Prevalence and anatomical sites of human papillomavirus, Epstein-Barr virus and herpes simplex virus infections in men who have sex with men, Khon Kaen, Thailand

Background Human papillomavirus (HPV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) cause sexually transmitted diseases (STDs) that are frequently found in men who have sex with men (MSM) with human immunodeficiency viral (HIV) infection. Methods This study investigated the prevalence of infection and anatomical site distribution of these viruses in asymptomatic MSM. DNA, extracted from cells collected from the anorectum, oropharynx and urethra of 346 participants, was investigated for the presence of EBV, HPV and HSV using real-time PCR. Demographic data from the participants were analyzed. Results All three viruses were found in all sampled sites. EBV was the commonest virus, being detected in the anorectum (47.7% of participants), oropharynx (50.6%) and urethra (45.6%). HPV and HSV were found in 43.9% and 2.9% of anorectum samples, 13.8% and 3.8% of oropharynx samples and 25.7% and 2% of urethra samples, respectively. HPV infection of the anorectum was significantly associated with age groups 21–30 (odds = 3.043, 95% CI = 1.643–5.638 and P = 0.001) and 46–60 years (odds = 2.679, 95% CI = 1.406–5.101 and P = 0.03). EBV infection of the urethra was significantly correlated with age group 21–30 years (odds = 1.790, 95% CI = 1.010–3.173 and P = 0.046). EBV/HPV co-infection of the anorectum (odds = 3.211, 95% CI = 1.271–8.110, P = 0.014) and urethra (odds = 2.816, 95% CI = 1.024–7.740, P = 0.045) was also associated with this age group. Among HIV-positive MSM, there was a significant association between age-group (odds = 21.000, 95% CI = 1.777–248.103, P = 0.016) in HPV infection of the anorectum. A failure to use condoms was significantly associated with HPV infection of the anorectum (odds = 4.095, 95% CI = 1.404–11.943, P = 0.010) and urethra (odds = 7.187, 95% CI = 1.385–37.306, P = 0.019). Similarly, lack of condom use was significantly associated with EBV infection of the urethra (odds = 7.368, 95% CI = 1.580–34.371, P = 0.011). Conclusion These results indicate that asymptomatic MSM in Northeast Thailand form a potential reservoir for transmission of STDs, and in particular for these viruses. Electronic supplementary material The online version of this article (10.1186/s12879-018-3406-0) contains supplementary material, which is available to authorized users.


Background
Men who have sex with men (MSM) are at higher risk of sexually transmitted diseases (STDs) than other groups. Incidence has been rising due to the practice of various sexual acts including penile-anal, oral-anal, and/or penile-oral contact [1,2]. HPV is the most common sexually transmitted viral infection and its prevalence is increasing [3]. Human papillomavirus (HPV) is a causative agent of genital warts [4] and the main risk factor for anal cancer in MSM [5]. The incidence of anal cancer is highest in HIV-infected MSM and is increasing annually. HPV is also associated with oropharyngeal and penile cancers, but at lower prevalence than anal cancer [6]. Detection of HPV infection in asymptomatic MSM can be used to monitor and follow-up HPV-persistent infection for HPV-related cancer intervention.
Other viruses are also found in MSM. Epstein-Barr virus (EBV) is one of the most common human viruses found in B-cells and epithelial cells of healthy persons [7]. The presence of EBV in sites such as the anus, oropharynx and urethra can be due not only to intimate contact but also to the movement of EBV-infected B-cells. Most people are infected in childhood and do not develop symptoms, or have very minor symptoms such as a mild infectious mononucleosis syndrome [8]. EBV has been frequently found in the non-genital and genital mucosa, ulcers and urethral discharges and associated with various malignancies including Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, breast cancer, gastric cancer, etc. [9]. It seems likely to be a co-factor in HPV-associated cancers such as anal and penile cancers [10,11]. Moreover, EBV infection is associated with HPV integration into the host genome, which is a relevant process in cervical cancer progression [12]. In contrast, EBV is more frequently found than HPV in oropharyngeal cancer [13,14]. Interestingly, the prevalence of EBV in isolated B-cells of MSM is significantly higher than in heterosexual men [15]. MSM appear to be at more risk of EBV infection. Although EBV causes various types of disease, including cancer, its co-prevalence with HPV among asymptomatic MSM at various anatomical sites has been little studied.
Herpes simplex virus (HSV) is one of the commonest sexually transmitted viral infections worldwide. The usual sites of HSV infection are skin and mucosal membranes. Primary infection sites of HSV-1 and HSV-2 are the oropharynx and genital tract, respectively. Infection is often asymptomatic. Even though HSV-2 is predominantly spread via the genital route (in contrast to HSV-1) and its seroprevalence is higher in HIV-positive (> 80%) than HIV-negative MSM [16], HSV-1 is causing an increasing proportion of anogenital herpes worldwide [17,18]. Anogenital HSV-1 is more common in MSM than heterosexual individuals [19]. Interestingly, HSV infection is associated with increased viral load of HIV in infected MSM [20]. Interestingly, co-infection of HSV and HPV16 in patients with head and neck carcinomas (HNSCC) has the worst disease outcome [21]. In addition, HSV-1 infection may modulate the radiation resistance of HPV16-positive HNSCC cells by improving cell survival after irradiation [22]. Therefore, HSV can be a co-factor of HPV-associated carcinogenesis and may be a main reservoir in MSM. We, therefore, investigated HSV in MSM.
To explore the prevalence and anatomical site distribution of HPV, EBV and HSV infecting asymptomatic MSM in Northeast Thailand, real-time polymerase chain reaction (RT-PCR) was used to detect these viruses from 346 participants at anatomical sites including the oropharynx, urethra and anorectum.

Specimen and data collection
In total, 358 asymptomatic MSM were enrolled under a cross-sectional study project title of "Factors associated to Neisseria gonorrhea infection by anatomic distributions among men who have sex with men, and multidrug resistant patterns of Neisseria gonorrhea" at M-Reach STDs clinic in Chatapadung Contracting Medical Unit, and the ARV Clinic, Khon Kaen Hospital, Khon Kaen Province, Thailand from September 2014 to July 2015. Prevalence of Neisseria gonorrhea in urethra was published [23,24], but not in oropharynx and anorectum. Cell samples from anorectum, oropharynx and urethra were collected using sterile Dacron swabs (Puritan, Hardwood Products, Guilford, USA). These swabs were immediately transferred into 2 ml of 10% formalin in normal saline solution and transported to laboratory on ice within 4 h. Three-hundred and forty-six asymptomatic MSM were included whereas 12 MSM were excluded because samples were not collected from all three anatomical sites. Participants provided basic demographic data and information concerning their sexual behavior, including number of sexual partners in the preceding 3 months, condom usage and HIV status. This was done by means of a self-reported questionnaire and data were recorded in an anonymous electronic file. The ethical approval for this study was obtained from Khon Kaen University Ethics Committee in Human Research, No. HE591377.

DNA extraction
Cells from swab samples were pelleted and washed with phosphate buffered saline by centrifugation at 2000 rpm for 5 min. Cells were lysed using lysis buffer (10 mM Tris HCl, 0.1 mM EDTA pH 7.5, 1% SDS and 0.5 M NaCl) supplemented with 50 mg/ml of proteinase K and then incubated at 60°C for 30 min. Protein was precipitated by addition of protein precipitation buffer (5 M potassium acetate, 11.5 ml of glacial acetic acid and 28.5 ml of distill water, pH 5.5), and then removed by centrifugation at 13,500 rpm for 5 min at 4°C. DNA was precipitated with an equal volume of isopropanol and collected by centrifugation at 13,500 rpm for 5 min at 25°C and washed with 70% ethanol. Finally, the DNA pellet was dried at 37°C for 15-30 min and then resuspended in distilled water. The quality of DNA was checked by amplifying the GAPDH gene using specific primers (GAPDH forward: 5′-TCATCAGCAATGCC TCCTGCA-3′ and reverse: TGGGTGGCAGTGAT GGCA-3′ by RT-PCR. Quantity of DNA was assessed using the NanoDrop™ (Thermo Scientific) [25].

Detection of HPV, HSV and EBV infection by RT-PCR
HPV infection was investigated using GP5+/GP6+ primers (forward: 5′-TTTGTTACTGTGGTAGATA CTAC-3′ and reverse: 5′-GAAAAATAAACTGTAAATC ATATTC-3′) by RT-PCR [26] to amplify a 141 bp portion of the L1 viral capsid gene. The reaction mixture had a final volume of 20 μl containing 1× SsoAdvan-cedTM Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA), 0.2 μM of forward primer, 0.2 μM of reverse primer and DNA template. Thermocycling conditions were a denaturation step of 5 min at 95°C followed by 45 cycles of 95°C for 10 s and 42°C for 30 s in an Applied Biosystems 7500 Fast real-time PCR Instrument (Applied Biosystems, Foster City, CA, USA). DNA from SiHa cells (an HPV16-positive cell line) was used as the positive control for HPV DNA detection.

Statistical analysis
Bivariate analysis (for comparisons of proportions) was used to investigate the association between EBV, HPV and/or HSV infection and age, number of sex partners, condom usage and HIV status of participants using SPSS software (SPSS Inc., Chicago, IL, USA). Any P-value < 0.05 was considered statistically significant. Table 1 shows demographic data and sexual behavior characteristics of 346 asymptomatic MSM participants. The age range was 18-60 years, with the mode being the 21-30-year-old group. Most participants had only one sexual partner, or no partners, in the previous 3 months and always used condoms. A total of 234 asymptomatic MSM self-revealed HIV status.

Prevalence of EBV, HPV or HSV infecting MSM and anatomical site distribution
Swab samples were obtained from each anatomical site (anorectum, oropharynx and urethra) of 346 asymptomatic MSM participants. HPV, EBV and HSV infection were detected in all three anatomical sites (Fig. 1). EBV infection was common at all three sites, with approximately 50% of samples returning a positive result from each (Fig. 1). The anorectum was the site where HPV infection was most common (43.9%), followed by the urethra (25.7%) and oropharynx (13.9%). HSV was the least prevalent (Fig. 1).
The prevalence of high-risk HPV types were higher than of low-risk HPV in all anatomical sites (Fig. 2). Double/multiple infections of high and low-risk types were particularly frequent in the anorectum (Fig. 2a), followed by the urethra (Fig. 2c), but a combination of risk types did not occur in the oropharynx (Fig. 2b). The anorectal site seems to be the main reservoir of infection of HPV high-and low-risk types in MSM.
Factors associated with prevalence of HPV, EBV and HSV in the anorectum, oropharynx and urethra among 346 asymptomatic MSM There were often significant differences between age groups in prevalence and anatomical sites, as shown in Table 2. EBV infection in the urethra (but not the anorectum or oropharynx) was significantly associated with the 21-30 years-old group (odds = 1.790, 95% CI = 1.010-3.173 and P = 0.046). HPV infection in the anorectum was significantly associated with the 21-30 years-old group (odds = 3.043, 95% CI = 1.643-5.638 and P = 0.001) and also the 46-60 years-old group (odds = 2.679, 95% CI = 1.406-5.101 and P = 0.03). HSV infection in the oropharynx was mostly found in the 46-60 years-old group. EBV infection in the oropharynx and urethra was significantly higher among HIV-positive MSM than among HIV-negative MSM (odds = 2.125, 95% CI = 1.257-3.594 and P = 0.005 and odds = 2.536, 95% CI = 1.496-4.298 and P = 0.001, respectively). Likewise, HPV infection in the anorectum was significantly associated with HIV-infected MSM (odds = 1.935, 95% CI = 1.150-3.257 and P = 0.013). In contrast, the incidence of HSV did not differ according to HIV status. This result suggested that HIV-infected MSM might act as reservoirs for transmission of EBV and HPV. Table 3

Co-infection with EBV, HPV and/or HSV in three anatomical sites
Co-infection with EBV and HPV was common, especially in the anorectum (17.3%) (Fig. 3). Reflecting its low prevalence generally, co-infection of HSV with either of the other two viruses was uncommon (Fig. 3). All three viruses were found in 0.9% (3/346) of MSM in the anorectum or urethra but not in the oropharynx (Fig. 3). These results demonstrate that co-infection of EBV and HPV common among northeast Thai MSM at all three anatomical sites. The association of demographic factors and co-infection of EBV, HPV and HSV in anorectal, oropharyngeal and urethal sites among asymptomatic MSM Co-infection of HPV with EBV or HSV in all three anatomical sites has not been reported previously. We investigated the association of EBV, HPV and/or HSV co-infection in anorectal, oropharyngeal or urethral sites with demographic factors. Interestingly, co-infection with EBV and HPV at both the anorectum and urethra was significantly associated with the 21-  Table S4).
Co-infections of EBV with HSV, and of HPV with HSV, and of all three viruses together, were not associated with any demographic factors in any anatomical sites (Additional file 2: Tables S2, Additional file 3: Table S3, and Additional file 4: Tables S4). These findings show that co-infection of EBV with HPV was predominately found in the 21-30 years-old group in the anorectum and urethra but not in the oropharynx.

Discussion
In this study, we addressed the prevalence of HPV, EBV and HSV as single and co-infections in different anatomical sites (anorectum, oropharynx and urethra) of 346 MSM in Northeast Thailand. Demographic information was collected from participants, and especially HIV status. Several previous reports exist about the prevalence of EBV in the same anatomical sites of MSM from other countries. EBV infection was found in the anorectum of 29.6% and 32% of HIV-positive German and Swedish MSM, respectively [30,31]. Oropharyngeal shedding of EBV was detected in 49-88.8% and 16-56% of HIV-seropositive and seronegative MSM, respectively [32,33]. In the urethral site, EBV was found in 30.7% of asymptomatic American MSM; notably, EBV prevalence (72.7%) was significantly associated with HIV shedding in semen [34], corresponding to our finding as shown in Table 2. EBV infection of the urethra ranged from 28 to 30.7% among American MSM [34,35]. Meanwhile, EBV infection of the urethra of American and Spanish men was ranged from 0.4-45% [36]. Most of these published articles demonstrated that EBV had the highest prevalence in the oropharynx, in contrast to the anorectum and urethra, corresponding to our results. Many studies have demonstrated a high prevalence of HPV infection in the anorectum, ranging from 34.8 to 65.3% [37][38][39], and was significantly associated with HIV-infected MSM [37,38], concordant with our results. Prevalence of HPV infection were lower in the oropharynx and urethra (9.6-13.0% and 10.2-16.3%, respectively) [37,[40][41][42]. As is the case for the anorectum, high prevalence of HPV infection at the oropharynx and urethra were significantly associated with HIV-positive MSM [41,42].
In Peruvian MSM, the five most common HPV genotypes from the anorectum were 53, 6, 16, 58 and 54 [43]. Similarly, HPV53 was the most frequently found genotype in anorectal samples from HIV-seropositive French MSM [44], but HPV16 was the most frequently found at    [39]. HPV16 was the most common genotype found in the anorectum of Italian MSM [45]. However, HPV18 was the most frequently found genotype in the anorectum and urethra of Northeast Thai MSM. In the urethral site, HPV58 was the most common in Italian MSM. HPV16 and HPV58 were the most common infections of the oropharynx of MSM in The Netherlands and Italy respectively [40,42]. Concordantly, our study has also demonstrated the highest proportion of HPV58 in the oropharynx. Although HPV16 was the most common genotype detected in these three anatomical sites among South African and Dutch MSM [42,46], there appears to be variation by region and nationality. Co-infections with two or more HPV genotypes are significantly more frequent than single infections [43,44], consistent with our findings and particularly in the anorectum. Unsurprisingly, high-risk HPV has been more frequently detected than low-risk genotypes among MSM worldwide [39,44].
Our report is the first to find that EBV infection is present in anorectal, oropharyngeal and urethral sites of northeast Thai MSM at higher prevalence than are HPV and HSV. A previous study found high-risk HPV at higher prevalence (90.8%) in the anorectal canal than low-risk-HPV (73.8%), HSV-1 (7.7%), HSV-2 (16.9%) and EBV (7.7%) among HIV-positive Brazilian men [11]. This agreed with a Swedish study of anal cell samples, which found higher HPV infection (76%) among HIV-infected and uninfected MSM than EBV (18.7%) and HSV (9.4%) [30].
HPV was also the most common virus found in anal swabs (44%), followed by semen (7.1%) and pharyngeal swabs (3.8%) in American MSM [35], consistent with our finding that HPV infection was more frequent in the anorectum than in the urethra and oropharynx.
It is well known that HIV-infected MSM have an increased risk of sexually transmitted infections (STIs) and STDs [52]. In addition, HIV infection not only increases susceptibility to persistent HPV but also increases the risk of acquisition of new HPV infections [53]. Similarity, EBV shedding was significantly associated with persistent HPV infection among HIV-infected MSM in the USA [35]. Our finding demonstrated that the presence of HPV in the anorectum was significantly associated with HIV infection in MSM (Table 2). We also found EBV infection of the oropharynx and urethra to be significantly associated with HIV-positive MSM (Table 2). Previous studies suggested that prevalence of EBV, HPV and HSV infection were associated with HIV-infected MSM but not HIV-uninfected MSM [30].
Different results in prevalence in anatomical sites of three viruses and in the association with any risk factors among various countries may depend on the technique used, site and equipment of sample collection, sample size, sexual behavior, nationality, geography, and particularly questionnaire pattern, etc. (such as a self-report or community based). The strength of our study is that 1) we used swab sample in urethra because this can increase sensitivity of Neisseria gonorrhea detection compared with urine sample [24] whereas many previous studies performed EBV, HPV and HSV detection in MSM semen [35,36]; additionally the cell samples can indicate true infection at each anatomical site instead of movement of EBV infected B cells to each sites; and 2) all participants recruited in our study are collected cell samples from all anatomical sites to compare each participants that can reduce the error of data. However, there was a limitation in our study including self-reporting MSM that may provide invalid answers such as HIV status [54]. This limitation may cause an inaccurate data analysis of demographic information.
Here, we also investigated HIV status among EBV, HPV or HSV-positive MSM. We found that HPV infection of the anorectum increased with age among HIV-infected MSM (Table 3). In addition, failure to use condoms by HIV-positive MSM was significantly associated with HPV infection of the anorectum and urethra as well as with EBV infection of the urethra (Table 3).

Conclusions
EBV and HPV were detected in asymptomatic MSM in Northeast Thailand and were found more frequently