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Abstract：
The diagnosis of pleural tuberculosis (TB) remains difficult due to the paucity of Mycobacterium tuberculosis in pleural fluid (PF). This study aimed to improve pleural TB diagnosis using highly sensitive digital PCR (dPCR) technique. A total of 310 patients with evidence of PF were consecutively enrolled, 183 of whom suffered from pleural TB and 127 from non-TB. PF samples were prospectively collected and total DNA was extracted. The copy numbers of M. tuberculosis insertion sequence (IS) 6110 and IS1081 in DNA were quantified using dPCR. The overall area under the curve of IS6110-dPCR was greater than that of IS1081-dPCR (0.85 versus 0.79). PF IS6110 OR IS1081-dPCR (according to their cut-off values, \"positive\" was defined as either of them was positive, while \"negative\" was defined as both of them were negative) had higher sensitivity and equal specificity compared with single target-dPCR. The sensitivity of PF IS6110 OR IS1081-dPCR for total, definite, and probable pleural TB was 59.0% (95% CI = 51.5% to 66.2%), 72.8% (95% CI = 62.6% to 81.6%), and 45.1% (95% CI = 34.6% to 55.8%), respectively. Its specificity was 100% (95% CI = 97.1% to 100.0%). PF IS6110 OR IS1081-dPCR showed a higher sensitivity than smear microscopy (57.4% versus 7.1%), mycobacterial culture (55.3% versus 31.8%), and Xpert MTB/RIF (57.6% versus 23.0%). Long antituberculosis treatment time (>1 month) was found to be associated with negative dPCR results in pleural TB patients. This study indicates that PF IS6110 OR IS1081-dPCR is an accurate molecular assay, which is more sensitive than routine etiological tests and has the potential to enhance the definite diagnosis of pleural TB. IMPORTANCE Pleural TB is one of the most frequent causes of pleural effusion, especially in areas with high burden of TB. Due to the paucibacillary nature of the disease, the diagnostic sensitivities of all available bacteriological and molecular tests remain poor. There is an urgent need to develop new efficient methods. Digital PCR (dPCR) is the third generation of PCR that enables the exact quantification of trace nucleic acids in samples. This study evaluates the diagnostic performance of pleural fluid (PF) dPCR analysis for pleural TB, and shows that PF IS6110 OR IS1081-dPCR has a higher sensitivity than routine etiological tests such as smear microscopy, mycobacterial culture, and Xpert MTB/RIF. This work provides a new choice for improving the definite diagnosis of pleural TB.
摘要：
由于胸膜液（PF）中结核分枝杆菌的缺乏，胸膜结核（TB）的诊断仍然很困难。本研究旨在使用高灵敏度数字PCR（dPCR）技术提高胸膜结核的诊断水平。连续纳入310例具有PF证据的患者，其中183例患有胸膜结核，127例非结核。前瞻性收集PF样品并提取总DNA。使用dPCR定量DNA中结核分枝杆菌插入序列(IS)6110和IS1081的拷贝数。IS6110-dPCR的曲线下总面积大于IS1081-dPCR(0.85对0.79)。PFIS6110或IS1081-dPCR(根据其截止值，“阳性”被定义为它们中的任何一个是阳性的，而“阴性”定义为两者均为阴性）与单靶dPCR相比具有更高的灵敏度和相同的特异性。PFIS6110或IS1081-dPCR的总灵敏度，确定，可能的胸膜结核为59.0％（95％CI=51.5％至66.2％），72.8%（95%CI=62.6%至81.6%），和45.1%(95%CI=34.6%至55.8%)，分别。其特异性为100%(95%CI=97.1%至100.0%)。PFIS6110或IS1081-dPCR显示出比涂片镜检更高的灵敏度（57.4%对7.1%），分枝杆菌培养（55.3%对31.8%），和XpertMTB/RIF（57.6%对23.0%）。在胸膜结核患者中，抗结核治疗时间长（>1个月）与dPCR结果阴性相关。这项研究表明，PFIS6110或IS1081-dPCR是一种准确的分子检测方法，比常规病因检查更敏感，有可能增强胸膜结核的明确诊断。重要性胸膜结核是胸腔积液的最常见原因之一，特别是在结核病负担较高的地区。由于这种疾病的低杆菌性质，所有可用的细菌学和分子测试的诊断敏感性仍然很差。迫切需要开发新的有效方法。数字PCR(dPCR)是能够精确定量样品中的痕量核酸的第三代PCR。这项研究评估了胸膜液（PF）dPCR分析对胸膜结核的诊断性能，并显示PFIS6110或IS1081-dPCR比常规病因检查如涂片显微镜检查具有更高的灵敏度，分枝杆菌培养，和XpertMTB/RIF。该工作为提高胸膜结核的明确诊断提供了新的选择。
