Narendra Hulikal

E6-E7 based nested multiplex PCR assay for genital HPV detection and simultaneous typing of 15 high and low-risk HPV types

Due to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping. HPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 10 The assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results. The designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.

HPV genotype prevalence in Indian women with cervical disease and estimation of the potential impact of HPV vaccines on prevention of cervical cancer

Human papillomavirus (HPV) causes genital and oropharyngeal cancers worldwide. There are significant gaps exist in the data on HPV genotype prevalence in this part of the country. HPV vaccination is one of the best preventive methods available currently. HPV genotyping plays an important role in the selection of appropriate vaccines and monitoring vaccine efficacy and coverage. The present study aimed to determine the HPV genotype prevalence and to estimate the potential impact of HPV vaccines on invasive cervical cancer. A total of 204 cervical biopsy samples collected from symptomatic women were subjected to an in-house designed and standardised nested multiplex PCR (NM-PCR) assay. The NM-PCR was designed to detect 38 Mucosal HPV types as a pooled result and genotyping of 15 HPV types. Further, the HPV genotype data was used to estimate the HPV vaccine bivalent, quadrivalent and nonavalent impact on the population using a mathematical formula. Out of 204 samples 188 were subjected to HPV-nested PCR. A total of 163 (86.7%) samples were positive for at least one HPV type. Multiple genotypes were identified in 30% of samples processed. HPV-16 (85.3%) was the most frequently detected genotype followed by HPV-18 (13.5%) and HPV-33 (11.0%). Other genotypes were observed less frequently. Based on the HPV prevalence observed in the study a mathematical model estimated the efficacy of bivalent, quadrivalent and nonavalent vaccines were 76.1%, 76.7%, and 91.1% (average) respectively. HPV-16 was the most prevalent (>85%) genotype detected in this study. Multiple infections observed in 30% of samples were quite high as compared to the majority of national, and global reference (15-25.4%) data. The Mathematical model showed that a nonavalent vaccine would give better protection.