ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, India.
Faculty of Health and Biological Sciences, Symbiosis International (Deemed University), Pune, India.
PLoS Negl Trop Dis. 2018 Nov 15;12(11):e0006922. doi: 10.1371/journal.pntd.0006922. eCollection 2018 Nov.
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.
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.
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.
世界卫生组织（WHO）已将 2020 年定为在印度次大陆（ISC）消除内脏利什曼病（VL）的目标。尽管在 ISC 中 VL 病例数量明显下降，但仍需要开发一种具有检测活动性感染和复发病例以及疗效检测的诊断测试。本研究验证了我们之前报道的基于 SYBR Green I 的封闭管环介导等温扩增（LAMP）检测方法诊断 VL 的敏感性和特异性。
验证研究在印度两个流行地区进行，分别位于位于巴特那的拉金德拉纪念医学科学研究所（RMRIMS）和瓦拉纳西的 Banaras Hindu 大学医学科学研究所（IMS）。这两个地点都提供了标准操作方案，用于对确诊的 VL 病例应用 LAMP 检测。通过接收者操作特征（ROC）曲线分析评估 LAMP 检测的诊断准确性。此外，还开发并验证了一种基于直接血裂解的简化 LAMP 检测方法（DBL-LAMP），以验证其诊断准确性。
共有 267 名符合条件的参与者入组研究，其中包括 179 例 VL 病例和 88 例对照。LAMP 检测的敏感性和特异性分别为 98.32%（95%置信区间[CI]：95.2-99.7%）和 96.59%（95%CI：90.4-99.3%）。ROC 曲线分析表明，LAMP 检测与 rK39 RDT 的曲线下面积（AUCROC）之间无显著差异，表明 LAMP 是一种优秀的诊断测试。对 67 例 VL 病例和 100 例对照样本进行的 DBL-LAMP 检测，敏感性为 93.05%（95%CI：84.75-97%），特异性为 100%（95%CI：96.30-100%）。
结论/意义：经验证的 VL 诊断用封闭管 LAMP 将为 ISC 正在进行的 VL 消除计划提供动力。基于直接血裂解的检测方法通过进一步减少时间和成本，促进了其在现场环境中的应用范围。
