Olson Alaina M, Wood Rachel C, Weigel Kris M, Yan Alexander J, Lochner Katherine A, Dragovich Rane B, Luabeya Angelique K, Yager Paul, Hatherill Mark, Cangelosi Gerard A
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA.
Department of Bioengineering, University of Washington, Seattle, Washington, USA.
J Clin Microbiol. 2025 Feb 19;63(2):e0114024. doi: 10.1128/jcm.01140-24. Epub 2024 Dec 31.
Tongue swab (TS) sampling combined with quantitative PCR (qPCR) to detect (MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-µL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS and IS. Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% ± 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum microbiological reference standard (MRS; sputum culture and/or Xpert Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 µL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS.
Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.
舌拭子（TS）采样联合定量聚合酶链反应（qPCR）检测结核分枝杆菌（MTB）DNA是一种有前景的替代痰液检测的结核病（TB）诊断方法。在先前的研究中，舌拭子采样的灵敏度通常低于痰液。在本研究中，我们评估了两种提高灵敏度的策略。一种策略是使用离心法从大容量泡沫拭子样本洗脱的2 mL悬浮液中浓缩舌背细菌。将沉淀重悬为500 μL悬浮液，然后在进行双靶点qPCR检测MTB插入元件IS和IS之前进行机械裂解。分级实验表明，临床拭子样本中大部分MTB DNA信号（99.22%±1.46%）存在于可沉淀部分。当应用于从124名疑似结核病的南非人收集的存档泡沫拭子时，相对于痰液微生物学参考标准（MRS；痰液培养和/或Xpert Ultra），该策略表现出83%的灵敏度（71/86）和100%的特异性（38/38）。第二种策略是使用序列特异性磁捕获（SSMaC）来浓缩MTB细胞释放的DNA。该方案在从128名疑似结核病的南非参与者收集的存档Copan FLOQSwabs植绒拭子样本上进行了评估。洗脱到500 μL缓冲液中的物质进行机械裂解。悬浮液用蛋白酶K消化，与生物素化的双靶点寡核苷酸探针杂交，然后使用磁分离浓缩约20倍。对浓缩物进行双靶点qPCR检测时，相对于痰液MRS，该策略表现出90%的灵敏度（83/92）和97%的特异性（35/36）。这些结果为在舌拭子中检测MTB DNA的自动化、高灵敏度方法指明了方向。
需要改进结核病（TB）检测方法。使用比痰液更容易获取的样本类型可能能够检测到更多病例，但对替代样本进行适当检测至关重要。在这里，我们描述了两种用于检测舌拭子中TB DNA的新型、高度准确的方法。
