Expanded HPV Genotyping by Single-Tube Nested-Multiplex PCR May Explain HPV-Related Disease Recurrence

The role of the human papillomavirus (HPV) in the establishment of cervical cancer has driven studies to find more effective methods of viral detection so that early intervention strategies can be performed. However, the methods still have limitations, especially regarding detecting the different genotypes simultaneously. We have developed a high-throughput system using a single-tube nested-multiplex polymerase chain reaction (NMPCR) for the detection of 40 HPV genotypes using capillary electrophoresis. The NMPCR assay was compared to the Hybrid Capture 2 assay (HC2) with 40 women from the Northeast of Brazil (São Luis, MA), a high endemic region, where the HPV positivity was 75% and 37.5%, respectively. These results were validated by performing a molecular epidemiological study on 5223 Brazilian women undergoing gynecological examinations from 2009 to 2017, who presented with an HPV prevalence of 59%. Multiple infections were found in 62.5% and 58% of the patients from the endemic region and from the Brazilian women population, respectively, mostly presenting high-risk genotypes (90.5% and 60%, respectively). Considering cervical intraepithelial neoplasia and adenocarcinomas, the sensitivity and specificity were 97.5% and 100%, respectively. The NMPCR assay was also capable of identifying viral subtypes in cases of multiple infections, even with low viral loads (10^-6 ng/µL of HPV DNA). The NMPCR test is a promising and robust tool for HPV diagnostics and a screening tool for prevention of cervical cancer.