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PLoS One. 2019 Apr 30;14(4):e0214131. doi: 10.1371/journal.pone.0214131. eCollection 2019.

Optimising fluorescein diacetate sputum smear microscopy for assessing patients with pulmonary tuberculosis.

Author information

Infectious Diseases & Immunity, Wellcome Trust Centre for Global Health Research, Imperial College London, United Kingdom.
IFHAD: Innovation For Health And Development, Laboratory of research and development, Universidad Peruana Cayetano Heredia, Lima, Peru.
Innovacion Por la Salud Y el Desarollo (IPSYD), Asociación Benéfica Prisma, Lima, Peru.
Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States of America.



Assessing Mycobacterium tuberculosis (TB) viability by fluorescein diacetate (FDA) microscopy can predict TB culture results, treatment response and infectiousness. However, diverse methods have been published. We aimed to optimise FDA microscopy, minimising sputum processing, biohazard and complexity for use in resource-constrained settings.


Optimization: Patients with smear-positive pulmonary TB before treatment and healthy control participants provided sputa. These were divided into equal aliquots that were tested directly or after NaOH centrifuge-decontamination. Each aliquot was cultured and used to prepare slides (n = 80). FDA microscopy used: 1 or 3 drops of sputum; with/out acid-alcohol wash; with/out phenol sterilization; with 0/30/60 seconds KMnO4 quenching. Control samples all had negative culture and microscopy results. FDA microscopy had higher sensitivity when performed directly (without centrifuge-decontamination) on 1 drop of sputum (P<0.001), because 3 drops obscured microscopy. Acid-alcohol wash and KMnO4 quenching made bacilli easier to identity (P = 0.005). Phenol sterilization did not impair microscopy (P>0.1). Validation: The 2 protocols that performed best in the optimization experiments were reassessed operationally by comparing duplicate slides (n = 412) stained with KMnO4 quenching for 30 versus 60 seconds. FDA microscopy results were similar (P = 0.4) and highly reproducible, with 97% of counts agreeing within +/-1 logarithm. Storage: Smear microscopy slides and aliquots of the sputum from which they were made were stored for 4 weeks. Twice-weekly, paired slides (n = 80) were stained with freshly prepared versus stored FDA and read quantitatively. Storing sputum, microscopy slides or FDA solution at 4°C or room temperature had no effect on FDA microscopy results (all P>0.2). Cost: Material costs for each slide tested by FDA microscopy using reagents purchased locally were USD $0.05 and required the same equipment, time and skills as auramine acid-fast microscopy.


We recommend a simple, bio-secure protocol for FDA microscopy that provides sensitive and repeatable results without requiring centrifugation.

Conflict of interest statement

All the authors declare that they have no competing interests exist in relation to this publication. The corresponding author had full access to all study data and was responsible for the decision to publish.

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