Whole-genome sequencing analysis reveals the spread of a vanB-carrying transposon among different vancomycin-resistant Enterococcus faecium clinical isolates in a non-endemic setting

Background: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements. Aim: To report prospective analysis of whole-genome sequencing (WGS) data, including both core-genome multi-locus sequence typing (cgMLST) and transposon analysis, during a vanB VREfm outbreak. Methods: Screening for vanB-positive VREfm isolates was performed by real-time polymerase chain reaction (PCR) on an overnight enriched broth and, if positive, subculture was performed. vanB-positive VREfm isolates underwent WGS. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics Workbench, or de-novo assemblies were used for BLASTN comparisons. Results: In total, 1358 real-time PCRs were performed. Two hundred and fifty-one specimens from 207 patients tested positive on PCR for vanB, of which 13 specimens obtained from six patients were identified as vanB VREfm positive on culture. These six patients harboured seven unique isolates belonging to four cluster types: CT118 (N=2), CT2483 (N=3), CT2500 (N=1) and CT2501 (N=1). Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all six patientsBackground: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements.Aim: To report prospective analysis of whole-genome sequencing (WGS) data, including both core-genome multi-locus sequence typing (cgMLST) and transposon analysis, during a vanB VREfm outbreak.Methods: Screening for vanB-positive VREfm isolates was performed by real-time polymerase chain reaction (PCR) on an overnight enriched broth and, if positive, subculture was performed. vanB-positive VREfm isolates underwent WGS. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics Workbench, or de-novo assemblies were used for BLASTN comparisons.Results: In total, 1358 real-time PCRs were performed. Two hundred and fifty-one specimens from 207 patients tested positive on PCR for vanB, of which 13 specimens obtained from six patients were identified as vanB VREfm positive on culture. These six patients harboured seven unique isolates belonging to four cluster types: CT118 (N=2), CT2483 (N=3), CT2500 (N=1) and CT2501 (N=1). Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all six patients that could be linked based on epidemiological data.Conclusion: A vanB VREfm outbreak occurred in the study hospital, including six patients with isolates belonging to four cluster types. In-depth transposon analysis revealed that &amp; nbsp;dissemination of transposon Tn1549 rather than clonal spread was the cause of the outbreak.(C)&amp; nbsp;2021 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.</p

Exhaled metabolite patterns to identify recent asthma exacerbations

Asthma is a chronic respiratory disease that can lead to exacerbations, defined as acute episodes of worsening respiratory symptoms and lung function. Predicting the occurrence of these exacerbations is an important goal in asthma management. The measurement of exhaled breath by electronic nose (eNose) may allow for the monitoring of clinically unstable asthma and exacerbations. However, data on its ability to perform this is lacking. We aimed to evaluate whether eNose could identify patients that recently had asthma exacerbations. We performed a cross-sectional study, measuring exhaled breath using the SpiroNose in adults with a physician-reported diagnosis of asthma. Patients were randomly divided into a training (n = 252) and validation (n = 109) set. For the analysis of eNose signals, principal component (PC) and linear discriminant analysis (LDA) were performed. LDA, based on PC1-4, reliably discriminated between patients who had a recent exacerbation from those who had not (training receiver operating characteristic (ROC)–area under the curve (AUC) = 0.76,95% CI 0.69–0.82), (validation AUC = 0.76, 95% CI 0.64–0.87). Our study showed that, exhaled breath analysis using eNose could accurately identify asthma patients who recently had an exacerbation, and could indicate that asthma exacerbations have a specific exhaled breath pattern detectable by eNose

Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids

Background: Pharmacotherapies for people with cystic fibrosis (pwCF) who have premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are under development. Thus far, clinical studies focused on compounds that induce translational readthrough (RT) at the mRNA PTC location. Recent studies using primary airway cells showed that PTC functional restoration can be achieved through combining compounds with multiple mode-of-actions. Here, we assessed induction of CFTR function in PTC-containing intestinal organoids using compounds targeting RT, nonsense mRNA mediated decay (NMD) and CFTR protein modulation. Methods: Rescue of PTC CFTR protein was assessed by forskolin-induced swelling of 12 intestinal organoid cultures carrying distinct PTC mutations. Effects of compounds on mRNA CFTR level was assessed by RT-qPCRs. Results: Whilst response varied between donors, significant rescue of CFTR function was achieved for most donors with the quintuple combination of a commercially available pharmacological equivalent of the RT compound (ELX-02-disulfate or ELX-02ds), NMD inhibitor SMG1i, correctors VX-445 and VX-661 and potentiator VX-770. The quintuple combination of pharmacotherapies reached swelling quantities higher than the mean swelling of three VX-809/VX-770-rescued F508del/F508del organoid cultures, indicating level of rescue is of clinical relevance as VX-770/VX-809-mediated F508del/F508del rescue in organoids correlate with substantial improvement of clinical outcome. Conclusions: Whilst variation in efficacy was observed between genotypes as well as within genotypes, the data suggests that strong pharmacological rescue of PTC requires a combination of drugs that target RT, NMD and protein function

Targeted exhaled breath analysis for detection of Pseudomonas aeruginosa in cystic fibrosis patients

Background Pseudomonas aeruginosa (PA) is an important respiratory pathogen for cystic fibrosis (CF) patients. Routine microbiology surveillance is time-consuming, and is best performed on expectorated sputum. As alternative, volatile organic compounds (VOCs) may be indicative of PA colonisation. In this study, we aimed to identify VOCs associated with PA in literature and perform targeted exhaled breath analysis to recognize PA positive CF patients non-invasively. Methods This study consisted of 1) a literature review to select VOCs of interest, and 2) a cross-sectional CF study. Definitions used: A) PA positive, PA culture at visit/chronically; B) PA free, no PA culture in ≥12 months. Exhaled VOCs were identified via quadrupole MS. The primary endpoint was the area under the receiver operating characteristics curve (AUROCC) of individual VOCs as well as combined VOCs against PA culture. Results 241 VOCs were identified in literature, of which 56 were further evaluated, and 13 could be detected in exhaled breath in our cohort. Exhaled breath of 25 pediatric and 28 adult CF patients, PA positive (n=16) and free (n=28) was available. 3/13 VOCs were significantly (p<0.05) different between PA groups in children; none were in adults. Notably, a composite model based on 5 or 1 VOC(s) showed an AUROCC of 0.86 (CI 0.71–1.0) and 0.87 (CI 0.72–1.0) for adults and children, respectively. Conclusions Targeted VOC analysis appears to discriminate children and adults with and without PA positive cultures with clinically acceptable sensitivity values

Long-Term Weight Changes After Starting Anti-IL-5/5Ra Biologics in Severe Asthma: The Role of Oral Corticosteroids

BACKGROUND Many patients with severe asthma are overweight or obese, often attributed to unintentional weight gain as a side effect of oral corticosteroids (OCSs). Anti-IL-5/5Ra biologics significantly reduce OCS use, but their long-term effects on weight are unknown. OBJECTIVES To examine (1) weight change up to 2 years after anti-IL-5/5Ra initiation in subgroups on the basis of maintenance OCS use at start of treatment and (2) whether cumulative OCS exposure before or changes in OCS exposure during treatment are related to weight change. METHODS Real-world data on weight and cumulative OCS dose from adults included in the Dutch Registry of Adult Patients with Severe asthma for Optimal DIsease management before and at least 2 years after starting anti-IL-5/5Ra were analyzed using linear mixed models and linear regression analyses. RESULTS For the included 389 patients (55% female; mean body mass index, 28 ± 5 kg/m$^{2}$; 58% maintenance OCS), mean weight decreased -0.27 kg/y (95% CI, -0.51 to -0.03; P = .03), with more weight loss in patients with maintenance OCS use than in those without maintenance OCS use (-0.87 kg/y [95% CI, -1.21 to -0.52; P < .001] vs +0.54 kg/y [0.26 to 0.82; P < .001]). Greater weight loss at 2 years was associated with higher cumulative OCS dose in the 2 years before anti-IL-5/5Ra initiation (β = -0.24 kg/g; 95% CI, -0.38 to -0.10; P < .001) and, independently, greater reduction in cumulative OCS dose during follow-up (β = 0.27 kg/g; 95% CI, 0.11 to 0.43; P < .001). CONCLUSIONS Anti-IL-5/5Ra therapy is associated with long-term weight reduction, especially in patients with higher OCS exposure before treatment and those able to reduce OCS use during treatment. However, the effect is small and does not apply to all patients, and so additional interventions seem necessary if weight change is desired

The relation between paracetamol use and asthma:a GA2LEN European case-control study

Studies from the UK and USA suggest that frequent use of paracetamol (acetaminophen) may increase the risk of asthma, but data across Europe are lacking. As part of a multicentric case-control study organised by the Global Allergy and Asthma European Network (GA(2)LEN), it was examined whether or not frequent paracetamol use is associated with adult asthma across Europe. The network compared 521 cases with a diagnosis of asthma and reporting of asthma symptoms within the last 12 months with 507 controls with no diagnosis of asthma and no asthmatic symptoms within the last 12 months across 12 European centres. All cases and controls were selected from the same population, defined by age (2045 yrs) and place of residence. In a random effects meta-analysis, weekly use of paracetamol, compared with less frequent use, was strongly positively associated with asthma after controlling for confounders. There was no evidence for heterogeneity across centres. No association was seen between use of other analgesics and asthma. These data add to the increasing and consistent epidemiological evidence implicating frequent paracetamol use in asthma in diverse populations

Forskolin-induced Organoid Swelling is Associated with Long-term CF Disease Progression

RATIONALE: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids (PDO) with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC). METHODS: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8 µM forskolin, quantified as area under the curve (AUC). We used linear mixed effect models and multivariable logistic regression to estimate the association of FIS with long-term FEV1pp decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC. RESULTS: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95%CI: 0.11%-0.54%; p=0.004) per 1000-points change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR: 0.18, 95%CI: 0.07-0.46, p<0.001), CF-related liver disease (adjusted OR: 0.18, 95%CI: 0.06-0.54, p=0.002) and diabetes (adjusted OR: 0.34, 95%CI: 0.12-0.97, p=0.044). These associations were absent for SCC. CONCLUSION: This study exemplifies the prognostic value of a PDO-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation

Rationale Asthma phenotyping requires novel biomarker discovery. Objectives To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs). Methods An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED. Results In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-β and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation. Conclusions The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA