title: 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.
authors.affiliation: WHO HPV LabNet Regional Reference Laboratory for Europe, Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, and University of Lausanne, 1011 Lausanne, Switzerland
authors.forename: C
authors.initials: C
authors.lastname: Estrade
authors.forename: P-A
authors.initials: PA
authors.lastname: Menoud
authors.forename: D
authors.initials: D
authors.lastname: Nardelli-Haefliger
authors.forename: R
authors.initials: R
authors.lastname: Sahli
mesh_terms: D000328:Adult; D019411:Clinical Laboratory Techniques / Q000191:economics* / Q000379:methods*; D005260:Female; D005838:Genotype; D006801:Humans; D008566:Membranes; D008875:Middle Aged; D009693:Nucleic Acid Hybridization / Q000379:methods; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D012680:Sensitivity and Specificity; D014773:Virology / Q000191:economics* / Q000379:methods*