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'Digital' Nucleic Acid Amplification Shows Promise for Rapid Antibiotic Susceptibility Testing


NEW YORK (GenomeWeb) – Testing bacterial cultures for antibacterial susceptibility takes days and the growing number of antibiotic-resistant bacterial infections is thought to be partly due to inappropriate antibiotic use that happens in the meantime.

To combat this problem, researchers led by Rustem Ismagilov at the California Institute of Technology have now demonstrated a method of antibacterial susceptibility testing that takes less than 30 minutes and can be performed directly from patient samples with no need for bacterial culture.

The method described in a Science Translational Medicine paper published this month, relies on digital loop-mediated isothermal amplification, or dLAMP, in which isothermal reactions are distributed into 1,280 compartments. The assay uses cycle threshold as a readout of bacterial replication after a brief antibiotic exposure and is made specific by amplifying nucleic acids of bacterial strains of interest.

Standard PCR-based molecular testing for DNA signatures of resistant strains is time-consuming, and since bacteria mutate frequently there is a risk of false negatives. Phenotypic testing — in which different antibiotics are applied to see if any is effective at killing the bacteria in question — is a common alternative to molecular testing, but it usually requires significant amounts of bacteria that are typically only available after culturing.

During the time it takes for a hospital lab to processes and culture samples and diagnose resistance, patients are often given empiric therapy, or a best-guess course of broad-spectrum antibiotics, and this potentially inappropriate antibiotic use can lead to selection in favor of resistant strains.

"From that point of view, the ability to get a phenotypic answer in 30 minutes from a clinical sample presents a tantalizing opportunity," Ismagilov said in an email.

"We must establish robust antimicrobial stewardship programs that move antibiotic prescriptions from guideline-driven approaches to rapid, point-of-care AST driven approaches," said Ismagilov, adding that a transition to employing POC AST would allow more tailored antibiotic prescribing, enable the pharmaceutical industry to justify deployment of narrow spectrum antibiotics, and preserve powerful broad-spectrum antibiotics for cases where they are truly needed.

The dLAMP AST method was demonstrated to be unaffected by levels of commensal bacteria, and, on a trial set of clinical patient urine samples, 53 of 54 dAST calls matched the gold standard AST call provided by a culture assay. The study also assessed the method using a slower commercial digital PCR system, and further notes that other amplification chemistries, such as recombinase polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), and helicase-dependent amplification (HDA), could potentially be optimized for this digital AST format.

The dAST method uses SlipChip technology that originated in the Ismagilov lab, with SlipChip Corp, a company founded in 2011, providing the injection molded chips that were used in the dAST research. The microfluidic SlipChip initiates reactions using a sliding motion to combine nanoliter volumes of reagents and samples, and the technology is currently available for licensing from Caltech. SlipChip Corp has also recently developed a new manufacturing method for the chips, Ismagilov said.

He and his colleagues previously demonstrated SlipChip-based digital LAMP could be used to detect HIV-1 RNA and have suggested it could be used for other applications, such as protein crystallization, cell culture, and immunoassays.

The dLAMP AST method builds on the lab's previous research, but the "real-time, rapid dLAMP with an [approximately] 7-min assay and dAST measurements are completely new," Ismagilov said.

The method uses a 15-minute antibacterial exposure step, followed by a brief, one-step extraction with QuickExtract DNA Extraction Solution from Epicentre, an Illumina company whose products are now distributed solely by Lucigen.

While the lab's previous work has focused more on infectious disease diagnostics for global health, it was motivated to move into the realm of AST because "it is a huge problem,” Ismagilov said. An increase in resistant bacteria could spell the end of modern medicine as we know it, he suggested, adding, "We, collectively, have to solve this problem now."

There are some commercially-available molecular ASTs, such as MRSA tests looking for the mecA gene in S. aureus, for example. "But they do not seem to be generalizable beyond a few specific examples in Gram positives," Ismagilov said.

Phenotypic tests are the gold standard, but there are no commercial tests that perform a phenotypic test directly from clinical samples without a culture step. Some tests do gain speed by shortening the culture times and using very sensitive amplification. For example, one particularly fast platform, the Pheno system from AccelerateDx, requires one day of bacterial culture and subsequent testing then takes seven hours. Although it provides an estimated 40 hours saved over the next fastest phenotypic method, some laboratorians have questioned whether that time savings is meaningful enough to make up for an increase in cost over existing systems.  

Interestingly, like the dLAMP AST, a few other rapid tests in development also targeted urinary tract infections as an initial rapid AST sample type. A group in Sweden has developed a method using padlock probes and another team recently reported a 30-minute AST test for UTIs that has been spun into a company called Astrego Diagnostics.

"Urine is certainly more tractable as a starting point than other sample types," Ismagilov explained. Furthermore, "UTIs are a major source of antibiotic prescriptions, and therefore a rapid [30-minute] point of care AST for UTIs is urgently needed to enable antimicrobial stewardship."

A 2017 Biomedical Microdevices review of microfluidic devices for AST points out that sample preparation, including culture time, is a major bottleneck for fast, phenotypic microfluidic ASTs. As such, the dLAMP method has a leg up on nearly all competitors. The review also suggests that development in this field may potentially be spurred by awards offered in the UK and the US for diagnostics focused on antibiotic stewardship.

The National Institutes of Health and Biomedical Advanced Research and Development Authority (BARDA) launched a $20 million competition called the Antimicrobial Resistance Diagnostic Challenge in 2016, and the agencies announced their 10 semifinalists in March, including industry-affiliated entries from Becton Dickinson, Philips, First Light Biosciences, Click Diagnostics, and Affinity Biosensor.

The Ismagilov lab still has some work to do to further develop its method. "While going from a sample to a phenotypic AST answer in 30 minutes is a tantalizing result, by no means is this a manufactured, FDA-approved method ready for deployment," Ismagilov emphasized.

"We need to expand this method to a number of other pathogens, antibiotics, and sample types, and then run formal clinical trials to understand the performance of this method," he said, but added that the lab does have "exciting preliminary results in these areas," including adapting the assay to run on other clinical pathogens.