The HPV Test Risk assay is an in vitro real-time PCR-based assay for the qualitative detection of human papillomavirus (HPV) DNA for the following 15 (probably) high-risk HPV genotypes, i.e., 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, and 68. The assay is intended to be used in the screening of women for cervical (pre)cancer.
HPVs are small, double-stranded DNA viruses and their circular genome consists of approximately 7,900 base pairs. More than 100 types of HPV have been identified, of which certain HPV types, known as high-risk HPV (hrHPV) like HPV16 and -18, are associated with the induction of mucosal lesions that can progress to malignancy. Cervical cancer and its precursor lesions (cervical intraepithelial neoplasia, CIN) are the most well known complication of a persistent infection with such a virus.
The viral genome contains early (E) and late (L) genes, which encode proteins necessary for early and late stages, respectively, of the HPV life cycle. The E6 and E7 gene products of hrHPV types have carcinogenic properties and are necessary for malignant transformation of the host cell. Malignant progression is often associated with viral integration into the genome of the host cell . Integration results in interruption of the viral genome in a region that may extend from the E1 to the L1 open reading frame. This may have consequences for PCR-mediated amplification of viral DNA in these regions. As not only the initiation but also maintenance of the transformed phenotype depends on continuous expression of the viral oncoproteins, the viral E6/E7 region is invariably retained in integrated viral genomes in cervical cancers. The HPV-Risk assay targets a conserved region within the E7 gene. The HPV-Risk assay has been clinically validated according to the international guidelines for HPV detection assays.
Principle of the Procedure
The HPV Test Risk assay is a multiplex, real-time PCR-based assay directed against the E7 gene of 15 (probably) hrHPV types that uses fluorescent probes for the detection of one or more accumulating PCR products. During each PCR cycle the fluorescent signal increases in a logarithmic manner resulting in an amplification curve. As soon as the amplification curve of the target comes above its threshold, the sample is considered positive for that target. The multiplex format allows the simultaneous detection of four different fluorescent dyes per reaction, with each fluorescent dye representing different targets. The four different targets are HPV16, HPV18, the 13 other hrHPV types as a pool, and the human β-globin gene. The HPV-Risk assay provides information on the presence of HPV16 and HPV18 each separately from the other 13 HPV genotypes that are detected as a pool. The human β-globin gene is used as the sample control determining both the quality of the sample DNA and the presence of potential inhibitory substances.
Limit of detection
The limit of detection (LOD) was determined using 10-fold dilution series of plasmids containing the HPV genome in a background of 100 ng human DNA per PCR. For HPV16, HPV18, and HPV51, the LOD testing was determined on 5-fold dilutions and tested 20 times. Duplicate testing was done for the HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, and 68. For β-globin the LOD was assessed on 3-fold dilution series of human placenta DNA and tested in duplicate.Analytical specificity
Analytical specificity analyses of the HPV-Risk assay were performed on 46,000 plasmids containing HPV genomes of 6, 11, 26, 40, 42, 43, 53, 61, and 70 and on 10,000 copies of the 3 most common vaginal micro organisms, i.e. Chlamydia trachomatis, Neisseria gonorrhoeae and Candida albicans. The HPV-Risk assay showed cross-reactivity with HPV70 of >17,000 copies input for the target ‘HPV Other’. HPV70 is considered probably carcinogenic on the basis of epidemiological, phylogenetic, and functional studies 11-13. The HPV-Risk assay did not show any reaction with the other non-targeted HPV types (6, 11, 26, 40, 42, 43, 53, and 61) or micro-organisms.
Clinical performance cervical scraping specimens
The clinical sensitivity and specificity of the HPV-Risk assay for cervical intraepithelial neoplasia grade 2 or higher (CIN2+ ) with cervical scraping specimens were validated according to the international guidelines for HPV detection assays 9. The clinical sensitivity for CIN2+ was 97,1% (67/69) and the clinical specificity for CIN2+ was 93,9% (774/824) 10. The clinical sensitivity and specificity were non-inferior to that the reference assay GP5+/6+-PCR, indicating a very good clinical performance.
The intra-laboratory reproducibility and inter-laboratory agreement of the HPV-Risk assay were validated according to the international guidelines for HPV detection assays . The intra-laboratory was 99,5% (544/547) with a kappa value of 0.98 and the inter-laboratory agreement was 99,2% (527/531) with a kappa value of 0.99, indicating very goodagreement .
Performance self-collected (cervico-)vaginal specimens
The performance of the HPV-Risk assay with self-collected (cervico-)vaginal specimens was validated for two different sampling methods, 1) self-collected lavage specimens, and 2) self-collected brush specimens. For self-collected lavage specimens the agreement with the GP5+/6+-PCR was 97% (59/61) with a CIN2+ sensitivity of 91.4% (21/23). For self-collected brush specimens the agreement with GP5+/6+-PCR was 93% (104/112) with a CIN2+ sensitivity of 93.9% (31/34).
Assay components and handling
The HPV-Risk assay is a ready-to-use kit for 96 reactions and consists of the HPV-Risk assay Master mix (purple cap), the HPV-Risk assay positive control (red cap) and the HPV-Risk assay negative control (blue cap). The HPV-Risk assay is a low-copy control.