Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, Lima, Peru.
J Clin Microbiol. 2010 May;48(5):1820-6. doi: 10.1128/JCM.01161-09. Epub 2010 Mar 3.
Pulmonary tuberculosis diagnosis is difficult when patients cannot produce sputum. Most sputum is swallowed, and tuberculosis DNA can survive intestinal transit. We therefore evaluated molecular testing of stool specimens for detecting tuberculosis originating from the lungs. Paired stool and sputum samples (n=159) were collected from 89 patients with pulmonary tuberculosis. Control stool samples (n=47) were collected from patients without tuberculosis symptoms. Two techniques for DNA extraction from stool samples were compared, and the diagnostic accuracy of the PCR in stool was compared with the accuracy of sputum testing by PCR, microscopy, and culture. A heminested IS6110-PCR was used for tuberculosis detection, and IS6110-PCR-positive stool samples then underwent rifampin sensitivity testing by universal heteroduplex generator PCR (heteroduplex-PCR) assay. For newly diagnosed pulmonary tuberculosis patients, stool IS6110-PCR had 86% sensitivity and 100% specificity compared with results obtained by sputum culture, and stool PCR had similar sensitivities for HIV-positive and HIV-negative patients (P=0.3). DNA extraction with commercially available spin columns yielded greater stool PCR sensitivity than DNA extraction with the in-house Chelex technique (P=0.007). Stool heteroduplex-PCR had 98% agreement with the sputum culture determinations of rifampin resistance and multidrug resistance. Tuberculosis detection and drug susceptibility testing by stool PCR took 1 to 2 days compared with an average of 9 weeks to obain those results by traditional culture-based testing. Stool PCR was more sensitive than sputum microscopy and remained positive for most patients for more than 1 week of treatment. In conclusion, stool PCR is a sensitive, specific, and rapid technique for the diagnosis and drug susceptibility testing of pulmonary tuberculosis and should be considered when sputum samples are unavailable.
当患者无法产生痰液时，肺结核的诊断较为困难。大部分痰液会被吞咽，而结核 DNA 可以在肠道转运中存活。因此，我们评估了粪便标本中用于检测源自肺部的结核病的分子检测。收集了 89 例肺结核患者的配对粪便和痰液样本（n=159）。收集了无肺结核症状的患者的对照粪便样本（n=47）。比较了两种从粪便样本中提取 DNA 的技术，并比较了粪便 PCR 检测的诊断准确性与痰液 PCR、显微镜检查和培养的准确性。使用 heminested IS6110-PCR 进行结核检测，然后对 IS6110-PCR 阳性的粪便样本进行 rifampin 敏感性检测，采用通用异源双链体生成器 PCR（异源双链体-PCR）法。对于新诊断的肺结核患者，粪便 IS6110-PCR 的敏感性为 86%，特异性为 100%，与痰培养的结果相当，且 HIV 阳性和 HIV 阴性患者的粪便 PCR 敏感性相似（P=0.3）。与使用自制 Chelex 技术相比，商用 spin 柱提取 DNA 可提高粪便 PCR 的敏感性（P=0.007）。粪便异源双链体-PCR 与痰培养对 rifampin 耐药和耐多药的检测结果有 98%的一致性。与传统基于培养的检测相比，粪便 PCR 检测肺结核和药物敏感性测试需要 1 到 2 天，而平均需要 9 周才能获得这些结果。粪便 PCR 比痰液显微镜检查更敏感，并且在接受治疗超过 1 周的时间内，大部分患者的粪便 PCR 仍呈阳性。总之，粪便 PCR 是一种敏感、特异、快速的肺结核诊断和药物敏感性检测技术，当无法获得痰液样本时应考虑使用。
