Early assessment of antibodies decline in Chagas patients following treatment using a serological multiplex immunoassay

Abstract

Chagas disease following infection with Trypanosoma cruzi is a major public health issue, with the disease spreading beyond endemic regions and becoming more global due to the migration of infected individuals. The currently available anti-parasitic drugs, nifurtimox and benznidazole, remain insufficiently evaluated for their efficacy in adult patients. A key challenge is the lack of markers for parasitological cure, which also precludes the development of new treatments. Consequently, there is a critical need for a practical method to assess drug performance within a short timeframe. In this retrospective analysis of the phase 2 randomized controlled BENDITA trial (ClinicalTrials.gov: NCT03378661), we report the potential of a serological multiplex method (MultiCruzi), combined with advanced statistical analytical methods, to measure the response to anti-parasitic treatment of adult Chagas patients. Applying this approach to serum samples from adult patients in the indeterminate chronic stage of Chagas disease, treated with different benznidazole regimens and combinations, we predict treatment efficacy after just 6 months of follow-up, in sharp contrast to data obtained with conventional and recombinant T. cruzi ELISA tests. The obtained results are also compared with the PCR data. We propose integrating MultiCruzi as a serological method endpoint in proof-of-concept clinical trials for Chagas disease.

Fig. 1. Dilution method showing the effect of treatment on the reactivity of antigen.

a Well images of serum samples diluted at 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400. These samples were collected at Baseline, 6 months and 12 months after the start of treatment with 300 mg of Benznidazole for 8 Weeks (patient ID3125). The curves showing the effect of treatment of the reactivity of Antigen 3 for (b) patient ID1053 and (c) patient ID3125. D0, Day 0; 6 M, 6 months following treatment; 12 M, 12 months following treatment; DF₅₀, Dilution Factor 50 at which 50% of the original reactivity remains. Bold numbers indicate the 3 selected dilution factors: 50, 400 and 3200. Each plate is imaged and analyzed using a colorimetric reader. An integrated software calculates the pixel intensity for each spot. To establish the net intensity for each antigen, the mean value of the duplicated spots is considered. Net signals correspond to the signal measured from each spot from which the background is subtracted.

Fig. 2. Linear predictor for the fit of the Log₂-based DF₅₀.

The Log₂DF₅₀ was modelled with a longitudinal random intercept nested Linear Mixed Model, against time, treatment and the interaction of time and treatment, taking into account 2 (baseline and 6 months) or 3 (baseline, 6 and 12 months) timepoints, nesting 15 antigens per patient, and 30 patients in each of 7 treatment subgroups. Nested models keep the connection between the patient and the antigens. a At 6 months, and (b) At 12 months after the start of treatment. Log₂ Binary logarithm, DF₅₀ Dilution Factor 50 at which 50% of the original reactivity remains, BZN Benznidazole.

Fig. 3. The ROC curve for the placebo versus treated groups analysis.

The slopes for log₂DF₅₀ against time (D0, 6 M, 12 M) were calculated for each patient and each antigen and pooled for all treatment groups. The ROC analysis was performed on the slopes from the placebo group versus the treated groups. ROC Receiver Operating Characteristic.

Fig. 4. The MultiCruzi monitoring results in each treatment Group, 12 Months after the start of treatment.

The result per patient was generated according to the defined algorithm and log₂DF₅₀ ratio threshold of −0.3, the percentage of each patients’ result (“Response to Treatment”, “Inconclusive” and “No Response to Treatment”) was then calculated per treatment arm. BZN Benznidazole.

Fig. 5. The percentage of patients responsive to treatment in function of the threshold for log₂DF₅₀ decrease between baseline and 12 months.

At all thresholds, the level of patients responsive to treatment in the Placebo group is significantly lower than in the treatment groups. The threshold is calculated according to the following formula: ${\log }_2\frac{{\mathrm{DF}50}_t}{{\mathrm{DF}50}_{\mathrm{baseline}}}$. For each patient, the number of antigens (N_t) with a change superior to the fixed threshold at 12 months was calculated and compared to the number of reactive antigens (N) at baseline. The results are set according to the following conditions: if $\frac{N_t}{N}\ge 0.5$, $“\mathrm{Response}\mathrm{to}\mathrm{Treatment}”$; if $0.3\le \frac{N_t}{N}<0.5$, “Inconclusive”; if $\frac{N_t}{N}<0.3$, “No Response to Treatment”. BZN Benznidazole.

Fig. 6. Percentage of patients with “Response to Treatment”, “Inconclusive” and “No Response to Treatment” results revealed by conventional, recombinant and MultiCruzi ELISA tests at 12 months following treatment.

For the conventional and recombinant ELISA, the percentage was calculated by setting a threshold of minimum 20% in seroreduction at 12 months post-treatment. For MultiCruzi, the threshold for the decrease of the log₂DF₅₀ is −0.3. Conv Conventional, Rec Recombinant, BZN Benznidazole.

Fig. 7. Agreement between MultiCruzi outcome and PCR results at a threshold for log₂DF₅₀ change of −0.7, 12 month post-treatment.

a Percentage of patients with « Response to Treatment », « Inconclusive » and « No Response to Treatment » results revealed by PCR and MultiCruzi ELISA tests at 12 months following treatment. b Classification of Placebo patients according to PCR and MultiCruzi. For PCR, patients having sustained negative PCR results after 12 months post-treatment are concluded to have « Parasitological clearance ». BZN Benznidazole.