Article Information

Mahmuda Naznin^{*, 1}, Md. Zahid Hossain², Mousomi Sarker³, Md. Shameem Yazdany⁴, Md. Anower Hussain Mian⁵, Farzana Ashrafi Neela⁶

¹Assistant professor, Department of Microbiology, Rajshahi Medical College, Rajshahi, Bangladesh

²Pathologist, Department of Pathology, Rajshahi Medical College, Rajshahi, Bangladesh

³Junior Consultant, Department of Gyneacology, Bagha Upazila Health Complex, Rajshahi, Bangladesh

⁴Brigadier General & Deputy DGMS, Ministry of Defence, Dhaka Cantonment, Dhaka, Bangladesh

⁵Professor & Dean, Faculty of Public Health, Bangladesh University of Health Sciences, Dhaka, Bangladesh

⁶Professor, Department of Botany, University of Rajshahi, Rajshahi, Bangladesh.

*Corresponding author: Mahmuda Naznin, Assistant professor, Department of Microbiology, Rajshahi Medical College, Rajshahi, Bangladesh.

Received: 07 November 2024; Accepted: 12 November 2024; Published: 16 December 2024

Citation: Mahmuda Naznin, Md. Zahid Hossain, Mousomi Sarker, Md. Shameem Yazdany, Md. Anower Hussain Mian, Farzana Ashrafi Neela. Prevalence and Molecular Characterization of High- Risk Human Papillomavirus among Patients with Cervical Intraepithelial Neoplasia and Carcinoma. Fortune Journal of Health Sciences. 7 (2024): 710-714.

Abstract

Keywords

Prevalence, High-risk human papillomavirus, HPV, Cervical carcinoma, CIN-III, Cancer

Article Details

Introduction

Cervical cancer stands as the second most prevalent malignancy, contributing to the highest number of cancer-related deaths in women globally [1]. Developing countries bear a substantial burden, accounting for an estimated 86% of the total global incidence of the disease [2,3]. An additional study [4] reported that on a global scale, cervical cancer ranks as the third most frequent cancer and the fourth leading cause of cancer-related mortality in women. This results in approximately 530,000 new cases and nearly 275,000 deaths annually. The World Health Organization (WHO) highlighted invasive cervical cancer as the second most common cancer among women in Sri Lanka and estimated 1,395 new cases and nearly 814 deaths each year [5].

Persistent infection with high-risk human papillomavirus (HPV) is widely recognized as a significant factor in the development of cervical cancer [6-8]. The presence of HPV is pervasive in cervical cancer specimens, representing the leading causative agent for this major human cancer globally [7]. The prevalence of HPV in women with cervical cancer is reported to be as high as 99.7% worldwide [7] and approximately 90% in Asia [8]. Notably, HPV-16 and HPV-18 are the most commonly identified HPV types, responsible for about 70% of cervical cancer cases globally [9]. Utilizing cytological screening such as the Pap smear test and the early detection of HPV are crucial elements in the secondary prevention of cervical cancer, contributing to a reduction in HPV-associated mortality [6]. In lower and middle-income countries, including Sri Lanka, the absence of effective screening programs poses challenges in detecting cervical abnormalities, contributing to elevated mortality rates from cervical cancer in these regions [10]. However, recent advancements in molecular biological techniques, such as HPV-DNA testing, offer effective screening methods for HPV and have the potential to enhance the early detection of cervical cancer in developing areas [11].

Several types of HPV therapeutic vaccines are under evaluation in both preclinical and clinical trials, encompassing live vector, protein or peptide, nucleic acid, and cell-based vaccines [12]. Currently, three HPV vaccines are included in vaccination programs in various countries: Cervarix (bivalent vaccine targeting HPV 16, 18), Gardasil (quadrivalent vaccine covering HPV 6, 11, 16, 18), and the nine-valent vaccine (addressing HPV 6, 11, 16, 18, 31, 33, 45, 52, 58). These vaccines are designed to target between 2 and 7 oncogenic HPV serotypes [13]. Over the long term (30-50 years), they hold the potential to reduce the incidence of diseases associated with the targeted HPV vaccine types [14].

A recent systematic review and meta-analysis affirmed the safety, tolerability, and efficacy of these vaccines against the vaccine-HPV types responsible for persistent infection and cervical disease in young women [15]. In Bangladesh, study on CIN and cervical carcinoma are very limited. Most of the study  restricted  mainly in Dhaka. Prevalence of CIN and cervical carcinoma in Rajshahi region was assessed in our previous study [16]. Molecular characterization is essential for further advancement in implementation of vaccination programme. So, the objective of this study was to investigate the prevalence and molecular characterization of high-risk HPV among patients with cervical intraepithelial neoplasia and carcinoma in a tertiary care hospital at Rajshahi, Bangladesh.

Methodology

This cross-sectional descriptive study was carried out at the Department of Pathology, Rajshahi Medical College, and the Department of Gynecology & Obstetrics, Rajshahi Medical College Hospital, Rajshahi, Bangladesh, spanning from July 2019 to June 2022. A total of 310 cases, identified as positive through the visual inspection with acetic acid (VIA) test, followed by histopathological examination were purposively enrolled as study subjects which were reported in our previous study [16]. The focus of the study was on CIN and carcinoma identified through histopathological assessment.

Cervical swab sample collection:

Cervical swab samples were collected by using commercially available digene HC2 DNA collection device and Kept at -20°C until HPV DNA detection were performed according to manufacturer`s instructions.

DNA extraction:

Extraction of DNA from cervical swab was performed by using QIA amp®DNA mini kit (QIAGEN Germany) according to the manufacturer's instructions. For further analysis the DNA samples were stored in AE buffer at -20°C.

Detection of HPV DNA Typing:

Geno typing of HPV DNA was done in real time PCR test by using QlAscreen HPV PCR test kit (QIAGEN, Germany) which targets a conserved region within the E7 gene. This test kit separately detects HPV-16, HPV-18 and the 13 other high-risk types as a pool. Subsequently, the DNA samples underwent analysis using MS Office tools.

Results

In this study, out of the total 310 participants in Figure-1, 17% had histopathologically diagnosed cases of cervical carcinoma. Additionally, patients with CIN-I, CIN-II, and CIN-III constituted 53%, 7%, and 23% of the total cases, respectively. Figure-2 showed among the total participants, HPV DNA was positive in 77% (n=240) of cases. In Figure-3 among cervical carcinoma patients, HPV DNA was detected in 98.1% (n=52) of cases. Conversely, among patients with CIN-I, CIN-II, and CIN-III, HPV DNA was present in 66.5% (n=109), 77.3% (n=17), and 87.3% (n=62) of cases, respectively. Figure-4(A) showed in HPV-positive carcinoma cases (n=52), HPV-16 was present in the highest proportion (32.7%) as both a single variant and in combination with HPV-18, found in 17.3% of cases. In Figure-4(B) we saw the highest proportion of HPV-positive CIN-I cases (n=109), other than HPV-16/18 was found as a single variant (39.4%), and as multiple variants, HPV-18 and other than HPV-16/18 (6.4%) were observed. In the highest proportion of HPV-positive CIN-II cases (n=17), HPV-16 was observed as a single variant (35.3%), and as multiple variants, HPV-16 & 18 (17.6%) were found in Figure-4(C) Similarly, in the highest proportion of HPV-positive CIN-III cases (n=62), HPV-16 was observed as a single variant (37.1%), and as multiple variants, HPV-16 & 18 (12.9%) were found in Figure-4(D).

Figure 1: Distribution of histopathologically diagnosed cases of cervical intraepithelial neoplasia and carcinoma (N=310)

Figure 2: Frequency of HPV DNA

Figure 3: Distribution of HPV DNA (N=310)

Figure 4: Characterization of high-risk HPV among HPV positive carcinoma n=52 (A), CIN-I n=109 (B), CIN-II n=17 (C) and CIN-III n=62 (D)

Discussion

In this study, among a total of 310 participants, 17% were diagnosed with cervical carcinoma. The prevalence of HPV DNA was found to be positive in 77% (n=240) of total cases. A similar study [17] reported an HPV prevalence of 84.7% (83/98). Another study [18] found a prevalence of 93%, this rate was higher than our findings. It is speculated that the previous study [18] utilized archival cervical tissue samples and was retrospective, which may explain the variation in results compared to our study. Specifically, among cervical carcinoma patients (n=53) in our study, HPV DNA was detected in 98.1% of cases. Among patients with CIN-I, CIN-II, and CIN-III, HPV DNA was present in 66.5%, 77.3%, and 87.3% of cases, respectively. In HPV-positive carcinoma cases, HPV-16 was present in 32.1% as a single variant, and a combination with HPV-18 was found in 17% of cases. In HPV-positive CIN-I cases, variants other than HPV-16/18 were found in 39.4% as a single variant, and as multiple variants, HPV-18 and other than HPV-16/18 (6.4%) were observed. For HPV-positive CIN-II cases, HPV-16 was detected in 35.3% as a single variant, and as multiple variants, HPV-16 and 18 (17.6%) were found. Similarly, in HPV-positive CIN-III cases, HPV-16 was observed in 37.1% as a single variant, and as multiple variants, HPV-16 & 18 (12.9%) were detected. A study [19] conducted in China reported that HPV-16 and HPV-33 have a stronger positive correlation with cervical lesion development than other variants, which was nearly similar to our findings. However, for limitation of our DNA detection kit design, we did not specify an association with HPV-33. Some other studies [20,21] have also reported that HPV16, 58, 52, and 33 are the most common types among women with persistent HR-HPV infections in China. Similarly, in a Bangladeshi study [22], HPV 16 was the most commonly identified in 28 (53.84%) cases, and HPV 18 was the second most common with 20 (38.46%) cases, either singly or in combination with other high-risk subtypes. A prospective study in India reported that HR HPV 16, 18, 31, 33, 35, 45, 58, and 86 were observed among CIN cases and cervical cancer cases [23]. A meta-analysis from the World Cancer Research Center indicated that HPV 16, 31, 51, and 53 were the most prevalent HPV genotypes [24,25]. Differences between our findings and the findings of these studies may be due to variations in diagnostic procedures and geographic locations. The high prevalence of HPV-16 and HPV-18 among our participants was also supported by additional studies [26,27] conducted in Bangladesh.

Conclusion

In the Rajshahi region of Bangladesh, a significant prevalence of high-risk human papillomavirus (HPV) is identified in CIN and carcinoma cases. Specifically, HPV-16 and HPV-18 emerge as the most prevalent viral causes of single or multiple HPV infections in the region. These findings underscore the critical role of HPV in cervical cancer development and highlight the importance of targeted interventions, including HPV vaccination and enhanced screening strategies, to mitigate the burden of cervical carcinoma in this particular geographical context. Comprehensive efforts focusing on the prevention and early detection of HPV infections are crucial for improving women's health outcomes in the Rajshahi region of Bangladesh.

Ethical approval and concent

Institutional review board (IRB) clearance was granted & the Ethical clearance Committee of Institute of Biological science (IBSC) Rajshahi University also approved all protocols. Written consent was obtained from all participants before sample collection.

Funding:

This research did not receive any specific grants from funding agencies in the public, commercial or not for profit sectors.

Conflict of interest:

The authors declare that they have no conflicts of interest.

Limitation:

For DNA detection we used commercial kits those gave specific curve for type 16 and type 18 but for other high-risk types, it gave single curve as a pool. So we could not identify other high risk types of HPV separately.

References

1. DM Parkin, F Bray, J Ferlay, and P Pisani. “Global cancer statistics, 2002,” Ca-A Cancer Journal for Clinicians 55 (2005): 74–108.
2. Parkin DM, Bray F. Chapter 2: the burden of HPV-related cancers. Vaccine 24 (2006): S3/11–25.
3. Parkin DM, Ferlay J, Hamdi-Cherif M, et al. Cancer in Africa: Epidemiology and Prevention. 4.3 Cervix Cancer. Lyon (2003).
4. Arbyn M, Castellsagué X, de Sanjosé S, Bruni L, Saraiya M, Bray F, Ferlay J: Worldwide burden of cervical cancer in 2008. Ann Oncol 22 (2011): 2675–2686.
5. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre): Human papillomavirus and related diseases report in Sri Lanka (2014).
6. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre): Human papillomavirus and related diseases report in world (2014).
7. Walboomers JMM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJF, Peto J, Meijer CJLMM, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189 (1999): 12–19.
8. Bao YP, Li N, Smith JS, Qiao YL, ACCPAB members: Human papillomavirus type distribution in women from Asia: a meta-analysis. Int J Gynecol Cancer 18 (2008): 71–79.
9. Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 384 (2003): 518–527.
10. World Health Organization. Wkly Epidemiol Rec 84 (2009): 117–132.
11. Qiao Y, Sellors JW, Eder PS, Bao Y, Lim JM, Zhao F, et al. A new HPV-DNA test for cervical-cancer screening in developing regions: a cross-sectional study of clinical accuracy in rural China. Lancet Oncol 9 (2008): 929–936.
12. Yang A, Jeang J, Cheng K, Cheng T, Yang B, Wu TC, et al. Current state in the development of candidate therapeutic HPV vaccines. Expert Rev Vaccines 15 (2016): 989-1007.
13. Laurent JS, Luckett R, Feldman S. HPV vaccination and the effects on rates of HPV-related cancers. Curr Probl Cancer 42 (2018): 493-506.
14. Dillner J, Arbyn M, Unger E, Dillner LJC, Immunology E. Monitoring of human papillomavirus vaccination. Clin Exp Immunol 163 (2011): 17-25.
15. Lu B, Kumar A, Castellsague X, Giuliano AR: Efficacy and safety of prophylactic vaccines against cervical HPV infection and diseases among women: a systematic review & meta-analysis. BMC Infect Dis 11 (2011): 13.
16. Naznin M, Hossain MZ, Saha S, Neela FA. Prevalence of cervical intraepithelial neoplasia and cervical carcinoma in a tertiary care hospital at Rajshahi. International Journal of Research in Medical Sciences 12 (2024): 16-31.
17. Karunaratne K, Ihalagama H, Rohitha S. et al. Human papillomavirus prevalence and type-distribution in women with cervical lesions: a cross-sectional study in Sri Lanka. BMC Cancer 14 (2014): 116.
18. Samarawickrema NA, Tabrizi SN, Hewavisenthi J, Leong T, Garland SM: Distribution of human papillomavirus genotypes in archival cervical tissue from women with cervical cancer in urban Sri Lanka. Int J Gynaecol Obstet 115 (2011): 180–182.
19. Li-li Zheng, Li-yuan Zheng, Chao Chen, Yi-ting Wang, Shuang-feng Chen, Qian-qian Zhong, et al., High-risk human papillomavirus distribution in different cytological classification women, Microbes and Infection 25 (2023): 105214.
20. Xiao S, Fan J, He S, et al. Analysis of human papillomavirus infection in 16,320 patients from gynecologic clinic in Central South China. Zhong Nan Da Xue Xue Bao Yi Xue Ban 40 (2015): 373–379.
21. Sun B, He J, Chen X, et al. Prevalence and genotype distribution of human papillomavirus infection in Harbin, Northeast China. Arch Virol 159 (2014): 1027–1032.
22. Shahida S, Chowdhury M, Shajahan F, Rifat J, Lubaba A, et al. Distribution of High-Risk Human Papillomavirus Genotypes among Women with Colposcopic Diagnosis of Cervical Intraepithelial Neoplasia in Bangladesh. Journal of Cancer Therapy 14 (2023): 277-290.
23. Srivastava S, Shahi UP, Dibya A, Gupta S and Roy JK. Distribution of HPV Genotypes and Involvement of Risk Factors in Cervical Lesions and Invasive Cervical Cancer: A Study in an Indian Population. International Journal of Molecular and Cellular Medicine 3 (2014): 61-73.
24. Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, et al. Worldwide Distribution of Human Papillomavirus Types in Cytologically Normal Women in the International Agency for Research on Cancer Hpv Prevalence Surveys: A Pooled Analysis. Lancet 366 (2005): 991-998.
25. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G and Pimenta JM. Human Papillomavirus Genotype Distribution in Low-Grade Cervical Lesions: Comparison by Geographic Region and with Cervical Cancer. Cancer Epidemiology, Biomarkers & Prevention 14 (2005): 1157-1164.
26. Jahan M, Islam T, Sultana S, Pervin M, Ashrafunnessa P, Tabassum S. Distribution of High-Risk Human Papilloma Virus Genotypes Among Cervical Cancer Patients in a Tertiary Level Hospital in Bangladesh. Bangladesh Medical Research Council Bulletin 45 (2019): 86-92.
27. Shahida S, Ansary NP, Begum A, Islam MA and Rifat ZA. Prevalence of High-Risk Human Papillomavirus (Type-16 and 18) in High-Grade Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer in a Tertiary Hospital of Bangladesh. Journal of Bangladesh College of Physicians and Surgeons 36 (2018): 112-117.