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

Abstract

Methods: A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum-infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum-infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings. Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91).

Conclusion: DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats.

Figure 1

Sera samples used to standardize ELISA and ICT and the origin of the samples (panel 1.1 and panel 1.2). n: number of samples; VL: visceral leishmaniasis; CPHL-Palmas: Central Public Health Laboratory of Palmas (Tocantins State); CPHL-Natal: Central Public Health Laboratory of Natal (Rio Grande do Norte State); UFMG: Universidade Federal de Minas Gerais of Belo Horizonte (Minas Gerais State); FIOCRUZ, MG: René Rachou Institute (FIOCRUZ, Minas Gerais State).

Figure 2

Sera samples used to validate the ELISA and ICT kits and the origin of the samples (panel 2.1 and panel 2.2). n: number of samples; VL: visceral leishmaniasis; VL/AIDS: coinfected patients; FIOCRUZ-MG: René Rachou Institute (FIOCRUZ, Minas Gerais State); IMT-FMUSP: Instituto de Medicina Tropical da Faculdade de Medicina, Universidade de São Paulo (São Paulo State). Aracaju, Bauru, Campo Grande, Natal, Rio de Janeiro, São Paulo are Brazilian cities; Piauí is a Brazilian state.

Figure 3

Representation of the ICT to detect IgG anti-Leishmania antibody. (a) The rapid test scheme developed for the detection of IgG anti-L. infantum. (b) Positive sample and (c) negative sample.

Figure 4

Dodecyl sulfate-polyacrylamide gel electrophoresis analysis of recombinant protein DTL-4. DTL-4 (~40 kDa) was purified in nickel affinity chromatography and submitted to SDS-PAGE analysis. Molecular weight markers are shown on the left.

Figure 5

Titration curves for the determination of protein concentration and sera dilutions for DTL-4-ELISA. Pools of ten VL-positive and ten negative sera were assayed. (a) Titration of DTL-4 concentration by adding successive decreasing amounts of protein to wells on ELISA plates. (b) Titration of anti-DTL-4, anti-A2, and anti-K39 antibodies in pools of sera, using 10 ng of each protein per well.

Figure 6

ELISA was performed to detect anti-DTL-4 IgG antibodies of sera. Sera from patients previously diagnosed with visceral leishmaniasis and healthy donors (panel 1.1). (a) DTL-4 discriminates significantly negative and positive samples (p < 0.0001; Mann–Whitney test). (b) ROC curve constructed from the absorbance values calculated for the 126 samples from patients with confirmed VL and 100 samples from healthy controls from the endemic area. Cut − off = 0.173. Area under the curve = 0.9727.

Figure 7

Cross-reactivity of DTL-4-ELISA with sera from patients diagnosed with other diseases. Sera from patients previously diagnosed with tegumentary leishmaniasis, Chagas' disease, malaria, toxoplasmosis, rheumatoid factor, lupus, tuberculosis, syphilis, and paracoccidioidomycosis were submitted for DTL-4-ELISA assay. Sera from VL patients (n = 155) and healthy donors (n = 140) were also included in this assay. The dotted line represents the cut-off.

Figure 8

Evaluation of the reactivity of samples from coinfected patients from different locations assayed with DTL-4-ELISA. Sera from HVL+/AIDS- patients (n = 167), sera from HVL+/AIDS+ patients (n = 62), and sera from healthy donors (n = 169) were included in this assay. Sera samples were diluted at 1 : 100. The dotted line represents the cut-off.

Figure 9

Illustrative results of detecting anti-Leishmania antibodies in sera and blood for VL diagnosis using the DTL-4-based ICT. (a) Sera samples from five VL different patients were included in this assay as a positive control (visible test and control line). (b) Sera samples from negative donors for LV with a previous diagnosis (ten samples of each disease) of Chagas' disease (CHA), malaria (MA), rheumatoid factor (RF), tegumentary leishmaniasis (TL), and healthy donor (HD) were tested. These assays presented negative results (only the control line was visible). (c) Representative test on the cassette. The sera sample from the VL patient was applied, and the control and test lines were visible.