Validation of a low-cost human papillomavirus genotyping assay based on PGMY PCR and reverse blotting hybridization with reusable membranes

Abstract

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

Fig. 1.

Cost evaluation. Costs in Swiss francs are shown as a function of the number of samples processed in parallel in a 96-well PCR plate with either a single reaction performed with buffer B per sample (PCR costs, line 1) or two reactions performed with buffer A and with buffer B per sample (PCR costs, line 2). Following gel electrophoresis, positive or doubtful samples were processed by RBH by sets of 40 hybridizations (RBH costs). The costs of RBH per sample are shown as a function of membrane reuse and the number of PCRs subjected to RBH: either one reaction per sample (1) or both reactions, in adjacent lanes, per sample (2). The membranes could be used a median of 11 times, with several being used more than 20 times (see File S2 in the supplemental material).