RESUMEN
Blinded rechecking is a method proposed for external quality assurance (EQA) of auramine-stained acid-fast bacilli (AFB) smears using fluorescence microscopy (FM), however, this procedure is not well developed and slides fading over time could compromise its implementation. Since bleaching of fluorescent molecules involves temperature-dependent chemical reactions, it is likely that low temperatures could slow down this process. We stored auramine-stained slides under different environmental conditions, including -20°C, and examined them over time. The slides stored in all the environments faded. At -20°C, fading was not reduced in relation to room temperature. Restaining and re-examining smears after five months showed that the slides containing saliva and storage at -20°C were associated with failure in AFB reappearance. In conclusion, the practice of freezing slides until they are viewed should be discouraged as it has a negative effect on blinded rechecking by reducing reading concordance after restaining. Specimen quality should be considered when interpreting FM-EQA results.
Asunto(s)
Benzofenoneido/efectos de la radiación , Colorantes Fluorescentes/efectos de la radiación , Microscopía Fluorescente/métodos , Fotoblanqueo , Garantía de la Calidad de Atención de Salud/métodos , Esputo/microbiología , Coloración y Etiquetado/métodos , Tuberculosis/diagnóstico , Argentina , Benzofenoneido/análisis , Criopreservación , Estudios de Factibilidad , Colorantes Fluorescentes/análisis , Humanos , Iluminación , Microscopía Fluorescente/instrumentación , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Preservación Biológica/métodos , Reproducibilidad de los Resultados , Método Simple Ciego , TemperaturaRESUMEN
Current methods for drug susceptibility testing of Mycobacterium tuberculosis are either costly or slow. As the prevalence of multidrug-resistant strains increases, the need for fast, reliable, and inexpensive methods that can also be applied in settings with scarce resources is obvious. We evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing of M. tuberculosis directly from clinical sputum samples with positive microscopy results for acid-fast bacilli with more than 10 acid-fast bacilli per high-power field. We have saved valuable time by omitting the preisolation step. The sensitivity (ability to detect true drug resistance) and specificity (ability to detect true drug susceptibility) of the direct NRA, using the direct proportion method as the reference, were 100 and 100%, 93 and 100%, 76 and 100%, and 55 and 99% for rifampin, isoniazid, streptomycin, and ethambutol, respectively, when tested on M. tuberculosis strains present in 121 samples. The results were in most cases available in 14 days. The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.