A two-hour antibiotic susceptibility test by ATP-bioluminescence.

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours.

New procedure for rapid identification of microorganisms causing urinary tract infection from urine samples by mass spectrometry (MALDI-TOF).

INTRODUCTION: Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry is widely established as a technique in clinical microbiology laboratories for the identification of microorganisms. Using this technique, it is also possible to obtain the identification of microorganisms from untreated urine samples. METHODS: In this study, a differential centrifugation protocol and a criterion for validation of the results in order to achieve microbial identification from untreated urine samples are proposed. Additionally, the sensitivity of the analytical procedure in monobacterial urine samples has been evaluated. RESULTS: A 90% sensitivity (confidence interval of 81.96%-94.84%) was obtained in urine samples with bacterial counts of ≥1×10(5)CFU/ml, and it was possible to improve the percentages of direct identifications from urine samples with bacterial counts of <1×10(5)CFU/ml. CONCLUSION: It is concluded that the MALDI-TOF system is both fast and reliable in the identification of individual microorganisms from untreated urine samples with counts of ≥1×10(5)CFU/ml.

Environmental Dissemination of SARS-CoV-2 in a University Hospital during the COVID-19 5th Wave Delta Variant Peak in Castile-León, Spain.

The dominant SARS-CoV-2 Delta variant (B.1.617.2) became the main circulating variant among countries by mid 2021. Attention was raised to the increased risk of airborne transmission, leading to nosocomial outbreaks even among vaccinated individuals. Considering the increased number of COVID-19 hospital admissions fueled by the spread of the variant, with Spain showing the highest COVID-19 rates in mainland Europe by July 2021, the aim of this study was to assess SARS-CoV-2 environmental contamination in different areas of a University Hospital in the region of Castile-León, Spain, during the peak of the 5th wave of COVID-19 in the country (July 2021). Air samples were collected from sixteen different areas of the Hospital using a Coriolis® µ air sampler. Surface samples were collected in these same areas using sterile flocked plastic swabs. RNA extraction followed by a one-step RT-qPCR were performed for detection of SARS-CoV-2 RNA. Of the 21 air samples, only one was positive for SARS-CoV-2 RNA, from the emergency waiting room. Of the 40 surface samples, 2 were positive for SARS-CoV-2 RNA, both from the microbiology laboratory. These results may be relevant for risk assessment of nosocomial infection within healthcare facilities, thus helping prevent and minimize healthcare staff's exposure to SARS-CoV-2, reinforcing the importance of always wearing appropriate and well-fit masks at all times and proper PPE when in contact with infected patients.

Bacterial/fungal infection in hospitalized patients with COVID-19 in a tertiary hospital in the Community of Castilla y León, Spain.

INTRODUCTION: Bacterial/fungal coinfection and superinfections contribute to the increased morbi-mortality of viral respiratory infections (RIs). The main objective of this study was to determine the incidence of these infections in hospitalized patients with COVID-19. METHOD: Retrospective observational study of all patients admitted for COVID-19 and bacterial/fungal infections at the Hospital Clínico Universitario of Valladolid, Spain (March 1-May 31, 2020). Demographic, clinical and microbiological data were compared based on Intensive Care Unit (ICU) admission and predictors of mortality by were identified using multivariate logistic regression analyses. RESULTS: Of the 712 COVID-19 patients, 113 (16%) presented bacterial/fungal coinfections or superinfections. Their median age was 73 years (IQR 57-89) and 59% were men. The profiles of ICU patients (44%) included male, SARS-CoV-2 pneumonia, leukocytosis, elevated inteleukin-6, with interferon ß-1b and tocilizumab and superinfection (p < 0.05). Coinfections were diagnosed in 5% (39/712) patients. Most common pathogens of respiratory coinfection (18) were Streptococcus pneumoniae (6) and Staphylococcus aureus (6). Superinfections were detected in 11% (80/712) patients. Urinary (53) and RI (39) constituted the majority of superinfections Acinetobacter baumannii multidrug-resistant was the main agent of IR and bacteremia. An outbreak of A. baumannii contributed to this result. Three patients were considered to have probable pulmonary aspergillosis. Mortality was higher in UCI patients (50% vs. 29%, p = 0.028). The predictive factors of mortality included being a male with various comorbidities, SARS-CoV-2 pneumonia, bacteremia and superinfections from A. baumannii. CONCLUSION: The outbreak of A. baumannii was a determining factor in the increases of the incidence of infection and the morbi-mortality of ICU patients.

Bacterial/fungal infection in hospitalized patients with COVID-19 in a tertiary hospital in the Community of Castilla y León, Spain. / Infección bacteriana/fúngica en pacientes con COVID-19 ingresados en un hospital de tercer nivel de Castilla y León, España.

INTRODUCTION: Bacterial/fungal coinfection and superinfections contribute to the increased morbi-mortality of viral respiratory infections (RIs). The main objective of this study was to determine the incidence of these infections in hospitalized patients with COVID-19. METHOD: Retrospective observational study of all patients admitted for COVID-19 and bacterial/fungal infections at the Hospital Clínico Universitario of Valladolid, Spain (March 1-May 31, 2020). Demographic, clinical and microbiological data were compared based on Intensive Care Unit (ICU) admission and predictors of mortality by were identified using multivariate logistic regression analyses. RESULTS: Of the 712 COVID-19 patients, 113 (16%) presented bacterial/fungal coinfections or superinfections. Their median age was 73 years (IQR 57-89) and 59% were men. The profiles of ICU patients (44%) included male, SARS-CoV-2 pneumonia, leukocytosis, elevated inteleukin-6, with interferon ß-1b and tocilizumab and superinfection (p < 0.05). Coinfections were diagnosed in 5% (39/712) patients. Most common pathogens of respiratory coinfection (18) were Streptococcus pneumoniae (6) and Staphylococcus aureus (6). Superinfections were detected in 11% (80/712) patients. Urinary (53) and RIs (39) constituted the majority of superinfections Acinetobacter baumannii multidrug-resistant was the main agent of IR and bacteremia. An outbreak of A. baumannii contributed to this result. Three patients were considered to have probable pulmonary aspergillosis. Mortality was higher in UCI patients (50 vs. 29%; p = 0.028). The predictive factors of mortality included being a male with various comorbidities, SARS-CoV-2 pneumonia, bacteremia and superinfections from A. baumannii. CONCLUSION: The outbreak of A. baumannii was a determining factor in the increases of the incidence of infection and the morbi-mortality of ICU patients.

Neurological Comorbidity Is a Predictor of Death in Covid-19 Disease: A Cohort Study on 576 Patients.

Introduction: Prognosis of Coronavirus disease 2019 (Covid-19) patients with vascular risk factors, and certain comorbidities is worse. The impact of chronic neurological disorders (CND) on prognosis is unclear. We evaluated if the presence of CND in Covid-19 patients is a predictor of a higher in-hospital mortality. As secondary endpoints, we analyzed the association between CND, Covid-19 severity, and laboratory abnormalities during admission. Methods: Retrospective cohort study that included all the consecutive hospitalized patients with confirmed Covid-19 disease from March 8th to April 11th, 2020. The study setting was Hospital Clínico, tertiary academic hospital from Valladolid. CND was defined as those neurological conditions causing permanent disability. We assessed demography, clinical variables, Covid-19 severity, laboratory parameters and outcome. The primary endpoint was in-hospital all-cause mortality, evaluated by multivariate cox-regression log rank test. We analyzed the association between CND, covid-19 severity and laboratory abnormalities. Results: We included 576 patients, 43.3% female, aged 67.2 years in mean. CND were present in 105 (18.3%) patients. Patients with CND were older, more disabled, had more vascular risk factors and comorbidities and fewer clinical symptoms of Covid-19. They presented 1.43 days earlier to the emergency department. Need of ventilation support was similar. Presence of CND was an independent predictor of death (HR 2.129, 95% CI: 1.382-3.280) but not a severer Covid-19 disease (OR: 1.75, 95% CI: 0.970-3.158). Frequency of laboratory abnormalities was similar, except for procalcitonin and INR. Conclusions: The presence of CND is an independent predictor of mortality in hospitalized Covid-19 patients. That was not explained neither by a worse immune response to Covid-19 nor by differences in the level of care received by patients with CND.

Identification of the DRB4*01:03:01:02N null allele with HLA-DRB1*04 in a Spanish donor.

The association between the DRB4*01:03:01:02N null allele and the HLA-DRB1*07~DQB1*03:03 haplotype has often been reported. Nevertheless, more unusual associations have also been found in other countries, such as its association with HLA-DRB1*04. HLA class I and II antigen typing is currently performed using DNA-based methods, making it more difficult to identify null alleles than if serological methods were used. Furthermore, the DRB3/4/5 loci are not usually studied. However, the identification of non-expressed HLA alleles is of great importance for transplantation so it is necessary to identify HLA antigen associations with null alleles and report these findings. In this paper, we describe the association of DRB4*01:03:01:02N null allele with DRB1*04 for the first time in Spain.

Utility of an immunocapture-agglutination test and an enzyme-linked immunosorbent assay test against cytosolic proteins from Brucella melitensis B115 in the diagnosis and follow-up of human acute brucellosis.

The utility of an immunocapture-agglutination (Brucellacapt, Vircell SL, Granada, Spain) test and an enzyme-linked immunosorbent assay IgG, IgA, and IgM (ELISA-IgG, ELISA-IgA, ELISA-IgM) against cytosolic proteins from Brucella melitensis B115 (R) was compared with ELISA-IgG, ELISA-IgA, and ELISA-IgM against smooth lipopolysaccharide (S-LPS) from B. melitensis 16M (S), serum agglutination test (SAT), and Coombs test in the diagnosis and follow-up for 10 months of 51 patients with acute brucellosis. The sensitivities of ELISA tests against cytosolic proteins varied from 49.0 % for ELISA-IgG to 64.7% for ELISA-IgM and were lower than the sensitivities showed by ELISA S-LPS (from 88.2% to 92.2%), SAT (88.2%), Coombs (96.1%), and Brucellacapt (98.0%) tests. Specificity was over 93% in all cases. The evolutionary behavior of the SAT, Coombs, and Brucellacapt tests was similar. There was a decrease of between 20% and 40% in antibody titer in the 10th month of evolution after treatment. The evolutional curves of IgG, IgA, and IgM against cytosolic protein increased slightly till the eighth month. The specific IgM and IgA antibodies against protein fractions began to show a drop from the eighth month on, showing levels slightly lower than the initial sera values by the end of the 10th month. In this month, titers of specific IgG against proteins fractions remained higher than the titers showed by the initial sera.

A two-hour procedure for determining the susceptibility of enterococci and staphylococci to antibiotics by a colourimetric method.

INTRODUCTION: Rapid determination of the antibiotic susceptibility test in bacteria remains a challenge for Clinical Microbiology laboratories. METHODS: An improvement in the colorimetric antimicrobial susceptibility testing performed with resazurin in enterococci and staphylococci has been carried out. The design of method was performed using two collection strains, which have a known susceptibility. This procedure was then validated against standard commercial methods on 15 strains of staphylococci and 15 strains of enterococci from patients. RESULTS: The essential agreement between the colorimetric method and commercial methods (E-test, MicroScan and VITEK2) was 100%. CONCLUSION: Resazurin allows us to obtain a reliable antibiotic susceptibility test in staphylococci and enterococci in less than two hours.

Susceptibility of the Acinetobacter calcoaceticus-A. baumannii complex to imipenem, meropenem, sulbactam and colistin.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex includes some of the most clinically relevant species of the genus Acinetobacter due to their capacity to cause epidemic nosocomial outbreaks as well as their increasing resistance to antibiotics. Susceptibility of Acinetobacter strains varies greatly depending on origin, thus highlighting the importance of local analyses of susceptibility profiles. Two hundred twenty-one strains of the A. calcoaceticus-A. baumannii complex were identified using biochemical tests and were biotyped. Strain susceptibility to imipenem, meropenem, colistin and sulbactam was studied using agar dilution. Eight different biotypes were found, type 1 accounting for 69.2% of the strains. MIC(50) and MIC(90) to imipenem, meropenem, colistin and sulbactam were 4 and 8 mg/l, 16 and 32 mg/l, 0.5 and 1mg/l, and 8 and 16 mg/l, with susceptibility rates of 64.3, 22.6, 98.2 and 73.8%, respectively. Biotype 1 was the most resistant. A statistically significant difference was observed for the mean MIC of the four predominant biotypes to imipenem, meropenem and sulbactam but not to colistin.

Procedure for microbial identification based on Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry from screening-positive urine samples.

Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry is a widely used proteomic technique in clinical microbiology laboratories, and enables microbial identification directly from clinical samples. This study seeks to establish a protocol for bacterial identification from monomicrobial urine samples that have tested positive in the screening with Sysmex UF-1000i (Sysmex Corporation, Kobe, Japan). Sysmex UF-1000i counts ≥1 × 10(7) bacteria/mL indicate a sufficient bacterial concentration to allow direct identification from urine, with 87.5% sensitivity. Microbial identification from urine with Sysmex UF-1000i counts between 1 × 10(5) and 1 × 10(7) bacteria/ml requires preincubation to obtain the adequate amount of bacteria needed for analysis, and 91.7% sensitivity thus being achieved.

Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures.

BACKGROUND: MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. METHODOLOGY/PRINCIPAL FINDINGS: We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. CONCLUSIONS/SIGNIFICANCE: MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

Association between self-replicating calcifying nanoparticles and aortic stenosis: a possible link to valve calcification.

AIMS: Among various hypotheses proposed for pathological tissue calcification, recent evidence supports the possibility that self-replicating calcifying nanoparticles (CNPs) can contribute to such calcification. These CNPs have been detected and isolated from calcified human tissues, including blood vessels and kidney stones, and are referred to as nanobacteria. We evaluated calcific aortic valves for the presence of CNP. METHODS AND RESULTS: Calcific aortic valves were obtained from 75 patients undergoing surgical valve replacement. The control group was formed by eight aortic valves corresponding to patients with heart transplants. In the microbiology laboratory, valves were screened for CNP using a 4-6 weeks specific culture method. The culture for CNP was positive in 48 of the 75 valves with aortic stenosis (64.0%) in comparison with zero of eight (0%) for the control group (P = 0.0005). The observation of cultures by way of scanning electron microscopy highlighted the resemblance in size and morphology of CNP. CONCLUSION: Self-replicating calcific nanometer-scale particles, similar to those described as CNP from other calcific human tissues, can be cultured and visualized from calcific human aortic valves. This finding raises the question as to whether CNP contribute to the pathogenesis of the disease or whether they are only innocent bystanders.