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Total laboratory automation (TLA) is a valuable component of microbiology laboratories and a growing number of publications suggest the potential impact of automation in terms of analysis standardization, streaking quality, and the turnaround time (TAT). The aim of this project was to perform a detailed investigation of the impact of TLA on the workflow of commonly treated specimens such as urine. This is a retrospective observational study comparing two time periods (pre TLA versus post TLA) for urine specimen culture processing. A total of 35,864 urine specimens were plated during the pre-TLA period and 47,283 were plated during the post-TLA period. The median time from streaking to identification decreased from 22.3 h pre TLA to 21.4 h post TLA (p < 0.001), and the median time from streaking to final validation of the report decreased from 24.3 h pre TLA to 23 h post TLA (p < 0.001). Further analysis revealed that the observed differences in TAT were mainly driven by the contaminated and positive samples. Our findings demonstrate that TLA has the potential to decrease turnaround times of samples in a laboratory. Nevertheless, changes in laboratory workflow (such as extended opening hours for plate reading and antibiotic susceptibility testing or decreased incubation times) might further maximize the efficiency of TLA and optimize TATs.
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Introduction: Since its emergence in late 2019, the SARS-CoV-2 virus has led to a global health crisis, affecting millions and reshaping societies and economies worldwide. Investigating the determinants of SARS-CoV-2 diffusion and their spatiotemporal dynamics at high spatial resolution is critical for public health and policymaking. Methods: This study analyses 194,682 georeferenced SARS-CoV-2 RT-PCR tests from March 2020 and April 2022 in the canton of Vaud, Switzerland. We characterized five distinct pandemic periods using metrics of spatial and temporal clustering like inverse Shannon entropy, the Hoover index, Lloyd's index of mean crowding, and the modified space-time DBSCAN algorithm. We assessed the demographic, socioeconomic, and environmental factors contributing to cluster persistence during each period using eXtreme Gradient Boosting (XGBoost) and SHapley Additive exPlanations (SHAP), to consider non-linear and spatial effects. Results: Our findings reveal important variations in the spatial and temporal clustering of cases. Notably, areas with flatter epidemics had higher total attack rate. Air pollution emerged as a factor showing a consistent positive association with higher cluster persistence, substantiated by both immission models and, to a lesser extent, tropospheric NO2 estimations. Factors including population density, testing rates, and geographical coordinates, also showed important positive associations with higher cluster persistence. The socioeconomic index showed no significant contribution to cluster persistence, suggesting its limited role in the observed dynamics, which warrants further research. Discussion: Overall, the determinants of cluster persistence remained across the study periods. These findings highlight the need for effective air quality management strategies to mitigate air pollution's adverse impacts on public health, particularly in the context of respiratory viral diseases like COVID-19.
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COVID-19 , SARS-CoV-2 , Análise Espaço-Temporal , Humanos , COVID-19/epidemiologia , COVID-19/transmissão , Suíça/epidemiologia , Poluição do Ar/estatística & dados numéricos , Pandemias , Fatores SocioeconômicosRESUMO
INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.
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Antibacterianos , Azitromicina , Proteínas de Bactérias , Farmacorresistência Bacteriana , Mutação , Neisseria gonorrhoeae , Proteínas Repressoras , Azitromicina/farmacologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Humanos , Proteínas Repressoras/genética , Farmacorresistência Bacteriana/genética , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Ribossômicas/genética , Gonorreia/microbiologia , Gonorreia/tratamento farmacológico , Masculino , FemininoRESUMO
PURPOSE: To determine predictors of mortality among patients with Pseudomonas aeruginosa bacteraemia. METHODS: Retrospective study. SETTING: This study conducted at the Lausanne University Hospital, Switzerland included adult patients with P. aeruginosa bacteraemia from 2015 to 2021. RESULTS: During the study period, 278 episodes of P. aeruginosa bacteraemia were included. Twenty (7%) isolates were multidrug-resistant. The most common type of infection was low respiratory tract infection (58 episodes; 21%). Sepsis was present in the majority of episodes (152; 55%). Infectious diseases consultation within 48 h of bacteraemia onset was performed in 203 (73%) episodes. Appropriate antimicrobial treatment was administered within 48 h in 257 (92%) episodes. For most episodes (145; 52%), source control was considered necessary, with 93 (64%) of them undergoing such interventions within 48 h. The 14-day mortality was 15% (42 episodes). The Cox multivariable regression model showed that 14-day mortality was associated with sepsis (P 0.002; aHR 6.58, CI 1.95-22.16), and lower respiratory tract infection (P < 0.001; aHR 4.63, CI 1.78-12.06). Conversely, interventions performed within 48 h of bacteraemia onset, such as infectious diseases consultation (P 0.036; HR 0.51, CI 0.27-0.96), and source control (P 0.009; aHR 0.17, CI 0.47-0.64) were associated with improved outcome. CONCLUSION: Our findings underscore the pivotal role of early infectious diseases consultation in recommending source control interventions and guiding antimicrobial treatment for patients with P. aeruginosa bacteraemia.
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In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.
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Inteligência Artificial , Bactérias , Sensibilidade e Especificidade , Humanos , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Bactérias/classificação , Algoritmos , Técnicas Bacteriológicas/métodos , Processamento de Imagem Assistida por Computador/métodos , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Bacteriologia , Automação Laboratorial/métodos , Meios de Cultura/químicaRESUMO
This retrospective study, conducted at Lausanne University Hospital (2015-2021), compared Staphylococcus aureus bacteraemia (SABA) patients with or without concomitant bacteriuria (SABU). Among 448 included bacteraemic patients, 62 (13.8%) had S. aureus concurrently isolated from urine. In multivariate analysis, there was a significant difference in the odds of community-onset bacteraemia (P 0.030), malignancy (P 0.002), > 1 pair of positive blood cultures (P 0.037), and persistent bacteraemia for at least 48 h (P 0.045) in patients with concurrent SABU. No difference concerning mortality was found. On the other hand, SABU was associated with higher rates of SABA recurrence after antibiotic cessation.
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Bacteriemia , Bacteriúria , Infecções Estafilocócicas , Humanos , Bacteriúria/complicações , Bacteriúria/microbiologia , Staphylococcus aureus , Estudos Retrospectivos , Relevância Clínica , Bacteriemia/complicações , Bacteriemia/microbiologia , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/microbiologiaRESUMO
Between March 2021 and February 2022, SARS-CoV-2 neutralizing antibodies dynamics was investigated in a prospective observational study in 903 healthcare workers of a hospital in Switzerland. A surrogate neutralization assay measuring the competitive inhibition of the angiotensin converting enzyme 2 (ACE2) binding to the spike protein (S) of the SARS-CoV-2 wild type virus and to five variants of concern (Alpha, Beta, Gamma, Delta, Omicron) was used. We observed a broad distribution of neutralization activity among participants and substantial differences in neutralizing titers against variants. Participants were grouped based on combinations of vaccination status (1, 2 or 3 doses) and/or prior or subsequent SARS-CoV-2 infection/reinfection. Triple vaccination resulted in the highest neutralization response, as did double vaccination with prior or subsequent infection. Double vaccination without infection showed an intermediate neutralization response while SARS-CoV-2 infection in non-vaccinated participants resulted in poor neutralization response. After triple vaccination or double vaccination plus infection, additional vaccination and/or reinfection had no impact on neutralizing antibody titers over the observed period. These results strongly support the booster dose strategy, while additional booster doses within short time intervals might not improve immunization. However, dynamics of neutralizing antibodies titers needs to be monitored individually, over time and include newly emerging variants.
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COVID-19 , SARS-CoV-2 , Humanos , Reinfecção , COVID-19/prevenção & controle , Pessoal de Saúde , Hospitais , Vacinação , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020-the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Saúde Pública , Suíça/epidemiologia , Controle de Doenças Transmissíveis , Genoma Viral/genética , FilogeniaRESUMO
Background: The need for effective public health surveillance systems to track virus spread for targeted interventions was highlighted during the COVID-19 pandemic. It spurred an interest in the use of spatiotemporal clustering and genomic analyses to identify high-risk areas and track the spread of the SARS-CoV-2 virus. However, these two approaches are rarely combined in surveillance systems to complement each one's limitations; spatiotemporal clustering approaches usually consider only one source of virus transmission (i.e., the residential setting) to detect case clusters, while genomic studies require significant resources and processing time that can delay decision-making. Here, we clarify the differences and possible synergies of these two approaches in the context of infectious disease surveillance systems by investigating to what extent geographically-defined clusters are confirmed as transmission clusters based on genome sequences, and how genomic-based analyses can improve the epidemiological investigations associated with spatiotemporal cluster detection. Methods: For this purpose, we sequenced the SARS-CoV-2 genomes of 172 cases that were part of a collection of spatiotemporal clusters found in a Swiss state (Vaud) during the first epidemic wave. We subsequently examined intra-cluster genetic similarities and spatiotemporal distributions across virus genotypes. Results: Our results suggest that the congruence between the two approaches might depend on geographic features of the area (rural/urban) and epidemic context (e.g., lockdown). We also identified two potential superspreading events that started from cases in the main urban area of the state, leading to smaller spreading events in neighboring regions, as well as a large spreading in a geographically-isolated area. These superspreading events were characterized by specific mutations assumed to originate from Mulhouse and Milan, respectively. Our analyses propose synergistic benefits of using two complementary approaches in public health surveillance, saving resources and improving surveillance efficiency.
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COVID-19 , Humanos , SARS-CoV-2/genética , Pandemias , Controle de Doenças Transmissíveis , Genômica , Análise por ConglomeradosRESUMO
Pseudomonas aeruginosa is a pathogen frequently encountered in healthcare-associated infections and immunocompromised patients. In bacteremia, this pathogen is associated with higher mortality than other Gram-negative pathogens. This increase in mortality was also found globally for multi-resistant compared to susceptible strains. Several factors have been associated with the development of resistance: previous ICU stay, use of carbapenems, and comorbidities were identified in multivariate analysis. In the therapeutic choice, previous antibiotic treatment remains the strongest driver suggesting a potential resistant strain. These risk factors will decide whether multi-resistant strains must be considered in the empiric coverage. For susceptible strains, a single agent can be used, ß-lactams are usually the first choice. Associations do not provide any advantage on mortality. Optimization of pharmacokinetic/pharmacodynamic parameters, such as prolonged infusion (for time-dependent antibiotics), increased dosage (for concentration-dependent antibiotics), and therapeutic drug monitoring, also influences the outcome. The increasing number of resistant strains led the clinician to use either recently approved new molecules but also associations. For multi-resistant strains, new molecules such as ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol have shown an adequate activity against P. aeruginosa. Older molecules like colistin and fosfomycin are also used in this indication. The complexity of the resistance and consequences on a larger scale of antibiotic prescription will probably lead to more individualized prescriptions.
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Fosfomicina , Infecções por Pseudomonas , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/induzido quimicamente , Colistina/uso terapêutico , Fosfomicina/uso terapêutico , Tazobactam/farmacologia , Tazobactam/uso terapêutico , Antibacterianos/farmacologia , Pseudomonas aeruginosa , Carbapenêmicos/farmacologia , Combinação de Medicamentos , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana MúltiplaRESUMO
Introduction. In early January 2020, the pandemic of COVID-19 (coronavirus disease 2019) rapidly spread from China and caused a worldwide pandemic.Hypothesis. Healthcare workers represent a high-risk group for acquiring COVID-19 and for nosocomial transmission of severe acute respiratory coronavirus 2 (SARS-CoV-2).Aim. We aimed to investigate over a 1 year period, across two pandemic waves, the SARS-CoV-2 seroprevalence in employees at a Western Switzerland public hospital.Methodology. A prospective observational SARS-CoV-2 seroprevalence study was proposed to all hospital employees who enrolled on a voluntary basis.Results. Out of 594 participants recruited on a voluntary basis, 269 volunteers (45.3â%) had anti-SARS-CoV-2 antibodies: this seroprevalence was twice higher than that reported in the local community. Healthcare workers with prolonged exposure to patients with COVID-19 showed a significantly higher odds ratio (OR) of having a positive SARS-CoV-2 serology [OR 3.19, 95â% confidence interval (CI) 2.16-4.74]. Symptoms showing the highest association with a positive serology were anosmia (OR 11.9, 95â% CI 5.58-30.9) and ageusia (OR 10.3, 95â% CI 4.8-26.3). A total of 17.1â% (95â% CI 12.2-21.1â%) of SARS-CoV-2 seropositive volunteers did not report a suspicion of COVID-19 in their personal history.Conclusion. Overall, we observed that the impact of the second SARS-CoV-2 pandemic wave was considerable and significantly affected healthcare workers with prolonged exposure to patients with COVID-19.
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COVID-19 , Pandemias , Anticorpos Antivirais , COVID-19/epidemiologia , Pessoal de Saúde , Hospitais , Humanos , Recursos Humanos em Hospital , SARS-CoV-2 , Estudos Soroepidemiológicos , Suíça/epidemiologiaRESUMO
BACKGROUND: In response to the current COVID-19 pandemic, multiple companies marketed serological tests. Rigorous, independent and comparative performances of these assays on defined clinical specimens are needed. METHODS: In a first preliminary phase, we investigated 16 IgG, IgM, IgA and pan Ig serological ELISA using a panel of 180 sera, comprising 97 sera from patients with a positive RT-PCR, and 83 negative sera sampled before November 1, 2019. In a second phase and to complete the evaluation on the full panel (100 positive and 300 negative), tests that passed pre-defined exclusion criteria of 90% sensitivity and 97% specificity were further evaluated on 220 additional sera chosen to assess possible cross-reactivity with other human viral infections. RESULTS: Among the 16 tests evaluated in the preliminary phase, two were excluded due to insufficient sensitivity at 15 days post-symptom onset and one was excluded due to poor specificity. Of the 13 tests evaluated using the full panel comprised of a diverse pool of sera including those reactive against known respiratory viruses, no systematic cross-reactivity was observed. However, heterogeneities across tests were found. Consistent with kinetics of antibody expression, maximal sensitivity was found two weeks post-symptom onset. CONCLUSION: In this independent evaluation, we compared the performance of 16 SARS-CoV-2 serological tests using well-characterized sera and found 13 tests with more than 90% sensitivity at 15 days post-symptom onset and 97% specificity across a diverse range of negative samples.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Imunoglobulina M , Pandemias , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Although many laboratories worldwide have developed their sequencing capacities in response to the need for SARS-CoV-2 genome-based surveillance of variants, only a few reported some quality criteria to ensure sequence quality before lineage assignment and submission to public databases. Hence, we aimed here to provide simple quality control criteria for SARS-CoV-2 sequencing to prevent erroneous interpretation of low-quality or contaminated data. We retrospectively investigated 647 SARS-CoV-2 genomes obtained over 10 tiled amplicons sequencing runs. We extracted 26 potentially relevant metrics covering the entire workflow from sample selection to bioinformatics analysis. Based on data distribution, critical values were established for 11 selected metrics to prompt further quality investigations for problematic samples, in particular those with a low viral RNA quantity. Low-frequency variants (<70% of supporting reads) can result from PCR amplification errors, sample cross contaminations, or presence of distinct SARS-CoV2 genomes in the sample sequenced. The number and the prevalence of low-frequency variants can be used as a robust quality criterion to identify possible sequencing errors or contaminations. Overall, we propose 11 metrics with fixed cutoff values as a simple tool to evaluate the quality of SARS-CoV-2 genomes, among which are cycle thresholds, mean depth, proportion of genome covered at least 10×, and the number of low-frequency variants combined with mutation prevalence data.
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COVID-19 , SARS-CoV-2 , Genoma Viral , Humanos , RNA Viral , Estudos RetrospectivosRESUMO
OBJECTIVES: New automated modules are required to provide fully automated solutions in diagnostic microbiology laboratories. We evaluated the performance of a Becton Dickinson Kiestra™ IdentifA/SusceptA prototype for MALDI-TOF identification (ID) and Phoenix™ antibiotic susceptibility testing (AST). METHODS: The performance of the IdentifA/SusceptA coupled prototype was compared with manual processing for MALDI-TOF ID on 1302 clinical microbial isolates or ATCC strains and for Phoenix™ M50 AST on 484 strains, representing 61 species. RESULTS: Overall, the IdentifA exhibited similar ID performances than manual spotting. Higher performances were observed for Gram-negative bacteria with an ID at the species level (score >2) of 96.5% (369/382) and 86.9% (334/384), respectively. A significantly better performance was observed with the IdentifA (95.2%, 81/85) compared with manual spotting (75.2%, 64/85) from colonies on MacConkey agar. Contrariwise, the IdentifA exhibited lower ID performances at the species level than manual processing for streptococci (76.1%, 96/126 compared with 92%, 115/125), coagulase-negative staphylococci (73.3%, 44/60 compared with 90%, 54/60) and yeasts (41.3%, 19/46 compared with 78.2%, 36/46). Staphylococcus aureus and enterococci were similarly identified by the two approaches, with ID rates of 92% (65/70) for the IdentifA and 92.7%, (64/69) for manual processing and 94.8%, (55/58) for the IdentifA and 98.2%, (57/58) for manual processing, respectively. The SusceptA exhibited an AST overall essential agreement of 98.82% (6863/6945), a category agreement of 98.86% (6866/6945), 1.05% (6/570) very major errors, 0.16% (10/6290) major errors, and 0.91% (63/6945) minor errors compared to the reference AST. CONCLUSIONS: Overall, the automated IdentifA/SusceptA exhibited high ID and AST performances.
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Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Automação Laboratorial , Enterococcus/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Streptococcus/efeitos dos fármacos , Leveduras/efeitos dos fármacosRESUMO
RT-PCRs to detect SARS-CoV-2 RNA is key to manage the COVID-19 pandemic. We analyzed SARS-CoV-2 viral loads from 22'323 RT-PCR results according to samples types, gender, age, and health units. Viral load did not show any difference across age and appears to be a poor predictor of disease outcome. SARS-CoV-2 viral load showed similar high viral loads than the one observed for RSV and influenza B. The importance of viral load to predict contagiousness and to assess disease progression is discussed.
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COVID-19/virologia , Infecções por Coronavirus/virologia , SARS-CoV-2/isolamento & purificação , Carga Viral/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19 , Criança , Pré-Escolar , Infecções por Coronavirus/diagnóstico , Progressão da Doença , Feminino , Humanos , Lactente , Recém-Nascido , Vírus da Influenza B/isolamento & purificação , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Pandemias , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Testes Sorológicos/métodos , Suíça/epidemiologiaRESUMO
C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.
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Interações Hospedeiro-Parasita , Manose/metabolismo , Parasitos/patogenicidade , Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Animais , Adesão Celular , Movimento Celular , Simulação por Computador , Feminino , Deleção de Genes , Glicosilação , Interações Hospedeiro-Parasita/imunologia , Humanos , Masculino , Camundongos , Parasitos/citologia , Parasitos/imunologia , Proteólise , Toxoplasma/citologia , Toxoplasma/imunologia , VirulênciaRESUMO
The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.
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Sistemas CRISPR-Cas/genética , Toxoplasma/patogenicidade , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Cryptosporidium/genética , Cryptosporidium/patogenicidade , Técnicas de Inativação de Genes , Genoma de Protozoário/genética , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Temperatura , Toxoplasma/genéticaRESUMO
Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.
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Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Ácidos Siálicos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Interleucina-12/imunologia , Camundongos , Camundongos Knockout , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Toxoplasmose Animal/genéticaRESUMO
Toxoplasma gondii possesses a limited set of actin-regulatory proteins and relies on only three formins (FRMs) to nucleate and polymerize actin. We combined filamentous actin (F-actin) chromobodies with gene disruption to assign specific populations of actin filaments to individual formins. FRM2 localizes to the apical juxtanuclear region and participates in apicoplast inheritance. Restricted to the residual body, FRM3 maintains the intravacuolar cell-cell communication. Conoidal FRM1 initiates a flux of F-actin crucial for motility, invasion and egress. This flux depends on myosins A and H and is controlled by phosphorylation via PKG (protein kinase G) and CDPK1 (calcium-dependent protein kinase 1) and by methylation via AKMT (apical lysine methyltransferase). This flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC). This study offers a coherent model of the key players controlling actin polymerization, stressing the importance of well-timed post-translational modifications to power parasite motility.
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Actinas/metabolismo , Comunicação Celular , Forminas/metabolismo , Locomoção , Organelas/metabolismo , Multimerização Proteica , Toxoplasma/fisiologia , Actinas/genética , Forminas/genética , Deleção de Genes , Técnicas de Inativação de GenesRESUMO
Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.