Comparative Evaluation of Fluorescein Diacetate/Ethidium Bromide Viability Microscopy and Ziehl-Neelsen Staining for Rapid Diagnosis of Pulmonary Tuberculosis.

Abstract

Background: Ziehl-Neelsen (ZN) staining is routinely used for tuberculosis (TB) diagnosis, but cannot differentiate viable from nonviable bacilli. This limitation is particularly relevant in smear-positive pulmonary TB patients, where assessment of treatment response is essential. Fluorescein diacetate/ethidium bromide (FDA/EtBr) viability microscopy detects live Mycobacterium tuberculosis based on metabolic activity. This study compared the diagnostic performance of FDA/EtBr microscopy with ZN staining, using culture and drug susceptibility testing (DST) as the reference standard.

Methods: A cross-sectional study was conducted on 300 smear-positive pulmonary TB patients at Santosh Medical College, Ghaziabad, and National Institute of TB and Respiratory Diseases, New Delhi. After n-acetyl-l-cysteine-sodium hydroxide decontamination, sputum samples were examined using ZN staining, FDA/EtBr viability microscopy, and mycobacteria growth indicator tube 960 culture/DST. Sensitivity, specificity, positive predictive value, negative predictive value (NPV), and diagnostic accuracy were calculated, and paired statistical comparision were performed and statistical significance ( P < 0.05) was assessed for diagnostic outcomes. Inter-test agreement was evaluated using Cohen's κ-statistic.

Results: FDA/EtBr microscopy demonstrated significantly higher sensitivity (94.4% vs. 88.9%; absolute increase 5.5%, P = 0.018) and higher diagnostic accuracy (91.0% vs. 86.7%; absolute increase 4.3%, P = 0.04) compared to ZN staining. While ZN staining showed marginally higher specificity (66.7% vs. 60.0%; P = 0.42), FDA/EtBr provided a significantly improved NPV (54.5% vs. 40.0%; absolute increase 14.5%, P = 0.03). Agreement with culture/DST was almost perfect for FDA/EtBr (κ = 0.85) and substantial for ZN staining (κ = 0.72).

Conclusion: FDA/EtBr viability microscopy offers statistically significant improvements in sensitivity, NPV, and diagnostic accuracy over ZN staining in smear-positive pulmonary TB patients, supporting its prioritized use for treatment monitoring and programmatic decision-making in resource-limited, high-burden settings.