FC 06-06CHARACTERIZATION OF GENOTYPE-SPECIFIC HPV PREVALENCE IN CUTANEOUS WART BIOPSIES

11. Genotyping
N. Redzic 1, S. Nouws 2, L. De Baere 3, I. Benoy 4, D. Vanden Broeck 4, J.P. Bogers 4, J. Jonckheere 4.
11. AMBIOR, Laboratory for Cell Biology & Histology, University of Antwerp, Antwerp, Belgium 2. Laboratory of Molecular Pathology, AML, Antwerp, Belgium (Belgium), 21. AMBIOR, Laboratory for Cell Biology & Histology, University of Antwerp, Antwerp, Belgium (Belgium), 31. Laboratory of Molecular Pathology, AML, Antwerp, Belgium (Belgium), 41. AMBIOR, Laboratory for Cell Biology & Histology, University of Antwerp, Antwerp, Belgium 2. Laboratory of Molecular Pathology, AML, Antwerp, Belgium 3. National Reference Centre for HPV, Brussels, Belgium (Belgium)

Background / Objectives

Cutaneous warts are a common, infectious and sometimes very painful problem, with a varying worldwide prevalence of 0.84-12.9%1. Warts are caused by infection with the human papillomavirus (HPV). The most frequently found HPV types in cutaneous warts are HPV1, 2, 3, 4, 7, 10, 27, 41, 57, 60, 63 and 65. The aim of this study was to develop a high-yield DNA extraction protocol for formalin-fixed paraffin-embedded biopsies and determine the prevalence of HPV in cutaneous warts in a Belgian population.


Methods

A total of 50 biopsies were included in this study. Before and after slicing of the sections predetermined  for DNA extraction (10x5µm), additional sections were made for haematoxylin-eosin (HE) staining to ensure that these were derived from wart epithelium. The optimized protocol involved overnight Proteinase K and EDTA digestion, followed by automated extraction on the NucliSENS® easyMAG® system (bioMérieux). A newly developed wart-associated HPV qPCR assay, capable of detecting the above mentioned cutaneous types, together with the in-house HPV Riatol genotyping assay, capable of detecting the most relevant mucosal types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67 and 68), were used to determine the HPV prevalence.


Results

The wart diagnosis was confirmed by HE staining. All samples tested positive for B-globin (cell control) and were considered valid. 24% [95%CI 12-36%] of the samples were negative for the above mentioned cutaneous as well as mucosal HPV types. Cutaneous HPV types 41, 60 and 63 were not detected. 8% [95%CI 0.5-16%] of the samples was infected with mucosal low-risk (HPV6 and 11) and high-risk (HPV16, 58 and 59) HPV types and 36% [95%CI 23-49%] contained multiple infections.


Conclusion

The most prevalent HPV types in the Belgian population were HPV1, 57, 4, 2, 27 and 7. Multiple HPV infections were detected in 36% of lesions, contradicting the current literature claiming that in immunocompetent patients only 0-16% of cutaneous warts exhibit multiple HPV infections2,3,4. Considering that cutaneous warts are very inconvenient disorders that can cause not only pain, but also diminish the quality of life of the affected individuals, a more efficient management should be implemented. Since it has been suggested that HPV type can influence natural course and response to treatment in certain subsets of verrucae2, genotype specific strategies should be considered, indicating an important role for future HPV genotyping.


References

1Hashmi,F., Torgerson,D., Fairhurst,C., Cockayne,S., Bell,K., Cullen,M., and Harrison-Blount,M. (2015). EVerT2-needling versus non-surgical debridement for the treatment of verrucae: study protocol for a single-centre randomised controlled trial. BMJ Open. 5, e009406.

2 Bruggink,S.C., Gussekloo,J., de Koning,M.N., Feltkamp,M.C., Bavinck,J.N., Quint,W.G., Assendelft,W.J., and Eekhof,J.A. (2013). HPV type in plantar warts influences natural course and treatment response: secondary analysis of a randomised controlled trial. J. Clin. Virol. 57, 227-232.

3Harwood,C.A., Spink,P.J., Surentheran,T., Leigh,I.M., de Villiers,E.M., McGregor,J.M., Proby,C.M., and Breuer,J. (1999). Degenerate and nested PCR: a highly sensitive and specific method for detection of human papillomavirus infection in cutaneous warts. J. Clin. Microbiol. 37, 3545-3555.

4de Koning,M.N., ter,S.J., Eekhof,J.A., Kamp,M., Kleter,B., Gussekloo,J., Feltkamp,M.C., Bouwes Bavinck,J.N., Purdie,K.J., Bunker,C.B., Proby,C.M., Meys,R., Harwood,C.A., and Quint,W.G. (2010). Evaluation of a novel broad-spectrum PCR-multiplex genotyping assay for identification of cutaneous wart-associated human papillomavirus types. J. Clin. Microbiol. 48, 1706-1711.