Introduction:Visceral leishmaniasis (VL), a neglected disease caused by Leishmania infantum, is endemic in more than 76 countries and 90% of the Latin American cases are found in Brazil. Dogs represent one of the main reservoirs of VL infection and play an important role in maintaining this zoonosis, with its control depending on more efficient diagnostic methods. The gold standard for the VL diagnosis still is the parasitological confirmation, but the efficiency of the technique depends on the sample used, resulting in a variable sensitivity. Serological methods are a viable alternative and new antigens have been studied to provide better essays, with different recombinant proteins being investigated to improve the performance of various tests.Objectives: This study reports the production and preliminary evaluation of a new chimerical recombinant protein, designed by joining fragments from five promising antigens previously described, with potential for the early diagnosis of canine VL.Methodology: This recombinant protein was produced in bacteria, affinity purified and evaluated in an ELISA test. The assay was standardized, and the best concentrations of antigen and primary antibody were established. The test was evaluated with a total of 261 canine sera with VL positivity confirmed using the DPP rapid test, mostly from asymptomatic animals, some symptomatic or lacking clinical information. These samples were further tested with the EIE-CVL ELISA, the confirmatory test recommended by the Brazilian Ministry of Health, with 133 having a second positive result while 128 were found to be negative. A total of 28 DPP negative sera were used as controls, all from endemic regions from Pernambuco. A ROC curve was used to determine sensitivity and specificity.Results: Values of 91.3% sensitivity and 100% specificity were observed for the new protein with the DPP and EIE-CVL positive sera, with 100% and 91.18% sensitivities observed within this group for the sera from symptomatic and asymptomatic dogs, respectively. A positive result was also observed for 48.4 % of the DPP positive, EIE-CVL negative sera. Conclusion:Supporting the use of this new recombinant protein for the early CVL diagnosis and as an efficient alternative to the currently recommended EIE-CVL assay.
Introduction: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum that has increased the number of cases in urban regions over the years in Brazil. This disease is of great epidemiological importance and has dogs as the main domestic reservoir, deeply influencing the maintenance of the biological cycle of the transmitting vector. Infected dogs may develop the disease either symptomatically or asymptomatically. Due to the proximity between humans and dogs, the early diagnosis of this neglected disease is of extreme importance for the well-being of both. The diagnostic test currently commercialized by the public service in Brazil is the EIE-LVC, which uses the extract of Leishmania major as capture antigen, conferring good sensitivity, however, its specificity can be variable since there is a possibility of cross-reaction with other species of the Trypanosomatidae family.Objectives: In this scenario, the development of a highly sensitive and specific test for detection of CLV antibodies in animals is very urgent. Therefore, this study aims to develop and validate a recombinant ELISA to diagnose CVL. Methodology:To achieve the objective, we selected a Q5 recombinant protein, provided by the project collaborators, and already described in literature by the research group. The Q5 recombinant test, in inhouse experiments, demonstrated greater efficiency when compared to current commercial tests available on the market. A comparison between the Q5 recombinant protein assay and the EIE-LVC kit was performed using 68 positive and 77 negative samples confirmed in our Dual-Path Platform technology (DPP® CVL rapid test). The sensitivity and specificity calculation was performed using MedCalc Software.Results: Preliminary results obtained with the Q5 recombinant protein showed satisfactory performance in the detection of CVL antibodies. The protein reached the same sensitivity and specificity values as the commercial kit presenting a result of 98% (92% to 99%) and 95% (87% to 98%) respectively. Conclusion:As future prospects, it is planned to scale up the production of prototypes so that a multicentric validation of the kits can be carried out.
Leishmaniasis is a neglected vector-borne disease with a worldwide distribution. Among its different clinical manifestations, the Visceral Leishmaniasis (VL) is the most severe form, which is caused by Leishmania infantum in Brazil. When not treated properly, it can evolve to death and for this reason, early diagnosis followed by adequate treatment is of upmost importance. Currently, the gold standard diagnostic is the parasitological examination, but it has several disadvantages such as low sensitivity. It is known that diagnostic tests can play a major role in patient management, disease surveillance and epidemiological studies, however, accurate human VL diagnosis remains a world problem.Objectives: Aiming to improve the national public health system, the objective of this study was to evaluate the performance of different antigens in immunochromatographic rapid test platform for serological diagnosis of human VL.Methodology: Three recombinant proteins (A1, B1 and C1) that displayed good performance in the enzyme immunoassay (ELISA) in previous studies were tested in a lateral flow platform. The optimal membrane, buffer and protein quantity per test were assessed to identify the best test condition for each protein. The C1 protein was cut off of the study in this phase due to the incompatibility with all tested buffers. After establishing the parameters for proteins A1 and B1, an internal assessment was conducted. After that, a prototype of each test was sent to external evaluation. Results:In the internal evaluation, 50 positive and 40 negative sera were tested. Another 10 samples positive for Trypanosoma cruzi and negative for VL were tested. The A1 protein obtained a sensitivity of 86% and specificity of 100%. The B1 protein obtained sensitivity of 88% and specificity of 100%. Both proteins did not cross reaction with T. cruzi. In the external evaluation, 48 negative and 52 positive sera were tested. The A1 protein obtained sensitivity of 98% and specificity of 90% and B1 obtained sensitivity of 98% and specificity of 92%. Conclusion:These results indicate a potential applicability of the protein B1 in the field. The rapid detection of L. infantum infection will certainly improve the patient's prognosis, preventing fatal resolutions.
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