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German Team Develops MALDI Assay for Rapid Blood Culture Susceptibility Testing

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NEW YORK (360Dx) – Researchers from University Hospital Münster and Bruker have developed a MALDI mass spec-based method for testing infected patient blood cultures for antimicrobial susceptibility.

The method, which was described in a paper published last week in the Journal of Clinical Microbiology, could allow clinicians to more rapidly determine the best course of treatment for patients with sepsis, said Evgeny Idelevich, a University Hospital Münster researcher and first author on the study. He added that he and his colleagues are collaborating with Bruker to develop a commercial version of the technique. That effort is being funded by a three-year, €900,000 ($1 million) grant from the German Federal Ministry of Education and Research.

The technique combines MALDI-based microbial identification with the direct-on-target microdroplet growth assay (DOT-MGA) previously developed by Idelevich and his colleagues. The DOT-MGA approach involves placing microdroplets containing suspensions of the bacteria of interest and the antibiotic being tested directly onto the MALDI target. Clinicians then incubate these microdroplet suspensions in an incubation chamber to let the bacteria grow. At the end of the incubation time, they remove the microdroplets using an absorbent material like filter paper or wipe, which leaves behind the bacterial cells that have sedimented atop the MALDI target during the course of the incubation period.

These cells can then be analyzed via mass spec as per a standard MALDI microbial identification assay. If the mass spec is able to make an identification, that is an indication that the cells grew well in the presence of the antibiotic being tested and are not susceptible. If no identification is made, that indicates that the cells did not grow and are susceptible to that antibiotic.

Idelevich said the approach is able to speed susceptibility testing to where results, which typically take a day to deliver, can be had the same day. That could allow doctors to more quickly move from broad-spectrum antibiotics to more targeted drugs, which could both improve patient outcomes and help stem the rise of antibiotic-resistance bacteria.

MALDI mass spec platforms identify microbes by matching the protein profiles of sample organisms generated via MALDI to profiles contained in a proprietary database. Compared to traditional biochemical methods of microbe detection, MALDI-based systems can offer significant improvements in speed, price, and accuracy.

The technique has seen significant uptake within clinical microbiology for organism identification, with Bruker's MALDI Biotyper and BioMérieux's Vitek MS systems now fairly standard platforms in clinical microbiology laboratories. Having established the technology for organism ID, these companies and other outside researchers now aim to apply them to susceptibility testing.

In recent years, significant effort has been put into molecular methods analyzing bacteria nucleic acids for genetic alterations indicative of different types of antibiotics, said Karsten Becker, a professor at the University of Münster and senior author on the study. However, he noted, one disadvantage of this approach is that it can only detect a subset of possible resistance mechanisms.

"There are mechanisms that go beyond simple DNA-based mechanisms, or mechanisms that are DNA-based but that are [still] unknown," he said. "There are a huge number of different [potential resistance] mechanisms, and so it is impossible to cover all of them with molecular approaches."

Many MALDI-based efforts have likewise used targeted analyses focused on detecting a limited number of resistance mechanisms. For instance, researchers have developed assays using MALDI to detect a mass spectral peak specific to the pKpQIL_p019 protein, which is often present on the same plasmid as the bkaKPC gene, which produces the protein carbapenemase KPC, which can confer resistance to carbapenems.

Bruker has developed CE-IVD kits for the Biotypers that take a broader approach, using the instrument to detect the hydrolysis products generated by resistant enterobacteria, like resistant strains of Klebsiella pneumoniae (KPC), when they break down a carbapenem. This has the advantage of being able to detect other carbapenem-resistance mechanisms as well as carbapenem resistance based on yet-to-be-discovered mechanisms. The company also offers a cephalosporin-resistance testing kit uses a similar approach.

The Münster assay casts an even wider net, however, allowing researchers to perform what are essentially traditional susceptibility testing assays in which growth of bacteria is tested in a range of antibiotics to assess which will be effective.

"We are on the way back, you could say, to a traditional culture approach, but combined with MALDI-TOF mass spectrometry," Becker said, adding that this could allow for the speed of newer approaches with the universality of conventional susceptibility testing.

Existing [non-MALDI] automated susceptibility testing systems typically require 16 to 20 hours to return a result, while even more recently developed rapid protocols take 10 to 12 hours, Idelevich said, meaning that targeted treatment often won't begin until the following day.

The DOT-MGA combined with MALDI can provide susceptibility testing results in as little as four hours, Idelevich said, noting that the DOT-MGA method contributes to shortened turnaround time by eliminating steps that would otherwise be needed to extract bacterial cells from their cultures and transferring them to the MALDI target for analysis.

The DOT-MGA approach is also compatible with automation, Idelevich said, and, because MALDI targets typically contain 96 spots, each of which can contain a bacteria-antibiotic suspension, it is also amenable to high levels of multiplexing.

Idelevich said the researchers currently run each condition in triplicate as they are still trying to determine the reproducibility of the approach, but he added that based on their research thus far he believes they will be able to run two or even one spot per condition. Typical susceptibility assays test no more than 18 or 19 antibiotics, "because you don't really have more antibiotics that would be relevant [to a given bacteria]," he said, which suggests the technique could let clinicians test full susceptibility panels for two to three bacteria per run.

The Münster researchers previously used the approach for susceptibility testing of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates. This month's JCM study was meant as a proof-of-principle showing the technique could be used for testing isolates from positive blood cultures, demonstrating its potential for managing sepsis cases.

In the study, the researchers spiked human blood with meropenem-resistant and non-resistant K. pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Proteus mirabilis, and Klebsiella oxytoca isolates collected from patients at University Hospital Münster. They then processed the positive blood cultures to produce bacterial suspensions that they combined with meropenem solution and spotted on the MALDI target where they were incubated, along with controls, for three or four hours before analysis using Bruker's MALDI Biotyper. The assay was able to identify meropenem-resistant organisms with a sensitivity of 92 percent and a specificity of 100 percent.

The researchers now aim to better standardize the assay, Idelevich said.

"Right now we use a plastic box for a humidity chamber, and we use Chem Wipes or filter papers as absorptive materials, and these things have to be standardized to provide people with small consumables that are standardized," he said. "The procedure itself also [needs to be] standardized. The MALDI-TOF measurements, the intensity of the device, internal standards to control for the quality of spectra, everything has to be controlled."

"It sounds like simple things, but we're very glad to have Bruker as a partner here, because they are very much aware of these [challenges] and very professional about them," he said.

Additional studies will also be needed to move the approach to the clinic, Idelevich said, adding that he expects the method would become available as a research-use-only kit initially with clinical versions following after that.