High-Throughput Analysis of Synthetic Peptides for the Immunodiagnosis of Canine Visceral Leishmaniasis

Abstract

BackgroundVisceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.Methodology/Principal FindingsImmunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs.Conclusions/SignificanceOur study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

“…Recently, Costa et al (27) performed ELISAs using single and mixed synthetic antigens from previously evaluated proteins, which showed high sensitivity in serum samples with low (95%) and intermediate (95%) antibody titers and high specificity (95%); the capacities of these synthetic antigen mixtures were not assayed against asymptomatic dogs, and crossreactivity assays were not performed. In addition, Faria et al (28) performed ELISAs using a mixture of synthetic antigens and obtained high sensitivity and specificity values (78.5% and 80%, respectively), but all of the synthetic antigens and the mixture exhibited high cross-reactivity with T. cruzi serum samples.…”

“…These antigens are relatively simpler and cheaper to produce than recombinant proteins. It also has been reported that the use of synthetic peptides (individually or in a mixture format), in comparison with the use of recombinant proteins, is able to increase the sensitivity and/or specificity of immunoassays for the serodiagnosis of parasitic diseases (26), such as canine and human visceral leishmaniasis (27,28).…”

Immunoproteomics and Immunoinformatics Analysis of Cryptococcus Gattii : Novel Candidate Antigens for Diagnosis

“…Only those future science group peptides that were simultaneously identified by the two programs were considered to be putative antigens for the development of immunoassays for cryptococcosis. This approach was based on the work of Faria et al, which showed that the use of the default scores of prediction software programs associated with the overlap predictions of more than one software program can be preferable to the use of a single type of prediction [40].…”

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

“…Moreover, no false-positive result was observed when ROC curves were plotted, despite regular cross-reactive conditions, such as dogs that were infected with T. cruzi or E. canis and animals immunized with Leishmune or Leish-Tec Brazilian commercial vaccine. Recently, Faria et al (62) performed an ELISA using a mixture of synthetic antigens and obtained high sensitivity and specificity values (78.5% and 80%, respectively); however, all of the synthetic antigens and the mixture presented high cross-reactivity with serum samples from T. cruzi-infected dogs. In addition, Marcondes et al (63), who performed a rapid ELISA, showed a high rate of false-positive results in which high percentages of cross-reactivity occurred between Leishmania spp.…”