cAST: Capillary-Based Platform for Real-Time Phenotypic Antimicrobial Susceptibility Testing

Abstract

Antimicrobial resistance is recognized as one of the greatest emerging threats to public health. Antimicrobial resistant (AMR) microorganisms affect nearly 2 million people a year in the United States alone and place an estimated $20 billion burden on the healthcare system. The rise of AMR microorganisms can be attributed to a combination of overprescription of antimicrobials and a lack of accessible diagnostic methods. Delayed diagnosis is one of the primary reasons for empiric therapy, and diagnostic methods that enable rapid and accurate results are highly desirable to facilitate evidence-based treatment. This is particularly true for clinical situations at the point-of-care where access to state-of-the-art diagnostic equipment is scarce. Here, we present a capillary-based antimicrobial susceptibility testing platform (cAST), a unique approach that offers accelerated assessment of antimicrobial susceptibility in a low-cost and simple testing format. cAST delivers an expedited time-to-readout by means of optical assessment of bacteria incubated in a small capillary form factor along with a resazurin dye. cAST was designed using a combination of off-the-shelf and custom 3D-printed parts, making it extremely suitable for use in resource-limited settings. We demonstrate that growth of bacteria in cAST is approximately 25% faster than in a conventional microplate, further validate the diagnostic performance with clinical isolates, and show that cAST can deliver accurate antimicrobial susceptibility test results within 4–8 h.

Visible colorimetric growth indicators of Neisseria gonorrhoeae for low-cost diagnostic applications

“…Although there is still more work to be done, we hope that the research discussed here provides a starting point to a colorimetric liquid microdilution or a paper microfluidic device susceptibility testing of N . gonorrhoeae , as has been developed for other organisms [ 35 , 58 ].…”

“…While this has never been explored for gonorrhea, XTT determined MIC values have been verified for Pseudomonas aeruginosa [55], Mycobacterium tuberculosis [56], and Helicobacter pylori [57]. Although there is still more work to be done, we hope that the research discussed here provides a starting point to a colorimetric liquid microdilution or a paper microfluidic device susceptibility testing of N. gonorrhoeae, as has been developed for other organisms [35,58].…”

“…The purple-colored resazurin (Fig 3A) is reduced by metabolic products of bacterial cells to produce pink-colored resorufin (Fig 3B and 3C), which can be further reduced to hydroresorufin, which is clear and colorless [33]. This color change is fluorescent at a excitation wavelength of 530-570nm with emissions at 585-590nm wavelength [34], but changes are also easily seen by eye [35], making it applicable to a variety of low-cost technologies [36][37][38]. Resazurin is typically diluted to a working concentration of~0.1mg/mL [39], but for PrestoBlue, the manufacturer recommends a standard 10% v/v dilution of the stock solution to obtain a working concentration.…”

Recent advances in biomedical, biosensor and clinical measurement devices for use in humans and the potential application of these technologies for the study of physiology and disease in wild animals

“…In a broad sense, phenotypic tests generally attempt to recreate classical microbiological strategies of growing bacteria and assessing their antibiotic susceptibility profiles. A number of innovative platforms have been developed which include gel modified electrode sensors capable of discriminating between Staphylococcus aureus and MRSA in under 1 h [78], the use of the redox agent potassium ferricyanide to report bacterial respiration in antibiotic-infused cultures [79], a capillary-based optical platform capable of aspartate aminotransferase (AST) results within 4-8 h [80], and the use of microfluidic platforms in combination with the commonly employed respiration rate sensitive compound resazurin to quickly determine antibiotic susceptibility from a sample [81,82]. Genotypic sensors have been developed for detection of AMR which directly probe for the presence of specific gene sequences indicative of bacterial species and any resistance genes [29,83].…”

Early Warning Diagnostics for Emerging Infectious Diseases in Developing into Late-Stage Pandemics

“…During the COVID-19 pandemic, inexpensive lateral flow assays were developed to rapidly detect viral RNA, antigens, and antibodies. − These technologies spanned multiple biological samples, target analytes, and sensing modalities, demonstrating the ability of this technology to be applied in various settings for numerous infectious diseases. For bacterial infections, antibiotic susceptibility testing has taken many forms, including in capillaries and other microfluidic platforms, , to improve the applicability to point-of-care settings.…”