Rapid Method Determines Antibiotic Susceptibility

A novel micro-fluidic device used a sensitive digital real-time loop-mediated isothermal amplification (dLAMP) assay to rapidly determine bacterial antibiotic susceptibility.

Rapid antimicrobial susceptibility testing (AST) is urgently needed to facilitate treatment decisions and prevent the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 minutes) directly from clinical samples.

To correct this lack, investigators at the California Institute of Technology (Palo Alto, CA, USA) designed a rapid (approximately seven minutes), microscale digital real-time loop-mediated isothermal amplification (dLAMP) assay to operate on the SlipChip microfluidic device.

The SlipChip is a microfluidic device manufactured by SlipChip Corporation (Pasadena, CA, USA) to perform multiplexed microfluidic reactions without pumps or valves. The device has two plates in close contact. The bottom plate contains wells preloaded with up to 48 reagents. These wells are covered by the top plate that acts as a lid for the wells with reagents. The device also has a fluidic path, composed of ducts in the bottom plate and wells in the top plate, which is connected only when the top and bottom plate are aligned in a specific configuration. Sample can be added into the fluidic path, filling both wells and ducts. Then, the top plate is "slipped", or moved, relative to the bottom plate so the complementary patterns of wells in both plates overlap, exposing the sample-containing wells of the top plate to the reagent-containing wells of the bottom plate, and enabling diffusion and reactions. Between the two plates, a lubricating layer of fluorocarbon is used to facilitate relative motion of the plates.

The current application used digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 minutes. To perform the assay, urine samples were diluted and incubated for 15 minutes before dLAMP analysis to calculate ratios of bacterial DNA concentrations between untreated (without antibiotics) and treated (with antibiotics) samples. Ratios below preset thresholds indicated sensitivity to the antibiotics, since treatment reduced bacterial abundance.

The performance of the assay platform was validated by analyzing 51 clinical samples that had been confirmed as containing bacteria that were either susceptible or resistant to one of two commonly used antibiotics (ciprofloxacin or nitrofurantoin).

The SlipChip dLAMP assay was described in detail in the October 4, 2017, online edition of the journal Science Translational Medicine.

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California Institute of Technology