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Study Finds that Cepheid's GeneXpert TB Test Performs Poorly On Extrapulmonary Samples

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NEW YORK (GenomeWeb) — Although the GeneXpert MTB/RIF assay from Cepheid is endorsed by the World Health Organization for diagnosing tuberculosis from expectorated sputum samples, its ability to detect TB in samples from other body compartments is unclear.

Co-infection of TB with HIV can be quite common in some parts of the world; in this population extrapulmonary TB is more frequently seen, and patients sometimes cannot mount the immune response needed to produce sputum. A recent study has now compared eight sample types in both HIV-positive and -negative individuals with TB, concluding that mycobacillary load varies across body compartments, thus diminishing the sensitivity of Xpert MTB/RIF in samples other than sputum.

The study, published this month in Scientific Reports, was led by Keertan Dheda, a professor of respiratory medicine and head of the Lung Infection and Immunity Unit at the University of Cape Town Lung Institute.

Dheda and his team compared time to positivity of bacterial cultures, MTB/RIF Ct values, and clinical characteristics of 1,321 patients. They distinguished between expectorated and induced sputum. They also tested tracheal aspirates, bronchoalveolar lavage fluid, and pleural fluids, as well as cerebral spinal fluid, pericardial fluid, and urine.

The study determined that time to positivity ─ a proxy of bacterial load ─ correlated to Ct values in pulmonary but not extrapulmonary specimens. It was also lower in pulmonary samples, indicating these had higher loads.

"Cycle threshold is a surrogate of the amount of starting PCR template and hence it is logical to assume that it would be a surrogate of TB bacterial load," Dheda told PCR Insider in an email, noting that his group previously reported this relationship in a paper in the American Journal of Respiratory and Critical Care Medicine.

Developed to run on Cepheid's GeneXpert platform, MTB/RIF simultaneously detects TB and resistance to a common TB treatment, rifampicin. The test launched in 2010, heavily subsidized by non-profits and endorsed by WHO in order to reach populations in the developing world where TB is endemic.

The current distribution agreement was negotiated by the Foundation for Innovative Diagnostics, which "has leveraged its investment in the development of Xpert MTB/RIF by negotiating a price reduction agreement with Cepheid … [that] fixes the pricing and defines the applicable market as the public sector in 145 high-burden and developing countries," according to a statement from October on FIND's website. And in August 2012 the US President's Emergency Plan for AIDS Relief, US Agency for International Development, Unitaid, and the Bill and Melinda Gates Foundation finalized an agreement with Cepheid to further reduce the negotiated price of the Xpert MTB/RIF test for eligible customers of the FIND country list to approximately $10. Meantime, the GeneXpert system costs about $17,000 for these customers.

The rollout is ongoing, but as of March 31 of this year, WHO reports 2,343 instruments and 6,291,330 cartridges have been procured under the concessional pricing that it helped to subsidize. In its first quarter earnings release earlier this year, Cepheid said sales of its GeneXpert system in developing countries continues to serve as an important revenue driver for the company.

Dheda told PCR Insider that he himself "had cautious optimism" about the MTB/RIF test when it launched. Now, however, he said that while it is a very good test for pulmonary TB when patients can expectorate sputum, it is "not the ideal test" in HIV-infected patients or those with certain types of extrapulmonary TB.

Lower sensitivity when the test is run on samples other than expectorated sputum has led WHO to only conditionally endorse MTB/RIF for extrapulmonary testing, according to the Scientific Reports study. PCR inhibition was previously thought to be the reason for the low sensitivity.

But Dheda's study also measured PCR inhibition, and demonstrated that inhibition played little or no role in explaining MTB/RIF-negative, culture-positive results.

"Although inhibition occurred in about 6 percent of patients with pulmonary samples, there was only one patient who was GeneXpert negative who had significant inhibition. Collectively these data suggest that inhibition is not a major factor explaining GeneXpert negative results in any compartment," Dheda said.

Rather than inhibition, the Scientific Reports study suggests low bacterial load may be the cause of sensitivity problems in extrapulmonary samples.

At the same time, in HIV positive patients, the MTB/RIF often gives negative results because of the low bacterial load in the paucity of available sputum, Dheda said. "Because of the attenuated immune system of HIV-infected patients, they are unable to mount a robust enough immune response to cause liquefactive necrosis in the lungs, and hence often fail to produce sputum," he said. "Inability to contain the TB infection [also] means spread from the lungs to extrapulmonary structures."

In extrapulmonary compartments, bacterial burden or concentration can be high, but is more variable.

This result is important, because "HIV/TB co-infection is very common in Africa and up to 80 percent of TB cases in many areas are HIV co-infected," Dheda said.

Children are yet another patient population in whom TB infection can be difficult to detect, since they are often physically unable to produce sputum for testing.

It is unclear whether Cepheid is working to address these shortcomings of the Xpert MTB/RIF assay. The company declined to comment for this article. Other commercial entities have taken note of the issue. Earlier this year, for instance, PCR Insider described a partnership between Akonni Biosystems and Harvard to develop a test appropriate to detect cell-free TB DNA in urine.

Ultimately, Dheda said MTB/RIF should still be used for suspected cases of TB if an adequate sputum sample can be obtained. In HIV-infected patients, if the test is negative, a further search for TB should be continued. Xpert MTB/RIF can still be used to investigate certain types of extrapulmonary TB, such as TB-lymphadenitis and TB-meningitis, where performance is modest to good, he said. By contrast, in other compartments, such as pleural and pericardial TB, "Xpert performs poorly, and in this context other investigations should be used."

Of note, Dheda said a University of Cape Town spinout company, Antrum Biotech, is currently developing a test for extrapulmonary TB that will be launched soon.

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