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Abstract Background Chagas disease, caused by the parasite Trypanosoma cruzi, is a zoonosis that affects more than seven million people. Current limitations on the diagnosis of the disease hinder the prognosis of patients and the evaluation of treatment efficacy, slowing the development of new therapeutic options. The infection is known to disrupt several host metabolic pathways, providing an opportunity for the identification of biomarkers. Methods The metabolomic and lipidomic profiles of a cohort of symptomatic and asymptomatic patients with T. cruzi infection and a group of uninfected controls were analysed using liquid chromatography/mass spectrometry. Differences among all groups and changes before and after receiving anti-parasitic treatment across those with T. cruzi infection were explored. Results Three lipids were found to differentiate between symptomatic and asymptomatic participants: 10-hydroxydecanoic acid and phosphatidylethanolamines PE(18:0/20:4) and PE(18:1/20:4). Additionally, sphinganine, 4-hydroxysphinganine, hexadecasphinganine, and other sphingolipids showed post-treatment abundance similar to that in non-infected controls. Conclusions These molecules hold promise as potentially useful biomarkers for monitoring disease progression and treatment response in patients with chronic T. cruzi infection. Graphical Abstract

Summary: Chagas disease, caused by Trypanosoma cruzi, affects millions worldwide. The 2030 WHO roadmap aims to eliminate it as a public health concern, emphasising the need for timely diagnosis to enhance treatment access. Current diagnostic algorithms, which rely on multiple tests, have prolonged turnaround times. This proves particularly problematic in resource-limited settings. Addressing this issue necessitates the validation and adoption of innovative tools. We explore recent developments in Chagas disease diagnosis, reviewing historical context and advancements. Despite progress, challenges persist. This article contributes to the understanding of current and future directions in this neglected healthcare area. Parasitological methods are simple but exhibit low sensitivity and require supplementary tests. Molecular methods, with automation potential, allow quantification and higher throughput. Serological tools show good performance but struggle with parasite antigenic diversity. Prioritising point-of-care tests is crucial for widespread accessibility and could offer a strategy to control disease impact. Ultimately, balancing achievements and ongoing obstacles is essential for comprehensive progress.

Chagas disease, caused by the parasite Trypanosoma cruzi, affects over 6 million people, mainly in Latin America. Two different clinical phases, acute and chronic, are recognised. Currently, 2 anti-parasitic drugs are available to treat the disease (nifurtimox and benznidazole), but diagnostic methods require of a relatively complex infrastructure and trained personnel, limiting its widespread use in endemic areas, and the access of patients to treatment. New diagnostic methods, such as rapid tests (RDTs) to diagnose chronic Chagas disease, or loop-mediated isothermal amplification (LAMP), to detect acute infections, represent valuable alternatives, but the parasite's remarkable genetic diversity might make its implementation difficult. Furthermore, determining the efficacy of Chagas disease treatment is complicated, given the slow reversion of serological anti-T. cruzi antibody reactivity, which may even take decades to occur. New biomarkers to evaluate early therapeutic efficacy, as well as diagnostic tests able to detect the wide variety of circulating genotypes, are therefore, urgently required. To carry out studies that address these needs, high-quality and traceable samples from T. cruzi-infected individuals with different geographical backgrounds, along with associated clinical and epidemiological data, are necessary. This work describes the framework for the creation of such repositories, following standardised and uniform protocols, and considering the ethical, technical, and logistic aspects of the process. The manual can be adapted according to the resources of each laboratory, to guarantee that samples are obtained in a reproducible way, favouring the exchange of data among different work groups, and their generalizable evaluation and analysis. The main objective of this is to accelerate the development of new diagnostic methods and the identification of biomarkers for Chagas disease.

The NHEPACHA Iberoamerican Network, founded on the initiative of a group of researchers from Latin American countries and Spain, aims to establish a research framework for Chagas disease that encompasses diagnosis and treatment. For this purpose, the network has created a questionnaire to gather relevant data on epidemiological, clinical, diagnostic, and therapeutic aspects of the disease. This questionnaire was developed based on a consensus of expert members of the network, with the intention of collecting high-quality standardized data, which can be used interchangeably by the different research centers that make up the NHEPACHA network. Furthermore, the network intends to offer a clinical protocol that can be embraced by other researchers, facilitating comparability among published studies, as well as the development of therapeutic response and progression markers.

Introduction Snakebite envenoming (SBE) is a public health problem in Paraguay where the presence of 15 medically important snake species has been reported. Blessed with large forested areas, its economy largely relies on agricultural production which increases the exposure of outdoor workers to the morbidity and mortality of SBE. Lack of sufficient and accurate epidemiological data highlights the importance of drawing an updated picture of SBE burden in the country.Methods We performed a retrospective descriptive study on secondary SBE data reported to the national surveillance system between 2015 and 2021. We addressed the availability and quality of the data and assessed its epidemiological and sociodemographic burden in Paraguay over that time period.Results In total, 1651 cases of SBE were reported between 2015 and 2021 representing an average of 235 cases per year (3.33 cases per 100 000 population). Overall, young males (68%, n=1125) of productive age (25 years old, IQR 29) in agricultural and/or livestock settings (47%, n=653) were the most affected population. Departments with a higher number of notifications were San Pedro (12%, n=191), Caazapá and Alto Paraná (10%, n=163). Regarding data quality, variables about clinical outcomes, treatment administration and case management were the worst reported.Conclusion SBE is a public health issue that affects young workers in rural areas in Paraguay. It mostly remains unattended and improvements in its reporting need to be done in order to gain a better insight into both the health and social burden of this neglected disease.

Background: Migrants are disproportionately affected by several infectious diseases differing the risk within migrant groups. This study aimed to estimate the prevalence of six chronic infections in asymptomatic migrants attended at primary care or specialized units where health assessments are offered. Methods: A multicentric cross-sectional study was conducted to estimate the prevalence of human immunodeficiency virus (HIV), hepatitis B and C virus, Strongyloides stercoralis, Schistosoma spp., and Trypanosoma cruzi infections in the migrant population who participated in a screening programme implemented at six primary health care centres, and two infectious diseases outpatient clinics in Catalonia, Spain. Results: Of the 314 recruited migrants, 284 (90.4 %) were tested for at least one infection. The prevalence of the infections was as follows: 1.8 % for HIV, 1.8 % for chronic hepatitis B virus, 14.1 % for previous exposure to a hepatitis B virus infection, 0.4 % for hepatitis C virus infection, 2.6 % for S.stercoralis infection, and 7.0 % for T.cruzi infection. No cases infected with Schistosoma spp. were reported. A higher prevalence of HIV, hepatitis B and C virus was observed in Sub-Saharan Africans, whereas a higher prevalence of S.stercoralis and T.cruzi infections was reported in migrants from Latin-America and the Caribbean. Conclusion: Findings suggest a high burden of the studied infections, including parasitic infections, in the migrant population with variations within migrant groups based on areas of birth.

BackgroundChagas 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 findingsWe 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/significanceProcedures 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.

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.

IntroductionChagas 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. MethodsWe 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.ResultsEight 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. DiscussionOur 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.

Abstract 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. Graphic abstract

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.

Abstract Background Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected disease that affects ~7 million people worldwide. Development of new drugs to treat the infection remains a priority since those currently available have frequent side effects and limited efficacy at the chronic stage. Natural products provide a pool of diversity structures to lead the chemical synthesis of novel molecules for this purpose. Herein we analyzed the anti-T. cruzi activity of nine alkaloids derived from plants of the family Amaryllidaceae. Methods The activity of each alkaloid was assessed by means of an anti-T. cruzi phenotypic assay. We further evaluated the compounds that inhibited parasite growth on two distinct cytotoxicity assays to discard those that were toxic to host cells and assure parasite selectivity. Results We identified a single compound (hippeastrine) that was selectively active against the parasite yielding selectivity indexes of 12.7 and 35.2 against Vero and HepG2 cells, respectively. Moreover, it showed specific activity against the amastigote stage (IC50 = 3.31 μM). Conclusions Results reported here suggest that natural products are an interesting source of new compounds for the development of drugs against Chagas disease.

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.

Chagas disease, caused by the parasite Trypanosoma cruzi, is the neglected tropical disease with a highest burden in Latin America. Its acute stage is mostly asymptomatic and goes unnoticed. Symptoms appear at the chronic stage, which is when diagnosis is usually made. This is based on the agreement of two conventional serological tests such as Enzyme-Linked Immunosorbent Assays (ELISAs). There are commercial kits with good sensitivity and specificity but their use is impractical in many highly endemic regions with poorly equipped laboratories. Luckily, several rapid diagnostic tests (RDTs) are available for the detection of anti-T. cruzi immunoglobulins. They are easy to operate, require no cold storage, provide fast turnaround of results, and some can work with a tiny volume of whole blood as sample. With the aim to field validate their use we compared an alternative algorithm based on a combination of RDTs with the standard based on ELISAs. In both cases a third test was available in case of discordance. RDTs were implemented by mobile teams in field campaigns to detect chronic T. cruzi-infections in the Chaco region of Bolivia. ELISAs were made in the reference laboratories located in the main hospitals of Yacuiba and Villa Montes, two major cities of the region. We enrolled 685 subjects who voluntarily participated in the study and had not been treated against the disease before. The agreement between the two main RDTs was 93.1% (638/685) (kappa index = 0.86; CI 95% 0.83-0.90). In comparison to the ELISAs algorithm, the combined use of the RDTs provided a sensitivity of 97.7% and a specificity of 96.1%. These results support the use of RDTs for the diagnosis of chronic Chagas disease in the studied region, and encourage their evaluation in other regions of Bolivia and other endemic countries.

Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ~30% of those chronically infected. By then, the drugs' efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8+ T cell, CD4+ T cell, and B cell epitopes that have

BackgroundDespite of a high disease burden, mainly in Latin America, Chagas disease (CD) is underdiagnosed and undertreated. Rapid diagnostic tests (RDTs) might improve the access to diagnosis. The aim of this study is to review the accuracy of commercially available RDTs used in field conditions for the diagnosis of chronic CD in populations at risk, in endemic and non-endemic countries.Methods/principal findingsWe undertook a comprehensive search of the following databases: PubMed, SCOPUS, LILACS (last up-date on the 01st July, 2017), without language or date limits. Non-electronic sources have been also searched. This review included clinical studies with cohort recruitment of individuals at risk of T. cruzi exposure, without age limits; adequate reference standards for the diagnosis of CD. We excluded case-control studies and those testing RDTs during acute CD. Data on test accuracies were pooled through a bivariate random-effects model. Only one index test was evaluated separately. Geographical area, commercial brand, disease prevalence, study size, and risk of bias were explored as possible source of heterogeneity. Values of sensitivity and specificity were computed to obtain summary positive/negative likelihood ratios, and summary diagnostic odds ratio. Ten studies were included on six different immunochromatographic RDTs. The pooled sensitivity and specificity of the RDTs resulted 96.6% (95% CI 91.3-98.7%) and 99.3% (95% CI 98.4-99.7%), respectively. Test accuracy was particularly good in endemic areas (98.07%/99.03% of sensitivity/specificity, respectively). One test (Stat-Pak) showed an overall sensitivity of 97% (95% CI 87.6-99.3) and specificity of 99.4% (95% CI 98.6-99.8).Conclusions/significanceRDTs demonstrated to be sufficiently accurate to recommend their use for screening in endemic areas, even as stand-alone tests. This approach might increase the accessibility to the diagnosis. However, an additional confirmatory test in case of positive result remains a prudent approach.

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.

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), affects more than six million people worldwide, with its greatest burden in Latin America. Available treatments present frequent toxicity and variable efficacy at the chronic phase of the infection, when the disease is usually diagnosed. Hence, development of new therapeutic strategies is urgent. Repositioning of licensed drugs stands as an attractive fast-track low-cost approach for the identification of safer and more effective chemotherapies. With this purpose we screened 32 licensed drugs for different indications against T. cruzi. We used a primary in vitro assay of Vero cells infection by T. cruzi. Five drugs showed potent activity rates against it (IC50 < 4 µmol L−1), which were also specific (selectivity index >15) with respect to host cells. T. cruzi inhibitory activity of four of them was confirmed by a secondary anti-parasitic assay based on NIH-3T3 cells. Then, we assessed toxicity to human HepG2 cells and anti-amastigote specific activity of those drugs progressed. Ultimately, atovaquone-proguanil, miltefosine, and verapamil were tested in a mouse model of acute T. cruzi infection. Miltefosine performance in vitro and in vivo encourages further investigating its use against T. cruzi.

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

African animal trypanosomiasis is caused by vector-transmitted parasites of the genus Trypanosoma. T. congolense and T. brucei brucei are predominant in Africa; T. evansi and T. vivax in America and Asia. They have in common an extracellular lifestyle and livestock tropism, which provokes huge economic losses in regions where vectors are endemic. There are licensed drugs to treat the infections, but adherence to treatment is poor and appearance of resistances common. Therefore, the availability of a prophylactic vaccine would represent a major breakthrough towards the management and control of the disease. Selection of the most appropriate antigens for its development is a bottleneck step, especially considering the limited resources allocated. Herein we propose a vaccine strategy based on multiple epitopes from multiple antigens to counteract the parasites´ biological complexity. Epitopes were identified by computer-assisted genome-wide screenings, considering sequence conservation criteria, antigens annotation and sub-cellular localization, high binding affinity to antigen presenting molecules, and lack of cross-reactivity to proteins in cattle and other breeding species. We ultimately provide 31 B-cell, 8 CD4 T-cell, and 15 CD8 T-cell epitope sequences from 30 distinct antigens for the prospective design of a genetic ensemble vaccine against the four trypanosome species responsible for African animal trypanosomiasis.

Chagas disease is caused by the parasite Trypanosoma cruzi. It affects several million people, mainly in Latin America, and severe cardiac and/or digestive complications occur in ~30% of the chronically infected patients. Disease acute stage is mostly asymptomatic and infection goes undiagnosed. In the chronic phase direct parasite detection is hampered due to its concealed presence and diagnosis is achieved by serological methods, like ELISA or indirect hemagglutination assays. Agreement in at least two tests must be obtained due to parasite wide antigenic variability. These techniques require equipped labs and trained personnel and are not available in distant regions. As a result, many infected people often remain undiagnosed until it is too late, as the two available chemotherapies show diminished efficacy in the advanced chronic stage. Easy-to-use rapid diagnostic tests have been developed to be implemented in remote areas as an alternative to conventional tests. They do not need electricity, nor cold chain, they can return results within an hour and some even work with whole blood as sample, like Chagas Stat-Pak (ChemBio Inc.) and Chagas Detect Plus (InBIOS Inc.). Nonetheless, in order to qualify a rapidly diagnosed positive patient for treatment, conventional serological confirmation is obligatory, which might risk its start. In this study two rapid tests based on distinct antigen sets were used in parallel as a way to obtain a fast and conclusive Chagas disease diagnosis using whole blood samples. Chagas Stat-Pak and Chagas Detect Plus were validated by comparison with three conventional tests yielding 100% sensitivity and 99.3% specificity over 342 patients seeking Chagas disease diagnosis in a reference centre in Sucre (Bolivia). Combined used of RDTs in distant regions could substitute laborious conventional serology, allowing immediate treatment and favouring better adhesion to it.

Epstein-Barr virus is a very common human virus that infects 90% of human adults. EBV replicates in epithelial and B cells and causes infectious mononucleosis. EBV infection is also linked to various cancers, including Burkitt’s lymphoma and nasopharyngeal carcinomas, and autoimmune diseases such as multiple sclerosis. Currently, there are no effective drugs or vaccines to treat or prevent EBV infection. Herein, we applied a computer-aided strategy to design a prophylactic epitope vaccine ensemble from experimentally defined T and B cell epitopes. Such strategy relies on identifying conserved epitopes in conjunction with predictions of HLA presentation for T cell epitope selection and calculations of accessibility and flexibility for B cell epitope selection. The T cell component includes 14 CD8 T cell epitopes from early antigens and 4 CD4 T cell epitopes, targeted during the course of a natural infection and providing a population protection coverage of over 95% and 81.8%, respectively. The B cell component consists of 3 experimentally defined B cell epitopes from gp350 plus 4 predicted B cell epitopes from other EBV envelope glycoproteins, all mapping in flexible and solvent accessible regions. We discuss the rationale for the formulation and possible deployment of this epitope vaccine ensemble.

BACKGROUND:Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. METHODOLOGY/PRINCIPAL FINDINGS:Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. CONCLUSIONS/SIGNIFICANCE:We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.