Identifying innovations produced by primary health care centers and evaluating their scalability: the SPRINT Occitanie cross-sectional study in France.

BACKGROUND: Practice-based research is one of the levers identified by the World Health Organization (WHO) to strengthen primary health care. The scaling of health and social care innovations has the potential to reduce inequities in health and to expand the benefits of effective innovations. It is now rapidly gaining the attention of decision-makers in health and social care, particularly in high-income countries. To meet the challenge of declining numbers of primary care physicians in France, Multi-professional Healthcare Centers (MHC) were created to bring together medical and paramedical professionals. They are a source of innovation in meeting the health challenges facing our populations. Specific methodology exists to identify health innovations and assess their scalability. A working group, including end-users and specialists, has adapted this methodology to the French context and the University department of general practice of Montpellier-Nîmes (France) launched a pilot study in Occitanie, a French region. OBJECTIVE: To identify and evaluate the scalability of innovations produced in pluri-professional healthcare centers in the Occitanie region. METHODS: A pilot, observational, cross-sectional study was carried out. The SPRINT Occitanie study was based on a questionnaire with two sections: MHC information and the modified Innovation Scalability Self-Administered Questionnaire (ISSaQ), version 2020. The study population was all 279 MHC in the Occitanie region. RESULTS: 19.3% (54) of MHC in the Occitanie region, responded fully or incompletely to the questionnaire. Four out of 5 U-MHCs were represented. Five MHC presented multiple innovations. The average per MHC was 1.94 (± 2.4) innovations. 26% of them (n = 9) had high scalability, 34% (n = 12) medium scalability and 40% (n = 14) low scalability. The main innovation represented (86%) were healthcare program, service, and tool. CONCLUSIONS: In our cross-sectional study, a quarter of the innovations were highly scalable. We were able to demonstrate the importance of MHC teams in working on primary care research through the prism of innovations. Primary-care innovations must be detected, evaluated, and extracted to improve their impact on their healthcare system.

Hospitalizations for Chronic Obstructive Pulmonary Disease Exacerbation During COVID-19.

This cross-sectional study investigates changes in the number of chronic obstructive pulmonary disease (COPD)­related admissions before, during, and after the COVID-19 pandemic in France.

1H, 13C and 15N backbone and side-chain resonance assignments of ∆∆BmSA1, the surface antigen of Babesia microti.

Human babesiosis is a vector-borne zoonotic infection caused mostly by the Apicomplexan parasite Babesia microti, distributed worldwide. The infection can result in severe symptoms such as hemolytic anemia, especially in immunodeficient patients. Also, asymptomatic patients continue transmission as unscreened blood donors, and represent a risk for Public Health. Early host-parasite interactions are mediated by BmSA1, the major surface antigen of Babesia microti, crucial for invasion and immune escape. Hence, a structural and functional characterization of the BmSA1 protein constitutes a first strategic milestone toward the development of innovative tools to control infection. Knowledge of the 3D structure of such an important antigen is crucial for the development of vaccines or new diagnostic tests. Here, we report the 1H, 15N and 13C NMR resonance assignment of ∆∆BmSA1, a truncated recombinant version of BmSA1 without the N-terminal signal peptide and the hydrophobic C-terminal GPI-anchor. Secondary structure prediction using CSI.3 and TALOS-N demonstrates a high content of alpha-helical structure. This preliminary study provides foundations for further structural characterization of BMSA1.

A systematic review of peptide-based serological tests for the diagnosis of leishmaniasis.

Serological methods should meet the needs of leishmaniasis diagnosis due to their high sensitivity and specificity, economical and adaptable rapid diagnostic test format, and ease of use. Currently, the performances of serological diagnostic tests, despite improvements with recombinant proteins, vary greatly depending on the clinical form of leishmaniasis and the endemic area. Peptide-based serological tests are promising as they could compensate for antigenic variability and improve performance, independently of Leishmania species and subspecies circulating in the endemic areas. The objective of this systematic review was to inventory all studies published from 2002 to 2022 that evaluate synthetic peptides for serological diagnosis of human leishmaniases and also to highlight the performance (e.g., sensitivity and specificity) of each peptide reported in these studies. All clinical forms of leishmaniasis, visceral and tegumentary, and all Leishmania species responsible for these diseases were considered. Following PRISMA statement recommendations, 1,405 studies were identified but only 22 articles met the selection criteria and were included in this systematic review. These original research articles described 77 different peptides, of which several have promising performance for visceral or tegumentary leishmaniasis diagnosis. This review highlights the importance of and growing interest in synthetic peptides used for serological diagnosis of leishmaniases, and their performances compared to some widely used tests with recombinant proteins.

Title: Revue systématique des tests sérologiques à base de peptides pour le diagnostic de la leishmaniose. Abstract: D'une sensibilité et d'une spécificité élevées, faciles à réaliser, économiques et adaptables à un format de test de diagnostic rapide, les méthodes sérologiques devraient répondre aux besoins du diagnostic de la leishmaniose. Actuellement, les performances des tests de diagnostic sérologique, malgré des améliorations avec les protéines recombinantes, varient fortement selon la forme clinique de la leishmaniose et les zones d'endémie. Les tests sérologiques à base de peptides sont prometteurs car ils pourraient compenser la variabilité antigénique et améliorer les performances, indépendamment des espèces et sous-espèces de Leishmania circulant dans les zones endémiques. L'objectif de cette revue systématique était d'inventorier toutes les études publiées de 2002 à 2022 qui évaluent les peptides synthétiques pour le diagnostic sérologique des leishmanioses humaines et également de mettre en évidence les performances (dont la sensibilité et la spécificité) de chaque peptide rapporté dans ces études. Toutes les formes cliniques de leishmanioses, viscérales et tégumentaires, et toutes les espèces de Leishmania responsables de ces maladies ont été considérées. Suite aux recommandations de la déclaration PRISMA, 1405 études ont été identifiées mais seuls 22 articles répondaient aux critères de sélection et ont été inclus dans cette revue systématique. Ces articles de recherche originaux décrivent 77 peptides différents, dont plusieurs sont prometteurs pour le diagnostic de la leishmaniose viscérale ou tégumentaire. Cette revue met en évidence l'importance et l'intérêt croissant accordés aux peptides synthétiques utilisés pour le diagnostic sérologique des leishmanioses, et leurs performances par rapport à certains tests largement utilisés avec des protéines recombinantes.

SILVI, an open-source pipeline for T-cell epitope selection.

High-throughput screening of available genomic data and identification of potential antigenic candidates have promoted the development of epitope-based vaccines and therapeutics. Several immunoinformatic tools are available to predict potential epitopes and other immunogenicity-related features, yet it is still challenging and time-consuming to compare and integrate results from different algorithms. We developed the R script SILVI (short for: from in silico to in vivo), to assist in the selection of the potentially most immunogenic T-cell epitopes from Human Leukocyte Antigen (HLA)-binding prediction data. SILVI merges and compares data from available HLA-binding prediction servers, and integrates additional relevant information of predicted epitopes, namely BLASTp alignments with host proteins and physical-chemical properties. The two default criteria applied by SILVI and additional filtering allow the fast selection of the most conserved, promiscuous, strong binding T-cell epitopes. Users may adapt the script at their discretion as it is written in open-source R language. To demonstrate the workflow and present selection options, SILVI was used to integrate HLA-binding prediction results of three example proteins, from viral, bacterial and parasitic microorganisms, containing validated epitopes included in the Immune Epitope Database (IEDB), plus the Human Papillomavirus (HPV) proteome. Applying different filters on predicted IC50, hydrophobicity and mismatches with host proteins allows to significantly reduce the epitope lists with favourable sensitivity and specificity to select immunogenic epitopes. We contemplate SILVI will assist T-cell epitope selections and can be continuously refined in a community-driven manner, helping the improvement and design of peptide-based vaccines or immunotherapies. SILVI development version is available at: github.com/JoanaPissarra/SILVI2020 and https://doi.org/10.5281/zenodo.6865909.

1H, 13C, 15 N backbone and side-chain NMR assignments for three MAX effectors from Magnaporthe oryzae.

Effectors are small and very diverse proteins secreted by fungi and translocated in plant cells during infection. Among them, MAX effectors (for Magnaporthe Avrs and ToxB) were identified as a family of effectors that share an identical fold topology despite having highly divergent sequences. They are mostly secreted by ascomycetes from the Magnaporthe genus, a fungus that causes the rice blast, a plant disease leading to huge crop losses. As rice is the first source of calories in many countries, especially in Asia and Africa, this constitutes a threat for world food security. Hence, a better understanding of these effectors, including structural and functional characterization, constitutes a strategic milestone in the fight against phytopathogen fungi and may give clues for the development of resistant varieties of rice. We report here the near complete 1H, 15 N and 13C NMR resonance assignment of three new putative MAX effectors (MAX47, MAX60 and MAX67). Secondary structure determination using TALOS-N and CSI.3 demonstrates a high content of ß-strands in all the three proteins, in agreement with the canonic ß-sandwich structure of MAX effectors. This preliminary study provides foundations for further structural characterization, that will help in turn to improve sequence predictions of other MAX effectors through data mining.

Combining High-Pressure NMR and Geometrical Sampling to Obtain a Full Topological Description of Protein Folding Landscapes: Application to the Folding of Two MAX Effectors from Magnaporthe oryzae.

Despite advances in experimental and computational methods, the mechanisms by which an unstructured polypeptide chain regains its unique three-dimensional structure remains one of the main puzzling questions in biology. Single-molecule techniques, ultra-fast perturbation and detection approaches and improvement in all-atom and coarse-grained simulation methods have greatly deepened our understanding of protein folding and the effects of environmental factors on folding landscape. However, a major challenge remains the detailed characterization of the protein folding landscape. Here, we used high hydrostatic pressure 2D NMR spectroscopy to obtain high-resolution experimental structural information in a site-specific manner across the polypeptide sequence and along the folding reaction coordinate. We used this residue-specific information to constrain Cyana3 calculations, in order to obtain a topological description of the entire folding landscape. This approach was used to describe the conformers populating the folding landscape of two small globular proteins, AVR-Pia and AVR-Pib, that belong to the structurally conserved but sequence-unrelated MAX effectors superfamily. Comparing the two folding landscapes, we found that, in spite of their divergent sequences, the folding pathway of these two proteins involves a similar, inescapable, folding intermediate, even if, statistically, the routes used are different.

Proteomic Analysis of the Promastigote Secretome of Seven Leishmania Species.

Leishmaniasis is one of the most impactful parasitic diseases worldwide, endangering the lives of 1 billion people every year. There are 20 different species of Leishmania able to infect humans, causing cutaneous (CL), visceral (VL), and/or mucocutaneous leishmaniasis (MCL). Leishmania parasites are known to secrete a plethora of proteins to establish infection and modulate the host's immune system. In this study, we analyzed using tandem mass spectrometry the total protein content of the secretomes produced by promastigote forms from seven Leishmania species grown in serum-free in vitro cultures. The core secretome shared by all seven Leishmania species corresponds to up to one-third of total secreted proteins, suggesting conserved mechanisms of adaptation to the vertebrate host. The relative abundance confirms the importance of known virulence factors and some proteins uniquely present in CL- or VL-causing species and may provide further insight regarding their pathogenesis. Bioinformatic analysis showed that most proteins were secreted via unconventional mechanisms, with an important role for vesicle-based secretion for all species. Gene Ontology annotation and enrichment analyses showed a high level of functional conservation among species. This study contributes to the current knowledge on the biological significance of differently secreted proteins and provides new information on the correlation of Leishmania secretome to clinical outcomes and species-specific pathogenesis.

Hfq and RNase R Mediate rRNA Processing and Degradation in a Novel RNA Quality Control Process.

RNA quality control pathways are critical for cell survival. Here, we describe a new surveillance process involved in the degradation of highly structured and stable ribosomal RNAs. The results demonstrated that the RNA chaperone Hfq and the 3'-5' exoribonuclease R mediate the elimination of detrimental rRNA fragments and are required for the correct processing of rRNA precursors. Escherichia coli cells lacking both Hfq and RNase R accumulate a high level of 16S- and 23S-derived rRNA fragments. Hfq and RNase R were also shown to participate in the maturation of 16S and 23S rRNA precursors. This correlates with the fact that in the absence of Hfq and RNase R, there are severe ribosome assembly defects and a sharp reduction in 70S ribosome levels. Hfq and RNase R may act independently or in a complex, as protein interaction studies revealed that these RNA-binding proteins can associate. This is the first demonstration that the well-conserved Hfq and RNase R proteins act on common regulatory pathways, unraveling previously unknown mechanisms of rRNA surveillance with important consequences for translation and cell survival.IMPORTANCE Quality control pathways that oversee the quality of stable RNA molecules are critical for the cell. In this work, we demonstrate, for the first time, a functional link between Hfq and RNase R in the processing and degradation of the highly structured rRNAs. These RNA-binding proteins are required for the maturation of 16S and 23S rRNAs and correct ribosome assembly. Furthermore, they participate in the degradation of rRNAs and clearance of toxic rRNA fragments from the cell. Our studies have also shown that Hfq and RNase R can form a complex. In summary, the cooperation between Hfq and RNase R in metabolic pathways of stable RNAs may represent a broader mechanism of RNA quality control, given the high conservation of these RNA-binding proteins throughout evolution.

Peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis.

Dogs are the main reservoir of zoonotic visceral leishmaniasis. Vaccination is a promising approach to help control leishmaniasis and to interrupt transmission of the Leishmania parasite. The promastigote surface antigen (PSA) is a highly immunogenic component of Leishmania excretory/secretory products. A vaccine based on three peptides derived from the carboxy-terminal part of Leishmania amazonensis PSA and conserved among Leishmania species, formulated with QA-21 as adjuvant, was tested on naive Beagle dogs in a preclinical trial. Four months after the full course of vaccination, dogs were experimentally infected with Leishmania infantum promastigotes. Immunization of dogs with peptide-based vaccine conferred immunity against experimental infection with L. infantum. Evidence for macrophage nitric oxide production and anti-leishmanial activity associated with IFN-γ production by lymphocytes was only found in the vaccinated group. An increase in specific IgG2 antibodies was also measured in vaccinated dogs from 2 months after immunization. Additionally, after challenge with L. infantum, the parasite burden was significantly lower in vaccinated dogs than in the control group. These data strongly suggest that this peptide-based vaccine candidate generated cross-protection against zoonotic leishmaniasis by inducing a Th1-type immune response associated with production of specific IgG2 antibodies. This preclinical trial including a peptide-based vaccine against leishmaniasis clearly demonstrates effective protection in a natural host. This approach deserves further investigation to enhance the immunogenicity of the peptides and to consider the possible engineering of a vaccine targeting several Leishmania species.

Pre-clinical evaluation of a P. berghei-based whole-sporozoite malaria vaccine candidate.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite P. berghei (Pb) that expresses the P. falciparum (Pf) circumsporozoite protein (PfCS), and showed that this parasite line (PbVac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human Pf parasite. Here, we analyzed PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables PbVac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation.

Trypanosomatid Infections: How Do Parasites and Their Excreted-Secreted Factors Modulate the Inducible Metabolism of l-Arginine in Macrophages?

Mononuclear phagocytes (monocytes, dendritic cells, and macrophages) are among the first host cells to face intra- and extracellular protozoan parasites such as trypanosomatids, and significant expansion of macrophages has been observed in infected hosts. They play essential roles in the outcome of infections caused by trypanosomatids, as they can not only exert a powerful antimicrobial activity but also promote parasite proliferation. These varied functions, linked to their phenotypic and metabolic plasticity, are exerted via distinct activation states, in which l-arginine metabolism plays a pivotal role. Depending on the environmental factors and immune response elements, l-arginine metabolites contribute to parasite elimination, mainly through nitric oxide (NO) synthesis, or to parasite proliferation, through l-ornithine and polyamine production. To survive and adapt to their hosts, parasites such as trypanosomatids developed mechanisms of interaction to modulate macrophage activation in their favor, by manipulating several cellular metabolic pathways. Recent reports emphasize that some excreted-secreted (ES) molecules from parasites and sugar-binding host receptors play a major role in this dialog, particularly in the modulation of the macrophage's inducible l-arginine metabolism. Preventing l-arginine dysregulation by drugs or by immunization against trypanosomatid ES molecules or by blocking partner host molecules may control early infection and is a promising way to tackle neglected diseases including Chagas disease, leishmaniases, and African trypanosomiases. The present review summarizes recent knowledge on trypanosomatids and their ES factors with regard to their influence on macrophage activation pathways, mainly the NO synthase/arginase balance. The review ends with prospects for the use of biological knowledge to develop new strategies of interference in the infectious processes used by trypanosomatids, in particular for the development of vaccines or immunotherapeutic approaches.

Inhibition of Plasmodium Liver Infection by Ivermectin.

Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids.

The Trypanosomatidae family includes the genera Trypanosoma and Leishmania, protozoan parasites displaying complex digenetic life cycles requiring a vertebrate host and an insect vector. Trypanosoma brucei gambiense, Trypanosoma cruzi, and Leishmania spp. are important human pathogens causing human African trypanosomiasis (HAT or sleeping sickness), Chagas' disease, and various clinical forms of Leishmaniasis, respectively. They are transmitted to humans by tsetse flies, triatomine bugs, or sandflies, and affect millions of people worldwide. In humans, extracellular African trypanosomes (T. brucei) evade the hosts' immune defenses, allowing their transmission to the next host, via the tsetse vector. By contrast, T. cruzi and Leishmania sp. have developed a complex intracellular lifestyle, also preventing several mechanisms to circumvent the host's immune response. This review seeks to set out the immune evasion strategies developed by the different trypanosomatids resulting from parasite-host interactions and will focus on: clinical and epidemiological importance of diseases; life cycles: parasites-hosts-vectors; innate immunity: key steps for trypanosomatids in invading hosts; deregulation of antigen-presenting cells; disruption of efficient specific immunity; and the immune responses used for parasite proliferation.