Desempenho das proteínas recombinantes rKLO8, rLb6H, rK28, rK39, 39r2 e 39r4 no diagnóstico sorológico da leishmaniose visceral canina: relação com apresentações clínicas da doença e vacinação

Abstract

A lei Visceral leishmaniasis (VL), a vector-borne zoonosis, is one of the major public health problems in Brazil and other tropical and subtropical countries. Since dogs represent an important element in transmission cycle, detection of infected dogs is an essential part of monitoring the disease. However, canine VL diagnosis (CVL) is hampered by the wide clinical variability and long incubation period. In order to increase the accuracy of CVL serological diagnosis, several recombinant Leishmania sp antigens have been developed and studied. The present study aimed to evaluate the diagnostic performance of rKLO8, rLb6H, 39r2 and 39r4 recombinant proteins in CVL diagnosis, compared to the reference antigens, rK28 and rK39. First, rK28, rKLO8, rLb6H and rK39 antigens were evaluated by IgG-ELISA for their ability to detect and differentiate TR-DPP® and EIE L.major-like® positive dogs (CVL group), TR-DPP® negative dogs (endemic control - CE group), both from Governador Valadares city (MG) and anti CVL vaccinated dogs (Leish-Tec® - VAC group), from Belo Horizonte city (MG). IgG reactivity against rK28, rKLO8, rK39 and rLb6H antigens was higher in sera from CVL group, with the highest sensitivity value obtained by rK28 (84%), followed by rK39 (82%), rKLO8 (75-77%) and rLb6H (52-57%). The most specific antigens were rK28 and rKLO8 (95-100% and 93- 100%, respectively), followed by rK39 (86-95%) and rLb6H (95%). In addition, vaccinated dogs sera reacted to all antigens, although in variable numbers, being higher for rLb6H (25%), followed by rKLO8 and rK39 (13%) and lower (4.5%) for the reference antigen, rK28. Then, the aforementioned antigens, and two other new recombinant proteins with 2 or 4 copies of the 39 amino acid repeat (39r2 and 39r4), were studied using serum samples from RIFI-negative dogs (SAE group) and dogs with parasitological confirmed Leishmania infection, asymptomatic (AD), oligosymptomatic (OD) and symptomatic (SD), from Belo Horizonte (MG). For all antigens, IgG and IgG2 antibody reactivity was higher in all clinical groups compared to the negative group. Although IgG1 was elevated (p <0.05) only in the symptomatic group for rK28, 39r2, 39r4 and rK39 antigens, serum levels of IgG, IgG1 and IgG2 correlated with clinical status for all antigens. Additionally, our results show that rLb6H and rKLO8 stood out in detection of asymptomatic dogs (100% and 81.81%, respectively), with higher sensitivity values, for rK28 and rLb6H (91,43%), followed by rKLO8 (85.71 %), 39r2 and 39r4 (both, 80%). Regarding the rK39 antigen, sensitivity values were similar in the two experimental stages (82% and 82.86%, respectively). Interestingly, symptomatic dogs with the highest IgG reactivity to rK28 had low reactivity to rLb6H. Regarding specificity, the maximum value (100%) was detected for rK28, 39r2, 39r4 and rK39, from homologous sequences between L. infantum and L. donovani, followed by rKLO8 (85.71%) and rLb6H (71.43%). The results show important characteristics of the rKLO8, rLb6H, 39r2 and 39r4 antigens for the diagnosis of CVL, as they present high performance, comparable to the reference antigens rK28 and rK39, allowing the improvement of CVL detection in groups with greater diagnostic difficulty (asymptomatic and oligosymptomatic).