NEW YORK – South Korean researchers reported on Wednesday that they have developed a culture-free method for blood-based pathogen identification and antimicrobial susceptibility profiling that they said could hasten the treatment of patients with suspected sepsis by reducing testing times by 40 to 60 hours compared with typical antimicrobial susceptibility testing workflows used in hospitals.
In an article published in Nature, the researchers from Seoul National University and Seoul-based in vitro diagnostics firm QuantaMatrix said that they used a synthetic peptide to recover bacterial pathogens from whole-blood samples for identification and susceptibility testing, with results achieved in about 13 hours. Lead author Tae Hyun Kim said by email that QuantaMatrix, which was founded in 2010 as a Seoul National University spinout, is licensing the ultrarapid antimicrobial susceptibility testing (URAST) method with plans to develop a fully automated instrument with integrated pathogen detection, identification, and antimicrobial susceptibility profiling.
The researchers said that they expect that the URAST system will "completely transform the current standards of bacteremia diagnostics," adding their methods have the potential to deliver phenotypic drug susceptibility information on the same day as the patient blood draw.
"With the potential to guide clinicians towards timely administration of optimal therapy, thereby saving many lives, our platform can also help de-escalate the spread of antimicrobial resistance and sustain the lifespan of existing antibiotics," they wrote.
Kim said that the researchers aimed to develop testing methods that forgo blood culture because it is the most time-consuming process in antimicrobial susceptibility testing. He said that many hospitals also do not provide 24-hour lab services, and their standard sepsis testing processes require manual steps that each introduce additional delays, further delaying test turnaround times beyond the critical treatment window for sepsis.
"Considering that patients' blood contains many growth inhibitors such as white blood cells and pre-administered antibiotics, we focused on approaches to directly isolate pathogens from whole blood," he said.
Developing faster methods for detecting blood-borne infections and identifying their causes has been an enduring problem that other firms have also sought to address by eliminating the need to culture blood.
T2 Biosystems has been a leader in the development of direct-from-blood sepsis tests with its T2Bacteria panel that is used for the detection of six common sepsis-causing pathogens within three to five hours. The firm has been developing a drug resistance panel with financial backing from the US Department of Health and Human Services.
German startup Noscendo recently began commercialization of a sequencing-based test, Disqver, that uses cell-free DNA in patient plasma samples, with an estimated 24 hours to receive definitive results.
Meanwhile, Oxford Nanopore Technologies and Day Zero Diagnostics formed late last year a collaboration to develop a whole-genome sequencing-based clinical diagnostic system for the same-day identification and antimicrobial susceptibility profiling of bloodstream infections without the need for blood culture, while Siemens Healthineers has been developing a next-generation sequencing-based test to identify bacteria and fungi in a patient's bloodstream within six hours.
Other firms including Immunexpress, Inflammatix, Bluejay Diagnostics, Diasorin, and Cytovale have taken a different approach to improving sepsis response through rapid tests that are meant to quickly indicate which patients likely have sepsis or other severe infections or to guide patient triage.
In the Nature article, the authors isolated bacterial pathogens from blood samples using magnetic nanoparticles with a synthetic version of human beta-2-glycoprotein 1 peptides, which bind to a diverse array of bacteria species. Kim said that the team also evaluated the use of other immune response-related substances, including mannose-binding lectin proteins, but found that nanoparticles coated with the synthetic beta-2-glycoprotein 1 peptides had the highest binding affinity.
The enriched bacteria were identified using a multiplex assay with single-stranded DNA probes that were immobilized on the surface of shape-encoded microdisks with unique hole patterns for each target gene. Bacterial cells were lysed for genomic DNA extraction followed by amplification using nested PCR and biotin labeling. The researchers used the hole patterns and fluorescent dyes to aid gene-based identification of pathogens and achieved results within three hours.
The authors reported that they also accelerated the antimicrobial susceptibility testing process by culturing the enriched bacteria in a purified medium. Under those conditions, rapidly dividing species such as Escherichia coli can reach the minimum cell count for URAST within four hours, which allowed antimicrobial susceptibility testing with results in less than eight hours.
To further reduce turnaround times, the researchers said that they evaluated URAST using custom-built 96-well chips that accommodate a low number of cellular inputs for minimum inhibitory concentration determination and susceptibility analysis.
The research team validated the URAST system in a 190-patient clinical study that paired the peptide-based enrichment methods with simultaneous tests for pathogen identification and susceptibility profiling. They reported that URAST identified bacterial pathogens with 100 percent concurrence with hospital-established testing protocols that used blood culture and matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry.
The team also performed retrospective antimicrobial susceptibility testing on six clinical isolates and found that their testing methods performed with 94.9 percent agreement and a 5.1 percent minor error rate in comparison with broth microdilution disk diffusion. They said that their tests in the study delivered antimicrobial susceptibility results an average of 48 hours more quickly than conventional hospital workflows.
While the researchers tested for 36 bacteria species during their study, Kim noted that QuantaMatrix intends to expand the target range to at least 37 bacterial species and six fungi, so that the test that could be used for the detection and characterization of more than 95 percent of pathogens that cause sepsis. He said that the highly multiplexed identification assay also allows for the expansion of the gene list without significant increases in testing costs or processes, and the company hopes to add testing for 27 genes that are connected with antimicrobial susceptibility and resistance.
Kim said that the team aims to build a system that would allow the seamless identification and characterization of pathogens within a blood sample with minimal involvement from clinicians so that the testing system can be implemented in clinical settings.
The Nature article notes that one of the patients in the clinical trial died during the testing period and a shorter turnaround time for testing may have helped healthcare providers to deliver optimal treatments and prevent renal failure. The patient had developed a fever 12 days after beginning chemotherapy for natural killer/T-cell lymphoma, and treatment began with the carbapenem antibiotic meropenem followed by Gilead's antifungal drug AmBisome (amphotericin B).
Two days after treatment began, test results identified the cause of the infection as a carbapenem-resistant strain of Klebsiella pneumoniae.
"We have been dedicated to AST research for over 10 years, dreaming of the day when our technology will reach the clinic and save many patients' lives," Kim said. "During the clinical pilot study, we witnessed numerous cases of sepsis patients passing away, which deepened our sense of responsibility towards this research."