Large scale multiplex PCR improves pathogen detection by DNA microarrays

<p>Abstract</p> <p>Background</p> <p>Medium 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.</p> <p>Results</p> <p>To 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.</p> <p>Conclusion</p> <p>Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.</p

Identification and Characterization of Bacterial Pathogens Causing Bloodstream Infections by DNA Microarray

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

Cost-Effectiveness Analysis of a Transparent Antimicrobial Dressing for Managing Central Venous and Arterial Catheters in Intensive Care Units

<div><p>Objective</p><p>To 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.</p><p>Design</p><p>This 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.</p><p>Patients</p><p>1000 patients per group simulated based on the source randomized controlled trial involving 1,879 adults expected to require intravascular catheterization for 48 hours.</p><p>Intervention</p><p>Chlorhexidine Gluconate-containing securement dressing compared to non-antimicrobial transparent dressings.</p><p>Results</p><p>The 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.</p><p>Conclusions</p><p>According to the base case scenario, the chlorhexidine gluconate dressing is more cost-effective than the reference dressing.</p><p>Trial Registration</p><p>This model is based on the data from the RCT registered with <a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a> (<a href="https://clinicaltrials.gov/ct2/show/NCT01189682" target="_blank">NCT01189682</a>).</p></div

Cost-effectiveness results for the probabilistic sensitivity analysis.

<p>The analysis uses 1,000 non-homogeneous Markov-Chain Monte Carlo simulations of 1,000 patients for each dressing strategy. The x axis represents the difference in effectiveness (number of CRBSI events in CHG versus non CHG dressing) and the y axis represents the difference in cost (mean cost per patient with CHG versus non CHG dressing) in €2013. The (0,0)-point indicates the reference dressing strategy (Non-CHG group). Each point in the graph represents the Incremental Cost-Effectiveness Ratio (ICER) of CHG-dressing strategy versus reference dressing. All but three points are at the left side of the graph, showing that CHG dressing strategy was 99.7% more effective than the comparator at the same costs per patient. The squared point in the center of the cloud represents the average CE ratio of all 1,000 simulations. CHG: chlorhexidine gluconate; CRBSI: catheter-related bloodstream infection.</p

Mean Cost for one patient in each dressing group.

<p>Time Horizon: 30-days ICU—1,000 NH-MCMC simulations of 1,000 patients (€2013).</p><p>CHG: Chlorhexidine Gluconate; CI: Confidence Interval; ICU: Intensive Care Unit; NH-MCMC: Non-Homogeneous Markov-Chain Monte Carlo simulation</p><p>Mean Cost for one patient in each dressing group.</p

Structure of the Markov Model showing the possible transition between health states from one Markov cycle to the next cycle.

<p>The costs per patient for each health state were calculated in both CHG and No-CHG dressing as respectively: State 1: €1,270 and €1,266; State 2: €1,364 and €1,361; State 3: €13,661 and €13,658; State 4: €13,756 and €13,752; State 5: €1,388 and €1,385; State 6: €1,266 and €1,266; State 7: €0 for both groups; State 8: €0 for both groups; CHG: chlorhexidine gluconate; CRBSI: catheter-related bloodstream infection; CT: catheter.</p

Occurrences per 1,000 patients as generated through 1,000 NH-MCMC of 1,000 patients in each dressing group, according to the base case scenario.

<p>CHG, Chlorhexidine Gluconate; CI, Confidence Interval; CRBSI, Catheter-related bloodstream infection; CT, Catheter; ICU, Intensive Care Unit; NH-MCMC, Non-Homogeneous Markov Chain Monte Carlo.</p><p>Occurrences per 1,000 patients as generated through 1,000 NH-MCMC of 1,000 patients in each dressing group, according to the base case scenario.</p

Health states defined from a multicentre randomized controlled trial [13].

<p>* New CT needed can mean either the replacement of the existing catheter, or the need for an additional catheter at a new site.</p><p>CRBSI, Catheter-related Bloodstream Infections; CT, Catheter (Central venous or radial / femoral arterial).</p><p>Health states defined from a multicentre randomized controlled trial [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130439#pone.0130439.ref013" target="_blank">13</a>].</p