Data Sheet 1_Evaluation of host-immune biomarker signatures as multiplex qPCR diagnostic assays: a pilot study toward meeting WHO target product profiles for TB diagnosis in India.xlsx

BackgroundTuberculosis (TB) remains the leading cause of death from a single infectious agent. Current diagnostic tools are limited, especially in low-resource settings. The World Health Organization's (WHO) Target Product Profiles (TPPs) call for rapid, non-sputum-based diagnostics with high sensitivity and specificity. This study evaluates our previously published TB-associated host-immune biomarkers alongside small-size signatures from other studies, in our previously published non-human primate (NHP) TB infection study dataset (GSE76703) and two previously published human datasets (GSE144127, GSE42834), which include other disease group comparators, including sarcoidosis. These were also evaluated in a small-scale, exploratory qPCR pilot study to assess the feasibility of implementing these previously validated signatures in a South Indian TB patient cohort, comparing their diagnostic performance against WHO TPP criteria.
MethodsTwenty-six genes from published signatures (INDUK, Roe1/Roe3, Sweeney3, RISK6) were analyzed in these NHP and human datasets, using network and machine learning approaches, prior to exploratory evaluation using single and multiplex qPCR assays. These were tested using peripheral blood sample RNAs from pulmonary TB (PTB) (n = 15) and extrapulmonary TB (EPTB) (n = 15) patients and high-incidence controls (n = 15). The diagnostic performance of biomarkers, prior signatures, and novel promising combinations were assessed against WHO TPPs for triage and confirmatory tests.
ResultsSeveral biomarker signatures successfully distinguished active TB ((ATB) PTB and EPTB combined) from controls. The minimal INDUK signature (GBP1 + IFIT3) met the optimal TPP criteria for both triage and confirmatory testing for PTB (100% sensitivity and specificity, area under the receiver operating characteristic curve (AUROC:1)) and achieved the 80% sensitivity, 100% specificity threshold for EPTB (AUROC: 0.92 CI: 0.8261–1.00). Combined signatures incorporating genes from INDUK, Roe1, and Sweeney3 further improved diagnostic accuracy for ATB overall (AUROC: 0.98 95% CI: 0.9472–1.00).
ConclusionThis preliminary pilot study demonstrates successful evaluation of biomarker signatures as diagnostic qPCR assays for TB diagnosis and, to our knowledge, is the first study to demonstrate the potential for combined host-immune biomarker signatures from different studies that meet WHO TPP benchmarks. These findings support the potential for the development of low-cost, field-adaptable diagnostic tools. Further validation is now under way on a larger cohort of TB patients and controls.

研究背景：结核病（TB）仍是单一传染源致死的首要病因。当前的结核病诊断工具存在局限，在资源匮乏地区尤为如此。世界卫生组织（WHO）的目标产品概况（Target Product Profiles, TPPs）呼吁开发快速、无需痰液样本且具备高灵敏度与高特异性的诊断方法。本研究针对我们既往发表的结核病相关宿主免疫生物标志物，结合其他研究中的小型特征集，在我们此前发布的非人灵长类（NHP）结核病感染研究数据集（GSE76703）以及两项既往发布的人类数据集（GSE144127、GSE42834）中进行了评估；上述数据集均纳入了包括结节病在内的其他疾病对照组别。此外，本研究还通过一项小型探索性定量聚合酶链反应（qPCR）预实验，评估了在印度南部结核病患者队列中应用上述既往验证过的特征集的可行性，并将其诊断性能与WHO目标产品概况标准进行了比对。

研究方法：本研究在上述非人灵长类与人类数据集中，采用网络分析与机器学习方法，对已发表特征集（INDUK、Roe1/Roe3、Sweeney3、RISK6）中的26个基因进行了分析，随后通过单重与多重qPCR实验开展探索性评估。本研究使用来自肺结核（PTB，n=15）、肺外结核（EPTB，n=15）患者以及高发对照人群（n=15）的外周血RNA样本开展上述实验。研究针对生物标志物、既往特征集以及新型潜在组合的诊断性能进行了评估，并对照WHO目标产品概况标准，明确其用于分诊检测与确认检测的适用性。

研究结果：多项生物标志物特征集可有效区分活动性结核病（ATB，即合并肺结核与肺外结核的病例）与对照组样本。简化版INDUK特征集（GBP1 + IFIT3）满足肺结核分诊与确认检测的最优目标产品概况标准（灵敏度与特异性均为100%，受试者工作特征曲线下面积AUROC=1）；针对肺外结核，该特征集达到了灵敏度80%、特异性100%的阈值标准（AUROC=0.92，置信区间CI：0.8261~1.00）。整合INDUK、Roe1与Sweeney3特征集基因的组合特征集进一步提升了整体活动性结核病的诊断准确率（AUROC=0.98，95%置信区间CI：0.9472~1.00）。

研究结论：本项预实验成功验证了可用于结核病诊断的qPCR检测生物标志物特征集；据我们所知，本研究首次证实了来自不同研究的宿主免疫生物标志物组合特征集具备符合WHO目标产品概况基准的潜力。上述研究结果支持开发低成本、可现场应用的结核病诊断工具的可行性。目前，针对更大规模结核病患者与对照组队列的进一步验证工作正在开展中。