Automated extraction improves multiplex molecular detection of infection in septic patients.

Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation.

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)

Falsa resistencia a carbapenemas en anaerobios / False carbapenem resistance in anaerobic microorganisms

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