BackgroundMedium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.ResultsTo increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.ConclusionOur data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in order to facilitate specific antimicrobial therapy. We developed a prototype DNA microarray for the identification and characterization of three important bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific in identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. We found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes bla TEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. This novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.

The gastrointestinal tract (GIT) of mammals is the main portal of entry for foreign DNA and proteins. We have documented the fate of orally administered DNA or protein in the GIT of the mouse. The gene for the Green Fluorescent Protein (GFP) (4.7 kb) and the genomes of bacteriophage M13 (7.25 kb) and adenovirus type 2 (Ad2; 35.9 kb) were used as test DNAs. Persistence of these DNAs in the GIT was monitored by Southern hybridization and fluorescent in situ hybridization (FISH) or by PCR. For studies on proteins, recombinant glutathione-S-transferase was fed to mice. Survival of the protein in the GIT was then assessed by Western blotting. Depending on feeding schedules and food regimens, but irrespective of mouse strain or DNA length, fragments of the GFP gene or other DNAs were detectable for up to 18 h after feeding by Southern blot analysis. The GFP DNA could be visualized by FISH in cecal epithelia. A high fiber diet reduced the time required for food to pass through the GIT, and foreign DNA was cleared more rapidly. A high fat diet or complexing of the foreign DNA with protamine or lipofectin did not extend DNA persistence times. Undegraded GST protein was detected only in foregut contents up to 30 min after feeding. At 15 and 30 min post feeding, trace amounts of GST were found in extracts of the kidney. The GIT is constantly exposed to highly recombinogenic fragments of foreign DNA and to intact foreign proteins. Our data have implications for studies on carcinogenesis and mutagenesis, and on the pathogenicity of infectious proteins such as prions.

ObjectiveTo model the cost-effectiveness impact of routine use of an antimicrobial chlorhexidine gluconate-containing securement dressing compared to non-antimicrobial transparent dressings for the protection of central vascular lines in intensive care unit patients.DesignThis study uses a novel health economic model to estimate the cost-effectiveness of using the chlorhexidine gluconate dressing versus transparent dressings in a French intensive care unit scenario. The 30-day time non-homogeneous markovian model comprises eight health states. The probabilities of events derive from a multicentre (12 French intensive care units) randomized controlled trial. 1,000 Monte Carlo simulations of 1,000 patients per dressing strategy are used for probabilistic sensitivity analysis and 95% confidence intervals calculations. The outcome is the number of catheter-related bloodstream infections avoided. Costs of intensive care unit stay are based on a recent French multicentre study and the cost-effectiveness criterion is the cost per catheter-related bloodstream infections avoided. The incremental net monetary benefit per patient is also estimated.Patients1000 patients per group simulated based on the source randomized controlled trial involving 1,879 adults expected to require intravascular catheterization for 48 hours.InterventionChlorhexidine Gluconate-containing securement dressing compared to non-antimicrobial transparent dressings.ResultsThe chlorhexidine gluconate dressing prevents 11.8 infections /1,000 patients (95% confidence interval: [3.85; 19.64]) with a number needed to treat of 85 patients. The mean cost difference per patient of €141 is not statistically significant (95% confidence interval: [€-975; €1,258]). The incremental cost-effectiveness ratio is of €12,046 per catheter-related bloodstream infection prevented, and the incremental net monetary benefit per patient is of €344.88.ConclusionsAccording to the base case scenario, the chlorhexidine gluconate dressing is more cost-effective than the reference dressing.Trial RegistrationThis model is based on the data from the RCT registered with www.clinicaltrials.gov (NCT01189682).