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The CONFIDENCE Assay consists of a Human papillomavirus test and a methylation biomarker: the Confidence HPV and the Confidence Marker tests.
The CONFIDENCE HPV is a TaqMan-based L1 region-specific multiplex real-time PCR assay for viral DNA detection. The test detects HPV16 and HPV18 separately and other high-risk types (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in group. The DNA extraction for HPV detection is performed on 200 ul LBC sample applying a silica- filter-plate-based purification method on a Tecan EVO liquid-handling platform using standardized, contamination safe workflow with high reliability. After the 96-channel automated PCR setup, 5-plex quantitative real-time PCR was performed on the QuantStudio 6 Flex platform in 384-well plate format in four reactions per sample. The sample quality is assured by the amplification of cellularity control in each sample. The DNA preparation process is controlled by the amplification of an artificial internal DNA control sequence added to the sample during the process.
In the samples collected from women over 25 years of age, the CONFIDENCE HPV was compared to cobas HPV on 3,150 samples resulting in 92.3% overall agreement. PPA was 83.3% (95% CI: 81.2–85.4) and NPA was 95% (95% CI: 94.4–95.6). Agreement levels for HPV16, HPV18 and other hrHPV types were comparable. The value of PABAK was 0.85 (95% CI: 0.83–0.86), while Cohen’s kappa was 0.78 (95% CI: 0.75–0.82). The clinical sensitivity and specificity for CIN2+ cases were found to be equivalent for the CONFIDENCE HPV and the cobas HPV tests.
The CONFIDENCE Marker test measures the methylation level of CpG sites in the promoter region of POU4F3 by qMSP. The quantitative measurement of the gene COL2A1 (type II collagen) is used as internal reference to normalize the methylation level of the POU4F3.
The M-index value ranges from 1 to 10,000 where the unit of M- index refers to a methylated ratio of 0.01% of the cells. The direct bisulfite treatment is performed on the LBC sample in 96-well plate format by using a Zymo Research kit. The PCR setup is implemented using the Tecan EVO liquid-handling system with standardized, highly reliable workflow. The amplification of the recovered bisulfite converted genomic target DNA and the internal reference sequence are performed using a TaqMan probe system on the QuantStudio 6 Flex Real-Time PCR platform in 384-well plate format. The sample quality is controlled by the amplification of the COL2A1 internal reference.
The clinical performance of POU4F3 methylation was evaluated by comparing the performance of methylation and cytology in triage of hrHPV positives (n = 1,287).
When comparing POU4F3 and cytology in triage, methylation was found to be significantly more sensitive than cytology: relative sensitivity and specificity yielded 1.67 (95% CI: 1.23–2.24) and 1.01 (95% CI: 0.7–1.22), respectively, in the age group of 25–65 for CIN2+. For CIN3+ endpoint in the age group of 25–65 and 30–65, relative sensitivities were found to be 1.74 (95% CI: 1.25–2.33) and 1.64 (95% CI: 1.08–2.27), respectively.