Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix / Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases

Introducción La detección de la multirresistencia a betalactámicos en Escherichia coli y Klebsiella pneumoniae es una cuestión clínicamente relevante. Por otro lado, es interesante diferenciar entre la producción 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 producción de BLEE en E. coli y K. pneumoniae. Material y métodos Se ensayaron 193 aislamientos clínicos fenotípicamente productores de BLEE (174 E. coli y 19K. pneumoniae) por Vitek 2 y BD Phoenix System y se utilizaron las tarjetas AST-N058 y los paneles UNMIC/ID-62, respectivamente. Se consideraron métodos fenotípicos de referencia la prueba de sinergia con doble disco y Etest. Como controles positivos y negativos se ensayaron 12 cepas genotípicamente caracterizadas. Resultados En el caso de los aislamientos clínicos, 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 ejecución del sistema experto elevó la sensibilidad del Phoenix al 100%. Sin embargo, el sistema experto de Vitek 2 consideró incoherentes los resultados obtenidos en 7 aislamientos con la prueba para BLEE positiva. Conclusión La sensibilidad de la prueba confirmatoria para la producción de BLEE es superior en las tarjetas N-058 de Vitek. No obstante, la actuación de los sistemas expertos sitúa a ambos sistemas a la misma altura en su capacidad de detección de BLEE en E. coli y K. pneumoniae (AU)

Introduction and Purpose Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. Material and Methods A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. Results In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. Conclusion Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumonia (AU)

Utilidad del medio chromID™ de betalactamasas de espectro extendido para detectar resistencia a cefalosporinas en enterobacterias inoculadas en frascos de hemocultivo / Utility of chromIDTM ESBL medium for detection of cephalosporin-resistant enterobacteria in inoculated blood culture bottles

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[Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases]. / Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix.

INTRODUCTION AND PURPOSE: Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. MATERIAL AND METHODS: A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. RESULTS: In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. CONCLUSION: Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumoniae.

Implication of p38 mitogen-activated protein kinase isoforms (alpha, beta, gamma and delta) in CD4+ T-cell infection with human immunodeficiency virus type I.

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 (p38alpha, p38beta, p38gamma and p38delta) 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 p38alphadn, p38gammadn and p38deltadn isoforms, but not by the p38betadn isoform. HIV-1 replication was delayed most by p38deltadn and to a lesser extent by p38alphadn and p38gammadn. Moreover, these three isoforms, p38alphadn, p38gammadn and p38deltadn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38alpha, p38gamma and p38delta, but not p38beta, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.

[CMV DNA detection in plasma using real-time PCR based on the SYBR-Green I dye method]. / Detección de ADN de CMV en plasma mediante PCR en tiempo real utilizando SYBR-Green I como señal de amplificación.

OBJECTIVE: The aim of this study is to assess a real-time PCR technique on the LightCycler 2.0 with SYBR-Green I detection as compared to another real-time PCR method based on detection with FRET (fluorescence resonance energy transfer) probes for the quantification of CMV DNA. METHODS: The two real-time PCR methods were used to test plasma samples from immunocompromised patients with clinically suspected CMV disease, patients under follow-up without symptoms, and healthy adults. A standard curve for quantitative analysis by the SYBR-Green I method was performed with 10-fold diluted solutions of DNA from the CMV Towne strain (ATCC VR-977) cultured in MRC-5 monolayer. In addition, frozen samples from patients positive for CMV pp65 antigenemia were also analyzed and results compared using the two real time PCR methods. RESULTS: The real-time PCR technique using SYBR-Green I on the LightCycler 2.0 was a highly specific, fast, simple and reliable test to quantify CMV; moreover, it was cost-effective. CONCLUSION: Quantification of CMV DNA in plasma using this sensitive, fast, low-cost method was advantageous for the diagnosis and follow up of patients with opportunistic CMV infection, which are increasingly more frequent in our daily hospital clinical practice.

Detección de ADN de CMV en plasma mediante PCR en tiempo real utilizando SYBR-Green I como señal de amplificación / CMV DNA detection in plasma using real-time PCR based on the SYBR-Green I dye method

Objetivo. El objetivo del presente estudio es evaluar una técnica rápida y sencilla de reacción en cadena de la polimerasa (PCR) en tiempo real en LightCycler 2.0 revelada con SYBR-Green I, comparándola con otra técnica de PCR en tiempo real que utiliza sondas FRET (fluorescence resonance energy transfer) para revelar la amplificación. Métodos. Los dos métodos de PCR en tiempo real se compararon utilizando muestras de plasma de pacientes inmunodeprimidos con sospecha clínica de enfermedad por citomegalovirus (CMV), de pacientes monitorizados sin sintomatología, y de adultos sanos. El estudio se completó con otras muestras de plasma congeladas de casos positivos por antigenemia pp65, y con ADN de CMV de la cepa Towne (ATCC VR-977) obtenido de cultivo en MRC-5, con el que elaboramos una curva estándar para su cuantificación. Resultados. La PCR revelada con SYBR-Green I resultó ser claramente la más rentable por su alta sensibilidad, rapidez y sencilla realización, además de su bajo coste. Conclusión. La determinación cuantitativa de ADN de CMV en plasma utilizando un método sensible, rápido y de bajo coste, como el que proponemos, supone una clara ventaja para el diagnóstico y seguimiento de estos cuadros, especialmente en hospitales como el nuestro, donde en los últimos años se ha incrementado sensiblemente el número de pacientes susceptibles de padecer esta infección oportunista (AU)

Objective. The aim of this study is to assess a real-time PCR technique on the LightCycler 2.0 with SYBR-Green I detection as compared to another real-time PCR method based on detection with FRET (fluorescence resonance energy transfer) probes for the quantification of CMV DNA. Methods. The two real-time PCR methods were used to test plasma samples from immunocompromised patients with clinically suspected CMV disease, patients under follow-up without symptoms, and healthy adults. A standard curve for quantitative analysis by the SYBR-Green I method was performed with 10-fold diluted solutions of DNA from the CMV Towne strain (ATCC VR-977) cultured in MRC-5 monolayer. In addition, frozen samples from patients positive for CMV pp65 antigenemia were also analyzed and results compared using the two real time PCR methods. Results. The real-time PCR technique using SYBR-Green I on the LightCycler 2.0 was a highly specific, fast, simple and reliable test to quantify CMV; moreover, it was cost-effective. Conclusion. Quantification of CMV DNA in plasma using this sensitive, fast, low-cost method was advantageous for the diagnosis and follow up of patients with opportunistic CMV infection, which are increasingly more frequent in our daily hospital clinical practice (AU)