Cascade filtration and droplet digital detection integrated microfluidic assay enables isolating culture-free phenotypic identification of carbapenem-resistant organisms

Carbapenem-resistant organisms (CROs) are characterized by high drug resistance, rapid transmission, and high lethality. Therefore, rapid detection for CROs is essential for appropriate applying antibiotics and implementing quarantine. Droplet digital chromogenic assays (DDCA) have been accepted as an effective means for rapid microbial detection as the small droplet volumes facilitate a significant enhancement in the local concentration of chromogenic factors and, therefore, reduce the required time of the test. Nevertheless, as their dependence on the time-consuming isolation culture, the DDCA is still associated with a long turnaround time. To overcome this limitation, we develop here a microfluidic chip-based CRO phenotypic identification method that integrates cascade filtration (CF) with DDCA (CF-DDCA). After a body fluid sample is introduced to the microfluidic chip, particles with sizes >5 μm are removed out by the primary filter, and Gram (+) cocci with sizes <1 μm removed out by the secondary filter so that only Gram (-) bacilli with sizes between 1.5 and 5 μm are selectively retained. The purified Gram (-) bacilli, along with chromogenic reagents and carbapenem antibiotics, are then subjected to the DDCA. We demonstrate that the CF can remove 99.9% of the interfering microorganisms and thus eliminates the isolating culture. Benefited from the isolating culture-free DDCA, phenotypic identification of CROs can be achieved within 3.5 h. Clinical urine sample testing shows that the sensitivity and specificity of the CF-DDCA for CRO identification are all 100%, and the total coincidence rate between CF-DDCA and the conventional assay is also 100%.