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ASU Team Using MALDI Mass Spec to Improve Detection, Monitoring of Active Tuberculosis


NEW YORK (360Dx) – A team led by researchers at Arizona State University's Biodesign Institute has developed a mass spec-based test for diagnosing and monitoring active tuberculosis.

Detailed in a study published this week in Proceedings of the National Academy of Sciences, the test uses MALDI mass spec combined with an antibody-based target enrichment device to measure the levels of two Mycobacterium tuberculosis peptides.

According to ASU researcher Ye Hu, one of the developers of the test and senior author on the study, the test could prove particularly useful for applications where existing tests struggle. He added that he and his colleagues are also collaborating with researchers at Purdue University on a portable mass spec-version of the test that could be used in resource-constrained locations.

Tuberculosis is commonly tested for using skin tests in a patient is injected with antigen from the organism and then monitored for an immune response. In addition, interferon-gamma release assays from companies like Qiagen and Oxford Immunotec have begun supplanting the decades-old skin test due to their higher accuracy. None of these assays, however, distinguish between latent and active forms of the disease, which makes them ineffective for diagnosing active cases and following their response to treatment.

For active disease management, clinicians typically use sputum-based tests that measure patient bacterial loads, but, Hu said, these tests have relatively poor sensitivity and are poorly suited to diagnosing and monitoring infections in patients with extrapulmonary infections, patients with co-infections like HIV, and pediatric patients. He estimated that such patients make up around 30 percent to 40 percent of tuberculosis cases.

The ASU-developed test measures the levels of two peptides in patient serum, one from the culture filtrate protein (CFP) and another from early secretory antigenic target (ESAT). Both proteins are well-established markers of Mycobacteria, Hu said, adding that he and his colleagues selected peptides from these proteins that they established as specific to M. tuberculosis.

To enable detection of these peptides at low levels, the researchers applied an upfront enrichment process using antibody-conjugated NanoDisks, a technology developed by Hu and his team that captures and enriches target analytes. The structure of the NanoDisks further increases the assay's performance by boosting the MALDI signal, leading to what Hu said was a sensitivity increase of up to 100-fold.

"That is quite important for antigens like these TB antigens," he said. "They are in very low concentrations. So this really addresses this major problem of diagnostic sensitivity."

In a cohort of 27 active pulmonary tuberculosis cases, 31 latent cases (LTBI), 32 non-tuberculosis mycobacterial infections (NTM), and 21 healthy controls, the test identified active cases with specificity of 87.1 percent and sensitivity of 90.6 percent. It registered no false positives in healthy controls but did in four of the 31 LTBI cases and three of the 32 NTM cases. It registered false negatives in two of the 27 active cases.

Looking in a cohort of HIV/tuberculosis co-infected patients, a group where extrapulmonary tuberculosis (EPTB) is particularly common, the test identified 91.3 percent and 82.4 percent, respectively, of culture-positive and culture-negative pulmonary cases, and 92.3 percent and 75 percent, respectively, of culture-positive and culture-negative EPTB cases.

This, the authors noted, improved upon the sensitivity of existing culture-based techniques and PCR-based tests.

The researchers also looked at a cohort of patients with active disease undergoing therapy and found that in 19 of 21 patients, the peptide levels had decreased or were undetectable in patients after six to 12 months of anti-tuberculosis therapy. One of the non-responsive patients did not receive a full treatment regimen due to alcohol-induced liver dysfunction, while the other, an HIV-positive patient, showed an initial decrease followed by a rebound, perhaps due to "a lack of leukocyte bactericidal activity associated with G6PD deficiency or a decrease in the proportion of CD4+ T lymphocytes," the authors suggested.

Hu said he and his colleagues are now working to evaluate the test in larger and expanded cohorts. They have a National Institutes of Health grant for investigating its performance in the pediatric population. They are also working to establish the test as a tool for rapid measurement of treatment efficacy, which Hu noted could prove useful for quickly identifying patients with drug-resistant strains that will not respond to conventional therapy.

"That's very important because in a lot of developing countries, there is a lot of multiple drug-resistant TB," he said. "How to identify those patients quickly during the treatment course is a big issue."

He added that progress was being made in portable mass spec instrumentation for running the test, which could also be key to implementing it in developing countries. Hu and his colleagues are working with scientists in the lab of Purdue University researcher Graham Cooks who are developing miniaturized DESI-mass specs. He said that while their work has to date focused mainly on analysis of small molecules, they have recently developed the capability to measure peptides on these instruments, opening up the possibility of using them for peptide-based tests like the ASU tuberculosis assay.

On the MALDI front, the NanoDisk technology could aid in diagnostics work beyond the tuberculosis test. Hu said he and his colleagues are now looking to use the technology to detect markers of other diseases like HIV and Ebola. He said they have recently applied for an NIH grant applying the method to real-time detection of different Ebola strains.

It could also prove useful for detection of protein biomarkers outside the microbiology space, particularly given the growing interest in MALDI as a clinical tool.

As Hu and his co-authors noted, the Bruker Microflex instrument they developed their assay on has already passed through US Food and Drug Administration clearance as part of Bruker's submission for its MALDI Biotyper clinical microbiology system. This could simplify the process for future assays using the device like the ASU tuberculosis test.

Additionally, the widespread adoption of MALDI-based clinical microbiology platforms in the US and Europe means that many hospitals and reference labs already have the instrumentation needed to run such a test.