Comparison of different sets of immunological tests to identify treatable immunodeficiencies in adult bronchiectasis patients.

Background: The reported prevalence of immunodeficiencies in bronchiectasis patients is variable depending on the frequency and extent of immunological tests performed. European Respiratory Society guidelines recommend a minimum bundle of tests. Broadening the spectrum of immunological tests could increase the number of patients diagnosed with an immunodeficiency and those who could receive specific therapy. The primary objective of the present study was to assess the performance of different sets of immunological tests in diagnosing any, primary, secondary or treatable immunodeficiencies in adults with bronchiectasis. Methods: An observational, cross-sectional study was conducted at the Bronchiectasis Program of the Policlinico University Hospital in Milan, Italy, from September 2016 to June 2019. Adult outpatients with a clinical and radiological diagnosis of bronchiectasis underwent the same immunological screening during the first visit when clinically stable consisting of: complete blood count; immunoglobulin (Ig) subclass tests for IgA, IgG, IgM and IgG; total IgE; lymphocyte subsets; and HIV antibodies. The primary endpoint was the prevalence of patients with any immunodeficiencies using five different sets of immunological tests. Results: A total of 401 bronchiectasis patients underwent the immunological screening. A significantly different prevalence of bronchiectasis patients diagnosed with any, primary or secondary immunodeficiencies was found across different bundles. 44.6% of bronchiectasis patients had a diagnosis of immunodeficiency when IgG subclasses and lymphocyte subsets were added to the minimum bundle suggested by the guidelines. Conclusion: A four-fold increase in the diagnosis of immunodeficiencies can be found in adults with bronchiectasis when IgG subclasses and lymphocyte subsets are added to the bundle of tests recommended by guidelines.

The Isoform GC1f of the Vitamin D Binding Protein Is Associated with Bronchiectasis Severity.

Vitamin D modulates immune responses and its deficiency has been observed in more than 60% of bronchiectasis patients. Vitamin D binding protein (DBP) is coded by the GC gene, is involved in the transport of vitamin D, and includes a number of isoforms based on single nucleotide polymorphisms (SNPs) in the coding region at rs7041 and rs4855. We evaluated the possible clinical impact of DBP polymorphisms and isoforms in an observational, cross-sectional study conducted in 116 bronchiectasis patients, who were genetically characterized for rs4588 and rs7041 SNPs. Results showed that the GC1f isoform (rs7041/rs4588 A/G) correlated with a more severe disease (18.9% vs. 6.3%, p = 0.038), a higher incidence of chronic infections (63.6% vs. 42%, p = 0.041), and a lower BACI score (0.0 (0.0, 2.5) vs. 3.0 (0.0, 3.0), p = 0.035). Moreover, blood concentration of vitamin D was higher in patients carrying GC1s (median (IQR): 20.5 (14.3, 29.7 vs. 15.8 (7.6, 22.4), p = 0.037)). Patients carrying GC1f isoform have a more severe disease, more chronic infections and lower asthmatic comorbidity in comparison to those without the GC1f isoform. Presence of the GC1s isoform (rs7041/rs4588 C/G) seems to be associated to a milder clinical phenotype with increased vitamin D levels and lower comorbidities score.

T2-High Endotype and Response to Biological Treatments in Patients with Bronchiectasis.

Although bronchiectasis pathophysiology has been historically understood around the presence of airway neutrophilic inflammation, recent experiences are consistent with the identification of a type 2 inflammation (T2) high endotype in bronchiectasis. In order to evaluate prevalence and clinical characteristics of bronchiectasis patients with a T2-high endotype and explore their response to biologicals, two studies were carried out. In a cross-sectional study, bronchiectasis adults without asthma underwent clinical, radiological, and microbiological assessment, along with blood eosinophils and oral fractional exhaled nitric oxide (FeNO) evaluation, during stable state. Prevalence and characteristics of patients with a T2- high endotype (defined by the presence of either eosinophils blood count ≥300 cells·µL-1 or oral FeNO ≥ 25 dpp) were reported. A case series of severe asthmatic patients with concomitant bronchiectasis treated with either mepolizumab or benralizumab was evaluated, and patients' clinical data pre- and post-treatment were analyzed up to 2 years of follow up. Among bronchiectasis patients without asthma enrolled in the cross-sectional study, a T2-high endotype was present in 31% of them. These patients exhibited a more severe disease, high dyspnea severity, low respiratory function, and high impact on quality of life. Among the five patients with severe eosinophilic asthma and concomitant bronchiectasis included in the series, treatment with either mepolizumab or benralizumab significantly reduced the exacerbation rate with an effect that persists for up to 2 years of follow up. If validated across different settings, our data suggest the need to design randomized controlled trials on biological treatments targeting the T2-high endotype in bronchiectasis patients.

Protease-Antiprotease Imbalance in Bronchiectasis.

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.

Diagnosis and Initial Investigation of Bronchiectasis.

Bronchiectasis refers to both the name of a disease and a single radiological appearance that may, or may not, be associated with disease. As chronic respiratory disease, bronchiectasis is characterized by a variable range of signs and symptoms that may overlap with other chronic respiratory conditions. The proper identification of bronchiectasis as a disease in both primary and secondary care is of paramount importance. However, a standardized definition of radiologically and clinically significant bronchiectasis is still missing. Disease heterogeneity is a hallmark of bronchiectasis and applies not only to radiological features and clinical manifestations but also to other aspects of the disease, including the etiological and microbiological diagnosis as well as the evaluation of pulmonary function. Although the guidelines suggest a "minimum bundle" of tests, the diagnostic approach to bronchiectasis is challenging and may be driven by the "treatable traits" approach based on endotypes and biological characteristics. A broad spectrum of diagnostic tests could be used to investigate the etiology of bronchiectasis as well as other pulmonary, extrapulmonary, and environmental traits. Individualizing bronchiectasis workup according to the site of care (e.g., primary, secondary, and tertiary care) could help optimize patients' management and reduce healthcare costs.

Prevalence and serotyping of S. pneumoniae in a large vaccine-naive cohort of adults with cystic fibrosis.

We conducted a prospective, observational study at the Adult CF Center, Ospedale Policlinico, Milano, Italy, from March 2017 to September 2019 to assess the prevalence and serotypes of Streptococcus pneumoniae (SP) in adults with CF naive to pneumococcal vaccination. Spontaneous sputum samples from 129 patients were analyzed for SP DNA and serotyped. SP was found in 24 subjects (19%) and the most common serotypes were 19F (16%), 4 (6%), and 9VA (3%). Higher FEV1 and non-pseudomonas infection significantly associate with SP on sputum. These results define a subgroup of patients that might deserve implementation of microbiological techniques directed to pneumococcal detection.

Integrative microbiomics in bronchiectasis exacerbations.

Bronchiectasis, a progressive chronic airway disease, is characterized by microbial colonization and infection. We present an approach to the multi-biome that integrates bacterial, viral and fungal communities in bronchiectasis through weighted similarity network fusion ( https://integrative-microbiomics.ntu.edu.sg ). Patients at greatest risk of exacerbation have less complex microbial co-occurrence networks, reduced diversity and a higher degree of antagonistic interactions in their airway microbiome. Furthermore, longitudinal interactome dynamics reveals microbial antagonism during exacerbation, which resolves following treatment in an otherwise stable multi-biome. Assessment of the Pseudomonas interactome shows that interaction networks, rather than abundance alone, are associated with exacerbation risk, and that incorporation of microbial interaction data improves clinical prediction models. Shotgun metagenomic sequencing of an independent cohort validated the multi-biome interactions detected in targeted analysis and confirmed the association with exacerbation. Integrative microbiomics captures microbial interactions to determine exacerbation risk, which cannot be appreciated by the study of a single microbial group. Antibiotic strategies probably target the interaction networks rather than individual microbes, providing a fresh approach to the understanding of respiratory infection.

Neutrophil extracellular traps, disease severity, and antibiotic response in bronchiectasis: an international, observational, multicohort study.

BACKGROUND: Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because little is understood of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response in bronchiectasis. METHODS: In this observational study, we did a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg of azithromycin 3 times per week for a year. FINDINGS: Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins most strongly associated with disease severity. This finding was validated in two observational cohorts, in which sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. INTERPRETATION: We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These data support the concept of targeting neutrophilic inflammation with existing and novel therapies. FUNDING: Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC).

Nasopharyngeal Microbiota Analysis in Healthy and Otitis-prone Children: Focus on History of Spontaneous Tympanic Membrane Perforation.

BACKGROUND: Recurrent acute otitis media (RAOM) is common in children, and it may result in spontaneous tympanic membrane perforation (STMP), management of which is often challenging. In the upper respiratory tract (URT), resident microorganisms play a pivotal role in otitis media pathogenesis and prevention, as they are able to inhibit the colonization process and otopathogens growth. In particular, Dolosigranulum spp. and Corynebacterium spp. have been associated with respiratory health in several studies. This study aims at comparing both nasopharyngeal microbiota of children with RAOM versus matched controls and nasopharyngeal microbiota of children with a history of RAOM with STMP. METHOD: Nasopharyngeal swabs were collected from 132 children, median age 3.51 (2.13-4.72), including 36 healthy children, 50 with RAOM without STMP, and 46 with RAOM with STMP. Bacterial DNA was subsequently extracted and 16S rRNA gene V3-V4 regions were polymerase chain reaction amplified and sequenced using Illumina MiSeq technology. RESULTS: A higher relative abundance of Dolosigranulum and Corynebacterium genera was detected in the nasopharynx of healthy children (16.5% and 9.3%, respectively) in comparison with RAOM without STMP (8.9% and 4.3%, respectively) and RAOM with STMP (5.2% and 2.8%, respectively). A decreasing pattern in relative abundance of these 2 pivotal genera through disease severity was detected. In all groups, the most abundant genera were Moraxella, Streptococcus and Haemophilus, followed by Dolosigranulum and Corynebacterium. CONCLUSIONS: Our study provides a characterization of the URT microbiota in otitis-prone children with and without history of recurrent STMP, suggesting that the role of Dolosigranulum and Corynebacterium in regulating the healthy URT microbiota should be further studied.

The Open Challenge of in vitro Modeling Complex and Multi-Microbial Communities in Three-Dimensional Niches.

The comprehension of the underlying mechanisms of the interactions within microbial communities represents a major challenge to be faced to control their outcome. Joint efforts of in vitro, in vivo and ecological models are crucial to controlling human health, including chronic infections. In a broader perspective, considering that polymicrobial communities are ubiquitous in nature, the understanding of these mechanisms is the groundwork to control and modulate bacterial response to any environmental condition. The reduction of the complex nature of communities of microorganisms to a single bacterial strain could not suffice to recapitulate the in vivo situation observed in mammals. Furthermore, some bacteria can adapt to various physiological or arduous environments embedding themselves in three-dimensional matrices, secluding from the external environment. Considering the increasing awareness that dynamic complex and dynamic population of microorganisms (microbiota), inhabiting different apparatuses, regulate different health states and protect against pathogen infections in a fragile and dynamic equilibrium, we underline the need to produce models to mimic the three-dimensional niches in which bacteria, and microorganisms in general, self-organize within a microbial consortium, strive and compete. This review mainly focuses, as a case study, to lung pathology-related dysbiosis and life-threatening diseases such as cystic fibrosis and bronchiectasis, where the co-presence of different bacteria and the altered 3D-environment, can be considered as worst-cases for chronic polymicrobial infections. We illustrate the state-of-art strategies used to study biofilms and bacterial niches in chronic infections, and multispecies ecological competition. Although far from the rendering of the 3D-environments and the polymicrobial nature of the infections, they represent the starting point to face their complexity. The increase of knowledge respect to the above aspects could positively affect the actual healthcare scenario. Indeed, infections are becoming a serious threat, due to the increasing bacterial resistance and the slow release of novel antibiotics on the market.

Sputum neutrophil elastase associates with microbiota and Pseudomonas aeruginosa in bronchiectasis.

INTRODUCTION: Neutrophilic inflammation is a major driver of bronchiectasis pathophysiology, and neutrophil elastase activity is the most promising biomarker evaluated in sputum to date. How active neutrophil elastase correlates with the lung microbiome in bronchiectasis is still unexplored. We aimed to understand whether active neutrophil elastase is associated with low microbial diversity and distinct microbiome characteristics. METHODS: An observational, cross-sectional study was conducted at the bronchiectasis programme of the Policlinico Hospital in Milan, Italy, where adults with bronchiectasis were enrolled between March 2017 and March 2019. Active neutrophil elastase was measured on sputum collected during stable state, microbiota analysed through 16S rRNA gene sequencing, molecular assessment of respiratory pathogens carried out through real-time PCR and clinical data collected. RESULTS: Among 185 patients enrolled, decreasing α-diversity, evaluated through the Shannon entropy (ρ -0.37, p<0.00001) and Pielou's evenness (ρ -0.36, p<0.00001) and richness (ρ -0.33, p<0.00001), was significantly correlated with increasing elastase. A significant difference in median levels of Shannon entropy as detected between patients with neutrophil elastase ≥20 µg·mL-1 (median 3.82, interquartile range 2.20-4.96) versus neutrophil elastase <20 µg·mL-1 (4.88, 3.68-5.80; p<0.0001). A distinct microbiome was found in these two groups, mainly characterised by enrichment with Pseudomonas in the high-elastase group and with Streptococcus in the low-elastase group. Further confirmation of the association of Pseudomonas aeruginosa with elevated active neutrophil elastase was found based on standard culture and targeted real-time PCR. CONCLUSIONS: High levels of active neutrophil elastase are associated to low microbiome diversity and specifically to P. aeruginosa infection.

Evaluation of active neutrophil elastase in sputum of bronchiectasis and cystic fibrosis patients: A comparison among different techniques.

Neutrophil elastase (NE) is a crucial marker of neutrophilic inflammation. We aimed to compare different techniques to detect active NE in sputum samples of 50 Bronchiectasis (BE) and 50 Cystic Fibrosis (CF) patients. Three methods including a ProteaseTag® Active NE Immunoassay (ELISA) and two enzymatic digestion assays (chromogenic -CS- and fluorogenic -FS- substrate) were compared. Results of active NE were also correlated with clinical data. The three methods provided statistically different values for NE activity in the same sputum samples in both cohorts. In the BE cohort, the highest correlations between NE activity and Bronchiectasis Severity Index (rho = 0.40, P < 0.0001), sputum purulence (AUC = 0.79), and chronic infections due to any pathogen (AUC = 0.76) and P. aeruginosa (AUC = 0.80) were found when NE was measured through the activity-based immunoassay. In the CF cohort, the highest correlations between NE activity and sputum quantity (rho = 0.71) and FEV1% (rho = 0.42, P = 0.03) were observed when the FS method was used, while similar correlations with chronic P. aeruginosa infection were identified with the FS and ELISA methods. NE activity in sputum correlates with clinical variables in both diseases. However, different methods to evaluate active NE in sputum lead to significantly different results, also in terms of correlation with clinical data.

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis.

RATIONALE: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease. OBJECTIVES: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis. METHODS: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk. MEASUREMENTS AND MAIN RESULTS: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months. CONCLUSIONS: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis.

A point-of-care neutrophil elastase activity assay identifies bronchiectasis severity, airway infection and risk of exacerbation.

INTRODUCTION: Neutrophil elastase activity in sputum can identify patients at high risk of airway infection and exacerbations in bronchiectasis. Application of this biomarker in clinical practice is limited, because no point-of-care test is available. We tested whether a novel semi-quantitative lateral flow device (neutrophil elastase airway test stick - NEATstik®) can stratify bronchiectasis patients according to severity, airway infection and exacerbation risk. METHODS: Sputum samples from 124 patients with stable bronchiectasis enrolled in the UK and Spain were tested using the NEATstik®, which scores neutrophil elastase concentration from 0 (<8 µg·mL-1 elastase activity) to 10 (maximum detectable neutrophil elastase activity). High neutrophil elastase activity was regarded as a NEATstik® grade >6. Severity of disease, airway infection from sputum culture and exacerbations over the 12 months were recorded. An independent validation was conducted in 50 patients from Milan, Italy. MEASUREMENTS AND MAIN RESULTS: Patients had a median age of 69 years and forced expiratory volume in 1 s (FEV1) 69%. High neutrophil elastase activity was associated with worse bronchiectasis severity using the bronchiectasis severity index (p=0.0007) and FEV1 (p=0.02). A high NEATstik® grade was associated with a significant increase in exacerbation frequency, incident rate ratio 2.75 (95% CI 1.63-4.64, p<0.001). The median time to next exacerbation for patients with a NEATstik® grade >6 was 103 days compared to 278 days. The hazard ratio was 2.59 (95% CI 1.71-3.94, p<0.001). Results were confirmed in the independent validation cohort. CONCLUSIONS: A novel lateral flow device provides assessment of neutrophil elastase activity from sputum in minutes and identifies patients at increasing risk of airway infection and future exacerbations.

Comparison of different conditions for DNA extraction in sputum - a pilot study.

BACKGROUND: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. METHODS: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. RESULTS: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. CONCLUSIONS: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.

How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis.

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

When and how ruling out cystic fibrosis in adult patients with bronchiectasis.

BACKGROUND: Bronchiectasis is the final result of different processes and most of the guidelines advocate for a careful evaluation of those etiologies which might be treated or might change patients' management, including cystic fibrosis (CF). MAIN BODY: CFTR mutations have been reported with higher frequency in bronchiectasis population. Although ruling out CF is considered as a main step for etiological screening in bronchiectasis, CF testing lacks of a standardized approach both from a research and clinical point of view. In this review a list of most widely used tests in CF is provided. CONCLUSIONS: Exclusion of CF is imperative for patients with bronchiectasis and CFTR testing should be implemented in usual screening for investigating bronchiectasis etiology. Physicians taking care of bronchiectasis patients should be aware of CFTR testing and its limitations in the adult population. Further studies on CFTR expression in human lung and translational research might elucidate the possible role of CFTR in the pathogenesis of bronchiectasis.

The Italian registry of pulmonary non-tuberculous mycobacteria - IRENE: the study protocol.

BACKGROUND: A substantial increase in pulmonary and extra-pulmonary diseases due to non-tuberculous mycobacteria (NTM) has been documented worldwide, especially among subjects suffering from chronic respiratory diseases and immunocompromised patients. Many questions remain regarding the epidemiology of pulmonary disease due to NTM (NTM-PD) mainly because reporting of NTM-PD to health authorities is not mandated in several countries, including Italy. This manuscript describes the protocol of the first Italian registry of adult patients with respiratory infections caused by NTM (IRENE). METHODS: IRENE is an observational, multicenter, prospective, cohort study enrolling consecutive adult patients with either a NTM respiratory isolate or those with NTM-PD. A total of 41 centers, including mainly pulmonary and infectious disease departments, joined the registry so far. Adult patients with all of the following are included in the registry: 1) at least one positive culture for any NTM species from any respiratory sample; 2) at least one positive culture for NTM isolated in the year prior the enrolment and/or prescribed NTM treatment in the year prior the enrolment; 3) given consent to inclusion in the study. No exclusion criteria are applied to the study. Patients are managed according to standard operating procedures implemented in each IRENE clinical center. An online case report form has been developed to collect patients' demographics, comorbidities, microbiological, laboratory, functional, radiological, clinical, treatment and outcome data at baseline and on an annual basis. An IRENE biobank has also been developed within the network and linked to the clinical data of the registry. CONCLUSIONS: IRENE has been developed to inform the clinical and scientific community on the current management of adult patients with NTM respiratory infections in Italy and acts as a national network to increase the disease's awareness. TRIAL REGISTRATION: Clinicaltrial.gov: NCT03339063.