Division of Infectious Diseases, Department of Internal Medicine, State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Clinical Epidemiology Unit, Peking Union Medical College, International Clinical Epidemiology Network, Beijing 100730, China.
Chin Med J (Engl). 2022 Jan 5;135(1):63-69. doi: 10.1097/CM9.0000000000001858.
Differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) has been a challenge for clinicians in high TB burden countries. The purpose of this study was to improve the accuracy of differential diagnosis of ATB and LTBI by using fluorescent immunospot (FluoroSpot) assay to detect specific Th1 cell immune responses. The novel mycobacterium tuberculosis (MTB) latency-associated antigens Rv1733c and synthetic long peptides derived from Rv1733c (Rv1733c SLP) were used based on virulence factors early secreting antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10).
Fifty-seven ATB cases, including 20 pathogen-confirmed ATB and 37 clinically diagnosed ATB, and 36 LTBI cases, were enrolled between January and December 2017. FluoroSpot assay was used to detect the interferon γ (IFN-γ) and interleukin 2 (IL-2) secreted by the specific T cells after being stimulated with MTB virulence factors ESAT-6 and CFP-10, MTB latency-associated antigens Rv1733c and Rv1733c SLP. The receiver operating characteristic (ROC) curve was used to define the best cutoff value of latency-associated antigens in the use of differentiating ATB and LTBI. The sensitivity, specificity, predictive value, and likelihood ratio of ESAT-6 and CFP-10-FluoroSpot combined with latency-associated antigen in the differential diagnosis of ATB and LTBI were also calculated.
Following the stimulation with Rv1733c and Rv1733c SLP, the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP had the largest area under the ROC curve, which was 0.766. With a cutoff value of 1 (spot-forming cells [SFCs]/2.5 × 105 peripheral blood mononuclear cells) for frequency, the sensitivity and specificity of distinguishing ATB from LTBI were 72.2% and 73.7%, respectively. ESAT-6 and CFP-10-FluoroSpot detected the frequency and proportion of single IFN-γ-secreting T cells; the sensitivity and specificity of distinguishing ATB from LTBI were 82.5% and 66.7%, respectively. Combined with the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP on the basis of ESAT-6 and CFP-10-FluoroSpot, the sensitivity and specificity increased to 84.2% and 83.3%, respectively.
Rv1733c SLP, combined with ESAT-6 and CFP-10, might be used as a candidate antigen for T cell-based tuberculosis diagnostic tests to differentiate ATB from LTBI.
在高结核负担国家，临床医生对活动性肺结核（ATB）和潜伏性结核感染（LTBI）的鉴别诊断一直存在挑战。本研究旨在通过荧光免疫斑点（FluoroSpot）检测来提高对 ATB 和 LTBI 进行鉴别诊断的准确性，该方法用于检测特定 Th1 细胞免疫反应。新型结核分枝杆菌（MTB）潜伏相关抗原 Rv1733c 和源自 Rv1733c 的合成长肽（Rv1733c SLP）是基于毒力因子早期分泌抗原靶 6（ESAT-6）和培养滤液蛋白 10（CFP-10）选择的。
2017 年 1 月至 12 月期间，纳入 57 例 ATB 病例，包括 20 例病原体确诊的 ATB 和 37 例临床诊断的 ATB，以及 36 例 LTBI 病例。采用 FluoroSpot 检测 MTB 毒力因子 ESAT-6 和 CFP-10 刺激后特异性 T 细胞分泌的干扰素 γ（IFN-γ）和白细胞介素 2（IL-2）。采用受试者工作特征（ROC）曲线来确定区分 ATB 和 LTBI 时使用潜伏相关抗原的最佳截断值。还计算了 ESAT-6 和 CFP-10-FluoroSpot 联合潜伏相关抗原在 ATB 和 LTBI 鉴别诊断中的灵敏度、特异性、预测值和似然比。
Rv1733c 和 Rv1733c SLP 刺激后，Rv1733c SLP 刺激的单 IL-2 分泌 T 细胞频率的 ROC 曲线下面积最大，为 0.766。以频率为 1（斑点形成细胞[SFC]/2.5×105 外周血单核细胞）作为截断值，区分 ATB 和 LTBI 的灵敏度和特异性分别为 72.2%和 73.7%。ESAT-6 和 CFP-10-FluoroSpot 检测到单 IFN-γ 分泌 T 细胞的频率和比例；区分 ATB 和 LTBI 的灵敏度和特异性分别为 82.5%和 66.7%。在 ESAT-6 和 CFP-10-FluoroSpot 的基础上，结合 Rv1733c SLP 刺激的单 IL-2 分泌 T 细胞频率，灵敏度和特异性分别提高至 84.2%和 83.3%。
Rv1733c SLP 联合 ESAT-6 和 CFP-10 可作为基于 T 细胞的结核诊断检测的候选抗原，用于区分 ATB 和 LTBI。
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