Debkishore Mitra

A Real-Time PCR Antibiogram for Drug-Resistant Sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.

Study of microscale hydraulic jump phenomenon for hydrodynamic trap-and-release of microparticles.

Easy trap-and-release of microparticles is necessary to study biological cellular behavior. The hydraulic jump phenomenon inspired us to conceive a microfluidic device for the hydrodynamic trap-and-release of microparticles. A sudden height increase in a microfluidic channel leads to a dramatic decrease in flow velocity, allowing effective trapping of the microparticles by energy conversion. The trapped particles can be released by stronger inertial force based on simply increasing the flow velocity. We present a systematic, numerical study of trap-and-release of the microparticles using multiphase Navier-Stokes equations. Effect of geometry flow velocity, particle diameter, and adhesion force on trap-and-release was studied.