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 geno- typic 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 ge- nome 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 oli- gonucleotide probe hybridization. The phylogenetic correlations in coinfections were an- alyzed 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 geno- type (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 geno- typing test, allow accurate diagnosis of monoinfections and coinfections. This may faciliate the development of specific viral tests and prophylactic anti-HPV vaccines.