Sexually transmitted infections (STIs) are strongly associated with HIV acquisition. These include ulcerating STIs such as Hemophilus ducreyi and Treponema pallidum, as well as nonulcerating STIs – gonorrhea, chlamydia, trichomoniasis, and herpes simplex virus (HSV) [1–4]. There is biological plausibility – cells susceptible to HIV infection such as CD4+ T cells and macrophages assemble in genital areas affected by STIs. Furthermore, STIs anatomically disrupt mucosal barriers, which can increase the exposure of HIV-susceptible cells to HIV. Attention to the synergistic role of STIs in HIV acquisition has led to systematic screening and treatment of many STIs in at-risk groups in an effort to decrease the risk of HIV acquisition [5,6]. However, there has been little information regarding the role of human papillomavirus (HPV) in HIV transmission.
HPV is the most common STI and can cause high-grade cervical intraepithelial neoplasia (CIN) and anal intraepithelial neoplasia (AIN), precursor lesions to cervical and anal cancer, respectively. Cervical cytology has been used for decades to identify women who require cervical colposcopy to visually identify CIN, biopsy it and treat it to prevent cervical cancer. Similarly, the goal of anal cytology is to seek cytological changes in anal canal epithelial cells to triage individuals to more sensitive high-resolution anoscopy (HRA). HRA allows high-grade lesions that contribute to abnormal cytological findings to be identified, biopsied for histopathologic assessment and treated before they progress to invasive anal cancer. Similar to cervical cytological abnormalities, anal cytological abnormalities associated with HPV infection include atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells – cannot rule out high-grade disease (ASC-H), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL). Similar to cervical cytology for CIN diagnosis, anal cytology has limited sensitivity to detect HPV-associated AIN. It is the cumulative sensitivity that makes cytology useful for cancer screening.
Although attention has been paid to the oncogenic potential of HPV, there is less information regarding HPV infection and disease as risk factors for HIV acquisition. We hypothesized that disease caused by HPV infection can enhance susceptibility to HIV infection because of increased tissue microvasculature, friability, and recruitment of CD4+ T cells and dendritic cells in response to HPV infection.
The aim of the study was to examine the association between the detection of anal HPV infection and HIV seroconversion in a cohort of sexually active HIV-negative men who have sex with men (MSM) in four US cities.
We recruited men who were participants in the EXPLORE trial in four cities (Boston, Denver, New York and San Francisco). The EXPLORE trial was a randomized clinical trial of the efficacy of a behavioral intervention to reduce the risk of HIV acquisition among sexually active HIV-negative MSM. Men were eligible for EXPLORE if they were HIV-negative, were 16 years of age or older, and reported insertive or receptive anal sex with men during the previous year. Details of the baseline methods have been previously published and are also available from the EXPLORE website (www.explorestudy.org) [7,8]. Men in monogamous relationships with other known HIV-negative men for 2 or more years were excluded from enrollment. Study personnel recruited EXPLORE participants using a variety of methods including outreach in streets, dance clubs, bars, bathhouses, sex clubs, health clubs, and video arcades. From January 2001 to October 2002, 1409 men were enrolled in the HPV ancillary study. We followed participants every 6 months from 12 to 48 months of follow-up of the EXPLORE study. Each participant provided written informed consent. The institutional review boards of each participating center approved the study protocol.
Sexual behavior and recreational drug use
We collected information on participants' sexual behaviors and drug use by Audio Computer-Assisted Self-Interview (ACASI) technology. Using ACASI, study participants were able to hear or read questions in English or Spanish and then enter answers on a keyboard. We also asked participants about sexual behaviors in the previous 6 months with partners of each HIV-serostatus type (HIV-negative, HIV-positive, and HIV-status unknown). ACASI interviews were conducted at baseline and at each 6-month follow-up visit.
Participants were HIV tested at each 6-month follow-up visit following HIV pretest counseling at the screening visit for the EXPLORE study. Antibodies to HIV in serum samples were detected using ELISA, and, if positive, were retested in duplicate. We confirmed HIV status using western blot or immunofluorescence assays and PCR if warranted. We referred all HIV-positive participants to medical and social services.
Anal sample collection
Trained personnel at each site collected anal specimens by rotating a water-moistened Dacron swab (Baxter Healthcare, McGraw Park, Illinois, USA) in the anal canal without direct visualization. Study staff then vigorously agitated each swab in a methanol-based fixative (PreservCyt; CYTYC, Boxborough, Massachusetts, USA) for the preparation and interpretation of thin-layer slides for cytology, as well as for HPV DNA testing using PCR.
Human papillomavirus testing
For specimen DNA preparation, we swirled the PreservCyt solution to suspend the cells. We used a pipette to transfer 1.5 ml of the solution to a microfuge tube that was spun at 16g for 15 min. The solution was decanted and dried overnight at room temperature or in a 65°C hot block for 1 h. The pellets were suspended in 100 μl sample transport medium (Digene, Silver Spring, Maryland, USA), and 200 μg/ml proteinase K (Boehringer Mannheim, Indianapolis, Indiana, USA). The samples were vortexed and digested at 56°C in a waterbath for 1 h and heated at 95°C for 10 min to inactivate proteinase K and frozen at −20°C until use. To perform PCR, we used 5 μl of sample for PCR amplification using a standard 40-cycle protocol to detect the presence of one or more HPV types using a generic probe set . PCR products from samples that were positive with the generic probe set were also typed by dot-blot hybridization using 29 individual type-specific probes and 10 individual HPV types combined into one probe set. HPV infection was classified as high risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, or 73) or low risk (types 6, 11, 53, 54, 55, 66, 83 or 84) based on the strength of association of specific HPV types with invasive cervical cancer [10,11].
Anal cytology testing
The study pathologist evaluated all anal cytological specimens and had no prior knowledge of the clinical status of the participants, their ACASI responses or other test results. Anal cytology specimens were classified as normal, ASC-US, ASC-H, LSIL, or HSIL using Bethesda criteria to evaluate cervical cytology .
The primary analysis assessed the association of HPV infection with the rate of acquisition of HIV infection. We used Cox proportional hazards regression modeling with a discrete time scale of twice-yearly visits. The analysis pertains to data collected from the 12–48 month visits from the EXPLORE trial. We constructed univariate and multivariate models to adjust for potential confounders. For multivariate models, we first assessed demographic and behavioral risk factors and self-reported STI covariates for confounding by examining their effects on the hazard ratios of the HPV predictors using bivariate proportional hazards models (i.e. the covariate and the HPV predictor variable). We considered the covariate a confounder if it altered the magnitude of the hazard ratio for the HPV predictor by at least 10% . Confounders were then included in the multivariate models with the corresponding HPV predictor.
Age [≤25, 26–30, 31–35, ≥36 years (reference)] and race [White (reference), Black, Hispanic, and other] were included as demographic variables in the models. We also included behavioral variables, assessed at every 6-month time interval and pertaining to the past 6 months of reported behavior via ACASI, as follows: male sexual partners [0–1 (reference), 2–3, 4–9 ≥ 10], unprotected receptive anal sex with HIV-positive, HIV-negative, and HIV-status unknown male partners, unprotected insertive anal sex with HIV-positive, HIV-negative, and HIV-status unknown male partners, primary partner status [no primary partner, HIV negative (reference), positive, unknown], and oral sex with HIV-positive or HIV-status unknown partners. Also included in the analyses was use of nonprescription drugs classified as ‘ever’ against ‘never’ (reference) reported in the past 6 months as follows: marijuana or hashish, poppers or inhaled nitrites, including ampules, smoked crack or rock cocaine or snorted or sniffed cocaine, swallowed, snorted, or smoked amphetamines such as speed, crystal, or crack, snorted or smoked heroin, use of hallucinogens such as phencyclidine (PCP), Special K, angel dust, acid, lysergic acid diethylamide (LSD), mushrooms or Ecstasy, and use of a needle to inject any drugs, including steroids, under the skin or into a vein. Alcohol use was defined as none (reference), light (≤three drinks/day on no more than 1–2 days/week), moderate (four to five drinks/day on no more than 1–2 days/week, or one to five drinks/day on 3–6 days/week, or one to three drinks/day daily), or heavy (four or more drinks every day or six or more drinks on a typical day when drinking). We based the use of alcohol or drugs before sex on the question, ‘In the past 6 months, about how often did you get high or have a few drinks immediately before or during sex?’ The responses to this question included the following: never, occasionally, often, all of the time. For purposes of this analysis, we coded ‘never’ as ‘no’, and all other responses as ‘yes.’ The EXPLORE questionnaire asked participants about depressive symptoms based on a shortened version (seven questions) of the Center for Epidemiologic Studies depression (CES-D) scale . We constructed an additive score and divided it into quartiles (with higher scores representing more depressive symptoms). We also included participants' self-reporting of herpes simplex, gonorrhea, and chlamydia. Apart from demographics, STIs, and each of the HPV indicator variables, we invoked the last-value-carried-forward convention to account for missing data. All models were stratified by randomization arm and study site to adjust for potential differences in HIV infection rates between the subgroups. Analyses were performed using SAS Version 9.1.3.
Table 1 shows various demographic, behavioral, and HPV-associated characteristics of the study participants at baseline. Of 1409 patients, 57% had anal HPV detected by PCR, and 32% were found to have any abnormal anal cytology.
Human papillomavirus as a risk factor for HIV seroconversion
Of the 1409 initially HIV-negative participants contributing 4375 person-years of follow-up, 51 HIV-seroconverted. The overall rate of HIV acquisition among this cohort was 1.17 per 100 person-years [95% confidence interval (CI) 0.87–1.53]. Of the HIV-seroconverters, 81% also tested positive for anal HPV.
In unadjusted analyses (Table 2), infection with one HPV type (hazard ratio 2.8, 95% CI 1.04–7.4, P = 0.04) and two or more HPV types (hazard ratio 3.6, 95% CI 1.5–8.4, P = 0.004) was associated with HIV seroconversion. In multivariable analyses (Table 2), we found evidence that infection with two or more HPV types was associated with HIV seroconversion (hazard ratio 3.5, 95% CI 1.2–10.6, P = 0.002) compared to those who were HPV uninfected.
Other than HPV, additional variables in the multivariable model evaluating predictors of HIV seroconversion that were statistically significant (P < 0.05) included unprotected receptive anal intercourse with HIV-unknown-status partners (hazard ratio 7.1, 95% CI 2.8–18.2) and history of methamphetamine use (hazard ratio 4.6, 95% CI 1.7–12.0), all in the previous 6 months.
Abnormal anal cytology as a risk factor for HIV seroconversion
There was some evidence for the association of abnormal anal cytology with the risk of HIV seroconversion in univariate analyses. The following were evaluated: atypical squamous cells versus benign cytology (hazard ratio 2.9, 95% CI 1.1–7.5, P = 0.03) and LSIL versus benign cytology (hazard ratio 1.8, 95% CI 0.69–4.6, P = 0.23). Due to low prevalence of HSIL, the univariate model hazard ratio estimate and 95% CI were not sufficiently stable to report. Additional predictors analyzed in univariate models that were statistically associated with HIV seroconversion were the presence of either atypical squamous cells or LSIL versus benign cytology (hazard ratio 2.1, 95% CI 1.0–4.5, P = 0.05). In multivariable analyses, there was no evidence of an independent effect of atypical squamous cells (hazard ratio 1.8, 95% CI 0.62–5.5, P = 0.27) or of LSIL (hazard ratio 1.2, 95% CI 0.44–3.23, P = 0.73) and HIV seroconversion. No estimates were attainable for the multivariate model for HSIL.
We report that among HIV-negative MSM in our study, anal HPV infection is associated with a higher risk of HIV acquisition after adjusting for potential confounders, compared with individuals who do not have HPV infection. Furthermore, in multivariate analysis that adjusted for sexual activity, the risk of HIV acquisition increased significantly with increasing number of HPV types isolated by PCR. The increasing risk of HIV seroconversion seen with increasing number of HPV types may reflect a higher number of anal lesions that facilitate HIV acquisition. Although the relationship between STIs and HIV acquisition is well established, and there have been multiple articles that report the magnitude of HPV disease in HIV-infected individuals [1,3], we are not aware of other studies that have investigated the relationship between HPV and HIV infection.
Several investigators have demonstrated that ulcerative and nonulcerative STIs are associated with HIV acquisition. In one of the earlier prospective studies examining the risk of HIV seroconversion from genital ulcer disease, investigators reported an adjusted risk of HIV of 4.7 among 293 heterosexual men in Nairobi, Kenya who acquired laboratory-confirmed STIs (primarily chancroid) from female sex workers . Studies among MSM have also shown generally consistent findings. In a nested case–control study of 104 men from San Francisco, California, Holmberg et al.  showed that men who had prevalent HSV-2 seropositivity had an adjusted odds ratio of 2.4 for HIV seroconversion compared to men who remained HSV-2 negative. In a meta-analysis of comparable cohort studies looking at the effect of HSV-2 infection in HIV acquisition, Freeman et al.  estimated a combined relative risk of 1.7 (95% CI 1.2–2.4) among MSM. Apart from chancroid and HSV-2, other STIs that have been demonstrated to be risk factors for HIV acquisition include syphilis [17,18], gonorrhea , chlamydia , and trichomonas . There have been few data on the contribution of HPV to HIV seroconversion.
Our study demonstrates that anal HPV infection in MSM is associated with HIV acquisition. The mechanisms are not yet clear, but HPV may be associated with lesions that are friable and susceptible to disruption of mucosal integrity in the HIV-negative individual [21,22] and may be associated with postcoital bleeding. Studies looking at the contribution of other STIs to HIV acquisition have also demonstrated that cells susceptible to HIV infection may be recruited to the surface of the STI-associated lesion . In one experimental model, individuals infected with H. ducreyi were demonstrated to have enrichment of T cells and macrophages after 48 h of infection, compared with individuals without infection. Immunohistochemistry showed increased expression of CCR5 and CXCR4 in macrophages and upregulation of CCR5 in CD4+ T cells. Immunohistochemistry studies of HPV-associated CIN have also shown local recruitment of T lymphocytes (including CD4+ T cells) by upregulation of vascular adhesion molecules such as ICAM-1 . In one cross-sectional study of 2758 MSM requesting anonymous HIV testing in Mexico City, men who reported both rectal bleeding and anal warts had an adjusted odds ratio (OR) of 3.5 (95% CI 2.1–5.8) of being diagnosed with HIV compared with men who reported neither postcoital bleeding nor anal warts . We propose that HPV infection in our population of MSM resulted in lesions that were susceptible to anatomic barrier disruption with increased vascularity, bleeding, and HIV-susceptible inflammatory cells.
We found less evidence for the role of abnormal anal cytology in predicting HIV seroconversion. Given that anal cytology is only about 50% sensitive in diagnosing cervical (CIN)  or AIN , in our study, it is likely that anal cytology underestimated the true prevalence of HPV-associated anal disease. Nondifferential misclassification bias would have likely resulted in the lack of effect seen. Inadequate numbers of cytological specimens would have contributed to not having enough statistical power to show an effect of the relationship between abnormal cytology and HIV seroconversion even if there was one. Studies that use HRA to identify and characterize HPV-associated lesions would have the highest sensitivity and precision in evaluating AIN as a risk factor for HIV disease.
Other risk factors that predict HIV seroconversion in the full EXPLORE cohort of six cities have been examined in detail in another report . In that analysis, as in ours, unprotected receptive anal intercourse and amphetamine use were independent risk factors for acquisition of HIV infection . HPV-associated variables were not examined in this previously reported analysis.
There are several potential limitations of this study. The first is that there were only 51 HIV seroconversions in the study, limiting power to evaluate the relationship between type-specific HPV infection or HPV persistence and HIV seroconversion. We also chose not to imput values for missing HPV or cytology data, and this also limited our statistical power. In addition, the men in the study may not be necessarily representative of MSM in general. The study specifically selected for men at high risk for HIV. Data from the overall EXPLORE study also indicate that African–American, Latino, younger men, and those of lower socioeconomic status were less likely to be study participants and were more likely to be HIV infected at study screening . However, even if data from our study were not generalizable to all MSM, we do not think that this would affect the biology of the relationship between HPV and HIV acquisition in other populations. Likewise, the association that we report between anal HPV and HIV acquisition in men is likely comparable to the relationship between HPV and incident HIV at other anogenital sites such as the cervix in women, which has not been studied. The cervix and the anus are embryologically similar and have similarities in histology such as a transformation zone where squamous and columnar cells meet and where HPV-associated disease often occurs . Further work in the cervix needs to be conducted to confirm the observation that we report here − that anal HPV is associated with HIV seroconversion among MSM. Finally, we do not have biologic measurements of other common STIs in this cohort, which may potentially confound the association between HPV and HIV acquisition. We did ask participants to report interval diagnosis or symptoms consistent with other STIs. It is interesting to note that though self-reported gonorrhea was associated with HIV seroconversion in the overall analysis of risk factors for HIV infection (HPV variables were not included) in the EXPLORE cohort previously reported, there was no statistically significant relationship between gonorrhea and HIV seroconversion noted in our analysis. This could be related to the low number of gonorrhea events overall (1.4–2.3% during any 6-month period) , which would not allow us to see an effect in our smaller sample size even if there was one. In contrast, the fact that we are able to still detect an independent association between HPV and HIV attests to the high prevalence of anal HPV in this population . Overall, the proportion of HIV seroconversions that would be attributable to HPV would likely be larger than the impact of other STIs, given the high prevalence of HPV in all sexually active populations worldwide .
In high HPV prevalence populations, we demonstrate that HPV is independently associated with HIV acquisition. With this observation more questions arise. More precise exploration of virologic factors such as type-specific HPV persistence and its temporal relationship with HIV acquisition is needed. Prospective studies that incorporate HRA for more sensitive and histological identification of AIN need to be performed. Work exploring the impact of anal and cervical HPV disease in women should be done to extend our observations seen here among men. With the recent US FDA approval of the first HPV prophylactic vaccine [31–34], immunization in HPV-unexposed individuals may not only prevent invasive cancer and anogenital warts, but also have the potential to reduce the risk of HIV acquisition as well. At a minimum, identification of HPV infection and HPV-associated lesions may improve assessment of HIV transmission risk.
Support to this study was provided by the University of California Universitywide AIDS Research Program [UARP-R97-SF-1030 (J.M.P.)], and by K23 AI054157 (P.V.C.H.), R01 CA54053 (J.M.P.), R01 CA/AI 88739 (J.M.P.), and by CYTYC Corporation. This work was also supported by the HIV Network for Prevention Trials – sponsored by the US National Institute of Allergy and Infectious Diseases (NIAID) and the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health (NIH), Dept of Health and Human Services (DHHS), through contract N01 AI35176 with Abt Associates, Inc.; contract N01 AI45200 with the Fred Hutchinson Cancer Research Center; and subcontracts with the Denver Dept of Health and Hospitals, the Fenway Community Health Center, the New York Blood Center and the Public Health Foundation, Inc. Support was also given by the HIV Prevention Trials Network, sponsored by NIAID, the National Institute of Child Health and Human Development, the National Institute on Drug Abuse, the National Institute of Mental Health, and the Office of AIDS Research of the NIH and DHHS, through a cooperative agreement with Family Health International (cooperative agreement 5 U01 AI46749) with a subsequent subcontract to Abt Associates, Inc., and subcontracts to the Denver Department of Health and Hospitals; cooperative agreement U01 AI48040 to the Fenway Community Health Center; cooperative agreement U01 AI48016 to Columbia University (including a subagreement with the New York Blood Center); cooperative agreement U01 AI47995 to the University of California, San Francisco. This publication was made possible by Grant Number UL1 RR024131-01 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research.
The authors thank Tom Coates, Daniel Cohen, Eric Vittinghoff, the HPV EXPLORE site coordinators (Louise Austin, Anthony Buckman, Ken Miller, Arnel Montenegro, David Pantalone, and Dave Ward) and counselors, Linda Ta, Jess Thompson, and Rossana Herrera from the UCSF HPV laboratory, Terry O'Donnell for administrative support, Maria Madison at Abt, Eileen Hess from the SCHARP Statistical Center at the Fred Hutchinson Cancer Research Center, and the study participants.
All authors contributed to the interpretation of the data and the overall intellectual content of the paper. Substantial contributions were made by the following in study conception and design (P.V.C.H., J.M.P.), and data analysis (M.H., P.V.C.H.).
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