Sequencing results among the discordant sample.

In India’s National Tuberculosis Elimination Program (NTEP), diagnosing tuberculosis (TB) and Drug-Resistant TB (DR-TB) involves collecting two sputum specimens from presumptive TB patients. Mycobacterium tuberculosis (MTB) is detected using the Truenat MTB - RIF assay at peripheral sites, with Trueprep for DNA extraction. In the case of a MTB positive, the second specimen is sent to a linked reference laboratory for Line Probe Assay (LPA) testing, requiring DNA extraction again. This study aimed to validate Trueprep-extracted DNA for First Line (FL) and Second Line (SL) LPA testing to assess real-time programmatic field conditions and DNA transportation across diverse Indian geographies. Four hundred MTB-positive Trueprep extracted DNA with ≥104 cfu/ml value from the first specimen, accompanied by the second sputum specimen, were transported from nine strategically selected Truenat sites (regions: South- 2, North- 2, East- 2, West- 3) to reference laboratories in a cool chain. The MTB-positive Trueprep-extracted DNA and the Genolyse-extracted DNA from the second specimen were subjected to FL and SL LPA testing, and the results were compared. Whole genome sequencing was performed for discordance resolution among mismatched Trueprep and Genolyse DNA results. Interpretable results were obtained in 397 (99.25%) of the 400 Trueprep-extracted DNA samples for FL LPA testing and 363 (90.75%) samples for SL-LPA testing. In comparison, Genolyse extracted DNA yielded 371 (99.46%) and 362 (97.05%) valid FL and SL LPA results, respectively, from 373 smear-positive specimens. Among 400 specimens, 160 were 105, 122 of 106, 101 of 104, 15 of 107, and 2 of 108 cfu/ml value. High concordance (97.7% for FL, 99.5% for SL LPA) was observed between Trueprep and Genolyse DNA. This study validates Trueprep-extracted DNA for reliable FL and SL-LPA testing in field settings, potentially streamlining NTEP’s diagnostic process, reducing reference laboratory workload, eliminating duplicate DNA extractions, and reducing the turnaround time (TAT).

在印度国家结核病消除规划（National Tuberculosis Elimination Program, NTEP）中，结核病（Tuberculosis, TB）与耐药结核病（Drug-Resistant TB, DR-TB）的诊断流程要求从疑似结核病患者体内采集两份痰液标本。基层检测机构采用Truenat MTB-RIF检测试剂盒完成结核分枝杆菌（Mycobacterium tuberculosis, MTB）的检测，核酸提取环节使用Trueprep试剂。若检测结果为MTB阳性，则将第二份痰液标本送至关联的参考实验室开展线性探针检测（Line Probe Assay, LPA），该环节需再次进行核酸提取。
本研究旨在验证经Trueprep试剂提取的核酸可用于一线（First Line, FL）与二线（Second Line, SL）LPA检测，以评估真实的项目现场实施条件，以及核酸在印度不同地域间的转运情况。研究纳入400份来自首份痰液标本的、菌落形成单位每毫升（cfu/ml）≥10^4的MTB阳性Trueprep提取核酸样本，同时配套第二份痰液标本，从9个经过策略性筛选的Truenat检测站点（地域分布：南部2个、北部2个、东部2个、西部3个）通过冷链转运至参考实验室。
随后分别对上述Trueprep提取的MTB阳性核酸，以及第二份标本经Genolyse试剂提取的核酸开展一线与二线LPA检测，并对检测结果进行比对。对于Trueprep与Genolyse核酸检测结果不一致的样本，采用全基因组测序开展不一致性解析。
在400份Trueprep提取核酸样本中，397份（99.25%）获得了可解读的一线LPA检测结果，363份（90.75%）获得了可解读的二线LPA检测结果。相较而言，针对373份涂片阳性标本，Genolyse提取的核酸可获得371份（99.46%）有效一线LPA检测结果与362份（97.05%）有效二线LPA检测结果。
全部400份样本中，160份的菌落形成单位浓度为10^5 cfu/ml，122份为10^6 cfu/ml，101份为10^4 cfu/ml，15份为10^7 cfu/ml，2份为10^8 cfu/ml。Trueprep与Genolyse提取的核酸检测结果具有高度一致性：一线LPA检测一致性达97.7%，二线LPA检测一致性达99.5%。
本研究验证了Trueprep提取的核酸可在现场环境中可靠用于一线与二线LPA检测，有望简化NTEP的诊断流程、减轻参考实验室的工作负担、省去重复的核酸提取步骤，并缩短检测周转时间（Turnaround Time, TAT）。