Journal of Clinical and Diagnostic Research. 2018 May, Vol-12(5): DC20-DC24 20 20 DOI: 10.7860/JCDR/2018/34187.11563 Original Article Nested PCR for Detection and Typing of Rare High Risk Human Papilloma Viruses like Genotype 73 Associated with Carcinoma Cervix Microbiology Section NIDHI GUPTA 1 , PRADYOT PRAKASH 2 , AMRITA GHOSH KAR 3 , NISHA RANI AGRAWAL 4 Keywords: Capsid gene, Cervical scrape, Cytology, Tissue, Uterus 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. INTRODUCTION Carcinoma Cervix is the fourth most common cancer among women with an estimated 5,28,000 new cases every year in the world [1]. Till date, out of more than 170 genotypes of HPVs, there are 40 mucosal genotypes which infect the genital tract and spread through sexual contact [2,3]. Based on their association with cervical and other anogenital cancers, mucosal HPVs are grouped as high risk and low risk genotypes. Low risk HPVs include genotypes 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, CP6108, probable high risk genotypes include 25, 53, 66 and, HR HPVs include genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 [4]. HPV- 16 and -18 together are responsible for about 70% cases of CaCx occurring in every region of the world [5]. Cervical HPV infection is contracted by mucosal contact during sexual intercourse and it is commonly present in young women after the onset of sexual activity. The majority of cervical HPV infections get spontaneously cleared within a few years [6]. However, the longer the duration of cervical HPV infection (persistence), the lesser are the chances that a patient can clear his/her infection. The progression from HPV infection to HPV persistence to the development of high grade lesions and ultimately cervical cancer appears to take, on an average, up to 15 years; making invasive CaCx a preventable disease [7]. Cervical Pap smear test is the routinely used screening method for identification of HSILs {Cervical Intraepithelial Neoplasia (CIN-2 and 3)} and CaCx. Since, its introduction, the Pap smear has helped reduce cervical cancer incidence and mortality rates by roughly half to two thirds [8]. However, the diagnostic confirmation is obtained on histopathological examination of cervical biopsy samples. SCC and adenocarcinoma are the two most common histological subtypes of CaCx and both are related to cervical HPV infection in more than 99% cases [9]. There are FDA approved HPV kits for detection of HR HPV in DNA isolated from cervical samples. The recently introduced kit Aptima HPV assay detects mRNA expressed by HR HPVs, thus making this test more specific than others [10]. In the cluster randomised trial, conducted by Sankaranarayanan R et al., it was concluded that in low resource settings, even a single round of HPV testing may lead to significant reduction in number of advanced cervical cancer and deaths from it [11]. Recently, an in house nested multiplex PCR has been devised to detect mucosal HPVs as a pool and simultaneously tried to type the two most common HPV genotypes-16 and -18, in CaCx [12]. In the study, lower HPV positivity (approximately 70%) in Formalin- Fixed Paraffin-Embedded (FFPE) samples of cervical SCC was attributed to formalin mediated degradation of DNA. In the present study, we have aimed at evaluating this in house nested PCR protocol for simultaneous detection of HPV, and typing of genotypes 16 and 18 in cervical scrape/biopsy samples and to correlate histological/cytological findings of these samples with the PCR findings. MATERIALS AND METHODS This prospective cohort study was done on 63 females in the age group 21-70 years attending outpatient Department of Gynaecology,