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

Migration des virus dans la plante. Revue bibliographique

Afin de générer une infection, un virus doit migrer dans sa plante hôte. Il existe 2 formes de migration des virus dans la plante : la migration à courte distance et la migration à longue distance. La migration à courte distance s'effectue de cellule à cellule. Au cours de ce processus, le virus migre des cellules infectées aux cellules adjacentes par les plasmodesmes. Plusieurs résultats indiquent que ce type de transport est sous le contrôle partiel du virus. En effet, chez différents phytovirus tels que le TMV, le TRV, le CPMV, l'AMV, le CaMV..., une ou plusieurs protéines, ayant une fonction de transport et dont les mécanismes d'action sont discutés, ont été caractérisées. La migration à longue distance qui s'effectue par les vaisseaux conducteurs est indispensable à l'établissement d'une infection systémique. Les mécanismes de la migration à longue distance restent l'objet de controverses car il n'est pas encore tout à fait établi si ce processus nécessite l'activité d'une fonction de transport ou si c'est un phénomène passif lié au transport des assimilats. Certains virus qui produisent normalement une infection subliminale ou qui restent localisés dans certains tissus se généralisent dans la plante lorsqu'ils sont co-inoculés avec d'autres qui sont eux systémiques. Apparemment, le virus assistant code pour une fonction qui manque au virus assisté dans cette plante, et cette fonction est probablement impliquée dans le transport. Cette revue présente une synthèse des différents aspects du transport des virus dans la plante de même que les mécanismes de la résistance des plantes à la migration des virus.The movement of viruses within the plant: a review. In order to generate a productive infection, a virus must move within its host plant. There are 2 forms of virus movement within a plant, namely, short distance and long distance. The short-distance movement is from cell to cell. During this process, the virus moves to an adjacent cell through the protoplasmic bridges, the plasmodesmata. Several results indicate that this form of spread is virus controlled. Indeed, proteins to which a movement function has been assigned have been identified in a number of plant viruses, such as TMV, TRV, CPMV, AMV and CaMV, to name a few. The long-distance movement takes place in the vascular tissues and it is a phenomenon essential in the establishment of a systemic infection. The mechanism of the long-distance spread is poorly understood. It is not yet clear whether movement in the vascular system requires the activity of a spread function or whether it is a passive process within the phloem elements. In certain combinations of viruses, one virus, which normally only establishes a subliminal or tissue-localized infection, invades other tissues along with the other virus. The "helper" virus apparently provides a function, presumably involved in spread, that the "helped" virus lacks in that plant. This review has sought to deal with the different aspects of virus spread within a plant and also with the mechanisms of plant resistance to virus movement