Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
PLoS Negl Trop Dis. 2011 Sep;5(9):e1310. doi: 10.1371/journal.pntd.0001310. Epub 2011 Sep 13.
Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.
METHODOLOGY/PRINCIPAL FINDINGS: Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs.
CONCLUSIONS/SIGNIFICANCE: Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.
内脏利什曼病是利什曼病中最严重的一种。全球约有 20%的人畜共患内脏利什曼病是由利什曼原虫引起的，在拉丁美洲也被称为恰加斯利什曼原虫，其在热带城市和城郊地区的发病率正在上升。在这种疾病形式中，狗是主要的储存宿主。用于识别感染利什曼原虫的动物的诊断方法无法检测到所有受感染的动物，这可能会影响疾病控制的效果。因此，为了有助于改进犬内脏利什曼病（CVL）的诊断方法，我们旨在使用高通量分析来识别和测试新的抗原。
方法/主要发现：利什曼原虫的免疫显性蛋白在计算机上被映射以预测 B 细胞表位，然后将 360 个预测肽合成在纤维素膜上。免疫测定用于选择最具反应性的肽，然后用犬血清进行研究。接下来，使用固相肽合成方案合成 10 个最具反应性的肽，并使用 ELISA 进行测试。还将这些肽的灵敏度和特异性与巴西卫生部推荐用于利什曼病控制计划的 EIE-LVC Bio-Manguinhos 试剂盒进行了比较。所选合成肽的灵敏度和特异性分别高达 88.70%和 95.00%，而 EIE-LVC 试剂盒的灵敏度和特异性分别为 13.08%和 100.00%。尽管基于合成肽的测试能够诊断高达 94.80%的无症状利什曼病犬，但 EIE-LVC 试剂盒未能检测到任何感染无症状犬的疾病。
结论/意义：我们的研究表明，使用合成肽的 ELISA 是一种具有很大潜力的诊断 CVL 的技术；此外，在其他诊断方法学中使用这些肽，如免疫层析试验，可能有助于 CVL 控制计划。
