Antimicrobial Susceptibility Testing Series: Traditional versus Rapid AST

Unlike traditional antimicrobial susceptibility testing (AST) workflows that take days, rapid AST methods allows MICs to be determined in a matter of hours. This means the time to optimal therapy, antibiotic DOT (days on therapy), and hospital stays can be shortened, with substantial direct cost savings. Emerging antimicrobial resistance (AMR) leads to a high incidence of antibiotic failure, especially for bloodstream infections. Rapid AST can also reduce exposure to potentially toxic broad-spectrum antibiotics.

There are two broad classes of rapid AST technology: genotypic and phenotypic susceptibility testing. Genotypic susceptibility testing detects resistance genes expressed in the organism using molecular methods like polymerase chain reaction (PCR)-based methods or similar.  PCR-based assays are designed to detect the presence of resistance genes, but it has limited utility because only a few resistance genes are firmly associated with phenotypic resistance (ie mecA, vanA, and vanB). There are hundreds of beta-lactamases and numerous mutations and expression mechanisms that result in resistance and too many to be detected by current molecular techniques. The absence of a resistance gene also does not necessarily predict susceptibility to a particular compound. On the other hand, phenotypic methods are based on the organism’s growth pattern and physiology when exposed to different antimicrobials. Phenotypic testing is the gold standard for guiding antimicrobial treatment decisions. Phenotypic methods include sensitive optical and biophysical methods since they must detect growth and/or metabolism with a low organism burden.

Several innovative technologies are available for rapid AST, and some examples of commercial systems are presented in Table 3. Microscopy-based systems confine the bacteria one focal plane with clear medium to allow them to be seen. Strategies like using electrophoretic of centrifugal immobilization into a surface covered by liquid or solid medium, inkjet printing onto a solid growth surface, or confining the bacteria to microfluidic channels. The Pheno™ system (Accelerate Diagnostics) electrophoreses organisms from positive blood cultures onto a solid surface. An automated microscope records their actions from individual organisms into microcolonies (every 10 minutes) and machine learning is used to evaluate their growth.

A number of microfluidic methods confine bacteria to channels or nanodroplets, and use traditional or fluorescence microscopy to visualize them. Microfluidics-based Captiver™ System (Astrego) captures individual bacterial cells on custom-designed microfluidic chips and measures their growth rate in real time, using single-cell imaging.

A challenge for microscopy-based methods is the need for higher magnification (usually > 400X) which requires costly optics and automation. The 215Dx UTI System (BacterioScan) uses laser microbial growth technology and measures forward laser light-scatter in a way similar to flow cytometry, but in bulk growth suspensions. This biophysical bacterial enumeration method is already commercialized for urine samples.

The ASTar® platform (Q-linea) uses high-speed time-lapse microscopy imaging of bacteria in broth.   The dRAST™ system (QuantaMatrix) uses time-lapse microsopy for MIC determination directly from positive blood cultures. The Reveal™ System (Specific) detects low concentrations of volatile organic compounds emitted by microorganisms as they grow.

The “FAST” (Flow Cytometry Antimicrobial Susceptibility Test)(FASTinov) incorporates fluorochromes (probes) that detect microbial cell lesions or metabolic changes and this detection is identifiable through flow cytometer analysis.

It is important to note that rapid AST methods must also factor in the time needed for pre-and post-analytic steps. In order for the potential for rapid AST platforms to be fully realized, results will need to be linked with robust decision support solutions that will lead to therapeutic changes in real time.

In the next article of the series, we will review how pharmaceutical-grade antibiotics are not optimized for AST platforms, and common ways that they can optimized.

Table 3. Examples of rapid AST platforms.

References

Bard JD, and Lee F (2018) Why can't we just use PCR? The role of genotypic versus phenotypic testing for antimicrobial resistance testing. Clin. Microbiol newsletter vol. 40(11):87-95

Smith KP and Kirby JE (2019) Rapid susceptibility testing methods. Clinics Lab. Med. 39(3):333-344 Link