Human papillomavirus (HPV) is a well-known cause of cervical cancer worldwide.1 It is a common sexually transmitted infection in women2–4 and also affects men.5 In men, HPV infections lead to genital warts and cancers, including cancer of the anus and penis.6 The prevalence of penile cancer is very low in developed countries and accounts only for 0.3% to 0.6% of cancers in these regions. However, it is a substantial health problem in developing countries.7 The incidence of penile cancer in India varies from 0.7/100,000 to 3.0/100,0008 and represents more than 6% of all cancers in Indian men.8
In developed countries, the prevalence of HPV-related cancers is higher in HIV-seropositive individuals than in the general population.9–11 Men who have sex with men (MSM) constitute a susceptible population, with studies showing a higher prevalence and incidence of HPV infection and related anogenital cancers than among HIV-seronegative men. It has been estimated that 95% of HIV-seropositive MSM in developed countries have anogenital HPV infection.12 One previous study reported a prevalence of penile HPV infection of 26% among HIV-seropositive MSM.13 Moreover, the prevalence of multiple high-risk HPV types in HIV-seropositive MSM is high.14 Although the risk of scrotal cancer is very low, the scrotum may be a potential reservoir for HPV infection and HPV transmission because the epithelium of the scrotum can harbor HPV.15,16 Human papillomavirus may be transmitted during insertive anal or vaginal intercourse, even with the use of a condom, and transmission may occur even among those who are circumcised.17 Knowledge of HPV infections in MSM in India is scarce and even less is known in Indian HIV-seropositive MSM.18 The reason for this gap in knowledge is the social stigma associated with this population.
We recently reported that the prevalence of anal HPV infection in Indian HIV-seropositive MSM was 95%,19 similar to the prevalence reported in HIV-seropositive men in the United States. A better understanding of penile and scrotal HPV infection and HPV-associated disease in Indian HIV-seropositive MSM is needed. Furthermore, MSM in India are largely behaviorally bisexual and bridging to women is a strong possibility. High penile HPV infection rates among HIV-seropositive MSM could therefore have consequences for both men and women's health. Therefore, we conducted a cross-sectional study of HIV-infected Indian MSM representing different ethnic and socioeconomic backgrounds to determine the prevalence of type-specific penile and scrotal HPV infection and to identify potentially modifiable risk factors for infection.
Three hundred HIV-seropositive MSM (150 per site) were recruited from 2 demographically distinct study sites, Christian Medical College Vellore, Tamil Nadu, India (a large teaching and referral hospital), and Humsafar Trust Mumbai, Maharashtra, India (a nongovernmental organization focusing on the health of MSM in India). Men were recruited through outreach workers and local HIV/AIDS support groups, and were also referred from other nongovernmental organizations. The study was approved by the institutional review boards of CMC Vellore, Humsafar Trust, and the University of California, San Francisco. Participants completed an interviewer-administered questionnaire on demographics, native place, socioeconomic status, medical history, number of sexual partners, sexual practices, and circumcision status.
The methodology for HPV DNA sampling was described by Weaver et al.20 Briefly, 600-grit emery paper was used to gently rub the penile skin and scrotal area followed by swabbing with a moist Dacron swab.20 Separate applicators were used to collect penile and scrotal cells, respectively. The 2 swabs were placed in separate vials of Sample Transport Medium (Qiagen, Inc, Gaithersburg, MD). Blood was collected to determine the CD4+ count and HIV viral load. CD4+ count was determined by flow cytometry (Becton Dickinson FACS Count). The plasma HIV viral load was determined by HIV-1 Amplicor (Amplicor HIV Monitor test, Version 1.5; Roche Diagnostics).
HPV DNA extraction was performed on all samples. Briefly, the samples were heat-inactivated followed by the addition of Proteinase K and ammonium acetate/ethanol mixture. They were frozen at −20°C overnight and then centrifuged to obtain the pellet. The samples were eluted in Tris-EDTA buffer. MY09/MY11 L1 consensus primers were used to amplify HPV sequences. Human β-globin primers were used as a positive internal control to test for the presence of human DNA as a measure of sample adequacy and absence of polymerase chain reaction inhibitors. Known positive, negative, and no-template controls were included in each assay. Samples were dot-blotted onto a membrane and probed for HPV DNA using a chemiluminescent procedure with a consensus probe mixture. Samples were then analyzed for the presence of 29 individual HPV types and a mix of 10 less common types.19,21 Samples that were negative for β-globin DNA amplification were considered to be uninterpretable and were excluded from analysis.
Descriptive statistics were calculated for participant characteristics and HPV status. Penile and scrotal HPV prevalences were calculated along with the corresponding exact 95% binomial confidence intervals (CIs). Descriptive estimates for concordance of HPV prevalence at 2 sites were calculated as percent agreement, with concordance assessed using McNemar test and the κ statistic.
Risk factors for any HPV infection and oncogenic HPV infection at penile and scrotal anatomic sites in Indian HIV-seropositive MSM were investigated using univariate methods (χ2 or Wilcoxon rank sum test). Multivariable logistic models were fit investigating the following independent variables: age, penile warts, lifetime number of insertive anal male partners (participant was insertive partner), circumcision, CD4 counts, currently on highly active antiretroviral therapy (HAART), number of female vaginal sex partners, and smoking status. In all models, the comparison group was those with no HPV infection. The final model was determined through backward elimination and maintained factors that were significant at P < 0.10 in the multivariable model.
The demographic characteristics of participants are shown in Table 1. The median age was 34 years. The demographic profile of the Tamil Nadu group differed from that of the Maharashtra group. In the Tamil Nadu site, 64% were married, 7% were circumcised, and 48% reported more than 5 lifetime female vaginal partners. Among the Maharashtra men, 31% were married, 27% were circumcised, and 12% reported more than 5 lifetime female vaginal partners.
Overall, receptive sex was reported by 86% of the men, with most having more than 200 lifetime male partners with whom they had receptive anal sex and 22% reporting almost always or always using a condom (data not shown). Sexual contact with women was reported by 61% of participants; 23% of these men reported almost always or always using a condom with their female partners (data not shown). Median (IQR) HIV viral load was 9090 (400–79400) copies/mL, and median (IQR) of self-reported nadir CD4 count was 243 (150–415) cells/mm3.
Of the 300 participants enrolled in the study, 299 provided samples for HPV testing.
Only samples that were β-globin positive were included for analysis. Of the 299 samples, 274 (92%) penile samples, 262 (88%) scrotal samples, and 251 of both penile and scrotal (84%) samples were β-globin positive, respectively.
The prevalence of penile or scrotal HPV infection stratified by age is shown in Figure 1. The prevalence of any type of HPV infection of the penis was 55% (95% CI, 49%–61%), whereas that of the scrotum was 54% (95% CI, 48%–60%). The prevalence of HPV infection at one or both sites was 69% (95% CI, 62%–74%; see Table, Supplemental Digital Content 1, https://links.lww.com/OLQ/A148) and increased with age, although the increase was not statistically significant in multivariate analysis. The prevalence of at least one oncogenic HPV type on the penis, scrotum, or either was 15%, 13%, and 23%, respectively. The prevalence of at least one nononcogenic HPV type on the penis and scrotum or either was 15%, 13%, and 20%, respectively. The most common HPV types seen in the genital region were HPV-35, HPV-16, HPV-6/11, and HPV-70, with prevalence of 8%, 6%, 6%, and 6% on the penis and scrotum or either, respectively (Fig. 2). Overall prevalance of HPV by anatomic site is presented in Figure 3.
There was good agreement between penile and scrotal HPV infection, with the concordance being 73% (κ = 0.45; see Table, Supplemental Digital Content2, https://links.lww.com/OLQ/A148). Thirty-four participants had at least one matching subtype on their penis and scrotum (excluding those positive with the 10-type probe mixture). Among the participants who had specific HPV types identified on the scrotum, 62% had at least one of the same types on their penis. Similarly, among the participants who had specific penile HPV types identified, 52% had at least one of the same types on their scrotum. The concordance was 84% for oncogenic HPV infection regardless of subtype (κ = 0.39).
Table 2A presents the univariate associations for penile HPV (any HPV and oncogenic HPV) infection and selects risk factors. Table 2B presents the univariate associations for scrotal HPV infection and select risk factors. Similarly, univariate association between risk factor and nononcogenic HPV is shown in Table, Supplemental Digital content 3, https://links.lww.com/OLQ/A148. Age in years, age at having first male partner, history of penile warts, circumcision, lifetime number of male partners with whom participant had insertive anal sex, lifetime number of female vaginal partners, and enrollment site were associated in univariate analysis with any penile HPV infection. In multivariate analysis, significant risk factors for any penile infection included lack of circumcision, having insertive sex with more than 100 male partners compared with those who had no insertive anal sex with male partners, and being a Tamil Nadu participant (Table 3A).
Age in years, lifetime number of partners with whom participants had insertive anal sex, lifetime number of female vaginal partners, and smoking were associated with oncogenic penile HPV infection in univariate analysis. In multivariate analysis, those 26 to 35 years old were more likely to have oncogenic penile HPV than those 18 to 25 years old, and those having insertive sex with more than 100 male partners were more likely to have oncogenic penile HPV compared with those with no insertive partners (Table 3A).
In univariate analysis of risks factors for any scrotal HPV infection, significant factors included history of penile warts, lifetime number of male partners with whom the participant had insertive anal sex, and lifetime number of female vaginal partners. In the final multivariable model, risk factors for any scrotal infection included a history of penile warts and having insertive anal sex with more than 100 lifetime male partners compared with having no insertive anal sex partners (Table 3B).
In univariate analysis of risk factors for oncogenic scrotal infection, significant factors included history of penile warts, lifetime number of male partners with whom participant had insertive anal sex, and lifetime number of female vaginal partners. With smoking, there was a higher trend toward scrotal oncogenic HPV infection. In the final multivariable model, those with a history of penile warts were more likely to have oncogenic scrotal HPV than those without a history, and those having insertive anal sex with 1 to 100 partners and more than 100 partners were more likely to have oncogenic scrotal HPV compared with those with no insertive partners (Table 3B).
This is the first study looking at genital HPV infection in an Indian MSM population and, in particular, the first study of Indian HIV-seropositive MSM. Participants from the 2 sites were different in some important demographic factors. Compared with the Maharashtra group, the median age of the Tamil Nadu participants was higher, a higher proportion was married, and a higher proportion was uncircumcised; Tamil Nadu participants also had a higher prevalence of any penile HPV infection, potentially due to the differences in the age, circumcision, and exposure to female partners. Modeling of HPV infection according to the individual study sites was not undertaken because of the high number of positive test results, particularly for oncogenic HPV types.
Men at both sites had many results in common, however. These include a high HPV prevalence of the penis (55%, summary of both sites) and scrotum (54% overall). Based on the high prevalence of HPV and the distribution of HPV genotypes, Indian HIV-seropositive MSM may be at high risk for genital warts, and possibly penile precancer or cancer.
Worldwide, the prevalence of high-risk HPV genotypes in penile and scrotal samples ranges between 22% and 34% among HIV-seronegative MSM.22 Although the prevalence of high-risk types in our study (40% and 35% on the penis and scrotum, respectively) was similar to those reported in studies of HIV-seropositive Western populations, if not slightly higher,16,17 the HPV type distribution was different in our study. In our study, HPV-35 was the most common oncogenic HPV type. This is consistent with our previously published study; HPV-35 was also the most common type of anal HPV infection in this population.19 None of the bivalent, quadrivalent, or recently licensed nonavalent HPV vaccines include HPV-35. Human papillomavirus type 35 is found in 6% to 10% of cervical specimens of Indian HIV-seropositive women23,24 and the percentage of cervical cancers attributable to HPV-35 is low (<2%).25 However, little is known of the prevalence of different HPV types in penile cancer among Indian HIV-seropositive men. Notwithstanding some amount of cross-protective immunity provided by the vaccines,26 these vaccines may not be fully effective in preventing cancers in the Indian MSM population. Even if some protection is provided, HPV vaccination is not currently offered through government programs for girls or boys in India23 and the overall uptake among young women is low. However, new initiatives are underway such as the public health program in the state of Delhi to vaccinate girls in schools.27
In multivariable analysis, men who were uncircumcised and who had more male anal sex partners with whom the participant was the insertive partner had higher rates of penile HPV infection. Other studies have reported similar findings, with increased detection of penile HPV infection associated with increasing number of lifetime sexual partners.28 Older men and men with more male sex partners were more likely to have oncogenic penile HPV infection. Our findings among Indian HIV-seropositive MSMs showed a strong independent association between sexual behavior and HPV detection in men similar to the data from other populations.5,28 In addition, those who had a larger number of insertive male anal sex partners and those who had female vaginal sex partners were more likely to have any scrotal HPV infection. The odds of having any scrotal HPV and oncogenic scrotal HPV infection increased when penile infection was also present.
In our study, a substantial number of participants reported having sex with female partners and a higher number of female sex partners were associated with a higher prevalence of genital HPV infection. This study highlights that Indian MSM may have more heterosexual partners than Western MSM populations, and thereby could potentially serve as a bridge population to women. High penile HPV infection rates among MSM could have consequences for women's health, and preventions designed to target MSM could also benefit women.
History of penile warts, number of male partners, and insertive anal sex were significantly associated with any genital HPV infection. When the sexual risk factors were analyzed, insertive anal sex was associated consistently with any penile/scrotal HPV infection and oncogenic HPV infection. This finding is similar to other published studies that have shown insertive anal sex as a risk factor for genital HPV infection.22 CD4 level, HIV viral load, and currently taking antiretroviral therapy were not associated with penile/scrotal or combined infection. This finding is similar to other published studies on anal and cervical cancer where the incidence of HPV infection had not declined despite antiretroviral therapy.29 Other risk factors such as condom usage and alcohol consumption were not associated with genital HPV infection. Smoking as a risk factor was associated in univariate analysis with oncogenic penile HPV infection but did not retain its significance when adjusted for other factors. Condom use did not protect against either penile or scrotal HPV infection, and this could be due to the presence of HPV in the genitalia not covered by the condom, such as the scrotum. Also, it is inherently difficult to assess condom use in a population because it is limited by the accuracy of reporting by participants.30 Circumcision is known to protect men from the acquisition of HPV, HIV, and other sexually transmitted infections.31,32 Similar to previous studies, we were not able to find any association between circumcision and scrotal HPV infection.33 Although circumcision may lead to reduced risk of cervical cancer and has important implications for public health practice, it is culturally problematic in India to study circumcision interventions.34
This study had several limitations. We did not have a history of other sexually transmitted infections in this population. Because the population was HIV seropositive, the results may not be generalizable to the population of Indian MSM at large. Our risk factor analysis was limited by the cross-sectional nature of the study, and only a prospective study will help to assess the effects of these variables on the incidence and clearance of HPV infection. Our study did not include assessment of HPV-associated disease of the penis and scrotum.
In conclusion, this study highlights the burden of genital HPV infection in Indian HIV-seropositive MSM. Although many of the oncogenic HPV infections detected would be prevented by currently available HPV vaccines, even the new nonavalent HPV vaccine may not be fully protective in this population because HPV-35 was the most commonly detected HPV type in this population. Further studies are needed to define the burden of HPV-associated disease in this population, including penile cancer, as well as the risk of HPV transmission to male and female sexual partners.
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