[Low risk of environmental contagion by SARS-CoV-2 in non-sanitary spaces]. / Bajo riesgo de contagio ambiental por SARS-CoV-2 en espacios no sanitarios.

Objective: To study the presence of SARS-CoV-2 on surfaces (high, medium and low contacts) and airs in non-sanitary spaces with high public influx to evaluate the risk of environmental contagion. Method: Surfaces and airs were analysed by RT-qPCR to detect the presence of SARS-CoV-2. Results: A total of 394 surfaces and air samples were obtained from spaces with high public influx such as offices, shopping centres and nursing homes. The virus was not detected in any of the samples analysed. Conclusion: Although we cannot emphatically conclude that there is no risk of environmental infection by SARS-CoV-2 in non-sanitary spaces, we can affirm that the risk is almost non- existent.

Development of an integrated method of concentration and immunodetection of bacteria.

The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s-1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria.

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.

Prevalence of colonization by methicillin-resistant Staphylococcus aureus ST398 in pigs and pig farm workers in an area of Catalonia, Spain.

BACKGROUND: A livestock-associated clonal lineage (ST398) of methicillin-resistant Staphylococcus aureus (MRSA) has been identified causing colonization or infection in farm workers. The aim of the study was to analyze the prevalence of MRSA-ST398 colonization in pigs and in pig farmers in an area with a high pig population (Osona, Barcelona province, Catalonia, Spain). METHODS: We performed a cross-sectional prevalence study in Osona (Catalonia, Spain), from June 2014 to June 2015. All pig farm workers from 83 farms were studied. Twenty of these farms were randomly selected for the study of both pigs and farmers: 9 fattening and 11 farrow-to-finish farms. All workers over the age of 18 who agreed to participate were included. Samples were analyzed to identify MRSA-ST398 and their spa type. RESULTS: Eighty-one of the 140 pig farm workers analyzed (57.9% (95% IC: 50.0-66.4%)) were MRSA-positive, all of them ST398. The mean number of years worked on farms was 17.5 ± 12.6 (range:1-50), without significant differences between positive and negative MRSA results (p = 0.763). Over 75% of MRSA-ST398 carriers worked on farms with more than 1250 pigs (p < 0.001). At least one worker tested positive for MRSA-ST398 on all 20 selected pig farms. Ninety-two (46.0% (95% IC: 39.0-53.0%)) of the nasal swabs from 200 pigs from these 20 farms were MRSA-positive, with 50.5% of sows and 41.4% of fattening pigs (p = 0.198) giving MRSA-positive results. All the isolates were tetracycline-resistant, and were identified as MRSA-ST398. The spa type identified most frequently was t011 (62%). Similar spa types and phenotypes of antibiotic resistance were identified in pigs and farmers of 19/20 tested farms. CONCLUSIONS: The prevalence of MRSA-ST398 among pig farm workers and pigs on farms in the studied region is very high, and the size of the farm seems to correlate with the frequency of colonization of farmers. The similar spa-types and phenotypes of resistance detected in pigs and workers in most of the farms studied suggest animal-to-human transmission.

[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.

Impact of the 2009 influenza A H1N1 pandemic on invasive pneumococcal disease in adults.

BACKGROUND: The incidence of invasive pneumococcal disease (IPD) appears to be associated with influenza. The objectives of this study were to evaluate the changes in IPD incidence and clinical data as well as the trends in Streptococcus pneumoniae serotype distribution in adults during the peak period of the 2009 influenza A H1N1 pandemic (IAP). METHODS: We performed a prospective multicentre study on IPD from week 42 to 48, 2009 in an area of Barcelona (Catalonia, Spain) covering 1,483,781 adult inhabitants. Serotyping was done by Quellung reaction. The data from 2009 were compared to those from the same periods in 2008 and 2010. RESULTS: Two hundred and three cases of IPD were detected during 2009, compared with 182 in 2008 and 139 in 2010. The incidence of IPD during the 7-week study period in 2009 (2.89) was statistically higher than that observed in 2008 (1.96) and 2010 (1.46). IAP was confirmed in 3/30 patients during the 2009 study period. Patients with IPD in 2009 were significantly healthier and younger than those in the other years, although the mortality was higher than in 2008 (p = 0.05) and 2010 (p > 0.05). Eleven (10 non-PCV-7) serotypes not present in 2008 appeared in 2009. CONCLUSIONS: During weeks 42 to 48, in which the 2009 IAP peaked in Catalonia, the incidence of IPD was statistically higher than that observed in the same time period in 2008 and 2010, with some differences in the epidemiological data, showing a close relationship between S. pneumoniae and influenza.

A novel assessment, diagnostic and treatment system for diabetic foot.

BACKGROUND: This report aims to present a novel system for the management of foot lesions in patients with diabetes. It was developed in the diabetic foot unit (DFU) of the Mutua de Terrassa University Hospital (HUMT) by primary care professionals, the Diabetic Foot Clinic (DFC), and during emergency cases treated by our group based on daily activities in patients with neuropathy or neuroischemia. BODY: This system considers five degrees of action based on two fixed variables: presence of infection and lesion depth. These two variables allowed the user to investigate aspects of the system until the overall action required by the pathology is made clear. These variables establish pathology stages of various severities that require different actions in aspects of care, management and treatment. CONCLUSION: This tool facilitates diagnosis, treatment, and coordination among different members of a multidisciplinary team working in specialized hospital units, primary care centers, and emergency settings.

Low risk of environmental contagion by SARS-CoV-2 in non-sanitary spaces.

OBJECTIVE: To study the presence of SARS-CoV-2 on surfaces (high, medium and low contact) and airs in non-sanitary spaces with high public influx to evaluate the risk of environmental contagion. METHODS: Surfaces and airs were analysed by RT-qPCR to detect the presence of SARS-CoV-2. RESULTS: 394 surfaces and air samples were obtained from spaces with high public influx such as offices, shopping centres and nursing homes. The virus was not detected in any of the samples analysed. CONCLUSION: Although we cannot emphatically conclude that there is no risk of environmental 27 infection by SARS-CoV-2 in non-sanitary spaces, we can affirm that the risk is almost non- existent.

Intervention to reduce the incidence of non-ventilator-associated hospital-acquired pneumonia: A pilot study.

BACKGROUND: Our aim was to evaluate the effectiveness of an intervention to reduce the incidence of non-ventilator-associated hospital-acquired pneumonia (NV-HAP) and determine compliance with preventive measures. METHODS: This was a quasi-experimental before-after study involving patients in the 53-bed Internal Medicine ward in a university hospital in Spain. The preventive measures included hand hygiene, dysphagia detection, head-of-bed elevation, withdrawal of sedatives in the event of confusion, oral care, and sterile or bottled water use. A prospective post-intervention study of the incidence of NV-HAP was carried out from February 2017 to January 2018 and compared with baseline incidence (May 2014 to April 2015). Compliance with preventive measures was analyzed with 3-point-prevalence studies (December 2015, October 2016, and June 2017). RESULTS: The rate of NV-HAP decreased from 0.45 cases (95% confidence interval 0.24-0.77) in the pre-intervention period to 0.18 cases per 1,000 patient-days (95% confidence interval 0.07-0.39) in the post-intervention period (P = .07). Compliance with most preventive measures improved after intervention and remained stable over time. CONCLUSIONS: The strategy improved the adherence to most of the preventive measures, with a decrease in the incidence of NV-HAP. Efforts to enhance adherence to such fundamental preventive measures are critical to lowering the incidence of NV-HAP.

[Diversity of Legionella pneumophila in cooling towers: coculture kinetics and virulence studies]. / Diversidad de subtipos de Legionella pneumophila en torres de refrigeración: estudios de cinética de crecimiento y virulencia.

BACKGROUND: Legionella pneumophila (L. pneumophila) was isolated from three cooling towers involved in three community outbreaks of Legionnaires disease. Each cooling tower had two different chromosomal DNA subtypes. However, only one matched identically to the clinical strains. To try to understand why only one of the environmental strains caused clinical cases we investigated the intrinsic virulence of these strains. METHODS: We selected six strains of L. pneumophila sg.1: two strains (A1 and B1) from cooling tower 1, two strains (A2 and B2) from tower 2 and two strains (A3 and B3) from tower 3. One of the two subtypes (A) exhibited the same chromosomal DNA subtype as the strains isolated from the patients in each outbreak and the other exhibited a different subtype. The replication within macrophages, the presence of lipopolysaccharide epitope recognized by MAb 3/1 and the growth kinetics in BCYE broth were investigated. Isolates were typed by pulsed field electrophoresis. RESULTS: The A strains did not have a higher virulence level, but were able to grow and survive better than strains B in BCYE broth. CONCLUSIONS: These results suggest that the strains better adapted to the environment will manage to displace the others and will be able to spread and infect humans. The adaptation to the environmental conditions could play an important role in the pathogenesis of the strains.

Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement.

Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2-3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL-1 with a detection limit (LoD) of 4 cfu·mL-1.

[Low mortality rate in an outbreak of Legionnaires' disease: correlation with the cytopathogenity study]. / Baja mortalidad en un brote de enfermedad del legionario: correlación con el estudio de citopatogenicidad.

BACKGROUND: In 2001 an outbreak of Legionnaires' diseases occurred in Murcia, Spain, with one of the lowest known rates of associated mortality. We describe the clinical data of a subgroup of patients, and present the results from molecular and virulence studies to correlate the lower mortality of the overall series with the strain virulence. PATIENTS AND METHODS: A subgroup of 86 patients from the outbreak of Legionnaires'disease was prospectively included. Demographic, risk factors and clinical evolution data were obtained. Moreover, we performed a pulsed field gel electrophoresis and cytopathogenicity assay of the Murcia outbreak that were compared with other unrelated Legionella isolates. RESULTS: Sixty-nine (77.9%) patients were males. The mean age of the patients was 58.2 years (range: 32-87). Smoking was the most frequent risk factor in 62 patients (71.7%) and 61 patients (70.2%) had underlying diseases. Clinical, laboratory and radiological manifestations were compatible with the atypical pneumonia syndrome. The mortality rate was 3.2%. All the clinical L. pneumophila isolates analyzed by PFGE showed the same subtype. When analyzing theses strains together with other Legionella strains, they were included in the group with lower virulence in the cytopathogenicity study. CONCLUSIONS: The most outstanding data in this subgroup of patients were: male-sex, smoking, atypical clinical manifestations and low mortality. The low virulence of this molecular genotype of L. pneumophila may be responsible, in part, for the low mortality observed in the outbreak in Murcia.

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.

Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areas.

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed >or= 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed >or= 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.

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.

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.

Prevalence of MRSA ST398 Carriage in Nursing Home Residents in an Area of Spain With a High Density of Pig Farming.

MRSA nasal carriage was detected in 15.7% of 204 residents from 6 nursing homes (NHs) in the Osona region (Barcelona, Spain), and the MRSA-ST398 lineage was identified in 15.6% of MRSA-positive residents and in 2.5% of all NH residents evaluated. Most MRSA-ST398 carriers (4 of 5) had direct or indirect contact with pig farms. Infect Control Hosp Epidemiol 2018;39:90-93.

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.