Molecular Methods for a Correct Diagnosis of Multiple HPV Infections and Clinical Implications for Vaccine

Introduction: The human papillomavirus (HPV) family is characterized by minimal genotypic differences corresponding to different virus types. The aim of this study was to detect the HPV coinfections and the inner genotype in a series of 336 cervical-vaginal samples. Methods: A total of 336 cervical-vaginal samples were taken from 2007 to 2009 using specific molecular techniques such as molecular sequencing and hybridizations. The genome amplification of the L1 open reading frame was analyzed by real-time polymerase chain reaction; direct sequencing was performed by SYBR green fluorescent molecule and degenerate primers MY09 and MY11. The HPV genotyping was accomplished via oligonucleotide probe hybridization. The phylogenetic correlations in coinfections were analyzed by sequence homology of the L1 genomic region. Identified genotypes were then compared. Results: Human papillomavirus positivity was observed in 125 cases (37.2%), with 21 cases (16.8%) of HPV presence in coinfections. Coinfections involved HPV 16 genotype (8 cases) and HPV 18 (5 cases). The HPV 16 infection was mainly associated with genotypes with a lower-than-broad sequence homology, so the HPV 18 was linked to genotypes represented in the opposite phylogenetic tree. Conclusions: The combined and steady use of diagnostic procedures, such as real-time polymerase chain reaction, molecular hybridization, direct sequencing, and HPV genotyping test, allow accurate diagnosis of monoinfections and coinfections. This may faciliate the development of specific viral tests and prophylactic anti-HPV vaccines.