Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.
Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.
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DOI：
10.1371/journal.pntd.0002845
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发表时间：
2014-05
影响因子：
3.8
通讯作者：
Geluk A
中科院分区：
文献类型：
--
作者：
Bobosha K;Tjon Kon Fat EM;van den Eeden SJ;Bekele Y;van der Ploeg-van Schip JJ;de Dood CJ;Dijkman K;Franken KL;Wilson L;Aseffa A;Spencer JS;Ottenhoff TH;Corstjens PL;Geluk A
迫切需要进行现场适用的测试来检测无症状麻风分枝杆菌（M. leprae）感染或预测麻风病的进展。由于麻风分枝杆菌感染的结果是由细胞和体液免疫决定的，因此我们的目标是开发检测促/抗炎和调节细胞因子以及针对麻风分枝杆菌的抗体的诊断测试。此前，我们开发了侧向层析测定（LFA）来检测细胞因子和抗 PGL-I 抗体。在这里，我们评估了新开发的 LFA 在资源匮乏环境中的应用进展。使用麻风分枝杆菌刺激的麻风患者和地方病对照（EC）的血液测试了IP-10、IL-10和抗PGL-I抗体的组合诊断价值。为了减少总体测试到结果的时间，研究了检测独特反应所需的最短全血测定时间。为了适应现场设置的 LFA，开发了用于 IP-10 和抗 PGL-I 抗体的干式 LFA，允许在环境温度下储存和运输。此外，应用多重LFA格式来同时检测抗PGL-I抗体和IP-10。为了提高灵敏度和定量，所有 LFA 均应用了上转换荧光体 (UCP) 报告技术。单一和多重 UCP-LFA 与 ELISA 具有良好的相关性。干试剂检测和便携式轻型 UCP-LF 条阅读器的性能表明其具有出色的现场稳定性。值得注意的是，检测刺激样品中的 IP-10 水平可以将全血测定时间从 24 小时缩短至 6 小时。此外，未刺激血浆中的 IP-10/IL-10 比率在患者和 EC 之间存在显着差异，表明在流行地区识别麻风分枝杆菌感染的可行性。干式 UCP-LFA 是一种低技术、稳健的检测方法，可以在现场检测针对麻风分枝杆菌的相关细胞因子和抗体。高水平的 IP-10 和所需的较短的全血测定时间，使得该细胞因子可用于区分麻风病患者和 EC。麻风病是世界卫生组织认为对发展中国家构成主要威胁的六种疾病之一，经常因诊断延迟而导致严重的终身残疾和畸形。及早发现麻风分枝杆菌（M. leprae）感染并采取有效干预措施对于阻断传播至关重要。因此，迫切需要检测无症状麻风分枝杆菌感染的现场友好测试。麻风分枝杆菌感染后的临床结果取决于促炎和抗炎细胞因子以及响应麻风分枝杆菌的抗体的平衡。在这项研究中，我们开发了侧向层析测定 (LFA)，用于检测促炎 (IP-10) 与抗炎/调节 (IL-10) 细胞免疫以及针对麻风分枝杆菌的抗体，并使用轻型便携式阅读器在埃塞俄比亚的现场环境中对其进行了评估。我们表明，IP-10 的检测可以将总体测试到结果的时间从 24 小时显着缩短至 6 小时。此外，未刺激血浆中的 IP-10/IL-10 比率在患者和 EC 之间存在显着差异，这可以提供识别麻风分枝杆菌感染的方法。因此，LFA 是一种低技术、稳健的检测方法，可应用于资源匮乏的环境中，测量对麻风分枝杆菌的免疫力，并可用作麻风病早期诊断的工具，从而实现及时治疗和减少传播。
Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC. Leprosy is one of the six diseases considered by WHO as a major threat in developing countries and often results in severe, life-long disabilities and deformities due to delayed diagnosis. Early detection of Mycobacterium leprae (M. leprae) infection, followed by effective interventions, is considered vital to interrupt transmission. Thus, field-friendly tests that detect asymptomatic M. leprae infection are urgently required. The clinical outcome after M. leprae infection is determined by the balance of pro- and anti-inflammatory cytokines and antibodies in response to M. leprae. In this study, we developed lateral flow assays (LFA) for detection of pro-inflammatory (IP-10) vs. anti-inflammatory/regulatory (IL-10) cellular immunity as well as antibodies against M. leprae and evaluated these in a field setting in Ethiopia using lightweight, portable readers. We show that detection of IP-10 allowed a significant reduction of the overall test-to-result time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, which can provide means to identify M. leprae infection. Thus, the LFAs are low-tech, robust assays that can be applied in resource-poor settings measuring immunity to M. leprae and can be used as tools for early diagnosis of leprosy leading to timely treatment and reduced transmission.
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影响因子：
9.1
作者：
Geluk, Annemieke;van Meijgaarden, Krista E.;Ottenhoff, Tom H. M.
通讯作者：
Ottenhoff, Tom H. M.
影响因子：
2.8
作者：
Corstjens, Paul L. A. M.;Zuiderwijk, Michel;Geluk, Annemiek
通讯作者：
Geluk, Annemiek
影响因子：
1.6
作者：
Franken, KLMC;Hiemstra, HS;Drijfhout, JW
通讯作者：
Drijfhout, JW
DOI：
10.4269/ajtmh.2012.12-0395
发表时间：
2012-11-01
影响因子：
3.3
作者：
Downs, Jennifer A.;van Dam, Govert J.;Fitzgerald, Daniel W.
通讯作者：
Fitzgerald, Daniel W.
影响因子：
2.2
作者：
Hong, Wenyan;Huang, Lihua;Zhou, Lei
通讯作者：
Zhou, Lei