期刊
PLOS NEGLECTED TROPICAL DISEASES
卷 14, 期 7, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pntd.0008488
关键词
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资金
- Brazilian Ministry of Health and Science and Technology through the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (MCTI/CNPq/MS-SCTIE - DECIT) [40/2012, 404259/2012-0]
- CNPq, Brazilian Ministry of Health
- State of Pernambuco Funding agency - FACEPE (MS/SES/FACEPE grant (PPSUS) [APQ-0815-2.13/17]
- FACEPE
- CNPq
- CAPES
Author summary Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, caused mainly byLeishmania infantumandL.donovani. Its control is still impaired by inefficient and/or expensive treatment and diagnostic methods. VL diagnosis is mostly dependent on serological assays made with bacterially expressedLeishmaniaproteins. Tests developed for the human form of the disease, however, are not efficient for its diagnosis in the canine host. Dogs are the major reservoir for the American VL and their infection also needs to be monitored, requiring a distinct diagnostic test. Here, we describe a new chimeric protein potentially able to be used for both human and canine VL diagnosis. Based on a systematic approach using threeLeishmaniaproteins known to be efficient for the diagnosis of either human or canine VL, fragments of each were joined in various combinations. The diagnostic potential of different chimeric proteins was then evaluated with human and canine sera from VL afflicted individuals. The best protein showed high levels of sensitivity in humans and dogs with no relevant false positive results with healthy controls or humans with tegumentary leishmaniasis. It is then potentially useful for the detection of both humans and dogs afflicted with VL in novel diagnostic tests. Background Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. Methodology/Principal findings Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both onin silicogene design and experimental data. Starting from the previous identification ofLeishmania infantumrecombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. Conclusion The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.
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