3.5% versus 11.3% with HHV-8 DNA.
These observations suggest that ART initiation may reduce seminal shedding of many herpesviruses, but CMV DNA seminal shedding, in particular, remains highly prevalent in treated HIV-infected subjects. This finding is in contrast with other reports, showing decreased frequency of CMV replication after ART initiation [29, 30], but these previous studies included subjects with lower CD4 counts and evaluated CMV replication in blood and not in semen.
Consistent with data from untreated subjects, we found that high-levels CMV replication (i.e. >4 log10 DNA copies/ml) was associated with HIV seminal shedding even after the initiation of ART. We also found an association between EBV shedding and detectable HIV in semen, though no dose response was observed. Similar to our previous findings [19, 23], CMV and EBV seminal shedding were positively associated with each other, and both viruses were associated with lower CD4 counts. Higher level CMV seminal shedding was positively associated with presence of pharyngeal STIs, with seminal HHV-8 and with use of PI-based ART regimens.
Interestingly, HIV seminal shedding in our cohort was not associated with non-specific genital inflammation or with any asymptomatic bacterial STIs. This may be a consequence of the relative low prevalence of urethral STIs in this asymptomatic cohort (3.5%). However, this also suggests that CMV may be a more common precipitant of HIV shedding in semen than bacterial STIs during ART even in this high-risk MSM population. It is also possible that HIV seminal shedding is derived from the proximal genital tract rather than distally and therefore asymptomatic (non-ulcerative) STIs would not contribute directly to an increase in HIV seminal shedding. Of note, low levels of HIV in blood plasma between 50 and 500 copies/ml were associated with HIV seminal shedding, suggesting that a complete suppression of HIV blood levels may minimize the risk of sexual HIV transmission.
A recent study [9] observed that frequent, isolated seminal HIV shedding among 25 subjects was associated with increased compartmentalized immune activation within the genital tract, but could not find any association between detectable HIV levels and any of the evaluated factors, including CMV replication. We previously described that seminal CMV replication is positively associated with levels of immune activation in the male genital tract [18], and our present study on a larger cohort, suggests that high-level CMV replication likely plays a role in HIV seminal shedding also in successfully treated HIV
infected individuals.
This study has several limitations. Most importantly, despite including seminal samples from 114 different subjects, the low frequency of seminal shedding for HIV limited our power to find additional associations. Moreover, in this observational study, the causality of the associations cannot be definitively determined. We did not assess pre- and post treatment levels in semen to address directly the impact of treatment in the same subject. However, since successful treatment of HIV in this cohort did not influence the rate of CMV replication compared to untreated subjects in our previous cohort, CMV replication is likely driving HIV shedding and not vice-versa. Lastly, since we only included MSM in this study, it needs to be determined if these associations remain true also in genital secretion of heterosexual men and women since this could have an impact on management of serodiscordant couples trying to conceive children.
In summary, the present study evaluated a large number of seminal samples (n=114) from HIV-infected sexually active MSM treated with ART with an extensive battery of tests for viral and bacterial co-infections. The association between isolated seminal HIV shedding and high-level CMV replication and EBV replication in the genital tract suggests that the presence of these viruses could play a role in HIV transmission not only in ART-naïve subjects as previously described [19, 21] but also in individuals on ART. These finding have important implications for the development of strategies to reduce HIV transmission.
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