Prevalence of Human Papillomavirus (HPV) and Other Sexually Transmitted Infections (STIs) among Italian Women Referred for a Colposcopy

Sexually transmitted infections (STIs) represent a major cause of morbidity in women and men worldwide. Human Papillomavirus (HPV) infections are among the most prevalent STIs and persistent infections with high-risk HPV (hrHPV) genotypes can cause cervical dysplasia and invasive cervical cancer. The association of other STIs with HPV cervical infection and/or dysplasia has however not yet been fully elucidated. The aim of this study was to assess the prevalence of HPV and other STIs among women presenting with an abnormal cervical cytology. Cervical infections with 28 HPV genotypes and seven other sexually transmitted pathogens were evaluated in 177 women referred for a colposcopy after an abnormal Pap smear. Positivity for at least one hrHPV genotype was shown in 87% of women; HPV 16 was the most prevalent (25.0%), followed by HPV 31 and HPV 51. The overall positivity for other STIs was 49.2%, with Ureaplasma parvum being the most prevalent microrganism (39.0%). Co-infections between hrHPV and other STIs were demonstrated in 17.5% of women; no significant association was demonstrated between multiple infections and the colposcopy findings. This study provides new epidemiological data on the prevalence of cervical infections associated with HPV and seven other common sexually transmitted pathogens in a population of women presenting with an abnormal cervical cytology.


Introduction
Human papillomaviruses (HPVs) are viruses known to be the primary cause of cervical cancer (CC), responsible of approximately 311,000 women's deaths in 2018 [1]. Even though incidences of CC have decreased in recent decades, a huge burden remains, especially in low-income countries.
Despite HPV infections being widespread among the female population, only a small fraction of women with a hrHPV infection will progress to pre-cancer or cancer. This indicates that additional risk factors are important for carcinogenesis, potentially including viral factors such as persistent infection with specific hrHPV genotypes or the presence of concomitant pathogens able to infect the genital tract.
It is well known that HPV 16 and 18 are the two most common genotypes related to cervical cancer development [4]. In countries where HPV vaccination has been implemented with a high coverage, these HPV types have significantly decreased [5]. However, concerns have been raised that the role vacated by the vaccine-targeted types could be taken over by the non-targeted high-risk HPV types [6,7]. It is therefore important to continue monitoring HPV types circulation to evaluate a possible type replacement.
There is growing evidence on the role of other Sexually Transmitted Infections (STIs) as co-factors for the development of cervical cancer in HPV-positive women [8,9]. Interactions between HPV and other pathogens that share similar mucosal sites could accelerate cancer progression, enhancing HPV replication and infection persistence. For example, recent studies have reported that there is an increased risk of cervical cancer in women co-infected with HPV and Chlamydia trachomatis (Ct) [8,10]. Ct infection seems to determine an increased inflammatory response that can facilitate HPV's entrance into the cervix basal membrane and favor persistent infection [11]. However, the specific role of Ct in the pathogenesis of CC is still controversial. Other microorganisms such as Trichomonas vaginalis (Tv), Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) have also been associated with cervical inflammatory processes, a situation that may facilitate the entrance of HPV [12].
The purpose of this study was to assess the prevalence and distribution of HPV genotypes among Italian women referred for a colposcopy following a recent diagnosis of abnormal cervical cytology. Moreover, the second aim was to investigate in this group of women the presence of other concomitant STIs and to determine their possible correlation with the grade of cervical lesion.

Study Design and Sample Collection
One hundred and seventy-seven women with a documented abnormal Pap smear were consecutively enrolled at the Gynaecology outpatient clinic of San Gerardo Hospital, Monza, Italy, between 2017 and 2019. The study protocol (Protocol: n. 305) was approved by the Ethics Committee of the University of Milano -Bicocca, Italy. A cervical sample was collected from all enrolled subjects for the molecular detection of HPV and STI pathogens. The cytological assessment was performed according to the 2001 Bethesda System [13].
The cervical samples were collected using a L-shaped FloqSwab (Copan, Brescia) and transported to the laboratory following resuspension in 20 mL of ThinPrep ® PreservCyt ® Solution (HOLOGIC™, Marlborough, MA, USA).

DNA Extraction, HPV and STI Detection
The DNA extraction was performed starting from 1 mL of resuspended cervical sample using NucliSENS ® easyMag ® (bioMérieux, Marcy-l'Étoile, France), an automated system for total nucleic acid extraction from clinical samples. All of the samples were processed using the "specific B" protocol, characterized by a higher final elution temperature (about 70 • C) and the use of silica beads diluted 1:2, in accordance with the manufacturer's instructions. The nucleic acid extraction system was fully automated, except for adding the initial sample and subsequently the silica beads (125 µL), 10 min after sample lysis, reducing the potential risk of sample cross-contamination. The nucleic acid extracts were eluted in 100 µL of NucliSENS ® easyMag ® elution buffer (bioMérieux).
The HPV and STI pathogen DNA detection was performed as recommended by the manufacturer using 5 µL of extracted DNA. The data recording and interpretation were done with the Seegene viewer software according to the manufacturer's instructions.

Statistical Analysis
The qualitative and quantitative variables were summarized with absolute and relative (percentage) frequencies and medians (interquartile ranges, IQR), respectively. A chi-squared or Fisher exact test was performed for the qualitative variables, when appropriate. A two-tailed p-value less than 0.05 was considered statistically significant. The statistical software STATA version 15 (StataCorp, College Station, TX, USA) was used for all statistical computations.

Discussion
HPV infection is the necessary but not sufficient cause for cervical cancer. Other cofactors can play a role, influencing HPV persistence and cervical disease progression.
The overall prevalence of both high-risk and low-risk HPVs in our study population of women referred for colposcopy after a recent abnormal Pap test was found to be 74.6%, with 65% (115/177) of them being positive for at least one hrHPV type. The high HPV positivity rates found in this selected population is in keeping with the range of HPV positivity previously reported among the patients with the same characteristics [14][15][16][17]. The prevalence rates are also influenced by the pre-analytical procedure for sample processing and the HPV test used for viral DNA detection [14][15][16][17]. For example, a Norwegian study reported an HPV DNA positivity of 82.7% using the same Anyplex™II HPV28 test [14]. Tewari and colleagues found a hrHPV prevalence of 58% using Cobas 4800 HPV test and 53% using Aptima HPV assay, respectively [17]. In two other studies conducted in our laboratory, using "in-house" real time assays able to detect only seven out of the 12 hrHPV genotypes, we observed an HPV prevalence of 44.2% and 34.3%, respectively [15,16]. HPV 16,31, 51 and 52 were found to be the most prevalent hrHPVs detected. Baasland et al. [14] reported similar results, with the exception of HPV 51, which appears to be more frequent in Italy. Previously published studies have in fact reported a high prevalence of the HPV 51 genotype in Italian women [15,18,19]. The high prevalence of HPV 16 is in keeping with the worldwide spread of this genotype, not only among women but also in the male population [6,9,14,20,21].
The majority of the women with a HPV 16 single infection were found to have a high-grade cervical abnormality at colposcopy examination (68.2%), demonstrating again the principal role of this genotype in precancerous lesion progression.
Overall, positivity for one or more of the seven STI pathogens under study was demonstrated in 49.2% of women, with Ureaplasma parvum found to be the most frequently identified pathogen (69/177, 39.0%), followed by Ureaplasma urealyticum (21/177, 11.9%).
Chlamydia trachomatis was identified in 2.8% of women and was always in co-infection with other STI pathogens. This positivity rate was low compared to the 12.8% of Ct prevalence that our group found in a previous study conducted on women with a recent history of cervical dysplasia [15] and other data reported in the literature regarding the diffusion of this pathogen among sexually active women (ranging from 1.8% to 10.4%) [22][23][24][25][26][27].
Several previously published studies reported a very high percentage of Ureaplasma spp. infection in female genital tracts. A study conducted in Japan showed an Ureaplasma parvum prevalence of 41.7% in women attending their first prenatal visit [23]; another study, carried out in Slovenia, showed that significantly more women aged 25 years and younger were infected with U. urealyticum (23.4%) compared to those aged above 25 years (9.2%), irrespective of symptoms [28]. Other studies conducted in Italy have reported positivity rates for Ureaplasma parvum of almost 25% in vaginal specimens [22] and 38.3% in cervical samples [29].
As a result of the high prevalence of Ureaplasma spp. and Mycoplasma spp. detection in the genital tracts of sexually active individuals and their unclear clinical role in lower urogenital tract infections, a recent position statement from the European STI Guidelines Editorial Board has indicated that the routine testing and treatment of asymptomatic or symptomatic men and women for M. hominis, U. urealyticum and U. parvum are not recommended [30].
However, several recent studies investigating the role of cervical co-infections of Ureaplasma spp. and other STIs in association with hrHPV, have supposed that these could be possible cofactors interacting with HPV in the development of precancerous and cancerous cervical lesions [31][32][33][34][35][36].
Moreover, a recent systematic review and meta-analysis aimed at investigating the association between female genital mycoplasmas and HPV infection, abnormal cervical cytopathology, and cervical cancer, concluded that Ureaplasma urealyticum and Ureaplasma parvum were associated with a significantly increased risk of overall HPV infection (OR 1.57; OR 3.02, respectively). Furthermore, infections with U. urealyticum, U. parvum, and Mycoplasma hominis were also found to be associated with a significantly increased risk of abnormal cervical cytopathology (OR 1.51; OR 1.41; OR 1.48, respectively) [37].
In the present study, infections with multiple hrHPV types were detected in 46.1% (53/115) of the HPV-positive patients; a total of 65 (36.7%) women were also found to be co-infected with HPV and STIs. In these co-infections, Ureaplasma parvum (37/65, 56.9%) was the most prevalent sexually transmitted pathogen, followed by Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis. In spite of the relatively high prevalence of hrHPV and STIs co-infections observed in the population studied, no statistically significant association was found between co-infections and positive colposcopy findings in these women.
Only in depth in vitro studies may confirm whether Ureaplasma spp. and/or other STIs are real cofactors or are just "followers", taking advantage of the immune tolerance and abnormal regulation of the cell cycle control generated by HPV. Moreover, future longitudinal clinical studies in women with an abnormal cervical cytology will allow us to better evaluate the relationship between lower genital tract infections, co-infections, and high-risk human papillomavirus infections.

Conclusions
The results of this study have demonstrated, as expected, a high prevalence of hrHPV infections in women referred for colposcopy, but also high rates of co-infections with Ureaplasma parvum and other STIs. Although a statistically significant association between co-infections between hrHPV and other STIs with positive colposcopy findings could not be demonstrated in the studied population, the high prevalence of STIs found in hrHPV-positive women, who are at higher risk of developing cervical disease, indicate that screening for genital infections in this population may be important. This would allow not only to prevent other STDs and their sequelae, but also reduce any potential influence of concomitant microorganisms on HPV infection. Future longitudinal studies investigating the role of STIs as cofactors in HPV mediated cervical carcinogenesis are warranted.

Conflicts of Interest:
The authors declare no conflict of interest.