By Ben Butkus
Qiagen and the Max Planck Institute for Infection Biology this week announced a collaboration to develop a real-time PCR-based molecular diagnostic test to assess the risk of patients with latent tuberculosis developing active TB during their lifetime.
According to Qiagen, the assay will be developed for use on its QIASymphony molecular testing platform; would ideally comprise two to five gene markers; and would serve as a reflex test following the company's Quantiferon-TB Gold test, a "pre-molecular" diagnostic assay that can be used to detect latent TB in patients.
In addition, researchers at both Qiagen and Max Planck are hoping that the qPCR-based whole-blood transcription profiling work they will be conducting to develop the test will also help reveal more about the biological pathways involved in the progression of latent TB to active TB.
"We are using expression profiling of white blood cells for a gene signature to find out more about risk assessment and how the progression to active disease happens," Ulrich Schriek, senior vice president of corporate business development at Qiagen, told PCR Insider this week.
"I would say this could be very similar to companies like XDx, who developed a blood-profiling assay [AlloMap] to monitor rejection of heart transplants; or the Genomic Health assay [Oncotype DX for cancer recurrence]. These kinds of gene signature or blood expression profiling tests can be extremely helpful to get more information about the potential progression of TB."
The collaboration will be based on research conducted over the last several years at the Max Planck Institute for Infection Biology, primarily using microarray analysis, to identify a set of a few hundred genes that "are highly interesting as candidate genes for a gene signature assay," Schriek said.
More specifically, the laboratory of MPIIB researcher Stefan Kaufmann has been comparing gene expression in healthy individuals with that of patients diagnosed with either latent or active TB, using primarily Agilent's Human Gene Expression 44K microarray kits.
During TB infection, changes occur in the transcriptional profiles of immune cells circulating through blood alterations in gene expression that reflect the body's immune response and help to distinguish between latent and active disease.
"We are looking at biological pathways that are affected, up- and down-regulated genes, clusters of genes, things like that," Jeroen Maertzdorf, a postdoc in the Kaufmann laboratory, told PCR Insider. "Combining those, we have a few hundred targets we're looking at."
The next immediate goal of the Qiagen-MPIIB collaboration will be to winnow those few hundred genes down to a handful of candidate genes that could serve as a sensitive and specific indicator of the potential transformation of latent TB to active TB.
To do this, Qiagen and MPIIB have developed qPCR assays for all differentially expressed genes from healthy and TB-infected individuals, and will be conducting high-throughput qPCR-based whole-blood transcription profiling on a Qiagen platform to survey the several hundred candidate genes for strong biomarkers for active TB progression.
"Our goal is now to reduce this number of genes to an amount that you can handle in a normal [qPCR] assay," Schriek said.
The expectations of Qiagen and MPIIB differ somewhat in this regard, as Qiagen hopes to identify a gene panel that could be adapted to its QIASymphony RGQ, a modular, automated, sample-to-answer platform featuring the company's Rotor-Gene Q real-time PCR machine. Because of the complexity of highly multiplexed real-time PCR, such a panel would likely have to constitute a half-dozen biomarkers at the most.
"If you're doing real-time PCR you should have an amount of genes that could be handled in real-time cyclers," Schriek said. "It would be ideal to reduce this test to an expression profile of between two and five genes."
Meantime, Maertzdorf agreed that such a small number of genes would be ideal for a diagnostic test, but noted that "from our point of view, I'm not sure that two to five genes are going to be specific enough. There may be other pulmonary diseases that give the same pattern. So it would be enough to distinguish a diseased from a healthy individual, but not specific enough to diagnose a particular disease."
Maertzdorf noted that a panel of a dozen or even two dozen genes might be more realistic, "although that's a rough estimate. How many it will be we cannot exactly say."
The ultimate goal of the project, according to Schriek, is to create a molecular diagnostic test that can be used to reflex patients who are found to be infected with TB using Qiagen's Quantiferon-TB Gold assay, which the company added to its portfolio when it acquired Australian biotech firm Cellestis in April 2011 for $355 million.
Quantiferon-TB gold, which is already commercially available, basically uses luminometry to measure the level of the cytokine IFN-γ circulating in a patient's blood as a result of an immune system challenge from an infectious agent. As such, the test can provide diagnostic information much earlier than DNA-based molecular tests, even when there are extremely low amounts of pathogen in the blood.
However, Quantiferon doesn't differentiate between latent and active TB infections. "If the Quantiferon test is positive, the result is that the patient has latent TB or possibly active TB," Schriek said. "In the reflex, you would use this molecular test for active TB … and if this can be excluded then the patient is latently infected, which means they are infected but not sick."
In the meantime, the molecular test can identify actively infected patients so they can be isolated and treated. This is important because only actively infected patients can transmit the infectious agent, Mycobacterium tuberculosis; thus, a highly sensitive molecular assay for active infections could go a long way toward outbreak prevention.
In addition, many patients diagnosed with latent TB are currently administered antibiotic treatment that is "a very tough procedure," Schriek said. "You are taking antibiotics for roughly six months, and there are reports of liver toxicity and so on. People have a reduced quality of life." The molecular assay for risk to develop active TB would help triage patients most in dire need of antibiotic treatment.
"Active TB testing, the molecular test, you would likely be using it in countries in the developing world," Schriek said. "In the developed world, you have more of a problem with latency. You don't have so many cases — really only a few cases — of active TB in Europe or the US."
Once developed, a molecular test for active TB infection would likely need to be conducted in a centralized laboratory, given the lack of portability of the QIAsymphony platform. Schriek said that the company might consider eventually creating a point-of-care version of the assay, but "right now we are focused on seeing how many genes will be relevant."
Qiagen did not provide a commercialization timeline for the molecular TB test.
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