Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain.

Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.

Whole-genome characterisation of Escherichia coli isolates from patients with bacteraemia presenting with sepsis or septic shock in Spain: a multicentre cross-sectional study.

BACKGROUND: Escherichia coli is the most frequent cause of bloodstream infections (BSIs). About one-third of patients with BSIs due to E coli develop sepsis or shock. The objective of this study is to characterise the microbiological features of E coli blood isolates causing sepsis or septic shock to provide exploratory information for future diagnostic, preventive, or therapeutic interventions. METHODS: E coli blood isolates from a multicentre cross-sectional study of patients older than 14 years presenting with sepsis or septic shock (according to the Third International Consensus Definitions for Sepsis and Septic Shock criteria) from hospitals in Spain between Oct 4, 2016, and Oct 15, 2017, were studied by whole-genome sequencing. Phylogroups, sequence types (STs), serotype, FimH types, antimicrobial resistance (AMR) genes, pathogenicity islands, and virulence factors were identified. Susceptibility testing was performed by broth microdilution. The main outcome of this study was the characterisation of the E coli blood isolates in terms of population structure by phylogroups, groups (group 1: phylogroups B2, F, and G; group 2: A, B1, and C; group 3: D), and STs and distribution by geographical location and bloodstream infection source. Other outcomes were virulence score and prevalence of virulence-associated genes, pathogenicity islands, AMR, and AMR-associated genes. Frequencies were compared using χ² or Fisher's exact tests, and continuous variables using the Mann-Whitney test, with Bonferroni correction for multiple comparisons. FINDINGS: We analysed 224 isolates: 140 isolates (63%) were included in phylogenetic group 1, 52 (23%) in group 2, and 32 (14%) in group 3. 85 STs were identified, with four comprising 44% (n=98) of the isolates: ST131 (38 [17%]), ST73 (25 [11%]), ST69 (23 [10%]), and ST95 (12 [5%]). No significant differences in phylogroup or ST distribution were found according to geographical areas or source of bloodstream infection, except for ST95, which was more frequent in urinary tract infections than in other sources (11 [9%] of 116 vs 1 [1%] of 108, p=0·0045). Median virulence score was higher in group 1 (median 25·0 [IQR 20·5-29·0) than in group 2 (median 14·5 [9·0-20·0]; p<0·0001) and group 3 (median 21 [16·5-23·0]; p<0·0001); prevalence of several pathogenicity islands was higher in group 1. No significant differences were found between phylogenetic groups in proportions of resistance to antibiotics. ST73 had higher median virulence score (32 [IQR 29-35]) than the other predominant clones (median range 21-28). Some virulence genes and pathogenicity islands were significantly associated with each ST. ST131 isolates had higher prevalence of AMR and a higher proportion of AMR genes, notably blaCTX-M-15 and blaOXA-1. INTERPRETATION: In this exploratory study, the population structure of E coli causing sepsis or shock was similar to previous studies that included all bacteraemic isolates. Virulence genes, pathogenicity islands, and AMR genes were not randomly distributed among phylogroups or STs. These results provide a comprehensive characterisation of invasive E coli isolates causing severe response syndrome. Future studies are required to determine the contribution of these microbiological factors to severe clinical presentation and worse outcomes in patients with E coli bloodstream infection. FUNDING: Instituto de Salud Carlos III.

Evaluation of the VITEK 2 system to test the susceptibility of Candida spp., Trichosporon asahii and Cryptococcus neoformans to amphotericin B, flucytosine, fluconazole and voriconazole: a comparison with the M27-A3 reference method.

We compared the susceptibilities of 302 isolates (209 Candida spp., 89 Cryptococcus neoformans and four Trichosporon asahii) against amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC) and voriconazole (VRC) obtained with an automated commercial system (VITEK 2, bioMérieux, Spain) and the Clinical and Laboratory Standards Institute (CLSI M27-A3) reference broth microdilution method (BMD). Reference BMD MIC endpoints were determined visually after 24-72 h of incubation, depending on the species, and VITEK 2 system MIC endpoints were determined spectrophotometrically by automated components of this equipment. For Candida spp. and T. asahii, the overall MIC agreement between of the results of the VITEK 2 system and the 24/48-h BMD was: 34/62% for AMB; 96.3% at 24/48-h for 5FC; 87.8/87.3% for FLC and 95.3/92% for VRC, respectively. The overall categorical agreement between both methods was: 98.5/97.6% for AMB at 24/48-h; 95.3% for 5FC at 24/48-h; 85.4/84.4% at 24/48-h for FLC; and 97.6/92.95% at 24/48-h for VRC. For C. neoformans, essential agreement was good for FLC (91%) and 5FC (84.2%) but not so good for AMB (69%). Excellent categorical agreement was obtained for all antifungal agents tested except for 5FC (69.7%). This new system could play an important role in the clinical laboratory, but more studies are necessary to verify its ability to identify resistant isolates.

[Designing a clinical microbiology laboratory]. / Diseño de un laboratorio de microbiología clínica.

The microbiology laboratory should be a safe, efficient, and comfortable place for those working there, and a pleasant place for visitors. According to the ISO 15189 standard, it should be spacious enough for the workload to be carried out without jeopardizing quality or the safety of the persons present, whether workers or visitors, and provide optimal comfort to all occupants. In addition, the setup should respect the privacy of patients, and provide controlled access to the different laboratory areas and a safe place for storing clinical samples, manuals, and reagents. In the design of the facilities, the needs of specialists, technicians, and other personnel must converge, without forgetting patients, their relatives, and other visitors. The clinical microbiology laboratory has certain characteristics that make it different from other diagnostic laboratories. Its main activity involves isolation, propagation, and handling of pathogenic microorganisms that pose a risk to the laboratory personnel. To minimize this risk, the laboratory must meet a certain level of biosafety. Moreover, correct interpretation of microbiological cultures depends on the capacity of the laboratory to avoid or minimize the presence of contaminants; hence, proper handling of samples and cultures (aseptic conditions, biosafety cabinet) is mandatory. A number of documents and regulations, from very general to highly specific (biosafety), affect the design of the microbiology laboratory. The aim of this report is to establish the minimum requirements and recommendations for designing clinical microbiology laboratories, based on a review of current regulations. It is contemplated as an aid to microbiology specialists who are designing or planning to reform their laboratories.

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10(1)-10(5) conidia ml(-1)), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 x 10(7) fg. The Tm ranged from 67.34 to 70.7 degrees C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.

Serotypes and genotypes of S. pneumoniae isolates from adult invasive disease in Spain: A 5-year prospective surveillance after pediatric PCV13 licensure. The ODIN study.

Serotypes/genotypes causing invasive pneumococcal disease (IPD) in adults are determined by vaccination strategies. The aim of this study was to assess the epidemiology of IPD in adults (≥18 years) after PCV13 introduction for children: serotypes, clonal complexes, antibiotic non-susceptibility and clinical presentations. We performed a prospective, clinical surveillance of hospitalized culture-confirmed IPDs in adults in nine Spanish hospitals (August 2010-June 2015). A total of 1087 culture-confirmed IPD episodes were included, of which 772 (71.0%) had bacteremic pneumonia (401 complicated/371 uncomplicated pneumonia), 122 (11.2%) meningitis, 102 (9.4%) non-focal bacteremia, 34 (3.1%) peritonitis and 57 (5.3%) others. The most common serotypes were: 3 (12.7%), 19A (8.5%), 8 (7.7%), 7F (6.3%), 1 (4.2%), 6C (4.2%), 11A (4.2%), 22F (4.2%) and 14 (4.0%). Vaccine types (PCV13 + 6C) caused 49.8% of IPD episodes, with a significant decrease over the 5-year period, and significant decreases in serotypes 6C and 7F. The most common genotypes were: CC180 (8.4%), CC191 (6.0%), and CC53 (5.0%). Vaccine types caused 53.9% (414/768) pneumonia episodes and 58.9% (235/399) complicated pneumonia, 53.4% IPD in adults <50 years (143/268), and 54.7% IPD in immunocompetent patients (337/616). Overall non-susceptibility was 25.9% to penicillin (1.1% for parenteral criteria), 24.9% to erythromycin and 2.7% to levofloxacin. CONCLUSIONS: Although the percentage of vaccine-types causing IPDs in adults significantly decreased, it remained high. Associations of vaccine types with pneumonia (with complicated pneumonia for specific serotypes), and immunocompetent patients point to the burden of IPD caused by PCV13 serotypes.

Comparison of the Sensititre YeastOne colorimetric microdilution panel and the NCCLS broth microdilution method for antifungal susceptibility testing against Candida species.

We evaluated the commercially prepared Sensititre YeastOne colorimetric antifungal panel to determine the susceptibility of 170 Candida spp isolates to amphotericin B, fluconazole, itraconazole, and flucytosine. The NCCLS reference microdilution method (M27-A document) was used as reference method. The YeastOne panel was performed according to the manufacturer's instructions. For the colorimetric method, MICs were determined at 24 h of incubation. MICs for the NCCLS reference method were read at 48 h of incubation. The overall agreement within +/-2 dilutions by both methods was calculated against the four antifungal agents. This agreement was 92.9, 68.2, 77.6 and 80% for amphotericin B, fluconazole, itraconazole, and flucytosine, respectively. Thirteen isolates (7.6%) showed very major discrepancies for fluconazole and 12 (7%) for itraconazole. We found that the reading of MIC with the YeastOne panel was somewhat easier than the reading of reference MIC, although the determination of endpoint was sometimes difficult, especially for azoles, because the trailing effect appeared in a high percentage of isolates.

Diseño de un laboratorio de microbiología clínica / Designing a clinical microbiology laboratory

El laboratorio de microbiología debe ser un lugar seguro, eficiente y cómodo para los trabajadores y agradable para los visitantes; según la norma ISO 15189, debe disponer de un espacio suficiente, de forma que su carga de trabajo se pueda realizar sin comprometer la calidad ni la seguridad de todo el personal, trabajador o visitante. Además, debe optimizar la comodidad de sus ocupantes, respetar la privacidad del paciente, controlar el acceso a las distintas zonas del laboratorio y contar con un lugar de almacenamiento que permita asegurar la continua integridad de las muestras, manuales y reactivos. En el diseño de las instalaciones deben converger las necesidades de los especialistas, técnicos y demás personal que desarrolla su actividad laboral en este entorno, sin olvidar a los pacientes, sus acompañantes y demás visitas. Resumen El laboratorio de microbiología clínica tiene unas peculiaridades que lo hacen diferente a otros laboratorios diagnósticos. Su objetivo fundamental es el aislamiento y cultivo de microorganismos patógenos, actividad que genera un riesgo para el personal y que, de acuerdo con los agentes biológicos que se manejen, obliga a un determinado nivel de bioseguridad. Por otro lado, la correcta interpretación de los cultivos microbiológicos depende de la capacidad del laboratorio de evitar o minimizar la presencia de flora contaminante, y es fundamental el correcto tratamiento de las muestras y cultivos (condiciones asépticas, cabinas de bioseguridad) (..) (AU)

The microbiology laboratory should be a safe, efficient, and comfortable place for those working there, and a pleasant place for visitors. According to the ISO 15189 standard, it should be spacious enough for the workload to be carried out without jeopardizing quality or the safety of the persons present, whether workers or visitors, and provide optimal comfort to all occupants. In addition, the setup should respect the privacy of patients, and provide controlled access to the different laboratory areas and a safe place for storing clinical samples, manuals, and reagents. In the design of the facilities, the needs of specialists, technicians, and other personnel must converge, without forgetting patients, their relatives, and other visitors. The clinical microbiology laboratory has certain characteristics that make it different from other diagnostic laboratories. Its main activity involves isolation, propagation, and handling of pathogenic microorganisms that pose a risk to the laboratory personnel. To minimize this risk, the laboratory must meet a certain level of biosafety. Moreover, correct interpretation of microbiological cultures depends on the capacity of the laboratory to avoid or minimize the presence of contaminants; hence, proper handling of samples and cultures (aseptic conditions, biosafety cabinet) is mandatory. A number of documents and regulations, from very general to highly specific (biosafety), affect the design of the microbiology laboratory. The aim of this report is to establish the minimum requirements and recommendations for designing clinical microbiology laboratories, based on a review of current regulations. It is contemplated as an aid to microbiology specialists who are designing or planning to reform their laboratories (AU)

Colecistitis aguda litiásica asociada a Brucella melitensis / Acute calculous cholecystitis associated with Brucella melitensis

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