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Cepheid Collaborator at Rutgers-NJMS Wins NIH Award to Further Develop Highly Multiplexed TB Assay

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The laboratory of David Alland at Rutgers Biomedical and Health Sciences' New Jersey Medical School has been awarded a five-year grant from the National Institutes of Health to further develop a highly multiplexed assay to detect multiple drug-resistant strains of tuberculosis using Cepheid's GeneXpert molecular testing platform.

Under the grant, the first year of which is worth $1.4 million, Alland's team will combine 10-color, real-time PCR detection making use of "novel large Stokes shift dyes," with "three-phase PCR fluidics, sloppy molecular beacon probes, the existing GeneXpert platform, and basic Xpert assay cartridge" to create a new assay, according to the grant's abstract.

This new "Xtend-XDR" assay will identify resistance to isoniazid (INH), fluoroquinolones (FQ), amikacin, and kanamycin — important drug resistances not currently detected by Cepheid's Xpert MTB/RIF assay — but will also complement the existing Xpert test by making it possible to select appropriate treatment and infection control approaches in TB patients.

As explained in the grant's abstract, in regards to drug-resistance detection, the current Xpert assay, despite having World Health Organization endorsement and being widely disseminated in developing countries, is "limited to identifying patients who potentially have [multi-drug resistance] by using RIF resistance as a surrogate. The Xpert assay cannot tell whether a patient with RIF resistance remains INH-susceptible, and thus could still be treated with an INH-containing regime."

Similarly, Xpert MTB/RIF can't identify which RIF-resistant patients can be treated with a FQ and/or aminoglycoside as opposed to those who have extensively drug-resistant TB. "Thus, patients are at risk for being either overtreated or undertreated, with potentially serious consequences," the grant's abstract states. "Furthermore, patients with undiagnosed XDR can remain infectious for prolonged periods, further spreading their disease."

Xtend-XDR will use "the same testing procedures and testing platform as the Xpert, and largely be restricted to patients who already test TB positive (and [rifampin]-resistant) in a primary Xpert test," the abstract states, adding that these features will make the new test highly cost effective.

"The combination of nested multiplex PCR, 10-color detection, molecular beacons, and melt curve analysis on the GeneXpert has proven in our lab to successfully identify drug-resistance mutations found in MDR and XDR TB strains," Alland wrote in a statement provided by Cepheid.

The new grant builds on work done by Alland under a previous NIH grant awarded in 2009. That grant, which was worth $7.5 million over five years, laid much of the groundwork for the current project, and in particular focused on developing the 10-color detection scheme for the GeneXpert, which currently supports six-color detection.

Alland's lab has now developed proof of principle for all essential assay components, and as such will now finalize assay development and create manufacturing processes, quality control, and assay software. Finally, the Alland lab will perform the analytic and pre-clinical studies necessary to prepare the assay for clinical trials.

"Initial field evaluations of this technology in South Korea and China will be underway shortly," Alland elaborated in the email statement.

"This program is an indication of Cepheid's ongoing commitment to exploring better diagnostic options for tuberculosis worldwide, with the potential for leveraging further our existing installed base of over 5,500 GeneXpert systems," David Persing, Cepheid's chief medical and technology officer, told PCR Insider in an email.

"To our knowledge, this program represents the first demonstration of successful discrimination of 10 independent detection channels on a real-time PCR instrument, and it shows that existing GeneXpert technology, rather than becoming obsolete, can be adapted to meet the needs of more complex next-generation molecular diagnostic tests," Persing added.

Alland, a long-time Cepheid collaborator, was also awarded a five-year grant worth several million dollars from NIAID in 2012 to develop sample prep methods and nested PCR assays to detect bloodstream infections. Although the overarching goal of that grant is to develop Xpert tests for bioterror applications, Cepheid said that it expects the blood-based sample prep methods to be of use in future molecular diagnostic products.

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