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| Author(s): |
Mahyumi Fujimori
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Medicina Tropical de São Paulo (IMT) |
| Defense date: | 2019-06-12 |
| Examining board members: |
Hiro Goto;
Roberto Mitsuyoshi Hiramoto;
Kelly Aparecida Kanunfre;
Marcia Dalastra Laurenti
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| Advisor: | Hiro Goto |
| Abstract | |
The dog is the main peridomiciliar reservoir of Leishmania (Leishmania) infantum, etiologic agent of visceral leishmaniasis (VL). In areas endemic for VL, the identification and euthanasia of infected dogs are considered crucial in controlling transmission of L. infantum to humans. Currently, the algorithm recommended by the Brazilian Ministry of Health consists in screening of dogs by the immunochromatographic test TR DPP® Leishmaniose Visceral Canina (DPP) and confirmation of positive cases by ELISA using lysate antigen of Leishmania major-like (ELISA-L. major-like). As parasite culture for the preparation of this ELISA kit is a limiting factor, the use of recombinant antigen (r) appears as an attractive alternative. In this study, the ELISA with the rK39, rK28, rKR95 and rK18 antigens (IDRI, USA) derived from Leishmania was evaluated. Using sera from 74 dogs from endemic areas for canine LV (CVL), with positive parasitological examination and 66 healthy dogs (DPP negative, ELISA L. major-like and DAT), collected in a city without LVC registry, the sensitivity of the ELISA-rK39, -rK28, -rK18 was, respectively, 97%; 96%; 96% and 89% and the specificity, 100%; 100%; 100% and 99%. Plates sensitized with each antigen maintained stability for up to 180 days when stored at 4°C and -20° C. The evaluation of repeatability and reproducibility presented coefficients of variation compatible with acceptable values. For the validation of the ELISA with the different recombinant antigens, 2,530 samples from epidemiological surveys of CVL endemic states in Brazil: Pará, Mato Grosso, São Paulo and Bahia were evaluated. Positivity with DPP combined with ELISA-L. major-like was 16% and the DPP combined with rK39 ELISA was 17%, suggesting the possibility of replacing the ELISA-L. major-like ELISA based on recombinant antigen. However, when assayed directly without DPP screening, the positivity of ELISA-rK39, rK28 and rKR95 was about 10% higher than with current assays in use, respectively, 28%; 27% and 27%, suggesting the early detection of infected animals. To answer this hypothesis, we analyzed 15 dogs (from a cohort of 600 dogs from Bauru-SP) that, according to the algorithm recommended by the Brazilian Ministry of Health, in the first intervention, had been considered negative for Leishmania infection and, 6 months after the initial evaluation were positive. In the samples that were supposedly negative in the initial evaluation, the ELISA-rK39, -rK28 and -rKR95 tests presented, respectively, 67%, 80% and 60% of positivity. These data suggest a significant amount of false negative results in the algorithm in use. The viability of the application of this ELISA based on recombinant antigen in areas endemic to CVL was also evaluated and agreement was obtained ranging from good to perfect, between the results obtained in all laboratories involved and those in the coordination laboratory. With the global analysis of this study, mainly considering the variability of the response in certain areas of Brazil, we consider the validation of the ELISA-rK39 for the diagnosis of canine infection by L. infantum. The data also point out the need to reassess the tests and the algorithm in routine use for the detection of dogs infected with L. infantum. (AU) | |
| FAPESP's process: | 17/03367-3 - Validation of Enzyme-Linked Immunosorbent Assay (ELISA) with recombinant antigens in serological diagnosis of Canine Visceral Leishmaniasis (VL) |
| Grantee: | Mahyumi Fujimori |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |