Evaluation of a Novel Broad-Spectrum PCR-Multiplex Genotyping Assay for Identification of Cutaneous Wart-Associated Human Papillomavirus Types

Abstract

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha (HPVs 2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 77, 91, and 94), gamma (HPVs 4, 65, 95, 48, 50, 60, and 88), mu (HPVs 1 and 63), and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.Papillomaviruses (PV) constitute a group of viruses associated with benign and malignant proliferative lesions of cutaneous and mucosal epithelia. The taxonomic family of PV includes 16 genera (5). More than 100 human papillomavirus (HPV) types have been fully sequenced. The number of HPV types that are not yet fully characterized is probably much larger. HPV types, distributed over five genera (alpha, beta, gamma, mu, and nu) and 16 species, infect the skin (5). HPV types belonging to species of three genera (alpha, gamma, and mu) have most frequently been detected in hyperkeratotic skin lesions (warts).The alphapapillomavirus (alpha-PV) types infecting the genital mucosa (e.g., HPV16, HPV18, HPV6, and HPV11) are the best understood. HPV16 and HPV18 are the most prevalent types involved in the pathogenesis of anogenital (e.g., cervical) cancer, and HPV types 6 and 11 cause genital warts and laryngeal papillomas. The alpha-PV types belonging to species 2, 4, and 8, which have been detected in cutaneous warts, have been studied less thoroughly. At present the most frequently detected alpha-PV types in cutaneous warts are HPV types 2, 3, 10, 27, and 57 (3, 10, 12-16, 18, 19). Gamma-PV types, including HPV4, HPV60, and HPV65, have also regularly been detected in cutaneous warts (4,10,12,15). Mu-PV types, HPV1 and HPV63, have also been detected in cutaneous warts (7). HPV types included in the beta and nu genera have rarely been detected in cutaneous warts. Only Harwood and coworkers (13) hav...

Infectious Diseases and Infestations of the Vulva

“…Frequently, large proportion of genital warts may spontaneously regress within 1-2 years. Genotype-specifi c immunity may develop and protect against reinfection in the patient [ 43 ]. In immunocompromised patients, these lesions are frequently overgrown, large masses with a prologue history of recurrences [ 44 , 45 ].…”

Evaluation of a Novel Multiplex Human Papillomavirus (HPV) Genotyping Assay for HPV Types in Skin Warts

“…Genotyping is performed with bead-based xMAP suspension array technology, which is able to simultaneously identify 23 wart-associated HPV types from the alpha (HPV2, -3, -7, -10, -27, -28, -29, -40, -43, -57, -77, -91, and -94), gamma (HPV4, -65, -95, -48, -50, -60, and -88), mu (HPV1 and -63), and nu (HPV41) genera (1). The assay design results in very low nonspecific background signals and therefore requires no background subtraction.…”

“…The panel of clinical samples consisted of swabs taken from 100 cutaneous warts derived from 100 individuals (ages, Ͼ4 years) with one or more cutaneous warts who self-presented to their general practitioner (1). The swabs were taken from one wart from each patient by firmly rubbing a prewetted cotton-tipped stick 5 times over the surface of the lesion.…”

“…However, in order to perform HPV genotyping, these methods rely on sequencing of PCR products or cloned PCR products, which is a time-intensive procedure. To overcome the drawbacks as referred to above, a novel high-throughput multiplex broad-spectrum HPV genotyping (MPG) procedure, the HSL-PCR/MPG assay, has been described recently to genotype 23 distinct HPV types, with a type-specific analytical sensitivity of 10 to 10,000 copies per reaction (1).…”

“…At the same time, a similar Luminex-based MPG assay, the multiplex BSwart-PCR/MPG (BSwart) assay for the detection of the genus alpha, mu, and nu HPV types 1,2,3,6,7,10,11,27,28,29,40,41,43,57,63,77,91,94, and 117, had been designed at the German Cancer Research Center. In this paper, the development of the BSwart assay and the cross-validation with the HSL-PCR/MPG assay, using 100 cutaneous wart swabs previously characterized for HPV by the HSL-PCR/ MPG assay, are reported.…”