A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay
A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay
复制标题
DOI：
10.1371/journal.pone.0151980
复制
发表时间：
2016-03-23
期刊：
影响因子：
3.7
通讯作者：
Alland, David
中科院分区：
文献类型：
--
作者：
Banada, Padmapriya P.;Naidoo, Uvistra;Alland, David
背景使用分子检测很难诊断儿童结核病（TB），因为儿童难以提供呼吸道样本。如果有足够的样本处理技术，粪便可以代替痰来进行结核病诊断检测。方法我们开发了一种快速方法来处理大量粪便，以便通过 Xpert MTB/RIF (Xpert) 结核病检测分析进行下游检测。该方法在添加了已知数量的结核分枝杆菌菌落形成单位 (CFU) 的粪便样本和结核分枝杆菌感染的食蟹猴 (Macaca fasciculis) 的粪便中进行了测试和优化。根据阳性 Xpert 测试数量、Xpert 样品处理对照 (SPC) 的循环阈值 (Cts) 以及结核分枝杆菌特异性 rpoB 探针的 Cts 来对性能进行评分。然后，该方法在南非德班的 20 名确诊结核病例和 20 名对照中进行了验证。结果该测定的分析检测限为 1,000 CFU/g 粪便。可以测试多达 1 克的加标粪便，而不会显示 PCR 抑制增加。在使用人体粪便的分析加标实验中，与少量样品相比，1g 样品提供了最佳灵敏度。然而，在患有结核病的猕猴中，0.6 克粪便样本的表现优于 0.2 克或 1.2 克样本。对儿童结核病嫌疑人和对照者的粪便进行检测表明，对于 0.6g 和 1.2g 粪便样本，检测灵敏度分别为 85% (95% CI 0.6-0.9) 和 84% (95% CI 0.6-0.96)，特异性为 100% (95% CI 0.77-1) 和 94% (95% CI 0.7-0.99)。结论这种新方法可以使用儿科粪便样本简单快速地检测结核病。
BackgroundTuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.MethodsWe developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.ResultsThe assay's analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6-0.9) and 84% (95% CI 0.6-0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77-1) and 94% (95% CI 0.7-0.99), respectively.ConclusionThis novel approach may permit simple and rapid detection of TB using pediatric stool samples.