Pharmacologic improvement of CFTR function rapidly decreases sputum pathogen density, but lung infections generally persist.

BackgroundLung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CF transmembrane conductance regulator (CFTR) modulators improve activity of dysfunctional CFTR channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections.MethodsWe performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI), on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR, and sequencing.ResultsMean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter spp., and Burkholderia spp. decreased by 2-3 log10 CFU/mL after 1 month of ETI. However, most participants remained culture positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria.ConclusionsTreatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment.Trial RegistrationClinicalTrials.gov NCT04038047.FundingThe Cystic Fibrosis Foundation and the NIH.

Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Combining Ivacaftor and Intensive Antibiotics Achieves Limited Clearance of Cystic Fibrosis Infections.

Drugs called CFTR modulators improve the physiologic defect underlying cystic fibrosis (CF) and alleviate many disease manifestations. However, studies to date indicate that chronic lung infections that are responsible for most disease-related mortality generally persist. Here, we investigated whether combining the CFTR modulator ivacaftor with an intensive 3.5-month antibiotic course could clear chronic Pseudomonas aeruginosa or Staphylococcus aureus lung infections in subjects with R117H-CFTR, who are highly ivacaftor-responsive. Ivacaftor alone improved CFTR activity, and lung function and inflammation within 48 h, and reduced P. aeruginosa and S. aureus pathogen density by ∼10-fold within a week. Antibiotics produced an additional ∼10-fold reduction in pathogen density, but this reduction was transient in subjects who remained infected. Only 1/5 P. aeruginosa-infected and 1/7 S. aureus-infected subjects became persistently culture-negative after the combined treatment. Subjects appearing to clear infection did not have particularly favorable baseline lung function or inflammation, pathogen density or antibiotic susceptibility, or bronchiectasis scores on CT scans, but they did have remarkably low sweat chloride values before and after ivacaftor. All persistently P. aeruginosa-positive subjects remained infected by their pretreatment strain, whereas subjects persistently S. aureus-positive frequently lost and gained strains. This work suggests chronic CF infections may resist eradication despite marked and rapid modulator-induced improvements in lung infection and inflammation parameters and aggressive antibiotic treatment. IMPORTANCE Recent work shows that people with CF and chronic lung infections generally remain persistently infected after treatment with drugs that target the CF physiological defect (called CFTR modulators). However, changes produced by modulators could increase antibiotic efficacy. We tested the approach of combining modulators and intensive antibiotics in rapid succession and found that while few subjects cleared their infections, combined treatment appeared most effective in subjects with the highest CFTR activity. These findings highlight challenges that remain to improve the health of people with CF.

Infants with cystic fibrosis have altered fecal functional capacities with potential clinical and metabolic consequences.

BACKGROUND: Infants with cystic fibrosis (CF) suffer from gastrointestinal (GI) complications, including pancreatic insufficiency and intestinal inflammation, which have been associated with impaired nutrition and growth. Recent evidence identified altered fecal microbiota taxonomic compositions in infants with CF relative to healthy infants that were characterized by differences in the abundances of taxa associated with GI health and nutrition. Furthermore, these taxonomic differences were more pronounced in low length infants with CF, suggesting a potential link to linear growth failure. We hypothesized that these differences would entail shifts in the microbiome's functional capacities that could contribute to inflammation and nutritional failure in infants with CF. RESULTS: To test this hypothesis, we compared fecal microbial metagenomic content between healthy infants and infants with CF, supplemented with an analysis of fecal metabolomes in infants with CF. We identified notable differences in CF fecal microbial functional capacities, including metabolic and environmental response functions, compared to healthy infants that intensified during the first year of life. A machine learning-based longitudinal metagenomic age analysis of healthy and CF fecal metagenomic functional profiles further demonstrated that these differences are characterized by a CF-associated delay in the development of these functional capacities. Moreover, we found metagenomic differences in functions related to metabolism among infants with CF that were associated with diet and antibiotic exposure, and identified several taxa as potential drivers of these functional differences. An integrated metagenomic and metabolomic analysis further revealed that abundances of several fecal GI metabolites important for nutrient absorption, including three bile acids, correlated with specific microbes in infants with CF. CONCLUSIONS: Our results highlight several metagenomic and metabolomic factors, including bile acids and other microbial metabolites, that may impact nutrition, growth, and GI health in infants with CF. These factors could serve as promising avenues for novel microbiome-based therapeutics to improve health outcomes in these infants.

Minimally invasive mitral valve replacement and concomitant Cox-Maze IV procedure using radiofrequency energy in situs inversus totalis: A case report.

INTRODUCTION: Situs inversus totalis (SIT) is an uncommon congenital condition characterized by total transposition of abdominal and thoracic viscera. Performing minimally invasive cardiac surgery on individuals with SIT requires different surgical planning because of the unfamiliar positions of the heart and great vessels. PRESENTATION OF CASE: A 52-year-old female was admitted to our center with palpitations and dyspnea on exertion. Chest X-ray showed dextrocardia. Echocardiography and chest computerized tomography (CT) revealed SIT with severe rheumatic mitral valve disease. DISCUSSION: Pre-operative three-dimensional (3D) chest CT reconstruction was helpful in surgical planning and management of cardiopulmonary bypass (CPB). Mitral valve replacement and concomitant atrial fibrillation (AF) ablation using radiofrequency (RF) energy via left mini-thoracotomy was successfully performed on the patient. CONCLUSION: Minimally invasive approach can be safely and effectively employed in patients with SIT.

Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.

RATIONALE: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments. OBJECTIVES: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics. METHODS AND MEASUREMENTS: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories. MAIN RESULTS: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. CONCLUSIONS: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.

Mitral prosthetic size predictor in minimally invasive mitral valve replacement.

BACKGROUND: Minimally invasive mitral valve replacement has become popular across the world. However, annular rupture and patient - prosthetic mismatch (PPM) are still problematic, particularly in the Asian population. To avoid this, a predictor model could be beneficial. Our study aimed to assess the value of mitral valve diameters measured on TTE and CT scan on predicting the actual mitral prostheses. METHODS: From January 2018 to December 2019, a total number of 96 patients underwent minimally invasive mitral valve replacement. The association between imaging measurements and the outcome was checked by scatter plot and Pearson's correlation coefficient. Univariable linear regression was used to build the prediction model. RESULTS: The three strongest correlations for the whole population are the following features: Mean TTE diameter (0.702), mean diameter on CT lowest plane through the mitral annulus (0.679), and area-derived diameter on CT highest plane through the mitral annulus (0.665). The prosthetic size of the tissue valve group was more correlated to the calculated annulus diameters than that of the mechanical valve group. Tissue valve size predictor models based on these calculated diameters were 16.19 + 0.27 × d (r = 0.744), 12.74 + 0.44 × d (r = 0.756) and 12.79 + 0.38 × d (r = 0.730), respectively. CONCLUSION: Mitral prosthetic size could be predicted based on the mitral diameters measured on TTE and CT scan. The overall correlation coefficient varied from 0.665 (CT Scan) to 0.702 (TTE). These models performed better when applied to bioprosthesis.

CFTR dysregulation drives active selection of the gut microbiome.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.

Fecal dysbiosis in infants with cystic fibrosis is associated with early linear growth failure.

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age1. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life1. Although this early linear growth failure is associated with worse long-term respiratory function and survival2,3, the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis4,5. As GI microbiome activities are known to affect endocrine functions6,7, the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.

Minimally Invasive Ozaki Procedure in Aortic Valve Disease: The Preliminary Results.

OBJECTIVE: The Ozaki procedure for aortic valve reconstruction was reported in 2014 with low mortality, a highly reproducible rate and a good midterm result. However, the procedure still requires conventional sternotomy to be accomplished. The aim of the study was to start an initial evaluation for the feasibility of the minimally invasive approach in combination with the Ozaki technique. METHODS: From January 06, 2017, to January 12, 2017, nine patients with severe aortic valve diseases underwent minimally invasive Ozaki procedure through an upper ministernotomy. The pericardium was harvested endoscopically using three trocars in different intercostal spaces. Then, a ministernotomy was performed and the Ozaki procedure was accomplished in a similar manner to the conventional technique. We analyzed the in-hospital mortality and complications of this group. RESULTS: The mean age was 47.4 years and 55.6% patients were female. The predominant pathology was chronic rheumatic valve disease (66.7%) and other patients were diagnosed with a bicuspid aortic valve. The mean aortic cross-clamp time was 106.8 minutes, the mean cardiopulmonary bypass time was 153.6 minutes, the mean ventilation time was 8.4 hours, and the mean intensive care unit time was 1.6 days. No mortality was recorded in our series, no conversion to full sternotomy was required, one patient experienced right hemothorax requiring drainage, and one patient required valve replacement. Intraoperative transesophageal echocardiography and predischarge transthoracic echocardiography showed five competent valves and three valves with trivial regurgitation, and no stenosis was detected. CONCLUSIONS: Ministernotomy combined with Ozaki procedure might be feasible, as well as an alternative to conventional sternotomy. This approach is associated with low mortality and morbidity and may be beneficial in younger populations.