Diagnostic Accuracy of Methylene Blue as a Counterstain For LED Fluorescence Microscopy in The Detection of Acid-Fast Bacilli: A Diagnostic Accuracy Study

Objectives
To evaluate the diagnostic accuracy and agreement of methylene blue as a secondary counterstain for auramine O-based light-emitting diode (LED) fluorescence microscopy in the detection and grading of acid-fast bacilli in sputum specimens.
Methods
A prospective diagnostic accuracy study was conducted at the Chest Diseases Laboratory, Lusaka, Zambia, between 6 January and 28 March 2026. The study included 20 direct sputum specimens, 20 concentrated sputum specimens processed using the 4% N-acetyl-L-cysteine-sodium hydroxide method, and 20 proficiency testing slides from the 2025 Round B external quality assessment programme. 2 smears were prepared from each specimen and stained with auramine O followed by either 0.3% methylene blue (index test) or 0.1% potassium permanganate (reference method). The primary outcome measures were sensitivity, specificity, positive predictive value, negative predictive value, and agreement between methylene blue and potassium permanganate for acid-fast bacilli detection and grading.
Results
Compared with potassium permanganate staining, methylene blue demonstrated 100% sensitivity and specificity across all specimen categories. For concentrated sputum specimens, sensitivity was 100% (95% CI 78.2% to 100%) and specificity was 100% (95% CI 47.8% to 100%). For direct sputum specimens, sensitivity was 100% (95% CI 66.4% to 100%) and specificity was 100% (95% CI 71.5% to 100%). Among PT slides, sensitivity and specificity were both 100% (95% CI 69.2% to 100%). Agreement between staining methods was complete across specimen categories (Cohen’s κ=1.0). Among positive smears, grading agreement within one grade was observed for all specimen types, with exact grading agreement of 66.7% for concentrated sputum, 88.9% for direct sputum and 100% for PT slides.
Conclusions
Methylene blue demonstrated excellent diagnostic agreement with potassium permanganate when used as a counterstain for auramine O LED fluorescence microscopy.

Tuberculosis, acid-fast bacilli, fluorescence microscopy, diagnostic accuracy, methylene blue

1. Chandra, T. J., Selvaraj, R., & Sharma, Y. V. (2015). Same day sputum smear microscopy for the diagnosis of pulmonary tuberculosis: Ziehl-Neelsen versus fluorescent staining. Journal of Family Medicine and Primary Care, 4(4), 525–528. doi.org/10.4103/2249-4863.174273 [Google Scholar] [Crossref]

2. Clinical and Laboratory Standards Institute. (2014). Evaluation of precision of quantitative measurement procedures (3rd ed., CLSI document EP05-A3). Clinical and Laboratory Standards Institute. [Google Scholar] [Crossref]

3. Forbes, B. A., Hall, G. S., Miller, M. B., Novak, S. M., Rowlinson, M. C., Salfinger, M., Somoskövi, A., Warshauer, D. M., & Wilson, M. L. (2018). Practice guidelines for clinical microbiology laboratories: Mycobacteria. Clinical Microbiology Reviews, 31(2), e00038-17. doi.org/10.1128/CMR.00038-17 [Google Scholar] [Crossref]

4. Hooja, S., Pal, N., Malhotra, B., Goyal, S., Kumar, V., & Vyas, L. (2011). Comparison of Ziehl-Neelsen and Auramine O staining methods on direct and concentrated smears in clinical specimens. Indian Journal of Tuberculosis, 58(2), 72-76. [Google Scholar] [Crossref]

5. Steingart, K. R., Henry, M., Ng, V., Hopewell, P. C., Ramsay, A., Cunningham, J., Urbanczik, R., Perkins, M., Aziz, M. A., & Pai, M. (2006). Fluorescence versus conventional sputum smear microscopy for tuberculosis: A systematic review. The Lancet Infectious Diseases, 6(9), 570–581. doi.org/10.1016/S1473-3099(06)70578-3 [Google Scholar] [Crossref]

6. World Health Organization. (2011). Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: Policy statement. World Health Organization. [Google Scholar] [Crossref]

7. World Health Organization. (2021). WHO operational handbook on tuberculosis. Module 3: Diagnosis-Rapid diagnostics for tuberculosis detection. World Health Organization. [Google Scholar] [Crossref]

8. World Health Organization. (2025). Global tuberculosis report 2025. World Health Organization. [Google Scholar] [Crossref]

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