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China's Zeesan Biotech Developing Portfolio of Melt Curve MDx Assays after SFDA Approves TB Tests

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This article has been updated from a previous version to clarify information regarding the development and commercialization of Zeesan Biotech's assays.

The Chinese State Food and Drug Administration recently approved a pair of tuberculosis drug-resistance assays developed by Xiamen-based molecular diagnostics firm Zeesan Biotech, PCR Insider has learned.

The new assays add to a rapidly growing portfolio of molecular diagnostic tests offered by Zeesan based on a real-time PCR-based method called multicolor melting curve analysis, or MMCA, developed by Xiamen University and licensed to Zeesan.

In addition to an MMCA-based assay for β-thalassemia mutations called MeltPro HBB, which Zeesan submitted to SFDA for approval last year, the company has submitted five other assays to the agency for approval. Among these tests, the furthest along in development is a human papillomavirus genotyping assay, Qingge Li, a professor of molecular diagnostics at Xiamen University who works closely with Zeesan, told PCR Insider this week.

Zeesan also plans to pursue CE IVD marking for the HPV genotyping assay later this year, Li said.

The new tuberculosis assays, called MeltPro TB/INH and MeltPro TB/RIF, are qualitative in vitro diagnostic kits designed to detect isoniazid resistance mutations and rifampin resistance mutations, respectively.

The assays are also notable because they can detect heteroresistant mutations — the co-existence of both wild-type and mutant Myocbacteriaum tuberculosis. In most cases, Li said, MeltPro TB/INH and TB/RIF would be administered together to determine whether or not a patient has multi-drug resistant tuberculosis.

Isoniazid "is one of the most effective first-line drugs in anti-TB therapy," Li said. "The detection of INH resistance is crucial for the treatment and control of MDR-TB. Although [rifampin] resistance was often regarded as a surrogate of MDR-TB, detection of RIF resistance alone could neglect those INH-monoresistant patients as well as those RIF-monoresistant patients."

This could lead to some patients losing an opportunity to initiate effective treatment or developing acquired resistance to RIF.

"Therefore, use of rapid tests to detect both RIF and INH resistance would have better outcomes than tests to detect RIF resistance alone," Li said. "Currently, INH and RIF resistance assays are equally required as reflected in various guidelines of drug resistance treatment and prevention." He noted that the World Health Organization also recently proposed early detection of both INH and RIF resistance for all patients before treatment.

The MMCA technique upon which the TB assays are based is an extension of melt curve analysis — a well-known and highly sensitive technique for analyzing the melting characteristics of different PCR products in order to determine the presence of mutations.

Unlike standard melt curve analysis, which uses adjacent probes that need dedicated Forster resonance energy transfer detection channels, the MMCA technique uses dual-labeled, self-quenched probes in the form of either a hairpin or linear probes, which allows for the use of common real-time PCR instruments, Li said.

Li and colleagues first described the method and a proof-of-principle RIF resistance assay based on it in a paper published in PLOS One in 2011. The described assay was able to detect all RIF-resistant mutations in the rpoB gene in any real-time PCR machine equipped with two fluorescence channels.

Since that time, Li and colleagues developed the aforementioned MeltPro TB/INH and TB/RIF assays, as well as three other TB drug-resistance tests, which Zeesan has submitted to the SFDA for approval.

Li said that all of the tests will compete in China primarily with the SFDA-approved GenoType MTBDRplus assay from German molecular diagnostics shop Hain. This test can detect both rifampin and isoniazid resistance mutations in a single assay and has been "extensively validated," Li said. However, he noted that the test relies on reverse hybridization, which requires multiple post-PCR manipulations and visual estimation of positive signals.

Another test on the horizon in China is Cepheid's Xpert MTB/RIF, which uses a fully automated, nested-PCR-based approach to detect TB and rifampin resistance in about two hours. This assay was endorsed by the World Health Organization for use in resource-poor areas of the world, and Cepheid currently sells the assay to dozens of countries under its high-burden developing country program.

However, Xpert MTB/RIF has not been approved for use in China, nor does it cover INH resistance. "Moreover, no data have shown this real-time PCR-based assay can detect heteroresistant mutations," Li said. It is worth noting that Cepheid is currently developing a new version of its Xpert TB test for highly multiplexed detection of extensively drug-resistant TB.

Li noted that Zeesan has no immediate plans to seek regulatory approval for its TB assays outside of China "simply because China … is now the biggest market of these assays."

A recent survey in China revealed that among patients with newly diagnosed TB, 5.7 percent had MDR-TB, and among previously treated patients, 25.6 percent had MDR-TB.

"Of note, the majority of the estimated 110,000 cases of MDR-TB in China annually are due to transmission of resistant strains to patients who had never before been treated for TB," Li said. "Such a severe situation prompts an urgent need for MDR-TB testing available to all patients."

To combat TB, he added, the Chinese government five years ago launched a national program for the development of assays for TB and drug resistance, stressing the need for rapid, easy-to-use, low-cost, and accurate molecular assays for TB resistance detection.

Although Zeesan plans to commercialize the TB assay only in its home country, it may seek regulatory approval both in and outside of China for its other assays that use the MMCA method. In 2011 PCR Insider reported on the company's β-thalassemia mutation assay, which researchers from the company and Xiamen University demonstrated could genotype 24 β-thalassemia mutations common in Chinese populations with 100 percent sensitivity and specificity (PCR Insider, 7/28/2011).

Zeesan submitted that test to the SFDA in July 2012, and Li said this week that the company expects it to be approved sometime this year.

The company has also developed a semi-automatic sample prep system called the Lab-Aid 820 that can be used for either the TB or β-thalassemia tests and can process up to 24 samples in about an hour.

Both tests can run on unadulterated clinical samples — for the β-thalassemia assay sample types are usually whole blood or amniotic fluid, while the TB tests generally use sputum samples. Including sample prep, the turnaround time to run a test is about three to four hours depending on the PCR instrument used, Li said.

Besides the three additional drug-resistance tests for TB, Zeesan has submitted to the SFDA a glucose-6-phosphate dehydrogenase deficiency mutation assay and an α-thalassemia mutation assay, Li said.

In addition, three targets currently under clinical validation at the company are inherited hearing loss, phenylketonuria, and HPV genotyping — the latter of which Zeesan plans to pursue a CE IVD mark for sometime this summer.

"One of the biggest advantages of this HPV assay is that it gives full genotyping of 15 high-risk HPVs in a single well by just a single DNA addition step," Li said. Researchers from Zeesan and Xiamen University demonstrated proof of principle for this test in a paper published in February in the Journal of Clinical Microbiology. They also demonstrated in a paper published in January in Nucleic Acids Research a potential next generation of the MMCA method that combines fluorescence color and melting temperature as a two-dimensional label for multiplex PCR detection, as well as an HPV genotyping assay based on the technique.

All of Zeesan's current and future assays based on the MMCA technique will be sold under the MeltPro brand name, Li said.

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