By Ben Butkus
A PCR-based molecular test for tuberculosis and rifampicin resistance developed by Cepheid and public sector partners has received a clinical vote of confidence for its ability to perform in remote and resource-poor regions of the world.
In a study published online yesterday in the New England Journal of Medicine, the test successfully identified 98 percent of all culture-confirmed TB cases, and accurately detected resistance to the anti-TB drug rifampicin in more than 97 percent of patients. The test also provided results in less than two hours while being run by relatively unskilled healthcare workers.
And, according to a study co-author, the test may even be superior to bacterial culture, the current gold standard for diagnosing TB and its resistance to rifampicin, one of the most powerful anti-TB drugs available.
"Whenever you are looking at a diagnostic test, you are always looking at the gold standard, so it can never be better than the gold standard," David Alland, a researcher at UMDNJ and co-author on the NEJM paper, told PCR Insider this week. "And Cepheid can't tell you that it's better than the gold standard, because they're not allowed to, but I can."
Cepheid developed the test, called the Xpert MTB/RIF, in concert with the Foundation for Innovative New Diagnostics and the University of Medicine and Dentistry of New Jersey, and with funding from FIND, the National Institutes of Health, and the Bill and Melinda Gates Foundation. The test runs on Cepheid's GeneXpert, a self-contained, fully-integrated and automated "sample-to-answer" molecular diagnostics platform.
Specifically, Xpert MTB/RIF integrates sample processing and PCR in a disposable plastic cartridge containing all reagents required for bacterial lysis, nucleic acid extraction, amplification, and amplicon detection. The only manual step is adding a buffer to sputum before transferring a defined volume to the cartridge. The test cartridge is then inserted into the GeneXpert device, which provides results within two hours.
Cepheid introduced the test and obtained the CE Mark for in vitro diagnostic use last year.
In the NEJM study, researchers from FIND, UMDNJ, Cepheid, and a number of other international public agencies and academic institutions assessed the performance of Xpert MTB/RIF in 1,730 patients from Peru, Azerbaijan, South Africa, and India with suspected drug-sensitive or multidrug-resistant pulmonary TB.
They examined separate specimens from each patient using smear microscopy, solid and liquid culture, and the Xpert MTB/RIF assay. Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561, or 98.2 percent of patients with smear-positive TB; and 124 of 171, or 72.5 percent of patients with smear-negative TB. The Xpert test was also specific in 604 of 609, or 99.2 percent of controls.
In addition, among patients with smear-negative, culture-positive TB, a second MTB/RIF test increased sensitivity by 12.6 percent and a third test increased sensitivity by 5.1 percent to a total of just over 90 percent. Lastly, the test also accurately detected resistance to rifampicin in more than 97 percent of patients.
Alland told PCR Insider that the study is notable as "the first large clinical validation" of Xpert MTB/RIF. "In a large, prospective, blinded clinical trial, at five separate sites with HIV-positive and HIV-negative patients, the assay performed well, both on smear positive and smear negatives. It performed well for predicting drug resistance as well."
Alland added that the study "shows that the assay is just about as sensitive as culture. And even in patients who, on microscopy, don't have any bacteria, it's still quite sensitive. That's a group that is very difficult to diagnose right now, and particularly in Africa, where many HIV-positive patients have microscopy-negative TB. They can have signs and symptoms that look just like TB but are something else; or TB, and it's hard to tell the difference."
The fact that Xpert MTB/RIF outperformed smear microscopy is crucial because that technique is currently the most widely used and "routinely misses half of all cases," according to an editorial accompanying the NEJM paper.
"Even though microscopy helps diagnose many cases of TB, it's very hard to do microscopy," Alland said. "People have to be trained, and look at 200 high-powered fields, at 20 minutes a slide. The microscopists, when they start using this test, are just delighted, because instead of looking at each slide for 20 minutes, they can spend two minutes processing a sample, and let the cartridge do the rest of the work."
In addition, Alland noted, "one test will pick up 70 percent of the smear microscopy negatives."
And even though the study didn’t make any specific claims about the performance of Xpert MTB/RIF compared to the diagnostic gold standard, bacterial culture, "there were a number of cases that were culture-negative, but Xpert positive, and all those patients — and the physicians didn't know about the status of the other tests — were being treated for TB. So, it's likely that in some cases the test is picking up TB even when the culture is negative," Alland said.
"And we've been doing some of our own calculations, and if you use an external standard that [involves] culture plus clinical suspicion of TB, [Xpert MTB/RIF] performs just as well as culture," he added. "Cepheid can't make those claims, but that's my interpretation of the data."
Other companies make molecular amplification-based tests for TB, including Roche, Becton-Dickinson, and Gen-Probe. "But you really can't do those tests outside a pretty technology-intensive laboratory," Alland said. "It's so easy to do the Xpert assay. It really can go anywhere there is electricity."
Nucleic acid testing for TB has existed for a number of years, "but because of the complexity of the testing, it was not something that was easy to be implemented in developing countries where there are high incidence rates," Cepheid CEO John Bishop told PCR Insider. Bishop added that previous tests have been "too complex to run, and relegated to central laboratories."
Bishop also pointed out that while other tests "generally have good sensitivity … on smear positives, the big difference here are those patients that are smear negative." Generally those patients would then have to be reflexed for culture-based testing, which might take six to eight weeks before a negative case can be called.
"We have very good sensitivity that has not really been seen previously with a first-run test," Bishop said. "In other words, you just pick up your smear negatives and your smear positives, and you're going to have good specificity and sensitivity. One of the primary reasons for that is because the test is running in the closed cartridge, and running nested PCR."
Although the Xpert MTB/RIF assay was designed with point-of-care treatment in developing countries in mind, both the NEJM paper and accompanying editorial still cited cost concerns as potential barriers to widespread adoption of the test.
"To achieve great simplicity of use, the MTB/RIF test uses sophisticated technology, which is costly to manufacture," the researchers wrote in their paper. "Although FIND has negotiated concessionary pricing for public-sector programs in low-income countries and is still working to further lower the costs of testing, the costs of instruments and tests will be considerably higher than those for microscopy, which is all that is currently available in peripheral healthcare settings in many countries."
According to a Cepheid spokesperson, the cost of the Xpert MTB/RIF test is about €50 ($64), but compassionate pricing will cut that figure in half. Meantime, the GeneXpert system normally costs in the neighborhood of $30,000, but the spokesperson said that Cepheid "now believes that we will be able to get the compassionate pricing for a single module system to under $10K."
"In the agreements with FIND, we said that we were committed to giving them compassionate pricing and bringing the price points down," Bishop said. "Even though everyone would like to see it a little lower than where it is right now … the overall medical impact and benefit is such that I would see the test being widely adopted, even at the current price points."
Bishop added that pricing can be compared to the chicken and egg analogy in that "as your volumes go up, and test demand goes up, the price point will move down, and will be much more attractive. As we're in the early days, we don't have that volume yet, but as the volume comes along, we're going to pass along some of that benefit to the developing market."
From Alland's point of view, the cost reduction to the markets that FIND serves "is really substantial. It's really to Cepheid's credit that they have agreed to a degree of cost reduction, and they are making a very small profit on the FIND market, which is the public health sector of high-TB-burden countries."
Alland echoed Bishop's comments that test volume is likely to drive down price, but also pointed out that the current price may counterbalance the current economic burden of TB infection.
"I've talked with some economists, and if you calculate the price of [a potential TB carrier] going to a city, staying with someone, buying their food, and days of lost work … the price of the test is actually less than the costs associated with not getting the test," Alland said.
The accompanying NEJM editorial also underscored the fact that Xpert MTB/RIF is limited to testing for rifampicin resistance while there are many new and promising drugs for TB that are also quickly being rendered useless by bacterial multidrug resistance.
Cepheid is working on that issue along with UMDNJ under a $7.5 million grant awarded to the partners last June by NIAID. The grant is being used to support development of a 10-color detection technology for GeneXpert, which is currently capable of detecting six colors.
Bishop said that there are "several" important drug-resistant types of TB, "which is why we are looking at multiplexing" under the NIAID grant. But Alland pointed out that even without multiplexing capabilities, rifampicin is "a good surrogate marker for multi-drug resistance. If someone has rifampicin-resistant TB, they're going to be the ones that are hard to treat."