Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)

Background
The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL.
Methodology
The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy.
Principal findings
A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%).
Conclusions/Significance
The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost.

背景
世界卫生组织（World Health Organization）曾设定目标，于2020年在印度次大陆（Indian subcontinent, ISC）消除内脏利什曼病（visceral leishmaniasis, VL）。尽管印度次大陆的VL病例数已出现显著下降，但目前仍亟需开发一种可用于检测现症感染、复发病例以及作为治愈验证指标的诊断工具。本研究对本团队此前报道的基于SYBR Green I的闭管环介导等温扩增（Loop-Mediated Isothermal Amplification, LAMP）检测方法，开展了用于VL诊断的灵敏度与特异度验证。
方法论
本验证研究在印度的两个流行疫区开展，分别位于巴特那的拉金德拉纪念医学科学研究所（Rajendra Memorial Research Institute of Medical Sciences, RMRIMS）以及瓦拉纳西的巴纳拉斯印度大学（Banaras Hindu University, BHU）医学科学研究所（Institute of Medical Sciences, IMS）。两个站点均按照标准操作流程，对确诊VL病例实施LAMP检测。采用受试者工作特征（Receiver Operating Characteristic, ROC）曲线分析，评估LAMP检测的诊断准确性。此外，本研究还开发并验证了一种基于直接血液裂解的简化LAMP检测方法（DBL-LAMP）的诊断效能。
主要发现
本研究共纳入267名符合入组标准的受试者，其中包括179名VL患者与88名健康对照。LAMP检测的灵敏度为98.32%（95%置信区间：95.2%~99.7%），特异度为96.59%（95%置信区间：90.4%~99.3%）。ROC曲线分析显示，LAMP检测与rK39快速诊断测试（rapid diagnostic test, RDT）的曲线下面积（AUC）无显著差异，表明LAMP是一种性能优异的诊断检测方法。针对67份VL患者样本与100份对照样本开展的DBL-LAMP检测，其灵敏度为93.05%（95%置信区间：84.75%~97%），特异度为100%（95%置信区间：96.30%~100%）。
结论与意义
经验证的闭管LAMP检测方法，将为印度次大陆当前正在推进的VL消除计划提供重要助力。基于直接血液裂解的DBL-LAMP检测进一步缩短了检测时长、降低了检测成本，拓展了其在现场环境中的应用场景。