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Study Suggests Feasibility of Immune-Response Biomarker Panels to Detect Latent Tuberculosis


NEW YORK (GenomeWeb) – Detecting latent tuberculosis, discriminating it from active infection, and predicting whether it will emerge into full-blown infection may prove critical to future TB eradication efforts. A small proof-of-principle study now suggests real-time RT-PCR panels detecting mRNA biomarkers in antigen-stimulated blood are a promising method.

Published this week in The Journal of Molecular Diagnostics, the study was accompanied by a commentary from David Persing, executive vice president and chief medical and technology officer at Cepheid.

Cepheid manufactures the GeneXpert platform, which runs cartridges capable of detecting Mycobacterium tuberculosis from various patient fluids.

"TB has been a big focus of ours and has been a big driver of the over 7,500 [GeneXpert] systems based worldwide; it's really been a driver behind democratization of molecular diagnostics in general," Persing told GenomeWeb.

While Cepheid does not have an active program in the latent TB space, the company is "certainly aware of the unmet need," he said.

The main purposes of the commentary was to draw attention to this need and to point out some of the shortcomings of current approaches, which are not able to distinguish between active and latent tuberculosis, or, among latent cases, to tell who is likely to reactivate to develop active infection, Persing noted.

This is critical because one third of the global population is latently infected with TB, and this population is a "ticking time bomb in terms of development of active tuberculosis," he said.

Latent TB is not contagious, but up to 10 percent of these cases will emerge from this reservoir and transition to active tuberculosis disease. The conversion rate is higher for people with HIV co-infection or who are otherwise immunocompromised.

"Any kind of tools that we can develop to identify those patients at greatest risk of reactivation go a long way toward ultimate complete control of tuberculosis," Persing said. Once detected, these patients could then be treated to lower their risk of reactivation.

In the JMD study, researchers at four different institutes in Korea stimulated 79 patient blood samples with TB antigens and measured mRNA expression levels of eight cytokines and chemokines ― IFN-γ, TNF-α, IL-2R, IL-4, IL-10, CXCL9, CXCL10, and CXCL11 ― using a multiplexed RT-qPCR TaqMan assay.

Two subsets of these biomarkers were able to detect all of the patients with active pulmonary TB infections, as well as 80 percent or more of the latent infections.

"This was a small study, it didn't include a lot of patients who can be confounding for these kinds of assays" such as those with autoimmune disease or other non-specific inflammatory conditions, Persing pointed out.

"But the data were really so promising, it suggests it needs to be expanded," he added.

In the study the researchers also compared the results of their assay to the Quantiferon-TB Gold In-Tube assay from Qiagen.

Known as an interferon gamma release assay (IGRA), that diagnostic was approved by the US Food and Drug Administration in 2001. It is also a measure of the response of patient blood to stimulation with TB antigens, but is ELISA-based rather than molecular. IGRAs are recommended by the US Centers for Disease Control to test people who received a type of vaccination that can cause a false positive TB skin test, and Quantiferon is projected to be a future growth driver for Qiagen.  

A multiplex panel of biomarkers, on the other hand, amounts to "multiple shots on goal," Persing said, which gives a lot of flexibility. "To me that was the real promise of this approach … [it is] a multiplexed immunoassay that is based on a molecular diagnostic readout of gene expression. There might be advantages to that just by virtue of the sheer number of markers you can look at."

He noted that these are theoretical advantages at this point, and the study should be expanded to a larger scale.

Interestingly, Cepheid's GeneXpert may be compatible with a multiplex biomarker panel such as this. Persing noted the platform currently uses six independent detection channels for the TB assay. A new research initative at the company has expanded that number to 10, he said, and that is being leveraged in development of new TB assays.

"TB Ultra, an ultra-sensitive version, will use six-color detection plus high-resolution melt," Persing said. Another test will look for markers of extreme-drug-resistant tuberculosis, or XDR-TB, and will use 10-color capability plus HRM, he said. "You end up with additional channels for detection and multiplexing, and you can also resolve more probe binding events within each color channel, [which] greatly expands our multiplexing capability."

The firm is also continuing to work on a "honeycomb" tube ― a microwell-based array that "expands the real-time detection capability to up to 1,000 individual reactions per GeneXpert cartridge," Persing said, and enables "individual nanoliter-level microwell real-time PCR reactions within the same Xpert cartridge configuration." Described in Genome Web previously, this program is also now being used to develop assays in Cepheid's oncology test pipeline.

"Multiplexing is clearly a direction we're moving in, both with the 10-color system and also with the honeycomb tube system," Persing said.

Additional studies will soon be published describing mRNA responses to TB antigen stimulation, Persing said, so this method "could represent a next-generation approach to evaluating TB latency and likelihood of reactivation."

Cepheid's current GeneXpert TB test, Xpert MTB/RIF, simultaneously detects TB and resistance to a common treatment, rifampicin. Launched in 2010, that assay is heavily subsidized by non-profits and endorsed by the World Health Organization in order to reach populations in TB-endemic areas of the developing world.

As of March 31 of last year, WHO reported 2,343 instruments and 6,291,330 cartridges have been procured under concessional pricing it helped to subsidize. The cumulative numbers grew to 3,553 instruments and 8,807,910 cartridges by late September of 2014. Cepheid also reported a 50 percent increase in commercial Xpert MTB/RIF revenue in the third quarter of last year, although the assay is part of an ongoing patent infringement lawsuit filed by Roche in July.

Meanwhile, a partnership between Akonni Biosystems and Harvard recently won $29 million in funding to develop an XDR-TB assay using microfluidics, a group in South Africa is developing a test specific for non-pulmonary TB, and a PCR-based test from Abbott was recently CE-marked. A group at Keck Graduate Institute of Life Sciences was also recently funded to develop a point-of-care isothermal test for low resource settings.