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Tuberculosis Detection With Cepheid Test Improved By Longhorn's Sample Transport Medium


NEW YORK (GenomeWeb) – In an effort to show the utility of its PCR reagents on a variety of platforms, Longhorn Vaccines and Diagnostics of San Antonio, Texas, has recently demonstrated that its proprietary sample transport medium can improve the sensitivity of the Cepheid GeneXpert assay for tuberculosis.

In a study published in the International Journal of Tuberculosis and Lung Disease, a team of researchers — including Longhorn's own scientists and researchers from the University of Pretoria and the Anova Health Institute in South Africa, the City of Milwaukee Health Department Laboratory, and the University of Texas at San Antonio — showed that the firm's PrimeStore Molecular Transport Medium improved Mycobacterium tuberculosis detection, enhancing detection of low concentrations of bacteria on the Xpert MTB/RIF assay.

"We are hopeful that this is going to be able to get good TB testing to many more people who currently don't have access to it," Gerald Fischer, the firm's president and CEO, told GenomeWeb.

Delivering molecular testing to TB patients in rural, low-resource settings is a challenge. Shipping specimens from these areas to centralized PCR labs has some advantages in terms of expertise of lab technicians, but transportation needs a medium that can preserve nucleic acids from low quantities of bacteria.

The Longhorn transport medium is a patented, proprietary blend of denaturants, surfactants, salts, and buffers. "When you put a specimen into PrimeStore, it lyses all the organisms and destroys all the enzymes — including nucleases — and preserves the naked RNA and DNA in an optimally buffered solution," Fischer said. The medium enables samples to be transported without a cold chain, and can stably preserve nucleic acids for 30 days at 40° C .

The reagent mixture was developed by the firm's CSO, Luke Daum, who had previously worked on molecular diagnostics for the US Air Force. Fischer, meanwhile, has an Army background and was trained as an infectious disease specialist at Walter Reed Army Medical Center.

"We were well aware that shipping specimens around the world is very difficult, particularly in getting a high-quality specimen back to the laboratory for testing," Fischer said.

The team realized that to develop excellent diagnostic tests, they would need to start with optimal specimens — so it began developing a one-step method to get the best nucleic acid sample. Other transport media is often designed with culture in mind, Fischer explained, "and that doesn't necessarily make it good for PCR or sequencing."

The firm has previously shown that the medium works with the ABI 7500 and Light Cycler PCR platforms, as well as multiple extraction methods, including Qiagen kits and robotic Magna Pure and ExMag, Fischer said.

"We've done a lot of studies with open PCR platforms ... but the Xpert platform is a cartridge platform, and we had not used PrimeStore with that platform before," he said.

The GeneXpert cartridge uses a filter to trap intact organisms from sputum samples, followed by lysis, so Longhorn was not sure its medium would work on that platform.

However, "It was not only detected, but it improved the GeneXpert by a log for detection," Fisher noted.

Longhorn is developing its reagents for use with any platform, since many laboratories, particularly in low-resource countries, will already have a molecular detection system in place. There are currently an estimated 10,000 GeneXpert systems in the global market, and the World Health Organization has encouraged their adoption in TB-endemic areas. Longhorn has not worked directly with Cepheid, however.

Tuberculosis bacteria are dangerous to transport and work with. But Longhorn's transport medium purports to kill the bacteria completely — a claim supported by a 2011 study in Epidemiology and Infection — and it also destroys all proteins, such as enzymes that can degrade nucleic acids.

Founded in 2008, Longhorn has been building a broader line of reagents for molecular detection. These include the PrimeStore reagents for collection and transport of nucleic acids; a spin column-based extraction reagent called PrimeXtract; an all-inclusive PCR reagent named PrimeMix; and a reagent designed particularly for samples that will be sequenced, called PrimeSeq. The latter is tailored to a variety of drug resistance-conferring genes of MTB.

Beyond just reagents, the company also offers assays. In 2010, Longhorn received Emergency Use Authorization from the US Food and Drug Administration for an H1N1 influenza assay, and it has developed MTB and malaria diagnostic assays since then, Fischer said.

The company is currently "lean and mean," and not trying to grow its staff, but sales of PrimeStore in particular are rising, especially in areas of global infectious disease surveillance, animal health surveillance, and support of clinical trials for vaccines and other products, Fischer said. 

The firm's all-inclusive PCR reagent mix, PrimeMix, includes primers, probes, and enzymes in a buffered solution, "so that even reverse transcriptase blended in is stable for a couple of months at room temperature," Fisher said. This makes it suitable for use in areas without reliable electricity, and the blend can be modified by customers using primers and probes of their choosing.

Customers in the US now include labs at the National Institutes of Health, the Centers for Disease Control and Prevention, and the Army, using the reagents "generally for surveillance studies around the world," Fischer said.

University and surveillance labs also use the reagents, he said, particularly noting Cincinnati Children's Hospital the National Institute of Communicable Diseases, and the University of Cape Town in South Africa.

In collaboration with researchers in South Africa, the company showed last year that the medium could be used to bring samples from rural areas to centralized labs, and "could provide an alternative in settings where Xpert testing is not available."

Longhorn also published a case study showing PrimeStore could be used for Ion Torrent sequencing to characterize resistance in clinical isolates within five days. In addition, it published results showing improved detection of pyrazinamide resistance in clinical samples via next-generation sequencing using the firm's PrimeXtract extraction reagent versus a phenotypic method involving bacterial culture.

Xpert MTB/RIF has faltered somewhat in pediatric and extrapulmonary TB testing, but Fischer said his firm's medium could help. "Since children don't cough up sputum, tests are relying on small numbers of organisms that may get up into their respiratory tract," Fisher noted. Having a more sensitive test that can detect fewer organisms will make detection more likely, he said.

The firm also presented a paper recently at the International Infectious Disease Conference in Hyderabad, India, showing that PrimeStore could also be used to detect organisms in blood and tissue, potentially allowing "more sensitive detection of MTB from any site."

The firm's hope is that organizations like the WHO that promote testing globally will now have a better way of collecting specimens in rural areas to be processed in centralized labs. Longhorn is "excited about helping groups get better testing," Fischer said, and is flexible with its pricing for low-resource settings.