Methylene blue is a good background stain for tuberculosis light-emitting diode fluorescence microscopy

SETTING: Damien Foundation Bangladesh tuberculosis (TB) control projects. OBJECTIVES: To compare blue ink, potassium permanganate and methylene blue background staining for transmitted light-emitting diode (LED) TB fluorescence microscopy (FM). DESIGN: Auramine smears made in triplicate from Ziehl-Neelsen (ZN) acid-fast bacilli (AFB) positive or negative sputum and stained with one of the background variations were read blind by LED FM. Reference laboratory rechecking of discordant series was used before and after auramine restaining as the gold standard. RESULTS: Of 1977 series evaluated, 991 (50.1%) were made from ZN-positive specimens. There were 919, 942 and 958 FM true-positives with blue ink, permanganate and methylene blue counterstaining, against respectively 12, 12 and 16 false-positives. Methylene blue counterstaining was more sensitive (95.6%, 95%CI 94.2-96.8) than blue ink or permanganate (92.7%, 95%CI 90.9-94.3 and 93.6%, 95%CI 91.9-95.0; respectively P < 0.01 and <0.05). No AFB could be found in 85% and 18% of 180 discordant series without and with restaining. CONCLUSIONS: Methylene blue is at least equivalent to potassium permanganate counterstaining for FM using blue LED transmitted excitation and is cheaper than blue ink. Restaining of all smears prior to first re-reading may be unavoidable for blinded rechecking of auramine-stained smears for external quality assessment.