Development and interlaboratory agreement of real-time PCR for HPV16 quantification in liquid-based cervical samples

Abstract

High risk HPV infection is the necessary cause for the development of precancerous and cancerous lesions of the cervix. Among HPV, HPV16 represents the most carcinogenic type. Since the determination of HPV16 DNA load could be clinically useful, we assessed quantitative real-time PCR targeting E6HPV16 and albumin genes on two different platforms. Series of SiHa cells diluted in PreservCyt were used to assess repeatability and reproducibility of two in-house real-time PCR techniques run in two different laboratories to determine HPV16 load. Furthermore, 97 HPV16 positive cervical samples were evaluated to estimate inter-center variability using Bland-Alman plots. As a whole, both techniques presented coefficients of variation for HPV16 load measurement similar to those established for other virus quantification with commercial kits. Moreover, the two real-time PCR techniques showed a very good agreement for HPV16 load calculation. Finally, we emphasize that robust HPV16 DNA quantification requires normalization of viral load by the cell number. Keywords: Papillomavirus, Viral load, Coefficient of variation, Agreement