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; 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; 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.

Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV Assay for Human Papillomavirus Genotyping

“…For this reason, we decided to compare H28 with our in-house PGMY-CHUV (PG) assay described in Chapter 5 of the WHO HPV Laboratory Manual (10). PG has been found to be highly comparable to the commercially available linear array (LA; Roche) with some advantages in terms of sensitivity and specificity toward HPV56 and HPV52 (20). PG has also been repeatedly proficient in several laboratories within the WHO HPV Laboratory Network at frequencies at least as good as those of LA (22).…”

“…PCR using the PGMY primers and reverse blotting hybridization (RBH) were performed as described in Chapter 5 of the WHO HPV Laboratory Manual and its validation study with 5 l DNA in a single 50-l PCR mixture containing 3 mM MgCl 2 (10,20). This assay allows genotyping of 31 HPV (6, 11, 16, 18, 26, Version 2 of PG (PGv2) was designed to resolve HPV68-discordant cases.…”

“…HPV51-and HPV68-discordant cases were assessed by DNA sequencing of PG amplicons and comparison to the nonredundant nucleic acid GenBank database as described in Estrade et al (20). DNA sequencing was also performed with the HMB01 primer for HPV51 (13).…”

“…An additional subset of 14 samples was also randomly selected among HPV-negative women with atypical squamous cells of undetermined significance (ASC-US). These DNA samples had been purified using MagNApure chemistry (Roche, Rotkreuz, Switzerland) as previously described (20).…”

“…They target at least 25 genotypes: the 14 high-risk HPV genotypes, indicated above, as well as low-risk (HPV6, -11, -42, -44, -53, -54, and -70) or potentially high-risk (HPV26, -69, -73, and -82) genotypes. These assays, including ours, are time-consuming and necessitate some degree of expertise (20). Owing to their ease of use and objective readout, real-time PCR assays capable of detecting a higher number of HPV genotypes are desirable.…”