Insight into the effect that combination antiretroviral therapies have upon HIV-1 RNA and DNA shedding at anal and genital mucosal sites is important since the amount of virus present at these sites is thought to be associated with risk of viral transmission to uninfected partners [1,2]. It is well established that combination antiretroviral therapies, particularly those that include a protease inhibitor, significantly reduce the concentration of HIV-1 RNA in plasma . Furthermore, studies have demonstrated that combination therapy reduces HIV-1 RNA levels in lymph nodes, tonsils, seminal fluid and vaginal secretions [4–8]. However, it appears that such therapies do not eliminate latently infected CD4 cells  and, more importantly, recent studies demonstrate that these HIV-infected mononuclear cells contain replication-competent HIV-1 provirus [10–14]. For a variety of reasons, only a portion of HIV-infected men under medical care currently receive therapies that include a protease inhibitor. Although many men on combination therapies initially achieve undetectable plasma levels, the ability to maintain low levels of viral replication has not been universal [15,16]. Data concerning HIV-1 detection in the anorectal and genital mucosa of persons receiving antiretroviral therapy are needed to counsel individuals about potential infectivity . At present, however, few data are available concerning HIV-1 in anorectal mucosa . In our previous study , conducted prior to the availability of potent combination antiretroviral therapies, we reported frequent detection of anorectal HIV-1 RNA and DNA. As part of an observational study of men who have sex with men (MSM), we assessed whether use of combination antiretroviral therapy is associated with decreased detection of both HIV-1 RNA and HIV-1 DNA in anorectal specimens.
Materials and methods
Between July 1996 and December 1997, 255 HIV-infected MSM who were at least 18 years of age were enrolled into a study of HIV-1 and the development of anal dysplasia. Men were recruited in Seattle, Washington through general practice and infectious disease outpatient clinics, advertisements in gay newspapers and distributed literature. Written informed consent was obtained according to a protocol approved by the University of Washington Human Subjects Review Committee. Subjects were not compensated for participation.
At the study visit, self-administered, structured questionnaires were used to obtain information concerning demographic characteristics, anal intercourse within the prior 4 months, the last occasion of unprotected receptive anal intercourse and medical and antiretroviral therapy histories (including medications taken during the previous week). Responses were reviewed with a clinician, who referred to photographs and samples of antiretroviral medications. General physical and colposcopic anorectal examinations were performed. HIV-1 disease staging was performed using standard Centers for Disease Control and Prevention (CDC) criteria; HIV-1 disease category reflects presence or history of symptomatic HIV-1 disease at the time of the survey (A, asymptomatic infection; B, non-AIDS symptomatic infection; C, AIDS) .
Specimen collection and laboratory testing
Serum and blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) were collected. Anorectal mucosal specimens for detection of HIV-1 DNA and HIV-1 RNA were collected with two dry Dacron swabs inserted separately 3–4 cm into the anorectal canal, rotated 360° then rotated again during removal. The first specimen was used for an anal Pap smear, then placed in 1 ml RNA reagent (Ultraspec; Biotecx Laboratories, Houston, TX, USA). The second swab was placed in specimen transport medium (STM; Digene Diagnostics, Silver Spring, MD, USA) for DNA assays and measurement of leukocyte esterase (LE), an indicator of anorectal inflammation (Chemstrip 2LN, Boehringer Mannheim, Indianapolis, IN, USA).
Anorectal swab specimens in STM were digested with proteinase K, precipitated with ethanol and suspended in Tris–EDTA buffer as previously described . A 5 μl sample of this specimen was qualitatively screened for HIV-1 DNA by polymerase chain reaction (PCR) using the SK431/462 primer pair to amplify a highly conserved 142 base pair HIV-1 gag sequence using a licensed assay (AMPLICOR Monitor HIV-1 Test, Roche Molecular Systems, Pleasanton, CA, USA). Screening for herpes simplex virus type 2 (HSV-2) and cytomegalovirus (CMV) DNA sequences were performed as previously described . Amplification of human β-globin DNA fragments was employed to confirm the integrity of specimen DNA and to detect the presence of PCR inhibitors . To reduce the likelihood of false-positive PCR results, appropriate negative controls were introduced to the sample collection, preparation and amplification stages of processing. For anorectal HIV-1 RNA analyses, cellular RNA was isolated from swab specimens and 1 μg was added to each internally standardized HIV-1 reverse transcriptase (RT) PCR reaction as previously described . The assay employs an internal standard to permit detection of RT-PCR inhibitors and has a limit of detection of 2 copies of HIV-1 RNA per PCR reaction.
Plasma HIV-1 RNA was measured using the AMPLICOR Monitor HIV-1 Test (Roche), which has a detection limit of 50 copies/ml. A licensed viral lysate serologic assay (Abbott HIV-1, Chicago, IL, USA) was used to confirm HIV-1 infection in the 42 subjects lacking detectable plasma and anorectal HIV-1 DNA and RNA.
Assessment of the replication competence of anorectal HIV-1 DNA
The replicative competence of HIV-1 DNA detected in anorectal swab specimens from men receiving highly active antiretroviral therapy who had < 50 copies HIV-1 RNA/ml in plasma was assessed. During follow-up clinic visits in July and August 1999, HIV-1 viral culture with p24 antigen assays were performed on pellets and supernatants prepared from 19 anorectal swab specimens collected from these men . The anorectal cultures were terminated after 5–7 days because of bacterial contamination despite the use of broad-spectrum antibiotics in the viral culture medium. To assess for evidence of viral infection, culture-derived cell pellets were screened using a modified, qualitative assay  for the presence of tandem long-terminal-repeat circular unintegrated (2-LTR) HIV-1 DNA . The presence of 2-LTR HIV-1 DNA indicates de novo infection of the mixed lymphocyte culture cells at a molecular detection level of 10 copies/150 000 cells and so provides evidence for ongoing HIV-1 replication in the anorectal canal.
Of the 255 men enrolled, 22 (9%) were excluded from analyses including: six men who were receiving antiretroviral monotherapy and three men who were receiving a protease inhibitor plus fewer than two reverse transcriptase inhibitors (to minimize heterogeneity within antiretroviral combination therapy categories); four men enrolled in blinded clinical trials of antiretroviral agents (to prevent misclassification of therapy received); and nine men reporting a history of unprotected receptive anal intercourse during the previous 48 hours with a partner not known to be HIV-1 negative (to minimize the possibility of detecting HIV-1 DNA or HIV-1 RNA from a sexual partner).
A value for HIV-1 RNA of 25 copies/ml was assigned to men with plasma HIV-1 RNA levels below the assay's limit of detection (50 copies/ml). In selected analyses, CD4 cell counts were grouped into clinically relevant categories (< 200, 200–500 and > 500 × 106 cells/l) and plasma HIV-1 RNA were grouped into sample quartiles. The LE variable was categorized as negative, trace, 1+, and 2+. Pearson's chi-square tests were used in univariate analyses of dichotomous and categorical variables; Mantel–Haenszel trend tests were used for ordered categorical variables (CD4 cell and plasma HIV-1 RNA groups). Mann–Whitney U tests were used for pairwise comparisons of continuous variables; pairwise comparisons were performed only when the overall test was significant. Potential confounding by anorectal inflammation of the association between therapy and detection of anorectal HIV-1 RNA and DNA was assessed by comparing variable coefficients before and after addition of LE indicator variables in multivariate logistic regression analyses.
Description of the study population
The mean age of the 233 participants included in analyses was 39 years (range 23–66); 89% were white and the mean number of years of education received was 15. The mean self-reported duration of HIV-1 infection was 7.0 years (range 0.1–13.3). Most men were sexually active, with 206 of 232 (89%) reporting sex with another male during the previous 12 months. Unprotected receptive anal intercourse (without a condom) since receipt of a positive HIV antibody test result was reported by 155 of 215 men (72%), and during the previous 12 months by 110 of 232 (47%).
Of the 233 men, no antiretroviral therapy during the prior week was reported by 89 (38%), with only 17 having ever received such therapy. Among the 72 men who had never received any form of antiretroviral therapy, 58 (81%) had at least one indication for treatment (CD4 cell count < 500 × 106 cells/l and/or plasma HIV-1 RNA level > 5000 copies/ml). A total of 144 of the 233 men (62%), received combination antiretroviral therapy during the week prior to their clinic visit, including 89 who received a protease inhibitor plus at least two reverse transcriptase inhibitors and 55 who received two or more reverse transcriptase inhibitors without a protease inhibitor. Of these 55, only four received a non-nucleoside reverse transcriptase inhibitor in addition to a nucleoside analog.
Men receiving therapy with a protease inhibitor plus two or more reverse transcriptase inhibitors had significantly more advanced HIV-1 disease (a greater proportion had symptomatic HIV-1 disease and lower median peripheral CD4 cell count) than men not receiving therapy (P < 0.001) and men receiving therapy that did not include a protease inhibitor (P < 0.001) (Table 1). Despite this profile of comparatively advanced disease, men receiving therapy that included a protease inhibitor had significantly lower plasma HIV-1 RNA levels (P < 0.001) than either of the other two groups.
Detection of anorectal HIV-1 DNA and HIV-1 RNA
Anorectal specimens collected from 207 (89%) of the 233 men were adequate for assessment of HIV-1 DNA, and specimens from 230 (99%) were adequate for HIV-1 RNA assay. Twenty-six specimens (11%) were inadequate for DNA assays because human β-globin sequences could not be amplified; anorectal specimens from three of these patients were insufficient in the RNA assay because of the presence of inhibitors of PCR amplification.
Overall, HIV-1 DNA was detected in 102 (49%) of 207 anorectal specimens and HIV-1 RNA in 74 (32%) of 230 specimens. Among the 207 specimens that were adequate for both HIV-1 DNA and HIV-1 RNA assessment, 127 (61%) had detectable anorectal HIV-1 DNA or HIV-1 RNA: 43 (21%) had both HIV-1 DNA and HIV-1 RNA detected, 59 (29%) had only HIV-1 DNA detected and 25 (12%) had only HIV-1 RNA detected.
Among the 105 men without detectable anorectal HIV-1 DNA, those with detectable anorectal HIV-1 RNA had significantly higher plasma HIV-1 RNA levels (median 44 237 copies/ml; range 346–1 470 912; n = 25) than those without detectable anorectal HIV-1 RNA (median 272 copies/ml; range 25–564 653; n = 80) (P < 0.001), suggesting that remote production of HIV-1 RNA was a likely source of anorectal HIV-1 RNA in the former patients.
Of 202 anorectal specimens for which HSV-2 and CMV DNA PCR results were available, four (2%) were HSV-2 positive and 14 (7%) were CMV positive. Of the former, two were HIV-1 DNA positive and three were HIV-1 RNA positive. Of the 14 CMV DNA-positive specimens, six were HIV-1 DNA positive and four were HIV-1 RNA positive.
Relationship of therapy to detection of anorectal HIV-1 DNA and HIV-1 RNA
HIV-1 DNA was detected in anorectal specimens from 48 (58%) of 78 men receiving no therapy, and only slightly less frequently in anorectal specimens obtained from men receiving protease inhibitors (35/81; 43%; P = 0.07), or two or more reverse transcriptase inhibitors alone (22/48; 46%; P = 0.20) (Table 2).
In contrast, HIV-1 RNA was detected in anorectal specimens collected from 43 of 88 men (49%) not on therapy, and significantly less often in specimens from men on two or more reverse transcriptase inhibitors alone (16/53; 30%;P = 0.03) and from men on two or more reverse transcriptase inhibitors plus a protease inhibitor (15/89; 17%;P < 0.001) (Table 2). The lower frequency of HIV-1 RNA detection in anorectal specimens from recipients of combination therapies including protease inhibitors, compared to men receiving other combination antiretroviral therapies, approached statistical significance (P = 0.06). Significant therapy-associated differences in detection of HIV-1 RNA were observed in specimens from men both with and without detectable HIV-1 DNA (Table 2).
To assess whether the association between therapy and detection of anorectal HIV-1 RNA and DNA might be confounded by anorectal inflammation, we examined the anorectal LE results that were available for 132 men. Anorectal HIV-1 RNA was detected in 5/51 (10%) men with negative or trace LE values, 8/33 (24%) men with anorectal LE values of 1+ and 22/48 (46%) men with 2+ values (P < 0.001). However, anorectal LE test results were not related to detection of anorectal HIV-1 DNA (P = 0.78). Despite the significant association of increasing LE levels with detection of anorectal HIV-1 RNA, adjustment for anorectal LE level did not appreciably change or eliminate the significance of the association of therapy with reduced detection of anorectal HIV-1 RNA (or the lack of a significant association between therapy and detection of anorectal HIV-1 DNA). Therefore, despite our previous observation of a positive association of detection of anorectal HIV-1 DNA and RNA with inflammatory cell counts  and of anorectal HIV-1 RNA and LE levels in the present study, we found no evidence of confounding by anorectal inflammation (as measured by LE testing) on the relation between therapy and detection of HIV-1 in anorectal specimens.
Anorectal HIV-1 DNA and HIV-1 RNA in men with undetectable plasma HIV-1 RNA
HIV-1 RNA was detected in anorectal specimens from 73/176 men (42%) with, and only 1/54 (2%) without, detectable plasma HIV-1 RNA (P < 0.001); the latter positive specimen was also HIV-1 DNA positive and was obtained from a subject receiving therapy that included a protease inhibitor. HIV-1 DNA was detected in anorectal specimens from 88/155 men (56%) with, and 14/50 men (28%) without, detectable plasma HIV-1 RNA (P < 0.001). Of the latter group, 12 were receiving a protease inhibitor and two were receiving only reverse transcriptase inhibitors.
HIV-1 cultures and p24 antigen assays (performed on anorectal swab material obtained from 19 men who lacked detectable plasma HIV-1 RNA but who had positive qualitative assays for HIV-1 viral DNA in anorectal specimens) were negative for HIV-1 p24 antigen and for 2-LTR viral DNA following 5–7 days culture.
The extent to which anorectal HIV-1 shedding occurs despite administration of combination antiretroviral therapies has implications for the control of HIV-1 transmission. In the present study, we found that anorectal HIV-1 RNA and DNA were frequently detected among both treated and untreated MSM. It is encouraging that HIV-1 RNA was detected less often in anorectal specimens obtained from men receiving combination antiretroviral therapies than in specimens obtained from untreated men. Only 2% of those with < 50 copies HIV-1 RNA/ml in plasma had detectable HIV-1 RNA in a single anorectal sample. Nevertheless, 17% of those on combination antiretroviral therapy that included a protease inhibitor had detectable anorectal HIV-1 RNA. Furthermore, close to half of all men examined, including 43% of men on combination antiretroviral therapy that included a protease inhibitor and 28% of those with < 50 copies/ml HIV-1 RNA in plasma, had HIV-1 DNA detected in a single anorectal swab.
It is important to determine whether cell-associated HIV-1 DNA detected in the anorectal mucosa of men with undetectable plasma viremia represents defective template or replication-competent provirus with potential for sexual transmission. In the substudy of 19 such men, we were unable to demonstrate replication-competent viral DNA in anorectal mucosa at assay levels of sensitivity. Nevertheless, it would be premature to conclude on the basis of these preliminary results that such patients are not infectious. HIV-1 culture of contaminated anorectal swab specimens represents a formidable technical challenge and few polymorphic blood mononuclear cells are usually present in anorectal swab specimens. Other investigators have repeatedly demonstrated replication-competent HIV-1 in latently infected T cells obtained from multiple compartments (including the rectum) in individuals receiving combination antiretroviral therapy who have had undetectable levels of plasma HIV-1 RNA for up to 2 years [10–14,24,25]. HIV-1 replication can result from activation of latently infected lymphocytes following exposure to proinflammatory cytokines  and allogeneic stimulation . The potential importance of cell-associated virus in transmission of lentiviruses, including HIV-1, has been reviewed elsewhere [28,29]. Therefore, even the absence of detectable HIV-1 RNA in anorectal swab specimens cannot assure the absence of infectivity.
To our knowledge, the only published study (of 15 subjects) examining the effect of combination antiretroviral therapy on detection of HIV-1 in rectal mucosa has been that of Kotler et al. , which showed significant reductions in HIV-1 RNA but did not assess presence of HIV-1 DNA. Other small, limited studies of the effect of various combination therapies are generally consistent in noting reductions in HIV-1 RNA in vaginal mucosa [8,30,31]. The present study is unique because of its large sample size, the measurement of both HIV-1 RNA and HIV-1 DNA in anorectal specimens and the assessment of the relationship between their detection and plasma HIV-1 RNA levels. In addition, because data from all men enrolled were analyzed, including men with and without effective suppression of plasma viremia, the results reflect the experience of a broad group of patients who receive such therapies. While it is difficult to assess the extent to which any cohort of HIV-infected MSM is representative, men in the present study are similar to those described elsewhere with respect to peripheral CD4 T cell counts and plasma HIV-1 RNA levels among untreated individuals [32,33], and the prevalence of protease inhibitor use among persons diagnosed with AIDS .
Since men in this study were not randomized to treatment categories, our assessment of therapy impact may be affected by correlates of anorectal HIV-1 detection that are also associated with initiation of therapy. Specifically, HIV-1 disease among men receiving combination therapy was likely to have been more advanced at the time therapy was initiated than among men not receiving therapy (higher plasma HIV-1 RNA levels, lower peripheral CD4 cell counts and greater frequency of HIV-related symptoms). Since high plasma HIV-1 RNA levels are associated with both advanced disease  and HIV-1 RNA detection in anorectal mucosa , our comparison of untreated with treated men may underestimate reductions in anorectal HIV-1 RNA that might be observed in a controlled assessment of therapy impact.
The frequent detection of HIV-1 RNA and DNA in anorectal swabs obtained from these men, regardless of therapy received, is of concern given that almost half of these MSM reported unprotected receptive anal intercourse during the previous year. Risk-reduction messages should emphasize the potential risk to HIV-seronegative insertive partners that accompanies exposure to HIV-1 infected cells in anorectal mucosa, particularly since some HIV-seronegative MSM report being more likely to engage in high-risk sexual encounters since the introduction of combination antiretroviral therapy [36,37].
In summary, we found lower rates of HIV-1 RNA detection in the anorectal mucosa of MSM receiving antiretroviral therapies, particularly among men receiving a protease inhibitor. However, frequent detection of anorectal HIV-1 DNA, even in men without detectable plasma or anorectal HIV-1 RNA, indicates that such men may nevertheless harbor a reservoir of infected cells in anorectal mucosa. Therefore, condom use during anal intercourse should continue to be encouraged, regardless of plasma measures of response to potent antiretroviral therapy.
We owe special thanks to the men who so generously participated in this study. We also thank Galen Miller, Robert Michael and Anne-Marie Williams for clinic support, and Drs Jeffrey Olliffe, Peter Shalit and Mary Starkebaum for assistance with patient recruitment.
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