Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Tracy L Meiring,Hans J Maree,Jennifer Moodley,Ed P Rybicki,Anna-Lise Williamson,Michael-John Freeborough,Beatrix Coetzee,Anna T Salimo,Inga I Hitzeroth

doi:10.1186/1743-422x-9-164

Abstract

BackgroundHuman papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.MethodsTotal DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.ResultsIllumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively.ConclusionsOur results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Highlights

Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts
Our results indicate that there are HPV types, with substantial prevalence, in Human immunodeficiency virus (HIV) positive women not being detected in molecular epidemiology studies using commercial kits
Total DNA was extracted from specimen HH015 and, prior to sequencing, the HPV circular DNA present was enriched using a randomly primed rolling circle amplification (RCA) method

Summary

Introduction

Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. In South Africa the incidence of HPV infection is high [2] resulting in cervical cancer being the leading cause of cancer-related death in women [1]. It has been conclusively established that infection with specific high-risk human papillomaviruses (HPV) is causally linked to the development of cervical cancer [3]. The largest worldwide HPV genotype distribution study carried out to date showed that the highest proportion of multiple HPV infections and infection with undetermined HPV types and species occurs in Africa [7]. There is limited information of this kind available for South Africa

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