Viral Load Levels Track Cervical Lesion Severity in HPV-Positive Indian Women

A cross-sectional study of 328 HPV-positive women in India reports that semi-quantitative viral load values from the Hybrid Capture 2 (HC2) assay show potential for identifying high-grade cervical lesions or invasive cervical cancer, while exploratory analyses suggest that adding HPV genotype information did not improve diagnostic discrimination. The research, conducted at the All India Institute of Medical Sciences in New Delhi, has been published in Frontiers in Global Women’s Health.

HC2 testing estimates the burden of high-risk human papillomavirus as relative light units divided by a cutoff ratio (RLU/CO), a semi-quantitative measure of viral load. The researchers evaluated whether these values were associated with the severity of cervical lesions and whether HPV genotyping could add value to triage among women who had already tested positive for high-risk HPV.

The study proceeded in two sequential phases. In the initial phase, 217 consecutive HPV-positive women underwent HC2 HPV DNA testing, colposcopy-guided biopsy, and histopathological assessment. In a subsequent phase, an additional 111 HPV-positive women received the same clinical and histopathological workup and additionally provided specimens for HPV genotyping. The full cohort of 328 women was analysed for age, histopathology, and viral load, while the genotyped subset was separately evaluated for genotype distribution, co-infection patterns, and exploratory genotype-integrated prediction models.

Statistical analysis included descriptive statistics, Kruskal-Wallis testing with Holm-adjusted Dunn’s post-hoc comparisons, and receiver operating characteristic (ROC) analyses reported with 95% confidence intervals and paired DeLong comparisons. RLU/CO values differed across the study-specific histopathological severity categories, with higher median values observed in low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and invasive cervical cancer than in normal histology and chronic cervicitis.

In the full cohort of 328 women, RLU/CO discriminated HSIL or invasive cervical cancer, defined as a severity score of 3 or higher, with an area under the ROC curve (AUC) of 0.798 (95% CI, 0.726-0.868). A study-derived cutoff of 12.08 RLU/CO, selected by the Youden index, yielded a sensitivity of 81.1% (95% CI, 69.5%-90.9%) and a specificity of 73.1% (95% CI, 68.0%-78.0%). The authors emphasise that this cutoff was derived within the study population and requires validation in independent populations before any clinical implementation.

Among the 111 genotyped participants, HPV16 was the most frequently detected genotype, and multiple-genotype infections occurred in 64 participants, representing 57.7% of the genotyped subgroup. This high proportion of co-infections reflects the recruitment of women who had already tested positive for high-risk HPV rather than the general population.

In the genotyped subgroup, comparative ROC analysis for detecting HSIL or invasive cervical cancer produced notably lower and more variable AUCs than in the full cohort: 0.659 for RLU/CO alone, 0.608 for RLU/CO combined with HPV16/18 genotyping, and 0.461 for RLU/CO combined with other high-risk HPV types. Neither genotype-integrated model improved discrimination compared with RLU/CO alone, and the HPV16/18 model performed comparably to viral load alone. The authors describe these genotype-integrated estimates as exploratory, given the small size of the genotyped subgroup, and note that they require validation in larger independent cohorts.

The findings indicate that HC2 RLU/CO values carry information relevant to lesion severity among HPV-positive women, but the authors caution that larger prospective studies are needed before the approach can be considered for clinical use. As a cross-sectional study in a single referral setting with a two-phase design, the results establish associations within this cohort rather than demonstrating performance in routine screening practice, and the study-derived cutoff and genotype-based models remain provisional pending external validation.

Subject of Research: The association between HPV viral load measured by Hybrid Capture 2 and cervical lesion severity in HPV-positive Indian women, with exploratory HPV genotyping analysis.

Article Title: Association of viral load values with cervical lesion severity and exploratory genotype analysis in HPV-positive Indian women

Article References: Singh, N., Zakiya, Z., Singhal, S., Meena, J., Kumari, R., Singh, A., Dandapath, I., Gaur, A., Tanwar, P., Mathur, S., Khan, M. A., & Malhotra, N. (2026). Association of viral load values with cervical lesion severity and exploratory genotype analysis in HPV-positive Indian women. Frontiers in Global Women's Health, 7, Article 1891484. https://doi.org/10.3389/fgwh.2026.1891484

Image Credits: AI Generated

DOI: 10.3389/fgwh.2026.1891484

Keywords: human papillomavirus, HPV viral load, Hybrid Capture 2, cervical cancer screening, cervical intraepithelial lesions, HPV genotyping, HPV16, colposcopy, cross-sectional study, India, diagnostic accuracy, ROC analysis