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Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein’s and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19.

Abstract Background Pre-exposure prophylaxis (PrEP) is a promising strategy to break COVID-19 transmission. Although hydroxychloroquine was evaluated for treatment and post-exposure prophylaxis, it is not evaluated for COVID-19 PrEP yet. The aim of this study was to evaluate the efficacy and safety of PrEP with hydroxychloroquine against placebo in healthcare workers at high risk of SARS-CoV-2 infection during an epidemic period. Methods We conducted a double-blind placebo-controlled randomized clinical trial in three hospitals in Barcelona, Spain. From 350 adult healthcare workers screened, we included 269 participants with no active or past SARS-CoV-2 infection (determined by a negative nasopharyngeal SARS-CoV-2 PCR and a negative serology against SARS-CoV-2). Participants allocated in the intervention arm (PrEP) received 400 mg of hydroxychloroquine daily for the first four consecutive days and subsequently, 400 mg weekly during the study period. Participants in the control group followed the same treatment schedule with placebo tablets. Results 52.8% (142/269) of participants were in the hydroxychloroquine arm and 47.2% (127/269) in the placebo arm. Given the national epidemic incidence decay, only one participant in each group was diagnosed with COVID-19. The trial was stopped due to futility and our study design was deemed underpowered to evaluate any benefit regarding PrEP efficacy. Both groups showed a similar proportion of participants experiencing at least one adverse event (AE) (p=0.548). No serious AEs were reported. Almost all AEs (96.4%, 106/110) were mild. Only mild gastrointestinal symptoms were significantly higher in the hydroxychloroquine arm compared to the placebo arm (27.4% (39/142) vs 15.7% (20/127), p=0.041). Conclusions Although the efficacy of PrEP with hydroxychloroquine for preventing COVID-19 could not be evaluated, our study showed that PrEP with hydroxychloroquine at low doses is safe. Trial registration ClinicalTrials.gov NCT04331834 . Registered on April 2, 2020.

Background: Chagas disease (CD) is endemic in Latin America and particularly common in Bolivia, but there is little information on the characteristics of chronic digestive involvement. Objectives: To determine the prevalence and characterize digestive manifestations in chronic CD patients in Cochabamba, Bolivia. Methods: Eighty-five T. cruzi-seropositive individuals with or without digestive symptoms (G1 group), and fifteen T. cruzi-seronegative patients with similar digestive symptoms to those seen in CD (G2 group) were included in the study. All patients underwent a detailed history including past medical history, epidemiological information, hygiene and dietary habits, a complete physical examination, two serological tests for T. cruzi, video endoscopy, barium swallow, and barium enema. Findings: We observed digestive manifestations in T. cruzi seropositive and seronegative patients. Colonic manifestations were detected in both groups, highlighting the relevance of other confounder factors in the region. Constipation was present in 52.9% of G1 patients, 62.4% presented two or more upper digestive tract symptoms, and 5.9% of them presented esophageal manifestations. Helicobacter pylori infection was detected in 58.8% of G1 patients, and all patients presented gastritis on endoscopy. Conclusions: Prevalence of digestive involvement in CD patients is higher than expected. However, digestive symptoms are not always caused by T. cruzi infection and require differential diagnoses.

Abstract Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells

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 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.

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.

Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease.

Chagas disease (CD) and tuberculosis (TB) are important health problems in Bolivia. Current treatments for both infections require a long period of time, and adverse drug reactions (ADRs) are frequent. This study aims to strengthen the Bolivian pharmacovigilance system, focusing on CD and TB. A situation analysis of pharmacovigilance in the Department of Cochabamba was performed. The use of a new local case report form (CRF) was implemented, together with the CRF established by the Unidad de Medicamentos y Tecnología en Salud (UNIMED), in several healthcare centers. Training and follow-up on drug safety monitoring and ADR reporting was provided to all health professionals involved in CD and TB treatment. A comparative analysis of the reported ADRs using the CRF provided by UNIMED, the new CRF proposal, and medical records, was also performed. Our results showed that out of all patients starting treatment for CD, 37.9% suffered ADRs according to the medical records, and 25.3% of them were classified as moderate/severe (MS). Only 47.4% of MS ADRs were reported to UNIMED. Regarding TB treatment, 9.9% of all patients suffered ADRs, 44% of them were classified as MS, and 75% of MS ADRs were reported to UNIMED. These findings show that the reinforcement of the Bolivian pharmacovigilance system is an ambitious project that should involve a long-term perspective and the engagement of national health workers and other stakeholders at all levels. Continuity and perseverance are essential to achieve a solid ADR reporting system, improving patient safety, drug efficacy and adherence to treatment.

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.