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South African University Developing MDR/XDR-TB Assays Using Brandeis' LATE-PCR Tech

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Scientists from Stellenbosch University in South Africa have been awarded $131,000 from the National Institutes of Health for the first year of a planned four-year project aimed at developing and evaluating a molecular assay for multi- and extreme-drug-resistant tuberculosis using so-called LATE-PCR and other PCR-related technologies developed at Brandeis University in the US.

Under the grant, administered by the National Institute of Allergy and Infectious Diseases, a team led by Robin Warren at Stellenbosch University will develop assays using the Brandies technologies to test well-characterized Mycobacterium tuberculosis samples held in repositories at the university, as well as routine clinical specimens from South Africa's National Health Laboratory service.

The core technology around which the researchers will based their assays is LATE, or linear-after-the-exponential, PCR, developed in the laboratory of Brandeis scientist Larry Wangh. In LATE-PCR only one side of a DNA duplex is amplified, resulting in a high proportion of single-stranded DNA, which allows probing over a much wider temperature range — and thus a greater degree of multiplexing — than is possible with symmetric PCR. The technique is also ideal for use with samples containing a low number of initial targets, according to its developers.

The Stellenbosch researchers will also explore the use of "Lights-On/Lights-Off" probes, which enable the detection of variations in several long target sequences using different fluorescence emissions; and PrimeSafe reagents, which are designed to suppress mis-priming during the amplification process and facilitate multiplexed reactions.

Using a combination of these technologies, the researchers anticipate being able to simultaneously detect, in a single-tube assay, mutations conferring resistance to isoniazid, rifampin, ethambutol, fluorquinolones, and aminoglucosides; as well as mycobacterial species causing disease.

The technology has the potential to help "rapidly and affordably define a profile of mutations which can be used to optimize the drug treatment at initial diagnosis … reduce morbidity and mortality … and help stem the global TB pandemic," the researchers wrote in a grant abstract describing their project.

Molecular methods for diagnosing TB and MD/XTB began to take hold in 2008, when the World Health Organization recommended the use of line probe assays to either supplement or replace traditional serological or culture- and microscopy-based methods.

In line probe assays, DNA is extracted from M. tuberculosis isolates or directly from clinical specimens. Then, resistance-determining regions are amplified using PCR, hybridized with oligonucleotide probes immobilized on a strip, and detected using colorimetric methods. However, according to the Stellenbosch researchers, these assays are technically demanding and require the use of sophisticated laboratories to reduce the risk of cross contamination.

More recently, in 2010 WHO endorsed the use of Cepheid's Xpert MTB/RIF platform in developing regions of the world after conducting an 18-month assessment of the system's field effectiveness to diagnose early TB, MDR-TB, and HIV-associated TB.

The Cepheid system is closed to reduce contamination risks, and is fully integrated and can be operated by relatively untrained technicians. It also has the advantage of being able to detect M. tuberculosis complex in some smear-negative cases. However, the Stellenbosch researchers point out in their grant abstract that the test "only measures rifampin resistance and thus currently only serves to prompt follow-up drug susceptibility testing by conventional cell culture methods, again delaying diagnosis" — although the company plans to commercialize a new version of its Xpert test that will enable highly multiplexed detection of extensively drug-resistant TB.

Now, under the recent NIAID grant, the South African research team will work with Brandeis to transfer the LATE-PCR and related technologies to Stellenbosch University to develop a similarly multiplexed assay.

Smiths Detection, a UK-based technology firm that counts among its specialties molecular diagnostic development, in the late 2000s took an exclusive license to LATE-PCR from Brandeis with sublicensing rights. In addition, in 2010 Smiths Detection said that its diagnostics business inked a collaboration and license agreement with Novartis Diagnostics to develop diagnostic tests using LATE-PCR for a variety of infectious diseases, beginning with healthcare-associated infections (PCR Insider, 3/25/2010).

It is unclear whether the Smiths Detection license is still in effect or whether the Stellenbosch University project would fall under the company's sublicensing rights. Brandeis' Wangh did not return e-mails seeking comment, and officials from Smiths Detection could not be reached prior to publication of this article.

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