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

Abstract

Background: 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.

Methods and results: 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.

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

Fig 1. Photograph demonstrating the characteristic cording colony of Mycobacterium tuberculosis.

There are 11 colonies in this image.

Fig 2. Optimisation study demonstrating the median colony forming units (CFU) or bacilli concentration per ml in each culture and microscopy technique.

Error bars indicate the interquartile range. All microscopy and quantitative culture concentration data were transformed to logarithmic (log) base 10 values. Note. FDA = fluorescein diacetate, AA = acid-alcohol, KMnO4 = potassium permanganate, s = seconds.

Fig 3. Optimisation study.

Bar graphs comparing the quantitative sensitivity of fluorescein diacetate (FDA) microscopy protocols A-G: (A) to identify acid-fast bacilli (AFB), shown as the difference in logarithmic base 10 counts between FDA bacilli/ml of sample versus AFB/ml in acid-fast (auramine) (B) to identify colony-forming units (CFU), shown as the difference in logarithmic base 10 counts between FDA bacilli/ml of sample versus CFU/ml in quantitative culture. Error bars indicate 95% confidence intervals.

Fig 4. Validation study.

A Bland-Altman plot demonstrating agreement between the bacilli count per 100 high powered fields in fluorescein diacetate (FDA) microscopy protocol F versus FDA microscopy protocol G. The difference between these 2 protocols is that the latter involves potassium permanganate quenching for 30 seconds longer. All count data were transformed to their logarithmic base 10 (log) value. The mean difference is -0.007 log (solid line), and the limits of agreement (dotted lines) are -0.70 to 0.71 log.

Fig 5. Storage study.

Graphs showing the number of bacilli per 100 fields at 8 time-points during the 4-week experiment, comparing fresh versus stored fluorescein diacetate (FDA) working solution in each patient sample storage condition and healthy control sample. Note: RT = room temperature.

Fig 6. Sensitivity of fluorescein diacetate (FDA) microscopy.

FDA sensitivity to detect Mycobacterium tuberculosis-positive slides using results from both the optimisation study, which compared FDA microscopy protocols A-G, and the storage study which only used FDA microscopy protocol F. Error bars indicate 95% confidence intervals. Box 1 describes the different FDA microscopy protocols used.