Singh Sarman, Singh Amit, Prajapati Suneel, Kabra Sushil K, Lodha Rakesh, Mukherjee Aparna, Singh Varinder, Hesseling Anneke C, Grewal Harleen M S
Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110 029, India.
Department of Paediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110 029, India.
BMC Microbiol. 2015 Sep 29;15:191. doi: 10.1186/s12866-015-0528-z.
Tuberculosis (TB) in children is neglected, mainly due to lack of sensitive diagnostic tools. Recently Xpert MTB/RIF assay has revolutionized the diagnostic field, but its usefulness in pediatric TB has not been reported from India and no report is available on its use on long term archived samples.
We recruited 130 pediatric patients with probable intrathoracic tuberculosis and their gastric aspirate (GA) and induced sputum (IS) samples on 2 consecutive days were collected between January 2009 and December 2012. All samples (n = 520) were subjected to smear examination, BACTEC-MGIT culture and in-house multiplex PCR. An aliquot of each sample was stored at -80 °C and tested in Xpert MTB/RIF assay in 2013.
Sample wise and patient wise detection rate of smear microscopy was 4.4 % and 10 %, while for BACTEC-MGIT culture this rate was 24.4 % and 46.9 %, respectively. Of the 130 day 1 GA samples, 31.5 % and 27.7 % day 2 GA samples were culture positive. Only 17.7 % GA samples were positive on both days. Of the 130 IS samples collected on day 1 and day 2, 15.4 % and 23.1 % samples were culture positive. A combination of GA and IS yielded best results. Combining both GA and IS, the overall sensitivity of Xpert MTB/RIF on smear and culture positive samples was 95.6 %. In smear negative and culture positive samples its sensitivity was 62.5 %. The duration of sample storage impacted the Xpert MTB/RIF test performance (p = 0.0001). In smear positive samples stored for 650-849 days, its sensitivity was 85.7 % and 77.1 % for IS and GA samples which dropped to 33.3 % and 50 %, respectively, if stored for more than 1050 days.
Confirmatory diagnosis of tuberculosis particularly in children is a medical challenge. No laboratory or radiological test can reach to a satisfactory level of diagnostic sensitivity. However, in this study we found that combination of multiple samples and multiple diagnostic tests can give much better yield, though not optimum. In present study, combination of 2 gastric aspirates (GA) and 2 induced sputum (IS) samples collected on two consecutive days, and tested on three diagnostic methods yielded a significantly high detection rate. Despite long term storage, the overall sensitivity of Xpert MTB/RIF on smear and -culture positive samples remained very high. But after storing these samples under subfreezing conditions the sensitivity of Xpert MTB/RIF decreased significantly. This is expected because even if the sample is smear and culture positive, the count of surviving mycobacteria goes down, after several years this count can reach to a undetectable level.
This report shows that smear and culture positive samples stored at subfreezing conditions for several years can be used in the Xpert MTB/RIF assay, while maintaining appreciable diagnostic test sensitivity and specificity.
儿童结核病受到忽视，主要是由于缺乏灵敏的诊断工具。最近，Xpert MTB/RIF检测方法给诊断领域带来了变革，但印度尚未报道其在儿童结核病诊断中的应用情况，也没有关于其在长期存档样本中应用的报告。
我们招募了130例可能患有胸内结核病的儿科患者，并在2009年1月至2012年12月期间连续两天收集他们的胃抽吸物（GA）和诱导痰（IS）样本。所有样本（n = 520）均进行涂片检查、BACTEC - MGIT培养和内部多重聚合酶链反应。每个样本的一份等分试样保存在-80°C，并于2013年进行Xpert MTB/RIF检测。
涂片显微镜检查按样本和患者计算的检出率分别为4.4%和10%，而BACTEC - MGIT培养的检出率分别为二十四点四%和46.9%。在130份第一天的GA样本中，31.5%的第二天GA样本培养呈阳性。只有17.7%的GA样本两天均为阳性。在第一天和第二天收集的130份IS样本中，15.4%和23.1%的样本培养呈阳性。GA和IS的组合产生了最佳结果。将GA和IS两者结合，Xpert MTB/RIF对涂片和培养阳性样本的总体敏感性为95.6%。在涂片阴性和培养阳性样本中，其敏感性为62.5%。样本储存时间影响Xpert MTB/RIF检测性能（p = 0.0001）。在储存650 - 849天的涂片阳性样本中，其对IS和GA样本的敏感性分别为85.7%和77.1%，如果储存超过1050天，则分别降至33.3%和50%。
结核病的确诊，尤其是儿童结核病的确诊是一项医学挑战。没有任何实验室或放射学检查能达到令人满意的诊断敏感性水平。然而，在本研究中我们发现，多种样本和多种诊断检测方法的组合能产生更好的结果，尽管并非最佳。在本研究中，连续两天收集的2份胃抽吸物（GA）和2份诱导痰（IS）样本，并采用三种诊断方法进行检测，产生了显著较高的检出率。尽管长期储存，但Xpert MTB/RIF对涂片和培养阳性样本的总体敏感性仍然很高。但在亚冰点条件下储存这些样本后，Xpert MTB/RIF的敏感性显著下降。这是可以预料的，因为即使样本涂片和培养呈阳性，存活的分枝杆菌数量也会下降，几年后这个数量可能会降至不可检测的水平。
本报告表明，在亚冰点条件下储存数年的涂片和培养阳性样本可用于Xpert MTB/RIF检测，同时保持可观的诊断检测敏感性和特异性。
