The present study aimed to assess the prevalence of STIs among Iranian women and investigate the associations between HPV/non-HPV STIs co-infection and cytological changes. Normal cytology result was observed in more than 90% of the study participants. The overall prevalence of HPV, HR-HPV, and LR-HPV genotypes was 39.1%, 27.5%, and 23.7%, respectively. It has been reported that more than 290 million people worldwide have been infected with HPV (12), and studies have described diverse HPV prevalence among Iranian women. This difference might result from the limited detection of each HPV assay, detectable HPV genotypes, and the studied groups. The total prevalence of HPV among Iranian women attending regular gynecological checkups was 31.1% by nested-PCR. Moreover, restriction fragment length polymorphism revealed that the most prevalent HPV genotypes were HPV-16 and -18, with a prevalence of 7.3% and 2.8%, respectively (13).
Commercially approved HPV kits, the Geno Array Test kit (Hybribio Limited, Hong Kong) and two versions of INNO-LiPA HPV Genotyping Extra (Fujirebio Europe, Belgium), were applied to detect and genotype HPV in 1387 archival liquid-based cytology and genital lesion samples of women without pre-malignant or malignant cervical disorders, some of whom had genital warts. The results showed that the prevalence of HPV was 40.59% and the most prevalent HPV genotypes were HPV-6 (9.95%), -16 (8.58%), -53 (4.61%), 11 (4.11%) and -31 (4.04%) (14). In the mentioned study (14), some samples had genital warts. Therefore, a higher rate of low-risk genotypes was observed.
In the present research, the most prevalent HPV genotype was HPV-53 (6.5%). According to the literature, this genotype was one of the most common genotypes in Iranian women (14-16). Moreover, in the aforementioned study (14), the authors showed that nearly 70% of individuals were infected with a single HPV genotype. In the current investigation, it was found that only about 40% of the cases were infected with a single genotype. The studied groups and the methods used in the two studies were different. However, the decrease in single genotype infections in our study compared to the mentioned study (14) indicates that the prevalence of multiple-HPV genotype infection among Iranian women is increasing, and the HPV infection status of a single genotype is changing to multiple genotype infections.
The most prevalent HR-HPV genotype in the present study was HPV-16 (6.1%), which was consistent with other studies that assessed HR-HPV genotypes. Jamdar et al. using the COBAS HPV test (Roche Molecular Systems, Pleasanton CA) reported that the most common HPV genotype in Iranian women was HPV-16 (3%) (17). Moreover, HPV APTIMA assay (Hologic Inc., USA) for detecting HR-HPV E6/E7 mRNA and real-time RT-PCR for the mRNA genotyping of HPV-16,-18, and -45 among Iranian women with normal cytology results showed that the most prevalent HR-HPV genotype was HPV-16 (2.2%) (18). The second most prevalent HR-HPV genotype in the present study was HPV-31 (5.5%), followed by HPV-52 (4.8%) and HPV-66 (4.6%). This pattern of HR-HPV prevalence was also observed in the research by Sohrabi et al. (HPV-31: 4%, HPV-52: 3.1%, HPV-66: 2.9%) (14). These findings provide valuable data for developing an HPV vaccine according to the prevalent HPV genotypes in our region.
In addition to assessing HPV prevalence, another main purpose of the present study was to evaluate non-HPV STI prevalence among Iranian women. No comprehensive molecular study about this topic has been conducted yet in our region. The rate of STIs is high, and more than one million new cases are daily diagnosed with either chlamydia, gonorrhea, syphilis or trichomonas’s infections (19). It is estimated that over 500 million people are infected with genital HSV-2 (20). We found that the overall prevalence of non-HPV STIs was 68.5%. The most prevalent non-HPV STIs were UP (42.7%), GBS (23.7%), CS (23.6%), UU (9.6%), and MH (7.1%). No infection with NG, TP, and HD was observed. The majority of the previous studies in the other regions demonstrated a lower rate of non-HPV STIs among women. For example, the overall prevalence of STIs in Korean (21), Mexican (22), and Saudi Arabian (23) women was 49.2%, 57.7%, and 27%, respectively. However, the studied populations differed from each other. The number of assessed STI pathogens should be considered one of the main reasons that may affect the total prevalence of STIs in each study. According to our and mentioned studies, the most prevalent non-HPV STI was UP (21, 22) or Ureaplasma species (23), with a prevalence of 42.7% in the present study and in Korea, 39.8% in Mexico, and 13% in Saudi Arabia. The assessed STI pathogens were not similar in our study and the mentioned investigations. As a result, the second and third most prevalent non-HPV STI pathogens varied. Briefly, the second and third prevalent non-HPV STIs in our study were GBS (23.7%) and Candida species (23.6%), while were MH (9.9%) and UU (7.6%) in Korea, Gardnerella vaginalis (25.9%) and CT (1.5%) in Mexico, and Ureaplasma species (21.1%) and MH (4.3%) in Saudi Arabia.
Although several studies showed a significant association between some non-HPV STIs and the presence of HPV, some others revealed no significant association (23-27). For example, Kim et al. used real-time PCR to detect seven STI pathogens and multiplex PCR for HR-HPV, and found that non-HPV STI-infected cases had a 1.47 times higher chance of HPV infection. However, no significant association was found when the STI pathogens were assessed separately (24). Displacement amplification for detecting CT and conventional methods for other pathogens showed a significant association between CT or UU and the presence of HPV. In contrast, HPV infection was not significantly correlated with bacterial vaginosis, GBS, CS, TV, and UU (27). In the present study, the overall prevalence of non-HPV STIs among HPV-positive and -negative cases was 83.2% and 59.2%, respectively (OR=3.14, P<0.001). When the non-HPV STI pathogens were evaluated separately, CT, MG, MH, UU, UP, CS, and GBS had a significant association with the presence of HPV, while TV, HSV-1, and HSV-2 did not have a significant association with HPV presence. It should be noted that the low prevalence of TV, HSV-1, and HSV-2 made it difficult to find statistical significance.
Previous studies found that the co-infection of HPV with some non-HPV STIs could increase the risk of cervical lesions progression. Briefly, de Abreu A. L. et al. found that co-infection of HPV with CT, NG, TV, and HSV-2 could raise the risk of ASC-US cytology. Moreover, it was found that HPV co-infection with CT, NG, and TV could augment the risk of HSIL (28). Magalhaes P. A et al. reported that CT infection did not increase the risk of HPV infection, while the CT/HPV co-infection could elevate the risk of LSIL development (29). It has also been found that HPV/Ureaplasma co-infection could increase the risk of developing low-grade and high-grade cytological findings (30-32). We assessed all variables that could independently affect cytological changes in all participants and found that in addition to HPV infection as the main factor for cervical cell changes, non-HPV STIs, UU, and GBS could influence the cytological changes. To clarify which of these identified non-HPV STIs may have synergistic interaction with HPV on the dysplastic transformation of cervical cells, these factors were evaluated in HPV-positive cases, and it was observed that HPV/GBS co-infection plays a significant role in the development of the cervical lesion. In other words, 50% of individuals with LSIL or worse cytological grades were infected with GBS. However, only 27.4% of individuals with the lower cytological result than LSIL were infected with GBS. This difference was statistically significant (P=0.016) and showed a greater chance of LSIL or worse cytological progression in the individuals infected with HPV and GBS at the same time. It was revealed that HPV/UU and HPV/non-HPV STIs had no significant association with the development of cervical lesions (Table 3). In the past, assessment of GBS as a non-HPV STI was less critical compared to other non-HPV STIs, such as CT, NG, TV, UU, UP, MG, and MH, and almost none of the research performed on the subject of our study have evaluated this bacteria as a non-HPV STI. Recently, a study used Illumina sequencing-based on 16S rRNA to determine cervical microbiota and suggested that HPV/GBS co-infection could increase the risk of cervical intraepithelial lesion progression (33). Our data confirmed this suggestion, and it was found that the co-infection of HPV with GBS could play a significant role in developing LSIL or, worse cytological grades.
Although this study updated the prevalence of HPV genotypes, revealed good information on non-HPV STIs prevalence, and assessed the association of HPV/non-HPV STIs co-infection in the cytological results of Iranian women, it had some limitations. We evaluated individuals who were referred for routine cervical cancer screening, the number of high-grade cytological results was low, and we were not able to assess the co-infection of HPV with non-HPV STIs in distinct cytological grades. Furthermore, the histopathological data of the participants were not available to investigate the association of HPV/non-HPV STI co-infections with histological changes. Further studies on cervical biopsies are highly recommended.
 

In conclusion, the sexually transmitted infections including HPV, UP, GBS, CS, UU, and MH were prevalent among the Iranian women participating in this study, and significant correlation between CT, MG, MH, UU, UP, CS, and GBS, and the presence of HPV was observed. Moreover, HPV/GBS co-infection could play a significant role in developing LSIL or, worse cytological grades, which needs further elucidation. Management of GBS infection among the HPV-positive cases is highly recommended.

 

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The authors declared no conflict of interest.