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

J Clin Microbiol. 2011 Oct;49(10):3474-81. doi: 10.1128/JCM.05039-11. Epub 2011 Aug 10.

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adult
  • Clinical Laboratory Techniques / economics*
  • Clinical Laboratory Techniques / methods*
  • Female
  • Genotype
  • Humans
  • Membranes
  • Middle Aged
  • Nucleic Acid Hybridization / methods
  • Papillomaviridae / classification*
  • Papillomaviridae / genetics*
  • Papillomaviridae / isolation & purification
  • Papillomavirus Infections / diagnosis
  • Papillomavirus Infections / virology*
  • Polymerase Chain Reaction / economics
  • Polymerase Chain Reaction / methods
  • Sensitivity and Specificity
  • Virology / economics*
  • Virology / methods*