Detection of active human papillomavirus (HPV) types 16/18 infections in endocervical brush samples using RNA in situ hybridization and flow-FISH assay

Background Although many human papillomavirus (HPV) infections resolve, persistent high-risk HPV infections can progress to cervical intraepithelial neoplasia (CIN) and invasive carcinoma. Current HPV screening strategies include DNA- and messenger RNA (mRNA) – based assays. This study evaluated the diagnostic performance of flow cytometry with fluorescence in situ hybridization (flow-FISH), integrating flow cytometry with fluorescence in situ hybridization (FISH), for transcriptionally active HPV infections through E6 mRNA expression.Methods Cervical scrape specimens were obtained from 31 women with HPV infection. Samples underwent HPV DNA testing, genotyped, and flow-FISH analysis for HPV-16 and 18 E6 mRNA detection. Probe specificity was validated using HeLa and CaSki cell lines, supported by RT-PCR.Results Flow-FISH demonstrated 100% specificity in detecting HPV mRNA in control cell lines. Among the clinical samples, flow-FISH detected HPV E6 mRNA transcripts in 18 of 31 cases (58%), including one PCR-negative sample and two Atypical Squamous Cells of Undetermined Significance (ASC-US) cases, indicating that transcriptionally active infections were missed by DNA testing.Conclusion Flow-FISH provides a direct assessment of viral oncogene expression at the single-cell level. It may complement DNA-based screening by identifying transcriptionally active high-risk HPV infections. Further studies with larger populations are needed to validate its clinical utility.