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IntroductionTuberculosis (TB) treatment typically involves a tailored combination of four antibiotics based on the drug resistance profile of the infecting strain. The increasing drug resistance of Mycobacterium tuberculosis (Mtb) requires the development of novel antibiotics to ensure effective treatment regimens. Gallium (Ga) is being explored as a repurposed drug against TB due to its ability to inhibit Mtb growth and disrupt iron metabolism. Given the potential interactions between Ga and established antibiotics, we investigated how a combination of Ga with levofloxacin (Lfx) or linezolid (Lzd) affects the growth and metabolome of a multidrug-resistant (MDR) Mtb clinical strain.MethodsMtb was cultured using a BACTEC 960 system with concentrations of Ga ranging from 125 to 1,000 μM and with 250 to 500 μM of Ga combined with 0.125 mg/L of Lfx or Lzd. For metabolome analysis, the antibacterials were used at concentrations that inhibited the growth of bacteria without causing cell death. Metabolites were extracted from Mtb cells and analyzed using chromatography-mass spectrometry.ResultsThe MDR Mtb strain exhibited a dose-dependent response to Ga. Notably, the enhancement in growth inhibition was statistically significant for the Ga/Lfx combination compared to Ga alone, while no such significance was observed for Ga/Lzd. Moreover, exposure to Ga/Lfx or Ga/Lzd resulted in distinct metabolite profiles. Ga treatment increased the level of aconitate, fumarate, and glucose in the cells, suggesting the inhibition of iron-dependent aconitase and fumarate hydratase, as well as disruption of the pentose phosphate pathway. The levels of glucose, succinic acid, citric acid, and hexadecanoic acid followed a similar pattern in cells exposed to Ga and Ga/Lfx at 500 μM Ga but exhibited different trends at 250 μM Ga.DiscussionIn the presence of Lfx, the Mtb metabolome changes induced by Ga are more pronounced compared to those observed with Lzd. Lfx affects nucleic acids and transcription, which may enhance Ga-dependent growth inhibition by preventing the metabolic redirection that bacteria typically use to bypass iron-dependent enzymes.

An accelerated local injection site reaction following Bacille Calmette-Guérin (BCG) vaccination has been associated with underlying active tuberculosis (TB) in high TB-prevalence settings. The clinical significance of this accelerated BCG reaction in individuals without TB symptoms, particularly in low TB-prevalence countries, is unclear.Using safety surveillance data and baseline interferon-gamma release assays (IGRA) within an international randomised trial of BCG vaccination in healthcare workers (the BRACE trial), we aimed to determine the incidence, and investigate for clinical implications, of an accelerated BCG reaction in asymptomatic adults in low and high TB-prevalence settings.An accelerated BCG reaction occurred in 755/1984 (38 %) of BCG-vaccinees. Although more frequently painful, tender, erythematous and/or swollen within the first fourteen days of vaccination, compared with non-accelerated reactions, the majority of injection site reactions were mild and did not meet criteria for an adverse event.Prior mycobacterial exposure, through prior BCG vaccination (OR 2.46, 95%CI 1.93–3.13, p

Summary: Background: Bacille Calmette-Guérin (BCG) vaccination has off-target (non-specific) effects that are associated with protection against unrelated infections and decreased all-cause mortality in infants. We aimed to determine whether BCG vaccination prevents febrile and respiratory infections in adults. Methods: This randomised controlled phase 3 trial was done in 36 healthcare centres in Australia, Brazil, the Netherlands, Spain, and the United Kingdom. Healthcare workers were randomised to receive BCG-Denmark (single 0.1 ml intradermal injection) or no BCG in a 1:1 ratio using a web-based procedure, stratified by stage, site, age, and presence of co-morbidity. The difference in occurrence of febrile or respiratory illness were measured over 12 months (prespecified secondary outcome) using the intention-to-treat (ITT) population. This trial is registered with ClinicalTrials.gov, NCT04327206. Findings: Between March 30, 2020, and April 1, 2021, 6828 healthcare workers were randomised to BCG-Denmark (n = 3417) or control (n = 3411; no intervention or placebo) groups. The 12-month adjusted estimated risk of ≥1 episode of febrile or respiratory illness was 66.8% in the BCG group (95% CI 65.3%–68.2%), compared with 63.4% in the control group (95% CI 61.8%–65.0%), a difference of +3.4 percentage points (95% CI +1.3% to +5.5%; p 0.002). The adjusted estimated risk of a severe episode (defined as being incapacitated for ≥3 consecutive days or hospitalised) was 19.4% in the BCG group (95% CI 18.0%–20.7%), compared with 18.8% in the control group (95% CI 17.4%–20.2%) a difference of +0.6 percentage points (95% CI −1.3% to +2.5%; p 0.6). Both groups had a similar number of episodes of illness, pneumonia, and hospitalisation. There were three deaths, all in the control group. There were no safety concerns following BCG vaccination. Interpretation: In contrast to the beneficial off-target effects reported following neonatal BCG in infants, a small increased risk of symptomatic febrile or respiratory illness was observed in the 12 months following BCG vaccination in adults. There was no evidence of a difference in the risk of severe disease. Funding: Bill & Melinda Gates Foundation, Minderoo Foundation, Sarah and Lachlan Murdoch, the Royal Children's Hospital Foundation, Health Services Union NSW, the Peter Sowerby Foundation, SA Health, the Insurance Advisernet Foundation, the NAB Foundation, the Calvert-Jones Foundation, the Modara Pines Charitable Foundation, the UHG Foundation Pty Ltd, Epworth Healthcare, the National Health and Medical Research Council, the Swiss National Science Foundation and individual donors.

Summary: Background: The COVID-19 pandemic led to the rapid development and deployment of several highly effective vaccines against SARS-CoV-2. Recent studies suggest that these vaccines may also have off-target effects on the immune system. We sought to determine and compare the off-target effects of the adenovirus vector ChAdOx1-S (Oxford-AstraZeneca) and modified mRNA BNT162b2 (Pfizer-BioNTech) vaccines on immune responses to unrelated pathogens. Methods: Prospective sub-study within the BRACE trial. Blood samples were collected from 284 healthcare workers before and 28 days after ChAdOx1-S or BNT162b2 vaccination. SARS-CoV-2-specific antibodies were measured using ELISA, and whole blood cytokine responses to specific (SARS-CoV-2) and unrelated pathogen stimulation were measured by multiplex bead array. Findings: Both vaccines induced robust SARS-CoV-2 specific antibody and cytokine responses. ChAdOx1-S vaccination increased cytokine responses to heat-killed (HK) Candida albicans and HK Staphylococcus aureus and decreased cytokine responses to HK Escherichia coli and BCG. BNT162b2 vaccination decreased cytokine response to HK E. coli and had variable effects on cytokine responses to BCG and resiquimod (R848). After the second vaccine dose, BNT162b2 recipients had greater specific and off-target cytokine responses than ChAdOx1-S recipients. Interpretation: ChAdOx1-S and BNT162b2 vaccines alter cytokine responses to unrelated pathogens, indicative of potential off-target effects. The specific and off-target effects of these vaccines differ in their magnitude and breadth. The clinical relevance of these findings is uncertain and needs further study. Funding: Bill & Melinda Gates Foundation, National Health and Medical Research Council, Swiss National Science Foundation and the Melbourne Children’s. BRACE trial funding is detailed in acknowledgements.

Introduction Syndromic panel assays, that is, using one test to simultaneously target multiple pathogens with overlapping signs and symptoms, have been integrated into routine paediatric care over the past decade, mainly for more severely ill and hospitalised patients. Their wider availability and short turnaround times open the possibility to apply them to non-hospitalised patients as well. In this context, it is important to trial how clinicians make use of pathogen detection data and if their early availability influences management decisions, particularly antibiotic use and hospitalisation.Methods and analysis Advanced Diagnostics for Enhanced QUality of Antibiotic prescription in respiratory Tract infections in Emergency rooms is an individually randomised, controlled, open-label effectiveness trial comparing the impact of a respiratory pathogen panel assay (BIOFIRE Respiratory Panel 2.1plus) used as a rapid syndromic test on nasopharyngeal swabs in addition to the standard of care versus standard of care alone. The trial will 1:1 randomise 520 participants under the age of 18 at 7 paediatric emergency departments in 5 European countries. Inclusion criteria for the trial consist of two sets, with the first describing respiratory tract infections in paediatric patients and the second describing the situation of potential management uncertainty in which test results may immediately affect management decisions. Enrolment started in July 2021 and is expected to be completed in early 2024. We will perform a two-sample t-test assuming a pooled variance estimate to compare the log-transformed mean time on antibiotic treatment (in hours) and number of days alive out of the hospital within 14 days after study enrolment between the control and intervention arms.Ethics and dissemination The trial protocol and materials were approved by research ethics committees in all participating countries. The respiratory pathogen panel assay is CE marked (assessed to meet European regulations) and FDA (United States Food and Drug Administration) cleared for diagnostic use. Participants and caregivers provide informed consent prior to study procedures commencing. The trial results will be published in peer-reviewed journals and at national and international conferences. Key messages will also be disseminated via press and social media where appropriate.Trial registration number NCT04781530.

Summary: Background: In preclinical studies, the use of double allogeneic grafts has shown promising results in promoting tissue revascularization, reducing infarct size, preventing adverse remodelling and fibrosis, and ultimately enhancing cardiac function. Building upon these findings, the safety of PeriCord, an engineered tissue graft consisting of a decellularised pericardial matrix and umbilical cord Wharton's jelly mesenchymal stromal cells, was evaluated in the PERISCOPE Phase I clinical trial (NCT03798353), marking its first application in human subjects. Methods: This was a double-blind, single-centre trial that enrolled patients with non-acute myocardial infarction eligible for surgical revascularization. Seven patients were implanted with PeriCord while five served as controls. Findings: Patients who received PeriCord showed no adverse effects during post-operative phase and one-year follow-up. No significant changes in secondary outcomes, such as quality of life or cardiac function, were found in patients who received PeriCord. However, PeriCord did modulate the kinetics of circulating monocytes involved in post-infarction myocardial repair towards non-classical inflammation-resolving macrophages, as well as levels of monocyte chemoattractants and the prognostic marker Meteorin-like in plasma following treatment. Interpretation: In summary, the PeriCord graft has exhibited a safe profile and notable immunomodulatory properties. Nevertheless, further research is required to fully unlock its potential as a platform for managing inflammatory-related pathologies. Funding: This work was supported in part by grants from MICINN (SAF2017-84324-C2-1-R); Instituto de Salud Carlos III (ICI19/00039 and Red RICORS-TERAV RD21/0017/0022, and CIBER Cardiovascular CB16/11/00403) as a part of the Plan Nacional de I + D + I, and co-funded by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) and AGAUR (2021-SGR-01437).

Summary: Background: Recurrences of herpes simplex virus (HSV) in the orofacial region (herpes labialis or cold sores) impact quality-of-life. We aimed to study whether the bacille Calmette-Guérin (BCG) vaccine can attenuate cold sore recurrences through off-target immunomodulatory effects. Methods: In this nested randomised controlled trial within the multicentre, phase 3 BRACE trial, 6828 healthcare workers were randomised in 36 sites in Australia, the Netherlands, Spain, the United Kingdom and Brazil, to receive BCG-Denmark or no BCG (1:1 ratio using a web-based procedure) and followed for 12 months with 3-monthly questionnaires. Exclusion criteria included contraindication to BCG vaccine or previous vaccination with BCG within the past year, any other live-attenuated vaccine within the last month, or any COVID-specific vaccine. The intervention group received one intradermal dose of 0.1 mL of BCG-Denmark corresponding to 2−8 x 105 colony forming units of Mycobacterium bovis, Danish strain 1331. The primary outcome was the difference in restricted mean survival time (i.e., time to first cold-sore recurrence), in participants with frequent recurrent herpes labialis (≥4 recurrences/year), analysed by intention-to-treat. Secondary outcomes addressed additional questions, including analyses in other sub-populations. Adverse events were monitored closely during the first 3 months and were reported in all participants who received one dose of study drug according to intervention received. The BRACE trial is registered with ClinicalTrials.gov, NCT04327206. Findings: Between March 30, 2020 and February 18, 2021, 84 individuals with frequent recurrent cold sores were randomly assigned to BCG (n = 38) or control (n = 46). The average time to first cold-sore recurrence was 1.55 months longer in the BCG group (95% CI 0.27–2.82, p = 0.02) than the control group (hazard ratio 0.54, 95% CI 0.32–0.91; intention-to-treat). The beneficial effect of BCG was greater in the as-treated population (difference 1.91 months, 95% CI 0.69–3.12, p = 0.003; hazard ratio 0.45, 95% CI 0.26–0.76). In prespecified subgroup analyses, only sex modified the treatment effect (interaction p = 0.007), with benefit restricted to males. Over 12 months, a greater proportion of participants in the BCG group compared with the control group reported a decrease in duration (61% vs 21%), severity (74% vs 21%), frequency (55% vs 21%), and impact on quality of life (42% vs 15%) of cold sore recurrences. In participants who had ever had a cold sore, there was also a decrease in self-reported burden of recurrences in the BCG group. In participants who had never had a cold sore, there was an increased risk of a first episode in the BCG group (risk difference 1.4%; 95% CI 0.3–2.6%, p = 0.02). There were no safety concerns. Interpretation: BCG-Denmark vaccination had a beneficial effect on herpes labialis, particularly in males with frequent recurrences, but may increase the risk of a first cold sore. Funding: Bill & Melinda Gates Foundation, the Minderoo Foundation, Sarah and Lachlan Murdoch, the Royal Children's Hospital Foundation, Health Services Union NSW, the Peter Sowerby Foundation, SA Health, the Insurance Advisernet Foundation, the NAB Foundation, the Calvert-Jones Foundation, the Modara Pines Charitable Foundation, the UHG Foundation Pty Ltd, Epworth Healthcare, and individual donors.

In this paper the authors show that the pathogenic bacterium Pseudomonas aeruginosa migrates between the gut and lungs of an ICU patient, and that differential evolutionary responses to antibiotic treatment occur in these organs.

BCG vaccination and revaccination are increasingly being considered for the protection of adolescents and adults against tuberculosis and, more broadly, for the off-target protective immunological effects against other infectious and noninfectious diseases. Within an international randomized controlled trial of BCG vaccination in healthcare workers (the BRACE trial), we evaluated the incidence of local and serious adverse events, as well as the impact of previous BCG vaccination on local injection site reactions (BCG revaccination). Prospectively collected data from 99% (5351/5393) of participants in Australia, Brazil, Spain, The Netherlands and the UK was available for analysis. Most BCG recipients experienced the expected self-limiting local injection site reactions (pain, tenderness, erythema, swelling). BCG injection site itch was an additional common initial local symptom reported in 49% of BCG recipients. Compared to BCG vaccination in BCG-naïve individuals, BCG revaccination was associated with increased frequency of mild injection site reactions, as well as earlier onset and shorter duration of erythema and swelling, which were generally self-limiting. Injection site abscess and regional lymphadenopathy were the most common adverse events and had a benign course. Self-resolution occurred within a month in 80% of abscess cases and 100% of lymphadenopathy cases. At a time when BCG is being increasingly considered for its off-target effects, our findings indicate that BCG vaccination and revaccination have an acceptable safety profile in adults.

The prevalence of scar formation following Bacille Calmette-Guérin (BCG) vaccination varies globally. The beneficial off-target effects of BCG are proposed to be stronger amongst children who develop a BCG scar. Within an international randomised trial (‘BCG vaccination to reduce the impact of coronavirus disease 2019 (COVID-19) in healthcare workers’; BRACE Trial), this nested prospective cohort study assessed the prevalence of and factors influencing scar formation, as well as participant perception of BCG scarring 12 months following vaccination . Amongst 3071 BCG-recipients, 2341 (76%) developed a BCG scar. Scar prevalence was lowest in Spain and highest in UK. Absence of post-injection wheal (OR 0.4, 95%CI 0.2–0.9), BCG revaccination (OR 1.7, 95%CI 1.3–2.0), female sex (OR 2.0, 95%CI 1.7–2.4), older age (OR 0.4, 95%CI 0.4–0.5) and study country (Brazil OR 1.6, 95%CI 1.3–2.0) influenced BCG scar prevalence. Of the 2341 participants with a BCG scar, 1806 (77%) did not mind having the scar. Participants more likely to not mind were those in Brazil, males and those with a prior BCG vaccination history. The majority (96%) did not regret having the vaccine.Both vaccination-related (amenable to optimisation) and individual-related factors affected BCG scar prevalence 12 months following BCG vaccination of adults, with implications for maximising the effectiveness of BCG vaccination.

Abstract Dual species interactions in co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization were examined. The genetic and phenotypic diversity between the isolates was high making the interactions detected strain-specific. Despite this, and the clinical origin of the strains, some interactions were common between some co-isolated pairs. For most pairs, P. aeruginosa exoproducts affected biofilm formation and reduced growth in vitro in its S. aureus counterpart. Conversely, S. aureus did not impair biofilm formation and stimulated swarming motility in P. aeruginosa. Co-culture in a medium that mimics respiratory mucus promoted coexistence and favored mixed microcolony formation within biofilms. Under these conditions, key genes controlled by quorum sensing were differentially regulated in both species in an isolate-dependent manner. Finally, co-infection in the acute infection model in Galleria mellonella larvae showed an additive effect only in the co-isolated pair in which P. aeruginosa affected less S. aureus growth. This work contributes to understanding the complex interspecies interactions between P. aeruginosa and S. aureus by studying strains isolated during acute infection.

The administration of extracellular vesicles (EV) from mesenchymal stromal cells (MSC) is a promising cell-free nanotherapy for tissue repair after myocardial infarction (MI). However, the optimal EV delivery strategy remains undetermined. Here, we designed a novel MSC-EV delivery, using 3D scaffolds engineered from decellularised cardiac tissue as a cell-free product for cardiac repair. EV from porcine cardiac adipose tissue-derived MSC (cATMSC) were purified by size exclusion chromatography (SEC), functionally analysed and loaded to scaffolds. cATMSC-EV markedly reduced polyclonal proliferation and pro-inflammatory cytokines production (IFNγ, TNFα, IL12p40) of allogeneic PBMC. Moreover, cATMSC-EV recruited outgrowth endothelial cells (OEC) and allogeneic MSC, and promoted angiogenesis. Fluorescently labelled cATMSC-EV were mixed with peptide hydrogel, and were successfully retained in decellularised scaffolds. Then, cATMSC-EV-embedded pericardial scaffolds were administered in vivo over the ischemic myocardium in a pig model of MI. Six days from implantation, the engineered scaffold efficiently integrated into the post-infarcted myocardium. cATMSC-EV were detected within the construct and MI core, and promoted an increase in vascular density and reduction in macrophage and T cell infiltration within the damaged myocardium. The confined administration of multifunctional MSC-EV within an engineered pericardial scaffold ensures local EV dosage and release, and generates a vascularised bioactive niche for cell recruitment, engraftment and modulation of short-term post-ischemic inflammation.

Respiratory tract infections (RTIs) are one of the most common reasons for seeking healthcare, but are amongst the most challenging diseases in terms of clinical decision-making. Proper and timely diagnosis is critical in order to optimise management and prevent further emergence of antimicrobial resistance by misuse or overuse of antibiotics. Diagnostic tools for RTIs include those involving syndromic and aetiological diagnosis: from clinical and radiological features to laboratory methods targeting both pathogen detection and host biomarkers, as well as their combinations in terms of clinical algorithms. They also include tools for predicting severity and monitoring treatment response. Unprecedented milestones have been achieved in the context of the COVID-19 pandemic, involving the most recent applications of diagnostic technologies both at genotypic and phenotypic level, which have changed paradigms in infectious respiratory diseases in terms of why, how and where diagnostics are performed. The aim of this review is to discuss advances in diagnostic tools that impact clinical decision-making, surveillance and follow-up of RTIs and tuberculosis. If properly harnessed, recent advances in diagnostic technologies, including omics and digital transformation, emerge as an unprecedented opportunity to tackle ongoing and future epidemics while handling antimicrobial resistance from a One Health perspective.

ObjectiveTo assess the arrhythmic safety profile of the adipose graft transposition procedure (AGTP) and its electrophysiological effects on post-myocardial infarction (MI) scar.BackgroundMyocardial repair is a promising treatment for patients with MI. The AGTP is a cardiac reparative therapy that reduces infarct size and improves cardiac function. The impact of AGTP on arrhythmogenesis has not been addressed.MethodsMI was induced in 20 swine. Contrast-enhanced magnetic resonance (ce-MRI), electrophysiological study (EPS), and left-ventricular endocardial high-density mapping were performed 15 days post-MI. Animals were randomized 1:1 to AGTP or sham-surgery group and monitored with ECG-Holter. Repeat EPS, endocardial mapping, and ce-MRI were performed 30 days post-intervention. Myocardial SERCA2, Connexin-43 (Cx43), Ryanodine receptor-2 (RyR2), and cardiac troponin-I (cTnI) gene and protein expression were evaluated.ResultsThe AGTP group showed a significant reduction of the total infarct scar, border zone and dense scar mass by ce-MRI (p = 0.04), and a decreased total scar and border zone area in bipolar voltage mapping (p < 0.001). AGTP treatment significantly reduced the area of very-slow conduction velocity (

BackgroundCurrent blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools.ObjectiveTo compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB.MethodsBlood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively.ResultsThe expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4+ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4+ T-cells, CD27− and CD38+ (ESAT-6/CFP-10-specific), and HLA-DR+ and Ki-67+ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4+ T-cells over Mtb-specific CD4+ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment.ConclusionOur study independently confirms that CD27−, CD38+, HLA-DR+ and Ki-67+ populations on Mtb-specific CD4+ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches.

Abstract Tuberculosis (TB) is a major cause of morbidity and mortality in children, and early diagnosis and treatment are crucial to reduce long-term morbidity and mortality. In this study, we explore whether urine nuclear magnetic resonance (NMR)-based metabolomics could be used to identify differences in the metabolic response of children with different diagnostic certainty of TB. We included 62 children with signs and symptoms of TB and 55 apparently healthy children. Six of the children with presumptive TB had bacteriologically confirmed TB, 52 children with unconfirmed TB, and 4 children with unlikely TB. Urine metabolic fingerprints were identified using high- and low-field proton NMR platforms and assessed with pattern recognition techniques such as principal components analysis and partial least squares discriminant analysis. We observed differences in the metabolic fingerprint of children with bacteriologically confirmed and unconfirmed TB compared to children with unlikely TB (p = 0.041 and p = 0.013, respectively). Moreover, children with unconfirmed TB with X-rays compatible with TB showed differences in the metabolic fingerprint compared to children with non-pathological X-rays (p = 0.009). Differences in the metabolic fingerprint in children with different diagnostic certainty of TB could contribute to a more accurate characterisation of TB in the paediatric population. The use of metabolomics could be useful to improve the prediction of TB progression and diagnosis in children.

Introduction BCG vaccination modulates immune responses to unrelated pathogens. This off-target effect could reduce the impact of emerging pathogens. As a readily available, inexpensive intervention that has a well-established safety profile, BCG is a good candidate for protecting healthcare workers (HCWs) and other vulnerable groups against COVID-19.Methods and analysis This international multicentre phase III randomised controlled trial aims to determine if BCG vaccination reduces the incidence of symptomatic and severe COVID-19 at 6 months (co-primary outcomes) compared with no BCG vaccination. We plan to randomise 10 078 HCWs from Australia, The Netherlands, Spain, the UK and Brazil in a 1:1 ratio to BCG vaccination or no BCG (control group). The participants will be followed for 1 year with questionnaires and collection of blood samples. For any episode of illness, clinical details will be collected daily, and the participant will be tested for SARS-CoV-2 infection. The secondary objectives are to determine if BCG vaccination reduces the rate, incidence, and severity of any febrile or respiratory illness (including SARS-CoV-2), as well as work absenteeism. The safety of BCG vaccination in HCWs will also be evaluated. Immunological analyses will assess changes in the immune system following vaccination, and identify factors associated with susceptibility to or protection against SARS-CoV-2 and other infections.Ethics and dissemination Ethical and governance approval will be obtained from participating sites. Results will be published in peer-reviewed open-access journals. The final cleaned and locked database will be deposited in a data sharing repository archiving system.Trial registration ClinicalTrials.gov NCT04327206

Abstract Despite efforts to improve tuberculosis (TB) detection, limitations in access, quality and timeliness of diagnostic services in low- and middle-income countries are challenging for current TB diagnostics. This study aimed to identify and characterise a metabolic profile of TB in urine by high-field nuclear magnetic resonance (NMR) spectrometry and assess whether the TB metabolic profile is also detected by a low-field benchtop NMR spectrometer. We included 189 patients with tuberculosis, 42 patients with pneumococcal pneumonia, 61 individuals infected with latent tuberculosis and 40 uninfected individuals. We acquired the urine spectra from high and low-field NMR. We characterised a TB metabolic fingerprint from the Principal Component Analysis. We developed a classification model from the Partial Least Squares-Discriminant Analysis and evaluated its performance. We identified a metabolic fingerprint of 31 chemical shift regions assigned to eight metabolites (aminoadipic acid, citrate, creatine, creatinine, glucose, mannitol, phenylalanine, and hippurate). The model developed using low-field NMR urine spectra correctly classified 87.32%, 85.21% and 100% of the TB patients compared to pneumococcal pneumonia patients, LTBI and uninfected individuals, respectively. The model validation correctly classified 84.10% of the TB patients. We have identified and characterised a metabolic profile of TB in urine from a high-field NMR spectrometer and have also detected it using a low-field benchtop NMR spectrometer. The models developed from the metabolic profile of TB identified by both NMR technologies were able to discriminate TB patients from the rest of the study groups and the results were not influenced by anti-TB treatment or TB location. This provides a new approach in the search for possible biomarkers for the diagnosis of TB.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is an emerging threat to TB control in Ukraine, a country with the third highest XDR TB burden globally. We used whole-genome sequencing of a convenience sample to identify bacterial genetic and patient-related factors associated with MDR/XDR TB in this country. MDR/XDR TB was associated with 3 distinct Mycobacterium tuberculosis complex lineage 2 (Beijing) clades, Europe/Russia W148 outbreak, Central Asia outbreak, and Ukraine outbreak, which comprised 68.9% of all MDR/XDR TB strains from southern Ukraine. MDR/XDR TB was also associated with previous treatment for TB and urban residence. The circulation of Beijing outbreak strains harboring broad drug resistance, coupled with constraints in drug supply and limited availability of phenotypic drug susceptibility testing, needs to be considered when new TB management strategies are implemented in Ukraine.

Objective: To analyse all cases of Nocardia pneumonia occurring between 2010 and 2016 in five Spanish hospitals. Methods: This was a retrospective observational analysis of clinical and microbiological data collected from 55 cases of Nocardia pneumonia. Results: There were one to 20 cases per hospital and six to nine cases per year. Chronic obstructive pulmonary disease, bronchiectasis, and asthma were the main predisposing underlying respiratory conditions. Thirty-four patients were receiving systemic and/or inhaled corticosteroids prior to infection, eight had neoplasia, and six had haematological malignancies. Clinical and radiological findings were common to pneumonia of other infectious aetiologies, except for the frequent presence of nodules and cavitation. Overall, the 1-year mortality was high (38.2%), and mortality was directly related to the pulmonary disease in 15 patients (27.3%). The most frequently identified species were N. cyriacigeorgica (n = 21), N. abscessus (n = 8), and N. farcinica (n = 5). All Nocardia isolates were susceptible to linezolid and all but two were susceptible to amikacin and trimethoprim–sulfamethoxazole. Conclusions: Nocardia pneumonia-associated mortality remains high, probably because of the debilitated status of patients in whom this pathogen is able to cause pulmonary infection. Keywords: Nocardia, Pneumonia, Multicentre study, Nocardia cyriacigeorgica, Spain, Respiratory infection

A systematic and ordered product development program, in compliance with current quality and regulatory standards, increases the likelihood of yielding a successful advanced therapy medicinal product (ATMP) for clinical use as safe and effective therapy. As this is a novel field, little accurate information is available regarding the steps to be followed, and the information to be produced to support the development and use of an ATMP. Notably, successful clinical translation can be somewhat cumbersome for academic researchers. In this article, we have provided a summary of the available information, supported by our experience in Spain throughout the development of an ATMP for myocardial infarction, from the pre-clinical stage to phase I clinical trial approval.

Background: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. Methods: The PeriCord is a clinical-size (12–16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. Findings: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability;

A quarter of the global human population is estimated to be latently infected by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). TB remains the global leading cause of death by a single pathogen and ranks among the top-10 causes of overall global mortality. Current immunodiagnostic tests cannot discriminate between latent, active and past TB, nor predict progression of latent infection to active disease. The only registered TB vaccine, Bacillus Calmette-Guérin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-γ centered approaches, have been proposed as targets for new TB-diagnostic tests or -vaccines. Recently, however, we identified novel Mtb antigens capable of eliciting multiple cytokines, including antigens that did not induce IFN-γ but several other cytokines. These antigens had been selected based on high Mtb gene-expression in the lung in vivo, and have been termed in vivo expressed (IVE-TB) antigens. Here, we extend and validate our previous findings in an independent Southern European cohort, consisting of adults and adolescents with either LTBI or TB. Our results confirm that responses to IVE-TB antigens, and also DosR-regulon and Rpf stage-specific Mtb antigens are marked by multiple cytokines, including strong responses, such as for TNF-α, in the absence of detectable IFN-γ production. Except for TNF-α, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-α+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-α+ cells clusters identified, those with CD8+TEMRA or CD8+CD4+ phenotypes, defined by the expression of multiple intracellular markers, were the most prominent in adult LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb.

Cigarette smoking and tuberculosis are a significant cause of death worldwide. Several epidemiological studies have demonstrated cigarette smoking is a risk factor for tuberculosis. Electronic cigarettes have recently appeared as a healthier alternative to conventional smoking, although their impact in tuberculosis is not well understood. The aim of this study was to explore the effect of electronic cigarettes in phagocytosis of Mycobacterium tuberculosis and cytokines production. In vitro infection was carried out by exposing THP-1 macrophages to four electronic vapor extracts and the intracellular burden of M. tuberculosis was determined. The percentage of infection was evaluated by confocal microscopy and the cytokine production by Luminex. A reduction of intracellular M. tuberculosis burden in THP-1 macrophages was found after its exposure to electronic vapor extract; the same trend was observed by confocal microscopy when Mycobacterium bovis BCG-GFP strain was used. Electronic cigarettes stimulate a pro-inflammatory cytokine response. We conclude that electronic cigarettes impair the phagocytic function and the cytokine response to M. tuberculosis.

Health Care Workers (HCW) may have an occupational risk of latent tuberculosis infection (LTBI) and TB disease. The objective of this study was to evaluate the performance of the 2-step strategy: tuberculin skin test (TST) followed by confirmation with Interferon (IFN)-γ- release assays (IGRAs) in HCW. A secondary objective was to determine the factors related to conversions and reversions. HCW at risk of occupational exposure who attended the Occupational Department of the Hospital Germans Trias i Pujol were included during the study period (2013-2016). All professionals testing negative for LTBI were included in a cohort study. These workers were followed up with the administration of a TST and an IGRA quantification at least one year after inclusion in the study. Workers with positive TST, regardless of the results of the IGRA tests, were followed-up with an IGRA. 255 workers were enrolled in the study and 108 workers from the same cohort were followed up. During the follow-up period, seven workers presented TST test conversion. One of these conversions was also confirmed by an IGRA test. There were 2 conversions of cases only testing positive with the IGRA. There have been only 2 reversions of cases testing negative with the IGRA. In this study, not all TST conversions were confirmed when using the IGRA test, which highlights the importance of the 2-step strategy. We have detected a low number of conversions and reversions. Our conclusions should be confirmed in studies with a longer follow-up time.

Abstract Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.

Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial–mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression.

Human cardiac progenitor cells (hCPC) are considered a good candidate in cell therapy for ischemic heart disease, demonstrating capacity to improve functional recovery after myocardial infarction (MI), both in small and large preclinical animal models. However, improvements are required in terms of cell engraftment and efficacy. Based on previously published reports, insulin-growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) have demonstrated substantial cardioprotective, repair and regeneration activities, so they are good candidates to be evaluated in large animal model of MI. We have validated porcine cardiac progenitor cells (pCPC) and lentiviral vectors to overexpress IGF-1 (co-expressing eGFP) and HGF (co-expressing mCherry). pCPC were transduced and IGF1-eGFPpos and HGF-mCherrypos populations were purified by cell sorting and further expanded. Overexpression of IGF-1 has a limited impact on pCPC expression profile, whereas results indicated that pCPC-HGF-mCherry cultures could be counter selecting high expresser cells. In addition, pCPC-IGF1-eGFP showed a higher cardiogenic response, evaluated in co-cultures with decellularized extracellular matrix, compared with native pCPC or pCPC-HGF-mCherry. In vivo intracoronary co-administration of pCPC-IGF1-eGFP and pCPC-HFG-mCherry (1:1; 40 × 106/animal), one week after the induction of an MI model in swine, revealed no significant improvement in cardiac function.

Outstanding progress has been achieved in developing therapeutic options for reasonably alleviating symptoms and prolonging the lifespan of patients suffering from myocardial infarction (MI). Current treatments, however, only partially address the functional recovery of post-infarcted myocardium, which is in fact the major goal for effective primary care. In this context, we largely investigated novel cell and TE tissue engineering therapeutic approaches for cardiac repair, particularly using multipotent mesenchymal stromal cells (MSC) and natural extracellular matrices, from pre-clinical studies to clinical application. A further step in this field is offered by MSC-derived extracellular vesicles (EV), which are naturally released nanosized lipid bilayer-delimited particles with a key role in cell-to-cell communication. Herein, in this review, we further describe and discuss the rationale, outcomes and challenges of our evidence-based therapy approaches using Wharton’s jelly MSC and derived EV in post-MI management.

Cardiology: Heart Failure Medication Reduces Heart Size The new wonder drug in heart failure management, Sacubitril/Valsartan, rejuvenates the heart by preventing its dilation. Using data from myocardial infarction and heart failure samples, we generated a mathematical model to better understand how Sacubitril/Valsartan modulates pathological heart resize and the combined effect of the drug. Our analysis revealed that Sacubitril/Valsartan mainly acts by blocking both, cell death and the pathological makeover of the outer-membrane of the cardiac cells. These two major processes occur after a heart attack. Most importantly, we discovered a core of 8 proteins that emerge as key players in this process. A better understanding of the mechanism of novel cardiovascular drugs at the most basic level may help decipher future therapies and indications.

Host susceptibility to respiratory tract infections (RTI) is dependent on both genetic and acquired risk factors. Repeated bacterial and viral RTI, such as pneumonia from encapsulated microorganisms, respiratory tract infections related to respiratory syncytial virus or influenza, and even the development of bronchiectasis and asthma, are often reported as the first symptom of primary immunodeficiencies. In the same way, neutropenia is a well-known risk factor for invasive aspergillosis, as well as lymphopenia for Pneumocystis, and mycobacterial infections. However, in the last decades a better knowledge of immune signaling networks and the introduction of next generation sequencing have increased the number and diversity of known inborn errors of immunity. On the other hand, the use of monoclonal antibodies targeting cytokines, such as tumor necrosis factor alpha has revealed new risk groups for infections, such as tuberculosis. The use of biological response modifiers has spread to almost all medical specialties, including inflammatory diseases and neoplasia, and are being used to target different signaling networks that may mirror some of the known immune deficiencies. From a clinical perspective, the individual contribution of genetics, and/or targeted treatments, to immune dysregulation is difficult to assess. The aim of this article is to review the known and newly described mechanisms of impaired immune signaling that predispose to RTI, including new insights into host genetics and the impact of biological response modifiers, and to summarize clinical recommendations regarding vaccines and prophylactic treatments in order to prevent infections.

The immunological characterization of different cell markers has opened the possibility of considering them as immune tools for tuberculosis (TB) management, as they could correlate with TB latency/disease status and outcome. CD4+ T-cells producing IFN-γ+ with a low expression of CD27 have been described as an active TB marker. In addition, there are unknown homing receptors related to TB, such as CCR4, which might be useful for understanding TB pathogenesis. The aim of our study is focused on the assessment of several T-cell subsets to understand immune-mechanisms in TB. This phenotypic immune characterization is based on the study of the specific immune responses of T-cells expressing CD27 and/or CCR4 homing markers. Subjects enrolled in the study were: (i) 22 adult patients with active TB, and (ii) 26 individuals with latent TB infection (LTBI). Blood samples were drawn from each patient. The expression of CD27 and/or CCR4 markers were analyzed within CD4+ T-cells producing: (i) IFN-γ+, (ii) TNF-α+, (iii) TNF-α+IFN-γ+, and (iv) IFN-γ+ and/or TNF-α+. The percentage of CD27− within all CD4+ T-cell populations analyzed was significantly higher on active TB compared to LTBI after PPD or ESAT-6/CFP-10 stimulation. As previously reported, a ratio based on the CD27 median fluorescence intensity (MFI) was also explored (MFI of CD27 in CD4+ T-cells over MFI of CD27 in IFN-γ+CD4+ T-cells), being significantly increased during disease (p < 0.0001 after PPD or ESAT-6/CFP-10 stimulation). This ratio was also assessed on the other CD4+ T-cells functional profiles after specific stimulation, being significantly associated with active TB. Highest diagnostic accuracies for active TB (AUC ≥ 0.91) were achieved for: (i) CD27 within IFN-γ+TNF-α+CD4+ T-cells in response to ESAT-6/CFP-10, (ii) CD27 and CCR4 markers together within IFN-γ+CD4+ T-cells in response to PPD, and (iii) CD27 MFI ratio performed on IFN-γ+TNF-α+CD4+ T-cells after ESAT-6/CFP-10 stimulation. The lowest diagnostic accuracy was observed when CCR4 marker was evaluated alone (AUC ≤ 0.77). CD27 and CCR4 expression detection could serve as a good method for immunodiagnosis. Moreover, the immunological characterization of markers/subset populations could be a promising tool for understanding the biological basis of the disease.

This study aimed to describe real-life microbiological testing of adults hospitalised with community-acquired pneumonia (CAP) and to assess concordance with the 2007 Infectious Diseases Society of America (IDSA)/American Thoracic Society (ATS) and 2011 European Respiratory Society (ERS) CAP guidelines. This was a cohort study based on the Global Initiative for Methicillin-resistant Staphylococcus aureus Pneumonia (GLIMP) database, which contains point-prevalence data on adults hospitalised with CAP across 54 countries during 2015. In total, 3702 patients were included. Testing was performed in 3217 patients, and included blood culture (71.1%), sputum culture (61.8%), Legionella urinary antigen test (30.1%), pneumococcal urinary antigen test (30.0%), viral testing (14.9%), acute-phase serology (8.8%), bronchoalveolar lavage culture (8.4%) and pleural fluid culture (3.2%). A pathogen was detected in 1173 (36.5%) patients. Testing attitudes varied significantly according to geography and disease severity. Testing was concordant with IDSA/ATS and ERS guidelines in 16.7% and 23.9% of patients, respectively. IDSA/ATS concordance was higher in Europe than in North America (21.5% versus 9.8%; p

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient’s study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections.

Background: Acute and chronic respiratory tract infections are a common cause of inappropriate antimicrobial prescription. Antimicrobial therapy leads to the development of resistance and the emergence of opportunistic pathogens that substitute the indigenous microbiota. Methods: This review explores the major challenges and lines of research to adequately establish the clinical role of bacteria and the indications for antimicrobial treatment, and reviews novel therapeutic approaches. Results: In patients with chronic pulmonary diseases and structural disturbances of the bronchial tree or the lung parenchyma, clinical and radiographic signs and symptoms are almost constantly present, including a basal inflammatory response. Bacterial adaptative changes and differential phenotypes are described, depending on the clinical role and niche occupied. The respiratory tract has areas that are potentially inaccessible to antimicrobials. Novel therapeutic approaches include new ways of administering antimicrobials that may allow intracellular delivery or delivery across biofilms, targeting the functions essential for infection, such as regulatory systems, or the virulence factors required to cause host damage and disease. Alternatives to antibiotics and antimicrobial adjuvants are under development. Conclusions: Prudent treatment, novel targets, and improved drug delivery systems will contribute to reduce the emergence of antimicrobial resistance in lower respiratory tract infections.

Myocardial infarction (MI) remains a dreadful disease around the world, causing irreversible sequelae that shorten life expectancy and reduce quality of life despite current treatment. Here, the authors engineered a cell-enriched myocardial graft, composed of a decellularized myocardial matrix refilled with adipose tissue-derived progenitor cells (EMG-ATDPC). Once applied over the infarcted area in the swine MI model, the EMG-ATDPC improved cardiac function, reduced infarct size, attenuated fibrosis progression, and promoted neovascularization of the ischemic myocardium. The beneficial effects exerted by the EMG-ATDPC and the absence of identified adverse side effects should facilitate its clinical translation as a novel MI therapy in humans.

OBJECTIVE:Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. METHODS:S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse-transcriptase real-time PCR (qRT-PCR). RESULTS:Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. CONCLUSIONS:Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay.

Clinical accuracy of IGRAs remains unclear on patients with immune-mediated inflammatory diseases (IMIDs). Here, we assess the impact of immunosuppressants and IMIDs on QuantiFERON-TB Gold In-Tube (QFN-G-IT) and T-SPOT.TB accuracy.Patients with IMIDs who required latent tuberculosis infection (LTBI) screening were enrolled and classified into: (i) 50 patients with inflammatory rheumatic diseases, (ii) 50 patients with psoriasis and (iii) 30 patients with Crohn's disease. A total of 44 healthy individuals without immunosuppression were also included as controls. Tuberculin skin test (TST), T-SPOT.TB and QFN-G-IT assays were performed. IGRAs were performed following manufacturer's instructions.Immunosuppressant's intake was more frequent on patients with Crohn's disease and psoriasis. Positive IGRAs and TST results were reduced in Crohn's disease patients, whereas rate of indeterminate T-SPOT.TB results was increased in this group with respect to the other IMIDs analysed and controls. When IFN-γ response was studied, the levels of this cytokine after mitogen stimulation were significantly lower in Crohn's and inflammatory rheumatic diseases than in psoriasis. Interestingly, psoriatic patients were the only ones not receiving corticosteroids. Furthermore, a negative correlation was observed between the IFN-γ secreted after mitogen stimulation and corticosteroids dose.IMIDs seem to negatively affect the clinical accuracy of IGRAs, being Crohn's disease patients the most affected individuals due to their concomitant drug-profile and impaired immune response.

Alicia Lacoma1,4, Cristina Prat1,4, Felipe Andreo2,4, Luis Lores3, Juan Ruiz-Manzano2,4, Vicente Ausina1,4, Jose Domínguez1,41Servei de Microbiologia, 2Servei de Pneumologia, Hospital Universitari Germans Trias i Pujol, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain; 3Servei de Pneumologia, Hospital de Sant Boi, Sant Boi de Llobregat, Spain; 4CIBER Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, SpainObjective: The identification of biological markers in order to assess different aspects of COPD is an area of growing interest. The objective of this study was to investigate whether levels of procalcitonin (PCT), C-reactive protein (CRP), and neopterin in COPD patients could be useful in identifying the etiological origin of the exacerbation and assessing its prognosis.Methods: We included 318 consecutive COPD patients: 46 in a stable phase, 217 undergoing an exacerbation, and 55 with pneumonia. A serum sample was collected from each patient at the time of being included in the study. A second sample was also collected 1 month later from 23 patients in the exacerbation group. We compared the characteristics, biomarker levels, microbiological findings, and prognosis in each patient group. PCT and CRP were measured using an immunofluorescence assay. Neopterin levels were measured using a competitive immunoassay.Results: PCT and CRP showed significant differences among the three patient groups, being higher in patients with pneumonia, followed by patients with exacerbation (P < 0.0001). For the 23 patients with paired samples, PCT and CRP levels decreased 1 month after the exacerbation episode, while neopterin increased. Neopterin showed significantly lower levels in exacerbations with isolation of pathogenic bacteria, but no differences were found for PCT and CRP. No significant differences were found when comparing biomarker levels according to the Gram result: PCT (P = 0.191), CRP (P = 0.080), and neopterin (P = 0.109). However, median values of PCT and CRP were high for Streptococcus pneumoniae, Staphylococcus aureus, and enterobacteria. All biomarkers were higher in patients who died within 1 month after the sample collection than in patients who died later on.Conclusions: According to our results, biomarker levels vary depending on the clinical status. However, the identification of the etiology of infectious exacerbation by means of circulating biomarkers is encouraging, but its main disadvantage is the absence of a microbiological gold standard, to definitively demonstrate their value. High biomarker levels during an exacerbation episode correlate with the short-term prognosis, and therefore their measurement can be useful for COPD management.Keywords: C-reactive protein, exacerbation, neopterin, procalcitonin, prognosis

Molecular mechanisms associated with pathophysiological changes in ventricular remodelling due to myocardial infarction (MI) remain poorly understood. We analyzed changes in gene expression by microarray technology in porcine myocardial tissue at 1, 4, and 6 weeks post-MI.MI was induced by coronary artery ligation in 9 female pigs (30-40 kg). Animals were randomly sacrificed at 1, 4, or 6 weeks post-MI (n = 3 per group) and 3 healthy animals were also included as control group. Total RNA from myocardial samples was hybridized to GeneChip® Porcine Genome Arrays. Functional analysis was obtained with the Ingenuity Pathway Analysis (IPA) online tool. Validation of microarray data was performed by quantitative real-time PCR (qRT-PCR).More than 8,000 different probe sets showed altered expression in the remodelling myocardium at 1, 4, or 6 weeks post-MI. Ninety-seven percent of altered transcripts were detected in the infarct core and 255 probe sets were differentially expressed in the remote myocardium. Functional analysis revealed 28 genes de-regulated in the remote myocardial region in at least one of the three temporal analyzed stages, including genes associated with heart failure (HF), systemic sclerosis and coronary artery disease. In the infarct core tissue, eight major time-dependent gene expression patterns were recognized among 4,221 probe sets commonly altered over time. Altered gene expression of ACVR2B, BID, BMP2, BMPR1A, LMNA, NFKBIA, SMAD1, TGFB3, TNFRSF1A, and TP53 were further validated.The clustering of similar expression patterns for gene products with related function revealed molecular footprints, some of them described for the first time, which elucidate changes in biological processes at different stages after MI.

Stem cell therapies are promising strategies to regenerate human injured tissues, including ischemic myocardium. Here, we examined the acquisition of properties associated with vascular growth by human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs), and whether they promoted vascular growth in vivo. UCBMSCs were induced in endothelial cell-specific growth medium (EGM-2) acquiring new cell markers, increased Ac-LDL uptake, and migratory capacity as assessed by qRT-PCR, Western blotting, indirect immunofluorescence, and invasion assays. Angiogenic and vasculogenic potentials could be anticipated by in vitro experiments showing self organization into Matrigel-mediated cell networks, and activation of circulating angiogenic-supportive myeloid cells. In mice, following subcutaneous co-injection with Matrigel, UCBMSCs modified to co-express bioluminescent (luciferases) and fluorescent proteins were demonstrated to participate in the formation of new microvasculature connected with the host circulatory system. Response of UCBMSCs to ischemia was explored in a mouse model of acute myocardial infarction (MI). UCBMSCs transplanted using a fibrin patch survived 4 weeks post-implantation and organized into CD31(+)network structures above the infarcted myocardium. MI-treated animals showed a reduced infarct scar and a larger vessel-occupied area in comparison with MI-control animals. Taken together, the presented results show that UCBMSCs can be induced in vitro to acquire angiogenic and vasculogenic properties and contribute to vascular growth in vivo.

BACKGROUND:Health care workers (HCWs) are a group at risk of latent tuberculosis infection (LTBI). The aims of this study were to determine IFN-gamma response by QuantiFERON-TB GOLD In Tube (QFN-G-IT) and T-SPOT.TB in HCWs, comparing the results with tuberculin skin test (TST); and to analyze the capacity of IFN-gamma tests to detect recent versus remote LTBI with a prolonged stimulation test (PST). METHODOLOGY/PRINCIPAL FINDINGS:A total of 147 HCWs were enrolled; 23 of whom were BCG vaccinated. 95 HCWs (64.6%) had a previous positive TST and were not retested; and 52 HCWs had a previous negative TST or were tested for the first time. When we analysed individuals without previous positive TST, the number of positive results for T-SPOT.TB was 12/52 (23.1%); and for QFN-G-IT, 9/52 (17.3%). The global concordance (kappa) between T-SPOT.TB and QFN-G-IT with TST was 0.754 and 0.929 respectively. Of individuals with previous positive TST, T-SPOT.TB and QFN-G-IT were negative in 51.6% (49/95) and 62.1% (59/95) respectively, decreasing the concordance to 0.321 and 0.288, respectively. In non-BCG vaccinated HCWs with previous positive TST a positive IFN-gamma test was associated with degree of exposure and diameter of TST. PST was performed in 24 HCW with previous positive TST and negative IFN-gamma tests. PST was developed in 3 cell cultures stimulated with medium alone, ESAT-6 and CFP-10, respectively. In the third and sixth day of incubation period, part of the supernatants were replaced with complete medium supplemented with (rIL)-2. On day 9, ELISPOT assay was performed. In 14 samples PST was not valid due to not having enough cells. In 8 cases, the response was negative, and in 2 cases positive, suggesting that these patients were infected with Mycobacterium tuberculosis in some point in the past. CONCLUSIONS:Both IFN-gamma tests showed a similar number of positive results, and concordance between the tests was excellent. None of the tests was affected by prior BCG vaccination. IFN-gamma tests are a useful tool for detecting recent infection in HCW population.

Abstract Background Patients undergoing high-risk surgeries with acid-based disorders are associated with poor outcomes. The screening of mixed acid-based metabolic disorders by calculating delta anion gap (AG)/delta bicarbonate (Bic) has a clinically relevant role in patients with high AG metabolic acidosis (MA), however its utility in individuals facing high-risk surgical procedures remains unclear. Objective Characterize metabolic acidosis using delta-AG/delta-Bic and its associations in patients undergoing high-risk surgeries with possible outcome-related complications. Design Prospective observational multicentric study. Setting Three tertiary hospitals in Brazil. Patients Patients undergoing high-risk surgeries, aged 18 years or older, requiring postoperative critical care. Main outcome measures Patients undergoing high-risk surgeries monitored during the postoperative phase across three distinct intensive care units (ICUs), with assessment encompassing laboratory analyses upon admission and 24 h thereafter. Patients with MA and with elevated AG within 24 h were separated into 3 subgroups: [G1 – delta-AG/delta-Bic 1.6] MA associated with alkalosis. Primary endpoint was 30-day mortality. The secondary endpoints were cardiovascular, respiratory, renal, neurological, coagulation and infective complications. Results From the 621 surgical patients admitted to ICU, 421 (51.7%) had any type of acidosis. After 24 h, 140 patients remained with MA with elevated AG (G1: 101, G2: 18, and G3: 21). When compared to patients from subgroups 1 and 3, the subgroup with no mixed disorders 2 showed higher 30-day mortality (adjusted HR = 3.72; 95% CI 1.11–12.89, p = 0.001), cardiovascular complications (p = 0.001), ICU mortality (p = 0.03) and sum of all complications during the ICU period (p = 0.021). Conclusion In the postoperative time, patients with metabolic acidosis and no mixed disorders present higher ICU-Mortality and higher cardiovascular postoperative complications when compared with patients with combined hyperchloremia or alkalosis. Delta-AG/delta-Bic can be a useful tool to evaluate major clinical outcomes in this population.