Search

Abstract Lactic acid bacteria (LAB) of the genus Lactiplantibacillus have been explored as potential mucosal vaccine vectors due to their ability to elicit an immune response against expressed foreign antigens and to their safety. However, tools for monitoring LAB distribution and persistence at the mucosal surfaces are needed. Here, we characterize Lactiplantibacillus plantarum bacteria expressing the infrared fluorescent protein IRFP713 for exploring their in vivo distribution in the mucosa and potential use as a mucosal vaccine vector. This bacterial species is commonly used as a vaginal probiotic and was recently found to have a niche in the human nose. Three different fluorescent L. plantarum strains were obtained using the nisin-inducible pNZRK-IRFP713 plasmid which contains the nisRK genes, showing stable and constitutive expression of IRFP713 in vitro. One of these strains was further monitored in BALB/c mice using near-infrared fluorescence, indicating successful colonization of the nasal and vaginal mucosae for up to 72 h. This study thus provides a tool for the in vivo spatiotemporal monitoring of lactiplantibacilli, allowing non-invasive bacterial detection in these mucosal sites. Key points • Stable and constitutive expression of the IRFP713 protein was obtained in different L. plantarum strains. • IRFP713+ L. plantarum 3.12.1 was monitored in vivo using near-infrared fluorescence. • Residence times observed after intranasal and vaginal inoculation were 24–72 h. Graphical abstract

Background: SARS-CoV-2 variation represents a serious challenge to current COVID-19 vaccines. Recent reports suggest that B.1.351 and other variants may escape the neutralization activity of the antibodies generated by current vaccines. Methods: Ninety-nine healthcare workers undertaking BNT162b2 mRNA vaccination were sampled at baseline, on the day of the second dose, and 14 days after the latter. Neutralization activity against SARS-CoV-2 B.1, B.1.1.7 and B.1.351 was investigated using a Vero-E6 model. Results: Eleven of the study participants had prior infection with SARS-CoV-2. Neutralization titers against the B.1 and the B.1.1.7 variants were not statistically different and were significantly higher than titers against the B.1.351 variant across pre-exposed and non-pre-exposed vaccinated individuals ( p1/80 after a single dose, while only 11% of non-exposed vaccinated individuals had titers >1/80. Conclusions: BNT162b2 mRNA-induced antibodies show a lower in vitro neutralizing activity against B.1.351 variant compared to neutralization against B.1.1.7 or B.1 variants. Interestingly, for individuals pre-exposed to SARS-CoV-2, one dose of BNT162b2 mRNA may be adequate to produce neutralizing antibodies against B.1.1.7 and B.1, while two doses of BNT162b2 mRNA provide optimal neutralizing antibody response against B.1.351 too.

Background: SARS-CoV-2 variant tracking is key to the genomic surveillance of the pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification.Case presentation: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike aminoacids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. Conclusions: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the targets of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

A detailed understanding of how and when SARS-CoV-2 transmission occurs is crucial for designing effective prevention measures. Other than contact tracing, genome sequencing provides information to help infer who infected whom. However, the effectiveness of the genomic approach in this context depends on both (high enough) mutation and (low enough) transmission rates. Today, the level of resolution that we can obtain when describing SARS-CoV-2 outbreaks using just genomic information alone remains unclear. In order to answer this question, we sequenced 49 SARS-CoV-2 patient samples from ten local clusters in NW Spain for which partial epidemiological information was available, and inferred transmission history using genomic variants. Importantly, we obtained high-quality genomic data, sequencing each sample twice and using unique barcodes to exclude cross-sample contamination. Phylogenetic and cluster analyses showed that consensus genomes were generally sufficient to discriminate among independent transmission clusters. However, levels of intrahost variation were low, which prevented in most cases the unambiguous identification of direct transmission events. After filtering out recurrent variants across clusters, the genomic data were generally compatible with the epidemiological information but did not support specific transmission events over possible alternatives. We estimated the effective transmission bottleneck size to be 1-2 viral particles for sample pairs whose donor-recipient relationship was likely. Our analyses suggest that intrahost genomic variation in SARS-CoV-2 might be generally limited and that homoplasy and recurrent errors complicate identifying shared intrahost variants. Reliable reconstruction of direct SARS-CoV-2 transmission based solely on genomic data seems hindered by a slow mutation rate, potential convergent events, and technical artifacts. Detailed contact tracing seems essential in most cases to study SARS-CoV-2 transmission at high resolution. Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Xunta de Galicia | Ref. ED431C2018/54 Banco Santander Consejo Superior de Investigaciones Científicas Conferencia de Rectores de las Universidades Españolas Servizo Galego de Saúde

SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in the urban wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check the safety of the water effluent and also of the sludge produced and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). Although more studies will be needed for confirmation, these results contribute to clarify the role of WWTPs in COVID-19 mitigation.

GAMES Investigators., [Background] Enterococcal endocarditis (EE) is a growing entity in Western countries. However, quality data from large studies is lacking., [Objectives] The purpose of this study was to describe the characteristics and analyze the prognostic factors of EE in the GAMES cohort., [Methods] This was a post hoc analysis of a prospectively collected cohort of patients from 35 Spanish centers from 2008 to 2016. Characteristics and outcomes of 516 cases of EE were compared with those of 3,308 cases of nonenterococcal endocarditis (NEE). Logistic regression and Cox proportional hazards regression analysis were performed to investigate risk factors for in-hospital and 1-year mortality, as well as relapses., [Results] Patients with EE were significantly older; more frequently presented chronic lung disease, chronic heart failure, prior endocarditis, and degenerative valve disease; and had higher median age-adjusted Charlson score. EE more frequently involved the aortic valve and prosthesis (64.3% vs. 46.7%; p < 0.001; and 35.9% vs. 28.9%; p = 0.002, respectively) but less frequently pacemakers/defibrillators (1.5% vs. 10.5%; p < 0.001), and showed higher rates of acute heart failure (45% vs. 38.3%; p = 0.005). Cardiac surgery was less frequently performed in EE (40.7% vs. 45.9%; p = 0.024). No differences in in-hospital and 1-year mortality were found, whereas relapses were significantly higher in EE (3.5% vs. 1.7%; p = 0.035). Increasing Charlson score, LogEuroSCORE, acute heart failure, septic shock, and paravalvular complications were risk factors for mortality, whereas prior endocarditis was protective and persistent bacteremia constituted the sole risk factor for relapse., [Conclusions] Besides other baseline and clinical differences, EE more frequently affects prosthetic valves and less frequently pacemakers/defibrillators. EE presents higher rates of relapse than NEE.

Purpose: To analyze the influence of adding gentamicin to a regimen consisting of β-lactam or vancomycin plus rifampicin on survival in patients suffering from Staphylococcal prosthetic valve endocarditis (SPVE). Methods: From January 2008 to September 2016, 334 patients with definite SPVE were attended in the participating hospitals. Ninety-four patients (28.1%) received treatment based on β-lactam or vancomycin plus rifampicin and were included in the study. Variables were analyzed which related to patient survival during admission, including having received treatment with gentamicin. Results: Seventy-seven (81.9%) were treated with cloxacillin (or vancomycin) plus rifampicin plus gentamicin, and 17 patients (18.1%) received the same regimen without gentamicin. The causative microorganism was Staphylococcus aureus in 40 cases (42.6%) and coagulase-negative staphylococci in 54 cases (57.4%). Overall, 40 patients (42.6%) died during hospital admission, 33 patients (42.9%) in the group receiving gentamicin and 7 patients in the group that did not (41.2%, P = 0.899). Worsening renal function was observed in 42 patients (54.5%) who received gentamicin and in 9 patients (52.9%) who did not (p = 0.904). Heart failure as a complication of endocarditis (OR: 4.58; CI 95%: 1.84–11.42) and not performing surgery when indicated (OR: 2.68; CI 95%: 1.03–6.94) increased mortality. Gentamicin administration remained unrelated to mortality (OR: 1.001; CI 95%: 0.29–3.38) in the multivariable analysis. Conclusions: The addition of gentamicin to a regimen containing vancomycin or cloxacillin plus rifampicin in SPVE was not associated to better outcome.

Objective: To investigate the rate of colorectal neoplasms (CRNs) in patients who have Enterococcus faecalis infective endocarditis (EFIE) with available colonoscopies and to assess whether this is associated with the identification of a focus the infection. Patients and Methods: Retrospective analysis of data from a prospective multicenter study involving 35 centers who are members of the Grupo de Apoyo para el Manejo de la Endocarditis en Espana [Support Group for the Management of Infective Endocarditis in Spain] cohort. A specific set of queries regarding information on colonoscopy and histopathology of colorectal diseases was sent to each participating center. Four-hundred sixty-seven patients with EFIE were included from January 1, 2008, to December 31, 2017, from whom data on colonoscopy performance and results were available in 411 patients. Results: One hundred forty-two (34.5%) patients had a colonoscopy close to the EFIE episode. The overall rate of colorectal diseases was 70.4% (100 of 142), whereas the prevalence of CRN (advanced adenomas and colorectal carcinoma) was 14.8% (21 of 142), with no significant differences between the group of EFIE of unknown focus and that with an identified focus. Conclusion: Our study adds to prior evidence suggesting a much higher rate of CRN among patients with EFIE than in the general population of the same age and sex. In addition, our findings suggest that this phenomenon might take place both in EFIE with an unknown and an identified source of infection.

SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in municipal wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check its presence in the water effluent and in the produced sludge and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP in 5 different days showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). These findings could be useful to develop a suitable strategy for early warning of COVID-19 incidence based on WWTP monitoring., Graphical abstract Unlabelled Image

La espectrometria de masas (EM) es una tecnica de analisis que permite caracterizar muestras midiendo las masas (estrictamente las razones masa-carga) de las moleculas componentes. Cuenta con mas de un siglo de historia y evolucion tecnologica y a lo largo de los anos ha ampliado su alcance desde los isotopos a moleculas pequenas, moleculas organicas mas complejas y, en las ultimas decadas, macromoleculas (acidos nucleicos y proteinas). La EM MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) es una variante que permite el analisis de mezclas complejas de proteinas y que se ha aplicado recientemente a la identificacion de microorganismos en cultivo, convirtiendose en una herramienta rapida y eficaz para el diagnostico microbiologico que ha conseguido entrar en poco tiempo en la rutina de muchos servicios de microbiologia clinica. El gran impacto que ha tenido esta impulsando el desarrollo de nuevas aplicaciones en el campo de la microbiologia clinica.

To evaluate the course of left-sided infective endocarditis (LsIE) in patients with liver cirrhosis (LC) analyzing its influence on mortality and the impact of surgery.Prospective cohort study, conducted from 1984 to 2013 in 26 Spanish hospitals.A total of 3.136 patients with LsIE were enrolled and 308 had LC: 151 Child-Pugh A, 103 B, 34 C and 20 were excluded because of unknown stage. Mortality was significantly higher in the patients with LsIE and LC (42.5% vs. 28.4%; p0.01) and this condition was in general an independent worse factor for outcome (HR 1.51, 95% CI: 1.23-1.85; p0.001). However, patients in stage A had similar mortality to patients without cirrhosis (31.8% vs. 28.4% p = NS) and in this stage heart surgery had a protective effect (28% in operated patients vs. 60% in non-operated when it was indicated). Mortality was significantly higher in stages B (52.4%) and C (52.9%) and the prognosis was better for patients in stage B who underwent surgery immediately (mortality 50%) compared to those where surgery was delayed (58%) or not performed (74%). Only one patient in stage C underwent surgery.Patients with liver cirrhosis and infective endocarditis have a poorer prognosis only in stages B and C. Early surgery must be performed in stages A and although in selected patients in stage B when indicated.

Background: There is little information concerning infective endocarditis (IE) in patients with bicuspid aortic valve (BAV) or mitral valve prolapse (MVP). Currently, IE antibiotic prophylaxis (IEAP) is not recommended for these conditions. Objectives: This study sought to describe the clinical and microbiological features of IE in patients with BAV and MVP and compare them with those of IE patients with and without IEAP indication, to determine the potential benefit of IEAP in these conditions. Methods: This analysis involved 3,208 consecutive IE patients prospectively included in the GAMES (Grupo de Apoyo al Manejo de la Endocarditis infecciosa en España) registry at 31 Spanish hospitals. Patients were classified as high-risk IE with IEAP indication (high-risk group; n = 1,226), low- and moderate-risk IE without IEAP indication (low/moderate-risk group; n = 1,839), and IE with BAV (n = 54) or MVP (n = 89). Results: BAV and MVP patients had a higher incidence of viridans group streptococci IE than did high-risk group and low/moderate-risk group patients (35.2% and 39.3% vs. 12.1% and 15.0%, respectively; all p < 0.01). A similar pattern was seen for IE from suspected odontologic origin (14.8% and 18.0% vs. 5.8% and 6.0%; all p < 0.01). BAV and MVP patients had more intracardiac complications than did low/moderate-risk group (50% and 47.2% vs. 30.6%, both p < 0.01) patients and were similar to high-risk group patients. Conclusions: IE in patients with BAV and MVP have higher rates of viridans group streptococci IE and IE from suspected odontologic origin than in other IE patients, with a clinical profile similar to that of high-risk IE patients. Our findings suggest that BAV and MVP should be classified as high-risk IE conditions and the case for IEAP should be reconsidered. Sin financiación 18.639 JCR (2018) Q1, 3/136 Cardiac & Cardiovascular Systems 9.280 SJR (2018) Q1, 1/365 Cardiology and Cardiovascular Medicine No data IDR 2018 UEM

Schizophrenia is a poorly understood chronic disease. Its pathophysiology is complex, dynamic, and linked to epigenetic mechanisms and microbiota involvement. Nowadays, correlating schizophrenia with the environment makes sense owing to its multidimensional implications: temporal and spatial variability. Microbiota involvement and epigenetic mechanisms are factors that are currently being considered to better understand another dimension of schizophrenia.This review summarises and discusses currently available information, focussing on the microbiota, epigenetic mechanisms, technological approaches aimed at performing exhaustive analyses of the microbiota, and psychotherapies, to establish future perspectives.The connection between the microbiota, epigenetic mechanisms and technological developments allows for formulating new approaches objectively oriented towards the development of alternative psychotherapies that may help treat schizophrenia.In this review, the gut microbiota and epigenetic mechanisms were considered as key regulators, revealing a potential new aetiology of schizophrenia. Likewise, continuous technological advances (e.g. culturomics), aimed at the microbiota-gut-brain axis generate new evidence on this concept.

OBJECTIVES The objective of this study was to analyse whether there is an association between reduced susceptibility to biocides in Acinetobacter baumannii and (i) antimicrobial resistance (co-resistance), (ii) prevalent (epidemic) clones, (iii) changes in the fitness or (iv) expression of genes related to efflux pumps and porins. METHODS Susceptibility to biocides and antimicrobials was determined in 49 clonally unrelated isolates of A. baumannii. Biological cost, in terms of mean generation time, was determined by spectrophotometry. Quantitative real-time RT-PCR was used to determine the relative expression of genes encoding several efflux pumps and porins. RESULTS Reduced susceptibility to chlorhexidine digluconate, benzalkonium chloride and Irgasan(®) was associated with resistance to aminoglycosides, tetracycline and ciprofloxacin (P

La espectrometría de masas (EM) es una técnica de análisis que permite caracterizar muestras midiendo las masas (estrictamente las razones masa-carga) de las moléculas componentes. Cuenta con más de un siglo de historia y evolución tecnológica y a lo largo de los años ha ampliado su alcance desde los isótopos a moléculas pequeñas, moléculas orgánicas más complejas y, en las últimas décadas, macromoléculas (ácidos nucleicos y proteínas). La EM MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) es una variante que permite el análisis de mezclas complejas de proteínas y que se ha aplicado recientemente a la identificación de microorganismos en cultivo, convirtiéndose en una herramienta rápida y eficaz para el diagnóstico microbiológico que ha conseguido entrar en poco tiempo en la rutina de muchos servicios de microbiología clínica. El gran impacto que ha tenido está impulsando el desarrollo de nuevas aplicaciones en el campo de la microbiología clínica.

Antibiotic treatment for septic shock is generally prescribed on an empirical basis using broad-spectrum antibiotics. Molecular diagnostic techniques can detect the presence of microbial DNA in blood within a few hours and facilitate early, targeted treatment. The aim of this study was to evaluate the economic impact of a real-time polymerase chain reaction technique, LightCycler SeptiFast (LSC), in patients with sepsis. A cost-minimisation study was carried out in patients admitted with a diagnosis of severe sepsis or septic shock to the intensive care unit of a university hospital. The stay in the intensive care unit, hospital admission, 28-day and six-month mortality, and the economic cost of the clinical process were also evaluated. The study involved 48 patients in the LSC group and 54 patients in the control group. The total cost was €42,198 in the control group versus €32,228 in the LCS group with statistically significant differences (P

Introduccion La infeccion por Candida se ha convertido en un importante problema de salud en todo el mundo. La epidemiologia de la candidemia se ha modificado considerablemente por la emergencia de las especies de Candida no-albicans. Esta variacion tiene especial importancia en la eleccion de la profilaxis y tratamiento empirico. Los metodos bioquimicos y los basados en biologia molecular presentan limitaciones para la identificacion correcta de las especies de Candida. El objetivo de este estudio es demostrar la capacidad del sistema de espectrometria de masas MALDI-TOF para la identificacion de estas especies y compararlo con la tecnologia utilizada en la actualidad. Metodos Se incluyeron todos los aislados recogidos durante dos anos (n=73) de Candida no-albicans procedentes de muestras invasivas. La identificacion se realizo mediante los sistemas Vitek-2 YST y API CAUX. Las identificaciones del MALDI-TOF se hicieron con el sistema Axima Confidence (Shimadzu Corporation), utilizando el software Shimadzu Launchpad y la base de datos SARAMIS (AnagnosTec GmbH). Las discrepancias se resolvieron mediante PCR multiplex LightCycler SeptiFast, PCR especifica de C. glabrata y digestion enzimatica con BanI del fragmento SADH en los aislados de C. parapsilosis. Resultados De los 73 aislados de Candida no-albicans, los metodos bioquimicos identificaron de forma concluyente 67 a nivel de especie y 6 a nivel de genero. El sistema MALDI-TOF obtuvo identificaciones a nivel de especie en todas ellas. La correlacion en la especie de todos los aislados estudiados fue del 85,07%, llegando al 94,52% si se estudia la correlacion entre la identificacion obtenida mediante metodos bioquimicos junto con los metodos empleados para el analisis de las discrepancias. En los aislados de C. parapsilosis, el sistema MALDI-TOF obtuvo una identificacion de C. orthopsilosis en tres de ellos, confirmandose por digestion con BanI del fragmento SADH. Conclusion En este estudio se ha demostrado la utilidad de la espectrometria de masas (sistema MALDI-TOF) para dotar al laboratorio de microbiologia de una mayor eficiencia y fiabilidad para la identificacion a nivel de especie de los aislados de Candida no-albicans. Ademas de mostrar su posible utilidad para la identificacion de especies relacionadas como C. parapsilosis, orthopsilosis y metapsilosis.

Infections with carbapenem-resistant enterobacteria are an emerging threat. This study reports the microbiologic, clinical, and epidemiologic features and the therapeutic outcomes of the infections caused by carbapenem- and pandrug-resistant Klebsiella emerged in our hospital. Fingerprinting analyses by automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry are also compared.Carbapenem-resistant Klebsiella spp. affecting 13 patients were investigated using automated rep-PCR (DiversiLab System) and MALDI-TOF. Species identification was performed by Vitek 2 System and MALDI-TOF. Antimicrobial susceptibility testing was made using Vitek 2 System and Etest. Screening for extended spectrum beta-lactamase (ESBL) and carbapenemase production was made by double disk synergy and Hodge tests, respectively. Synergy studies were performed using Etest. DNA array was used for detection of KPC and ESBLs. bla(VIM-1) gene was amplified by PCR and sequencing. Use of carbapenems in the hospital was studied.A total of 13 patients were found to be colonized/infected with carbapenem-resistant Klebsiella. All patients were previously submitted to surgery and/or presented with severe underlying disease. After carbapenem-resistant Klebsiella isolation, the majority of the patients were treated with amikacin plus carbapenem, tigecycline, or fosfomycin. All Klebsiella isolates (n = 14), except two, had the bla(VIM-1) gene and all Klebsiella pneumoniae also had bla(SHV) gene associated with ESBL production. DiversiLab system showed higher discriminatory power than MALDI-TOF for strain typing.The risk of a rapid dissemination and the persistence of these multidrug-resistant strains through the time determine the need to implement routine procedures for metallo-beta-lactamase detection and measures for prevention of the spread of these microorganisms. The combined use of MALDI-TOF for species identification and DiversiLab System for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks. The potential clinical utility of fosfomycin in this matter should be considered in future studies.

Between February 2006 and October 2009, 38 patients in different wards at the A Coruña University Hospital (northwest Spain) were either infected with or colonized by an epidemic, multidrug-resistant (MDR), and extended-spectrum-β-lactamase (ESBL)-producing strain of Enterobacter cloacae (EbSF), which was susceptible only to carbapenems. Semiautomated repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the E. cloacae isolates belonged to the same clone. Cloning and sequencing enabled the detection of the SFO-1 ESBL in the epidemic strain and the description of its genetic environment. The presence of the ampR gene was detected upstream of bla SFO-1 , and two complete sequences of IS 26 surrounding ampR and ampA were detected. These IS 26 sequences are bordered by complete left and right inverted repeats (IRL and IRR, respectively), which suggested that they were functional. The whole segment flanked by two IS 26 copies may be considered a putative large composite transposon. A gene coding for aminoglycoside acetyltransferase (gentamicin resistance gene [ aac3 ]) was found downstream of the 3′ IS 26 . Despite the implementation of strict infection control measures, strain EbSF spread through different areas of the hospital. A case-control study was performed to assess risk factors for EbSF acquisition. A multivariate analysis revealed that the prior administration of β-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization were statistically associated with SFO-1-producing E. cloacae acquisition. This study describes for the first time an outbreak in which an SFO-1-producing E. cloacae strain was involved. Note that so far, this β-lactamase has previously been isolated in only a single case of E. cloacae infection in Japan.

Antecedentes Las herramientas genotipicas basadas en el analisis de la region V3 de la envuelta viral se perfilan como la alternativa a los ensayos fenotipicos para la determinacion del tropismo del VIH por los receptores de quimiocinas CCR5 y CXCR4 en la practica clinica. Este trabajo evalua la concordancia entre los distintos algoritmos de interpretacion genotipica actualmente disponibles en pacientes VIH infectados con subtipo B versus subtipos no-B. Metodos Se seleccionaron pacientes VIH positivos, procedentes del Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), Espana. A partir de muestras de plasma, se amplifico y secuencio la region V3 de la envuelta viral. El tropismo viral se determino usando 8 algoritmos genotipicos distintos. La concordancia entre los distintos predictores se evaluo calculando el indice de concordancia kappa. El subtipo genetico fue determinado por analisis filogenetico. Resultados Se incluyeron un total de 92 pacientes, 72 infectados por subtipo B y 20 por no-B. En pacientes con subtipo B, se obtuvieron valores significativos de kappa para todas combinaciones posibles (n=28) entre los algoritmos genotipicos analizados. La mejores valores entre predictores no relacionados se obtuvieron para webPSSMSINSI/WetcatPART (k: 0,771) y webPSSMSINSI/geno2pheno (k: 0,574). En subtipos no-B solo se obtuvieron valores significativos para 13 combinaciones, correspondiendo los mejores a PSSMX4R5/WetcatPART (k: 0,600) y PSSMSINSI/Charge rule (k: 0,590). Conclusion Existe una alta concordancia entre los distintos algoritmos genotipicos utilizados para la determinacion del tropismo viral en pacientes infectados con subtipo B, especialmente con webPSSMSINSI y geno2pheno o Wetcat. Sin embargo, la concordancia general de los algoritmos para subtipos no-B es menor. Esta falta de consenso podria justificarse por la baja prevalencia de los subtipos no B tienen en las bases de datos empleadas para el entrenamiento de los predictores genotipicos.

Un nuevo test multiplex basado en una reaccion en cadena de polimerasa en tiempo real LightCycler Septi- Fast® permite la identificacion de 25 especies bacterianas y fungicas directamente desde la sangre. El test SeptiFast® ha sido utilizado para el diagnostico etiologico rapido de la endocarditis infecciosa, pero no ha sido ensayado directamente sobre las vegetaciones cardiacas de pacientes intervenidos por endocarditis infecciosa. Se realizo un estudio prospectivo para analizar 15 muestras de tejido valvular con endocarditis infecciosa activa utilizando SeptiFast® y comparando su sensibilidad con el hemocultivo, el cultivo del tejido valvular y el SeptiFast® en sangre. La sensibilidad del SeptiFast® del tejido valvular fue del 100%, confirmo el diagnostico obtenido mediante hemocultivo en 13 casos y proporciono el diagnostico etiologico en 2 casos con hemocultivo negativo. La sensibilidad de SeptiFast en el tejido valvular fue superior al cultivo convencional de las vegetaciones y al SeptiFast en sangre.

The Spanish HIV-2 and HTLV Group was founded in 1989. Since then, a total of 144 cases of HTLV-1 and 717 cases of infection with HTLV-2 have been reported. Most patients infected with HTLV-1 are immigrants from Latin America and Sub-Saharan Africa. A total of 34 patients had developed diseases associated with HTLV-1: 21 tropical spastic paraparesis and 13 leukemias. The profile of patients infected with HTLV-2 is of males, native Spaniards, intravenous drug users and coinfected with HIV-1. The majority of transfusion centres in Spain have recently introduced anti-HTLV screening of blood donors, at the moment only among persons coming from HTLV-1 endemic areas. In 2009 a total of 30 new HIV-2 cases were reported, making a total of 216 so far. Most are male, originating from Sub-Saharan Africa.

With a novel real-time multiplex polymerase chain reaction test, the LightCycler SeptiFast® test, 25 bacterial and fungal species can be identified directly in blood. The SeptiFast® test has been used for rapid etiologic diagnosis in infectious endocarditis using blood samples but has not been evaluated directly on cardiac vegetations in patients being treated for infectious endocarditis. We prospectively analyzed 15 samples of heart valve tissue with active infectious endocarditis using the SeptiFast® test and compared the test's sensitivity with that of blood culture, valve tissue culture, and the SeptiFast® test in blood. The sensitivity of the SeptiFast test in heart valve tissue was 100%. The test results confirmed the diagnosis obtained using blood culture in 13 cases and identified the pathogen in 2 cases where blood culture tested negative. The sensitivity of the SeptiFast® test in heart valve tissue was greater than that obtained with conventional culture of vegetations or with the SeptiFast test in blood.

OBJECTIVES: To evaluate the potential improvement of antimicrobial treatment by utilizing a new multiplex polymerase chain reaction (PCR) assay that identifies sepsis-relevant microorganisms in blood. DESIGN: Prospective, observational international multicentered trial. SETTING: University hospitals in Germany (n = 2), Spain (n = 1), and the United States (n = 1), and one Italian tertiary general hospital. PATIENTS: 436 sepsis patients with 467 episodes of antimicrobial treatment. METHODS: Whole blood for PCR and blood culture (BC) analysis was sampled independently for each episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of gainable days on early adequate antimicrobial treatment attributable to PCR findings was assessed. MEASUREMENTS AND MAIN RESULTS: Sepsis criteria, days on antimicrobial therapy, antimicrobial substances administered, and microorganisms identified by PCR and BC susceptibility tests. RESULTS: BC diagnosed 117 clinically relevant microorganisms; PCR identified 154. Ninety-nine episodes were BC positive (BC+); 131 episodes were PCR positive (PCR+). Overall, 127.8 days of clinically inadequate empirical antibiotic treatment in the 99 BC+ episodes were observed. Utilization of PCR-aided diagnostics calculates to a potential reduction of 106.5 clinically inadequate treatment days. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (confidence interval 15-31) and 36.4 days/100 tests in the intensive care and surgical ward populations (confidence interval 22-51). CONCLUSIONS: Rapid PCR identification of microorganisms may contribute to a reduction of early inadequate antibiotic treatment in sepsis.

Resumen Introduccion La deteccion de la multirresistencia a betalactamicos en Escherichia coli y Klebsiella pneumoniae es una cuestion clinicamente relevante. Por otro lado, es interesante diferenciar entre la produccion de betalactamasas de espectro extendido (BLEE) y otros mecanismos de resistencia para evitar el tratamiento inadecuado de infecciones causadas por este tipo de cepas. El objetivo del presente estudio fue comparar la capacidad de las pruebas confirmatorias de los sistemas automatizados Vitek 2 y Phoenix para detectar la produccion de BLEE en E. coli y K. pneumoniae. Material y metodos Se ensayaron 193 aislamientos clinicos fenotipicamente productores de BLEE (174 E. coli y 19 K. pneumoniae) por Vitek 2 y BD Phoenix System y se utilizaron las tarjetas AST-N058 y los paneles UNMIC/ID-62, respectivamente. Se consideraron metodos fenotipicos de referencia la prueba de sinergia con doble disco y Etest. Como controles positivos y negativos se ensayaron 12 cepas genotipicamente caracterizadas. Resultados En el caso de los aislamientos clinicos, la sensibilidad fue del 99,5% para Vitek 2 y del 95,3% para Phoenix. En las cepas control no hubo diferencias entre ambos sistemas. La ejecucion del sistema experto elevo la sensibilidad del Phoenix al 100%. Sin embargo, el sistema experto de Vitek 2 considero incoherentes los resultados obtenidos en 7 aislamientos con la prueba para BLEE positiva. Conclusion La sensibilidad de la prueba confirmatoria para la produccion de BLEE es superior en las tarjetas N-058 de Vitek. No obstante, la actuacion de los sistemas expertos situa a ambos sistemas a la misma altura en su capacidad de deteccion de BLEE en E. coli y K. pneumoniae.

The purpose of this paper was to investigate the occurrence of carbapenem-resistant Enterobacter cloacae in our institution, to detect the carbapenemase-associated resistance and to determine the genetic relatedness of the isolates. Species identification and antimicrobial susceptibility testing were performed using the Vitek 2 system and Etest. Multiplex polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) was used for the detection of extended-spectrum beta-lactamase (ESBL)-producers. The bla (IMP) and bla (VIM) genes were amplified by PCR and sequenced. The DiversiLab System was used for strain-typing. During the period 2006-2008, 12 different isolates of carbapenem-resistant E. cloacae (2.3 %) were recovered in our laboratory. Only two positive isolates for the bla (VIM) gene were detected. The minimum inhibitory concentration (MIC) values were higher for all carbapenems in the group of non-metallo-beta-lactamase (MBL)-producers. All isolates showed MIC valuesor=2 against this tigecycline. The two bla (VIM-1)-carrying isolates showed different genotypes. For non-MBL-producers, two clonally related clusters were observed. Different mechanisms can be associated with carbapenem-resistance in E. cloacae. MBL-producing strains are less prevalent than those with other mechanisms of resistance. The clonal relationship confirms the risk of spread of these organisms with the transfer of patients to different wards and the persistence of these clones over time or the 'de novo' acquisition of the resistance caused by the selective pressure exerted by antibiotics treatments.

The CD4+ T-cell reduction characteristic of human immunodeficiency virus type 1 (HIV-1) infection is thought to result, in addition to infected T-cell death, mainly from uninfected bystander T-cell apoptosis. Nevertheless, the immunological and virological mechanisms leading to T-cell death during HIV-1 infection are not yet fully understood. In the present study, we analysed the individual implication of the p38 mitogen-activated protein kinase (MAPK) isoforms (p38α, p38β, p38γ and p38δ) during apoptosis induced by HIV-1, taking into account that HIV-1 replication is known to be blocked by p38 inhibitors. For this purpose, we used the SupT1 cell line, where death induced by HIV-1 mainly occurs by uninfected bystander cell apoptosis. A variety of SupT1-based cell lines were constructed constitutively expressing, under the control of cytomegalovirus promoter (PCMV), each dominant-negative (dn) p38 isoform and each wild-type p38 isoform as a control. An enhanced green fluorescent protein marker gene, under the control of the HIV-1 promoter, was inserted in all of them. These cell lines were infected with HIV-1 and analysed by flow cytometry. We found that survival in SupT1-based cell lines infected by HIV-1 was increased by the p38αdn, p38γdn and p38δdn isoforms, but not by the p38βdn isoform. HIV-1 replication was delayed most by p38δdn and to a lesser extent by p38αdn and p38γdn. Moreover, these three isoforms, p38αdn, p38γdn and p38δdn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38α, p38γ and p38δ, but not p38β, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.

This study was partially financed by the CIBER de Enfermedades Respiratorias (CIBERES) from the ISCIII (CB06/06/0058) and by Fundación BBVA-FundacioóCarolina (research fellowship of Maricela Valerio)., Muñoz, P; Kestler, M; De Alarcon, A; Miro, JM; Bermejo, J; Rodriguez-Abella, H; Farinas, MC; Belaustegui, MC; Mestres, C; Llinares, P; Goenaga, M; Navas, E; Oteo, JA; Tarabini, P; Bouza, E.

Viral hepatitis is an infectious disease affecting the liver. At this time, five different human hepatitis viruses are recognized and characterized in detail: Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). The hepatitis viruses differ widely in their modes of transmission and clinical features. Whereas all of them can cause acute hepatitis, only HBV, HDV, and HCV cause chronic hepatitis. This article reviews the epidemiology and clinical manifestations of the different types of viral hepatitis.

los campos de la biomedicina. Una via previamente desconocida en la que fragmentos de doble hebra de ARN de 21 a 23 residuos (ARNpi) median la degradacion especifica de secuencias de ARN mensajero (RRNm) se esta convirtiendo en una de las herramientas mas poderosas en la investigacion en biologia cellular y genetica. Existen grandes expectativas en el uso terapeutico del silenciamiento genetico por medio de ARNi que se basan en su potencia, especificidad y fisiologia. La comunicacion de las primeras evidencias sobre la efectividad del ARNi como supresor de la replicacion de virus de la inmunodeficiencia humana (VIH) ha estimulado de nuevo el desarrollo de estrategias de terapia genica o molecular para el tratamiento de la infeccion por VIH.

field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection.

The present study assesses the clinical usefulness of the hepatitis C core antigen assay for monitoring of patients being treated for chronic hepatitis C virus (HCV) infection. Eighty-six serum samples were selected at random from 16 patients and levels of HCV RNA and HCV core antigen were determined simultaneously and in parallel to compare both techniques. The data obtained were compared by Pearson's correlation and the coefficients calculated by Fisher transformation and by calculating the difference and standard error. A good linear correlation was observed between both techniques. Maximum correlation, with significant difference, was found between patients infected with the 1a genotype and other genotypes. In conclusion, the HCV core antigen assay is useful for the diagnosis of early infection; however, its use for determining the exact timing of viral elimination during treatment is clearly unsuitable.

Hepatitis B virus (HBV) genotyping and treatment resistances analysis provide key information for the study of infected patients. Among the existing HBV genotyping methods, restriction fragment length polymorphism (RFLP) based methods are used widely, but HBV genetic variability may lead to wrong results. A simple method for HBV genotyping is described. This single assay provides information related to both genotyping and lamivudine resistance. measuring genotyping reliability. A short region including the YMDD motif of the polymerase gene was analyzed using cluster analysis of 55 isolates. To discriminate the seven HBV genotypes, a selected fragment was used as representative of each genotype (consensus sequences). Comparison between complete genomes from GenBank and from the YMDD analysis using PHILIP package was used for method testing. Sequencing of 102 different serum samples was carried out, and the results were compared with representative sequences. Consensus sequences were chosen corresponding to the different HBV genotypes. Statistical comparison between our method and others gives more than 90% of coincidences with a critical level of 0.056. Comparison between RFLP and the method described gives 3% of discordance results and 3% of untypables samples by RFLP. All the discordant results observed had a change in the pre-S sequence which introduced a new restriction site.

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.

Aims Surgery for infective endocarditis (IE) is associated with high mortality. Our objectives were to describe the experience with surgical treatment for IE in Spain, and to identify predictors of in-hospital mortality. Methods Prospective cohort of 1000 consecutive patients with IE. Data were collected in 26 Spanish hospitals. Results Surgery was performed in 437 patients (43.7%). Patients treated with surgery were younger and predominantly male. They presented fewer comorbid conditions and more often had negative blood cultures and heart failure. In-hospital mortality after surgery was lower than in the medical therapy group (24.3 vs 30.7%, p = 0.02). In patients treated with surgery, endocarditis involved a native valve in 267 patients (61.1%), a prosthetic valve in 122 (27.9%), and a pacemaker lead with no clear further valve involvement in 48 (11.0%). The most common aetiologies were Staphylococcus (186, 42.6%), Streptococcus (97, 22.2%), and Enterococcus (49, 11.2%). The main indications for surgery were heart failure and severe valve regurgitation. A risk score for in-hospital mortality was developed using 7 prognostic variables with a similar predictive value (OR between 1.7 and 2.3): PALSUSE: prosthetic valve, age ≥ 70, large intracardiac destruction, Staphylococcus spp, urgent surgery, sex [female], EuroSCORE ≥ 10. In-hospital mortality ranged from 0% in patients with a PALSUSE score of 0 to 45.4% in patients with PALSUSE score > 3. Conclusions The prognosis of IE surgery is highly variable. The PALSUSE score could help to identify patients with higher in-hospital mortality. 4.036 JCR (2014) Q1, 29/123 Cardiac and cardiovascular systems UEM

Fundamento La diarrea, aguda o cronica, es una complicacion comun en los pacientes inmunodeprimidos tales como los infectados por el virus de la inmunodeficiencia humana (VIH), sometidos a trasplantes de medula osea o de organo solido y aquellos con leucemias u otras alteraciones del sistema inmunitario. Dada la importancia de reconocer las posibles etiologias de la diarrea a al ahora de administrar una terapia antimicrobiana especifica o evitar un diagnostico incorrecto de rechazo postrasplante, hemos investigado la presencia de antigeno de astrovirus y adenovirus en las heces de pacientes inmunodeprimidos. Pacientes Y METodos Se analizaron las heces de 258 pacientes inmunodeprimidos hospitalizados en el Complejo Hospitalaria Universitario de Santiago de Compostela y diagnosticados de diarrea durante 1997- 99. La deteccion de los antigenos viricos se realizo mediante enzimoinmunoanalisis (EIA). Tambien se analizaron para otros enteropatogenos comunes. Resultados El antigeno de adenovirus fue positivo en 5 casos (2%) y el astrovirus en 12 (5%). Los pacientes mas frecuentemente afectados fueron los prematuros y los hematologicos. Se detecto antigeno de astrovirus en tres pacientes infectados por VIH. La mayoria de los casos positivos presentaban una alteracion de la flora intestinal o presencia de toxina de Clostridium difficile, ambas situaciones relacionadas con tratamiento antibiotico prolongado. Conclusiones Los astrovirus y los adenovirus son enteropatogenos a considerar en individuos inmunocomprometidos hospitalizados. Es, pues, conveniente realizar un diagnostico certero de la etiologia de la diarrea de cara a la administracion de un tratamiento antimicrobiano, en los casos en que este sea necesario, o evitar un diagnostico incorrecto de rechazo postrasplante.