Genome-Wide Association Study of Susceptibility to Pseudomonas aeruginosa Infection in Cystic Fibrosis.

QUESTION: Pseudomonas aeruginosa (Pa) is a common pathogen that contributes to progressive lung disease in Cystic Fibrosis (CF). Genetic factors other than CF-causing CFTR variations contribute approximately 85% of the variation in chronic Pa infection age in CF according to twin studies, but the susceptibility loci remain unknown. Our objective is to advance understanding of the genetic basis of host susceptibility to Pa infection. MATERIALS AND METHODS: We conducted a genome-wide association study (GWAS) of chronic Pa infection age in 1037 Canadians with CF. We subsequently assessed the genetic correlation between chronic Pa infection age and lung function through polygenic risk score (PRS) analysis and inferred their causal relationship through bi-directional Mendelian Randomization analysis. RESULTS: Two novel genome-wide significant loci with lead SNPs rs62369766 (chr5p12; p-value= 1.98 ×10-8) and rs927553 (chr13q12.12; p-value= 1.91 × 10-8) were associated with chronic Pa infection age. The rs62369766 locus was validated using an independent French cohort (N=501). Furthermore, PRS constructed from CF lung function-associated SNPs was significantly associated with chronic Pa infection age (p-value=0.002). Finally, our analysis presented evidence for a causal effect of lung function on the chronic Pa infection age (Beta=0.782 years, p-value= 4.24 × 10-4). In the reverse direction, we observed a moderate effect (Beta=0.002, p-value=0.012). CONCLUSIONS: We identified two novel loci that are associated with chronic Pa infection age in individuals with CF. Additionally, we provided evidence of common genetic contributors and a potential causal relationship between Pa infection susceptibility and lung function in CF. Therapeutics targeting these genetic factors may delay the onset of chronic infections which accounts for significant remaining morbidity in CF.

Practical Guidance for Clinical Microbiology Laboratories: Updated guidance for processing respiratory tract samples from people with cystic fibrosis.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.

Effect of L-arginine on cystic fibrosis Pseudomonas aeruginosa biofilms.

Cystic fibrosis (CF) airways are L-arginine deficient which may affect susceptibility to infection with certain pathogens. The potential impact of L-arginine supplementation on Pseudomonas aeruginosa, a common CF airway pathogen, is unclear. This study investigated the effects of L-arginine on P. aeruginosa biofilm cultures, using the laboratory strain PAO1 and multi-drug resistant CF clinical isolates. P. aeruginosa biofilms were grown in a chambered cover-glass slide model for 24 h, then exposed to either L-arginine alone or combined with tobramycin for an additional 24 h. Biofilms were visualized using confocal microscopy, and viable cells were measured via plating for colony-forming units. Increasing concentrations of L-arginine in bacterial culture medium reduced the biovolume of P. aeruginosa in a dose-dependent manner. Notably, L-arginine concentrations within the physiological range (50 mM and 100 mM) in combination with tobramycin promoted biofilm growth, while higher concentrations (600 mM and 1200 mM) inhibited growth. These findings demonstrate the potential of L-arginine as an adjuvant therapy to inhaled tobramycin in treating P. aeruginosa infections in people with CF.

Lack of correlation between in vitro and within patient measures of P. aeruginosa biofilms in cystic fibrosis.

Current in vitro biofilm modelling of the opportunistic pathogen, Pseudomonas aeruginosa (PA) in people with cystic fibrosis (PwCF) is limited in its ability to mimic the complexities of the cystic fibrosis (CF) lung environment. Recent adaptations of the Microbial Identification after Passive CLARITY Technique (MiPACT) in CF research have allowed for the direct imaging of PA biofilm spatial organization and structure in expectorated sputum. Here, we performed a comparative analysis of in vitro and within patient (ex vivo) measures of PA biofilms using sputa from new onset infected children with CF. MiPACT-fluorescent in situ hybridization (FISH) and fluorescent anti-Psl monoclonal antibody (mAb) staining was performed to directly visualize PA and Psl (exopolysaccharide in PA biofilm matrix) in 11 CF sputum specimens. Corresponding PA isolates, recovered from the same sputum samples, were grown as biofilms in a glass slide chamber model, then visualized by fluorescent live-cell and anti-Psl mAb staining. We observed that PA biovolume, aggregation and Psl antibody binding (normalized per PA biovolume) in CF sputum did not correlate with the in vitro model, although a trend towards significance in the biovolume relationship was observed with the addition of sputum supernatant to the in vitro model.

A randomised trial of oral prednisone for cystic fibrosis pulmonary exacerbation treatment.

BACKGROUND: Elevated markers of systemic and pulmonary inflammation are associated with failure to recover lung function following pulmonary exacerbations in people with cystic fibrosis (pwCF). Our aim was to determine whether adjuvant oral prednisone treatment would improve recovery of forced expiratory volume in 1 s (FEV1) % pred in CF pulmonary exacerbations not responding to antibiotic therapy. METHODS: This was a randomised, double-blind, placebo-controlled trial in pwCF treated with intravenous antibiotics for a pulmonary exacerbation. At day 7, those who had not returned to >90% baseline FEV1 % pred were randomised to adjuvant prednisone 1 mg·kg-1 twice daily (maximum 60 mg·day-1) or placebo for 7 days. The primary outcome was the difference in proportion of subjects who recovered >90% baseline FEV1 % pred at day 14 of i.v. antibiotic therapy. RESULTS: 173 subjects were enrolled, with 76 randomised. 50% of subjects in the prednisone group recovered baseline FEV1 on day 14 compared with 39% of subjects in the placebo group (difference of 11%, 95% CI -11-34%; p=0.34). The mean±sd change in FEV1 % pred from day 7 to day 14 was 6.8±8.8% predicted in the prednisone group and 4.6±6.9% predicted in the placebo group (mean difference 2.2% predicted, 95% CI -1.5-5.9%; p=0.24). Time to subsequent exacerbation was not prolonged in prednisone-treated subjects (hazard ratio 0.83, 95% CI 0.45-1.53; p=0.54). CONCLUSIONS: This study failed to detect a difference in FEV1 % pred recovery between adjuvant oral prednisone and placebo treatment in pwCF not responding at day 7 of i.v. antibiotic therapy for pulmonary exacerbations.

Predictive modeling of antibiotic eradication therapy success for new-onset Pseudomonas aeruginosa pulmonary infections in children with cystic fibrosis.

Chronic Pseudomonas aeruginosa (Pa) lung infections are the leading cause of mortality among cystic fibrosis (CF) patients; therefore, the eradication of new-onset Pa lung infections is an important therapeutic goal that can have long-term health benefits. The use of early antibiotic eradication therapy (AET) has been shown to clear the majority of new-onset Pa infections, and it is hoped that identifying the underlying basis for AET failure will further improve treatment outcomes. Here we generated machine learning models to predict AET outcomes based on pathogen genomic data. We used a nested cross validation design, population structure control, and recursive feature selection to improve model performance and showed that incorporating population structure control was crucial for improving model interpretation and generalizability. Our best model, controlling for population structure and using only 30 recursively selected features, had an area under the curve of 0.87 for a holdout test dataset. The top-ranked features were generally associated with motility, adhesion, and biofilm formation.

Factors associated with lung function response with oral antibiotic treatment of pulmonary exacerbations in cystic fibrosis.

Pulmonary exacerbations treated with oral antibiotics (oPEx) have a significant effect on lung function decline in people with cystic fibrosis (CF). However, factors associated with lung function response with oPExs are not well defined. We performed a retrospective cohort study of pediatric and adult patients with CF followed in the Toronto CF Database. Lung function response was measured both as the change in forced expiratory volume in 1 second (FEV1) from Day 0 of antibiotic therapy to end of treatment as well as from baseline to end of treatment. Drop from baseline to Day 0 FEV1 was strongly associated with lung function response (p<0.001). Greater FEV1 improvements were associated with longer antibiotic treatment durations. Older, female patients had less improvements in FEV1 at end of treatment compared to younger, male patients.

Treatment response to pulmonary exacerbation in primary ciliary dyskinesia.

INTRODUCTION: Pulmonary exacerbation (Pex) are common in pediatric primary ciliary dyskinesia (PCD), however changes in forced expiratory volume in 1 s precent predicted (FEV1pp) during Pex are not well described. AIM: To assess the evolution of FEV1pp during Pex and to define factors associated with failure to return to baseline lung function. METHOD: This was a retrospective study of patients with PCD between 2010 and 2022. Pex were defined as the presence of increased respiratory symptoms treated with intravenous (IV) antibiotics. The main outcomes were the changes in FEV1 during therapy and the proportion of patients (responders) achieving ≥90% of baseline FEV1pp values at the end of admission. RESULTS: The study included 52 Pex events in 28 children with PCD. The rate of responders was 32/41 (78%) at the end of admission. Nonresponse was associated with lower median body mass index (BMI) Z-score (-2.4 vs. -0.4, p < .01) and with a history of IV treated Pex in the previous year (p = .06). For the 22 Pex with available FEV1pp measurements at mid admission, the median relative and absolute improvement from admission to Day 7 was 9.1% and 6.2%, respectively (p- .001), and from Days 7 to 14 was 4.4% and 2.8%, respectively (p = .08). CONCLUSION: In children with PCD treated with IV antibiotics, the majority of lung function recovery happens during the first week of IV therapy. Lower BMI was associated with nonresponse to therapy.

Exhaled breath profiles to detect lung infection with Staphylococcus aureus in children with cystic fibrosis.

BACKGROUND: An electronic nose (eNose) can be used to detect volatile organic compounds (VOCs). Exhaled breath contains numerous VOCs and individuals' VOCs mixtures create distinct breath profiles. Previous reports have shown that eNose can detect lung infections. Whether eNose can detect Staphylococcus aureus airway infections in breath of children with cystic fibrosis (CF) is currently unclear. METHODS: In this cross-sectional observational study, a cloud-connected eNose was used for breath profile analysis of clinically stable paediatric CF patients with airway microbiology cultures positive or negative for CF pathogens. Data-analysis involved advanced signal processing, ambient correction and statistics based on linear discriminant and receiver operating characteristics (ROC) analyses. RESULTS: Breath profiles from 100 children with CF (median predicted FEV1 91%) were obtained and analysed. CF patients with positive airway cultures for any CF pathogen were distinguishable from no CF pathogens (no growth or usual respiratory flora) with accuracy of 79.0% (AUC-ROC 0.791; 95% CI: 0.669-0.913) and between patients positive for Staphylococcus aureus (SA) only and no CF pathogen with accuracy of 74.0% (AUC-ROC 0.797; 95% CI: 0.698-0.896). Similar differences were seen for Pseudomonas aeruginosa (PA) infection vs no CF pathogens (78.0% accuracy, AUC-ROC 0.876, 95% CI: 0.794-0.958). SA- and PA-specific signatures were driven by different sensors in the SpiroNose suggesting pathogen-specific breath signatures. CONCLUSIONS: Breath profiles of CF patients with SA in airway cultures are distinct from those with no infection or PA infection, suggesting the utility of eNose technology in the detection of this early CF pathogen in children with CF.

Comparative analysis of respiratory symptom scores to detect acute respiratory events in children with cystic fibrosis.

BACKGROUND: Patient-reported outcomes (PROs) are important outcome measures in research and clinical practice. This study describes the longitudinal variability the Cystic Fibrosis Questionnaire-Revised (CFQ-R) Respiratory score and the Chronic Respiratory Infection Symptom Score (CRISS), as well as their ability to identify acute respiratory events in children with CF. METHODS: In this prospective observational study, the parent-proxy (6 -13 years) and self-reported (6-18 years) CFQ-R Respiratory score and CRISS (6-18 years) were measured every 3 months over 2 years. The lung clearance index (LCI) and FEV1 were also measured. We compared the diagnostic accuracy of the PROs in distinguishing acute respiratory events and clinically stable visits, using the minimal important difference of each PRO as the threshold. RESULTS: A total of 98 children with CF were included. On average, the symptom scores did not change between clinically stable visits. The positive predictive value (PPV) and negative predictive value (NPV) of a ≥8.5-point worsening in the parent-proxy CFQ-R score to identify acute respiratory events (n=119) (PPV 70.2% and NPV 87.0%) were higher than for the self-reported CFQ-R score (PPV 58.9% and NPV 72.2%). The PPV and NPV of an ≥11-point change in the CRISS for acute respiratory events (n=137) was 56.5% and 79.6%, respectively. The PPV and NPV of all PROs were increased when combined with the LCI and/or FEV1pp. CONCLUSION: Symptoms scores differ in their ability to identify acute respiratory events in children with CF; PPV and NPV of all PROs were improved when combined with lung function outcomes.

Evaluation of clinically relevant changes in the lung clearance index in children with cystic fibrosis and healthy controls.

BACKGROUND: The limits of reproducibility of the lung clearance index (LCI) are higher in children with cystic fibrosis (CF) compared with healthy children, and it is currently unclear what defines a clinically meaningful change. METHODS: In a prospective multisite observational study of children with CF and healthy controls (HCs), we measured LCI, FEV1% predicted and symptom scores at quarterly visits over 2 years. Two reviewers performed a detailed review of visits to evaluate the frequency that between visit LCI changes outside ±10%, ±15%, ±20% represented a clinically relevant signal. In the setting of acute respiratory symptoms, we used a generalised estimating equation model, with a logit link function to determine the ability of LCI worsening at different thresholds to predict failure of lung function recovery at follow-up. RESULTS: Clinically relevant LCI changes outside ±10%, ±15% and ±20% were observed at 25.7%, 15.0% and 8.3% of CF visits (n=744), respectively. The proportions of LCI changes categorised as noise, reflecting biological variability, were comparable between CF and HC at the 10% (CF 9.9% vs HC 13.0%), 15% (CF 4.3% vs HC 3.1%) and 20% (CF 2.4% vs HC 1.0%) thresholds. Compared with symptomatic CF visits without a worsening in LCI, events with ≥10% LCI increase were more likely to fail to recover baseline LCI at follow-up. CONCLUSION: The limits of reproducibility of the LCI in healthy children can be used to detect clinically relevant changes and thus inform clinical care in children with CF.

Impact of antibiotic eradication therapy of Pseudomonas aeruginosa on long term lung function in cystic fibrosis.

INTRODUCTION: While antibiotic eradication therapy (AET) of early Pseudomonas aeruginosa infection is considered standard of care, its long-term effect on the subsequent course of cystic fibrosis (CF) lung disease remains unclear. METHODS: CF patients who were P. aeruginosa-free for at least a year and had a minimum of 10 years of pulmonary function measurements were included. Subjects were categorized as Never if they never had P. aeruginosa isolated from a respiratory tract sample. Subjects changed to the Eradicated group if they had a P. aeruginosa infection, were treated with AET, and subsequently cleared their infection. Subjects changed to the Chronic group if AET did not clear their P. aeruginosa infection. The primary outcome was absolute FEV1 decline over time, with age as the time variable. Mixed-effects linear regression models were used to account for the repeated lung function measurements over time within each patient. RESULTS: 205 CF subjects (48% female) were included; the median (IQR) age at first infection was 9.6 (5.6, 14.6) years. The median (IQR) follow-up was 10.2 (5.7, 14.7) years for the Eradicated group, 8.8 (4.5, 14.9) years for the Chronic group and 2.8 (1.0, 5.7) years for the Never group was among those patients that had at least one P. aeruginosa infection over the study period, annual lung function decline of FEV1 was significantly less (-1.11% predicted/year; 95% CI: -1.18, -1.04) in the Eradication group compared to the Chronic group (-1.57%; -1.64, -1.50) (p<0.001). CONCLUSIONS: AET against P. aeruginosa improves lung function trajectory in CF patients.

Pseudomonas aeruginosa aggregation and Psl expression in sputum is associated with antibiotic eradication failure in children with cystic fibrosis.

We previously demonstrated that P. aeruginosa isolates that persisted in children with cystic fibrosis (CF) despite inhaled tobramycin treatment had increased anti-Psl antibody binding in vitro compared to those successfully eradicated. We aimed to validate these findings by directly visualizing P. aeruginosa in CF sputum. This was a prospective observational study of children with CF with new-onset P. aeruginosa infection who underwent inhaled tobramycin eradication treatment. Using microbial identification passive clarity technique (MiPACT), P. aeruginosa was visualized in sputum samples obtained before treatment and classified as persistent or eradicated based on outcomes. Pre-treatment isolates were also grown as biofilms in vitro. Of 11 patients enrolled, 4 developed persistent infection and 7 eradicated infection. P. aeruginosa biovolume and the number as well as size of P. aeruginosa aggregates were greater in the sputum of those with persistent compared with eradicated infections (p < 0.01). The amount of Psl antibody binding in sputum was also greater overall (p < 0.05) in samples with increased P. aeruginosa biovolume. When visualized in sputum, P. aeruginosa had a greater biovolume, with more expressed Psl, and formed more numerous, larger aggregates in CF children who failed eradication therapy compared to those who successfully cleared their infection.

Genomic epidemiology of Mycobacterium abscessus in a Canadian cystic fibrosis centre.

The Mycobacterium abscessus complex causes significant morbidity and mortality among patients with Cystic Fibrosis (CF). It has been hypothesized that these organisms are transmitted from patient to patient based on genomics. However, few studies incorporate epidemiologic data to confirm this hypothesis. We longitudinally sampled 27 CF and 7 non-CF patients attending a metropolitan hospital in Ontario, Canada from 2013 to 2018. Whole genome sequencing along with epidemiological data was used to evaluate the likelihood of transmission. Overall, the genetic diversity of M. abscessus was large, with a median pairwise distance (IQR) of 1,279 (143-134) SNVs between all Ontario M. abscessus isolates and 2,908 (21-3,204) single nucleotide variants (SNVs) between M. massiliense isolates. This reflects the global diversity of this pathogen, with Ontario isolates widely dispersed throughout global phylogenetic trees of each subspecies. Using a maximum distance of 25 SNVs as a threshold to identify possible transmission, we identified 23 (of 276 total) pairs of closely-related isolates. However, transmission was probable for only one pair based on both genomic and epidemiological data. This suggests that person-to-person transmission of M. abscessus among CF patients is indeed rare and reinforces the critical importance of epidemiological data for inferences of transmission.

Opportunistic Pathogens in Cystic Fibrosis: Epidemiology and Pathogenesis of Lung Infection.

Cystic fibrosis (CF) is one of the most common life-shortening genetic diseases in Caucasians. Due to abnormal accumulation of mucus, respiratory failure caused by chronic infections is the leading cause of mortality in this patient population. The microbiology of these respiratory infections includes a distinct set of opportunistic pathogens, including Pseudomonas aeruginosa, Burkholderia spp., Achromobacter spp., Stenotrophomonas maltophilia, anaerobes, nontuberculous mycobacteria, and fungi. In recent years, culture-independent methods have shown the polymicrobial nature of lung infections, and the dynamics of microbial communities. The unique environment of the CF airway predisposes to infections caused by opportunistic pathogens. In this review, we will highlight how the epidemiology and role in disease of these pathogens in CF differ from that in individuals with other medical conditions. Infectious diseases (ID) physicians should be aware of these differences and the specific characteristics of infections associated with CF.

The effect of antibiotic changes during treatment of cystic fibrosis pulmonary exacerbations.

BACKGROUND: Antibiotics are often changed during treatment of pulmonary exacerbations (PEx) in people with cystic fibrosis (CF) who have a poor clinical response. We aimed to characterize the reasons CF providers change antibiotics and examined the effects of antibiotic changes on lung function recovery. METHODS: This was a retrospective cohort study using the Toronto CF Database from 2009 to 2015 of adults and children with CF PEx treated with intravenous antibiotics. The co-primary outcome measure was absolute and relative change in forced expiratory lung volume in 1 s (FEV1) at end of treatment and follow-up. Secondary outcome assessed the proportion of patients returning to > 90% or > 100% previous baseline FEV1. RESULTS: A total of 399 PEx were included of which 105 had antibiotic changes. Reasons for antibiotic changes included change in antibiotic route prior to discharge (26%), drug reactions (20%), poor FEV1 response (25%), targeting additional microbes (16%) and lack of symptom improvement (13%). In our multivariable analysis, among non-responders (< 90% FEV1 recovery to baseline or lack of symptom improvement at the interim time point), a change in antibiotics was not associated with any significant difference in absolute or relative FEV1 at end of treatment or at follow-up. Antibiotic change in non-responders was not associated with improved return to 90% or 100% baseline FEV1 at end of treatment or follow-up. CONCLUSIONS: Changing antibiotics during CF PEx treatment in those with poor clinical response was not associated with any improved FEV1 response or return to baseline lung function.