Analytical validation of a novel high multiplexing real-time PCR array for the identification of key pathogens causative of bacterial ventilator-associated pneumonia and their associated resistance genes.

OBJECTIVES: Rapid diagnosis and appropriate empirical antimicrobial therapy before the availability of conventional microbiological results is of pivotal importance for the clinical outcome of ventilator-associated pneumonia (VAP). We evaluated the VAPChip, a novel, closed cartridge molecular tool aiming to identify directly from clinical samples and within a working day the principal bacteria causative of VAP as well as clinically relevant ß-lactam resistance genes. METHODS: The Real-time Array PCR for Infectious Diseases (RAP-ID) is a novel technology that combines multiplex PCR with real-time microarray detection. The VAPChip is a closed cartridge kit adapted to the RAP-ID instrument that targets 13 key respiratory pathogens causative of VAP and 24 relevant antimicrobial resistance genes that mediate resistance to ß-lactam agents, including extended-spectrum cephalosporins and carbapenems. Analytical validation of the VAPChip was carried out blindly on a collection of 292 genotypically characterized bacterial reference and clinical isolates, including 225 isolates selected on the basis of their species identification and antimicrobial resistance profiles and 67 bacterial isolates belonging to the oropharyngeal flora not targeted by the array. RESULTS: The limit of detection of the assay lies between 10 and 100 genome copies/PCR and the dynamic range is five orders of magnitude permitting at least semi-quantitative reporting of the results. Sensitivity, specificity and negative and positive predictive values ranged from 95.8% to 100% for species identification and detection of resistance genes. CONCLUSIONS: VAPChip is a novel diagnostic tool able to identify resistant bacterial isolates by RAP-ID technology. The results of this analytical validation have to be confirmed on clinical specimens.

Immobilization of the thrombin inhibitor r-hirudin conserving its biological activity.

Surface immobilization of the thrombin inhibitor r-hirudin was carried out on two different polymers. Linkage to poly(urethane-graft-acrylic acid) (PAC/PU) was done via carboxylic acid groups, using a water soluble carbodimide, while the immobilization on a modified poly[(ethene-co-vinyl acetate)-graft-vinyl chloride] (PVC/EVA) was achieved via the alcohol groups of the polymer using HDI as spacer. Direct immobilization of r-hirudin leaded to a remarkable loss of thrombin activity. As proved by means of protein chemical analysis, loss of activity was due to a selective coupling via the N-terminal amino group of r-hirudin, which is essential for its thrombin activity. Based on these results we developed an immobilization method via an epsilon-amino group of r-hirudin preserving full biological activity of the r-hirudin coated surface.