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BackgroundThe 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.MethodsAn 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.ResultsA 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.ConclusionA 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.

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.

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.

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.

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.

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.

RationaleRecently 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.ObjectivesTo determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis.MethodsA 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 resultsAMP 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.ConclusionsPatients 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.

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. Ministry of Health (MOH) Nanyang Technological University This research is supported by the Singapore Ministry of Health's National Medical Research Council under its Transition Award (NMRC/TA/0048/2016 to S.H.C.); the Clinician-Scientist Individual Research Grant (MOH-000141 to S.H.C.); NTU Integrated Medical, Biological and Environmental Life Sciences (NIMBELS) (NIM/03/2018 to S.H.C.); the British Lung Foundation through the GSK/British Lung Foundation Chair of Respiratory Research (to J.D.C.) and the Scottish Government Chief Scientist Office (SCAF/17/03). K.T.-A. gratefully acknowledges the financial support of the EPSRC via grant EP/N014391/1.

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. Published version

Summary 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).

Bronchiectasis is a chronic respiratory disease with neutrophilic airway inflammation playing a prominent role in its pathophysiology [1]. The inflammatory process depends on the release of neutrophil elastase (NE) and subsequent formation of neutrophil extracellular traps to facilitate the neutralisation of pathogens. An excessive release of NE can lead to several damaging lung effects, including mucus gland stimulation, increase in sputum production, impairment in ciliary beat frequency, and extracellular matrix and airway epithelia destruction. The activity of NE (aNE) has been previously evaluated in sputum samples of a Scottish cohort of bronchiectasis patients [2]. The authors demonstrated that increased levels of aNE in sputum are associated with disease severity and poor clinical outcomes. This experience identified NE as one of the most promising biomarkers in bronchiectasis and, subsquently, a point-of-care assay for aNE was validated [3]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Gramegna has nothing to disclose. Conflict of interest: Prof. Aliberti reports grants and personal fees from Bayer Healthcare, grants and personal fees from Aradigm Corporation, grants and personal fees from Grifols, personal fees from Astra Zeneca, personal fees from Basilea, personal fees from Zambon, personal fees from Novartis, personal fees from Raptor, grants and personal fees from Chiesi, personal fees from Actavis UK Ltd, personal fees from Horizon, grants and personal fees from INSMED, outside the submitted work. Dr. Aliberti reports grants and personal fees from Bayer Healthcare, grants and personal fees from Aradigm Corporation, grants and personal fees from Grifols, personal fees from Astra Zeneca, personal fees from Basilea, personal fees from Zambon, personal fees from Novartis, personal fees from Raptor, grants and personal fees from Chiesi, personal fees from Actavis UK Ltd, personal fees from Horizon, grants and personal fees from INSMED, outside the submitted work. Dr. Aliberti reports grants and personal fees from Bayer Healthcare, grants and personal fees from Aradigm Corporation, grants and personal fees from Grifols, personal fees from Astra Zeneca, personal fees from Basilea, personal fees from Zambon, personal fees from Novartis, personal fees from Raptor, grants and personal fees from Chiesi, personal fees from Actavis UK Ltd, personal fees from Horizon, grants and personal fees from INSMED, outside the submitted work. Conflict of interest: Dr. Sibila has nothing to disclose. Conflict of interest: Dr. Di Francesco has nothing to disclose. Conflict of interest: Dr. Sotgiu has nothing to disclose. Conflict of interest: Dr. Perea has nothing to disclose. Conflict of interest: Dr. Terranova has nothing to disclose. Conflict of interest: Dr. Oriano has nothing to disclose. Conflict of interest: Dr. Pilocane has nothing to disclose. Conflict of interest: Dr. Saderi has nothing to disclose. Conflict of interest: Dr. Chalmers reports grants and personal fees from Astrazeneca, grants and personal fees from Boehringer Ingelheim, grants and personal fees from Glaxosmithkline, grants and personal fees from Insmed, personal fees from Zambon, grants from Gilead Sciences, outside the submitted work. Conflict of interest: Dr. Marchisio has nothing to disclose. Conflict of interest: Dr. Blasi reports grants and personal fees from astrazeneca, grants from bayer, grants and personal fees from chiesi, grants and personal fees from gsk, personal fees from guidotti, personal fees from grifols, grants and personal fees from insmed, personal fees from menarini, personal fees from mundipharma, personal fees from novartis, grants and personal fees from pfizer, personal fees from zambon, outside the submitted work.

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.

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.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.Among 185 patients enrolled, decreasing α-diversity, evaluated through the Shannon entropy (ρ -0.37, p0.00001) and Pielou's evenness (ρ -0.36, p0.00001) and richness (ρ -0.33, p0.00001), was significantly correlated with increasing elastase. A significant difference in median levels of Shannon entropy as detected between patients with neutrophil elastase ≥20 µg·mLHigh levels of active neutrophil elastase are associated to low microbiome diversity and specifically to

Background: Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because of a lack of understanding of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a mode of host defence that has been implicated in multiple inflammatory diseases. Methods: We conducted a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. Nano LC/MS-MS was used to identify proteomic markers associated with disease severity, defined using the bronchiectasis severity index (BSI) (n=40). These were validated in two cohorts (n=175 and n=275) using a validated immunoassay to measure NETs. 20 patients with acute exacerbations of bronchiectasis were treated with intravenous antibiotics for 14 days and proteomics used to identify proteins associated with treatment response. Two studies of long-term macrolide treatment, one in bronchiectasis and a post-hoc analysis of the AMAZES trial in asthma, investigated the effect of macrolide treatment on NETs. Findings: 96 proteins were differentially expressed in sputum between severe and mild bronchiectasis. The most abundant and most discriminating were proteins known to be NET proteins. This was validated in two observational cohorts where sputum NETs were associated with BSI, quality of life, lung function, future risk of hospital admissions and mortality. Antibiotic treatment in 20 patients was associated with 23 significantly down-regulated and 16 up-regulated proteins, with the “neutrophil degranulation” pathway being most strongly implicated in response. Patients with Pseudomonas aeruginosa infection had a blunted proteomic and clinical response to treatment. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Interpretation: NETs are identified as a key marker of disease severity and treatment response in bronchiectasis. Funding: Supported by the Scottish Government Chief Scientist Office (Senior Clinical Fellowship to JDC) and British Lung Foundation through the BLF Chair of Respiratory Research. Supported by the European Bronchiectasis Network (EMBARC), a European Respiratory Society Clinical Research Collaboration. Declaration of Interests: None to declare Ethics Approval Statement: The studies were approved by local research ethics committees and patients provided informed consent.

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.

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.

Prevalence and role of S. pneumoniae (SP) as a chronic pathogen in cystic fibrosis (CF) deserves new evidence in the adult population. In order to investigate prevalence and serotypes of SP in sputum of vaccine-naive subjects, as well as their clinical impact, a prospective, observational study enrolling adults (≥18 years) with CF during stable state was conducted at the Adult CF Centre, Policlinico Hospital, Milan, Italy, between 2017 and 2018. SP DNA was extracted from sputum samples and screened in duplicate by Real Time PCR using specific TaqMan assay. Positive samples were serotyped by Real Time PCR using serotype specific TaqMan assay (13 serotypes). Among the 128 patients (median age: 40 yrs; females 41%) enrolled, SP was found in 24 (19%) and the most represented serotypes were 19F (16%), 4 (6%), 9VA (3%) and 5 (2%). Co-infections by different serotypes were reported in 9% of the patients. SP was less prevalent in patients on inhaled antibiotics (12% vs. 31%, p=0.008), ICS/LABA use (16% vs. 46%, p=0.008) and long-term macrolide therapy (3% vs. 26%, p=0.001) than those who did not receive these treatments. SP was less prevalent in patients with chronic infection due to any bacteria (15% vs. 75%, p=0.001) and to P. aeruginosa (8% vs. 32%, p=0.001) than in patients with no chronic infection. No differences were identified in terms of comorbidities and history of exacerbations between those with vs. without SP. No association between the presence of SP and exacerbations during follow up was identified (RR:0.53, p=0.142). Prospective studies are needed to investigate the persistence of SP in this population as well as the association with relevant clinical outcomes.

Neutrophil elastase (NE) is a crucial marker of neutrophilic inflammation in bronchiectasis (BE) and cystic fibrosis (CF). We aimed at comparing different techniques to detect active NE in sputum samples of 50 BE and 50 CF adults recruited during stable state at the Bronchiectasis and CF Programs of the Policlinico Hospital in Milan, Italy. 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 different clinical data. The three methods provided statistically different values for NE activity in the same sputum samples in both cohorts. The three methods were comparable in terms of consistency and agreement. In the BE cohort, the highest correlation between NE activity and Bronchiectasis Severity Index (rho=0.40, P

While bacterial infection has been widely studied in cystic fibrosis (CF), evidence of a pathogenic role of viruses is scarce. The aim of this study was to investigate the role of respiratory viruses (RV) in adults with CF during stable state (SS) and exacerbation (EX). A prospective, observational study on adults with CF was conducted at the Adult CF Centre, Policlinico Hospital, Milan, Italy, between 2017 and 2018. DNA and RNA were extracted from sputa collected during SS and at first EX to identify 18 RV (Luminex Nx TAG®). Among 129 patients on SS (median age: 41 yrs; female 42%), RV were detected in 27% including: Rhinovirus/Enterovirus (HRV/EV, 16%), Metapneumovirus (hMPV, 8%), Coronaviruses (CoV, 2%), Adenovirus (1.5%), RSV (1.5%) and Parainfluenza viruses (1.5%). Co-infection of different RV was detected in 5 patients. Prevalence of RV during SS was higher in Autumn/Winter than in Spring/Summer (33% vs. 23%, p=0.218). During the 1-year follow up, 38 patients had exacerbations with RV detection in 42%, including HRV/EV (13%), hMPV (11%), CoV (8%) and Influenza B virus (5.3%). Only 1 patient showed the same viral species in SS and EX, while 9 patients (23.7%) previously negative in SS acquired RV infection in EX. A higher prevalence of RV detection in EX was present among patients with a history of chronic sinusitis (p=0.021) and at least one EX in the previous year (p=0.003). No association between the presence of RV during SS and EX during follow-up was reported (RR 1.24, p=0.46). Prospective studies should investigate persistence of RV in CF sputa as well as the association with clinical outcomes.

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. Electronic supplementary material The online version of this article (10.1186/s40248-018-0166-z) contains supplementary material, which is available to authorized users.

IntroductionNeutrophilic 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.MethodsAn 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.ResultsAmong 185 patients enrolled, decreasing α-diversity, evaluated through the Shannon entropy (ρ −0.37, p−1 (median 3.82, interquartile range 2.20–4.96) versus neutrophil elastase −1 (4.88, 3.68–5.80; pPseudomonas 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.ConclusionsHigh levels of active neutrophil elastase are associated to low microbiome diversity and specifically to P. aeruginosa infection.

Introduction: There is no consensus on which is the best method to extract bacterial DNA from sputum. We aimed at comparing 20 different DNA extraction techniques on both mock community and sputum samples. Methods: One mock was built and 5 sputa from cystic fibrosis and bronchiectasis patients were evaluated at the Policlinico Hospital in Milan, Italy. 20 techniques for sputum bacterial DNA extraction were compared, including two enzyme-based (Roche and Zymo) and two beads-based (Mobio and QIAGEN) techniques which were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) steps. Extractions were performed in duplicate to evaluate the variability among samples. DNA was quantified and, then, tested using Real time PCR for 16S rDNA and S. aureus. Results: Although 16S rDNA was similarly detected across all the different techniques in 20 ng of extract (range 19-21 Ct), Roche kit gave the highest DNA yield in both mock and sputa (see figure). The mean standard deviation between replicates was lower in DTT-treated vs non-treated samples (0.17 vs 0.47 Ct, respectively). In both enzymatic-based techniques, the lowest Ct values for Real time PCR for S. aureus in both mock and sputa was identified when lysostaphin was added. Conclusion: An enzyme-based kit with the addition of both DTT treatment and lysostaphin steps seems to be the preferred technique for DNA bacterial extraction on sputum.

Rationale: In translational medicine the research on systemic and local biomarkers for disease activity in cystic fibrosis (CF) is still an unmet need. Sputum glucose is one of the most promising and it has already been evaluated in different chronic lung diseases. This study aimed to investigate if sputum glucose was associated with clinical markers of lung disease severity in CF adults. Methods: This was an observational, prospective study of consecutive adults with CF enrolled at the Adult CF Center in Milan, Italy, between April and June 2017. Patients with a diagnosis of CF according to last guidelines underwent glucose evaluation on sputum during stable state and demographics, clinical and microbiological data were collected. Sputum glucose assay uses the glucose oxidase-peroxide reaction for the determination of glucose concentrations. CF-related diabetes (CFRD) was evaluated as a confounder through stratification. Results: A total of 28 patients (median age 33 years; females 64%) were enrolled; median BMI was 22, median FEV1 %predict was 76% and 29% had CFRD. Glucose was identified in sputum of all patients with a median level of 13.36 uM (6.72-24.09). Sputum glucose was significantly higher in patients with 3+ exacerbation (PEx)/year in comparison to those with no PEx (P Conclusion: Sputum glucose seems to act as a biomarker of disease severity which may complement patient clinical assessment and be a target for treatment.

Traditionally regarded as single cell organisms, bacteria naturally and preferentially build multicellular communities that enable them to react efficiently to external stimuli in a coordinated fashion and with extremely effective outcomes. These communities are bacterial biofilms, where single cells or microcolonies are embedded in self-built Extracellular Polymeric Substance (EPS), composed of different macromolecules, e.g., polysaccharides, proteins, lipids, and extracellular DNA (eDNA). Despite being the most common form in nature and having many biotechnologically useful applications, biofilm is often regarded as a life-threatening form of bacterial infection. Since this form of bacterial life is intrinsically more resistant to antibiotic treatment and antimicrobial resistance is reaching alarming levels, we will focus our attention on how nanotechnology made new tools available to the medical community for the prevention and treatment of these infections. After a brief excursus on biofilm formation and its main characteristics, different types of nanomaterials developed to prevent or counteract these multicellular forms of bacterial infection will be described. A comparison of different classifications adopted for nanodrugs and a final discussion of challenges and future perspectives are also presented.

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. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.