Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster. METHODS: Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3. RESULTS: Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3. CONCLUSIONS: During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis.

Risk of Multidrug-Resistant Pathogens in Severe Community-Acquired Pneumonia.

Severe community-acquired pneumonia (SCAP) is difficult to treat when caused by difficult-to-treat (DTR) pathogens because of limited treatment options and poorer clinical outcomes. Over time, several predictive scoring systems based on risk factors for infection with multidrug resistant pathogens have been developed. We reviewed the available tools for identifying DTR pathogens as the cause of SCAP, both predictive scoring systems and rapid diagnostic methods, to develop management strategies aimed at early identification of DTR pathogens, reducing broad-spectrum antibiotic use and improving clinical outcomes. The scoring systems reviewed show considerable heterogeneity among them at the level of the region studied, the definition of risk factors, as well as which DTR pathogens are the target pathogens. The models described have shown limited effectiveness in reducing inappropriate antibiotic treatment or improving patient outcomes by themselves. However, predictive models could serve as a first step in identifying DTR pathogen infections as part of a larger detection algorithm. Rapid diagnostic tools, such as multiplex polymerase chain reaction, would be useful for the rapid identification of pneumonia-causing pathogens and their resistance mechanisms. In resource-limited settings, rapid tests should be limited to patients at high risk of developing SCAP due to DTR pathogens. We propose an integrative algorithm based on the different scores, taking into account local epidemiological data, where ideally each center should have an antimicrobial stewardship program.

Relationship between Respiratory Microbiome and Systemic Inflammatory Markers in COPD: A Pilot Study.

The respiratory microbiome may influence the development and progression of COPD by modulating local immune and inflammatory events. We aimed to investigate whether relative changes in respiratory bacterial abundance are also associated with systemic inflammation, and explore their relationship with the main clinical COPD phenotypes. Multiplex analysis of inflammatory markers and transcript eosinophil-related markers were analyzed on peripheral blood in a cohort of stable COPD patients (n = 72). Respiratory microbiome composition was analyzed by 16S rRNA microbial sequencing on spontaneous sputum. Spearman correlations were applied to test the relationship between the microbiome composition and systemic inflammation. The concentration of the plasma IL-8 showed an inverted correlation with the relative abundance of 17 bacterial genera in the whole COPD cohort. COPD patients categorized as eosinophilic showed positive relationships with blood eosinophil markers and inversely correlated with the degree of airway obstruction and the number of exacerbations during the previous year. COPD patients categorized as frequent exacerbators were enriched with the bacterial genera Pseudomonas which, in turn, was positively associated with the severity of airflow limitation and the prior year's exacerbation history. The associative relationships of the sputum microbiome with the severity of the disease emphasize the relevance of the interaction between the respiratory microbiota and systemic inflammation.

Reduced airway levels of fatty-acid binding protein 4 in COPD: relationship with airway infection and disease severity.

BACKGROUND: For still unclear reasons, chronic airway infection often occurs in patients with Chronic Obstructive Pulmonary Disease (COPD), particularly in those with more severe airflow limitation. Fatty-acid binding protein 4 (FABP4) is an adipokine involved in the innate immune response against infection produced by alveolar macrophages (Mɸ). We hypothesized that airway levels of FABP4 may be altered in COPD patients with chronic airway infection. METHODS: In this prospective and controlled study we: (1) compared airway FABP4 levels (ELISA) in induced sputum, bronchoalveolar lavage fluid (BALF) and plasma samples in 52 clinically stable COPD patients (65.2 ± 7.9 years, FEV1 59 ± 16% predicted) and 29 healthy volunteers (55.0 ± 12.3 years, FEV1 97 ± 16% predicted); (2) explored their relationship with the presence of bacterial airway infection, defined by the presence of potentially pathogenic bacteria (PPB) at ≥103 colony-forming units/ml in BALF; (3) investigated their relationship with the quantity and proportion of Mɸ in BALF (flow cytometry); and, (4) studied their relationship with the severity of airflow limitation (FEV1), GOLD grade and level of symptoms (CAT questionnaire). RESULTS: We found that: (1) airway levels of FABP4 (but not plasma ones) were reduced in COPD patients vs. controls [219.2 (96.0-319.6) vs. 273.4 (203.1-426.7) (pg/ml)/protein, p = 0.03 in BALF]; (2) COPD patients with airway infection had lower sputum FABP4 levels [0.73 (0.35-15.3) vs. 15.6 (2.0-29.4) ng/ml, p = 0.02]; (3) in COPD patients, the number and proportion of Mɸ were positively related with FABP4 levels in BALF; (4) BALF and sputum FABP4 levels were positively related with FEV1, negatively with the CAT score, and lowest in GOLD grade D patients. CONCLUSIONS: Airway FABP4 levels are reduced in COPD patients, especially in those with airway infection and more severe disease. The relationship observed between Mɸ and airway FABP4 levels supports a role for FABP4 in the pathogenesis of airway infection and disease severity in COPD.

Differential Proteome Between Patient-Related and Non-related Environmental Isolates of Legionella pneumophila.

Molecular epidemiologic studies of Legionella have shown different molecular types coexisting in the same environment, with only one having the ability to trigger an outbreak. We therefore studied the proteome of isolates of these different molecular types in search of the proteins responsible for infection. In this study, we performed a differential proteomic analysis between patient-related and non-patient-related environmental isolates using two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry. Sixty-three spots were observed as being different between the two groups; 31 spots were identified corresponding to 23 different proteins. Patient-related isolates overexpressed proteins associated with metabolism, with enzymes of the tricarboxylic acid cycle and the degradation pathways being the most abundant proteins identified. However, the largest group of non-patient-related proteins was associated with stress response. Furthermore, the MOMP protein was located in different spots depending on their patient-related or non-patient-related origin, suggesting different post-translational modifications. According to these results, different bacterial adaptation pathways are activated in stress conditions which influence their ability to produce infection.

[Proteomics in infectious diseases]. / Proteómica en enfermedades infecciosas.

Infectious diseases have a high incidence in the population, causing a major impact on global health. In vitro culture of microorganisms is the first technique applied for infection diagnosis which is laborious and time consuming. In recent decades, efforts have been focused on the applicability of "Omics" sciences, highlighting the progress provided by proteomic techniques in the field of infectious diseases. This review describes the management, processing and analysis of biological samples for proteomic research.

Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012-2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.

Comparative molecular and antibody typing during the investigation of an outbreak of Legionnaires' disease.

An outbreak of Legionnaires' disease with 113 confirmed cases was reported in the town of Mataró, Spain, in August 2002. In this study, we compared three different typing methods and characterized the clinical isolates by comparing them with other clinical isolates with the same ST from our own database to further characterize the outbreak. In the outbreak, a total of 16 clinical (nine patients) and 32 environmental (from four environmental sources) Legionella pneumophila isolates were analyzed by pulsed-field electrophoresis (PFGE), sequence-based typing (SBT), and monoclonal antibody typing (MAb). We compared the MAb and SBT profiles of the outbreak clinical isolates and other unrelated clinical isolates showing the same ST profile. We obtained seven different PFGE and SBT profiles and six MAb patterns from the outbreak isolates. PFGE and SBT showed 100% concordance during the outbreak. SBT proved to be highly discriminatory, particularly with the addition of the new neuA gene. One PFGE, SBT (ST-37), and Philadelphia profile was observed among the clinical isolates. Using PFGE, this ST37 Philadelphia profile was closely related to other unrelated clinical isolates. These findings suggest that the ST37 Philadelphia profile could be a virulence marker in our area. The combination of the three methodologies was useful to further characterize and obtain additional information on a very explosive outbreak. Despite the minor discrimination of PFGE versus SBT, the two genetic methods are recommended in outbreak investigations. Further studies are currently underway in this area to obtain more definitive conclusions.

Evaluation of the Kinetics of Pancreatic Stone Protein as a Predictor of Ventilator-Associated Pneumonia.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a severe condition. Early and adequate antibiotic treatment is the most important strategy for improving prognosis. Pancreatic Stone Protein (PSP) has been described as a biomarker that increases values 3-4 days before the clinical diagnosis of nosocomial sepsis in different clinical settings. We hypothesized that serial measures of PSP and its kinetics allow for an early diagnosis of VAP. METHODS: The BioVAP study was a prospective observational study designed to evaluate the role of biomarker dynamics in the diagnosis of VAP. To determine the association between repeatedly measured PSP and the risk of VAP, we used joint models for longitudinal and time-to-event data. RESULTS: Of 209 patients, 43 (20.6%) patients developed VAP, with a median time of 4 days. Multivariate joint models with PSP, CRP, and PCT did not show an association between biomarkers and VAP for the daily absolute value, with a hazard ratio (HR) for PSP of 1.01 (95% credible interval: 0.97 to 1.05), for CRP of 1.00 (0.83 to 1.22), and for PCT of 0.95 (0.82 to 1.08). The daily change of biomarkers provided similar results, with an HR for PSP of 1.15 (0.94 to 1.41), for CRP of 0.76 (0.35 to 1.58), and for PCT of 0.77 (0.40 to 1.45). CONCLUSION: Neither absolute PSP values nor PSP kinetics alone nor in combination with other biomarkers were useful in improving the prediction diagnosis accuracy in patients with VAP. CLINICAL TRIAL REGISTRATION: Registered retrospectively on August 3rd, 2012. NCT02078999.

Methicillin-resistant and methicillin-sensitive Staphylococcus aureus in pork industry workers, Catalonia, Spain.

Background: Methicillin-resistant S. aureus (MRSA) especially ST398, is a zoonotic agent. This study aimed to determine the prevalence of methicillin-susceptible S. aureus (MSSA) and MRSA among workers in the pork production chain. Methods: 659 workers associated with 123 pig farms, livestock transporters, one pig slaughterhouse, pork transporters and 23 pork butcheries were studied for S. aureus recovery, and all isolates were characterized (antibiotic resistance, MLST and spa-typing). Results: The prevalence of S. aureus was 35.5%, 75.6% of isolates being MRSA. The prevalence of MRSA was 68.7% (149/217) among pig farm, 33.9% (19/56) livestock transporters, 2.9% (9/306) slaughterhouse, 0% in pork transporters (0/36) and butchery workers (0/44). Of the 234 S. aureus-positive workers, 100% (149/149) of pig farm workers, 82.6% (19/23) of livestock transporters, and 16.4% (9/55) of slaughterhouse workers carried MRSA isolates (p < 0.001). Of the workers who had contact with live swine, 61.8% (178/288) were S. aureus-positive, MRSA being detected in 96.1% of cases (p < 0.001). The most frequent lineage among MRSA were: ST398 (97.7%; 173/177) and ST1 (1.7%; 3/177); and among MSSA were ST30 (19.2%; 11/57) and ST5 (10.5%; 6/57). The most frequent spa-types among MRSA were t011 (93.8%, 166/177) and t1451 (2.25%, 4/177), and among MSSA: t084 (10.5%, 6/57) and t021 (7.0%, 4/57). All MRSA isolates showed resistance to tetracycline, 92.7% to clindamycin, 81.9% to erythromycin and 40.1% to cotrimoxazole. Conclusions: Pig industry workers having occupational contact with live animals present a high risk of colonization of MRSA, especially by MRSA-ST398. Prevention measures should be intensified in any employment sector involving live animals.

Clinical and Epidemiological Characteristics of Streptococcus suis Infections in Catalonia, Spain.

Introduction: Streptococcus suis (S. suis) is a human zoonotic pathogen of occupational origin, with infection acquired through contact with live pigs or pig meat. Pig farming is one of Catalonia's biggest industries and as a result this region of Spain has one of the highest density pig populations per km2. The aim of our study was to describe the infections caused by S. suis occurring in that area over a 9-year period. Materials and Methods: A retrospective, multi-center study was carried out by searching records from 15 hospitals in Catalonia for the period between 2010 and 2019. Results: Over the study period altogether nine cases of S. suis infection were identified in five hospitals, with five of these cases occurring in the 2018-2019 period. The mean age of patients was 48 ± 8.9 years and all of them were males. Five patients (55.6%) worked in pig farms. The most frequent manifestation of infection was meningitis (5 cases; 55.6%) followed by septic arthritis (3 cases; 33.3%). None of the patients died at 30 days; nonetheless, 4 developed hearing loss as a long-term complication. Conclusion: The most commonly identified S. suis infection was meningitis. Over 50% of the episodes occurred in the last 2 years and have affected pig farm workers. Further surveillance is needed in order to know its prevalence.

The Respiratory Microbiome in Cystic Fibrosis: Compartment Patterns and Clinical Relationships in Early Stage Disease.

We compared the bacterial microbiomes lodged in the bronchial tree, oropharynx and nose of patients with early stage cystic fibrosis (CF) not using chronic antibiotics, determining their relationships with lung function and exacerbation frequency. CF patients were enrolled in a cohort study during stability and were checked regularly over the following 9 months. Upper respiratory samples (sputum [S], oropharyngeal swab [OP] and nasal washing [N]) were collected at the first visit and every 3 months. 16S rRNA gene amplification and sequencing was performed and analyzed with QIIME. Seventeen CF patients were enrolled (16.6 SD 9.6 years). Alpha-diversity of bacterial communities between samples was significantly higher in S than in OP (Shannon index median 4.6 [IQR: 4.1-4.9] vs. 3.7 [IQR: 3-1-4.1], p = 0.003/Chao 1 richness estimator median 97.75 [IQR: 85.1-110.9] vs. 43.9 [IQR: 31.7-59.9], p = 0.003) and beta-diversity analysis also showed significant differences in the microbial composition of both respiratory compartments (Adonis test of Bray Curtis dissimilarity matrix, p = 0.001). Dominant taxa were found at baseline in five patients (29.4%), who showed lower forced expiratory volume in the first second (FEV1%, mean 74.8 [SD 19] vs. 97.2 [SD 17.8], p = 0.035, Student t test). The Staphylococcus genus had low RAs in most samples (median 0.26% [IQR 0.01-0.69%]), but patients with RA > 0.26% of Staphylococcus in bronchial secretions suffered more exacerbations during follow-up (median 2 [IQR 1-2.25] vs. 0 [0-1], p = 0.026. Mann-Whitney U test), due to S. aureus in more than a half of the cases, microorganism that often persists as bronchial colonized in these patients (9/10 [90%] vs. 2/7 [28.6%], p = 0.034, Fisher's exact test). In conclusion, the bronchial microbiome had significantly higher diversity than the microbial flora lodged in the oropharynx in early stage CF. Although the RA of the Staphylococcus genus was low in bronchial secretions and did not reach a dominance pattern, slight overrepresentations of this genus was associated with higher exacerbation frequencies in these patients.

Role of hot water temperature and water system use on Legionella control in a tertiary hospital: An 8-year longitudinal study.

Although measures to minimize Legionella colonization in sanitary hot water installations are well established, there is little evidence of their long-term effectiveness in hospital buildings. During an 8-year period, hot water in a large hospital building was sampled monthly in areas with suitable dimensioning and recirculation and in areas with dead legs and low-use taps. In the former areas, the percentage of Legionella-negative samples was 83.2% when the temperature was ≥55%, 64.9% when between 50.1 °C and 54.0 °C, and 51.6% when ≤50 °C (p for trend <0.001). In the highest temperature group, no samples with ≥103 cfu/L were observed. In poorly designed areas, only 44.7% of samples were negative, and 28.9% presented ≥103 cfu/L although reaching 55 °C. In these areas, multivariate analysis showed that if hot water supplies were not used daily, the risk of Legionella colonization was greater than two-fold (odds ratio: 2.84; 95% confidence interval: 1.26-6.41), and the risk of finding Legionella concentrations ≥103 cfu/L was more than three-fold (odds ratio: 3.18; 95% confidence interval: 1.36-7.46), regardless the temperature. These findings indicate that the effectiveness of maintaining sanitary hot water at a minimum temperature of 55 °C is significantly better than that at 50 °C for the environmental control of Legionella but only in installations with suitable dimensioning and recirculation. In installations that do not meet these conditions, high temperatures alone result in insufficient control.

Legionella SBT applied directly to respiratory samples as a rapid molecular epidemiological tool.

Legionnaires' disease (LD) is an atypical pneumonia caused by the inhalation of Legionella. The methods used for the diagnosis of LD are direct culture of respiratory samples and urinary antigen detection. However, the sensitivity of culture is low, and the urinary antigen test is specific only for L. pneumophila sg1. Moreover, as no isolates are obtained, epidemiological studies cannot be performed. The implementation of Nested-sequence-based typing (Nested-SBT) makes it possible to carry out epidemiological studies while also confirming LD, especially in cases caused by non-sg 1. Sixty-two respiratory samples from patients with Legionella clinically confirmed by positive urinary antigen tests were cultured and tested by Nested-SBT, following the European Study Group for Legionella Infections (ESGLI) protocol. Only 2/62 (3.2%) respiratory samples were culture-positive. Amplification and sequencing of Nested-SBT genes were successfully performed in 57/62 samples (91.9%). The seven target genes were characterised in 39/57 (68.4%) respiratory samples, and the complete sequence type (ST) was obtained. The mip gene was the most frequently amplified and sequenced. Nested-SBT is a useful method for epidemiological studies in culture-negative samples, achieving a 28.7-fold improvement over the results of culture studies and reducing the time needed to obtain molecular epidemiological results.

New system for the detection of Legionella pneumophila in water samples.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Antibody test for Legionella pneumophila detection.

Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.

Population structure of Environmental and Clinical Legionella pneumophila isolates in Catalonia.

Legionella is the causative agent of Legionnaires' disease (LD). In Spain, Catalonia is the region with the highest incidence of LD cases. The characterisation of clinical and environmental isolates using molecular epidemiology techniques provides epidemiological data for a specific geographic region and makes it possible to carry out phylogenetic and population-based analyses. The aim of this study was to describe and compare environmental and clinical isolates of Legionella pneumophila in Catalonia using sequence-based typing and monoclonal antibody subgrouping. A total of 528 isolates were characterised. For data analysis, the isolates were filtered to reduce redundancies, and 266 isolates (109 clinical and 157 environmental) were finally included. Thirty-two per cent of the clinical isolates were ST23, ST37 and ST1 while 40% of the environmental isolates were ST284 and ST1. Although the index of diversity was higher in clinical than in environmental ST isolates, we observed that clinical STs were similar to those recorded in other regions but that environmental STs were more confined to particular study areas. This observation supports the idea that only certain STs trigger cases or outbreaks in humans. Therefore, comparison of the genomes of clinical and environmental isolates could provide important information about the traits that favour infection or environmental persistence.

Specific IgA against Pseudomonas aeruginosa in severe COPD.

BACKGROUND: The bronchial mucosa is protected by a specialized immune system focused on the prevention of colonization and infection by potentially pathogenic microorganisms (PPMs). Immunoglobulin A (IgA) is the principal antibody involved in this mechanism. A defective immune barrier may facilitate the recurrent presence of PPMs in COPD. PURPOSE: The aim of this study was to determine IgA-mediated bronchial specific immune responses against Pseudomonas aeruginosa in stable patients with severe disease. METHODS: COPD patients with good-quality sputum samples obtained during stability were included and classified according to the presence or absence of chronic bronchial colonization by P. aeruginosa. Levels of specific IgA for P. aeruginosa in sputum were determined by ELISA and expressed as ratios, using the pooled level of 10 healthy subjects as reference (optical density450 patient/control). RESULTS: Thirty-six stable COPD patients were included, 15 of whom had chronic colonization by P. aeruginosa. Levels of specific IgA against P. aeruginosa in stable non-colonized patients were lower than those in healthy subjects (IgA ratio: median =0.15 [interquartile range {IQR} 0.05-0.36]). Colonized patients had higher levels, (1.56 [IQR 0.59-2.79]) (p<0.001, Mann-Whitney U test), with figures equivalent but not exceeding the reference value. CONCLUSION: IgA-based immune response against P. aeruginosa was low in severe COPD patients. Levels of specific IgA against this microorganism were higher in colonized patients, but did not attain clear-cut levels above the reference. An impaired local response against P. aeruginosa may favor chronic colonization and recurrent infections in severe COPD.

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

AIM: To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. MATERIALS & METHODS: Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. RESULTS: The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. CONCLUSION: SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.