In the present study, we examined 346 MSM, with a high risk for HIV infection and other STI, as other studies have already shown [15, 16], to assess the prevalence of anal cytological abnormalities and their association with anal HPV infection, socio-demographic and behavioral factors. Notably, this is one of the few studies conducted on relatively young MSM (median age 32 years), while most of the previous studies focused on older MSM [17, 18]. Only a recent investigation was conducted on very young HIV-negative MSM, their median age being 22 years . In addition, most of the research data on HPV and anal lesions have been collected in HIV-infected individuals, while this study focused on HIV-uninfected patients. It also represents, to our knowledge, the largest investigation conducted in Italy on this type of population.
This study was conducted on individuals with no visible HPV-related lesions in the anogenital area, which was assessed through the careful inspection of the external genitalia and perianal area. However, since no intra-anal examination was performed, neither through a digital rectal exam nor with high-resolution anoscopy (HRA), it cannot be excluded that a proportion of the individuals enrolled had intra-anal disease.
Our study showed that most of the subjects were infected by HPV in the anal canal, with an overall prevalence of 72.5%, which is a higher value compared to other cohorts of HIV-negative MSM [17, 18]. The HPV testing methods used may account for differences between these studies. In addition, our results may also depend on the characteristics of the participants. In fact, as already mentioned above, they showed a high risk for HIV-1 infection and other STI [15, 16]. HPV 16 was the most prevalent type in the present study, confirming that this genotype is the most frequently detected in the anal canal of both men and women [18, 20–23].
The same samples used to assess anal HPV infection were also employed for liquid-based cytology. A proportion of these samples was not adequate for the cytological interpretation (13.6%), either because of scant cellularity or the massive presence of anucleated squamous cells. Due to the modality of the present study, conducted on STI Unit attendees who are not seen by scheduled appointments, it must be noted that it was not possible to ask the participants to adopt particular precautions prior to the collection of the anal sample, as otherwise indicated by the New York State Department of Health AIDS Institute  and by the Anal Neoplasia Clinic of the UCSF Comprehensive Cancer Center . Therefore, patient behavior in the 24 hours preceding the exam, such as use of lubricants, might have compromised the quality of the specimen and might explain the observed proportion of inadequate samples.
Notably, a valid HPV test result was obtained for all samples, independently of the adequacy for the morphological evaluation, a fact that has also been observed in other studies . This finding suggests that, although some samples lacked a sufficient number of nucleated cells to allow the cytological interpretation, the material was enough to obtain a valid HPV test result. This is probably due to the high sensitivity of PCR-based methods, which are able to detect virtually one copy of DNA.
Among the participants with a valid cytology report, we found abnormal anal cytology (ASC-US+) in 29.8% of the cases. This finding is consistent with results of previous studies conducted on HIV-uninfected MSM that report a prevalence of anal cytological abnormalities between 15 and 30% [26, 27]. Notably, no H-SIL cases were found in the present study. This finding is probably due to the relatively young age of the participants, although a previous study conducted on a cohort of 262 HIV-negative MSM with a mean age of 45 years also failed to evidence any H-SIL cases at baseline . Even though only anal H-SIL is considered the real precursor of anal cancer, it is worth noting that L-SIL may be clinically important. Previous studies have shown that about 40% of HIV-negative homo/bisexual men with L-SIL at baseline progressed to H-SIL within 2-4 years [26, 27]. A diagnosis of ASC-US may also hide SILs, a fact also evidenced in earlier studies [28, 29]. These data underline that all patients with a cytological report of ASC-US or worse need a follow-up or accurate diagnostic investigations, which may include high-resolution anoscopy and biopsy . In fact, it is worth noting that one third of ASC-US and L-SIL reports may be associated with high-grade AIN diagnosed on a biopsy . In addition, a recent study evidenced AIN2+ in 20% of patients with a negative cytology .
Importantly, infection by any HPV type was associated with a 4-fold increased risk of having abnormal anal cytology. MSM with a concurrent infection by LR-HPV and HR-HPV types had an increased prevalence of ASC-US+ compared to HPV-negative patients, but the highest rate of cytological abnormalities was found in patients with an infection by HPV16 and/or 18, which showed the strongest association with abnormal anal cytology (OR=5.62). Notably, previous studies have shown that infections with HPV16 or 18 are significant risk factors for the development of and progression to high-grade AIN (AIN-2, 3) [33, 34].
We found a borderline correlation between abnormal anal cytology and HPV multiple infection. A previous study showed that a multiple infection was present with comparable frequency in men with normal cytology and those with ASC-US/L-SIL . However, other reports have demonstrated an association between multiple HPV types and the presence of anal lesions [29, 36]. Therefore, the possible role of multiple infections in the development of anal dysplasia remains unclear.
No association of abnormal cytology and age was found, a fact already observed in other studies that evidenced a similar prevalence of anal cytological abnormalities in all age groups . We previously showed that HIV-negative MSM with a higher number of lifetime and recent sexual partners and having receptive anal sex were at increased risk of HPV infection . Yet, none of these factors was significantly associated with abnormal anal cytology in the present study. It must be noted that receptive anal intercourse was indicated as a factor associated with anal cytological abnormalities in other studies . However, it has been previously shown that anal HPV infection is not exclusively present in men reporting receptive sex [13, 39, 40], because the anal infection can also be acquired through other sexual practices. Therefore, it is not surprising to find a similar prevalence of abnormal anal cytology among MSM having and not having receptive intercourse. In addition, data collected for the present study referred to sexual behavior in the six months preceding the enrollment, thus it cannot be excluded that MSM not reporting receptive sex at the time of the interview had previously engaged in this practice. Finally, it is possible that our ability to identify statistically significant associations for some of the variables analyzed may have been limited by the small number of patients included in each group after stratification.