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

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Abstract:

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.

Keywords: acid-fast bacilli; fluorescence microscopy; light-emitting diode; methylene blue; tuberculosis

Document Type: Regular Paper

Affiliations: 1: International Union Against Tuberculosis and Lung Disease, Paris, France; and Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium 2: Damien Foundation, Dhaka, Bangladesh 3: Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium

Publication date: 2010-12-01

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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