Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing

Abstract

In the face of looming threats from multi-drug resistant microorganisms, there is a growing need for technologies that will enable rapid identification and drug susceptibility profiling of these pathogens in health care settings. In particular, recent progress in microfluidics and nucleic acid amplification is pushing the boundaries of timescale for diagnosing bacterial infections. With a diverse range of techniques and parallel developments in the field of analytical chemistry, an integrative perspective is needed to understand the significance of these developments. This review examines the scope of new developments in assay technologies grouped by key enabling domains of research. First, we examine recent development in nucleic acid amplification assays for rapid identification and drug susceptibility testing in bacterial infections. Next, we examine advances in microfluidics that facilitate acceleration of diagnostic assays via integration and scale. Lastly, recentdevelopments in biosensor technologies are reviewed. We conclude this review with perspectives on the use of emerging concepts to develop paradigm-changing assays.

Keywords: PCR; antibiotic susceptibility testing; high-resolution melting analysis; microfluidics; molecular diagnostics; point-of-care; polymerase chain reaction.

Figure 1

Emerging demonstrations of NAAT-based pathogen identification. (a) With HRMA, species were identified by their unique melting curves. After PCR amplification and HRMA, the raw melting curve of an unknown species was normalized and transformed into a derivative curve. An adaptive algorithm was then matched the unknown curve (green line) against an archived melting curve database (gray lines) to find the best fit. (b) Schematic of the SHERLOCK platform. The DNA or RNA target extracted from biological samples is amplified by RPA (RT-RPA or RPA, respectively). RPA products are detected in a reaction mixture containing T7 RNA polymerase, Cas13, a target-specific crRNA, and an RNA reporter that fluoresces when cleaved. Figure adapted with permission from Reference . Copyright 2017, AAAS. Abbreviations: crRNA, CRISPR RNA; HRMA, high-resolution melting analysis; NAAT, nucleic acid amplification technology; PCR, polymerase chain reaction; RFU, reflective fluorescence unit; RT, reverse transcription; RPA, recombinase polymerase amplification; SHERLOCK, specific high-sensitivity enzymatic reporter unlocking.

Figure 2

Emerging demonstrations of NAAT-based, pheno-molecular AST. Pheno-molecular AST uses NAAT to measure bacterial growth under phenotypic AST and takes advantage of the speed and sensitivity of NAAT to achieve rapid and robust AST. Typical assays start with the brief incubation of a bacteria sample with and without antibiotics. AST can then be determined by measuring the differences of DNA quantity between reactions with and without antibiotics, via either quantitative PCR or digital PCR, as bacteria that do not grow in the presence of the antibiotic (i.e., susceptible) would have less bacterial DNA than the no-antibiotic control. (a) The pheno-molecular AST concept has been coupled with broad-based, real-time PCR and HRM. In this case, unknown bacteria in the sample can be identified by matching the newly generated melt curve to the database of previously built melt curves via a machine-learning algorithm. AST is determined via a measurable ΔCq between the antibiotic sample and the no-antibiotic control. Panel adapted with permission from Reference . Copyright 2017, American Chemical Society. (b) The pheno-molecular AST concept has also been coupled with digital PCR. Following the incubation of samples with and without ABX for different time courses, antibiotic susceptibility of the pathogen tested is determined via digital PCR based on the fold change relative to time 0. Panel adapted with permission from Reference . Copyright 2016, John Wiley & Sons. Abbreviations: ABX, antibiotics; AST, antibiotic susceptibility testing; Cq, cycle number; HRM, high-resolution melting; NAAT, nucleic acid amplification technology; PCR, polymerase chain reaction; qPCR, quantitative PCR; RFU, relative fluorescence unit; SVM, support vector machine.

Figure 3

Emerging technologies for microfluidic assay integration. (a) Example of droplet magnetofluidic assay integration. (Top) A magnetofluidic assay cartridge with aqueous reagents for DNA extraction and amplification. (Middle) Overview of droplet magnetofluidic manipulation. Left panel shows a schematic of aqueous droplet anchored by a hydrophilic poly(methyl methacrylate) (PMMA) substrate. Magnetic particles are actuated on a hydrophobically coated surface via a rare-earth neodymium (NdFeB) magnet. Magnetic particles can be extracted from droplets via translational motion of the magnet. (Bottom) Overview of cartridge operation. DNA extraction, particle washing, elution, and amplification are all achieved on a single cartridge, with each process linked via translocation of magnetic particles between each reagent droplet. Panel adapted with permission form Reference under the terms of the Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0. (b) Example of a paper analytical device–based integration. (Top) Three-dimensional rendering of an analytical device for loop-mediated amplification (LAMP) assay. (Bottom) Overview of device operation. Each reagent for DNA extraction, washing, and amplification is loaded sequentially into a moving paper matrix at each step of the assay. Panel adapted with permission form Reference . Copyright 2015, American Chemical Society. (c) Example of vacuum-driven integration. (Top) Image of a vacuum-driven device with interdigitating vacuum batteries highlighted in blue and green. (Middle) Principle of operation. The vacuum pulls air out of fluidic channels through gas-permeable walls, allowing fluids to be pulled through via negative pressure. (Bottom) Equipment-free loading and automatic sample compartmentalization. Panels adapted with permission from Reference under the terms of the Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0.

Figure 4

Emerging microfluidic devices for scale-driven enhancement of antibiotic susceptibility testing (AST). (a) A microfluidic device with fully integrated droplet generation, incubation, and in-line fluorescence detection is developed to perform single-cell bacterial growth detection and AST. Scaling the reaction chambers in the form of 20-pL droplets within this device enabled detection of single-cell Escherichia coli growth and its susceptibility/resistance to gentamicin in as little as 1 h. Panel adapted with permission from Reference . Copyright 2017, Elsevier. (b) A microfluidic digital array device for antimicrobial susceptibility testing. The microfluidic chip adopts a modular and scalable design for testing multiple antibiotic conditions in the same chip. Bacteria can be reliably digitized in 250-pL chambers via vacuum-assisted loading and oil-driven digitization. MICs (minimum inhibitory concentrations) for E. coli and Staphylococcus aureus against various antibiotics were measured using the digital chip. Panel adapted with permission from Reference . Copyright 2016, Chemical & Biological Microsystems Society.

Figure 5

Rapid antibiotic susceptibility testing (AST) via microfluidics-enhanced single-cell imaging. (a) Cells are first seeded into the device via a large fluidic reservoir, which feeds the cells into microfluidic channels. The channels are connected to an outlet via a sieve, which keeps the cells retained in the channels. (b) Scanning electron microscope image reveals bacterial growth in the microfluidic channels. Panel adapted with permission from Reference . Copyright 2017, PNAS.

Figure 6

(a) Cantilever biosensor for detecting bacterial susceptibility to antibiotics. (Top) The cantilever is conjugated with a bacteria-specific receptor to capture the target bacteria. The attachment of bacteria to the cantilever leads to a change in cantilever fluctuation. (Bottom) Deflection of the cantilever for the antibiotic susceptibility testing (AST) experiments involving Escherichia coli. Panel adapted with permission form Reference . Copyright 2013, Springer Nature. (b) Microfluidic cantilever for detecting bacteria and their antibiotic susceptibility. (Top) The microfluidic channel filled with bacteria supported on a silicon substrate and irradiated with a specific wavelength of tunable infrared light. (Bottom) The inner surface of the cantilever’s microchannel was functionalized with a bacteria-targeted receptor. Shown on the right are fluorescent and scanning electron microscope images from the top side of the microchannel. Panel adapted with permission from Reference under the terms of the Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0. Abbreviations: LB, lysogeny broth; PBS, phosphate buffered saline.