Background: Tuberculosis (TB) remains a significant public health challenge in rural India, where access to advanced diagnostics is limited. The Crystal Violet Decolorization (CVD) assay offers a potential low-cost, microscopy-based alternative for detecting Mycobacterium tuberculosis and drug resistance.

Methods: The CVD assay was assessed for M. tuberculosis detection against CBNAAT and for rifampicin and isoniazid resistance against CBNAAT and culture-based drug susceptibility testing, respectively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated, with feasibility assessed via turnaround time and cost.

Results: Of 350 samples, CBNAAT identified 82 (23.4%) as M. tuberculosis-positive. The CVD assay demonstrated a sensitivity of 92.68% (95% CI: 84.76–97.30%) and specificity of 98.13% (95% CI: 95.70–99.43%) for TB detection, comparable to ZN staining (sensitivity 89.02%, specificity 97.76%). For drug resistance, the CVD assay achieved sensitivities of 85.71% (95% CI: 57.19–98.22%) for rifampicin and 81.25% (95% CI: 54.35–95.95%) for isoniazid, with specificities of 98.53% and 96.77%, respectively. The assay’s turnaround time was 2.5 hours for TB detection and 26.5 hours for resistance testing, with a cost of INR 150 per test, significantly lower than CBNAAT (INR 2000) and culture (INR 2500).
Conclusion: The CVD assay offers high diagnostic accuracy for M. tuberculosis detection and good performance for drug resistance screening, with cost-effectiveness and feasibility for rural settings. The assay’s simplicity and affordability make it a promising tool for enhancing TB control in resource-limited regions, supporting India’s National Strategic Plan for Tuberculosis Elimination.

Keywords: Mycobacterium tuberculosis, Crystal Violet Decolorization assay, Drug resistance, Point-of-care diagnostics, Resource-limited settings