International Journal of Research and Scientific Innovation (IJRSI)
Diagnostic Accuracy of Methylene Blue as a Counterstain For LED Fluorescence Microscopy in The Detection of Acid-Fast Bacilli: A Diagnostic Accuracy Study
Published August 6, 2026 • Vol. 13, Issue 7, pp. 2567–2577Open Access
DOI: 10.51244/IJRSI.2026.1307000188
Abstract
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.
Keywords: Tuberculosis, acid-fast bacilli, fluorescence microscopy, diagnostic accuracy, methylene blue
| Journal | International Journal of Research and Scientific Innovation (IJRSI) |
|---|---|
| ISSN | 2321-2705 |
| Volume / Issue | Volume 13, Issue 7 |
| Pages | 2567–2577 |
| Publication date | August 6, 2026 |
| DOI | 10.51244/IJRSI.2026.1307000188 |
| Publisher | RSIS International |
| License | Open Access |
How to cite this article
Wina Mutende, Mwenya Jonathan, Alan Musunsa, James Mukamwembo, Nyirenda Wilson, Esther Kalito Nyendwa, Beene Muleza Manchishi, Jonathan Simpemba, John Muzyamba, Kasamba Ng’andu, Phenister Mwale, Wenga Namutowe, David Kajoba Mumena, & Phiri Faliton (2026). Diagnostic Accuracy of Methylene Blue as a Counterstain For LED Fluorescence Microscopy in The Detection of Acid-Fast Bacilli: A Diagnostic Accuracy Study. International Journal of Research and Scientific Innovation (IJRSI), 13(7), 2567-2577. https://doi.org/10.51244/IJRSI.2026.1307000188
BibTeX
@article{Wina2026,
title = {Diagnostic Accuracy of Methylene Blue as a Counterstain For LED Fluorescence Microscopy in The Detection of Acid-Fast Bacilli: A Diagnostic Accuracy Study},
author = {Wina Mutende and Mwenya Jonathan and Alan Musunsa and James Mukamwembo and Nyirenda Wilson and Esther Kalito Nyendwa and Beene Muleza Manchishi and Jonathan Simpemba and John Muzyamba and Kasamba Ng’andu and Phenister Mwale and Wenga Namutowe and David Kajoba Mumena and Phiri Faliton},
journal = {International Journal of Research and Scientific Innovation (IJRSI)},
volume = {13},
number = {7},
pages = {2567--2577},
year = {2026},
doi = {10.51244/IJRSI.2026.1307000188},
publisher = {RSIS International}
}