Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

Abstract

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy.OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL).METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection.FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis.MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka

“…A high (>70%) serological response was seen in the majority of CL infections in all age groups, both genders, and in all studied lesion types in this study. But it is usually the VL infection that gives high seroprevalence [ 31 – 33 ]. Compared to seroprevalence rates reported for CL in other endemic settings in the world, the new assay described here reported a high value [ 34 – 36 ].…”

Improved Performance of ELISA and Immunochromatographic Tests Using a New Chimeric A2-Based Protein for Human Visceral Leishmaniasis Diagnosis

“…Anti-Leishmania antibodies' detection is an important diagnostic alternative and may be achieved using several different test platforms. The most used serological methods are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests [9][10][11][12]. Nevertheless, on serological assays, the diagnosis of cases with low or undetectable anti-Leishmania antibodies is a drawback and causes a drop in the test sensitivity.…”

“…To overcome the limitations in serological diagnosis, several groups have proposed the use of recombinant antigens comprising mapped and repetitive epitopes that can improve specificity and sensitivity of antibody detection [9,11,[14][15][16][17][18]. In agreement, incorporating the recombinant rK39 or K28 antigens on immunochromatography platforms for VL serological diagnosis (rapid tests) represented a significant improvement since besides being faster and easier to perform, rapid tests with recombinant antigens are more accurate compared with assays based on total antigens [19,20].…”

“…The recombinant protein A2 has also emerged as a promising antigen for VL serodiagnosis, which could be, in part, attributed to the repetitive structure of B cell epitopes present in A2 [9,[21][22][23][24]. Based on these previous studies, we designed a new chimeric protein containing part of the repetitive epitopes from A2 and other repetitive proteins expressed only by the visceralizing species of Leishmania, named DTL-4.…”

A spotlight on the diagnostic methods of a fatal disease Visceral Leishmaniasis

“…A few samples from healthy controls are also immunoblotted with CL patient samples. rFc, rC9 and rA2 are three recombinant L infantum proteins that could be promising candidates for VL diagnosis and used for immunoblotting tests for detection 93 . Thus, a positive immunoblotting test is interpreted in the light of the patient's clinical status.…”