Microbial Interface Biology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany.
Institute for Medical Biometry and Statistics, University of Lübeck, University Hospital Schleswig-Holstein, Kiel, Germany.
PLoS One. 2022 Jul 15;17(7):e0265554. doi: 10.1371/journal.pone.0265554. eCollection 2022.
Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test.
Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types.
We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts.
Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.
肺结核（TB）通过痰液样本进行诊断。由于儿童和消瘦患者的痰液采样具有挑战性，因此开发使用唾液的诊断测试似乎很有前景，但由于细菌载量低和灵敏度差而受到阻碍。在这里，我们提出了一种从唾液中富集结核分枝杆菌（Mtb）的新颖而快速的方法，该方法可能为诊断性唾液测试提供基础。
脂质体生物素化磁珠（LMB）与含 Mtb 的 PBS 和健康供体的唾液以及 TB 患者的唾液孵育。流式细胞术用于评估珠子结合 Mtb 的能力，而实时定量聚合酶链反应（qPCR）用于检测 Mtb 并确定不同样本类型中分枝杆菌 DNA 的量。
与非功能化珠子相比，我们发现 LMB 可有效结合 Mtb。基于 LMB 开发的 qPCR 检测方法（LMB 检测法）使我们能够在添加样本类型中富集分枝杆菌 DNA，包括 PBS 和健康供体的唾液（富集倍数高达约 8.7 倍）。在 Mtb 污染的唾液样本中，我们发现 LMB 检测法将 5ml 体积中 102 个细菌的检测率从 15 个样本中 0 个（0%）提高到 15 个样本中的 6 个（40%）。与此一致，LMB 检测法增加了两个独立队列中 TB 患者唾液样本的正确识别率。
实施基于 LMB 的检测方法的原理可能会提高现有诊断技术的灵敏度，例如通过功能化从口腔中采样的材料来提高 Mtb 采样的效率。
