Antonello Maria, Scutari Rossana, Lauricella Calogero, Renica Silvia, Motta Valentina, Torri Stefania, Russo Cristina, Gentile Leonarda, Cento Valeria, Colagrossi Luna, Mattana Giordana, Codecasa Luigi Ruffo, Vismara Chiara, Scaglione Francesco, Veronese Silvio Marco, Bonoldi Emanuela, Bandera Alessandra, Gori Andrea, Mazzola Ester, Perno Carlo Federico, Alteri Claudia
Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.
Department of Experimental Medicine, University of Rome "Tor Vergata,"Rome, Italy.
Front Microbiol. 2021 Sep 13;12:727774. doi: 10.3389/fmicb.2021.727774. eCollection 2021.
Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available. MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/10 cells. IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values ( : 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/10 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550-8,444) copies/10 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples ( = 0.003 and 0.01 for rpoB and = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease. ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.
由于分枝杆菌数量较少且常常缺乏用于培养的新鲜样本，在肺外结核感染的不同部位快速、可靠地诊断结核病是一项诊断挑战。在此，我们评估了基于自制液滴数字PCR（ddPCR）的检测方法在89份活检样本中相对于培养的性能，这些样本有新鲜的以及福尔马林固定石蜡包埋（FFPE）的子样本。新鲜子样本中的结核分枝杆菌（MTB）诊断通过培养进行。新鲜子样本还进行了抗酸杆菌涂片显微镜检查（AFB）和Xpert MTB/RIF检测（Xpert）。通过针对IS6110和rpoB的内部ddPCR检测方法对89份FFPE子样本进行盲法重复MTB检测。使用H37Rv菌株的系列稀释液评估ddPCR检测方法的分析灵敏度。通过概率分析计算检测限（LOD）。结果以每10个细胞中的拷贝数表示。IS6110和rpoB的ddPCR检测方法在预期值和观察值之间显示出良好的线性相关性（分别为：0.9907和0.9743）。概率分析预测IS6110和rpoB的MTB DNA的LOD分别为每10个细胞17和40个拷贝。在89份活检样本中，68份培养阳性，21份培养阴性。以分枝杆菌培养作为参考方法，IS6110检测在67/68份培养阳性样本中产生阳性结果，每10个细胞的中位数四分位间距（IQR）为1680（550 - 8444）拷贝（灵敏度：98.5%；准确性：98.9%）。这些性能优于FFPE子样本中rpoB检测方法所报告的性能（灵敏度：66.20%；准确性：74.1%），甚至优于新鲜子样本中Xpert和AFB所报告的性能（灵敏度分别为：79.4%和33.8%；准确性分别为：84.3%和49.4%）。当根据rpoB和IS6110 ddPCR检测到的分枝杆菌载量对Xpert和AFB结果进行分层时，相对于Xpert和AFB阳性样本，Xpert和AFB阴性样本中的细菌载量较低（rpoB分别为 = 0.003和0.01，IS6110分别为 = 0.01和0.11），证实了这些方法在少菌型疾病中的低灵敏度。ddPCR在FFPE样本中提供了高度灵敏、准确且快速的MTB诊断，这由IS6110检测方法与培养结果之间的高度一致性所定义。这种方法可以安全地引入临床常规操作中，以加速MTB诊断，主要是在培养结果仍然不可用时。
