Micro- and Nanotechnologies
Microdevices for high-throughput cellular and biomolecular profiling
Bacterial infections, such as bloodstream infections (BSI), ventilator-associated infection (VAI), and urinary tract infections (UTI), are a common cause of patient morbidity and mortality. Rapid identification of the causative pathogens and their antimicrobial susceptibility profiles will improve the clinical workflow for clinical management, accelerate clinical decision-making, and improve patient outcomes. However, definitive clinical microbiological analysis of samples obtained from patients requires several days, hindering proper management of infection and driving the overuse and misuse of broad-spectrum antibiotics. Novel precision technologies for rapidly identifying the pathogens and their antibiotic resistance are highly sought-after.
To address this clinical unmet need, we develop a nanotube assisted microwave electroporation (NAME) technique for intracellular detection of species-specific bacterial 16s rRNA in 30 minutes. NAME allows amplification-free pathogen identification at the single cell level. Unlike typical sensing techniques that lyse the bacteria and dilute the intracellular content, NAME directly detects species-specific regions of the 16S rRNA inside the cells. Due to the small volume of a bacterium, the target molecule in the cell has a high effective concentration, which creates a strong signal for single cell detection without amplification. Intracellular detection of bacterial 16S rRNA in viable cells also facilitates subsequent antimicrobial susceptibility testing (AST). By incorporating an adaptable microfluidic design, we demonstrate a phenotypic AST system that rapidly determines the existence of bacteria, classifies major classes of bacteria, detects polymicrobial samples, and identifies antimicrobial susceptibility directly from clinical samples at the single-cell level. The adaptable microfluidic system can dramatically accelerate the workflow of the microbiological analysis. Pathogen classification, which is based on microfluidic separation and microscopic inspection, eliminate the slow culture step. This approach rules out negative samples, classifies bacteria according to size and shape in as few as 5 minutes, and identifies samples with multiple pathogens for polymicrobial infection diagnosis. By monitoring the bacterial growth directly, AST results can be reported in as few as 30 minutes or in a time scale similar to the doubling times of the bacteria.
In this study, we report the integrated microfluidic system for rapid pathogen classification and AST. We demonstrate the NAME technique for identifying bacteria that commonly cause BSI, VAI, and UTI. In collaboration with our clinical and industrial partners, we are developing an integrated ID-AST platform for rapid diagnosis of bacterial infections. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST. Our results demonstrate the analytical and clinical feasibilities of the integrated ID-AST platform for rapid microbiological analysis.