Pro-resolving and pro-inflammatory fatty acid-derived mediators in sputum of stable state bronchiectasis patients.

BACKGROUND: Bronchiectasis is characterized by neutrophilic inflammation and frequent exacerbations often associated with infections. Lipid mediators play critical roles in the inflammatory response, and the balance between anti-inflammatory and pro-inflammatory mediators could drive to chronic inflammation. The aim of this study was to evaluate the metabolites of docosahexaenoic acid and arachidonic acid in sputum of adults with bronchiectasis defining their associations with clinical data, bacterial load and neutrophil elastase. METHODS: An observational, cross-sectional study was conducted at the bronchiectasis program of the Policlinico Hospital in Milan, Italy, where patients were enrolled. Active neutrophil elastase was measured by enzyme-linked immunosorbent assay, pro-resolving and pro-inflammatory fatty acid-derived mediators were evaluated by mass spectrometry and respiratory pathogens were assessed by real-time PCR. Analysis were performed on sputum collected during stable state and clinical data were also collected. RESULTS: Levels of pro-inflammatory mediators derived from arachidonic acid metabolism showed association with neutrophil elastase, were proportional to Pseudomonas aeruginosa identifications and were linked with radiological gravity index, while the concentrations of pro-resolution mediators derived from docosahexaenoic acid were associated with a better health status, highlighted by the inverse correlation with radiological gravity index, bacterial infections and sputum volume production. CONCLUSION: Pro-inflammatory mediators derived from FA metabolisms are associated with severity of bronchiectasis while DHA-derived metabolites are inversely associated with severity of the disease, which may be used for personized treatment of bronchiectasis.

Prognostic parameters of in-hospital mortality in COVID-19 patients-An Italian experience.

Background During COVID-19 outbreak, Italy was the first country in Europe to be heavily affected with an intensive care unit mortality of 26%. In order to reduce this percentage, physicians should establish clear and objective criteria to stratify COVID-19 patients at high risk of in-hospital death. Thus, the aim has been to test a large spectrum of variables ranging from clinical evaluation to laboratory biomarkers to identify which parameter would best predict all-cause in-hospital mortality in COVID-19 patients. Design observational study. Results Multivariate Cox regression analysis showed that each 5 years of increase in age corresponded to a hazard ratio (HR) of 1.28 (95% CI 1.00-1.65, P = .050); each increment of 803 ng/L of N-terminal pro-B-type natriuretic peptide (NT-proBNP) corresponded to a HR of 1.24 (95% CI 1.11-1.39, P < .001); each increment of 58 ng/L of interleukin (IL)-6 corresponded to a HR of 1.23 (95% CI 1.09-1.40, P < .001), and each increment of 250 U/L of lactate dehydrogenase (LDH) corresponded to a HR of 1.23 (95% CI 1.10-1.37, P < .001). According to the calculated cut-points for age (≥70 years), NT-proBNP (≥803 ng/L), IL-6 (≥58 ng/L) and LDH (≥371 U/L) when 2 out of these 4 were overcome, the HR was 2.96 (95% CI 1.97-4.45, P < .001). Conclusion In COVID-19 patients, besides age, the evaluation of three biochemical parameters, available in few hours after hospital admission can predict in-hospital mortality regardless of other comorbidities.

Trends and risk factors of SARS-CoV-2 infection in asymptomatic blood donors.

BACKGROUND: A large proportion of SARS-CoV-2-infected individuals does not develop severe symptoms. Serological tests help in evaluating the spread of infection and disease immunization. The aim of this study was to prospectively examine the trends and risk factors of SARS-CoV-2 infection in blood donors. STUDY DESIGN AND METHODS: We screened 8798 asymptomatic donors presenting in Milan from July 2020 to February 2021 (10,680 presentations) before the vaccination campaign for anti-nucleoprotein (NP) antibodies, and for anti-spike receptor-binding domain (RBD) antibodies and nasopharyngeal swab PCR in those who tested positive. RESULTS: The prevalence of anti-NP+/RBD+ tests increased progressively with time up to ~15% (p < .0001), preceded by a peak of PCR+ tests. Anti-RBD titers were higher in anti-NP IgG+/IgM+ than in IgG+/IgM- individuals and in those with a history of infection (p < .0001); of these 197/630 (31.2%) displayed high titers (>80 AU/ml). Anti-RBD titers declined during follow-up, depending on baseline titers (p < .0001) and time (p = .025). Risk factors for seroconversion were a later presentation date and non-O ABO blood group (p < .001). A positive PCR was detected in 0.7% of participants in the absence of SARS-CoV-2 viremia. CONCLUSIONS: During the second wave of SARS-CoV-2 infection in Northern Italy, we detected an increase in seroprevalence in healthy blood donors from ~4% to ~15%, with a trend paralleling that observed in the general population. Seroconversion was more frequent in carriers of non-O blood groups. The persistence of anti-RBD antibodies was short-lived.

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.

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.

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.

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.

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.

Neutrophil elastase in bronchiectasis.

The role of neutrophil elastase (NE) is poorly understood in bronchiectasis because of the lack of preclinical data and so most of the assumptions made about NE inhibitor potential benefit is based on data from CF. In this context, NE seems to be a predictor of long-term clinical outcomes and a possible target of treatment. In order to better evaluate the role of NE in bronchiectasis, a systematic search of scientific evidence was performed.Two investigators independently performed the search on PubMed and included studies published up to May 15, 2017 according to predefined criteria. A final pool of 31 studies was included in the systematic review, with a total of 2679 patients. For each paper data of interest were extracted and reported in table.In this review sputum NE has proved useful as an inflammatory marker both in stable state bronchiectasis and during exacerbations and local or systemic antibiotic treatment. NE has also been associated with risk of exacerbation, time to next exacerbation and all-cause mortality. This study reviews also the role of NE as a specific target of treatment in bronchiectasis. Inhibition of NE is at a very early stage and future interventional studies should evaluate safety and efficacy for new molecules and formulations.

Acute flaccid myelitis associated with enterovirus-D68 infection in an otherwise healthy child.

BACKGROUND: Reporting new cases of enterovirus (EV)-D68-associated acute flaccid myelitis (AFM) is essential to understand how the virus causes neurological damage and to characterize EV-D68 strains associated with AFM. CASE PRESENTATION: A previously healthy 4-year-old boy presented with sudden weakness and limited mobility in his left arm. Two days earlier, he had an upper respiratory illness with mild fever. At admission, his physical examination showed that the child was febrile (38.5 °C) and alert but had a stiff neck and weakness in his left arm, which was hypotonic and areflexic. Cerebrospinal fluid (CSF) examination showed a mild increase in white blood cell count (80/mm3, 41% neutrophils) and a slightly elevated protein concentration (76 gm/dL). Bacterial culture and molecular biology tests for detecting viral infection in CSF were negative. The patient was then treated with intravenous ceftriaxone and acyclovir. Despite therapy, within 24 h, the muscle weakness extended to all four limbs, which exhibited greatly reduced mobility. Due to his worsening clinical prognosis, the child was transferred to our Pediatric Intensive Care Unit; at admission he was diagnosed with acute flaccid paralysis of all four limbs. Brain magnetic resonance imaging (MRI) was negative, except for a focal signal alteration in the dorsal portion of the medulla oblongata, also involving the pontine tegmentum, whereas spine MRI showed an extensive signal alteration of the cervical and dorsal spinal cord reported as myelitis. Signal alteration was mainly localized in the central grey matter, most likely in the anterior horns. Molecular biology tests performed on nasopharyngeal aspirate and on bronchoalveolar lavage fluid were negative for bacteria but positive for EV-D68 clade B3. Plasmapheresis was performed and corticosteroids and intravenous immunoglobulins were administered. After 4 weeks of treatment, the signs and symptoms of AFM were significantly reduced, although some weakness and tingling remained in the patient's four limbs. MRI acquired after 3 weeks showed that the previously reported alterations were no longer present. CONCLUSION: This case suggests that EV-D68 is a neurotropic agent that can cause AFM and strains are circulating in Europe. EV-D68 disease surveillance is required to better understand EV-D68 pathology and to compare various strains that cause AFM.

Streptococcus pneumoniae colonisation in children and adolescents with asthma: impact of the heptavalent pneumococcal conjugate vaccine and evaluation of potential effect of thirteen-valent pneumococcal conjugate vaccine.

BACKGROUND: The main aim of this study was to evaluate Streptococcus pneumoniae carriage in a group of school-aged children and adolescents with asthma because these results might indicate the theoretical risk of invasive pneumococcal disease (IPD) of such patients and the potential protective efficacy of the 13-valent pneumococcal conjugate vaccine (PCV13). METHODS: Oropharyngeal samples were obtained from 423 children with documented asthma (300 males, 70.9%), and tested for the autolysin-A-encoding (lytA) and the wzg (cpsA) gene of S. pneumoniae by means of real-time polymerase chain reaction. RESULTS: S. pneumoniae was identified in the swabs of 192 subjects (45.4%): 48.4% of whom were aged <10 years, 46.9% aged 10-14 years, and 4.7% aged ≥15 years (p < 0.001). Carriage was significantly less frequent among the children who had received recent antibiotic therapy (odds ratio [OR 0.41]; 95% confidence interval [95% CI] 0.22-0.76). Multivariate analyses showed no association between carriage and vaccination status, with ORs of 1.05 (95% CI 0.70-1.58) for carriers of any pneumococcal serotype, 1.08 (95% CI 0.72-1.62) for carriers of any of the serotypes included in 7-valent pneumococcal conjugate vaccine (PCV7), and 0.76 (95% CI 0.45-1.28) for carriers of any of the six additional serotypes of PCV13. Serotypes 19 F, 4 and 9 V were the most frequently identified serotypes in vaccinated subjects. CONCLUSIONS: These results showed that carriage of S. pneumoniae is relatively common in all school-aged children and adolescents with asthma, regardless of the severity of disease and the administration of PCV7 in the first years of life. This highlights the problem of the duration of the protection against colonisation provided by pneumococcal conjugate vaccine, and the importance of re-colonization by the same pneumococcal serotypes included in the previously used vaccine.

Pneumococcal colonization in older adults.

BACKGROUND: Little is known about pneumococcal carrier states in older adults. The main aim of this study was to evaluate pneumococcal colonization patterns among older adults in two centres in Milan, Italy, before the widespread use of the 13-valent pneumococcal vaccine (PCV13) in this age group, to investigate demographic and clinical features that are associated with pneumococcal colonization and to estimate the potential coverage offered by PCV13. RESULTS: Among 417 adults ≥65 years old (171, 41.1 %, ≥75 years), 41 (9.8 %) were pneumococcal carriers. Univariate and multivariate analyses revealed that pneumococcal colonization was significantly less common among individuals with underlying co-morbidities than among those without (odds ratio [OR] 0.453, 95 % confidence interval [CI] 0.235-0.875, p = 0.018; adjusted OR 0.503, 95 % CI 0.255-0.992, p = 0.047). Moreover, among these patients, those with cardiac disease had a significantly lower risk of colonization (OR 0.308, 95 % CI 0.119-0.795, p = 0.015; adjusted OR 0.341, 95 % CI 0.13-0.894, p = 0.029). Only one vaccinated subject who received 23-valent polysaccharide pneumococcal vaccine (PPV23) was colonized. Twenty-five (89.3 %) of the subjects who were <75 years old and 9 (75.0 %) of those who were ≥75 years old were colonized by at least one of the serotypes that is included in PCV13, with serotype 19 F being the most common. Respiratory allergies as well as overall co-morbidities were more common in subjects who were positive for only non-PCV13 serotypes compared with negative subjects and those who were carriers of only PCV13 serotypes. CONCLUSIONS: Although this study included a relatively small number of subjects and has been performed in a limited geographic setting, results showed that pneumococcal colonization in older people is common, and the monitoring of carriers can offer useful information about the circulation of this pathogen among older people and the potential protective effect of pneumococcal vaccines. Because the colonization in most cases involves the strains that are included in PCV13, this vaccine could be useful in the prevention of pneumococcal infections in the overall population of older people. In subjects with respiratory allergies and in those with co-morbidities, the addition of the PPV23 to PCV13 should be recommended. Due to the low vaccination coverage, urgent educational programmes are required to inform older adults and their medical doctors of the risks of pneumococcal infection and the efficacy and safety of the available pneumococcal vaccines.

Measurement of lipocalin-2 and syndecan-4 levels to differentiate bacterial from viral infection in children with community-acquired pneumonia.

BACKGROUND: In this study, we evaluated the lipocalin-2 (LIP2) and syndecan-4 (SYN4) levels in children who were hospitalized for radiologically confirmed CAP in order to differentiate bacterial from viral infection. The results regarding the LIP2 and SYN4 diagnostic outcomes were compared with the white blood cell (WBC) count and C reactive protein (CRP) levels. METHODS: A total of 110 children <14 years old who were hospitalized for radiologically confirmed CAP were enrolled. Serum samples were obtained upon admission and on day 5 to measure the levels of LIP2, SYN4, and CRP as well as the WBC. Polymerase chain reaction of the respiratory secretions and tests on blood samples were performed to detect respiratory viruses, Streptococcus pneumoniae, and Mycoplasma pneumoniae. RESULTS: CAP was considered to be due to a probable bacterial infection in 74 children (67.3 %) and due to a probable viral infection in 16 children (14.5 %). Overall, 84 children (76.4 %) were diagnosed with severe CAP. The mean values of the WBC count and the LIP2 and SYN4 levels did not differ among the probable bacterial, probable viral, and undetermined cases. However, the CRP serum concentrations were significantly higher in children with probable bacterial CAP than in those with probable viral disease (32.2 ± 55.5 mg/L vs 9.4 ± 17.0 mg/L, p < 0.05). The WBC count was the best predictor of severe CAP, but the differences among the studied variables were marginal. The WBC count was significantly lower on day 5 in children with probable bacterial CAP (p < 0.01) and in those with an undetermined etiology (p < 0.01). The CRP and LIP2 levels were significantly lower 5 days after enrollment in all of the studied groups, independent of the supposed etiology of CAP (p < 0.01 for all comparisons). No statistically significant variation was observed for SYN4. CONCLUSIONS: Measuring the LIP2 and SYN4 levels does not appear to solve the problem of the poor reliability of routine laboratory tests in defining the etiology and severity of pediatric CAP. Currently, the CRP levels and WBC, when combined with evaluation of clinical data, can be used to limit the overuse of antibiotics as much as possible and to provide the best treatment to the patient.

Interaction between Streptococcus pneumoniae and Staphylococcus aureus in paediatric patients suffering from an underlying chronic disease.

Little is known about the interaction between Streptococcus pneumoniae and Staphylococcus aureus in school-age children and adolescents suffering from an underlying chronic disease. To increase our knowledge in this regard, an oropharyngeal swab was obtained from school-age children and adolescents suffering from asthma (n = 423), cystic fibrosis (CF) (n = 212) and type 1 diabetes mellitus (DM1) (n = 296). S. pneumoniae detection and serotyping were performed using a real-time polymerase chain reaction, and S. aureus detection was performed using the RIDAGENE MRSA system. Among asthmatic, CF and DM1 patients, both pathogens were identified in 65/423 (15.4%), 21/212 (9.9%) and 62/296 (20.9%) children, respectively; S. pneumoniae alone was identified in 127/434 (30.0%), 21/212 (9.9%) and 86/296 (29.1%), respectively; S. aureus alone was identified in 58/434 (13.7%), 78/212 (36.8%) and 49/296 (16.6%), respectively. S. pneumoniae colonisation rates were higher in younger children and declined with age, whereas the frequency of S. aureus colonisation was quite similar in the different age groups. Among asthmatic and CF patients aged 6-9 years, S. aureus carriage was significantly higher in children who were positive for S. pneumoniae (P <0.05). No significant association emerged between S. aureus carriage and carriage of S. pneumoniae serotypes included in the pneumococcal conjugate vaccines (PCVs). This study shows for the first time that school-age children and adolescents with asthma, CF and DM1 are frequently colonised by S. pneumoniae and S. aureus and that no negative relationship seems to exist between these pathogens. Moreover, the supposed protection offered by PCV administration against S. aureus colonisation was not demonstrated.

Oropharyngeal and nasal Staphylococcus aureus carriage by healthy children.

BACKGROUND: As healthy children are the main reservoir of respiratory pathogens and the main cause of bacterial diffusion in the community, it could be interesting to investigate the type of screening that should be used during the early years of life in order to obtain a more precise estimate of Staphylococcus aureus circulation. The aim of this study was to evaluate oropharyngeal and nasal S. aureus carriage in otherwise healthy children and adolescents aged 6-17 years. METHODS: The oropharyngeal and nasal samples were collected in December 2013 from 497 healthy students attending five randomly selected schools in Milan, Italy, using an ESwab kit, and S. aureus was identified using the RIDA®GENE methicillin-resistant S. aureus (MRSA) system. RESULTS: Two hundred and sixty-four subjects (53.1%) were identified as S. aureus carriers: 129 (25.9%) oropharyngeal carriers and 195 (39.2%) nasal carriers, of whom 60 (12.1%) were both oropharyngeal and nasal carriers. Oropharyngeal carriage increased with age (p < 0.001), whereas nasal carriage decreased. There was little or no agreement between oropharyngeal and nasal carriage in any of the age groups. MRSA was identified in only three cases (0.6%), always in nasal samples. There were no differences between the carriers and non-carriers in terms of the distribution of age, gender, ethnicity, the number of siblings in the household, exposure to passive smoking, previous clinical history, allergic sensitisation, or previous influenza, pneumococcal and meningococcal vaccinations. The frequency of male children was higher among the subjects with positive nasal and oropharyngeal swabs (66.7%) than among those with positive oropharyngeal swabs alone (46.4%; p = 0.02). CONCLUSIONS: The oropharyngeal carriage of mainly methicillin-sensitive S. aureus is frequent in otherwise healthy children, including a relatively high proportion of those without nasal colonisation. These findings highlight the importance of adding throat to nasal screening when monitoring the circulation of S. aureus in the community.

Genetic polymorphisms and risk of recurrent wheezing in pediatric age.

BACKGROUND: Wheezing during early life is a very common disorder, but the reasons underlying the different wheezing phenotypes are still unclear. The aims of this study were to analyse the potential correlations between the risk of developing recurrent wheezing and the presence of specific polymorphisms of some genes regulating immune system function, and to study the relative importance of the associations of different viruses and genetic polymorphisms in causing recurrent episodes. METHODS: The study involved 119 otherwise healthy infants admitted to hospital for a first episode of wheezing (74 of whom subsequently experienced recurrent episodes) and 119 age- and sex-matched subjects without any history of respiratory problem randomly selected from those attending our outpatient clinic during the study period. All of the study subjects were followed up for two years, and 47 single nucleotide polymorphisms (SNPs) in 33 candidate genes were genotyped on whole blood using an ABI PRISM 7900 HT Fast Real-time instrument. RESULTS: IL8-rs4073AT, VEGFA-rs833058CT, MBL2-rs1800450CT and IKBKB-rs3747811AT were associated with a significantly increased risk of developing wheezing (p = 0.02, p = 0.03, p = 0.05 and p = 0.0018), whereas CTLA4-rs3087243AG and NFKBIB-rs3136641TT were associated with a significantly reduced risk (p = 0.05 and p = 0.04). IL8-rs4073AT, VEGFA-rs2146323AA and NFKBIA-rs2233419AG were associated with a significantly increased risk of developing recurrent wheezing (p = 0.04, p = 0.04 and p = 0.03), whereas TLR3-rs3775291TC was associated with a significantly reduced risk (p = 0.03). Interestingly, the study of gene-environment interactions showed that rhinovirus was significantly associated with recurrent wheezing in the presence of IL4Ra-rs1801275GG and G (odds ratio [OR] 6.03, 95% confidence interval [CI]: 1.21-30.10, p = 0.03) and MAP3K1-rs702689AA (OR 4.09, 95% CI: 1.14-14.61, p = 0.03). CONCLUSIONS: This study shows a clear relationship between the risk of wheezing and polymorphisms of some genes involved in the immune response. Although further studies are needed to confirm the results, these findings may be useful for the early identification of children at the highest risk of developing recurrent episodes and possibly subsequent asthma.

Genomic Characterization of Carbapenem-Resistant Acinetobacter baumannii (CRAB) in Mechanically Ventilated COVID-19 Patients and Impact of Infection Control Measures on Reducing CRAB Circulation during the Second Wave of the SARS-CoV-2 Pandemic in Milan, Italy.

COVID-19 has significantly affected hospital infection prevention and control (IPC) practices, especially in intensive care units (ICUs). This frequently caused dissemination of multidrug-resistant organisms (MDROs), including carbapenem-resistant Acinetobacter baumannii (CRAB). Here, we report the management of a CRAB outbreak in a large ICU COVID-19 hub Hospital in Italy, together with retrospective genotypic analysis by whole-genome sequencing (WGS). Bacterial strains obtained from severe COVID-19 mechanically ventilated patients diagnosed with CRAB infection or colonization between October 2020 and May 2021 were analyzed by WGS to assess antimicrobial resistance and virulence genes, along with mobile genetic elements. Phylogenetic analysis in combination with epidemiological data was used to identify putative transmission chains. CRAB infections and colonization were diagnosed in 14/40 (35%) and 26/40 (65%) cases, respectively, with isolation within 48 h from admission in 7 cases (17.5%). All CRAB strains belonged to Pasteur sequence type 2 (ST2) and 5 different Oxford STs and presented blaOXA-23 gene-carrying Tn2006 transposons. Phylogenetic analysis revealed the existence of four transmission chains inside and among ICUs, circulating mainly between November and January 2021. A tailored IPC strategy was composed of a 5-point bundle, including ICU modules' temporary conversion to CRAB-ICUs and dynamic reopening, with limited impact on ICU admission rate. After its implementation, no CRAB transmission chains were detected. Our study underlies the potentiality of integrating classical epidemiological studies with genomic investigation to identify transmission routes during outbreaks, which could represent a valuable tool to ensure IPC strategies and prevent the spread of MDROs. IMPORTANCE Infection prevention and control (IPC) practices are of paramount importance for preventing the spread of multidrug-resistant organisms (MDROs) in hospitals, especially in the intensive care unit (ICU). Whole-genome sequencing (WGS) is seen as a promising tool for IPC, but its employment is currently still limited. COVID-19 pandemics have posed dramatic challenges in IPC practices, causing worldwide several outbreaks of MDROs, including carbapenem-resistant Acinetobacter baumannii (CRAB). We present the management of a CRAB outbreak in a large ICU COVID-19 hub hospital in Italy using a tailored IPC strategy that allowed us to contain CRAB transmission while preventing ICU closure during a critical pandemic period. The analysis of clinical and epidemiological data coupled with retrospective genotypic analysis by WGS identified different putative transmission chains and confirmed the effectiveness of the IPC strategy implemented. This could be a promising approach for future IPC strategies.

Increase in invasive group A streptococcal infections in Milan, Italy: a genomic and clinical characterization.

Background: Group A Streptococcus (GAS) causes multiple clinical manifestations, including invasive (iGAS) or even life-threatening (severe-iGAS) infections. After the drop in cases during COVID-19 pandemic, in 2022 a sharp increase of GAS was reported globally. Methods: GAS strains collected in 09/2022-03/2023 in two university hospitals in Milan, Italy were retrospectively analyzed. Clinical/epidemiological data were combined with whole-genome sequencing to: (i) define resistome/virulome, (ii) identify putative transmission chains, (iii) explore associations between emm-types and clinical severity. Results: Twenty-eight isolates were available, 19/28 (67.9%) from adults and 9/28 (32.1%) from pediatric population. The criteria for iGAS were met by 19/28 cases (67.9%), of which 11/19 (39.3%) met the further criteria for severe-iGAS. Pediatric cases were mainly non-invasive infections (8/9, 88.9%), adult cases were iGAS and severe-iGAS in 18/19 (94.7%) and 10/19 (52.6%), respectively. Thirteen emm-types were detected, the most prevalent being emm1 and emm12 (6/28 strains each, 21.4%). Single nucleotide polymorphism (SNP) analysis of emm1.0 and emm12.0 strains revealed pairwise SNP distance always >10, inconsistent with unique transmission chains. Emm12.0-type, found to almost exclusively carry virulence factors speH and speI, was mainly detected in children and in no-iGAS infections (55.6 vs. 5.3%, p = 0.007 and 66.7 vs. 0.0%, p < 0.001, respectively), while emm1.0-type was mainly detected in severe-iGAS (0.0 vs. 45.5%, p = 0.045). Conclusions: This study showed that multiple emm-types contributed to a 2022/2023 GAS infection increase in two hospitals in Milan, with no evidence of direct transmission chains. Specific emm-types could be associated with disease severity or invasiveness. Overall, these results support the integration of classical epidemiological studies with genomic investigation to appropriately manage severe infections and improve surveillance.

Role of polymorphisms of toll-like receptor (TLR) 4, TLR9, toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A genes in malaria susceptibility and severity in Burundian children.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs)-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs) of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A could modulate malaria susceptibility and severity. METHODS: This study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument. RESULTS: A total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9%) were considered as suffering from uncomplicated and 49 (8.1%) from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p=0.03), although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in genotype frequencies between malaria cases and uninfected controls as well as between uncomplicated and severe malaria cases. CONCLUSIONS: TLR9 T1237C seems to condition susceptibility to malaria in Burundian children but not its severity, whereas none of the assessed SNPs of TLR4, TIRAP and FCGR2A seem to influence susceptibility to malaria and disease severity in this population.