HPV types HPV2, HPV27, and HPV57 are etiologically associated with more than 65% of common warts (verrucae vulgares), the most frequent HPV-associated benign lesions of the skin. When common warts appear in the anogenital region they can be misdiagnosed as anogenital warts (condylomata acuminata), which are etiologically associated with HPV6 and HPV11. In children the mentioned misdiagnosis could have potentially serious legal consequences, since the appearance of novel wart(s) in a child’s anal or genital region can be considered as an indicator of sexual abuse and can potentially trigger legal action against the parents or household members. Thus, although routine detection of HPV types present in tissue specimens or swabs of common and anogenital warts is not generally recommended, it could be very helpful in some clinical circumstances and/or for legal purposes, especially in children. To the best of our knowledge, no quantitative real-time PCR (RT-PCR) allowing simultaneous amplification and differentiation of HPV2, HPV27, and HPV57 has been developed so far. The present study describes the development and evaluation of the first multiplex type-specific quantitative RT-PCR, enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of HPV types HPV2, HPV27, and HPV57 in a single PCR reaction.
The novel HPV2/27/57 multiplex RT-PCR was designed and optimized on plasmid standards and clinical samples of common warts.
The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children’s warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional low-risk Alpha-PV PCR.
The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.