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Introduction. Bacteriemia is a major cause of morbidity and mortality among hospitalized patients worldwide. Early identification of microorganisms from blood culture can lead to improvement of treatment and outcomes. Methods. The study was divided into two phases. The first phase when a comparison of the methods was made to check the concordance between them, using as a reference the standard method implemented in the laboratory. In a second phase, both methods are combined. We used the rapid identification method and when it could not identify we used the standard method. The microorganisms that were not identified by either of the two methods were identified from colony at 24 hours Results. A total of 589 microbial positive blood cultures have been included in the present study. With the rapid method we obtained 96% and 88% identification results for Gram-negative bacilli (GNB) and Gram-positive cocci (GPC) respectively. In this study we observed that the combination of the rapid and standard method achieved identifications of 98% and 97% for GNB and GPC respectively. Conclusions. The data analysed shows that both methods combined perform better than individually. We achieved an optimization of the identification of microorganisms directly from positive blood cultures by MALDI-TOF. This combination identified 98% of the microorganisms in between ten minutes to one hour and a half since the blood culture flagged positive.

The epidemiology of community-acquired pneumonia (CAP) has changed, influenced by sociosanitary conditions and vaccination status. We aimed to analyze the recent epidemiology of bacterial CAP in hospitalized children in a setting with high pneumococcal vaccination coverage and to describe the clinical characteristics of pediatric Staphylococcus aureus CAP.Children17 years old hospitalized from 2008 to 2018 with bacterial CAP in 5 tertiary hospitals in Spain were included. Cases with pneumococcal CAP were randomly selected as comparative group following a case-control ratio of 2:1 with S. aureus CAP.A total of 313 bacterial CAP were diagnosed: Streptococcus pneumoniae CAP (n = 236, 75.4%), Streptococcus pyogenes CAP (n = 43, 13.7%) and S. aureus CAP (n = 34, 10.9%). Throughout the study period, the prevalence of S. pyogenes increased (annual percentage change: +16.1% [95% CI: 1.7-32.4], P = 0.031), S. pneumoniae decreased (annual percentage change: -4.4% [95 CI: -8.8 to 0.2], P = 0.057) and S. aureus remained stable. Nine isolates of S. aureus (26.5%) were methicillin-resistant. Seventeen cases (50%) with S. aureus CAP had some pulmonary complication and 21 (61.7%) required intensive care. S. pneumoniae CAP showed a trend toward higher prevalence of pulmonary complications compared with S. aureus CAP (69.1% vs. 50.0%, P = 0.060), including higher frequency of pulmonary necrosis (32.4% vs. 5.9%, P = 0.003).The incidence of S. aureus CAP in children remained stable, whereas the prevalence of pneumococcal CAP decreased and S. pyogenes CAP increased. Patients with S. aureus presented a high frequency of severe outcomes, but a lower risk of pulmonary complications than patients with S. pneumoniae.

Objectives. Congenital cytomegalovirus infection (cCMV) has been considered more prevalent among HIV-exposed children during pregnancy. Spanish national guidelines recommend the cCMV screening in these newborns. Nowadays, pregnant women have a better control of HIV infection compared to previous decades. We aim to analyze the prevalence and associated risk factors to cCMV in these children. Patients and methods. A retrospective cross-sectorial study was performed. All newborns exposed to HIV were assisted in a third-level hospital (2014-2020). Epidemiological and clinical data of the mother and newborn were recorded. Shell vial urine culture and/or CRP were performed along the two first weeks of life for the neonatal screening of cCMV. Results. Overall 69 newborns were enrolled. A high proportion (82.4%) of the mothers had been diagnosed with HIV before getting pregnant. All women received ART during the pregnancy. Median T-CD4 lymphocytes before delivery was 641/mm3 (IQR: 480-865) and the viral load was undetectable in 83.6%. Serological test for CMV along the first trimester of pregnancy was performed in 73.5% (positive IgG in 96%). There were no congenital cases of HIV neither cCMV (CI 95%:0-5.3%). Conclusions. The cCMV prevalence in newborns exposed to HIV was 0%, lower than reported before, probably related to a better and earlier ART during pregnancy, leading to a better immunological status.

Herbaspirillum species are Gram-negative bacteria belonging to the class Betaproteobacteria, order Burkholderiales. The phylogenetic and phenotypic similarities among these groups easily lead to species misidentification. Herbaspirillum bacteraemia is an uncommon clinical entity. The objective of this review is to collect information to contribute to the management of this infection. We describe our own case series and review the cases reported in the literature. Cancer appears as the major underlying disease. The main source of bacteraemia was respiratory. Phenotypic identification methods often misidentify this species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular methods identify at genus level, but species assignment is not reliable. Herbaspirillum spp. showed a highly susceptible antimicrobial profile. β-Lactams showed good activity with low MIC values, except ampicillin. All isolates were resistant to colistin, suggesting an intrinsic resistance mechanism. Herbaspirillum spp. is an uncommon pathogen. MALDI-TOF MS or molecular methods are necessary to achieve a reliable genus identification. These species are not multidrug resistant. Piperacillin/tazobactam or ceftazidime might be a good treatment for this microorganism.

Background In Spain, data of candidemia are limited to surveys conducted in specific areas or tertiary care centers. Also, in recent years, attention has shifted toward episodes of candidemia in non-ICU wards. Aims We reviewed the cases of Candida isolates recovered from the blood of patients admitted to the Emergency Room (ER) in our tertiary care hospital. Methods The patients selected for this study had an isolation of Candida in the blood culture. All data were collected retrospectively from the clinical records of a 11-year period. Results Candida albicans and other species of the genus were present in 10 and 18 patients, respectively. The patients did not present different clinical features in comparison with other reports of hospitalized patients. All patients had several risk factors for candidemia. Only two patients had received previous antifungal therapy before admission. All the isolates of C. albicans, Candida glabrata and the only isolate of Candida tropicalis were susceptible to all the antifungal agents tested. Only one isolate of Candida parapsilosis was susceptible dose-dependent to fluconazole, and the only isolate of Candida metapsilosis was resistant to fluconazole. Conclusions It is essential to evaluate the risk factors, underlying conditions and clinical features in non-hospitalized patients in order to determine whether an empirical treatment for candidemia is appropriate.

Frontline health care workers (HCW) have higher risk than the general population to become infected by SARS.CoV.2, so they were a priority group for Covid-19 vaccine administration. We compared the incidence and prevalence of HCW infected pre-and post-vaccination with the BNT162b2 mRNA COVID-19 vaccine.Prospective observational study carried out between 01/12/20 and 07/03/21 in La Paz University Hospital, Madrid (Spain). SARS.CoV.2 positive cases in HCW after vaccination were collected and compared to those hospitalized COVID-19 patients at the same hospital.Two weeks after finishing the first round of vaccinations daily new cases of HCW infections (symptomatic and asymptomatic) decreased substantially and cumulative cases of infected HCW and hospitalized COVID-19 patients started to diverge. No new positive cases of HCW infection were registered seven days after the second dose of BNT162b2 mRNA COVID-19 vaccine.BNT162b2 mRNA COVID-19 vaccine is highly effective in Spanish HCW.Los trabajadores sanitarios (TS) de primera línea tienen mayor riesgo de infectarse de SARS-CoV-2 que la población general, por lo que han sido un grupo prioritario para la vacunación frente a COVID-19. Comparamos la incidencia y prevalencia de TS infectados antes y después de la vacunación con BNT162b2 mRNA frente a COVID-19.Estudio prospectivo observacional realizado entre 01 de diciembre de 2021 en el Hospital Universitario La Paz, Madrid, España. Se registraron los casos positivos para SARS-CoV-2 en TS y se compararon con los hospitalizados por COVID-19.Dos semanas tras la primera ronda de vacunación las nuevas infecciones en TS (sintomáticas y asintomáticos) disminuyeron sustancialmente y los casos acumulados de TS infectados y pacientes hospitalizados por COVID-19 empezaron a divergir. No hubo nuevas infecciones en TS vacunados a los siete días de la segunda dosis de la vacuna.La vacuna BNT162b2 mRNA frente a SARS-CoV-2 es altamente eficaz en TS españoles.

The multisystem inflammatory syndrome in children (MIS-C) is a novel and concerning entity related to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Although MIS-C has been the subject of intensive research efforts, its pathophysiology and optimal treatment remain elusive. We studied the clinical features, laboratory findings, and immunoinflammatory profiles of seven children prospectively admitted to a pediatric intensive care unit (PICU) during the first wave of the pandemic. All patients had immunoglobulin (Ig)-G against SARS-CoV-2, four of seven patients had both IgM and IgG, and in one of the 7 SARS-CoV-2 was detected in a respiratory sample. All patients received intravenous fluid boluses (median: 15 mL/kg) and norepinephrine. The most common form of respiratory support was supplemental oxygen via nasal cannula. None of the patients needed mechanical ventilation. The cardiovascular system was frequently involved. All patients had an elevated troponin-I (median: 107.3 ng/L). Four out of seven patients had coronary artery abnormalities, and two of seven had both abnormal electrocardiogram (EKG) findings and evidence of left ventricular dysfunction on echocardiogram. Ig levels and complement function were normal. Peripheral blood phenotyping with flow cytometry showed decreased T-cell numbers at the expense of CD8+ T-cells. Cytokine profiling showed a heterogeneous increase in interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-18, IL-2Ra, IL-10, and IL-1Ra that tended to normalize after treatment. Our study shows that children with MIS-C have elevated plasma levels of pro- and anti-inflammatory cytokines in the acute phase of the disease without other relevant immunologic disturbances. These findings suggest the presence of a mixed antagonist response syndrome (MARS) similar to that present in pediatric sepsis. Combining a meticulous differential diagnosis with cautiously coordinated immunomodulatory therapy and high-quality supportive care can help clinicians avoid causing iatrogenic harm in patients with MIS-C.

REIPI-SEIMC COVID-19 group and COVID@HULP group., [Background] The clinical presentation of COVID-19 in patients admitted to hospital is heterogeneous. We aimed to determine whether clinical phenotypes of patients with COVID-19 can be derived from clinical data, to assess the reproducibility of these phenotypes and correlation with prognosis, and to derive and validate a simplified probabilistic model for phenotype assignment. Phenotype identification was not primarily intended as a predictive tool for mortality., [Methods] In this study, we used data from two cohorts: the COVID-19@Spain cohort, a retrospective cohort including 4035 consecutive adult patients admitted to 127 hospitals in Spain with COVID-19 between Feb 2 and March 17, 2020, and the COVID-19@HULP cohort, including 2226 consecutive adult patients admitted to a teaching hospital in Madrid between Feb 25 and April 19, 2020. The COVID-19@Spain cohort was divided into a derivation cohort, comprising 2667 randomly selected patients, and an internal validation cohort, comprising the remaining 1368 patients. The COVID-19@HULP cohort was used as an external validation cohort. A probabilistic model for phenotype assignment was derived in the derivation cohort using multinomial logistic regression and validated in the internal validation cohort. The model was also applied to the external validation cohort. 30-day mortality and other prognostic variables were assessed in the derived phenotypes and in the phenotypes assigned by the probabilistic model., [Findings] Three distinct phenotypes were derived in the derivation cohort (n=2667)—phenotype A (516 [19%] patients), phenotype B (1955 [73%]) and phenotype C (196 [7%])—and reproduced in the internal validation cohort (n=1368)—phenotype A (233 [17%] patients), phenotype B (1019 [74%]), and phenotype C (116 [8%]). Patients with phenotype A were younger, were less frequently male, had mild viral symptoms, and had normal inflammatory parameters. Patients with phenotype B included more patients with obesity, lymphocytopenia, and moderately elevated inflammatory parameters. Patients with phenotype C included older patients with more comorbidities and even higher inflammatory parameters than phenotype B. We developed a simplified probabilistic model (validated in the internal validation cohort) for phenotype assignment, including 16 variables. In the derivation cohort, 30-day mortality rates were 2·5% (95% CI 1·4–4·3) for patients with phenotype A, 30·5% (28·5–32·6) for patients with phenotype B, and 60·7% (53·7–67·2) for patients with phenotype C (log-rank test p, [Interpretation] Patients admitted to hospital with COVID-19 can be classified into three phenotypes that correlate with mortality. We developed and validated a simplified tool for the probabilistic assignment of patients into phenotypes. These results might help to better classify patients for clinical management, but the pathophysiological mechanisms of the phenotypes must be investigated., [Funding] Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA., Funding: Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA.

BackgroundCarbapenem-resistant Enterobacteriaceae (CRE) are an emerging problem in the paediatric population worldwide with high mortality rates in bloodstream infection (BSI).ObjectivesTo evaluate predictors of 30 day mortality in CRE BSI in a paediatric cohort.MethodsA retrospective observational single-centre study (December 2005–August 2018) was conducted. Cases of CRE BSI in children 0 to 16 years were included. Microbiological identification (MALDI Biotyper) and antimicrobial susceptibility testing (Vitek2® and MicroScan panel NBC44) according to EUCAST breakpoints were performed. PCR OXVIKP® was used to confirm carbapenemase genes (OXA-48, VIM, KPC, NDM). Demographic characteristics, underlying diseases, source of bacteraemia, antimicrobial therapy and outcomes were collected from medical records. Survival analysis to establish predictors of 30 day mortality was performed.ResultsThirty-eight cases were included; 76.3% were hospital-acquired infections and 23.7% related to healthcare. All patients had at least one underlying comorbidity and 52.6% were recipients of an organ transplant. VIM carbapenemase was the predominant mechanism (92.1%). Previous CRE colonization or infection rate was 52.6%. Intestinal tract (26.3%) and vascular catheter (21.1%) were the most common sources of infection. Crude mortality within 30 days was 18.4% (7/38); directly related 30 day mortality was 10.5%. Conditions associated with an increment in 30 day mortality were intensive care admission and inadequate empirical therapy (P ConclusionsCRE BSI mortality rate is high. The most important factor related to 30 day survival in our CRE BSI cohort in children was empirical treatment that included at least one active antibiotic.

The aim of this work was to study the epidemiology of candidemia in our hospital in order to determine whether the T2MR system might be a useful tool for early diagnosis of candidemia in selected units. We perform a retrospective review of all candidemia episodes registered in the last 12 years in selected units of our hospital in adult and pediatric patients. Candida species and antifungal susceptibility patterns were registered. A total of 686 isolates were registered, of which 625 were infections due to the five most common species of Candida. C. albicans (45.6%) and C. parapsilosis (33.1%) were the predominant species found in our institution. In adults these species were closely followed by C. glabrata (12–21%) in all units. While in pediatric medical and intensive care units (PICU), these species were followed by other uncommon yeasts. Resistance rates to triazoles were low in C. albicans and C. parapsilosis. In C. glabrata and C. tropicalis the resistance rates to fluconazole ranged from 10.86 to 6.67%. Resistance rates for echinocandins were very low and all strains were susceptible to amphotericin B. T2Candida® might be useful to guide antifungal targeted treatment and discontinuation of antifungal empirical treatment in those units where the five most common Candida species represent more than the ninety percent of the isolates. The selection of medical and surgical units should be based on local epidemiology and antifungal susceptibility patterns. Incidence should be taken into account in order to make clinical decisions based on negative results. Lay Abstract T2Candida® might be useful selectively in clinical units according to their local epidemiology, antifungal resistance patterns, and incidence of candidemia. It optimizes the clinical value of positive results supporting decisions about targeted therapies or discontinuations based on negative results.

Objectives Early diagnosis of invasive Candida infections is a challenge for pediatricians, intensivists, and microbiologists. To fill this gap, a new nanodiagnostic method has been developed using manual application of T2 nuclear magnetic resonance to detect Candida species. The aim of this study was to evaluate, prospectively, the usefulness as a tool diagnosis of the T2Candida panel in pediatric patients admitted at the PICU compared with blood culture. Design This is a prospective, observational, and unicentric study to compare T2Candida results with simultaneous blood cultures for candidemia diagnose. Setting This study was carried out in a 1,300-bed tertiary care hospital with a 16-bed medical-surgical PICU. Patients Sixty-three patients from 0 to 17 years old were enrolled in this study, including those undergoing solid organ transplantation (kidney, liver, pulmonary, multivisceral, intestinal, and heart) and hematopoietic stem cell transplantation. Measurements and main results Seven patients were positive by the T2Candida test. Only two of them had the simultaneous positive blood culture. T2Candida yielded more positive results than blood cultures. Conclusions T2Candida might be useful for the diagnosis of candidemia in PICUs. The prevalence of candidemia might be underestimated in this pediatric population. The use of this diagnostic tool in these units may help clinicians to start adequate and timely antifungal treatments.

Early detection and identification of pathogens in bloodstream infections (BSI) is important to initiate or adjust antibiotic therapy as soon as possible. The current gold standard for diagnostic of BSI infection is the blood culture, that has a turnaround time of one to few days. Molecular tests performed directly in blood samples have promised faster diagnostics, with response times of a few hours, but their implementation into the clinical routine has been hampered by critical technical and procedural problems. Assay integration into laboratory workflows with random-access loading mode and minimal hands-on time is essential to meet rapid response times. Decreasing assay costs will favor fair clinical evaluations and might increase the applicability of the assays. Control of background contamination with bacterial DNA is one of the most difficult problems and might be avoided with pathogen-specific real-time PCR designs oriented to particular patient groups, or perhaps by quantitative, next-generation sequencing approaches.

Anaerobiospirillum succiniciproducens is a gram-negative anaerobic spiral rod which is part of the normal flora of dogs and cats and can produce bacteraemia and diarrhoea in humans. In this report we describe two cases of bacteraemia caused by A. succiniciproducens which was successfully identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a comprehensive literature review of 48 cases of A. succiniciproducens bacteraemia in which we describe previous underlying conditions, clinical presentations, identification methodology and antibiotic susceptibility data.

Extraintestinal pathogenic Escherichia coli (ExPEC) are among the most frequently isolated bacterial pathogens in hospitals. They are considered opportunistic pathogens and are found mostly in urinary and bloodstream infections. They are genetically diverse, and many studies have sought associations between genotypes or virulence genes and infection site, severity, or outcome, with varied, often contradictory, results. To understand these difficulties, we have analyzed the diversity patterns in the core genomes and virulomes of more than 500 ExPEC isolates from 5 different collections. The core genome was analyzed using a multilocus sequence type-based single-nucleotide polymorphism (SNP) pyrosequencing approach, while the virulence gene content (the virulome) was studied by polymerase chain reaction detection of 25 representative genes. SNP typing showed a similar population structure in the different collections: half of the isolates belong to a few sequence types (5 to 8), while the other half is composed of a large diversity of sequence types that are found once or twice. Sampling analysis by rarefaction plots of SNP profiles showed saturation curves indicative of a limited diversity. Contrary to this, the virulome shows an extremely high diversity, with almost as many gene profiles as isolates, and linear, nonsaturating, rarefaction plots, even within sequence types. These data show that genetic exchange rates are very heterogeneous along the chromosome, being much higher in the virulome fraction of the genome than in the core genome.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a frequent cause of bacteremia and sepsis, but the role of ExPEC genetic virulence factors (VFs) in sepsis development and outcome is ill-defined. Prospective study including 120 adult patients with E. coli bacteremia to investigate the impact of bacterial and host factors on sepsis severity and mortality. Patients' clinical and demographic data were registered. Phylogenetic background of E. coli isolates was analyzed by SNP pyrosequencing and VFs by PCR. The E. coli isolates presented an epidemic population structure with 6 dominant clones making up to half of the isolates. VF gene profiles were highly diverse. Multivariate analysis for sepsis severity showed that the presence of cnf and blaTEM genes increased the risk of severe illness by 6.75 (95% confidence interval [CI] 1.79-24.71) and 2.59 (95% CI 1.04-6.43) times respectively, while each point in the Pitt score increased the risk by 1.34 (95% CI 1.02-1.76) times. Multivariate analysis for mortality showed that active chemotherapy (OR 17.87, 95% CI 3.35-95.45), McCabe-Jackson Index (OR for rapidly fatal category 120.15, 95% CI 4.19-3446.23), Pitt index (OR 1.78, 95% CI 1.25-2.56) and presence of fyuA gene (OR 8.05, 95% CI 1.37-47.12) were associated to increased mortality while the presence of P fimbriae genes had a protective role (OR 0.094, 95%IC 0.018-0.494). Bacteremic E. coli had a high diversity of genetic backgrounds and VF gene profiles. Bacterial VFs and host determinants had an impact on disease evolution and mortality.

We have performed a retrospective, before-after comparison of turnaround time and therapy adjustment parameters before and after the introduction of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) plus mecA polymerase chain reaction (PCR) for the identification of methicillin-resistant Staphylococcus aureus (MRSA) in positive blood cultures. There were 227 episodes of S. aureus bacteremia during the study periods. The pre-MALDI-TOF and post-MALDI-TOF groups included 133 and 94 patients, respectively. The two rapid methods performed sequentially decreased the turnaround time of MRSA identification by nearly 50% (2.06 ± 1.95 vs. 3.95 ± 1.70 days). There was no significant reduction in the length of hospitalization (28.27 ± 32.16 vs. 28.62 ± 28.75 days). In both groups, the adequacy of the empirical antibacterial therapy was similar (59.49% vs. 51.31%), but the optimization of the therapy was more frequent in the post-MALDI-TOF group. Routine implementation of these techniques provides results earlier than conventional methods and increases the proportion of episodes with adequate change of empirical to directed antimicrobial therapy.

Human cytomegalovirus (HCMV) may cause severe or fatal disease among immunocompromised patients. The first line prophylaxis and systemic HCMV disease therapy is ganciclovir (GCV). The presence of GCV-resistant virus has been linked to fatal HCMV disease. The implementation of rapid and sensitive techniques for the early detection and monitoring of GCV-resistance may be helpful to support antiviral therapy management. A pyrosequencing assay for the detection and quantitation of the most frequent mutations conferring moderate- and high-grade GCV resistance was implemented. The pyrosequencing achieved an analytical sensitivity for adequate interpretation of ≥103 copies/ml. The assay was validated with 18 whole blood samples taken over a 6-month period from an umbilical cord blood recipient infected persistently with HCMV and allowed the detection and monitoring of the M460I and A594V GCV-resistant mutations. The percentage of resistant quasispecies ranged from 7.9% to 55.2% for the M460I mutation and from 19.8% to 43% for the A594V mutation. Clearance of the M460I mutation occurred in parallel with a decrease in the HCMV viremia, while the A594V mutation persisted. The pyrosequencing method for detection of GCV is sensitive enough to be used directly on clinical samples for the early identification of resistance mutations and allows the quantitation of resistant and wild type virus quasispecies within hours. The quantitation of minor resistant variants is an important issue to understand their relationship with viral load modification, and potentially anticipate treatment adjustment. J. Med. Virol. 85:1250–1257, 2013. © 2013 Wiley Periodicals, Inc.

Bacteraemia due to carbapenemase-producing Enterobacteriaceae is an emerging medical problem. Management of this entity is complicated by the difficulty in Identifying resistance patterns and the limited therapeutic options. A cohort study was performed including all episodes of bloodstream infection due to OXA-48-producing Enterobacteriaceae (O48PE), occurring between July 2010 and April 2012. Data on predisposing factors, clinical presentation, therapy and outcome were collected from medical records. There were 40 cases of bacteraemia caused by O48PE, 35 Klebsiella pneumoniae and five Escherichia coli. Patients were elderly with significant comorbidities (57.5% underlying malignancy). Thirty-five cases (87.5%) were nosocomial, and five (12.5%) were healthcare-associated. Patients had frequently been exposed to antibiotics and to invasive procedures during hospitalization. The most common source of bacteraemia was the urinary tract followed by deep intra-abdominal surgical site infection. Clinical presentation was severe sepsis or shock in 18 cases (45%). Extended-spectrum β-lactamase production was detected in 92.5% of isolates. MIC90 for ertapenem, imipenem and meropenem were 32, 16 and 16 mg/L, respectively. Most frequently preserved antibiotics were amikacin, colistin, tigecycline and fosfomycin. These antibiotics combined are the basis of targeted therapies, including carbapenem in selected cases. Median delay in starting clinically adequate and microbiologically appropriate treatment was 3 days. Crude mortality during admission and within 30 days from bacteraemia was 65% and 50%, respectively. Bloodstream infections caused by O48PE have a poor prognosis. Delay in diagnosis and in initiation of optimal antimicrobial therapy is frequent. Suspicion and rapid identification could contribute to improving outcomes.

Background. Because most infections caused by carbapenemase-producing Enterobacteriaceae (CPE) begin during hospitalization, there are limited data about community-onset (CO) infections caused by CPE. Our aim is to describe the frequency of CO infections caused by CPE as well as the clinical features of CO bloodstream infections (CO-BSIs). Methods. This study includes retrospective case series of CO infections caused by CPE in a tertiary hospital from January 2010 to July 2014. Any clinical sample with a positive culture for CPE that had been ordered by primary care doctors or by doctors at the emergency room (ER) were classified as CO. Epidemiological and microbiological features of CO cases were assessed as were clinical features of CO-BSIs. Results. Of 780 clinical samples with CPE, 180 were requested at the ER or by primary care doctors (22.9%), 150 of which were produced by Klebsiella pneumoniae (83.3%). The blaOXA-48 gene was detected in 149 isolates (82.8%) followed by the blaVIM gene, 29 (16.1%). Sixty-one patients (33.9%) had a prior history of CPE infection/colonization. Thirty-four of the 119 (28.6%) patients without prior history of CPE infection/colonization did not fulfill Friedman criteria for healthcare-associated infections (HAIs). Considering previous hospitalization of up to 12 months as a criterion for defining HAI, only 16 (13.4%) cases were identified as community-acquired infections. The most frequent positive sample was urine (133 of 180; 73.9%). Twenty-one (11.7%) patients had a BSI, 9 of them secondary to urinary tract infections (42.9%). Thirty-day crude mortality among patients with BSI was 23.8% (5 of 21). Conclusions. Community-onset infections caused by CPE are an important subgroup of all CPE infections. The urinary tract is the main source. Bloodstream infections accounted for more than 10% of the cases.

This article describes the development and validation with clinical samples of an internally controlled multiplex quantitative real-time PCR (QRT-PCR) for human polyomaviruses BK (BKV) and JC (JCV). Blood and urine samples from renal transplant recipients with suspected nephropathy, and cerebrospinal fluid (CSF) specimens from AIDS, natalizumab-treated and HIV-negative patients with suspected progressive multifocal leukoencephalopathy, previously checked for BKV and JCV by conventional PCR, were tested by QRT-PCR. All samples positive by conventional PCR were confirmed by QRT-PCR. Four cases of JCV-associated neurological infection, including all those detected in natalizumab-treated patients, and one case of BKV-related neurological infection were only identified by QRT-PCR. BKV was quantified in the CSF of neurological patients for the first time. Analyses of the Quality Control for Molecular Diagnostics 2010 panel were "highly satisfactory" for BKV and "satisfactory" for JCV. The QRT-PCR is specific and reproducible. It improves the sensitivity of conventional PCR for the diagnosis of BKV and JCV infection in various diseases.

Please cite this paper as: del Rosal et al. (2011) Pandemic H1N1 influenza-associated hospitalizations in children in Madrid, Spain. Influenza and Other Respiratory Viruses 5(6), e544–e551. Objective To describe the epidemiological and clinical characteristics of children hospitalized with 2009 pandemic influenza (pH1N1) in Madrid, Spain. Patients/Methods We included patients less than 14 years of age admitted to one of 18 hospitals in Madrid, Spain, between May 1 and November 30, 2009 and diagnosed with pH1N1 by polymerase chain reaction. A retrospective chart review was conducted and data were compared by age, presence of high-risk medical conditions, and pediatric intensive care unit (PICU) admission. Results A total of 517 pH1N1 cases were included for final analysis. One hundred and forty-two patients (27·5%) had predisposing underlying illnesses, with immunosuppression (36 children, 7%) and moderate persistent asthma (34, 6·6%) being the most common ones. Patients with underlying medical conditions had longer hospital stays [median 5, interquartile range (IQR) 3–8 days, versus median 4, IQR 3–6, P

It is not known whether influenza-like illnesses (ILI) in pregnant women caused by influenza virus, specifically, those caused by the 2009 Influenza A H1N1 virus (nH1N1), can be clinically distinguished from those caused by other agents. From 1st July 2009 until 20th September 2009, an observational study including all pregnant women presenting at Hospital Universitario La Paz with an ILI was carried out. A specific reverse-transcriptase polymerase chain reaction (RT-PCR) for nH1N1 in nasopharyngeal swabs was prospectively carried out in all patients. Retrospectively, samples were analysed for multiple respiratory virus panel (RT-PCR microarray). Clinical, demographical and other microbiological variables were evaluated as well. A total of 45 pregnant women with ILI were admitted. Of these, 14 (31.1%) women had nH1N1 infection and 11 with a non-influenza ILI (35.48%) were positive for other viruses (five rhinovirus, four parainfluenza virus, one bocavirus and one adenovirus). In 20 patients, no aetiologic agent was identified. The clinical course of nH1N1 was mild, without deaths or severe complications. No significant differences were found when comparing the clinical presentation and course of patients with and without nH1N1 infection. Six women with nH1N1 infection received oseltamivir. Influenza and non-influenza ILI were clinically indistinguishable among pregnant women. Many ILI in pregnant women remain undiagnosed, despite undergoing an RT-PCR microarray for several respiratory viruses.

Objectives: To describe: 1) the main features of antimicrobial stewardship activites (ASA) in Spanish hospitals and 2) the perceptions of the Spanish Infectious Diseases (ID) community on ASA. Methods: An online survey was designed and distributed through the e-mailing lists of several working groups of the Spanish Clinical Microbiology and Infectious Diseases Society.

We have developed a rapid protocol for the identification of Candida species from positive blood cultures by combining a simple method for nucleic acid extraction and preparation using microbial storage cardboards with polymerase chain reaction (PCR) and pyrosequencing of a small region of the 18 S rRNA gene. The protocol is robust and easy to implement and can be performed in 4 h. The method was tested against a collection of clinical blood cultures. Agreement of sequence identifications with standard microbiological methods was 100%.

We present a case of bacteraemia due to meticillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene. The susceptibility to meticillin of Staphylococcus aureus was investigated directly from one blood culture bottle using GenomEra MRSA/SA (Abacus Diagnostica Oy) test. This test identified S. aureus but the presence of the mecA gene result was negative, and the isolate was reported as meticillin-sensitive Staphylococcus aureus (MSSA). Susceptibility studies were done using VITEK 2 AST-P588 susceptibility cards (bioMérieux). The strain was identified as MRSA by the VITEK 2 system, although oxacillin MIC was low (0.5 µg ml−1). In view of these results, the isolate was tested for the presence of the mecC gene by a specific PCR and was verified as MRSA carrying mecC. The emergence of this new mecA homologue could have important consequences for the detection of MRSA when routine PCR methods are used as an identification method or provisional detection of MRSA, as in the case reported in this article, because S. aureus carrying the mecC gene will be wrongly diagnosed as meticillin susceptible. Negative results must be interpreted with caution and should be followed by conventional culture, and antimicrobial susceptibility testing or detection of mecC gene by a specific PCR.

We describe 12 cases of linezolid-resistant Enterococcus faecalis. The present study was done in 2 wards of Hospital Universitario La Paz in Madrid, Spain. The 2 wards involved were the intensive care unit (ICU) and reanimation unit. Twelve clinical strains of E. faecalis reported by the clinical laboratory as linezolid resistant based on MICs determined by E-test (AB Biodisk, Solna, Sweden) were collected between September 2005 and October 2006. The MIC of linezolid for all the resistant isolates was >128 microg/mL. The isolates were analyzed for the presence of the G2576T mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and pyrosequencing. Pyrosequencing showed that the first isolate had G and T at position 2576 in a 1:1 ratio, whereas the remaining ones had a wild type to mutant ratio of 1:3. PCR-RFLP showed that the mutations were in alleles 1, 3, and 4. The 12 isolates under investigation came from different patients but were indistinguishable by pulsed-field gel electrophoresis (n = 7) and repetitive extragenic palindromic sequence (REP)-PCR (n = 12). This is the first report of a clonal outbreak of linezolid-resistant E. faecalis in Spain. To prevent or minimize the emergence of resistance, we should use linezolid strictly after the therapeutic indications, courses of treatment should be kept as short as possible, and risk factors for resistance development should be considered before starting. In addition, we suggest that susceptibility testing of clinically significant Gram-positive pathogens should be done in all cases of treatment failure, and, depending on the local epidemiology of each ICU, it might be advisable to do it before starting treatment with linezolid.

BinaxNOW® PBP2a rapid immunochromatographic assay is a novel test for the identification of methicillin resistance in Staphylococcus aureus from clinical blood culture samples based on detection of penicillin binding protein 2a. We have evaluated the utility of this assay to do a rapid diagnostic of methicillin susceptibility directly from blood culture bottles after identification of S. aureus in positive bottles by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Twenty of 21 methicillin-resistant S. aureus (MRSA) samples from blood cultures were positive on direct immunochromatographic testing (sensitivity 95.24%, 95% confidence interval [CI] 74.13% to 99.75%), whereas 37 methicillin-susceptible S. aureus (MSSA) samples were negative (specificity 100%, 95% CI 88.99% to 99.75%). The combined use of MALDI-TOF mass spectrometry and BinaxNOW® PBP2a test is useful for the rapid identification of both MRSA and MSSA from blood cultures.