Multi-antigen print immunoassay (MAPIA)-based evaluation of novel recombinant Leishmania infantum antigens for the serodiagnosis of canine visceral leishmaniasis

Abstract

Background: Domestic dogs are the principal reservoir hosts of Leishmania infantum in regions where visceral leishmaniasis (VL) is endemic. Although serologic methods are frequently used for the screening of infected dogs, antibody-based tests require further assessment, due to lack of sensitivity and specificity. In this study, we employed a multi-antigen printing immunoassay (MAPIA) to compare the antibody responses to novel recombinant proteins of L. infantum with the potential for the detection of canine VL.

Findings: MAPIA strips were prepared employing 12 recombinant proteins. Antibody reactivity to these antigens was compared using a panel of sera collected from clinically asymptomatic (n = 16) and symptomatic (n = 41) culture-positive animals. Our findings showed that the canine immune response to antigen differs between dogs and depends on infection status. Using this screening assay, when five out of the 12 antigens were combined, an overall 81% detection rate of L. infantum-infected dogs was achieved.

Conclusions: We conclude that MAPIA is an effective screening tool to rapidly select multiple antigens of diagnostic utility to be used in a more sensitive point of care diagnostic test such as the Dual-Path Platform (DPP) multiplex test for the rapid detection of infected dogs.

Figure 1

MAPIA with recombinant Leishmania infantum antigens. Images of strips containing printed individual antigens. Strips were incubated with serum samples from L. infantum-positive dogs (lanes 1, 3–13), serum from a normal control (uninfected) dog (lane 2), and the positive standard control serum pool (lane 14). The optimum concentration of each antigen wereLci1 = 0.236 mg/mL, Lci2 = 0.222 mg/mL, Lci3 = 0.530 mg/mL, Lci4 = 0.055 mg/mL, Lci5 = 0.139 mg/mL, Lci6 = 0.347 mg/mL, Lci7 = 0.097 mg/mL, Lci8 = 0.125 mg/mL, Lci10 = 0.139 mg/mL, Lci11 = 0.055 mg/mL, Lci12 = 0.236 mg/mL, Lci13 = 0.180 mg/mL, L. major lysate = 0.7620 mg/mL, recombinant CRA&FRA T. cruzi proteins = 0.290 mg/mL and protein A solution = 0.200 mg/mL. Antibody reactivity was detected as described in Methods. The test bands were visually read by two independent operators. Any visible band in the test area (in addition to the control line) was considered a positive reaction.

Figure 2

Increased diagnostic sensitivity by combining the individual recombinant antigens for detecting clinically symptomatic and asymptomatic Leishmania infantum infected dogs.