Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis
Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis
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DOI：
10.1128/jcm.00386-18
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发表时间：
2018-07-01
影响因子：
9.4
通讯作者：
Schallig, Henk
中科院分区：
文献类型：
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作者：
Adams, Emily Rebecca;Schoone, Gerard;Schallig, Henk
开发了一种新的泛利什曼原虫环介导等温扩增（LAMP）检测方法，用于诊断皮肤和内脏利什曼病（CL和VL），可用于近患者环境。根据185个核糖体DNA（rDNA）和小环动基体DNA（kDNA）的保守区设计引物，根据高拷贝数选择。在哥伦比亚西南部105例患者的前瞻性队列试验中，评价了LAMP检测法用于CL诊断。采集疑似CL患者的病变拭子样本，并使用LAMP检测试剂盒进行检测，将结果与显微镜检查和/或培养的复合参考结果进行比较，以计算诊断准确度。对来自埃塞俄比亚的50名疑似VL患者的样品（包括全血、外周血单核细胞和血沉棕黄层）进行LAMP测定。根据脾脏或骨髓穿刺物显微镜检查的参考标准计算诊断准确性。为了计算分析特异性，对100份临床样本和发热病原体（包括疟疾寄生虫、虫媒病毒和细菌）分离株进行了检测。我们发现，LAMP检测法诊断CL的灵敏度为95%（95%置信区间[CI]，87.2%至98.5%），特异性为86%（95% CI，67.3%至95.9%）。对于VL可疑者，在全血中LAMP测定的灵敏度为92%（95% CI，74.9%至99.1%），特异性为100%（95% CI，85.8%至100%）。对于CL，LAMP检测是一种灵敏的诊断工具，与替代CL诊断相比，需要更少的设备、时间和专业知识。对于VL，使用微创样品的LAMP测定比金标准更灵敏。分析特异性为100%。
A novel pan-Leishrnania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 185 ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.