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.
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‐
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‐
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 this site in Chinese MSM where it was not associated with HIV status. However, HPV6, 18, 31, 39, 45 and 66 were significantly associated with HIV infection in China [
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].
Most studies of HSV infection in MSM have reported only seroprevalence (82.5–90%) [
47,
48]. However, a few studies have reported prevalence of HSV DNA in the anorectum (ranging from 7.0 to 16.9%), oropharynx (0–7.2%) and urethra (2.3–49.5%) among MSM [
11,
34,
49‐
51]. Consistent with previous studies, we found very low prevalence of HSV in all three sites (Fig.
1).
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].
A few studies have reported co-infection of EBV, HPV and/or HSV in asymptomatic MSM. For example, high-risk HPV/HSV-2 co-infections were present in the anorectum of 55% of Brazilian MSM. Corresponding values for other combinations in that study were 27% (high-risk HPV/low-risk HPV/EBV), 9% (high-risk HPV/low-risk HPV/HSV2/EBV) and 9% (high-risk HPV/low-risk HPV/HSV-1/HSV-2/EBV) [
11]. HPV and HSV co-infections were found in 4.3% of oral samples from Finnish males [
50]. We have demonstrated that co-infection of EBV with HPV was the most common in the anorectum (17.3%), oropharynx (6.4%) and urethra (12.4%) and was significantly associated with the 21–30 years-old group (Fig.
3 and Additional file
2: Tables S2, Additional file
3: Table S3, and Additional file
4: Tables S4).
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).