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Validation of Enzyme-Linked Immunosorbent Assay (ELISA) with recombinant antigens in serological diagnosis of canine Visceral Leishmaniasis (VL)

Grant number: 15/03117-1
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): June 01, 2015
Effective date (End): May 31, 2017
Field of knowledge:Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
Acordo de Cooperação: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal Investigator:Hiro Goto
Grantee:Mahyumi Fujimori
Host Institution: Instituto de Medicina Tropical de São Paulo (IMT). Universidade de São Paulo (USP). São Paulo , SP, Brazil


In Brazil, Visceral Leishmaniasis (VL) is booming, present in all regions, with a prevalence of 3,253 cases in 2013. In the last three decades, VL has reached urban and peri-urban centers also. In the transmission of VL in Brazil, the sand fly Lutzomyia longipalpis is the main vector, and the dog (Canis familiaris) and the fox (Dusycion vetulus), involved in domestic and sylvatic cycle, respectively, are the main reservoirs. Consequently, the measures to control transmission consider these elements, but in this project, the focus will be the dog infected with L. (L.) infantum. Dogs infected with L. (L.) infantum may present, from a clinical point of view, as asymptomatic, oligosymptomatic or symptomatic. Since these dogs have a strong parasitism on the skin, it is believed that this permits an easy vector infection and constitutes the important link that maintains the epidemiological chain. Thus, the identification of infected dogs is considered a crucial step in the control of disease transmission, and it is recommended to perform sample or census survey, depending on the epidemiological situation regarding the risk of transmission in the area to be investigated.Although the positive parasitological examination in bone marrow aspirate material or lymph nodes is the undisputed parameter, it is impractical in canine surveys, and serological methods for the diagnosis of canine VL (CVL) are used, instead. In this regard, the Ministry of Health recommends the immunochromatographic assay DPPTM-Leishmania test for screening dogs, and the confirmation of positive cases by ELISA based on total lysate of Leishmania major-like. Since the parasite culture for preparation of total antigen constitutes a limiting factor, it would be desirable to have an ELISA based on the use of recombinant antigen, which we propose in this project.Based on survey data in Spain, for detection of anti-Leishmania antibodies using the heat shock protein=Hsp, L. (L.) infantum Hsp83 in CVL diagnosis, we tested this antigen in human leishmaniasis, using ELISA, which showed good sensitivity and specificity. In preliminary studies of ELISA-rHsp83, with sera from dogs submitted to parasitological examination, we obtained high sensitivity (88.9%) and high specificity (94.4%). It is noteworthy that we have the plasmid containing the hsp83 gene of L. (L.) infantum and have standardized the expression and production of recombinant antigen rHsp83, and we are trying its larger-scale, pre-industrial production in our laboratory.The rK28 antigen was originally built as a chimeric fusion protein based on epitopes rK9, rK26 and rK39 of L. infantum, and have showed high sensitivity (99%) and specificity (96%) in Italy dogs tested by ELISA. Recently, the rK28 antigen was produced as fusion polyprotein from the synthesis of the K28 gene. It comprises regions of L. donovani haspb1 (L. infantum K26 homologue), L. donovani kinesin (L. infantum K39 homologue) and L. donovani haspb2 (L. infantum K9 homologue). In samples of human VL, the ELISA-rK28 had a sensitivity of 96.8 to 99.6 % and a specificity of 96.2 to 100.0%.Therefore, based on published reports and studies from our group, we believe that the ELISA-rk28 and ELISA-rHsp83 tests are good candidates for use in the diagnosis of CVL in endemic areas serological surveys.Thus, the present study aims to validate the serological methods ELISA-rK28 and ELISA-rHsp83 for the diagnosis of canine VL, replacing the ELISA based on total lysate of Leishmania major-like as the confirmatory test, post immunochromatographic DPP "Leishmania test. Validate alternatively serological methods ELISA-rK28 and ELISA-rHsp83 for the diagnosis of canine VL as a confirmatory test, regardless of the result of DPP " Leishmania test. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MAHYUMI FUJIMORI; ARLEANA DO BOM PARTO FERREIRA DE ALMEIDA; STELLA MARIA BARROUIN-MELO; LUIZ RICARDO PAES DE BARROS CORTEZ; MALCOLM SCOTT DUTHIE; ROBERTO MITSUYOSHI HIRAMOTO; FLAVIANE ALVES DE PINHO; STEVEN GREGORY REED; VALÉRIA RÉGIA FRANCO SOUSA; NAZARÉ FONSECA SOUZA; et al. Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection. Memórias do Instituto Oswaldo Cruz, v. 116, . (15/22075-8, 15/03117-1, 17/03367-3)

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