Nested PCR for Detection and Typing of Rare High Risk Human Papilloma Viruses like Genotype 73 Associated with Carcinoma Cervix

Abstract

Introduction: There are 15 high risk genotypes of Human Papilloma Viruses (HPV) which are associated with Carcinoma Cervix (CaCx), of which genotype 16 and 18 are the commonest. Aim: To evaluate nested multiplex Polymerase Chain Reaction (PCR) protocol for simultaneous detection of HPV, and typing of high risk genotypes mainly genotypes 16 and 18 in cervical scrape/biopsy samples. Materials and Methods: This prospective cohort study was done on 63 females in the age group 21-70 years attending outpatient Department of Gynaecology, Sir Sunderlal Hospital, BHU, Uttar Pradesh, India from March 2015 to August 2016. Cervical biopsy (n=14) and cervical scrape (n=49) samples from 63 females were subjected to histopathology/cytology and nested multiplex PCR for detection and typing of HPV. Further, in HPV positive tissue samples, in which virus was not typed as genotype 16 and 18, 142 bp consensus sequence of L1 capsid gene was amplified by nested PCR employing MY/GP+ primers and subjected for sequencing to ascertain the HPV genotypes. Results: On histological examination, 13 (92.8%) out of 14 biopsies were diagnosed as cases of Squamous Cell Carcinoma (SCC). Of the 13 cases of SCC of uterine cervix, all were HPV positive and yielded either 134 bp L1 capsid amplicons (n=12) or HPV 16 specific amplicon (n=1). On subjecting cervical scrape samples for the PCR, 24.3%, 100%, 57.1%, and 66.7 % cases with Negative for Intraepithelial Lesion or Malignancy (NILM), Atypical Squamous Cells of Undetermined Significance (ASCUS), Low Grade Intraepithelial Lesion (LSIL), and High Grade Intraepithelial Lesion (HSIL) respectively were positive for HPV. Conclusion: Detection of High Risk HPVs (HR HPV) in all the tissue samples of SCC of uterine cervix indicates the usefulness of the nested multiplex PCR protocol in detection and typing of HPVs in tissue samples of CaCx. Further, this protocol may be used to detect HPVs in scrape samples along with cervical cytology as a tool for screening CaCx.

Keywords

• capsid gene
• cervical scrape
• cytology
• tissue
• uterus