Mutations related to Antiretroviral Resistance identified by ultra-deep sequencing in HIV-1 infected children under Structured Interruptions of HAART

Altres ajuts: CONACYT/GCPS/44519Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI

Heterogeneous Infectivity and Pathogenesis of SARS-CoV-2 Variants Beta, Delta and Omicron in Transgenic K18-hACE2 and Wildtype Mice

The emerging SARS-CoV-2 variants of concern (VOCs) may display enhanced transmissibility, more severity and/or immune evasion; however, the pathogenesis of these new VOCs in experimental SARS-CoV-2 models or the potential infection of other animal species is not completely understood. Here we infected K18-hACE2 transgenic mice with B.1, B.1.351/Beta, B.1.617.2/Delta and BA.1.1/Omicron isolates and demonstrated heterogeneous infectivity and pathogenesis. B.1.351/Beta variant was the most pathogenic, while BA.1.1/Omicron led to lower viral RNA in the absence of major visible clinical signs. In parallel, we infected wildtype (WT) mice and confirmed that, contrary to B.1 and B.1.617.2/Delta, B.1.351/Beta and BA.1.1/Omicron can infect them. Infection in WT mice coursed without major clinical signs and viral RNA was transient and undetectable in the lungs by day 7 post-infection. In silico modeling supported these findings by predicting B.1.351/Beta receptor binding domain (RBD) mutations result in an increased affinity for both human and murine ACE2 receptors, while BA.1/Omicron RBD mutations only show increased affinity for murine ACE2

Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used

Development and Evaluation of an NTM-IGRA to Guide Pediatric Lymphadenitis Diagnosis

Background: Diagnosis of nontuberculous mycobacteria (NTM) infections remains a challenge. In this study, we describe the evaluation of an immunological NTM-interferon (IFN)-γ release assay (IGRA) that we developed using glycopeptidolipids (GPLs) as NTM-specific antigens. Methods: We tested the NTM-IGRA in 99 samples from pediatric patients. Seventy-five were patients with lymphadenitis: 25 were NTM confirmed, 45 were of unknown etiology but compatible with mycobacterial infection and 5 had lymphadenitis caused by an etiologic agent other than NTM. The remaining 24 samples were from control individuals without lymphadenitis (latently infected with M.Tuberculosis, uninfected controls and active tuberculosis patients). Peripheral blood mononuclear cells were stimulated overnight with GPLs. Detection of IFN-γ producing cells was evaluated by enzyme-linked immunospot assay. Results: NTM culture-confirmed lymphadenitis patient samples had a significantly higher response to GPLs than the patients with lymphadenitis of unknown etiology but compatible with mycobacterial infection (P < 0.001) and lymphadenitis not caused by NTM (P < 0.01). We analyzed the response against GPLs in samples from unknown etiology lymphadenitis but compatible with mycobacterial infection cases according to the tuberculin skin test (TST) response, and although not statistically significant, those with a TST ≥5 mm had a higher response to GPLs when compared with the TST <5 mm group. Conclusions: Stimulation with GPLs yielded promising results in detecting NTM infection in pediatric patients with lymphadenitis. Our results indicate that the test could be useful to guide the diagnosis of pediatric lymphadenitis. This new NTM-IGRA could improve the clinical handling of NTM-infected patients and avoid unnecessary misdiagnosis and treatments

Sección abierta de homenaje a Laura Rubio

Sección abierta de homenaje a Laura Rubi

COVID-19 in children and adolescents in Europe: a multinational, multicentre cohort study

Background To date, few data on paediatric COVID-19 have been published, and most reports originate from China. This study aimed to capture key data on children and adolescents with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection across Europe to inform physicians and health-care service planning during the ongoing pandemic. Methods This multicentre cohort study involved 82 participating health-care institutions across 25 European countries, using a well established research network—the Paediatric Tuberculosis Network European Trials Group (ptbnet)—that mainly comprises paediatric infectious diseases specialists and paediatric pulmonologists. We included all individuals aged 18 years or younger with confirmed SARS-CoV-2 infection, detected at any anatomical site by RT-PCR, between April 1 and April 24, 2020, during the initial peak of the European COVID-19 pandemic. We explored factors associated with need for intensive care unit (ICU) admission and initiation of drug treatment for COVID-19 using univariable analysis, and applied multivariable logistic regression with backwards stepwise analysis to further explore those factors significantly associated with ICU admission. Findings 582 individuals with PCR-confirmed SARS-CoV-2 infection were included, with a median age of 5·0 years (IQR 0·5–12·0) and a sex ratio of 1·15 males per female. 145 (25%) had pre-existing medical conditions. 363 (62%) individuals were admitted to hospital. 48 (8%) individuals required ICU admission, 25 (4%) mechanical ventilation (median duration 7 days, IQR 2–11, range 1–34), 19 (3%) inotropic support, and one (<1%) extracorporeal membrane oxygenation. Significant risk factors for requiring ICU admission in multivariable analyses were being younger than 1 month (odds ratio 5·06, 95% CI 1·72–14·87; p=0·0035), male sex (2·12, 1·06–4·21; p=0·033), pre-existing medical conditions (3·27, 1·67–6·42; p=0·0015), and presence of lower respiratory tract infection signs or symptoms at presentation (10·46, 5·16–21·23; p<0·0001). The most frequently used drug with antiviral activity was hydroxychloroquine (40 [7%] patients), followed by remdesivir (17 [3%] patients), lopinavir–ritonavir (six [1%] patients), and oseltamivir (three [1%] patients). Immunomodulatory medication used included corticosteroids (22 [4%] patients), intravenous immunoglobulin (seven [1%] patients), tocilizumab (four [1%] patients), anakinra (three [1%] patients), and siltuximab (one [<1%] patient). Four children died (case-fatality rate 0·69%, 95% CI 0·20–1·82); at study end, the remaining 578 were alive and only 25 (4%) were still symptomatic or requiring respiratory support. Interpretation COVID-19 is generally a mild disease in children, including infants. However, a small proportion develop severe disease requiring ICU admission and prolonged ventilation, although fatal outcome is overall rare. The data also reflect the current uncertainties regarding specific treatment options, highlighting that additional data on antiviral and immunomodulatory drugs are urgently needed. Funding ptbnet is supported by Deutsche Gesellschaft für Internationale Zusammenarbeit

Design of 3D Metric Geometry Study and Research Activities within a BIM Framework

This paper presents research work in which an innovative didactic proposal was designed for the study of 3D metric geometry in the second year of A-level courses (secondary education) in the specialty of the sciences. The designed didactic proposal has the format of a workshop of geometry practices and is framed in the Didactic Situations Theory and in the Anthropological Theory of the Didactic. Although the mentioned theories, which the French school has developed since the seventies, are widely known and studied by secondary school mathematics teachers during their training in pedagogy, especially in Spanish- and French-speaking countries, their innovative approach has not been fully implemented in the field of algebraic geometry. Mathematics textbooks follow the traditional approach in which the teacher simply provides the contents and instructs the student, who captures these concepts and reproduces them as they have been supplied. In the presented didactic proposal, the approach proposed by Brousseau is followed, in which three fundamental elements take part: student, teacher, and the didactic environment. The teacher is the one who facilitates the environment in which the student builds his knowledge. In the proposal the didactic contract is stablished, the didactic situations are designed, and the means and didactic variables for the study of 3D metric geometry are chosen. The methodology followed in this study consisted of identifying educational problems; describing the theoretical framework; developing the didactic proposal; and analysis, reflection, and criticism of this training product. The contextualization of the geometry workshop in the field of the construction of structures and the use of deductive reasoning techniques that are applied in synthetic geometry and also in the building information modeling (BIM) methodology may be a way of showing the use of analytical geometry in the industries of engineering and architecture; they also serve as an opportunity for students to better understand this mathematical work

Design of 3D Metric Geometry Study and Research Activities within a BIM Framework

This paper presents research work in which an innovative didactic proposal was designed for the study of 3D metric geometry in the second year of A-level courses (secondary education) in the specialty of the sciences. The designed didactic proposal has the format of a workshop of geometry practices and is framed in the Didactic Situations Theory and in the Anthropological Theory of the Didactic. Although the mentioned theories, which the French school has developed since the seventies, are widely known and studied by secondary school mathematics teachers during their training in pedagogy, especially in Spanish- and French-speaking countries, their innovative approach has not been fully implemented in the field of algebraic geometry. Mathematics textbooks follow the traditional approach in which the teacher simply provides the contents and instructs the student, who captures these concepts and reproduces them as they have been supplied. In the presented didactic proposal, the approach proposed by Brousseau is followed, in which three fundamental elements take part: student, teacher, and the didactic environment. The teacher is the one who facilitates the environment in which the student builds his knowledge. In the proposal the didactic contract is stablished, the didactic situations are designed, and the means and didactic variables for the study of 3D metric geometry are chosen. The methodology followed in this study consisted of identifying educational problems; describing the theoretical framework; developing the didactic proposal; and analysis, reflection, and criticism of this training product. The contextualization of the geometry workshop in the field of the construction of structures and the use of deductive reasoning techniques that are applied in synthetic geometry and also in the building information modeling (BIM) methodology may be a way of showing the use of analytical geometry in the industries of engineering and architecture; they also serve as an opportunity for students to better understand this mathematical work

Transoral endoluminal approach to Zenker's diverticulum using LigasureTM: early clinical experience

The development of minimally invasive procedures has rekindled interest in endoluminal techniques for the management of Zenker's diverticulum. Tissue sealers as employed in laparoscopic surgery have not been previously used for Zenker's diverticulum septotomy. Supported by the established safety of linear cutters, bipolar forceps, and ultrasonic scalpels, we have started a procedure using the Ligasure 5TM tissue sealer. Safety and efficacy results in our early clinical experience are shown for a prospective series of 5consecutive Zenker's diverticulum cases that were perorally managed with tissue sealing. The procedure was quickly and safely performed in the endoscopy room under sedation. Mean number of seals per patient was2, and mean procedure duration was 33minutes. No complications developed during or after the procedure, and patients were discharged with immediate dysphagia relief and adequate oral tolerance. No diverticular relapses occurred after a mean follow-up of 21months (range18-30). This procedure may be repeated as often as desired with no need for hospital admission. Safety should be prospectively assessed by further studies using a higher number of procedures