Molecular Evidence of the High-risk Human Papillomavirus 56 Genotype in Cervical Abnormalities using Multiplex Nested PCR Assay: A Cross-sectional Study from a Tertiary Care Center in Puducherry, India

Introduction: Human Papillomavirus (HPV) is a group of Deoxyribonucleic Acid (DNA) viruses linked to both benign and malignant diseases. High-Risk (HR) HPV genotypes, especially HPV 16 and HPV 18, are key contributors to cervical cancer. Aim: To determine the prevalence of HR HPV genotypes, including HPV 16, 18, 31, 45, 51 and 56, in cervical abnormalities in women by comparing cytological Pap smear results with inhouse Multiplex Nested Polymerase Chain Reaction (PCR) to refine diagnostic protocols. Materials and Methods: A cross-sectional study was conducted in the Department of Obstetrics and Gynaecology, tertiary care hospital and the Mahatma Gandhi Medical Advanced Research Institute, Puducherry, India over a period of six months, from November 2023 to May 2024. The study included 100 symptomatic women presenting with complaints such as vaginal discharge, abnormal bleeding and pelvic pain. OBG endocervical samples were cross checked against cytological pap smear and multiplex nested PCR. The PCR amplified products were further confirmed by bidirectional Sanger sequencing. The sequenced data were analysed and annotated using MEGA software version 10.0 and submitted to the National Centre for Biotechnology Information (NCBI) database. Results: A total of 100 samples were analysed, with 14 (14%) detected as HPV DNA in symptomatic women. HPV 16 was observed in four patients (28.57%), followed by HPV 56 in four patients (28.57%). HPV 18 and HPV 31 were detected in two patients each (14.29%), while HPV 45 and HPV 51 were observed in one patient each (7.14%). Symptoms such as abnormal vaginal bleeding, pelvic pain and vaginal discharge were predominant with specific HPV genotypes. The detection limit of Multiplex Nested PCR was higher than that of the Pap smear. Co-infections with organisms like Candida albicans and Trichomonas vaginalis were noted in HPV-negative cases. The sequenced samples were published with GenBank accession numbers PQ518860 – PQ518863. Conclusion: While nested PCR exhibited higher sensitivity compared to the Pap smear, this study concludes that routine HPV genotyping and cytology screening for HR HPV strains are essential to improve early diagnosis and prompt treatment outcomes in HR groups.