Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing 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 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen’s susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

The diagnosis of infectious diseases is ineffective when the diagnostic test does not meet one or more of the necessary standards of affordability, accessibility, and accuracy. The World Health Organization further clarifies these standards with a set of criteria that has the acronym ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users). The advancement of the digital age has led to a revision of the ASSURED criteria to REASSURED: Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free or simple, and Deliverable to end-users. Many diagnostic tests have been developed that aim to satisfy the REASSURED criteria; however, most of them only detect a single target. With the progression of syndromic infections, coinfections and the current antimicrobial resistance challenges, the need for multiplexed diagnostics is now more important than ever. This review summarizes current diagnostic technologies for multiplexed detection and forecasts which methods have promise for detecting multiple targets and meeting all REASSURED criteria.

This communication shows that the concept of Brownian trapping with drift can be applied to improve quantitative molecular measurements. It has the potential to combine the robustness of end-point spatially resolved readouts, the ultrasensitivity of digital single-molecule measurements, and the large dynamic range of qPCR; furthermore, at low concentrations of analytes, it can provide a direct comparison of the signals arising from the analyte and from the background. It relies on the finding that molecules simultaneously diffusing, drifting (via slow flow), and binding to an array of nonsaturable surface traps have an exponentially decreasing probability of escaping the traps over time and therefore give rise to an exponentially decaying distribution of trapped molecules in space. This concept was tested with enzyme and protein measurements in a microfluidic device.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial Graphical AbstractDigital counting reveals antibiotic susceptibility: Single molecule counting detects subtle changes in the amount and the assembly state of bacterial chromosomes after short exposure of live bacteria to antibiotics. Keywordsantibiotics; DNA; antimicrobial susceptibility testing; digital PCR; chromosome segregationThe increasingly liberal use and misuse of antibiotics (ABX) has led to widespread development of antibiotic resistance. [1] To address this crisis, we need rapid and reliable tests of a pathogen's susceptibility to the drugs available (antimicrobial susceptibility test, AST) to provide correct, life-saving treatment, facilitate antibiotic stewardship [2] and drastically decrease hospital costs. [1a,3] Having a rapid AST that provides results within the period of a doctor visit would lead to improved patient outcomes and reduced spread of antibiotic resistance. [4] Development of a rapid AST is currently the focus of significant research efforts [5] that aim to supplant traditional clinical methods. To reduce the spread of resistance, one urgently needed AST is for urinary tract infections (UTIs), which are among the most common bacterial infections, yet can progress to pyelonephritis or sepsis. [6] Two types of ASTs are currently used in clinical settings: traditional culture-based methods and genotypic methods. Culture-based tests remain the gold standard for determining antibiotic susceptibility because they detect phenotypic susceptibility to a drug, however these tests require a long period of antibiotic exposure (typically 16-24 h). [7] We [8] and others [5a,5b,5k,9] have proposed using confinement of single, or a small number of, bacterial cells in small volumes to reduce the duration of antibiotic exposure required to read out the phenotype of the target pathogen. However, these methods typically do not differentiate between the pathogen and the potential contamination of the sample with commensal bacteria. Alternative genotypic methods (detecting genes responsible for known mechanisms of resistance) are more rapid than culture-based approaches. [10] However, these resistance genes constitute only a fraction of all possible mechanisms of resistance, [11] and new forms of resistance evolve quickly. [12] Therefore, p...