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Mass Spec Struggles to Demonstrate Utility as Platform for SARS-CoV-2 Dx

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NEW YORK – While mass spectrometry has played a substantial role in research into the biology of SARS-CoV-2, it has been largely absent as a tool for diagnosing infections.

Some researchers and studies, suggest, though, that the technology could carve out a niche within the COVID-19 testing world, even as molecular assays and, to a growing extent, immunoassay-based rapid antigen testing, will likely continue to dominate the diagnostic scene.

Among the potential advantages mass specs could offer are rapid turnaround time, the ability to circumvent supply chain limitations, and, in the case of MALDI-based testing, a large pre-existing install base of clinical instruments around the world.

At the same time, questions remain about whether MALDI-based approaches will provide the sensitivity required for diagnosing infections. And while LC-MS/MS methods are typically more sensitive and have shown promise in some studies, such instruments don't provide as high a level of throughput as MALDI and are also not commonly used for clinical infectious disease work.

Led by Bruker and BioMérieux, which offer, respectively, the MALDI Biotyper and Vitek MS systems, MALDI mass spec has become a widely used technology in the clinical microbiology lab for microbial identification and is becoming increasingly used for applications like antibiotic resistance testing.

Compared to conventional microbial ID assays, MALDI is less expensive and faster to results, which has allowed it to make rapid inroads into the field. It is also more user friendly and streamlined than LC-MS/MS approaches, which has contributed to its greater uptake for clinical microbiology work, despite the fact that the latter technology is typically more sensitive and can offer deeper analysis.

MALDI has not typically been used for virus identification, but as clinicians struggled with supply chain issues and other problems as they worked to scale molecular SARS-CoV-2 testing this spring, a number of parties began exploring if MALDI might prove useful for diagnosing the virus.

UK-based testing firm MAP diagnostics announced in April that it was working to develop a MALDI-based SARS-CoV-2 test. The company and collaborators published a paper in Diagnostics in September demonstrating that they could detect spiked virus in saliva samples and that detection of viral proteins could potentially be combined with detection of host proteins to diagnose COVID-19 infections. The company is currently running trials in the US and UK to determine how their assay performs in gargle samples in patients whose infection status has been confirmed by PCR, said CEO Ray Illes.

In July, researchers from the Autonomous University of Chile, Talca and the University of Talca published a study in Nature Biotechnology describing a test that combined MALDI mass spec with machine learning to diagnose COVID-19 infections.

Also in July, the French Defence Health Service launched a clinical study that aimed to enroll 600 subjects with the goal of developing a MALDI-based test for diagnosing SARS-CoV-2 infections and to correlate protein markers with the clinical course of the infected subjects.

"A high sensitivity, high specificity MALDI assay that requires limited sample processing steps could greatly improve testing capacity in laboratories that already have a MALDI instrument, particularly those without a high-throughput automated molecular platform," said Laura Filkins, assistant professor of pathology at UT Southwestern Medical Center and director of microbiology at Children's Health/Children's Medical Center Dallas. She added that such an approach could also "free laboratories from some of the current supply chain shortages for nucleic acid extraction and PCR."

It's not clear, however, that MALDI mass spec offers the level of performance required for an effective SARS-CoV-2 test, Filkins said, noting that the methods published thus far appear to lack the sensitivity needed for direct detection of the viral peptides and that approaches using measurement of host response proteins to diagnose infections may lack the required specificity.

Filkins and colleagues published a commentary in Clinical Chemistry looking at the MALDI assay developed by the Chilean researchers. The Chilean team developed their test using MALDI mass spec data from 362 samples (211 SARS-CoV-2 positive and 151 SARS-CoV-2 negative by RT-PCR). Using machine learning to analyze 88 MALDI peaks collected across the samples, the researchers developed a model that they found diagnosed COVID-19 cases with 93 percent specificity and 95 percent sensitivity.

The Clinical Chemistry authors noted, however, that the study did not look at samples with other viruses or bacteria that might be mistaken for SARS-CoV-2 and that some of the samples were used both for the development of the diagnostic model and its validation. Additionally, they observed that only one of the peaks examined by the Chilean team was consistently reproducible, and that this peak was lower in infected patients, suggesting that their assay was not primarily based on detection of SARS-CoV-2 virus proteins but was in fact picking up some aspect of the host response.

Filkins said that looking at host response was a potentially interesting diagnostic approach but one that required more thorough evidence to support it. Leonardo Santos, a researcher at the University of Talca and senior author on the Nature Biotechnology paper did not respond to requests for comment.

Given MALDI's challenges on the sensitivity front, some researchers have explored whether higher performance LC-MS/MS systems could be useful for diagnosing SARS-CoV-2. In June, a team led by researchers at the Technical University of Munich developed a parallel reaction monitoring (PRM) assay on a Thermo Fisher Scientific Fusion Lumos Tribrid that measured 23 SARS-CoV-2 peptides in nasopharyngeal swab samples from 91 subjects (37 negative and 54 positive by RT-PCR). They found that they were able to identify only 20 percent of the PCR-positive cases with their PRM assay.

"For detecting the virus in patients, it is not particularly successful and in many ways not competitive with the PCR-based detection technologies that are out there," said Bernhard Küster, chair of proteomics and analytics at TUM and senior author on the study."

Jean Armengaud, a researcher at the French Frédéric Joliot Life Science Institute (CEA), said, however, that he believed some groups had struggled to detect SARS-CoV-2 proteins in nasopharyngeal swab samples in part because the transport media used to store such samples often has proteins in it that can interfere with mass spec analysis.

Filkins likewise noted that many transport media "contain fetal bovine serum, which can predominate the mass spectrum, making detection of low abundance viral peptides more challenging."

She added that this was an important consideration for clinical tests generally, including mass spec-based tests, and said that some institutions have used saline buffers during the pandemic due to supply chain issues and that these buffers might prove a suitable alternative for mass spec-based tests.

In a study published in July, Armengaud and his colleagues used nasopharyngeal swabs spiked with different amounts of purified SARS-CoV-2 protein to develop an LC-MS/MS test on a Thermo Fisher Q-Exactive HF instrument that they then tested on nine clinical samples from COVID-19 patients in the remission phase of their infections. Of those samples, they identified only two of five patients who were positive by PCR but the authors noted that the three cases that were undetected had very low viral loads when they were sampled.

In August, a team led by Mayo Clinic researchers published a MedRxiv preprint that combines antibody enrichment of the SARS-CoV-2 nucleocapsid with high-field asymmetric ion mobility spectrometry (FAIMS) PRM mass spec on a Thermo Fisher Orbitrap Exploris 480 instrument. Using a machine-learning model to identify COVID-19 positive samples, they were able to identify RT-PCR-positive patients with 98 percent sensitivity and 100 percent specificity.

The results suggest that LC-MS/MS could provide the level of performance needed to effectively test for SARS-CoV-2. However, the complexity of the Mayo assays, which involves antibody enrichment and ion mobility separation, makes it less amenable to rapid deployment than would be the case for a MALDI-based assay.

Additionally, throughput would be relatively low. Filkins suggested that a MALDI assay could potentially run as many as 1,000 samples per day. The Mayo researchers said their assay could run around 100 samples per day.

Armengaud suggested that his team's assay was intended more as a demonstration of the potential of LC-MS/MS for infectious disease work than a test meant to be deployed in time to play a significant role in the current pandemic. He said his team was currently exploring several approaches to improve the sensitivity of their assay, though he declined to provide further detail.

"It's a demonstration of the power of mass spectrometry, which has the power of being very discriminative and being quick," he said. "However, it can't be applicable in terms of a diagnostic today because the instruments are too costly and only specialists are able to operate these kinds of instruments today."

He said he hoped that demonstrations like his group's paper would push vendors toward the development of more affordable and streamlined analytical platforms that would be better suited to the diagnostic market.

"Maybe it won't be applicable to COVID-19, but it would be interesting to get it ready for the next pandemic," he said.

There has been one mass spectrometry-based test to received Emergency Use Authorization from the US Food and Drug Administration, Ethos Laboratories' SARS-CoV-2 MALDI-TOF Assay, which received EUA in August. The test runs on Agena's MassArray System, which uses MALDI mass spec to measure not proteins but PCR products and so is more analogous to molecular testing than most of the other mass spec tests under development.