The worldwide incidence of human papillomavirus (HPV)–associated anal cancer is increasing in both men and women, with current annual rates in the United States approximating 2/100,000.1 In men who have sex with men (MSM), the incidence is similar to that of cervical cancer before the introduction of cervical cytology screening and is approximately 2 to 3 times this rate in HIV-infected MSM.2,32,3
Approximately 40 HPV types infect the anogenital area, and these can be broadly differentiated into high-risk (HR) and low-risk types based on their historical association with cervical cancer. More than 80% of all anal cancers are related to persistent infection with HR-HPV infection, with HPV type 16 being the most commonly isolated.4
Anal HPV–associated disease due to HPV types 6 and 11 (low-risk) and types 16 and 18 (HR) may be prevented in men with the use of the quadrivalent HPV vaccine (qHPV) Gardasil (Merck and Co, Whitehouse Station, NJ).5,65,6 This vaccine is indicated for males and females aged 9 to 26 years, with optimal efficacy seen in individuals without evidence of current or past HPV infection.6 A vaccine with increased valency that additionally incorporates HR-HPV types 31, 33, 45, 52, and 58 (nHPV) Gardasil 9 (Merck and Co) has recently been approved by the US Food and Drug Administration for boys aged 9 to 15 years.
In Western populations, the prevalence of anal HPV infection in MSM varies from study to study. Several studies report anal HPV prevalence rates of 45% to 70% in HIV-uninfected and 65% to 96% in HIV-infected MSM,7–97–97–9 and in a recent meta-analysis of HIV-infected MSM, HPV16 was the most frequently detected type at 35.4% (95% confidence interval, 32.9–37.9).10 In general, HIV-infected MSM also have an increased prevalence of all HPV infections, HR-HPV, and multiple HPVs, compared with HIV-uninfected MSM.10,1110,11
Although the qHPV was approved by the Thai Food and Drug Administration in 2007, limited information is available regarding HPV vaccine types prevalent in Southeast Asia, with few studies of anal HPV in Thai MSM specifically. One study in Bangkok found that any HPV type was present in 58.5% and 85.0%, and HR-HPV in 36.6% and 57.5% of 123 HIV-uninfected and 123 HIV-infected MSM, respectively, with HPV16 being the most common HR-HPV detected.12
This study aimed to increase the information available on anal HPV infection in Thai MSM, and in particular to define prevalence of HPV infection and risk factors as they relate to both qHPV and nHPV vaccines. These data, although of general interest in this understudied population, are also of local interest to help inform national immunization policy and guidelines in a population of HIV-infected and HIV-uninfected Thai MSM at risk for developing anal cancer.
MATERIALS AND METHODS
The Bangkok MSM Cohort study was established in 2006 as part of the US Centers for Disease Control and Prevention (CDC) collaboration with the Thailand Department of Disease Control of the Ministry of Public Health.13,1413,14 The main study protocol was reviewed and approved by the Thailand Ministry of Public Health Ethical Review Committee and by a CDC institutional review board. Written informed consent was obtained from all participants. For the HPV substudy, approval was sought and obtained from the CDC to ship a convenience sample of frozen anal swab specimens obtained between 2006 and 2010 from 200 HIV-infected and 200 HIV-uninfected participants to the University of Pittsburgh for HPV testing. Approval was also obtained for this study from the University of Pittsburgh Institutional Review Board.
The study location was a dedicated men's sexual health clinic in a central Bangkok hospital. Eligible men were at least 18 years old, were Thai national, were resident of Bangkok or adjacent provinces, had a history of penetrative male-to-male sex in the 6 months before recruitment, and were available for follow-up visits every 4 months for 3 to 5 years. Men were recruited from entertainment venues (bars, discos, and saunas), parks, community-based organizations, the Internet, and by word of mouth. Men received pretest and posttest HIV and risk behavior counseling during every study visit. Those testing positive for HIV infection were referred for antiretroviral treatment according to Thai national guidelines. Those with active sexually transmitted infections were treated, and those without hepatitis B virus immunity were offered hepatitis B virus vaccination, free of charge. A total of 1744 men were recruited, all of whom had anal swab samples taken at the initial visit.
Demographic, behavioral, and clinical data were collected by computer-assisted self-interview. Data on concomitant infections were available from the parent Bangkok MSM Cohort Study as previously described.13,1413,14 It should be noted that no information was collected on history of HPV vaccination, or on any clinical markers of HPV infection such as anal warts or other anal conditions; neither was anal cytology collected.
Anal HPV Sample Collection
Specimens for HPV testing were obtained by study clinical staff once at the main study baseline visit by inserting a water-moistened polyester swab into the anal canal, rotating the swab and then withdrawing. The swab was immediately placed into a Digene Sampler tube and frozen at −80°C. For this study, a convenience sample of 400 anal swab samples were chosen from 200 HIV-infected and 200 HIV-uninfected study participants. The HPV media was thawed at room temperature for 30 minutes and vortexed for at least 5 seconds, and then an aliquot of 200 μL of fluid was removed. The aliquot was immediately refrozen at −80°C. Samples were shipped on dry ice from Bangkok to Pittsburgh, where they were again stored at −80°C before processing.
DNA Extraction and β-Globin Quantification. DNA was extracted from anal swabs using the QIAamp DNA Investigator Kit (Qiagen, Valencia, CA). DNA content was assessed by optical density on the NanoVue spectrophotometer (GE Healthcare Bio-Sciences Corp, Piscataway, NJ), and human β-globin content was determined by quantitative real-time polymerase chain reaction (PCR). β-Globin was quantified as a measure of sample collection efficiency, DNA extraction efficiency, and verification of the absence of PCR inhibitors in the extract.15
Reverse Line Blot Assay. Samples that tested positive for β-globin and/or HPV by the EvaGreen assay were analyzed for HPV subtype by the reverse line blot assay.16 Probes specific for 37 different HPV subtypes (HPV types 6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 68, 70, 71, 72, 73, 81, 82 (MM4), 82 (IS39), 83, 84, and CP6108) were covalently attached to negatively charged Biodyne C membranes (Pall Corp, Port Washington, NY) with the aid of a blotting apparatus with 45 channels (Miniblotter 45; Immunetics, Boston, MA). Probes for the individual subtypes, labeled at the 5′ end with an amino group and 6-carbon spacer, were covalently attached to the membrane through the use of a coupling reagent (EDC; Thermo Scientific, Pittsburgh, PA). In preparation for hybridization of denatured, biotinylated GP5+/GP6+ PCR products to the covalently attached probes, the blot was rotated in the apparatus 90° to result in a checkerboard pattern. Thus, each sample was hybridized simultaneously to 37 covalently applied probes for HPV subtypes and β-globin. Ten-microliter aliquots of the PCR reactions were denatured and hybridized to the blots. After hybridization, the blots were washed to remove nonspecifically bound products and incubated with streptavidin-horseradish peroxidase conjugate (GE Healthcare). Specifically bound PCR products were detected by enhanced chemiluminescence, and signals were detected on a Chemi-Doc XRS+ Molecular Imager (Bio-Rad, Hercules, CA).
Herpes simplex virus (HSV) type 2 antibody testing used the HerpeSelect 2 ELISA IgG (Focus Diagnostic, Cypress, CA) and results were reported as index value relative to the cutoff calibrator (index value = specimen optical density [OD] divided by mean of cutoff calibrator OD). A positive result for the presence of IgG antibodies to HSV-2 defined by an index value greater than 1.10. Herpes simplex virus type 1 antibodies were detected using the HerpeSelect 1 ELISA IgG kit (Focus Diagnostic). Results were reported as index values relative to the cutoff calibrator (where index value = specimen optical density divided by mean cutoff calibrator optical density). An index value greater than 1.10 was used to identify positive results, according to the manufacturer's instructions. To reduce the likelihood of false-positive results using this index for HSV types 1 and 2, samples with an index value greater than 1.10 but less than 3.5 were repeated in duplicate. In cases where 1 or both of 2 repeat tests remained greater than 1.10, a positive result was confirmed. If both repeated tests' index values were less than 1.10, the test was considered to be negative. Hepatitis A virus antibody testing used the Monolisa Total Anti-HAV Plus (Bio-Rad, Marnes-la-Coqueete, France) using an inhibition enzyme immunoassay. Results were reported as ratio value relative to the calculated cutoff value (ratio value = mean of cutoff OD divided by mean of sample OD). Samples with a ratio value greater than or equal to 1.10 are considered to be positive. Hepatitis B surface antigens and antibodies were determined qualitatively using a hemagglutination assay (Serodia, FUGIREBIO Inc, Tokyo Japan), or 1 of 2 enzyme immunoassays (Murex [Abbott Diagnostics, Kyalami, South Africa] or Monolisa [Bio-Rad, Marnes-la-Coquette, France]) depending on kit availability. Hepatitis B virus core antibodies were determined using a competitive enzyme immunoassay (ETI-AB-COREK PLUS; DiaSorin, Saluggia, Italy) or 1 of 2 enzyme immunoassays (Murex [Abbott Diagnostics] or Monolisa [Bio-Rad]).
Data were summarized as mean and SD for continuous variables and percentages for categorical variables. Participant demographic characteristics, proportions with serologic evidence of selected infections (hepatitis A, hepatitis B, and HSV types 1 and 2), and HPV prevalence among HIV-infected participants versus HIV-uninfected participants were compared using t tests for continuous variables and χ2 tests for categorical variables. Because of the observed differences between HIV-infected and HIV-uninfected participants and the ensuing likelihood that HIV status will influence relationships between various risk factors, behaviors, and HPV status, all further analyses were stratified by HIV status. For each HPV type investigated, χ2 tests were used to compare the proportion of participants with and those without each of the concomitant infections (based on serostatus). Relationships between categorical variables on sexual partners/behavior and HPV types were also assessed using χ2 tests. P values less than 0.05 were considered statistically significant; no adjustments were made for multiple comparisons. All statistical analyses were carried out using SAS version 9.4 (SAS Institute, Cary, NC).
Study participants were generally young (mean age, 25.6 years; range, 18–55 years), and most had at least a high school education (88.5%). Drug use in the last 4 months was significantly more common in HIV-infected participants (49.5% vs. 35.6%, P < 0.01) than in HIV-uninfected participants. Although 20.2% of participants reported drinking alcohol once or more per week, this was similar regardless of HIV status. Among HIV-infected men, the mean CD4+ T cell count was 410 cells/μL, 47.9% were on antiretroviral therapy, and 69.8% had a detectable HIV plasma viral load. In addition, the HIV-infected men were significantly more likely to be positive for hepatitis B core antibody (65.6% vs. 33.0%, P < 0.01), HSV type 1 antibody (65.4% vs. 47.9%, P < 0.01), and HSV type 2 antibody (39.1% vs. 16.6%) than HIV-uninfected men (Table 1).
Of the 400 samples, 386 (96.5%) swabs (192 from HIV-infected and 194 from HIV-uninfected persons) had adequate β-globin for HPV genotype testing. Overall, 171 (44.3%) of participants had single or multiple HPVs detected. One hundred forty-three participants (37.0%) had at least one of the nHPV types; 126 patients (32.6%) had at least one of the qHPV types. Among HPV types identified, the most common genotype was HPV11 (15.5% prevalence), followed by HPV16 (13.2%), HPV18 (5.7%), and HPV45 (4.1%).
Prevalence of qHPV and nHPV virus types was higher among HIV-infected men (Table 1). Individual HPV types HPV11 (20.8% vs. 10.3%, P < 0.01), HPV16 (16.7% vs. 9.8%, P = 0.04), HPV18 (9.4% vs. 2.1%, P < 0.01), HPV33 (5.7% vs. 1.0%, P < 0.01), and HPV45 (6.3% vs. 2.1%, P = 0.04) were significantly more prevalent in HIV-infected than in HIV-uninfected men. There was a significant difference in both qHPV types (42.2% vs. 23.2%, P < 0.01) and nHPV types (50.0% vs. 24.2%, P < 0.01) between HIV-infected men and uninfected men.
Prevalence statistics of qHPV and nHPV virus types in different groups by age, alcohol use, and drug use are shown in Table 2. Both qHPV and nHPV virus types were most common in participants' age 22 to 26 years, but there were no statistically significant differences in HPV prevalence across age groups. Increased alcohol consumption was not associated with greater prevalence of qHPV, nor was injectable drug use in either HIV subgroup. Prevalence of qHPV and nHPV virus types also did not vary significantly by employment status or living situation (data not shown).
Prevalence of each HPV virus type according to other concomitant infectious conditions is shown in Table 3. Generally, concomitant infectious diseases (hepatitis A, hepatitis B, HSV types 1 and 2) did not seem strongly associated with qHPV and nHPV type prevalence in either HIV-infected or HIV-uninfected participants. The prevalence of qHPV was not higher in individuals with any of the concomitant infections than it was in the overall study cohort; the same was true for nHPV types.
Table 4 shows the prevalence of anal HPV by self-reported sexual positioning. Notably, there was no significant relationship between sexual positioning and prevalence of qHPV or nHPV types. There were also no significant relationships when testing for relationships between other sexual behaviors (i.e., sexual partners and frequency of condom usage) and HPV prevalence (data not shown).
Among HIV-infected participants, lower CD4+ T cell count (<200 cells/mm3) was associated with a higher prevalence of vaccine-preventable HPV types (Table 5), although this association was not statistically significant (P = 0.22). There was no association between HIV plasma ribonucleic acid (RNA) quantification and prevalence of qHPV or nHPV types.
Anal vaccine type HPV is prevalent in this population of Thai MSM. Although it should be acknowledged that testing was done on a convenience sample of swab specimens from the Bangkok MSM Cohort Study and may not be generalizable to the larger population of MSM in Bangkok, the overall rates and distribution by HIV serostatus are comparable to both Western populations and to findings from a previous study in Bangkok.11,1211,12
Although HIV-infected men were more likely to have serological evidence of natural infection with hepatitis B, in addition to infection with both HSV types 1 and 2, they were no more likely to have prevalent anal HPV infection than the HIV-uninfected men. The prevalence of these coinfections suggests increased sexual activity in this group, but this was not borne out in the detailed risk behavior information collected from this cohort and may indicate previous and not ongoing risk.
A higher prevalence of anal HPV with lower CD4+ T cell counts has previously been reported, and although there was a trend in this study, it did not reach statistical significance.17 Lower CD4+ T cell counts are a marker of progressive immunodeficiency in untreated HIV infection. A higher anal HPV prevalence may therefore be explained by decreased cell-mediated mucosal immunity and its inability to control new, persistent, or recurrent HPV infection. The introduction of combination antiretroviral treatment (CART) in Thailand coincided with the initiation of the current study in 2006. Although the number of CD4+ T cells has been shown to increase incrementally after the initiation of CART, plasma HIV RNA usually declines quickly and becomes undetectable. This temporal difference between these 2 markers in responding to CART may, to some extent, explain the lack of consistency in their reported associations with HPV prevalence.
Similar to findings from the EXPLORE study,18 where HPV prevalence was evenly distributed over a wide age range, there was no statistically significant difference in the age ranges examined, although HPV types were more commonly seen at a younger age in our population. Anal HPV is often rapidly acquired after sexual debut and thereafter becomes undetectable over a period, which in HIV-infected MSM is reported to approximate 3 years for qHPV types.19 The higher HPV prevalence in the 22- to 26-year age group likely reflects continued HPV exposure and infection after sexual debut.
The lack of association between social behaviors (alcohol and drug use), sexual behaviors (condom usage, sexual partners, and sexual positioning), and HPV prevalence is surprising, but may be explained by the biases associated with conducting a selective population study such as this one. We cannot assume that the reported behaviors accurately reflect the lifetime exposure to risk behaviors such as alcohol, drug use, and sexual behavior that could result in acquisition of sexually transmitted infections.
The prevalence of qHPV and nHPV types in both HIV serostatus groups indicates that Thai MSM would benefit from targeted HPV vaccination in a similar way to Western MSM. This would not only prevent anal HPV if given before exposure but also reduce the incidence of sequelae such as warts, dysplasia, and anogenital cancers. Furthermore, there is also evidence that HPV vaccination may protect against HPV types, most commonly HPV16, that are found in the oropharynx and are associated with cancer at this site.20 And finally, because anal HPV infection may be a risk for incident HIV in MSM, vaccination could have the potential to mitigate this risk.21
Human papillomavirus vaccination is one example of an available intervention for MSM. However, a broader concern relates to addressing how access to comprehensive strategies that may include HPV, and multiple other vaccines can be developed and implemented to reduce the overall level of morbidity and mortality in this vulnerable population.
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