Epidemiological Study of Various HPV Strains in Cervical Fluid Samples in South-Eastern Iran, 2018–2020

1Pathology and Stem Cell Research Center, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran 2Department of Obstetrics and Gynecology, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran 3Immunology of Infectious Diseases Research Center, Research Institute of Basic Medical Sciences, and Department of Laboratory Sciences, Faculty of Paramedicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 4Department of Biostatistics and Epidemiology, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran 5Department of Pathology, Resident NYU Langone Health, Mineloa, NY, USA 6Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran


Introduction
Cervical malignancy ranks the third in prevalence and the fourth in and cause of death among malignancies of women. It has been stated that chronic infection with human papillomavirus (HPV) types is the main cause of cervical dysplasia and then cervical cancer. 1,2 HPV is a virus with several limited genes and proteins that can have long-term, significant and irreversible effects on infected human cells and tissues. 3 Many strains of HPV have been identified, and it has been estimated that 40 of these strains are sexually transmitted. So far, the identified strains associated with a variety of malignancies are 14 types that can cause cervical, oropharyngeal, tonsil, and tongue cancers. 4,5 A high percentage of cervical cancers are caused by HPV16 and HPV18, accounting for nearly 70% of cases. 6,7 HPV infections are categorized as the most common infections worldwide; however, most cases are transient and do not lead to cervical anomaly: approximately 54% and 91% disappear in 1 and 2 years, respectively. 8 HPV infections are very common: approximately 75%-80% of people who are sexually active become infected with the virus during their lifetime. 9,10 The HPV infection prevalence varies in females of different ages, but a considerable number of reports have been linked to the women between the ages of 20-30. 10,11 In many countries, there is a reverse relation between women's age and the rate of HPV spread. Additionally, it has been reported that low-income areas for all age groups were associated with increases in HPV spread. 12 Previous studies have shown that the prevalence of HPV has a two-point peak: the first is observed in women under 30 years and the second is seen in women aged 55-64 years. 13 Actually, the distribution of HPV infections is different across countries and various states. 14 The global report of the spread of HPV in females without cervical disorders Open Access http://www.aimjournal.ir is 10.4%, which is 8% in Asia, and in Iran, the variable has been reported from 0.63% to 36.2%. 15,16 Due to the significant difference in reports in Iran from different provinces and the increasing speed of infection of people via different ways (genital, oral, gastrointestinal tract and skin), there is a need for further research and a more detailed examination of samples related to exposed individuals. In order to more accurately identify HPV transmission methods, it is necessary to identify and control infected individuals associated with this dangerous virus. 17,18 Therefore, this project was designed to determine the prevalence of different HPV strains among Iranian women, who were undergoing routine screening for cervical cancer using INNO-LiPA HPV Genotyping EXTRA II Assay.

Materials and Methods
In this cross-sectional study, all women who referred to Kerman University clinics in order to have Pap liquid test specimens test and who met the inclusion criteria were included in the study from 2018 to 2020. A total of 1079 cases of Pap liquid test specimens were collected and delivered to the Pathology and Stem Cell Research Center in Afzalipour School of Medicine, during the period 2018-2020. Participants were women selected with the age limit of 15 to 60 years. The exclusion criteria were as follows: previous hysterectomy, vaginal bleeding, symptomatic cervical or vaginal infection, a history of cervical cancer, pregnancy, radiation therapy, and using immunosuppressive drugs. 19,20 All the women provided written informed consent at the beginning of the research. The cytological criteria included: 1) the presence of koilocytes with raisin type of nuclei, 2) the presence of dysplastic nuclei (LSIL to HSIL), 3) the presence of parakeratosis and 4) inflammatory process.

DNA Extraction and PCR Condition
HPV-DNA extraction, evaluating and genotyping were performed by experts. Women's specimens were prepared by a gynecologist from the cervix with the Cytobrush, and fixed in liquid base fixative. For DNA derivation, DNA purification and amplification MN kits (MACHEREY-NAGEL GmbH, Germany) were used according to the manufacturer's directions and the concentration of extract. DNA concentration was assessed in NanoDrop 2000c Spectrophotometer (Isogen Life Science, Veldzigt, the Netherlands). HPV-DNA polymerase chain reaction (PCR) was performed using 5′-CGTCCMAARGGAWACTGATC-3′ as forward primer and 5′-GCMCAGGGWCATAAYAATGG-3′ as reverse primer. The PCR duplication steps included denaturation stage for 5 minutes at 95 o C, 45 cycles at 90 o C for 30 seconds, 45 seconds at 56 o C for, 45 seconds at 72 o C and an ultimate step at 72⁰C temperature for 5 minutes. In this project, β-globin were used as the control to validate the PCR condition. Negative (distilled water) and positive controls were used during the PCR test for every 20 samples to exclude possible contamination and to validate the accuracy. For further validation, the PCR products were run on the 2% agarose gel and visualized by ethidium bromide. Additionally, the positive samples were selected for more analysis. 16,21 Human Papilloma Virus Screening The HPV-DNA genotyping was performed using the INNO-LiPA HPV Genotyping EXTRA II Assay kit from Fujirebio Europe N.V. Company (Belgium). The kit principle was reverse hybridization. In summary, a fragment of the L1 region called SPF10 of the HPV DNA was protracted by special primers, afterward the biotinylated amplimers were denatured first and then hybridized with particular probes. In addition, to check the quality of the extracted DNA, the human HLA-DPB1 gene specific primers were added. Subsequently, streptavidin-conjugated alkaline phosphatase was used and then incubated with BCIP/NBT chromogen, to be confirmed by vision. 16

Statistical Analysis
Statistical analysis was done using SPSS version 20 software. The relation between categorical variables was evaluated with chi-square and Fisher exact tests. P < 0.05 was considered to be statistically significant.
Menopause status was not significantly related to HPV infection (P = 0.859). Genotype had a significant relation with HPV infection (P < 0.001). Genotype 1 was the most related category among five genotypes. From the cytological point of view in this study, positive predictive correlation between cytological abnormalities (ASCUS, LSIL, HSIL, SCC) and HPV molecular analysis was 87%-93%.

Discussion
Different types of HPV are responsible for 70%, 90%, 60% and 70% of vaginal/vulvar, anal, penile and oropharyngeal cancers, respectively. 2,22 The growth rate of cervical cancer  in developing countries appears to be gradually increasing due to the lack of effective screening programs. 16 Given that high-risk HPVs appear to be the leading cause of these cancers, rapid diagnosis and treatment of this infection can prevent the progression of the lesions to cancer. The importance of diagnosing this infection was highlighted in routine screening programs and clinical practice. 5 Global research has shown that HPV-16 and HPV-18 are the most associated single and multi-type infections in cervical cancer. In studies performed on pap smears in Iran, HPV-16 and 18 were identified as the main underlying genotypes. 6,8,16 Based on a global study on female uterine cervical screening programs that showed an HPV prevalence of 11.7%, 22 the prevalence rate of HPV in the present study was (37.7%) with rates of 47%, 19% and 34 % of subjects respectively placed in the HR, PHR and LR groups. Jamdar et al reported that the prevalence of HR-HPV infection was 10.3% 23 in Tehran for cervical cancer routine screening. Sjoeborg et al mentioned that development of the cancerous area was significantly different in HPV genotypes, with the highest pertaining to genotype HPV-16 and then to HPV-33. 24 HPV distribution varies in diverse areas and people, even in different sections of single provinces. According to the Iran National Cancer Registry Center, cervical cancer is the 12th most common malignancy among Iranian women. 15 In Iran, a study by Shafaghi et al aimed to determine the genotypes of HPV in cervical samples in the Tehran province: HPV was detected in 265 of 851 patients (31.1%) and the HPV genotypes detected in the infected samples included HPV-16 (7.3%) and HPV-18 (2.8%). 25 In a study by the Isfahan University of Medical Sciences on pap smear of women aged 18-60 years, HPV was diagnosed in 46 samples; 7 HPV-16 (15%) samples were found. 33 While HPV-16 is the most common genotype in many countries, HPV-39 in the Northern region of Portugal and HPV-52 and 58 in Shanghai province are predominant genotypes. 16,26,27 In China, a study by Wang et al to determine the genotypes of HPV revealed that the total HPV positive rate was 14.1%. 28 The investigation reported that the most prevalent genotypes were HPV-16, HPV-52, HPV-58, HPV-53 and HPV-31 with rates of 3.8, 2.5, 1.8, 1.4 and 0.8%, respectively. 28 The HR-HPV infection has been reported in studies from neighboring Middle-Eastern countries such as Qatar (5.8%), Saudi Arabia (6.9%), Kuwait (2.4%), Pakistan (2.8%), and Bahrain (9.8%). 29,30 However, we found that the positive rate for HPV was 37.7% and the most prevalent genotypes were HPV-16 (10.9%), HPV-52 (4.8%), HPV-53 (9.6%) and HPV-31 (2.5%), Single genotype infection was (38.5%) and HR-HPV infection (47%). In comparison to our results, a study by Bruni and colleagues demonstrated that the five most prevalent genotypes among a Spanish population were HPV genotypes 16, 18, 52, 31 and 58, 15 compared to this research, which showed HPV-16 (10.9%), HPV-18 (1.2%), HPV-52 (4.8%), HPV-31 (2.5%), and HPV-58 (1.8%). Various studies have been mentioned, 15,29,30 that are not consistent with our results which may be due to the conditions of the person's sexual partner, the difference in sample size, regional vaccination programs, test methods, etc.
In the present survey, HPV infection peaked at the age of 32 years. There was an elevated occurrence which is probably due to the following: the deficiency of immunity defense versus HPV, absence of an effective vaccination plan at an early age, dangerous unprotected and risky marital sexual behaviors by husband and/or wife, the increasing age of marriage, more coming of women in public swimming pools, increasing cohabitation, having multiple sexual partners, and performing multiple types of waxing and skin abrasions in illegal and unsanitary beauty salons. 16,[31][32][33] The decrease in the single and multiple HPV infection percentages in middle-aged and older groups, more likely resulting from host immune reaction. 1,33 The limitations of this study should be noted. The study could not be representativeness of the target population because only the southeastern region was included. Moreover, adjustment of confounders was not done because of the lack of information (like the number of sexual partners, water-pipe smoking, cigarette smoking, etc). Therefore, larger and prospective studies are required to validate our results.
In conclusion, there is a high spread of HPV in Iranian females and like other parts of the world, it might be one of the most important reasons for various cellular changes in the uterine cervical lesion screening programs (ASCUS, AGUS, CIN1, CIN2, CIN3, carcinoma). Therefore, the use of new and different diagnostic methods has the ability to prevent and control early changes in proliferative disorders. Also, by identifying common HPV genotypes in each region of Iran, a suitable vaccine could be developed to control and prevent this type of cellular change.