Diagnostic utility of HPV-16/18 E6 mRNA fold change measured by in-house semi-quantitative real-time PCR in cervical cancer triage for differentiation of CIN3 and cervical cancer from lower-grade lesions.

Cervical cancer remains a major public health concern in low- and middle-income countries, where screening and triage resources are limited. Although HPV DNA testing is highly sensitive, it cannot distinguish transient infections from transforming infections, highlighting the need for complementary molecular markers. In Phase I, an in-house semi-quantitative SYBR Green-based real-time PCR (qPCR) assay was developed and validated for detection of HPV-16/18 E6 and E7 mRNA. The assay was optimized using genotype-specific primers targeting HPV-16/18 E6 and E7 and the housekeeping gene ribosomal protein S9 (RPS9). Primer concentration, cycling conditions, melt-curve specificity, and Ct cutoffs were optimized. Ct thresholds were established using 40 HPV DNA-positive clinical samples, including 25 E6/E7 mRNA-positive and 15 negative samples, using Receiver Operating Characteristic (ROC) analysis and Youden's index. In Phase II, 70 HPV DNA-positive cervical swab samples with histopathologically confirmed diagnoses of chronic cervicitis (CC, n=14), CIN1 (n=14), CIN2 (n=14), CIN3 (n=14), and invasive carcinoma (IC, n=14) were evaluated. E6 mRNA positivity increased significantly with disease severity (p < 0.001) and showed a strong inverse correlation with HPV DNA load (Spearman's ρ=-0.89, p < 0.001). ROC analysis demonstrated that E6 fold change (E6 FC) provided the highest diagnostic accuracy for distinguishing CIN3+ from lower-grade lesions (AUC=0.9329; 95% CI: 0.8540-1.0000; p < 0.0001). An E6 FC cutoff of ≥0.648 yielded 95.24% sensitivity and 86.36% specificity. These findings support E6 FC as a promising molecular triage marker and demonstrate the potential utility of the in-house qPCR assay for strengthening cervical cancer screening and triage strategies.