Evaluation of bleach-sedimentation for sterilising and concentrating Mycobacterium tuberculosis in sputum specimens

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. Methods This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. Results M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. Conclusions Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.Peer Reviewe

Niveles de resistencia a drogas antituberculosas en pacientes con infección VIH / Tuberculosis, Lima, 1998-2001

Objetivos: Determinar los niveles de resistencia a drogas antituberculosas de primera línea en muestras clínicas de pacientes con coinfección por el virus de la inmunodeficiencia humana y tuberculosis (VIH-TB) en cinco hospitales de Lima en el periodo 1998-2000. Materiales y métodos: Estudio descriptivo que incluyó las muestras de cultivos de Mycobacterium tuberculosis de pacientes con coinfección VIH-TB, a los que se realizó las pruebas de sensibilidad por el método de las proporciones para isoniacida (H), estreptomicina (S), etambutol (E) y rifampicina (R), y el método de Wayne para pirazinamida (H). Resultados: De 523 muestras de pacientes incluidos, 78,2% correspondieron a varones, 72,7% fueron de pacientes sin antecedentes de tratamiento previo. Los valores de resistencia global primaria y multidrogorresistencia (MDR) primaria fueron 55,8% y 32,1%, respectivamente; en tanto que los valores de resistencia global adquirida y MDR adquirida fueron 93,0% y 74,8%. La resistencia primaria por drogas fue H (42,1%), R (35,0%), S (35,3%), E (19,0%) y Z (24,5%) respectivamente; y la resistencia adquirida por drogas fueron H (85,3%), R (78,3%), S (64,4%), E (42,0%) y Z (46,2%). Conclusión: Los niveles resistencia a drogas antituberculosas y la MDR en pacientes con coinfección VIH-TB provenientes de hospitales de Lima son elevados