Corantes alternativos para uso no diagnóstico baciloscópico de Tuberculose.

Abstract

In almost all Tuberculosis control programs in the world, sputum smear analyze is the first choice diagnosis tool of the disease. The World Health Organization estimates that 150 million people, between the years 2002 and 2020, would have Tuberculosis, and this would be the minimum number of sputum smears to be performed during this period. The most widely stain used in smear staining is Ziehl-Neelsen s, described for over 125 years. This stain is based on the property that mycobacterias have, including Mycobacterium tuberculosis main causing tuberculosis, to withhold a dye solution composed of fuchsin and phenol (carbolfuchsin) in its cell wall, even after washing with acid-alcohol solution. However, carbolfuchsin is recognized as a carcinogenic substance in rodents, when introduced into diet. There is reference to danger of great harm to human health by prolonged exposure, with irreversible consequences. It is flammable, but heating is recommended for implementation of main coloring technique, which releases combustion gases and vapors hazardous if inhaled. No other less toxic substance was proposed yet. Thus, experimental study of evaluation of twelve alternate dyes on coloring smears was carried out, aimed at implementing in clinical samples suspected of Tuberculosis. In-test dyes solutions were prepared, at different concentrations and without phenol addition. For testing, positive acid-fast bacilli sputum smears and clinical isolate of M. tuberculosis smears were stained without heating, for 5 minutes, bleached with alcohol-acid solution for 1 minute, and counterstained with methylene blue, for 1 minute. The microscopic readings were held in transmitted light and in fluorescence (three filters), at immersion objective (100 x). The most promising dye was the alkaloid from vegetable species (AEV), which allowed fluorescence reading, at green and blue filters, without excessive aggregation to background cells and/or to mucous material present in sputum samples. Sensitivity and specificity tests, and comparison with current bacteriological method confirmed the potential use of AEV. Optimization tests for traditional microscopy reading were made with no success, a fact that indicates other testing. Three purified alkaloids and four alkaloids fractions from amazonian plant species were tested and considered promising for use in mycobacterial stain. They could be added to AEV, providing the ideal dye for Tuberculosis diagnosis, allowing both traditional and fluorescence microscopy.