A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB). We found no published evidence assessing whether this is beneficial. Objective. To evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, 6 smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The number of acid-fast bacilli (AFB)/100 fields, the number of precipitates/100 fields, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used generalized linear regression models adjusted by staining technique with a random effects term to correct for between-sample variability. Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process. Conclusions: Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

背景：部分痰涂片显微镜检查规程建议，在结核分枝杆菌（Mycobacterium tuberculosis, TB）染色过程中，将滤纸覆盖于痰涂片之上。目前尚未有公开研究评估该操作是否具有益处。目的：评估滤纸对痰涂片显微镜检查结果的影响。方法：从30例确诊肺结核患者与4名健康对照受试者中采集痰液样本。每份痰液样本制备6张涂片，总计204张，分别采用齐-尼染色法（Ziehl-Neelsen, ZN）、金胺染色法或二乙酸荧光素（fluorescein diacetate, FDA）活性染色。对每种染色规程下的半数玻片进行随机分组：在染色液涂布前，将沃特曼（Whatman）3级滤纸覆盖于干燥后的涂片之上，并在染色液冲洗前移除滤纸。记录每100视野下的抗酸杆菌（acid-fast bacilli, AFB）数量、每100视野下的沉淀物数量，以及涂片被冲洗掉的比例。统计分析采用经染色技术校正的广义线性回归模型，并引入随机效应项以校正样本间的变异性。结果：在染色规程中使用滤纸，可显著降低显微镜检查的阳性率，且该效应不受ZN染色、金胺染色或FDA染色的影响（p=0.01）。与此一致的是，使用滤纸的涂片其镜检分级低于未使用滤纸的组别（p=0.04）；且无论采用何种染色技术，使用滤纸均可使AFB计数降低0.28个对数单位（95%置信区间，CI=0.018, 0.54，p=0.04）。所有分析均显示，金胺染色的敏感性始终高于ZN染色，AFB计数更高（p=0.001）；而FDA染色的敏感性与AFB计数均更低（p<0.0001）。在染色过程中，滤纸的使用与沉淀物的出现（p=0.5）或涂片被冲洗掉的概率（p=0.6）均无关联。结论：滤纸会降低抗酸杆菌显微镜检查的敏感性，且未发现可检测到的益处，因此不建议使用该操作。