Joaquim Gascon

Background: Chagas disease, caused by the parasite Trypanosoma cruzi, remains a major neglected tropical disease, with millions of people living with the infection worldwide. Current treatments are effective in the acute stage of the disease, but are poorly tolerated and show reduced efficacy in chronic infections, highlighting an urgent need for novel therapeutic strategies. A key bottleneck in early-stage drug discovery is target identification, which is traditionally dependent on costly and low-throughput experimental methods. Computational approaches offer a cost-effective and fast alternative to traditional methods. Methods: In this study, we present an integrated in silico pipeline that combines ligand-based and structure-based computational approaches to prioritize potential molecular targets for bioactive compounds against T. cruzi. The ligand-based component performed similarity searches across curated bioactivity databases containing known ligand–protein associations, and the most similar candidates were then further evaluated using a structure-based approach through pairwise structural alignment against the T. cruzi proteome from AlphaFold. Results: The pipeline was validated using eight compounds with known targets, successfully recovering the correct target in six cases. Additionally, two compounds with anti-T. cruzi activity but unknown mechanisms of action were analyzed to hypothesize their potential targets. Conclusions: Overall, the pipeline demonstrated moderate success, with limitations arising from challenges in handling novel chemotypes and poorly annotated targets. Nevertheless, its modular nature allows for an easy adaptation to other neglected tropical diseases, providing a flexible and cost-effective framework for early-stage target prioritization.

Background: Trypanosoma cruzi, the causative agent of Chagas disease, infects approximately 7 million people worldwide, predominantly in Latin America, and is a growing public health concern in non-endemic regions due to global migration. Most chronically infected individuals remain asymptomatic; however, 20–30% develop cardiac or gastrointestinal complications. Current treatments, benznidazole (BZN) and nifurtimox (NFX), are limited by poor tolerability and frequent drug-related adverse events (DRAEs), resulting in high discontinuation rates. We evaluated alternative BZN and NFX regimens to improve tolerability and adherence.Methods: This open-label, randomised, non-inferiority phase 2 trial enrolled 450 adults with chronic T. cruzi infection across six treatment arms: BZN 150 mg twice daily for 60 days (BZN-60, standard of care [SoC]), BZN 150 mg once daily for 30 (BZN-30) or 90 (BZN-90) days; and NFX 240 mg twice daily for 60 days (NFX-60, SoC) or 30 days (NFX-30), or NFX 240 mg once daily for 90 days (NFX-90). DRAEs were assessed by severity, causality, and organ system.Findings: DRAEs occurred in 273 (61%) of 450 participants, totalling 1052 events, with significantly fewer in the BZN group than in the NFX group (118/225 [52%] vs 155/225 [69%]; p<0·0001). The BZN-30 arm had the lowest DRAE rate (29/75 [39%]; p<0·0001 vs BZN-60 SoC). Severe DRAEs occurred in both groups, including neutropenia and hypersensitivity reactions. An unexpected pattern of lymphopenia with neutrophilia affected 19 (8%) NFX-treated participants. Most DRAEs occurred within 10–14 days regardless of treatment duration.Interpretation: BZN showed superior tolerability compared with NFX, with significantly lower DRAE frequency and fewer events per patient. The BZN-30 regimen significantly improved tolerability compared with standard care, supporting its potential to enhance treatment completion. The early, duration-independent onset of DRAEs suggests idiosyncratic rather than cumulative toxicity mechanisms. Reducing NFX duration from 60 to 30 days conferred no tolerability advantage.

Chagas disease is a neglected tropical infection that affects millions of people. This study explores transcriptomic changes in T. cruzi-infected subjects before and after treatment. Using total RNA sequencing, gene transcription was analyzed in peripheral blood mononuclear cells from asymptomatic (n=19) and symptomatic (n=8) T. cruzi-infected individuals, and non-infected controls (n=15). Differential expression was compared across groups, and before/after treatment in infected subgroups. Untreated infection showed 12 upregulated and 206 downregulated genes in all T. cruzi-infected subjects, and 47 upregulated and 215 downregulated genes in the symptomatic group. Few differentially expressed genes were found after treatment and between the different infected groups. Gene set enrichment analysis highlighted immune-related pathways activated during infection, with therapy normalizing immune function. Changes in the kynurenine/tryptophan ratio, increased pre-treatment, suggested chronic immune fatigue, which was restored post-treatment. These differentially expressed genes offer insights for potential biomarkers and pathways associated with disease progression and treatment response.

Background: The current antiparasitic treatment for chronic Chagas disease of 8 weeks daily benznidazole or nifurtimox is poorly tolerated and reaches only a small minority of those with chronic infections. Defining parasitological cure is compromised by the low blood trypomastigote densities, which fluctuate close to or below the limit of qPCR detection. Methods: To address this limitation and improve the assessment of parasitological cure we developed a probabilistic model of therapeutic efficacy based on serial qPCR data. We pooled clinical and laboratory data from two prospective trials in Bolivian adults with chronic indeterminate Chagas disease. In both trials randomised arms included placebo or standard of care benznida-zole (300mg/day for 8 weeks). In the first trial, the experimental arms were fosravuconazole monotherapies (400mg/week for 4 or 8 weeks, or 200mg/week for 8 weeks); in the second trial the experimental arms were shorter or lower dose benznidazole regimens (300mg/day for 2 or 4, or 150mg/day for 4 weeks), or combinations of fosravuconazole 300mg weekly for 8 weeks with either benznidazole 150mg/day for 4 weeks or benznidazole 300mg/week for 8 weeks. Serial parasite densities were estimated from triplicate qPCRs targeting T. cruzi satellite DNA taken from one to three 5 or 10ml blood samples at 8-12 visits over one year. Treatment efficacies were estimated under a hierarchical Bayesian model, taking as input serial cycle threshold (Ct) data grouped by time point, blood draw and technical replicate. The primary analysis was done in a per-protocol population defined as patients randomised to placebo or patients who took an active treatment >80% of the allocated treatment duration. Results: The two trials randomised 441 patients. 34,804 qPCR Ct values were recorded over 5,402 unique visits, comprising 449 participant years follow-up. In a per-protocol population ( n =424), an estimated 81% (95% Credible Interval [CrI] 70 to 89%, n =69) had parasitological cure following benznidazole 300mg/day for 8 weeks. All other benznidazole regimens had similar estimated cure proportions (95% CrIs >63%) except the 2-week regimen (63% cured [95%CrI 43-81%], n =27, probability of inferiority relative 8-week: 0.95). Recurrent parasitaemias following benznidazole were at substantially lower densities than at baseline. In comparison, only 3.9% of patients allocated to placebo were cured (95%CrI 1 to 9%, n =77). Fosravuconazole was relatively ineffective: 23% cured following 400mg for 8 weeks (95%CrI, 10 to 40%, n =45); 9% following 400mg for 4 weeks (95%CrI 3 to 21%, n =46); and 2% following 200mg for 8 weeks (95%CrI 0 to 11%, n =48). Recurrent parasitaemias one year after fosravuconazole treatment were only slightly lower than at baseline. Fosravuconazole caused dose-dependent increases in liver transaminases. Conclusions: Therapeutic assessments in Chagas disease must account probabilistically for qPCR test performance and low density post treatment parasitaemias. In Bolivian chronic Chagas disease, weekly dosing for eight weeks or daily dosing over four weeks both appear as effective as the current eight weeks daily regimen. The total dose of benznidazole in the current standard of care regimen is excessive.

Chagas disease, caused by parasite Trypanosoma cruzi, is the most important neglected tropical disease in the Americas. Two drugs are available for treatment, but access to them is challenging, in part due to complex diagnostic algorithms. These are stage-dependent, involve multiple tests and are ill-adapted to the reality of vast areas where the disease is endemic. Molecular and serologic tools are used to detect acute and chronic infections, with the performance of the latter showing geographic differences. Breakthroughs in the development of new diagnostic tools include the validation of a loop-mediated isothermal amplification assay for acute infections (T. cruzi-LAMP), and the regional validation of several rapid diagnostic tests (RDTs) for chronic infection, which simplify testing in resource-limited settings. The literature search was carried out in the MEDLINE database until NaN Invalid Date NaN. This review outlines existing algorithms, and proposes new ones focused on point-of-care testing. Integrating point-of-care testing into existing diagnostic algorithms in certain endemic areas will increase the access to timely diagnosis and treatment. However, additional research is needed to validate the use of these techniques across a wider geography, and to better understand the cost-effectiveness of their large-scale implementation.

Chagas disease is the most important protozoan infection in the Americas, and constitutes a significant public health concern throughout the world. Development of new medications against its etiologic agent, Trypanosoma cruzi, has been traditionally slow and difficult, lagging in comparison with diseases caused by other kinetoplastid parasites. Among the factors that explain this are the incompletely understood mechanisms of pathogenesis of T. cruzi infection and its complex set of interactions with the host in the chronic stage of the disease. These demand the performance of a variety of in vitro and in vivo assays as part of any drug development effort. In this review, we discuss recent breakthroughs in the understanding of the parasite’s life cycle and their implications in the search for new chemotherapeutics. For this, we present a framework to guide drug discovery efforts against Chagas disease, considering state-of-the-art preclinical models and recently developed tools for the identification and validation of molecular targets.

Mounting a balanced and robust humoral immune response is of utmost importance for reducing the infectivity of Trypanosoma cruzi. While the role of such a response in controlling the infection is well known, there is a lack of tools that can be used to quickly evaluate it. We developed a serum parasite inhibition assay (to evaluate changes in the parasite infection after exposing infective T. cruzi trypomastigotes to serum samples from infected patients). It is based on Vero cells as the hosts and the Tulahuen β-galactosidase parasite strain, genetically engineered to be quantifiable by spectrophotometry. In parallel, we developed an in-house ELISA to correlate the anti-T. cruzi antibody titres of the clinical samples with their observed anti-parasitic effect in the serum parasite inhibition assay. Serum samples from chronically T. cruzi-infected patients significantly inhibited parasite invasion in a titre-dependant manner, regardless of the patient’s clinical status, compared to samples from the non-infected controls. In addition, there was a clear correlation between the reactivity of the samples to the whole-parasite lysates by ELISA and the inhibitory effect. The results of this work confirm the previously described anti-parasitic effect of the serum of individuals exposed to T. cruzi and present a framework for its large-scale evaluation in further studies. The serum parasite inhibition assay represents a reproducible way to evaluate the intensity and anti-parasitic effect of humoral responses against T. cruzi, which could be applied to the evaluation of candidate antigens/epitopes in the design of Chagas disease vaccine candidates.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Snakebite envenomation is a neglected tropical disease that causes over 100,000 deaths each year. The only effective treatment consists of antivenoms derived from animal sera, but these have been deemed with highly variable potency and are usually inaccessible and too costly for victims. The production of antivenoms by venom-independent techniques, such as the immunization with multi-epitope constructs, could circumvent those drawbacks. Herein, we present a knowledge-based pipeline to prioritize potential epitopes of therapeutic relevance from toxins of medically important snakes in West Sub-Saharan Africa. It is mainly based on sequence conservation and protein structural features. The ultimately selected 41 epitopes originate from 11 out of 16 snake species considered of highest medical importance in the region and 3 out of 10 of those considered as secondary medical importance. Echis ocellatus, responsible for the highest casualties in the area, would be covered by 12 different epitopes. Remarkably, this pipeline is versatile and customizable for the analysis of snake venom sequences from any other region of the world.