Fig 1.
Antigens used in multiplex Chagas assay.
(A) List of antigens. (B) Arrangement of antigens spotted in duplicate into the wells of 96-well microtiter plates. Four Positive Control (PC) spots (Methods) were printed to validate the test and give the spatial orientation of the matrix.
Table 1.
Summary of the linear mixed model (LMM) regression output for each biomarker and in each subgroup.
Both the intercept and slope of the linear regression of each antigen corresponds to an average over the 15 mice in each group. Intercept > 10. Blue: significant p-values (< 0.05); Red: high intensity. We have data on several mice where we have treatment group and biomarker reactivity measured at different time points. Where all mice have the same slope and intercept relating reactivity to time and treatment group, a regular multiple linear regression model can be fitted with time and treatment group as the predictor, and reactivity as the response. Biomarker reactivities < 10 were considered below the positive reactivity threshold and were regarded as non-reactive. Slopes compared between treatment groups were assigned as different based on a 5% significance level.
Fig 2.
Multiplex immunoassay antigen screening with serum from benznidazole treated and non-treated T. cruzi infected mice.
(A) Images of multiplex assays screened with serum from a vehicle treated infected BALB/c mouse (BN201-4). (B) Images after screening with serum from an infected mouse (BN206-4) that had been subjected to benznidazole treatment (100 mg kg-1, 5 days), initiated 101 days post-infection (dpi). The dpi are indicated. Antigens were arrayed in duplicate as in Fig 1. For illustrative purposes, specific antigens are framed as follows: IBAG39 (green), IBAG257 (blue), IBAG108 (red), IBAG37 (black), IBAG38 (yellow), IBAG101 (orange), IBAG131 (purple). (C) Longitudinal data showing the proportion of serum samples reacting with selected antigens at a net intensity >10 (minimum set threshold) at each time point (n = 45). Each IBAG is indicated by an arrow. The curves of the two most reactive antigens IBAG257 (blue) and IBAG39 (green) overlap.
Fig 3.
Average change of IBAG reactivity over time, based on the linear prediction model for mice treated with vehicle, and benznidazole at 30 and 100 mg kg-1.
The fits correspond to the intercept and slopes in Table 1. The reactivity of (A) IBAG38 and (B) IBAG39 remains stable in mice treated with vehicle and benznidazole at 30 mg kg-1 and sharply decreases in 100 mg kg-1 benznidazole treated mice. (C) IBAG257 declines in reactivity in the three groups. The reactivities of (D) IBAG36, (E) IBAG37 and (F) IBAG101 increase only in vehicle-treated animals. The vertical axis is representative for the real intensity of the IBAGs.
Fig 4.
In vivo and ex vivo bioluminescence imaging of benznidazole-treated Trypanosoma cruzi infected mice.
BALB/c mice were infected with bioluminescent T. cruzi (CL Brener strain) (Methods). 101 days post-infection (dpi), they were treated with benznidazole (BZ) at 30 or 100 mg kg-1, once daily for 5 days by the oral route (n = 15 per group). In parallel, a control cohort was administered with the HPMC vehicle (Methods). The mice were monitored regularly by bioluminescence imaging. (A) Representative ventral images of two mice per group (see also S1 Fig). (B) At the experimental end-point, mice were euthanized and organs and tissues were arrayed in a square petri dish, bathed in d-luciferin, and subjected to ex vivo imaging (Methods). The organs and tissues are organized as shown in the inset (right). See S2 Fig for other examples. All images use the same log10 scale heat-map with minimum and maximum radiance values as indicated. Mice that were bioluminescence negative at the experimental end-point by both in vivo and ex vivo imaging were designated as cured.
Fig 5.
Bioluminescence total flux and IBAG39 intensity of reactivity for each of the 15 mice treated with 100 mg kg-1 benznidazole (BZ).
The linear correlation coefficient R is given in each graph. The red curve is intensity of IBAG39 and the black curve is log (total flux (p/s)).
Table 2.
Linear correlation coefficients of each antigen for each infected mouse treated with 100 mg kg-1 benznidazole.
Correlation coefficients were calculated, for log10 (total flux) and biomarkers, for each mouse separately (n = 6 observations post-treatment). Statistically significant correlation coefficients are marked in red. Red: linear correlation coefficients > 0.8 (p-value < 0.05).