Relationship between hybrid capture II ratios and DNA amplification of E1, E6 and L1 genes used for the detection of human papillomavirus in samples with different cytological findings.

ABSTRACT

The hybrid capture II (HCII) assay is widely used in the detection of human papillomavirus virus (HPV). However, due to the limited number of HPV genotypes, it does not permit a comprehensive typing of viruses and “grey zone” (borderline negative or positive results) are often difficult to interpret. As such, polymerase chain reaction (PCR) should be used in parallel with HCII assays, and consensus PCR detection is capable of covering a wider detection range than with the HCII method. We examined the relationship between HCII relative light unit/cut-off (RLU/CO) ratios and PCR amplification results. This was done using previously described primer sets (MY/GP) as well as with our primers for HPV E1, L1 and E6 gene amplification, and performed on samples exhibiting differ-ent cytological findings. Together, 243 samples were divided into three groups having RLU/CO ratios of < 0.4 (n = 21), 0.4-4 (n = 64) and ≥ 4 (n = 158), respectively. All samples were subjected to PCR amplification using MY/GP and the newly designed E1, L1 and E6 primers. Results were verified by direct sequencing. PCR amplification sen-sitivities were higher when using the E1 primers than for the MY/GP, E6 or L1 primers. The E1 assay can be used for HPV detection with a sensitivity of 102 copies μl-1. Samples with RLU/CO ratios exceeding 4, and grey zone samples of 0.4-4, were amplified using E1 primers in 79.74% and 26.56% of the total cases, respectively. Cytologi-cal data of grey zone samples were primarily found to be normal (77%) whereas those with RLU/CO ratios > 4 were found in any of the cytological data categories. We concluded that HPV screening by HCII for grey zone sam-ples should be analyzed together with cytological data, as well as with a PCR screening tool that incorporates the E1 primers.