Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis
Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis
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DOI：
10.1128/jcm.02076-18
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发表时间：
2019-05-01
影响因子：
9.4
通讯作者：
Campos-Neto, Antonio
中科院分区：
文献类型：
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作者：
Abeijon, Claudia;Alves, Fabiana;Campos-Neto, Antonio
内脏利什曼病是由利什曼原虫引起的一种严重的致死性疾病。VL的金标准诊断试验是脾、淋巴结或骨髓穿刺液中寄生虫或其DNA的证明。血清学检测存在，但不能区分活动VL从任何以前暴露于寄生虫或以前治疗的VL疾病。使用质谱，我们以前已经确定了三个L。VL患者尿液中的婴儿蛋白质生物标志物（Li-isd 1、Li-txn 1和Li-ntf 2），并开发了一种灵敏且特异的基于尿液的抗原检测测定法，用于诊断在巴西发生的VL（其中VL由L. infantum）。然而，我们在DetectoGen实验室未发表的观察结果表明，这些生物标志物仅在印度和肯尼亚的VL患者中检测到55%至60%，其中该疾病由L. donovani。在这里，我们报告的发现和表征的两个新的生物标志物的L。来自这两个国家的VL患者的尿液中存在Donovani（Ld-mao 1和Ld-ppi 1）。建立了特异性兔IgG和鸡IgY的捕获酶联免疫吸附试验，检测Ld-mao 1和Ld-ppi 1的灵敏度分别为44.4%和28.8%。相比之下，设计用于同时检测所有五种利什曼原虫生物标志物的多重测定显著地将测定灵敏度提高至82.2%。这些结果验证了在VL患者的尿液中发现的利什曼原虫蛋白生物标志物作为开发该疾病的准确诊断测试的有力工具的效用。
Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani. Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.