RESUMEN
OBJECTIVES: To assess the quality of molecular detection of respiratory viruses in clinical diagnostic laboratories. STUDY DESIGN: Respiratory virus proficiency panels were produced from diluted stocks of respiratory viruses provided and tested by four reference laboratories. The panels consisted of strong positive, positive, low positive and negative samples for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1 and 3, adenovirus serotypes 4 and 7, human rhinovirus serotypes 16, 72 and 90, human coronaviruses OC43 and 229E. The panels were sent to 17 participants; results and information on methodology was collected. RESULTS: All laboratories returned results, of which five submitted complete data sets. So, for analysis all results were combined. Samples were correctly identified by participants in 93.75%, 76.75% and 47.03% for the high positive, positive and low positive samples, respectively. One false positive was reported for all data sets (1.1%). The overall score for all assays using different methodologies was 78.8%. Laboratory performance was not dependant on methodology as all in-house methodologies could achieve optimal results, but dependant on careful optimisation and procedures specific to the laboratory. CONCLUSIONS: The first proficiency panel showed that in general all participants performed well. Although, it also highlights areas for improvement for all participants in order to generate robust results for use in clinical diagnostics.
Asunto(s)
Laboratorios/normas , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Virosis/diagnóstico , Virosis/virología , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Humanos , Control de Calidad , Virus ARN/genética , Virus ARN/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
The effect of Bordetella bronchiseptica infection on the viability of murine macrophage-like cells and on primary porcine alveolar macrophages was investigated. The bacterium was shown to be cytotoxic for both cell types, particularly where tight cell-to-cell contacts were established. In addition, bvg mutants were poorly cytotoxic for the eukaryotic cells, while a prn mutant was significantly less toxic than wild-type bacteria. B. bronchiseptica-mediated cytotoxicity was inhibited in the presence of cytochalasin D or cycloheximide, an inhibitor of microfilament-dependent phagocytosis or de novo eukaryotic protein synthesis, respectively. The mechanism of eukaryotic cell death was examined, and cell death was found to occur primarily through a necrotic pathway, although a small proportion of the population underwent apoptosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Bordetella bronchiseptica/patogenicidad , Macrófagos/patología , Operón/fisiología , Factores de Virulencia de Bordetella , Adhesinas Bacterianas/fisiología , Apoptosis , Línea Celular , Cicloheximida/farmacología , Citocalasina D/farmacología , Hemaglutininas/fisiologíaRESUMEN
The uptake and persistence of Bordetella bronchiseptica was characterized in murine phagocytes by using a novel bioluminescence-based reporter system. A mini-Tn5 promoter probe carrying the intact lux operon from the terrestrial bacterium Photorhabdus luminescens which allowed measurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of bioluminescent fusion strains of B. bronchiseptica. The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of B. bronchiseptica was monitored by luminometry and by fluorescence and electron microscopy. The number of bacteria internalized, in a microfilament-dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI). At an MOI of <500:1 (bacteria to macrophages), viable numbers of intracellular bacteria declined over a 4-day period. However, at an MOI of >/=500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfection with little decline in numbers, indicating that a critical population size may have been essential for intracellular persistence. No evidence of macrophage killing by intracellular bacteria was detected over the 4-day period. Intracellular bioluminescent B. bronchiseptica organisms in mouse peritoneal cells were detected at 24 and 48 h after intraperitoneal injection of mice. Bioluminescence is shown to act as a convenient real-time technique for monitoring of intracellular survival of B. bronchiseptica in vitro and may provide a suitable means for examining the role of long-term intracellular survival of the bacterium in the host.