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Trypanosomatids are a diverse group of uniflagellate protozoa that include globally important pathogens such asTrypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase (FAS)-I system typically used forde novosynthesis of long chain fatty acids (LCFA) in other eukaryotes. Instead, these microbes have evolved a modular fatty acid elongase (ELO) system comprised of individual ELO enzymes that operate processively. The role of the ELO system in maintaining lipid homeostasis in trypanosomes has not been determined. Here we demonstrate that ELO2 and ELO3 are required for global lipidome maintenance in the insect stage ofT. cruziwhereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that merits examination of the trypanosomatid ELO pathway as critical for central metabolism.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate’s skin.

Chronic Chagas cardiomyopathy (CCC) is one of the leading causes of morbidity and mortality due to cardiovascular disorders in endemic areas of Chagas disease (CD), a neglected tropical illness caused by the protozoan parasite Trypanosoma cruzi. CCC is characterized by parasite persistence and inflammatory response in the heart tissue, which occur parallel to microRNA (miRNA) alterations. Here, we investigated the miRNA transcriptome profiling in the cardiac tissue of chronically T. cruzi-infected mice treated with a suboptimal dose of benznidazole (Bz), the immunomodulator pentoxifylline alone (PTX), or the combination of both (Bz+PTX), following the CCC onset. At 150 days post-infection, Bz, PTX, and Bz+PTX treatment regimens improved electrocardiographic alterations, reducing the percentage of mice afflicted by sinus arrhythmia and second-degree atrioventricular block (AVB2) when compared with the vehicle-treated animals. miRNA Transcriptome profiling revealed considerable changes in the differential expression of miRNAs in the Bz and Bz+PTX treatment groups compared with the control (infected, vehicle-treated) group. The latter showed pathways related to organismal abnormalities, cellular development, skeletal muscle development, cardiac enlargement, and fibrosis, likely associated with CCC. Bz-Treated mice exhibited 68 differentially expressed miRNAs related to signaling pathways like cell cycle, cell death and survival, tissue morphology, and connective tissue function. Finally, the Bz+PTX-treated group revealed 58 differentially expressed miRNAs associated with key signaling pathways related to cellular growth and proliferation, tissue development, cardiac fibrosis, damage, and necrosis/cell death. The T. cruzi-induced upregulation of miR-146b-5p, previously shown in acutely infected mice and in vitro T. cruzi-infected cardiomyocytes, was reversed upon Bz and Bz+PTX treatment regimens when further experimentally validated. Our results further our understanding of molecular pathways related to CCC progression and evaluation of treatment response. Moreover, the differentially expressed miRNAs may serve as drug targets, associated molecular therapy, or biomarkers of treatment outcomes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively

Microangiopathy may worsen the clinical outcome of Chagas disease. Given the obstacles to investigating the dynamics of inflammation and angiogenesis in heart tissues parasitized by

Introduction Patients hospitalized due to heart failure (HF) constitute a heterogeneous population whose prognosis is difficult to forecast. The purpose of this study was to create a model based on simple bedside recordable echocardiographic, analytical and objective clinical parameters that could accurately predict mortality and/or rehospitalization risk in different stages of HF course. Methods A retrospective analysis of 347 patients admitted to a Cardiology ward due to decompensated HF was performed. The echocardiographic variables pulmonary artery systolic pressure (PSAP) and E/e' ratio, and the analytical/clinical variables systolic blood pressure (SBP), urea and brain natriuretic peptide (BNP) were selected for inclusion. Subgroups were created for each variable and an odds ratio (OR) for the risk of in-hospital mortality (IHM) was calculated. A numerical value proportional to the OR was attributed to each subgroup. A score was created, ranging from 0–47 points, corresponding to the sum of the classification attributed to each variable. ROC curve analysis was used to assess predictive value of the score for IHM. Kaplan-Meyer and Cox-regression plots were used to assess mortality (24MM) and the composite endpoint of HF rehospitalization or death at 24 months (24HM). Results Mean patient age was 78 (±9) years; 51% were men. Score variable means were - PSAP: 47 (±15) mmHg; E/e': 16.8 (±7.8); SPB: 138 (±31) mmHg; Urea: 71 (±35) mg/dl; BNP: 911 (±995) pg/ml. Mean ejection fraction (EF) was 48% (±16). 35% of patients had EF Conclusion AHFM score is an accurate model for predicting IHM and long-term risk of HF death. Its use may help to identify patients with high risk of mortality, in need of specialized care, and those with lower risk of death, who might be candidates for early discharge or lenient follow-up. Funding Acknowledgement Type of funding sources: None.

Introduction Patients hospitalized due to acute heart failure (AHF) compose a heterogeneous population whose prognosis is difficult to forecast. Previously, BAUN score has proven to be able to accurately predict in-hospital mortality (IHM) in AHF. We aimed to evaluate BAUN score performance in the prediction of long-term outcomes in this population, comparing it to the recently validated Get With The Guidelines (GWTG) score. Methods A retrospective analysis of 1052 patients admitted to a Cardiology ward due to AHF was performed. 268 patients were excluded due to data omission or therapy with sacubitril/valsartan. Using the variables systolic blood pressure, urea, brain natriuretic peptide and sodium at admission, BAUN score was calculated, ranging from 0–28 points. GWTG score was also calculated at the index event. ROC curve analysis was used to compare the predictive value of the two scores for IHM. Kaplan-Meyer and Cox-regression analysis were performed to evaluate BAUN score prediction ability for 24-month mortality (24-MM) and for the composite endpoint of 24-month rehospitalization or death (24-MH). Results Mean patient age was 77 (±10) years; 51% were men. Mean left ventricle ejection fraction (EF) was 49% (±16.4). An EF Conclusion BAUN outperforms GWTG score for IHM prediction in AHF. It also independently predicts 24-MM and 24-MH. Its use may identify patients with high risk of mortality/readmission, in need of specialized care, and those patients with low risk of death, who might be candidates for lenient surveillance. Funding Acknowledgement Type of funding sources: None.

Introduction Global longitudinal strain (GLS) is considered a more sensitive marker of systolic dysfunction than other measures commonly used in clinical practice, such as left ventricle ejection fraction (EF). Our objective was to evaluate the impact of reduced GLS in death and cardiovascular events in patients hospitalized due to heart failure with mid-range or preserved ejection fraction, with previous history of acute myocardial infarction. Methods A retrospective analysis of 170 patients admitted to a Cardiology ward due to acute heart failure (AHF) was performed. Patients with reduced EF (Simpson biplane method - EF Results A total of 127 patients were included. Mean patient age was 64 (±14) years; 72% were men. 48% of patients had history of ST elevation AMI. Mean EF was 54% (±8) and mean GLS was −14.3 (±3.8). Rates of 12MM and 12CV M were 14.2% and 19.3%, respectively. A statistically significant association between 12MM and 12MCV was found in univariate analysis for GLS (p0.05). However, when considering a severely compromised GLS ( Conclusion GLS is an independent predictor of 12MM and 12CVM in patients hospitalized due to AHF, with an EF ≥40% and previous history of acute myocardial infarction. In the subgroup of patients with heart failure with mid-range EF, a severely compromised GLS ( Funding Acknowledgement Type of funding sources: None.

We study the effects of bond and site disorder in the classical $J_{1}$-$J_{2}$ Heisenberg model on a square lattice in the order-by-disorder frustrated regime $2J_{2}>\left|J_{1}\right|$. Combining symmetry arguments, numerical energy minimization and large scale Monte Carlo simulations, we establish that the finite temperature Ising-like transition of the clean system is destroyed in the presence of any finite concentration of impurities. We explain this finding via a random-field mechanism which generically emerges in systems where disorder locally breaks the same real-space symmetry spontaneously globally broken by the associated order parameter. We also determine that the phase replacing the clean one is a paramagnet polarized in the nematic glass order with non-trivial magnetic response. This is because disorder also induces non-collinear spin-vortex-crystal order and produces a conjugated transverse dipolar random field. As a result of these many competing effects, the associated magnetic susceptibilities are non-monotonic functions of the temperature. As a further application of our methods, we show the generation of random axes in other frustrated magnets with broken SU(2) symmetry. We also discuss the generality of our findings and their relevance to experiments., 17 pages, 14 figures. (v2) References and further discussions added; Final version as published

All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galpα1,3Galfβ and Galpα1,3Galfβ1,3Manpα glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2 (126-, 44-, and 18-fold change, respectively) and the T-cell chemoattractants CCL3 and CCL5 (23- and 16-fold change, respectively). Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.

SARS-CoV-2 extensively N-glycosylates its surface spike (S) proteins. This post-translational modification is essential to modulate protein conformation and host cell invasion. Each S monomer can be modified with up to 22 N-glycans. To meet the high demand of protein glycosylation during virus replication, SARS-CoV-2 upregulates the expression of host N-glycosylation genes. Although a substantial amount of detail is known about the structure of S protein N-glycans, the role of N-glycosylation in SARS-CoV-2 infection remains largely undetermined. Here, we investigated the essentiality of the host N-glycosylation pathway and viral N-glycans for SARS-CoV-2 infection. When either monkey or human cells were preincubated with glycosylation inhibitors, including FDA-approved iminosugars, virus infection was significantly reduced. This infection phenotype was confirmed after RNAi knockdown of several glycosylation genes. In addition, enzymatic deglycosylation of whole viral particles confirmed that accessible oligosaccharides on the SARS-CoV-2 surface are essential for host cell infection. Altogether, we show evidence that the normal functioning of the host N- glycosylation machinery is essential not only for SARS-CoV-2 to infect, but also to produce new functional virions. These findings open the door for developing new approaches targeting N-glycosylation against COVID-19.

The ability to undergo morphological changes during adaptation to distinct environments is exploited byCandida albicansand has a direct impact on virulence. In this study, we investigated the influence of fungal extracellular vesicles (EVs) during yeast growth, biofilm formation, and morphogenesis inC. albicans. Addition ofC. albicansEVs (CaEVs) to the culture medium positively affected yeast growth. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated thatCaEVs inhibited biofilm formation byC. albicans in vitro. By time-lapse microscopy and SEM, we showed thatCaEV-treatment stops filamentation promoting pseudohyphae formation with multiple sites for yeast budding. The ability ofCaEVs to regulate dimorphism was further compared to EVs isolated from differentC. albicansstrains,Saccharomyces cerevisiae, andHistoplasma capsulatum.CaEVs from distinct strains robustly inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 hours. A minor inhibitory effect was promoted by EVs fromS. cerevisiaeandH. capsulatumonly after 24 hours of incubation. The inhibitory effect ofCaEVs was promoted by a combination of lipid compounds identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably,CaEVs were also able to reverse filamentation, transforming hyphal growth to yeast forms. Transcriptomic analysis demonstrated that treatment withCaEVs modified the expression of more than 300 genes. The most effectively upregulated pathways were related to DNA metabolism. The downregulated genes were mostly associated with extracellular and adhesion proteins. Finally, yeast cells treated withCaEVs for 24 hours lost their agar invasive ability and were avirulent when inoculated inGalleria mellonellalarvae. In summary, our results indicate that fungal EVs can profoundly modifyC. albicansgrowth and regulate yeast-to-hypha differentiation inhibiting biofilm formation and virulence.

Current treatment for Chagas disease with the only available drugs, benznidazole or nifurtimox, has substantial limitations, including long treatment duration and safety and tolerability concerns. We aimed to evaluate the efficacy and safety of new benznidazole monotherapy regimens and combinations with fosravuconazole, in the treatment of Chagas disease.We did a double-blind, double-dummy, phase 2, multicentre, randomised trial in three outpatient units in Bolivia. Adults aged 18-50 years with chronic indeterminate Chagas disease, confirmed by serological testing and positive qualitative PCR results, were randomly assigned (1:1:1:1:1:1:1) to one of seven treatment groups using a balanced block randomisation scheme with an interactive response system. Participants were assigned to benznidazole 300 mg daily for 8 weeks, 4 weeks, or 2 weeks, benznidazole 150 mg daily for 4 weeks, benznidazole 150 mg daily for 4 weeks plus fosravuconazole, benznidazole 300 mg once per week for 8 weeks plus fosravuconazole, or placebo, with a 12-month follow-up period. The primary endpoints were sustained parasitological clearance at 6 months, defined as persistent negative qualitative PCR results from end of treatment, and incidence and severity of treatment-emergent adverse events, serious adverse events, and adverse events leading to treatment discontinuation. Primary efficacy analysis was based on the intention-to-treat and per-protocol populations and secondary efficacy analyses on the per-protocol population. Safety analyses were based on the as-treated population. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT03378661.Between Nov 30, 2016, and July 27, 2017, we screened 518 patients, and 210 were enrolled and randomised. 30 patients (14%) were assigned to each treatment group. All 210 randomised patients were included in the intention-to-treat population, and 190 (90%) were included in the per-protocol population. In the intention-to-treat analysis, only one (3%) of 30 patients in the placebo group had sustained parasitological clearance at 6 months of follow-up. Sustained parasitological clearance at 6 months was observed in 25 (89%) of 28 patients receiving benznidazole 300 mg daily for 8 weeks (rate difference vs placebo 86% [95% CI 73-99]), 25 (89%) of 28 receiving benznidazole 300 mg daily for 4 weeks (86% [73-99]), 24 (83%) of 29 receiving benznidazole 300 mg daily for 2 weeks (79% [64-95]), 25 (83%) of 30 receiving benznidazole 150 mg daily for 4 weeks (80% [65-95]), 23 (85%) of 28 receiving benznidazole 150 mg daily for 4 weeks plus fosravuconazole (82% [67-97]), and 24 (83%) of 29 receiving benznidazole 300 mg weekly for 8 weeks plus fosravuconazole (79% [64-95]; p0·0001 for all group comparisons with placebo). Six patients (3%) had ten serious adverse events (leukopenia [n=3], neutropenia [n=2], pyrexia, maculopapular rash, acute cholecystitis, biliary polyp, and breast cancer), eight had 12 severe adverse events (defined as interfering substantially with the patient's usual functions; elevated alanine aminotransferase [n=4], elevated gamma-glutamyltransferase [n=2], elevated aspartate aminotransferase [n=1], neutropenia [n=3], leukopenia [n=1], and breast cancer [n=1]), and 15 (7%) had adverse events that led to treatment discontinuation (most of these were in the groups who received benznidazole 300 mg daily for 8 weeks, benznidazole 300 mg once per week for 8 weeks plus fosravuconazole, and benznidazole 150 mg daily for 4 weeks plus fosravuconazole). No adverse events leading to treatment discontinuation were observed in patients treated with benznidazole 300 mg daily for 2 weeks or placebo. There were no treatment-related deaths.Benznidazole induced effective antiparasitic response, regardless of treatment duration, dose, or combination with fosravuconazole, and was well tolerated in adult patients with chronic Chagas disease. Shorter or reduced regimens of benznidazole could substantially improve treatment tolerability and accessibility, but further studies are needed to confirm these results.Drugs for Neglected Diseases initiative (DNDi).For the Spanish translation of the abstract see Supplementary Materials section.

descripción no proporcionada por scopus, ISGlobal work is supported by the Departament d’Universitats i Recerca de la Generalitat de Catalunya, Spain (AGAUR; 017SGR00924) and by the Instituto de Salud Carlos III (ISCIII) RICET Network for Cooperative Research in Tropical Diseases (ISCIII; RD16/0027/0004 - PI1290) and FEDER. MJP research is supported by the Ministry of Health, Government of Catalonia (PERIS 2016-2010 SLT008/18/00132). ICA, JG, and FT are supported by the grant number U01AI129783 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). ICA is also partly supported by the grant number 5U54MD007592 from the National Institute on Minority Health and Health Disparities (NIMHD), NIH. MCL and MCT were supported by ISCIII RICET grant RD16/0027/0005 - PI1290 and FEDER and by grants SAF2016-81003-R and SAF2016-80998-R from the Spanish “Programa Estatal I+D+i (MINECO)”. AA's work was supported by the Italian Ministry of Health “Fondi Ricerca Corrente - Linea 3, progetto 9” to IRCCS Sacro Cuore Don Calabria Hospital. JR was supported by CONACyT Fossis grant #261006. The Drugs for Neglected Diseases initiative (DNDi) is grateful to its donors, public and private, who have provided funding to DNDi since its inception in 2003. A full list of DNDi's donors can be found at http://www.dndi.org/donate/donors/. FIND is grateful to its donors, public and private, who have helped bring innovative new diagnostics for diseases of poverty. A full list of FIND’s donors can be found at: https://www.finddx.org/partners-donors/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50–300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers. Barcelona Institute for Global Health (ISGlobal) receives support from the Spanish Ministry of Science, Innovation and Universities through the Centro de Excelencia Severo Ochoa 2019–2023 Program (CEX2018-000806-S). ISGlobal and Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP) are members of the Centres de Recerca de Catalunya (CERCA Program), Generalitat de Catalunya. Work in the laboratory of C.F.B. is funded by Fundació La Marató de TV3 (reference 566/U/2018) and Fundación Mundo Sano. This project was co-financed by the European Union through the European Regional Development Fund with the support of Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya. N.C., M.G., J.G., and M.J.P. receive funds from the Redes temáticas de investigación cooperativa en salud (RETICS), Spanish Tropical Diseases Network “RD12/0018/0010” and from the Agencia de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya; grant “2017 SGR 00924.” M.G., C.B., J.G., M.J.P., and I.C.A. belong to the Ibero-American Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas network. I.C.A. is partially supported by grants no. 2G12MD007592 and 5U54MD007592 from the National Institute on Minority Health and Health Disparities of the US National Institutes of Health. We are grateful to the Biomolecule Analysis Core Facility at University of Texas at El Paso, Border Biomedical Research Center, funded by National Institute on Minority Health and Health Disparities grants 2G12MD007592 and 5U54MD007592. M.T.M. received a PhD fellowship from the Science Without Borders Program, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. Sí

Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galβ (NGP9B), Galα(1,6)[Galα(1,2)]Galβ (NGP11B), and Galα(1,3)Galβ(1,4)Glcβ (NGP1B) that are differentially recognized in sera from individuals withLeishmania majorinfection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.

Background: Chagas disease is a major neglected vector-borne disease. In this study, we investigated the safety and efficacy of three oral E1224 (a water-soluble ravuconazole prodrug) regimens and benznidazole versus placebo in adult chronic indeterminate Chagas disease. Method: In this proof-of-concept, double-blind, randomised phase 2 clinical trial, we recruited adults (18–50 years) with confirmed diagnosis of Trypanosoma cruzi infection from two outpatient units in Bolivia. Patients were randomised with a computer-generated randomisation list, which was stratified by centre and used a block size of ten. Patients were randomly assigned (1:1:1:1:1) to five oral treatment groups: high-dose E1224 (duration 8 weeks, total dose 4000 mg), low-dose E1224 (8 weeks, 2000 mg), short-dose E1224 (4 weeks + 4 weeks placebo, 2400 mg), benznidazole (60 days, 5 mg/kg per day), or placebo (8 weeks, E1224-matched tablets). Double-blinding was limited to the E1224 and placebo arms, and assessors were masked to all treatment allocations. The primary efficacy endpoint was parasitological response to E1224 at the end of treatment, assessed by PCR. The secondary efficacy endpoints were parasitological response to benznidazole at end of treatment, assessed by PCR; sustainability of parasitological response until 12 months; parasite clearance and changes in parasite load; incidence of conversion to negative response in conventional and non-conventional (antigen trypomastigote chemiluminescent ELISA [AT CL-ELISA]) serological response; changes in levels of biomarkers; and complete response. The primary analysis population consisted of all randomised patients by their assigned treatment arms. This trial is registered with ClinicalTrials.gov, number NCT01489228. Findings: Between July 19, 2011, and July 26, 2012, we screened 560 participants with confirmed Chagas disease, of whom 231 were enrolled and assigned to high-dose E1224 (n=45), low-dose E1224 (n=48), short-dose E1224 (n=46), benznidazole (n=45), or placebo (n=47). Parasite clearance was observed with E1224 during the treatment phase, but no sustained response was seen with low-dose and short-dose regimens, whereas 13 patients (29%, 95% CI 16·4–44·3) had sustained response with the high-dose regimen compared with four (9%, 2·4–20·4) in the placebo group (p

IntroductionChagas disease (CD) affects ~7 million people worldwide. Benznidazole (BZN) and nifurtimox (NFX) are the only approved drugs for CD chemotherapy. Although both drugs are highly effective in acute and paediatric infections, their efficacy in adults with chronic CD (CCD) is lower and variable. Moreover, the high incidence of adverse events (AEs) with both drugs has hampered their widespread use. Trials in CCD adults showed that quantitative PCR (qPCR) assays remain negative for 12 months after standard-of-care (SoC) BZN treatment in ~80% patients. BZN pharmacokinetic data and the nonsynchronous nature of the proliferative mammal-dwelling parasite stage suggested that a lower BZN/NFX dosing frequency, combined with standard or extended treatment duration, might have the same or better efficacy than either drug SoC, with fewer AEs.Methods and analysisNew ThErapies and Biomarkers for ChagaS infEctiOn (TESEO) is an open-label, randomised, prospective, phase-2 clinical trial, with six treatment arms (75 patients/arm, 450 patients). Primary objectives are to compare the safety and efficacy of two new proposed chemotherapy regimens of BZN and NFX in adults with CCD with the current SoC for BZN and NFX, evaluated by qPCR and biomarkers for 36 months posttreatment and correlated with CD conventional serology. Recruitment of patients was initiated on 18 December 2019 and on 20 May 2021, 450 patients (study goal) were randomised among the six treatment arms. The treatment phase was finalised on 18 August 2021. Secondary objectives include evaluation of population pharmacokinetics of both drugs in all treatment arms, the incidence of AEs, and parasite genotyping.Ethics and disseminationThe TESEO study was approved by the National Institutes of Health (NIH), U.S. Food and Drug Administration (FDA), federal regulatory agency of the Plurinational State of Bolivia and the Ethics Committees of the participating institutions. The results will be disseminated via publications in peer-reviewed journals, conferences and reports to the NIH, FDA and participating institutions.Trial registration numberNCT03981523.

The microneme organelles of Toxoplasma gondii tachyzoites release protein complexes (MICs), including one composed of the transmembrane protein MIC6 plus MIC1 and MIC4. In this complex, carbohydrate recognition domains of MIC1 and MIC4 are exposed and interact with terminal sialic acid and galactose residues, respectively, of host cell glycans. Recently, we demonstrated that MIC1 and MIC4 binding to the N-glycans of Toll-like receptor (TLR) 2 and TLR4 on phagocytes triggers cell activation and pro-inflammatory cytokine production. Herein, we investigated the requirement for TLR2 heterodimerization and co-receptors in MIC-induced responses, as well as the signaling molecules involved. We used MICs to stimulate macrophages and HEK293T cells transfected with TLR2 and TLR1 or TLR6, both with or without the co-receptors CD14 and CD36. Then, the cell responses were analyzed, including nuclear factor-kappa B (NF-&kappa, B) activation and cytokine production, which showed that (1) only TLR2, among the studied factors, is crucial for MIC-induced cell activation, (2) TLR2 heterodimerization augments, but is not critical for, activation, (3) CD14 and CD36 enhance the response to MIC stimulus, and (4) MICs activate cells through a transforming growth factor beta-activated kinase 1 (TAK1)-, mammalian p38 mitogen-activated protein kinase (p38)-, and NF-&kappa, B-dependent pathway. Remarkably, among the studied factors, the interaction of MIC1 and MIC4 with TLR2 N-glycans is sufficient to induce cell activation, which promotes host protection against T. gondii infection.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

The α-Gal antigen [Galα(1,3)Galβ(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.