PCR-RFLP assay as an option for primary HPV test

Area of Science:

• Molecular biology
• Oncology
• Virology

Background:

• Persistent human papillomavirus (HPV) infection is a primary driver of cervical cancer development.
• Accurate HPV detection and genotyping are crucial for cervical cancer prevention and management.
• Existing diagnostic methods vary in performance and cost, necessitating evaluation of alternative approaches.

Purpose of the Study:

• To evaluate the analytical performance of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in identifying human papillomavirus (HPV) in cervical cells.
• To compare the HPV genotyping capabilities of PCR-RFLP with the PapilloCheck® microarray assay.
• To assess the suitability of PCR-RFLP as a primary HPV screening tool, particularly in resource-limited settings.

Main Methods:

• Cervical samples from 325 women were analyzed using conventional cytology and HPV testing.
• HPV-positive samples (n=80) identified by PCR-Multiplex were further analyzed by PCR-RFLP and PapilloCheck® microarray.
• Discordant results were resolved using DNA sequencing.

Main Results:

• A genotyping agreement of 71.25% (57/80) was observed between PCR-RFLP and PapilloCheck® for at least one HPV type.
• HPV 16 was the most prevalent type detected by both methods.
• PCR-RFLP identified 25% HPV coinfection, while PapilloCheck® identified 62.5%, indicating differences in sensitivity for multiple infections.
• Cohen's kappa analysis showed perfect agreement for HPV 33 and 58, very good for HPV 51, and good for several other HPV types.

Conclusions:

• The in-house HPV genotyping assay (PCR-RFLP) demonstrates acceptable analytical performance and can be considered for primary HPV screening, especially in low-income countries.
• PapilloCheck® microarray offers more comprehensive detection of HPV coinfections.
• A tiered approach, using PCR-RFLP for initial screening and PapilloCheck® for confirmation of coinfections, may be an effective strategy for HPV testing.