Evaluation and validation of a PrintrLab-based LAMP assay to identify Trypanosoma cruzi in newborns in Bolivia: a proof-of-concept study.

BACKGROUND: Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. METHODS: In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months. FINDINGS: Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. INTERPRETATION: The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. FUNDING: Inter-American Development Bank.

Five-year serological and clinical evolution of chronic Chagas disease patients in Cochabamba, Bolivia.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, is a neglected infectious disease that exerts the highest public health burden in the Americas. There are two anti-parasitic drugs approved for its treatment-benznidazole and nifurtimox-but the absence of biomarkers to early assess treatment efficacy hinders patients´ follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a longitudinal, observational study among a cohort of 106 chronically T. cruzi-infected patients in Cochabamba (Bolivia) who completed the recommended treatment of benznidazole. Participants were followed-up for five years, in which we collected clinical and serological data, including yearly electrocardiograms and optical density readouts from two ELISAs (total and recombinant antigens). Descriptive and statistical analyses were performed to understand trends in data, as well as the relationship between clinical symptoms and serological evolution after treatment. Our results showed that both ELISAs documented average declines up to year three and slight inclines for the following two years. The recorded clinical parameters indicated that most patients did not have any significant changes to their cardiac or digestive symptoms after treatment, at least in the timeframe under investigation, while a small percentage demonstrated either a regression or progression in symptoms. Only one participant met the "cure criterion" of a negative serological readout for both ELISAs by the final year. CONCLUSIONS/SIGNIFICANCE: The study confirms that follow-up of benznidazole-treated T. cruzi-infected patients should be longer than five years to determine, with current tools, if they are cured. In terms of serological evolution, the single use of a total antigen ELISA might be a more reliable measure and suffice to address infection status, at least in the region of Bolivia where the study was done. Additional work is needed to develop a test-of-cure for an early assessment of drugs´ efficacy with the aim of improving case management protocols.

Practical diagnostic algorithms for Chagas disease: a focus on low resource settings.

INTRODUCTION: 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 1 August 2023. AREAS COVERED: This review outlines existing algorithms, and proposes new ones focused on point-of-care testing. EXPERT OPINION: Integrating point-of-care testing into existing diagnostic algorithms in certain endemic areas will increase 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.

State-of-the-Art in the Drug Discovery Pathway for Chagas Disease: A Framework for Drug Development and Target Validation.

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.

Computational Prediction of Trypanosoma cruzi Epitopes Toward the Generation of an Epitope-Based Vaccine Against Chagas Disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is considered a Neglected Tropical Disease. Limited investment is assigned to its study and control, even though it is one of the most prevalent parasitic infections worldwide. An innovative vaccination strategy involving an epitope-based vaccine that displays multiple immune determinants originating from different antigens could counteract the high biological complexity of the parasite and lead to a wide and protective immune response. In this chapter, we describe a computational reverse vaccinology pipeline applied to identify the most promising peptide sequences from T. cruzi proteins, prioritizing evolutionary conserved sequences, to finally select a list of T and B cell epitope candidates to be further tested in an experimental setting.

Candimine from Hippeastrum escoipense (Amaryllidaceae): Anti-Trypanosoma cruzi activity and synergistic effect with benznidazole.

BACKGROUND: Chagas disease (CD), caused by Trypanosoma cruzi, represents a health threat to around 20 million people worldwide. Side effects of benznidazole (Bzn) cause 15-20% of patients to discontinue their treatment. Evidence has increased in favor of the use of drug combinations to improve the efficacy and tolerance of the treatment. Natural products are well known to provide structures that could serve as new drugs or scaffolds for CD treatment. Spp of the Amaryllidoideae sub family of Amaryllidaceae family are known by their bioactives alkaloids, which have been reported by their antiparasitic activities. PURPOSE: To evaluate the anti-T. cruzi activity of the isolated alkaloid candimine (Cnd) from Hippeastrum escoipense Slanis & Huaylla; and to assess the combination effect between Cnd and Bzn against different life stages of T. cruzi parasites. METHODS: The chemical profile of H. escoipense alkaloids extract (AE-H. escoipense), including quantitation of Cnd was performed through GC/MS and UPLC-MS/MS techniques. Subsequently, Cnd was isolated using Shephadex LH-20. Then, the AE-H. escoipense and Cnd were tested against T. cruzi, (epimastigotes, trypomastigotes, and amastigotes) by in vitro proliferation and viability assays. The cytotoxicity was evaluated against Vero and HepG2 mammalian cells. The ultrastructural analysis was perform by transmission electron microscopy (TEM) and mitochondrial activity was carried out by MTT assay. Drug combination assay between Cnd and Bzn was evaluated using the Chou-Talalay method. RESULTS: The AE-H. escoipense and Cnd showed high and specific anti-T. cruzi activity, comparable to Bzn. Cnd induces ultrastructural changes in T. cruzi, such as vacuolization, membrane blebs, and increased mitochondrial activity. Regarding the interaction between Cnd and Bzn, it generates synergism in the combinations of 0.25×IC50 in epimastigotes, 2×IC50 in trypomastigotes+amastigotes, and 0.25, 2, and 4×IC50 in amastigotes. CONCLUSION: The synergism between Cnd and Bzn indicates that the combination at the concentration of 4×IC50 could be useful as an effective new therapy against CD in the chronic stage. Thus, Cnd isolated from the leaves of H. escoipense emerges as potential candidate for the development of a new drug for the treatment of CD.

Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots.

BACKGROUND: Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. METHODOLOGY/PRINCIPAL FINDINGS: We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. CONCLUSIONS/SIGNIFICANCE: Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field.

Development and Application of an Assay to Evaluate the Anti-Parasitic Effect of Humoral Responses against Trypanosoma cruzi.

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 ß-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.

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease.

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)]Galß. 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.

The Use of AlphaFold for In Silico Exploration of Drug Targets in the Parasite Trypanosoma cruzi.

Chagas disease is a devastating neglected disease caused by the parasite Trypanosoma cruzi, which affects millions of people worldwide. The two anti-parasitic drugs available, nifurtimox and benznidazole, have a good efficacy against the acute stage of the infection. But this is short, usually asymptomatic and often goes undiagnosed. Access to treatment is mostly achieved during the chronic stage, when the cardiac and/or digestive life-threatening symptoms manifest. Then, the efficacy of both drugs is diminished, and their long administration regimens involve frequently associated adverse effects that compromise treatment compliance. Therefore, the discovery of safer and more effective drugs is an urgent need. Despite its advantages over lately used phenotypic screening, target-based identification of new anti-parasitic molecules has been hampered by incomplete annotation and lack of structures of the parasite protein space. Presently, the AlphaFold Protein Structure Database is home to 19,036 protein models from T. cruzi, which could hold the key to not only describe new therapeutic approaches, but also shed light on molecular mechanisms of action for known compounds. In this proof-of-concept study, we screened the AlphaFold T. cruzi set of predicted protein models to find prospective targets for a pre-selected list of compounds with known anti-trypanosomal activity using docking-based inverse virtual screening. The best receptors (targets) for the most promising ligands were analyzed in detail to address molecular interactions and potential drugs' mode of action. The results provide insight into the mechanisms of action of the compounds and their targets, and pave the way for new strategies to finding novel compounds or optimize already existing ones.

Molecular detection and parasite load of Trypanosoma cruzi in digestive tract tissue of Chagas disease patients affected by megacolon.

Chagas disease, caused by the Trypanosoma cruzi parasite in the Americas affects ∼ 7 million people, 30% with cardiac tissue damage and 10-15% with digestive disorders. In this study, we have developed a protocol to detect the presence of the parasite and estimate its load in resected dysfunctional tissue segments of chronically infected patients with digestive megacolon. We have included samples from 43 individuals, 38/5 with positive/negative serology for Chagas disease and digestive syndromes. Samples of 1.5 to 2.0 cm2 were taken from different points of the dysfunctional digestive tract in specialized centres in Cochabamba, Bolivia. T. cruzi cultures were performed by inoculation with NNN-LIT culture medium, and genomic material was obtained from the samples for multiplex qPCR with TaqMan probes targeting satellite nuclear DNA. Cultures failed to isolate T. cruzi but qPCR reached a sensitivity of 42.1% (16/38) with all three spots and in triplicate. A new quantification methodology using synthetic satellite DNA as quantitation standard revealed parasite loads ranging from 2.2 × 102 to 1.0 × 106 satellite DNA copies/µl. Positive samples from the distal end showed a higher parasite load. The results of the present study strengthen and add further evidence to previous findings in an experimental mouse model of chronic T. cruzi infection, providing a valuable tool to improve scientific knowledge on the relevance of the digestive tract in parasite persistence, and underline the need of a better understanding of host-pathogen interaction in digestive tissues, considering pathophysiology, disease immunology and response to treatment.

Computer-Aided Analysis of West Sub-Saharan Africa Snakes Venom towards the Design of Epitope-Based Poly-Specific Antivenoms.

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.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory.

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Anti-Trypanosoma cruzi activity of alkaloids isolated from Habranthus brachyandrus (Amaryllidaceae) from Argentina.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, affects over six million people worldwide, mainly in Latin American countries. Currently available drugs have variable efficacy in the chronic phase and significant side effects, so there is an urgent need for safer chemotherapeutic treatments. Natural products provide privileged structures that could serve as templates for the synthesis of new drugs. Among them, Amaryllidaceae plants have proved to be a potential natural source of therapeutical agents due to their rich diversity in alkaloids. PURPOSE: To identify alkaloids with anti-T. cruzi activity from Habranthus brachyandrus (Baker) Sealy (Amaryllidaceae, subfamily Amaryllidoideae) collected in Argentina. METHODS: An H. brachyandrus alkaloid extract was tested against T. cruzi, and its cytotoxicity profile was evaluated against two mammalian cell lines to ascertain its selectivity against the parasite and potential liver toxicity. It was also assessed by a stage-specific anti-amastigote assay and analysed by GC/MS to determine its alkaloid profile. The isolated alkaloids were also tested using the aforementioned assays. RESULTS: The extract showed high and specific activity against T. cruzi. The alkaloids lycoramine, galanthindole, 8-O-demethylmaritidine, 8-O-demethylhomolycorine, nerinine, trisphaeridine, deoxytazettine, and tazettamide were identified by means of GC-MS. In addition, hippeastidine (also named aulicine), tazzetine, ismine, and 3-epimacronine were isolated. The alkaloid ismine was specifically active against the parasite and had low toxicity against HepG2 cells, but did not show anti-amastigote activity. CONCLUSION: The extract had specific anti-T. cruzi activity and the isolated alkaloid ismine was partially responsible of it. These results encourage further exploration of H. brachyandrus alkaloids in search of novel starting points for Chagas disease drug development.

Compounds targeting GPI biosynthesis or N-glycosylation are active against Plasmodium falciparum.

The emergence of resistance to first-line antimalarials, including artemisinin, the last effective malaria therapy in some regions, stresses the urgent need to develop new effective treatments against this disease. The identification and validation of metabolic pathways that could be targeted for drug development may strongly contribute to accelerate this process. In this study, we use fully characterized specific inhibitors targeting glycan biosynthetic pathways as research tools to analyze their effects on the growth of the malaria parasite Plasmodium falciparum and to validate these metabolic routes as feasible chemotherapeutic targets. Through docking simulations using models predicted by AlphaFold, we also shed new light into the modes of action of some of these inhibitors. Molecules inhibiting N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase (GlcNAc-PI de-N-acetylase, PIGL/GPI12) or the inositol acyltransferase (GWT1), central for glycosylphosphatidylinositol (GPI) biosynthesis, halt the growth of intraerythrocytic asexual parasites during the trophozoite stages of the intraerythrocytic developmental cycle (IDC). Remarkably, the nucleoside antibiotic tunicamycin, which targets UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (ALG7) and N-glycosylation in other organisms, induces a delayed-death effect and inhibits parasite growth during the second IDC after treatment. Our data indicate that tunicamycin induces a specific inhibitory effect, hinting to a more substantial role of the N-glycosylation pathway in P. falciparum intraerythrocytic asexual stages than previously thought. To sum up, our results place GPI biosynthesis and N-glycosylation pathways as metabolic routes with potential to yield much-needed therapeutic targets against the parasite.

Identification of compounds with activity against Trypanosoma cruzi within a collection of synthetic nucleoside analogs.

Introduction: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and it is the most important neglected tropical disease in the Americas. Two drugs are available to treat the infection, but their efficacy in the chronic stage of the disease, when most cases are diagnosed, is reduced. Their tolerability is also hindered by common adverse effects, making the development of safer and efficacious alternatives a pressing need. T. cruzi is unable to synthesize purines de novo, relying on a purine salvage pathway to acquire these from its host, making it an attractive target for the development of new drugs. Methods: We evaluated the anti-parasitic activity of 23 purine analogs with different substitutions in the complementary chains of their purine rings. We sequentially screened the compounds' capacity to inhibit parasite growth, their toxicity in Vero and HepG2 cells, and their specific capacity to inhibit the development of amastigotes. We then used in-silico docking to identify their likely targets. Results: Eight compounds showed specific anti-parasitic activity, with IC50 values ranging from 2.42 to 8.16 µM. Adenine phosphoribosyl transferase, and hypoxanthine-guanine phosphoribosyl transferase, are their most likely targets. Discussion: Our results illustrate the potential role of the purine salvage pathway as a target route for the development of alternative treatments against T. cruzi infection, highlithing the apparent importance of specific substitutions, like the presence of benzene groups in the C8 position of the purine ring, consistently associated with a high and specific anti-parasitic activity.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Novel Purine Chemotypes with Activity against Plasmodium falciparum and Trypanosoma cruzi.

Malaria and Chagas disease, caused by Plasmodium spp. and Trypanosoma cruzi parasites, remain important global health problems. Available treatments for those diseases present several limitations, such as lack of efficacy, toxic side effects, and drug resistance. Thus, new drugs are urgently needed. The discovery of new drugs may be benefited by considering the significant biological differences between hosts and parasites. One of the most striking differences is found in the purine metabolism, because most of the parasites are incapable of de novo purine biosynthesis. Herein, we have analyzed the in vitro anti-P. falciparum and anti-T. cruzi activity of a collection of 81 purine derivatives and pyrimidine analogs. We firstly used a primary screening at three fixed concentrations (100, 10, and 1 µM) and progressed those compounds that kept the growth of the parasites < 30% at 100 µM to dose-response assays. Then, we performed two different cytotoxicity assays on Vero cells and human HepG2 cells. Finally, compounds specifically active against T. cruzi were tested against intracellular amastigote forms. Purines 33 (IC50 = 19.19 µM) and 76 (IC50 = 18.27 µM) were the most potent against P. falciparum. On the other hand, 6D (IC50 = 3.78 µM) and 34 (IC50 = 4.24 µM) were identified as hit purines against T. cruzi amastigotes. Moreover, an in silico docking study revealed that P. falciparum and T. cruzi hypoxanthine guanine phosphoribosyltransferase enzymes could be the potential targets of those compounds. Our study identified two novel, purine-based chemotypes that could be further optimized to generate potent and diversified anti-parasitic drugs against both parasites.

Serological reactivity against T. cruzi-derived antigens: Evaluation of their suitability for the assessment of response to treatment in chronic Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 6 million people worldwide. Following a mostly asymptomatic acute phase, the disease progresses to a long-lasting chronic phase throughout which life-threatening disorders to the heart and/or gastrointestinal tract will manifest in about 30% of those chronically infected. During the chronic phase, the parasitemia is low and intermittent, while a high level of anti-T. cruzi antibodies persist for years. These two features hamper post-chemotherapeutic follow-up of patients with the tools available. The lack of biomarkers for timely assessment of therapeutic response discourages a greater use of the two available anti-parasitic drugs, and complicates the evaluation of new drugs in clinical trials. Herein, we investigated in a blinded case-control study the serological reactivity over time of a group of parasite-derived antigens to potentially address follow up of T. cruzi chronically infected subjects after treatment. We tested PFR2, KMP11, HSP70, 3973, F29 and the InfYnity multiplexed antigenic array, by means of serological assays on a multi-national retrospective collection of samples. Some of the antigens exhibited promising results, underscoring the need for further studies to determine their potential role as treatment response biomarkers.

Amaryllidaceae plants: a potential natural resource for the treatment of Chagas disease.

BACKGROUND: Chagas disease is a neglected zoonosis caused by the parasite Trypanosoma cruzi. It affects over six million people, mostly in Latin America. Drugs available to treat T. cruzi infection have associated toxicity and questionable efficacy at the chronic stage. Hence, the discovery of more effective and safer drugs is an unmet medical need. For this, natural products represent a pool of unique chemical diversity that can serve as excellent templates for the synthesis of active molecules. METHODS: A collection of 79 extracts of Amaryllidaceae plants were screened against T. cruzi. Active extracts against the parasite were progressed through two cell toxicity assays based on Vero and HepG2 cells to determine their selectivity profile and discard those toxic to host cells. Anti-T. cruzi-specific extracts were further qualified by an anti-amastigote stage assay. RESULTS: Two extracts, respectively from Crinum erubescens and Rhodophiala andicola, were identified as highly active and specific against T. cruzi and its mammalian replicative form. CONCLUSIONS: The results retrieved in this study encourage further exploration of the chemical content of these extracts in search of new anti-T. cruzi drug development starting points.