Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats / Abundancia, distribución y expresión de genes de proteínas cristalinas nematicidas en cepas de Bacillus thuringiensis de diversos hábitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.(AU)

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters

BackgroundAlmost two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted, nor new tests identified to improve the prediction and management of SARS-CoV-2 infection. MethodsRetrospective observational analysis of the predictive performance of clinical parameters and laboratory tests in hospitalised patients with COVID-19. Outcomes were 28-day survival and maximal severity in a cohort of 1,579 patients and two validation cohorts of 598 and 434 patients. A pilot study conducted in a patient subgroup measured 17 cytokines and 27 lymphocyte phenotypes to explore additional predictive laboratory tests. Findings1) Despite a strong association of 22 clinical and laboratory variables with the outcomes, their joint prediction power was limited due to redundancy. 2) Eight variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the statistical predictive power. 3) The interpretation of clinical and laboratory variables was improved by grouping them in categories. 4) Age and organ damage-related tests were the best predictors of survival, and inflammatory-related tests were the best predictors of severity. 5) The pilot study identified several immunological tests (including chemokine ligand 10, chemokine ligand 2, and interleukin 1 receptor antagonist), that performed better than currently used tests. ConclusionsCurrently used tests for clinical management of COVID-19 patients are of limited predictive value due to redundancy, as all measure aspects of two major processes: inflammation, and organ damage. There are no independent predictors based on the quality of the nascent adaptive immune response. Understanding the limitations of current tests would improve their interpretation and simplify clinical management protocols. A systematic search for better biomarkers is urgent and feasible. This study was funded by Instituto de Salud Carlos III, Madrid, Spain, grants COV20/00416, Cov20/00654 and COV20/00388 to R.P-B, ATS and JBM respectively and co-financed by the European Regional Development Fund (ERDF). DA-S is recipient of a doctoral fellowship from the Vall dHebron Research Institute, Barcelona, Spain. ASM was supported by a postdoctoral grant "Juan Rodes" (JR18/00022) from Instituto de Salud Carlos III through the Ministry of Economy and Competitiveness, Spain

Monitoring emergence of SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19) from December 2020 to March 2021

BackgroundSince its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2, become dominant in several European countries raising great concern. AimThe aim of this study was to develop a duplex real-time RT-qPCR assay to detect, discriminate and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the {Delta}HV69/70 deletion, to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). ResultsB.1.1.7 variant was first detected in sewage from the Southern city of Malaga (Andalucia) in week 20_52, and multiple introductions during Christmas holidays were inferred in different parts of the country, earlier than clinical epidemiological reporting by the local authorities. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from WWTPs which reached B.1.1.7 prevalences higher than 90% for [≥] 2 consecutive weeks showed that 8.1{+/-}1.8 weeks were required for B.1.1.7 to become dominant. ConclusionThe study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern (VOCs) as soon as they are identified by clinical sequencing, and its integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

T-cell responses as a correlate of COVID-19 vaccination. A pilot study in Health Care Workers.

BackgroundIt is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses. MethodsTwenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined. ResultsIGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (R>0.8) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between specific antibody levels and the IGRA assay in the ability to detect immune response to SARS-CoV-2, there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one, with Ig(S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA-test. ConclusionWhole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers.

Epidemiological dynamics of the incidence of COVID-19 in children and the relationship with the opening of schools in Catalonia (Spain)

Here we analyse the epidemiological trend of the incidence of COVID-19 in children in Catalonia (Spain) during the first 20 weeks of the 2020-2021 school year. This study demonstrates that while schools were open the incidence rate among children remained significantly lower than in general population, despite a greater diagnostic effort in children. These results suggest that schools have not played a significant role in the SARS-CoV-2 dissemination in Catalonia.

Reduction of respiratory syncytial virus burden of disease observed in primary care diagnosis of children under 5 years old during the COVID-19 pandemic: a time-series analysis using routinely collected data from primary care electronic health records in Catalonia (Spain).

We observed an unusual pattern of respiratory syncytial virus (RSV) in children under 5 years in Catalonia (Spain). We observed a nearly absence during winter months and a subsequent surge late spring. Primary care electronic health records combined with hospital RSV laboratory confirmations could be a useful surveillance system to monitorize trends of respiratory pathogens.

Functional profile, homing and residency of protective T cell immune responses against SARS-CoV-2

Considering that SARS-CoV-2 interacts with the host at the respiratory tract mucosal interface, T cells strategically placed within these surfaces, namely resident memory T cells, will be essential to limit viral spread and disease. Importantly, these cells are mostly non-recirculating, which reduces the window of opportunity to examine circulating lymphocytes in blood as they home to the lung parenchyma. Here, we demonstrate that viral specific T cells can migrate and establish in the lung as resident memory T cells remaining detectable up to 10 months after initial infection. Moreover, focusing on the acute phase of the infection, we identified virus-specific T cell responses in blood with functional, migratory and apoptotic patterns modulated by viral proteins and associated with clinical outcome. Our study highlights IL-10 secretion by virus-specific T cells associated to a better outcome and the persistence of resident memory T cells as key players for future protection against SARS-CoV-2 infection.

Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site, generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We propose that natural selection has favored a "Dont burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.

Early outcomes of tocilizumab in adults hospitalized with severe COVID19. An initial report from the Vall dHebron COVID19 prospective cohort study.

BackgroundModulation of the immune system to prevent lung injury is being widely used against the new coronavirus disease (COVID-19) despite the scarcity of evidence. MethodsWe report the preliminary results from the Vall dHebron prospective cohort study at Vall dHebron University Hospital, in Barcelona (Spain), including all consecutive patients who had a confirmed infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and who were treated with tocilizumab until March 25th. The primary endpoint was mortality at 7 days after tocilizumab administration. Secondary endpoints were admission to the intensive care unit, development of ARDS and respiratory insufficiency among others. Results82 patients with COVID-19 received at least one dose of tocilizumab. The mean ({+/-} SD) age was 59.1 (19.8) years, 63% were male, 22% were of non-Spanish ancestry, and the median (IQR) age-adjusted Charlson index at baseline was 3 (1-4) points. Respiratory failure and ARDS developed in 62 (75.6%) and 45 (54.9%) patients, respectively. Median time from symptom onset to ARDS development was 8 (5-11) days. The median time from symptom onset to the first dose of tocilizumab was 9 (7-11) days. Mortality at 7 days was 26.8%. Hazard ratio for mortality was 3.3; 95% CI, 1.3 to 8.5 (age-adjusted hazard ratio for mortality 2.1; 95% CI, 0.8 to 5.8) if tocilizumab was administered after the onset of ARDS. ConclusionTime from lung injury onset to tocilizumab administration may be critical to patient recovery. Our preliminary data could inform bedside decisions until more data from clinical trials becomes available. Summary of the articles main pointIn patient with COVID-19 and lung injury, time from lung injury onset to tocilizumab administration may be critical to patient recovery. Early administration of host-directed therapies may improve patient outcome.

First Enterovirus D68 (EV-D68) cases detected in hospitalised patients in a tertiary care university hospital in Spain, October 2014 / Primeras detecciones de Enterovirus D68 (EV-D68) en población hospitalizada en España, Octubre 2014

Several outbreaks of Enterovirus 68 (EV-D68) have recently been reported in the USA and Canada, causing substantial hospitalisation of children with severe respiratory disease. The acute flaccid paralysis detected in the USA and Canada among children with EV-D68 infection has raised concerns about the aetiological role of this EV serotype in severe neurological disease. The circulation of EV-D68 in the general European population seems to be low, but European Centre for Disease Prevention and Control (ECDC) recommends being vigilant to new cases, particularly in severely ill hospitalised patients. In October 2014, enteroviruses were detected in respiratory samples collected from five hospitalised patients, children and adults. Phylogenetic analysis of partial VP1 sequences confirmed that the detected enteroviruses belonged to the D68 serotype, which were also similar to strains reported in USA (2014). However, all five patients developed respiratory symptoms, but only one required ICU admission. None of the patients described had symptoms of neurological disease. Other considerations related to the detection methods used for the diagnosis of respiratory enteroviruses are also discussed. In conclusion, additional evidence has been provided that supports the role of EV-D68 in respiratory infections in hospitalised patients

En EEUU y Canadá, desde el pasado verano, se han descrito varios brotes causados por EV-D68 afectando a pacientes, principalmente niños, con enfermedad respiratoria grave. En algunos de estos casos la infección por EV-D68 se ha asociado a enfermedad neurológica grave. Aunque en población europea la circulación de este serotipo parece ser baja, el ECDC recuerda la necesidad de reforzar la vigilancia, especialmente en pacientes hospitalizados. En octubre de 2014, en las muestras respiratorias de 5 pacientes ingresados en el Hospital Universitario Vall d’Hebron de Barcelona, se pudieron detectar enterovirus. Estos fueron caracterizados como EV-D68 mediante análisis filogenético de las secuencias parciales codificantes para la proteína viral VP1. Estas secuencias eran además similares a las de las cepas aisladas en los últimos meses en EEUU. Estos 5 pacientes presentaron síntomas respiratorios, pero sólo uno requirió ingreso en la Unidad de Cuidados Intensivos. Sin embargo, ninguno de los pacientes presentó síntomas de enfermedad neurológica. En este trabajo se comentan también consideraciones relacionadas con los métodos de diagnóstico para enterovirus, especialmente para este serotipo. En conclusión, en este trabajo se confirma el posible papel etiológico del EV-D68 en la infección respiratoria del paciente ingresado

[Microbiological diagnosis of viral respiratory infections in the adult patient]. / Diagnóstico microbiológico de las infecciones virales respiratorias en el paciente adulto.

Conventional techniques for the diagnosis of respiratory viruses are still being used, although molecular methods are now considered as a gold standard in this field. Molecular techniques have a great number of advantages such as an excellent sensitivity, specificity, adaptability to emerging viruses, capability for multiplex and for automation. With all the available repertoire of techniques for microbiological diagnosis, the knowledge relative to respiratory viruses is growing up not only for new aetiological agents but also for its epidemiology. The advances in molecular and non-molecular fast diagnostic methods for one or more respiratory viruses allow quick decisions in the management of the patient. However, there are also disadvantages. The great sensitivity of molecular techniques has meant a significant increase in the rate of multiple detections of respiratory viruses, whose clinical involvement is difficult to interpret. Finally, it remains to show whether the use of new techniques, of high cost, in the microbiological routine diagnosis of acute respiratory viral infections in the hospitalized patient, is cost effective.