IFHAD: Innovation For Health And Development, London, UK.
BMC Infect Dis. 2011 Oct 11;11:269. doi: 10.1186/1471-2334-11-269.
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.
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.
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.
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.
漂白沉淀法通过对痰液进行消毒和浓缩结核分枝杆菌，可以提高显微镜检查诊断结核病的效果。我们研究了重力漂白沉淀法对痰涂片镜检的安全性、敏感性、速度和可靠性的影响。
这项盲法、对照研究使用了 72 例结核病患者的痰液标本。确定了消毒痰液所需的漂白剂浓度和暴露时间（n=31）。根据这些结果，比较了 5 种可使痰液标本消毒的重力漂白沉淀技术（n=16）的性能。表现最好的一种漂白沉淀技术是向 1 份痰液中加入 1 份体积的 5%漂白剂，震荡 10 分钟，用 8 份蒸馏水稀释，过夜沉淀后再进行显微镜检查。该技术进一步通过比较在痰标本漂白沉淀前后（n=25）肉眼可见抗酸杆菌数量、玻片阅读速度和重复性进行了评估。制作了 3 份涂片以提高精度，并使用齐-尼染色法进行染色。
向 1 份痰液中加入 15%漂白剂 1 分钟、6%漂白剂 5 分钟或 3%漂白剂 20 分钟，可成功地对痰液中的 M. tuberculosis 进行消毒。与常规涂片相比，漂白沉淀法显著减少了肉眼可见的抗酸杆菌数量（100 个显微镜视野中每 100 个抗酸杆菌的几何平均值分别为 166（95%CI 68-406）和 346（95%CI 139-862）；p=0.02）。漂白沉淀法对低菌载量标本的稀释作用小于对高浓度可见抗酸杆菌标本的稀释作用（p=0.02）。从漂白沉淀后的痰液中制作的涂片比常规涂片阅读速度更快（分别为 9.6 分钟和 11.2 分钟，p=0.03）。常规抗酸杆菌计数的观察者间一致性很高（r=0.991），但通过漂白沉淀后显著降低（p=0.03）至 r=0.707，因为偶尔强烈阳性的漂白沉淀涂片会被误读为阴性。
重力漂白沉淀法通过对痰液进行消毒提高了实验室安全性，但降低了显微镜下可见的抗酸杆菌浓度，特别是对含有高浓度 M. tuberculosis 的痰液标本。漂白沉淀法允许在可用的时间内检查更多的标本，但降低了观察者间阅读涂片的可靠性。因此，漂白沉淀法的效果取决于标本特征以及显微镜检查是按规定时间进行还是规定时间内阅读完规定数量的显微镜视野。这些发现解释了先前研究结果相互矛盾的原因。
