The involvement of HPV in development of mucosal disorders has been widely established. Specific cutaneous disorders have also been associated with HPV, however the exact role of HPV remains largely unknown. The lack of optimization and standardisation of the pre-analytical phase forms a major obstacle. The aim of this study was to develop an accurate/patient friendly sampling method for skin disorders, with cutaneous warts as a case study. An additional aim was to create an HPV qPCR genotyping assay capable of detecting the most prevalent wart-associated HPV types (i.e. HPV1, 2, 3, 4, 7, 10, 27, 41, 57, 60, 63 and 65).
Several sampling methods were examined, i.e. skin scrapings (n=5), swabs (n=6) and a tape-based sampling method (n=6). In addition, the performance of two different swabs, i.e. cotton (Abbott MC Specimen Collection) and flocked (FLOQSwab Copan Diagnostics) was analysed. In total 32 warts were sampled by both types of swabs in an alternating order. The optimized DNA extraction protocol involved overnight Proteinase K and EDTA digestion, followed by automated extraction on the NucliSENS® easyMAG® system (bioMérieux). Quantification of the DNA yield was achieved by B-globin qPCR (cell control) and Kruskal-Wallis and Paired Student T-test were used to compare Ct-values. A wart-associated HPV genotyping qPCR assay, able to detect the above mentioned cutaneous HPV types, was developed, containing type-specific primers and consensus probes capable of detecting multiple types.
All samples tested positive for B-globin and were considered valid. Skin scrapings had significantly higher yield than both swab and tape-based methods (p<0.01), the latter two did not significantly differ from each other (p>0.05). When comparing B-globin Ct values no significant difference was found between cotton and flocked swabs irrespective of sampling sequence (p >0.05). All swabs were HPV positive, however there were some discrepancies in HPV type-specific detection but these were not statistically significant and can be attributed to the assay detection limit (Pearson’s Χ2 test p>0.05; κ=0.79 [95%CI, 0.73-0.86]).
Although somewhat better DNA yield was found in skin scrapings, the patient discomfort was an important limitation of this method. Considering that, in combination with our optimized DNA extraction procedure, all samples gave valid results with the less invasive swab method this technique is preferred. An additional advantage of swabs is the option for automated pre-analytical processing, which is not feasible with the alternative methods. Performance of both types of swabs was demonstrated to be equal.