Limited effects of azithromycin on the oropharyngeal microbiome in children with CF and early pseudomonas infection.

BACKGROUND: Tobramycin inhalation solution (TIS) and chronic azithromycin (AZ) have known clinical benefits for children with CF, likely due to antimicrobial and anti-inflammatory activity. The effects of chronic AZ in combination with TIS on the airway microbiome have not been extensively investigated. Oropharyngeal swab samples were collected in the OPTIMIZE multicenter, randomized, placebo-controlled trial examining the addition of AZ to TIS in 198 children with CF and early P. aeruginosa infection. Bacterial small subunit rRNA gene community profiles were determined. The effects of TIS and AZ were assessed on oropharyngeal microbial diversity and composition to uncover whether effects on the bacterial community may be a mechanism of action related to the observed changes in clinical outcomes. RESULTS: Substantial changes in bacterial communities (total bacterial load, diversity and relative abundance of specific taxa) were observed by week 3 of TIS treatment for both the AZ and placebo groups. On average, these shifts were due to changes in non-traditional CF taxa that were not sustained at the later study visits (weeks 13 and 26). Bacterial community measures did not differ between the AZ and placebo groups. CONCLUSIONS: This study provides further evidence that the mechanism for AZ's effect on clinical outcomes is not due solely to action on airway microbial composition.

A Phase 3 Open-Label Study of Elexacaftor/Tezacaftor/Ivacaftor in Children 6 through 11 Years of Age with Cystic Fibrosis and at Least One F508del Allele.

Rationale: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was shown to be efficacious and safe in patients ≥12 years of age with cystic fibrosis and at least one F508del-CFTR (cystic fibrosis transmembrane conductance regulator) allele, but it has not been evaluated in children <12 years of age. Objectives: To assess the safety, pharmacokinetics, and efficacy of ELX/TEZ/IVA in children 6 through 11 years of age with F508del-minimal function or F508del-F508del genotypes. Methods: In this 24-week open-label phase 3 study, children (N = 66) weighing <30 kg received 50% of the ELX/TEZ/IVA adult daily dose (ELX 100 mg once daily, TEZ 50 mg once daily, and IVA 75 mg every 12 h) whereas children weighing ⩾30 kg received the full adult daily dose (ELX 200 mg once daily, TEZ 100 mg once daily, and IVA 150 mg every 12 h). Measurements and Main Results: The primary endpoint was safety and tolerability. The safety and pharmacokinetic profiles of ELX/TEZ/IVA were generally consistent with those observed in older patients. The most commonly reported adverse events included cough, headache, and pyrexia; in most of the children who had adverse events, these were mild or moderate in severity. Through Week 24, ELX/TEZ/IVA treatment improved the percentage of predicted FEV1 (10.2 percentage points; 95% confidence interval [CI], 7.9 to 12.6), Cystic Fibrosis Questionnaire-Revised respiratory domain score (7.0 points; 95% CI, 4.7 to 9.2), lung clearance index2.5 (-1.71 units; 95% CI, -2.11 to -1.30), and sweat chloride (-60.9 mmol/L; 95% CI, -63.7 to -58.2); body mass index-for-age z-score increased over the 24-week treatment period when compared with the pretreatment baseline. Conclusions: Our results show ELX/TEZ/IVA is safe and efficacious in children 6 through 11 years of age with at least one F508del-CFTR allele, supporting its use in this patient population. Clinical trial registered with www.clinicaltrials.gov (NCT03691779).

Application of gap time analysis with flexible hazards to pulmonary exacerbations in the EPIC observational study.

Cystic fibrosis and other chronic lung disease clinical trials often use time to first pulmonary exacerbation (PEx) or total PEx count as endpoints. The use of these outcomes may fail to capture patterns or timing of multiple exacerbations and how covariates influence the risk of future exacerbations. Analysis of gap times between PEx provides a useful framework to understand risks of subsequent events, particularly to assess if there is a temporary increase in a hazard of a subsequent PEx following the occurrence of a PEx. This may be useful for estimating the amount of time needed to follow patients after a PEx and predicting which patients are more likely to have multiple PEx. We propose a smoothed hazard for gap times to account for elevated hazards after exacerbations. A simulation study was conducted to explore model performance and was able to appropriately estimate parameters in all situations with an underlying change point with independent or correlated recurrent events. Models with different change-point structures and trends are compared using Early Pseudomonas Infection Control (EPIC) observational study data, using a quasi-likelihood modification of the Akaike information criterion; a model with a change-point provided a better fit than a model without one. The analysis suggests that the change point may be 1.8 years (SE 0.09) after the end of a PEx. Models including covariates in the hazard function revealed that having one or two copies of the Δ$\Delta$ F508 mutation, female sex, and higher numbers of previous PEx were significantly associated with increased risk of another PEx.

Influence of Acid Blockade on the Aerodigestive Tract Microbiome in Children With Cystic Fibrosis.

BACKGROUND: Acid blockade is commonly prescribed in patients with cystic fibrosis (CF). Growing concerns, however, exist about its possible role in the pathophysiology of pulmonary infections. We aimed to investigate if acid blockade alters esophageal and respiratory microbiota leading to dysbiosis and inflammation. METHODS: We performed a cross sectional study of children with CF who were either prescribed acid blockade or not. Samples from the gastrointestinal and respiratory tracts were obtained and microbiome analyzed. Mixed effect models were used to compare outcomes between cohorts and across sampling sites. A random subject intercept was included to account for the multiple sampling sites per individual. RESULTS: A cohort of 25 individuals, 44% girls with median age of 13.8 years [IQR 11.2--14.8] were enrolled. Alpha diversity, total bacterial load, and beta diversity were similar across anatomic compartments, across the upper gastrointestinal tract, and in respiratory samples. Similar alpha diversity, total bacterial load, and beta diversity results were also observed when comparing individuals on versus those off acid blockade. IL-8 was elevated in the distal versus proximal esophagus in the whole cohort (P < 0.01). IL-8 concentrations were similar in the distal esophagus in patients on and off acid blockade, but significantly greater in the proximal esophagus of subjects on treatment (P < 0.01). CONCLUSIONS: On the basis of these data, acid blockade use does not appear to influence the microbiome of the aerodigestive tract in children with cystic fibrosis suggesting a complex interplay between these medications and the bacterial composition of the esophagus and lung.

Nanodiagnostics to monitor biofilm oxygen metabolism for antibiotic susceptibility testing.

In clinical environments, many serious antibiotic-resistant infections are caused by biofilm-forming species. This presents issues when attempting to determine antimicrobial dosing as traditional antibiotic susceptibility tests (ASTs) are typically designed around planktonic bacteria and thus offer information that is not relevant to the biofilm phenotype present in the patient. Even the popular Calgary biofilm device may provide inaccurate minimum biofilm inhibitory concentrations (MBICs) and can be time- and material-intensive. In this work, we present a method utilizing oxygen-sensitive nanosensor technology to monitor the oxygen consumption dynamics of living biofilms as they are exposed to antibiotics. We incorporated our nanosensors into biofilms grown from P. aeruginosa strains of varying sensitivity to traditional classes of antibiotics. Through measuring nanosensor response under antibiotic administration we determined the concentrations able to cease biofilm metabolism. This method provides information on the MBIC as well as kinetic response information in a manner that requires fewer materials and is more reflective of biofilm behavior than a traditional AST.

Luminescent Nanosensors for Ratiometric Monitoring of Three-Dimensional Oxygen Gradients in Laboratory and Clinical Pseudomonas aeruginosa Biofilms.

Bacterial biofilms can form persistent infections on wounds and implanted medical devices and are associated with many chronic diseases, such as cystic fibrosis. These infections are medically difficult to treat, as biofilms are more resistant to antibiotic attack than their planktonic counterparts. An understanding of the spatial and temporal variation in the metabolism of biofilms is a critical component toward improved biofilm treatments. To this end, we developed oxygen-sensitive luminescent nanosensors to measure three-dimensional (3D) oxygen gradients, an application of which is demonstrated here with Pseudomonas aeruginosa biofilms. The method was applied here and improves on traditional one-dimensional (1D) methods of measuring oxygen profiles by investigating the spatial and temporal variation of oxygen concentration when biofilms are challenged with antibiotic attack. We observed an increased oxygenation of biofilms that was consistent with cell death from comparisons with antibiotic kill curves for PAO1. Due to the spatial and temporal nature of our approach, we also identified spatial and temporal inhomogeneities in the biofilm metabolism that are consistent with previous observations. Clinical strains of P. aeruginosa subjected to similar interrogation showed variations in resistance to colistin and tobramycin, which are two antibiotics commonly used to treat P. aeruginosa infections in cystic fibrosis patients.IMPORTANCE Biofilm infections are more difficult to treat than planktonic infections for a variety of reasons, such as decreased antibiotic penetration. Their complex structure makes biofilms challenging to study without disruption. To address this limitation, we developed and demonstrated oxygen-sensitive luminescent nanosensors that can be incorporated into biofilms for studying oxygen penetration, distribution, and antibiotic efficacy-demonstrated here with our sensors monitoring antibiotic impacts on metabolism in biofilms formed from clinical isolates. The significance of our research is in demonstrating not only a nondisruptive method for imaging and measuring oxygen in biofilms but also that this nanoparticle-based sensing platform can be modified to measure many different ions and small molecule analytes.

Prevention of chronic infection with Pseudomonas aeruginosa infection in cystic fibrosis.

PURPOSE OF REVIEW: This review provides an update on definitions of chronicity of infection, approaches to airway sampling to detect infection, strategies for Pseudomonas aeruginosa eradication, impact of cystic fibrosis transmembrane regulator protein (CFTR) modulators and future challenges for clinical trials. RECENT FINDINGS: Rates of P. aeruginosa have decreased over the past two decades with establishment of effective eradication protocols. Definitions of chronic P. aeruginosa infection have required adaptation for healthier populations. Although molecular (PCR) approaches to early P. aeruginosa detection are sensitive, to date, earlier diagnosis has not impacted on clinical outcomes. Despite eradication regimens, some people with early P. aeruginosa fail to clear their infection. Most people also experience a recurrence and eventual transition to chronic infection. Several recent studies sought to address this gap. CFTR modulators (predominantly ivacaftor) demonstrated reduced P. aeruginosa density, although infection may persist or recur demonstrating the need for continued antiinfective therapies in the modulator era. SUMMARY: Future studies of approaches to P. aeruginosa eradication will be complex due to expanded availability and ongoing competitive clinical trials of CFTR modulators. Studies to address optimal eradication therapy, particularly in adults, will be required, though adequate recruitment to power these studies may prove challenging.

Microbiological efficacy of early MRSA treatment in cystic fibrosis in a randomised controlled trial.

OBJECTIVE: To evaluate microbiological effectiveness, that is, culture negativity of a non-blinded eradication protocol (Rx) compared with observation (Obs) in clinically stable cystic fibrosis participants with newly positive methicillin resistant Staphylococcusaureus (MRSA) cultures. DESIGN: This non-blinded trial randomised participants ages 4-45 years with first or early (≤2 positive cultures within 3 years) MRSA-positive culture without MRSA-active antibiotics within 4 weeks 1:1 to Rx or Obs. The Rx protocol was: oral trimethoprim-sulfamethoxazole or if sulfa-allergic, minocycline plus oral rifampin; chlorhexidine mouthwash for 2 weeks; nasal mupirocin and chlorhexidine body wipes for 5 days and environmental decontamination for 21 days. The primary end point was MRSA culture status at day 28. RESULTS: Between 1 April 2011 to September 2014, 45 participants (44% female, mean age 11.5 years) were randomised (24 Rx, 21 Obs). At day 28, 82% (n=18/22) of participants in the Rx arm compared with 26% (n=5/19) in the Obs arm were MRSA-negative. Adjusted for interim monitoring, this difference was 52% (95% CI 23% to 80%, p<0.001). Limiting analyses to participants who were MRSA-positive at the screening visit, 67% (8/12) in the Rx arm and 13% (2/15) in the Obs arm were MRSA-negative at day 28, adjusted difference: 49% (95% CI 22% to 71%, p<0.001). Fifty-four per cent in the Rx arm compared with 10% participants in the Obs arm remained MRSA-negative through day 84. Mild gastrointestinal side effects were higher in the Rx arm. CONCLUSIONS: This MRSA eradication protocol for newly acquired MRSA demonstrated microbiological efficacy with a large treatment effect. TRIAL REGISTRATION NUMBER: NCT01349192.

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Pulmonary exacerbations and clinical outcomes in a longitudinal cohort of infants and preschool children with cystic fibrosis.

BACKGROUND: Pulmonary exacerbations (PEx) in school aged children and adults with cystic fibrosis (CF) lead to increased morbidity and lung function decline. However, the effect of exacerbations in young children with CF is not fully understood. We sought to characterize the frequency and clinical impact of PEx in a pilot study of infants and pre-school aged children with CF. METHODS: Thirty young children with CF [median (range) 1.5 years (0.2-4.9)] were prospectively followed for 2 years. Exacerbation frequency (hospitalizations and outpatient antibiotic use) was determined. Chest radiographs were performed at enrollment and study completion and assigned a Brasfield score. Lung function at age 7 years was assessed in a subset of children. The association between PEx frequency, chest radiograph score, and lung function was determined using Spearman correlation coefficients and corresponding 95% confidence intervals. Correlations with an absolute magnitude of 0.3 or greater were considered clinically significant. RESULTS: Over 2 years, participants experienced a median of two PEx (range 0-13). Chest radiograph scores at enrollment and study completion were inversely associated with PEx frequency (R = -0.48 and R = -0.44, respectively). The association between frequency of PEx and lung function [forced expiratory volume in 1 s (FEV1)] at age 7 years was small (R = 0.20). Higher forced vital capacity (FVC) at 7 years was associated with more frequent PEx during the study (R = 0.44). CONCLUSIONS: Children with worse chest radiograph scores had more frequent PEx over the subsequent 2 years, suggesting a group of patients at higher risk for PEx. Frequent PEx in infants and young children with CF were not associated with lower FEV1 and FVC at 7 years, although spirometry in this age group may not be a sensitive marker of mild lung disease and disease progression.

Cystic fibrosis: a model system for precision medicine.

PURPOSE OF REVIEW: Development of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, small molecule therapies that target the basic defect in cystic fibrosis (CF), represents a new era in CF treatment. This review highlights recent progress in CF therapeutics as an example of precision medicine and personalized approaches to test CFTR modulators using preclinical model systems. RECENT FINDINGS: CFTR modulators are now clinically available for approximately 50% of the United States CF population. The CFTR potentiator, ivacaftor, is approved for people with CF ages 2 years and older with at least one gating mutation (G551D, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, or S549R) or the R117H conductance mutation. The recent Food and Drug Administration approval of the corrector/potentiator combination, lumacaftor/ivacaftor, expands modulator therapy to people with CF homozygous for the F508del mutation, ages 12 years and older. Ivacaftor and lumacaftor, however, do not fully restore CFTR activity. Thus, next-generation correctors and potentiators are in development. Read-through agents targeting nonsense mutations and genotype agnostic treatments (gene-editing and gene therapy) are also in various phases of clinical development. SUMMARY: CFTR modulators promise to transform the therapeutic landscape in CF in a precision based fashion. Areas of ongoing research include developing drugs for all mutation classes so that all persons with CF can benefit from these therapies, and refining preclinical assays that allow the selection of the most effective treatments on an individual basis.

Remote endpoints for clinical trials in cystic fibrosis: Report from the U.S. CF foundation remote endpoints task force.

The COVID-19 pandemic necessitated a rapid shift in clinical research to perform virtual visits and remote endpoint assessments, providing a key opportunity to optimize the use of remote endpoints for clinical trials in cystic fibrosis. The use of remote endpoints could allow more diverse participation in clinical trials while minimizing participant burden but must be robustly evaluated to ensure adequate performance and feasibility. In response, the Cystic Fibrosis Foundation convened the Remote Endpoint Task Force (Supplemental Table 1), a multidisciplinary group of CF researchers with remote endpoint expertise and community members tasked to better understand the current and future use of remote endpoints for clinical research. Here, we describe the current use of remote endpoints in CF clinical research, address key unanswered questions regarding their use and feasibility, and discuss the next steps to determine clinical trial readiness.

Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis.

RATIONALE: Progressive lung function decline is a defining feature of cystic fibrosis (CF). Because airway inflammation plays a central role in CF lung disease, inflammatory biomarkers that can be used to monitor disease activity would be valuable. OBJECTIVES: Examine longitudinal relationships between sputum biomarkers and lung function. METHODS: In this prospective, longitudinal cohort study, sputum induction was performed annually over 3 years in 35 children with CF. Sputum was assayed for mediators related to proteolysis and a panel of inflammatory cytokines. MEASUREMENTS AND MAIN RESULTS: Sputum neutrophil elastase, tissue inhibitor of metalloproteinase-1, and TNF-α increased over time, whereas neutrophil elastase antiprotease complexes (NEAPCs) and secretory leukoprotease inhibitor (SLPI) significantly decreased over time. Higher detectable baseline neutrophil elastase was associated with more rapid lung function decline. Similar results for neutrophil elastase were observed in a validation cohort. When categorizing subjects as "rapid" or "slow" decliners, logistic regression demonstrated that the initial measurement of neutrophil elastase had the highest individual predictive value for subsequent lung function decline, whereas neutrophil elastase, IL-8, and IL-6 had the highest combined predictive value. Lung function decline was associated with increases in neutrophil counts, neutrophil elastase, and IL-1ß and declines in NEAPCs and SLPI. CONCLUSIONS: In children with CF, a single determination of sputum biomarkers, particularly neutrophil elastase, has predictive value for subsequent lung function decline, and longitudinal changes in sputum inflammatory biomarkers are related to lung function changes. Based on our results, sputum neutrophil elastase was the most informative biomarker to monitor disease activity.

Infection, Allergy, and Inflammation: The Role of Aspergillus fumigatus in Cystic Fibrosis.

Aspergillus fumigatus (Af) is a mold frequently detected in airway samples from people with cystic fibrosis (pwCF). Abnormal airway mucus may allow Af to germinate, resulting in airway infection or an allergic response. While Af is known to increase morbidity in pwCF, individual responses and the degree of impact on lung disease vary. Improved approaches to diagnosis, treatment, and prevention of Af, particularly the persistent Af infection, are needed. This update highlights our current understanding of Af pathophysiology in the CF airway, the effects of Af on pwCF, and areas of research needed to improve clinical outcomes.

Managing cystic fibrosis in children aged 6-11yrs: a critical review of elexacaftor/tezacaftor/ivacaftor combination therapy.

INTRODUCTION: Cystic fibrosis is a life-limiting, autosomal recessive genetic disorder resulting in multi-organ disease due to CF transmembrane conductance regulator (CFTR) protein dysfunction. CF treatment previously focused on mitigation of disease signs and symptoms. The recent introduction of highly effective CFTR modulators, for which ~90% of people with CF are CFTR variant-eligible, has resulted in substantial health improvements. AREAS COVERED: In this review, we will describe the clinical trials leading to approval of the highly effective CFTR modulator, elexacaftor-tezacaftor-ivacaftor (ETI), with a focus on the safety and efficacy of this treatment in children aged 6-11 years. EXPERT OPINION: The use of ETI in variant-eligible children aged 6-11 is associated with marked clinical improvements with a favorable safety profile. We anticipate that introduction of ETI in early childhood may result in the prevention of pulmonary, gastrointestinal, and endocrine complications from CF, consequently leading to previously unimaginable gains in the quality and quantity of life. However, there is an urgent need to develop effective treatments for the remaining 10% of people with CF who are not eligible or unable to tolerate ETI treatment, and to increase access of ETI to more pwCF across the world.

Integrating airway microbiome and blood proteomics data to identify multi-omic networks associated with response to pulmonary infection.

Host response to airway infections can vary widely. Cystic fibrosis (CF) pulmonary exacerbations provide an opportunity to better understand the interplay between respiratory microbes and the host. This study aimed to investigate the observed heterogeneity in airway infection recovery by analyzing microbiome and host response (i.e., blood proteome) data collected during the onset of 33 pulmonary infection events. We used sparse multiple canonical correlation network (SmCCNet) analysis to integrate these two types of -omics data along with a clinical measure of recovery. Four microbe-protein SmCCNet subnetworks at infection onset were identified that strongly correlate with recovery. Our findings support existing knowledge regarding CF airway infections. Additionally, we discovered novel microbe-protein subnetworks that are associated with recovery and merit further investigation.

Therapeutic beta-lactam dosages and broad-spectrum antibiotics are associated with reductions in microbial richness and diversity in persons with cystic fibrosis.

Persons with cystic fibrosis (PwCF) suffer from pulmonary exacerbations (PEx) related in part to lung infection. While higher microbial diversity is associated with higher lung function, the data on the impact of short-term antibiotics on changes in microbial diversity is conflicting. Further, Prevotella secretes beta-lactamases, which may influence recovery of lung function. We hypothesize that sub-therapeutic and broad spectrum antibiotic exposure leads to decreasing microbial diversity. Our secondary aim was to evaluate the concerted association of beta-lactam pharmacokinetics (PK), antibiotic spectrum, microbial diversity, and antibiotic resistance on lung function recovery using a pathway analysis. This was a retrospective observational study of persons with CF treated with IV antibiotics for PEx between 2016 and 2020 at Children's National Hospital; respiratory samples and clinical information were collected at hospital admission for PEx (E), end of antibiotic treatment (T), and follow-up (F). Metagenomic sequencing was performed; PathoScope 2.0 and AmrPlusPlus were used for taxonomic assignment of sequences to bacteria and antibiotic resistance genes (ARGs). M/W Pharm was used for PK modeling. Comparison of categorical and continuous variables and pathway analysis were performed in STATA. Twenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years. Only 12/43 beta-lactam courses had therapeutic PK, and 18/43 were broad spectrum. A larger decrease in richness between E and T was seen in the therapeutic PK group (sufficient - 20.1 vs. insufficient - 1.59, p = 0.025) and those receiving broad spectrum antibiotics (broad - 14.5 vs. narrow - 2.8, p = 0.030). We did not detect differences in the increase in percent predicted forced expiratory volume in one second (ppFEV1) at end of treatment compared to PEx based on beta-lactam PK (sufficient 13.6% vs. insufficient 15.1%) or antibiotic spectrum (broad 11.5% vs. narrow 16.6%). While both therapeutic beta-lactam PK and broad-spectrum antibiotics decreased richness between PEx and the end of treatment, we did not detect longstanding changes in alpha diversity or an association with superior recovery of lung function compared with subtherapeutic PK and narrow spectrum antimicrobials.