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CHUQ Researchers Develop LC-MS/MS Assay for Direct Detection of Bacteria in Urine

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NEW YORK – Researchers from the Centre Hospitalier Universitaire de Québec (CHUQ) have developed a mass spec-based method for identifying bacteria directly from urine specimens.

Described in a study published this month in Molecular & Cellular Proteomics, the method could offer faster and more quantitative analyses of patient urine samples, said Arnaud Droit, formerly an assistant professor at CHUQ (now an associate professor at Université Laval) and senior author on the paper.

The study represents one of several ongoing mass spec-based efforts to detect and identify bacteria directly from urine samples.

Mass spec, and MALDI mass spec specifically, has made significant inroads into the clinical microbiology market, with Bruker's MALDI Biotyper and bioMérieux's VITEK MS platforms becoming standard equipment in large clinical microbiology labs in the US and Europe. The systems, which identify microbes via their protein profiles, have seen quick uptake due to their relative ease of use and their ability to provide results much more quickly than traditional biochemical assays and a lower cost than many nucleic acid based tests.

These systems still require a culturing step to generate the isolates they use for their analysis, however, and this step can add a day or more to turnaround times. It can also present challenges in the case of organisms that are difficult to culture.

This has led researchers to explore whether direct mass spec analysis of patient samples might be possible. Most commonly this has been done using MALDI instruments. The CHUQ team, however, opted for an LC-MS/MS approach due to the higher performance of such platforms, said Florence Roux-Dalvai, a CHUQ researcher and first author on the paper.

Specifically, Roux-Dalvai said she and her colleagues believed LC-MS/MS might offer better discrimination between closely related species as well as a more quantitative measurement.

Quantitation is important, she said, because patients with urinary tract infections often receive antibiotic treatment only if their specimen grows above a certain number of colonies.

In the MCP study, the researchers combined several mass spec techniques to develop their assay, starting with shotgun mass spec assays of pure bacterial colonies to develop mass spectral libraries for use in subsequent data-independent acquisition (DIA) assays. They used those DIA assays to detect bacterial peptides in urine samples, quantifying 31,000 peptides from 190 samples containing 15 bacterial species that cause 84 percent of all UTIs.

The researchers then used machine learning to develop peptide signatures specific to each of these species and then built targeted proteomic assays to these peptide signatures.

They tested these targeted assays in urine samples inoculated with the four most commonly found causes of UTIs (Escherichia coli, Streptococcus agalactiae, Enterococcus faecalis, and Klebsiella pneumonia) at five different concentrations running the experiments with 90-minute LC gradients on a Thermo Fisher Scientific Orbitrap Fusion. They also ran the experiments on a Thermo Fisher Q Exactive HF-X using a 30-minute LC gradient.

The assays showed 100 percent accuracy in all inoculations at concentrations above the standard clinical threshold and 97 percent accuracy overall.

The researchers also compared their direct detection approach to a standard MALDI-TOF workflow, finding that in a set of 27 patients, the two methods agreed on 19 of the samples (seven of which were not infected and nine of which were infected with E. coli), while disagreeing on eight samples, seven of which the MALDI-TOF method identified as infected while the LC-MS/MS approach identified as not infected, though these seven were identified by the MALDI-TOF at the genus but not species level.

The results suggest the CHUQ team's approach may not be as sensitive as a traditional culture-based MALDI-TOF method. Roux-Dalvai said the researchers are investigating whether a short, broth-based culturing period (of perhaps two to four hours) might boost the sensitivity of the method while still retaining much of its advantage in turnaround time.

While LC-MS/MS has the potential to enable direct analysis of clinical microbiology samples, there are questions around the practicability of the approach in a clinical setting. The Orbitrap Fusion and Q Exactive HF-X instruments used in the MCP study are research platforms that are considerably more expensive and require more expertise than the MALDI-TOF systems currently in clinical use.

Roux-Dalvai said that the researchers are working to move their assays to a triple quadrupole platform, which is more commonly used in clinical labs.

She said they are also considering applying the technique to other sample types, including blood cultures, and for non-clinical applications like food safety testing — specifically detecting contamination of milk products.

The CHUQ researchers are not alone in their efforts to develop mass spec assays for direct detection of bacteria.

While Bruker's MALDI Biotyper workflows generally require culturing isolates for analysis, its MALDI Biotyper Sepsityper assay allows for direct detection of pathogens from positive blood culture bottles in 15 to 20 minutes, according to the company.

In January, a team led by researchers at the IHU Méditerranée Infection published a study in the Journal of Clinical Microbiology using a MALDI Biotyper for direction identification of bacteria in UTIs. Using a centrifugation-based sample prep technique with a standard MALDI Biotyper workflow and custom-built database, the researchers were able to correctly diagnose 90 percent of 500 infected monobacterial samples.

Leiden, Netherlands-based microbiology firm BiosparQ is likewise developing a MALDI-based approach for identifying infections directly from patient samples. The company is currently evaluating the technology's usefulness for assessing urinary tract infections with researchers at Amsterdam University Hospital and aims to launch a CE-IVD-marked instrument for UTI detection in 2021.

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