Pitfalls of commercially available HPV tests in HPV68a detection

BACKGROUND: Human papillomavirus 68 (HPV68) is a probable carcinogenic HPV genotype which is included in almost all HPV screening assays and exists as two genetically variable subtypes (HPV68a and HPV68b). Routine HPV sample testing has shown that the cobas 4800 HPV Test (Roche) provides higher false-negative rates for HPV68 status than PapilloCheck HPV-Screening (Greiner Bio-One). The aim of our study was to evaluate the efficacy of cobas 4800 in HPV68 detection. METHODS: A total of 2,145 cervical/cervicovaginal samples from women aged 17–88 were tested for HPV68 status using the cobas 4800 and PapilloCheck HPV tests. Viral load was assessed by quantitative PCR in all of the HPV68-positive cases. HPV68a/b subtyping was performed with real-time PCR followed by high resolution melting curve analysis, and was subsequently confirmed by Sanger sequencing. RESULTS: Cobas 4800 detected HPV positivity in only 13/33 HPV68 single-genotype infection cases. Viral load was comparable across both tested subgroups. HRM analysis and Sanger sequencing identified the HPV68a subtype in all of the 20 instances of cobas 4800 false negatives. HPV68a and HPV68b were detected in 3/13 and 10/13 cases identified as other HPV-positive by cobas 4800. CONCLUSION: The HPV68a subtype was missed by cobas 4800 in more than 85% of all HPV68a-positive cases. Therefore, commercially available assays may underestimate HPV68 prevalence.