Fig 1.
Patients and healthy controls from different geographical regions in Brazil.
Samples were collected from 122 VL patients, 64 VL-AIDS patients, and 83 healthy individuals living in endemic areas. n—number of samples; VL—visceral leishmaniasis; VL-AIDS—co-infection; HC—healthy controls. The base map was obtained from https://apps.nationalmap.gov/viewer/.
Fig 2.
Receiver operating characteristic (ROC) curves to determine ELISA performance.
ROC curves were constructed using the values of ABS% Positive obtained from testing samples of VL patients (n = 90) and healthy endemic controls (n = 90) (Panel 1).
Fig 3.
Percentage of absorbance of the positive standard (ABS% Positive) obtained by ELISA using recombinant antigens in sera from VL patients and healthy controls (Panel 1).
The horizontal black lines represent the median ABS% Positive Control values with a 95% confidence interval. Friedman test: p < 0.0001 (VL patients and Healthy controls). Dunn’s multiple comparisons test: VL patients: **—p = 0.0049; ***—p = 0.0002; ****—p < 0.0001. Healthy controls: **—p = 0.0044 (rK28 x rK39) and p = 0.0013 (rK18 x rK39); ****—< 0.0001; ns—p = 0.0517 (rK39 x rKR95) and p > 0.9999 (rK28 x rKR95).
Table 1.
Diagnostic performance parameters obtained by ELISA with recombinant antigens determined by ROC curves (Panel 1).
Fig 4.
Reactivity indices obtained by ELISA with recombinant antigens with 122 samples from VL patients, 64 samples from VL / AIDS patients, 83 samples from healthy controls (Panel 2), and 190 samples from patients with other diseases (Panel 3).
The horizontal black lines represent the median ABS% Positive Control values with a 95% confidence interval. Friedman test: p < 0.0001 (for the four groups of samples). Dunn’s multiple comparisons test: VL patients: *—p = 0.0282; ***—p = 0.0004; ****—p < 0.0001; ns—p = 0.2522 (rK18 x rKR95) and p = 0.3187 (rK28 x rK18). VL / AIDS patients: *—p = 0.0300; **—p = 0.0077; ****—p < 0.0001; ns—p = 0.6920 (rK28 x rK18), p > 0.9999 (rk28 x rKR95) and p = 0,2825 (rK39 x rKR95). Healthy controls: *—p = 0.0374; ***—p = 0.0003; ****—p < 0.0001; ns—p = 0.4002 (rK28 x rKR95) and p = 0.1692 (rK39 x rKR95). Other diseases patients: *—p = 0.0152; ****—p < 0.0001; ns—p = 0.1646 (rK28 x rK39) and p > 0.9999 (rK39 x rKR95). The four antigens discriminated significantly between negative (healthy controls) and positive (VL patients) samples (Mann-Whitney test: p < 0.0001).
Fig 5.
Reactivity indices obtained by rK28-ELISA, rK39-ELISA, rK18-ELISA, and rKR95-ELISA with 122 samples from VL patients, 83 samples from healthy controls, 190 samples from patients with other diseases, and 64 samples from VL / AIDS patients.
The horizontal black lines represent the median ABS% Positive Control values with a 95% confidence interval. Kruskal-Wallis’s: p < 0.0001 (for the four antigens). Dunn’s multiple comparisons test: rK28: *—p = 0.0327; ****—p < 0.0001; ns—p = 0.6551. rK39: *—p = 0.0262; ****—p < 0.0001; ns—p = 0.0515. rK18: **—p = 0.0025; ***—p = 0.0003; ****—p < 0.0001; ns—p > 0.9999. rKR95: ****—p < 0.0001; ns—p > 0.9999 (control x other diseases) and p = 0.1357 (VL x VL / AIDS).
Table 2.
Performance obtained by rK18-ELISA, rK28-ELISA, rK39-ELISA, and rKR95-ELISA in sera from VL patients, VL / AIDS patients, healthy controls (Panel 2), and patients with other diseases (Panel 3).
Fig 6.
Antigen-specific antibodies in sera patients with potentially confounding infectious diseases (Panel 3) developed for recombinant antigens.
The horizontal black lines represent the median ABS% Positive Control values with a 95% confidence interval. Friedman test (p values are in the graphic for each disease), followed by Dunn’s multiple comparisons test: Cutaneous leishmaniasis: **—p = 0.0038; ***—p = 0.0002. Malaria: *—p = 0.0342 (rK28 x rK18) and p = 0.0170 (rK18 x rKR95); **—p = 0.0011. Mucosal leishmaniasis: *—p = 0.0325; ***—p = 0.0002. Paracoccidioidomycosis: *—p = 0.0225 (r18 x rKR95) and p = 0.0368 (rK28 x rKR95); ****—p < 0.0001. Syphilis: *—p = 0.0291. Toxoplasmosis: *—p = 0.0351; **—p = 0.0057; ***—p = 0.0001. Tuberculosis: *—p = 0.0207; ***—p = 0.0003.