RESUMEN
Although a whole-cell pertussis vaccine was introduced in Pakistan in 1980, little is known about the pertussis prevalence and circulating strains in Pakistan. The aim of this study was to analyze Bordetella parapertussis isolates circulating between 2005 and 2009 in Pakistan and to compare them with those found in other countries during different periods. A total of 59 (7.35%) B. parapertussis isolates from 802 subjects (median age, 3 years) from Pakistan, with pertussis-like symptoms were investigated. We carried out genotyping and DNA microarray analyses on these isolates and compared them with some international isolates of B. parapertussis. We found that the allele for pertactin (prn) found in strains studied from Pakistan was identical to the predominant type found in Europe. We showed that B. parapertussis isolates circulating in Pakistan are part of the same pulsed-field gel electrophoresis group to those circulating in Finland during the period of 1982-2007. Finally, microarray analysis confirmed that the isolates collected in Pakistan, were quite similar to international strains. Overall, these results confirm that B. parapertussis is extremely monomorphic. The high isolation rate of B. parapertussis (7.35%) compared to Bordetella pertussis (0.5%) may suggest that the whole-cell vaccine used in Pakistan is effective against B. pertussis (0.5% infections detected), but much less so against B. parapertussis.
Asunto(s)
Infecciones por Bordetella/epidemiología , Infecciones por Bordetella/microbiología , Bordetella parapertussis/clasificación , Bordetella parapertussis/genética , Tipificación Molecular , Proteínas de la Membrana Bacteriana Externa/genética , Bordetella parapertussis/aislamiento & purificación , Niño , Preescolar , Electroforesis en Gel de Campo Pulsado , Genotipo , Humanos , Lactante , Análisis por Micromatrices , Epidemiología Molecular , Pakistán/epidemiología , Factores de Virulencia de Bordetella/genéticaRESUMEN
Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.