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Higher Levels of HIV in Rectal Secretions Among MSM
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"Higher Concentration of HIV RNA in Rectal Mucosa Secretions than in Blood and Seminal Plasma, among Men Who Have Sex with Men, Independent of Antiretroviral Therapy"
The Journal of Infectious Diseases 2004;190:156-161
Richard A. Zuckerman,1 William L. H. Whittington,1 Connie L. Celum,1 Tarquin K. Collis,1,a Aldo J. Lucchetti,5 Jorge L. Sanchez,5 James P. Hughes,2 Jose L. Sanchez,4 and Robert W. Coombs1,3
Departments of 1Medicine, 2Biostatistics, and 3Laboratory Medicine, University of Washington, Seattle; 4US Military HIV Research Program, Walter Reed Army Institute of Research, Rockville, Maryland; 5Asociación Civil Impacta Salud y Educación, Lima, Peru
"...HIV RNA was often found at high levels in rectal secretions, even in men receiving ART, and paired HIV RNA levels in rectal secretions were greater than those in either the blood or seminal plasma among HIV-infected MSM... ART had a greater direct effect on levels of HIV in seminal plasma than in rectal secretions... The observation that ART has an independent effect on HIV RNA in seminal plasma but not in rectal mucosal secretions, relative to blood plasma, may be due to differential levels of antiretroviral drugs or to anatomic and immunologic differences in the male genital tract versus rectal mucosa... Higher rectal and seminal HIV levels likely increase the risk of HIV transmission, although anogenital VL transmission thresholds have not been demonstrated. Future studies to define the relationship between seminal and mucosal HIV levels and HIV transmission will need to take into account the differential effects of ART, as well as viral and bacteriologic coinfections, genetic polymorphisms, and behavioral factors..."
ABSTRACT
High levels of human immunodeficiency virus (HIV) in rectal secretions and semen likely increase the risk of HIV transmission.
HIV-infected men who have sex with men made 2--3 study visits, over 4 weeks, to assess rectal, seminal, and plasma levels of HIV RNA. Mixed-effects models estimated the effect of factors on HIV shedding.
Twenty-seven (42%) of 64 men were receiving antiretroviral therapy (ART); regardless of ART use, median HIV RNA levels were higher in rectal secretions (4.96 log10 copies/mL) than in blood plasma (4.24 log10 copies/mL) or seminal plasma (3.55 log10 copies/mL; P < .05, each comparison).
ART was associated with a 1.3-log10 reduction in rectal HIV RNA in a model without plasma HIV RNA; with and without plasma RNA in models, ART accounted for a >1-log10 decrease in seminal HIV RNA levels.
Thus, controlling for plasma HIV RNA, ART had an independent effect on seminal, but not rectal, HIV levels.
INTRODUCTION
Even though the sexual transmission of HIV is relatively inefficient, the majority of HIV infections worldwide are acquired sexually. The association between serum HIV levels and HIV transmission likely reflects the infectiousness of genital and rectal secretions, and models have suggested that higher seminal HIV levels are associated with an increased risk of transmission. However, actual transmission thresholds of virus load (VL) in semen or in cervical or rectal secretions have not been determined, because prospective partner studies that can define transmission thresholds are difficult to conduct. In addition, HIV transmission is affected by other cofactors, such as trauma, inoculum size, stage of disease and VL in the source partner, concomitant bacterial or viral sexually transmitted infection (STI) in either the infected or susceptible sex partner, and host coreceptor and genetic polymorphisms.
Factors that influence rectal HIV shedding and infectiousness may differ from cofactors that affect seminal HIV levels, given the abundance of activated rectal lymphocytes and the absence, in the intestinal tract, of a immunologic equivalent to the blood-testes barrier. The pathogenesis of HIV replication in the rectal mucosa, including CD4 cell depletion, apoptosis, and cells associated with latent infection, has been the focus of a number of studies. However, little is known about the factors that influence rectal HIV RNA levels, a potential surrogate for infectiousness. Thus, we characterized HIV RNA levels in rectal secretions and compared RNA levels and their correlates in paired seminal plasma and blood plasma specimens from HIV-infected men who have sex with men (MSM) in Seattle, Washington, and Lima, Peru.
Study Design
The study population has been described elsewhere [19]. In brief, between December 1999 and January 2001, HIV-infected MSM were enrolled for a study of the effects of STIs on HIV shedding, at the Harborview Medical Center sexually transmitted disease (STD) clinic in Seattle and the Raul Patrucco STD clinic in Lima. Eligible participants were HIV-infected MSM >18 years of age and were either naive for antiretroviral therapy (ART) or had been receiving a stable regimen for ⩾30 days before enrollment.
Fifteen men from Seattle were scheduled for 2 study visits (at enrollment and 2 weeks later) under a pilot protocol. All other participants were scheduled for 3 visits (at enrollment and 2 and 4 weeks later). The institutional review boards of the University of Washington (UW), Seattle, and the Universidad Peruana Cayetano Heredia and Asociación Via Libre, Lima, approved the protocol. Study participants provided written, informed consent and were compensated for travel and related expenses. Men included in this analysis completed >2 visits and had negative STI screening results, no genitourinary or rectal symptoms suggestive of an STI, and normal physical examination results.
At all study visits, a physical examination, including anoscopy, was performed and a questionnaire was administered about risk behaviors, ART, STI symptoms, and recent STI diagnoses. Semen was collected in sterile containers by study participants and transported to the laboratory within 2 h. Participants were not excluded from the study if a semen specimen was not provided.
RESULTS
Participant characteristics and completeness of samples. Sixty-four of 125 enrolled men met the criteria for inclusion in the analysis; 39 of the remaining 61 were excluded because of a concurrent STI, and 22 did not complete 2 visits. Men from Seattle and Peru did not differ by baseline median CD4 cell count, frequency of receptive anal intercourse, or condom or lubricant use with anal intercourse. However, the men from Seattle were significantly older and were more likely to be receiving ART and to report more sex partners during the preceding 2 months than were the men from Peru. The 64 participants made 164 study visits (64 made 2 visits, and 36 made 3 visits), from which 157 plasma samples (96%), 159 rectal samples (97%), and 133 semen samples (81%) were collected and analyzed. There were no significant differences in clinical site, age, CD4 cell count, or plasma or rectal VL between the visits when the men did or did not provide semen samples (data not shown).
Baseline HIV RNA levels. Regardless of ART use, median HIV RNA levels were higher in rectal secretions (4.96 log10 copies/mL) than in blood plasma (4.24 log10 copies/mL) or seminal plasma (3.55 log10 copies/mL; P < .05, each comparison). In men who were receiving ART, median baseline HIV RNA levels were significantly higher in rectal secretions than in blood plasma or seminal plasma (P < .05), but levels did not differ between blood plasma and seminal plasma (P = .9). In men who were not receiving ART, median baseline HIV RNA levels were highest in rectal secretions and differed significantly among all 3 samples (P < .05, each comparison).
Within-subject variability. During all study visits, HIV RNA levels in blood plasma were correlated with seminal plasma HIV levels and rectal secretions. Within-subject variability for HIV RNA levels in seminal plasma (SD = 0.6) and rectal secretions (SD = 0.6) exceeded within-subject variability for blood plasma (SD = 0.3).
Patterns of HIV detection. Among the 27 participants who were receiving ART at the time of enrollment in the study, 9 (35%) of 26 had detectable HIV RNA in the plasma, 10 (37%) of 27 in rectal secretions, and 11 (55%) of 20 in seminal plasma. In comparison, men not receiving ART had higher rates of HIV RNA detection in blood plasma (37/37 [100%]; P < .05 vs. men receiving ART), rectal secretions (35/37 [95%]; P < .05), and seminal plasma (28/36 [78%]; P = .08). Regardless of ART use, HIV RNA detection in blood plasma and rectal secretions was usually concordant (57/63 [90.5%]), whereas agreement between detection in blood plasma and seminal plasma was weaker (39/56 [69.6%]; P < .01, vs. plasma and rectal secretions).
HIV RNA in the plasma was either persistently detectable or undetectable during all study visits for 62 (96.9%) of 64 participants over the course of the average 4 weeks of the study. In contrast, 8 participants had intermittently detectable rectal HIV RNA (2 receiving ART and 6 not), and 11 participants had intermittently detectable seminal HIV RNA (7 receiving ART and 4 not).
Correlates of rectal and seminal HIV shedding. Univariate predictors of HIV shedding in rectal secretions and seminal plasma were similar. ART use, a higher CD4 cell count, Seattle study site, and lower plasma VL were associated with lower seminal and rectal VLs (data not shown, all P < .05, for each factor and anatomic site). Mixed-effects models for rectal and seminal HIV loads were developed with and without plasma VL.
Without plasma VL in the model, ART was associated with a 1.3-log reduction in rectal VL, and CD4 cell count and Peru study location also were independently associated with rectal VL. When the plasma VL was included in the model (table 2), each log change in plasma VL was associated with a 0.5-log parallel change in rectal VL, and ART use and CD4 cell count were no longer significantly associated with rectal VL. Peru study site was independently associated with higher rectal VL after controlling for the other factors in both models. This effect of study location on rectal HIV RNA levels was not explained by the friability of rectal mucosa on examination, frequency of receptive anal intercourse, or condom or lubricant use with anal intercourse (data not shown).
In the model that did not include plasma VL, ART and CD4 cell count were associated with seminal HIV RNA level. When plasma VL was included, ART and plasma VL were significantly associated with seminal VL; however, CD4 cell count was not associated with seminal VL. In both models, ART use accounted for a decrease of >1 log10 in seminal HIV RNA level, and study location was not associated with HIV levels in the seminal plasma.
DISCUSSION
HIV RNA was often found at high levels in rectal secretions, even in men receiving ART, and paired HIV RNA levels in rectal secretions were greater than those in either the blood or seminal plasma among HIV-infected MSM from Seattle, Washington, and Lima, Peru. ART had a greater direct effect on levels of HIV in seminal plasma than in rectal secretions. Mixed-effects models with and without blood plasma VL were included to describe the differential effect of ART in these 2 anatomical sites. In models that included blood plasma VL, ART had no significant independent effect on HIV levels in rectal secretions, which suggests that the effect of ART on rectal VL was through its effect on blood plasma VL. In contrast, ART was independently associated with a significant decrease in HIV levels in the seminal plasma when the blood plasma VL was included in the model. The observation that ART has an independent effect on HIV RNA in seminal plasma but not in rectal mucosal secretions, relative to blood plasma, may be due to differential levels of antiretroviral drugs or to anatomic and immunologic differences in the male genital tract versus rectal mucosa.
Most studies of HIV RNA levels in rectal tissues have used biopsy specimens, and HIV has been frequently detected, even in persons with suppressed plasma HIV levels. However, HIV RNA levels from biopsy specimens may not represent the quantity of HIV in mucosal secretions to which the insertive sex partner is exposed. Sno-strips, which were previously used to sample cervical fluid for HIV, were used in the present study to sample secretions of the rectal mucosa. We detected HIV RNA at rates similar to those of a study that used rectal biopsy specimens and at a higher rate (70%) than that previously reported (32%) in a study in which anal swabs were used. Unlike the investigators in that study, we obtained samples through an anoscope positioned 3--4 cm above the dentate line that likely sampled HIV RNA on the columnar epithelium of the rectal mucosa, rather than the stratified squamous epithelium sampled by anal swabs.
Research of the effects of ART on rectal HIV RNA levels has largely focused on persons with suppressed plasma HIV RNA loads and on the identification and characterization of the virus reservoir. We recruited a heterogeneous population of MSM, including those having varying durations of ART with probable variable compliance, given that 35% of participants who reported current ART use had detectable plasma VLs at the baseline visit. The present study is the first large, prospective study to compare rectal HIV levels between persons who are and are not receiving ART and to show variable levels of suppression among men receiving ART.
ART was associated with lower seminal HIV RNA levels, which is consistent with previous reports. Although our baseline detection rate of 78% in seminal plasma from untreated patients is similar to those of previous reports (66%--96%), our baseline rate of 55% detection in patients receiving ART was much higher than those of previous studies of treated patients since highly active antiretroviral regimens have been available (0%--33%). Most of these studies obtained their follow-up samples between 10 weeks and 18 months after the initiation of ART, and many of our participants had been receiving ART longer and may have had lower adherence or higher rates of antiretroviral resistance, given that they were recruited from clinic attendees rather than from clinical trial participants. ART regimens varied considerably among the participants, with all men receiving at least 1 nucleoside reverse-transcriptase inhibitor, 80% (20/25) receiving a protease inhibitor, and 36% (9/25) receiving a nonnucleoside reverse-transcriptase inhibitor.
Although seminal HIV levels did not differ between the study sites, levels of HIV in rectal secretions were 0.5 log10 higher in Lima participants than in Seattle participants after we controlled for ART use, CD4 cell count, and plasma VL, which could not be explained by the examination findings or behavioral factors that we assessed. Inflammation, as measured by anorectal Pap smear, has been associated with the increased detection of HIV, and it is possible that there was some clinically undetectable difference in inflammation that may have been mediated by viral cofactors such as herpes simplex virus--2, which could explain this geographic difference. The present study was not designed to examine differences in rectal HIV shedding caused by possible viral cofactors (such as herpesviruses). However, such factors may influence rectal HIV shedding and require further study.
There were several limitations to the study. Although it is likely that mostly cell-free HIV in secretions was measured by the rectal Sno-strips, it is possible that some cellular material was soaked up by them. There is no consensus with regard to whether cell-free HIV, cell-associated HIV, or both contribute to HIV transmission; however, the atraumatic sampling of secretions by use of Sno-strips likely represents mucosal HIV RNA levels (cell-free and -associated) to which the insertive partner may be exposed in the absence of rectal bleeding. The high within-person variability of HIV levels in rectal secretions and seminal plasma, which likely is a function of the biological variability, suggests that more-frequent measurements are needed to further elucidate patterns of rectal and seminal HIV shedding [34]. However, despite such high within-person variability, significant differences in HIV RNA levels were detected between the anatomic sites studied.
HIV levels varied by anatomical site and were affected differently by ART. Higher rectal and seminal HIV levels likely increase the risk of HIV transmission, although anogenital VL transmission thresholds have not been demonstrated. Future studies to define the relationship between seminal and mucosal HIV levels and HIV transmission will need to take into account the differential effects of ART, as well as viral and bacteriologic coinfections, genetic polymorphisms, and behavioral factors.
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