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National University of Ireland Group Develops Multiplex Real-Time PCR Test for TB Complex


A team from the National University of Ireland Galway's Molecular Diagnostics Research Group has developed and validated a two-stage multiplex real-time PCR assay to identify all members of the Mycobacterium tuberculosis complex.

The test could prove useful to centralized clinical reference labs for the purposes of tracking and conducting epidemiological studies on the various Mycobacterium species comprising the complex, which are currently not routinely tested for, but which recent studies have shown may be more responsible for human disease than previously thought.

In addition, the assay is an example of pushing multiplex real-time PCR to its limit using current technologies, according to some of its developers.

Several companies or academic research groups have developed real-time PCR-based assays to diagnose Mycobacterium tuberculosis, which is responsible for a majority of cases of human TB. Many of these assays also test for drug resistance, and some are conducive to deployment in resource-poor areas of the world that are affected most by TB. Cepheid's Xpert MTB/RIF is the most successful commercial such test to date.

However, several other species of Mycobacterium comprise the Mycobacterium tuberculosis complex, or MTC, and are responsible for some cases of TB in humans or can cause complications when co-infecting with HIV. The MTC members are M. tuberculosis, M. canettii, M. africanum, M. bovis, M. caprae, M. microti, M. pinnipedii, and the attenuated M. bovis BCG vaccine strain.

Because routine testing for these members of the MTC is not routinely performed, their global frequency, distribution, and role in disease is not fully understood.

"Essentially it's unknown what impact the other members of the TB complex are causing, or [contribute] to TB infection worldwide," Thomas Barry, research director and founding member of the Molecular Diagnostics Research Group at NUI and one of the developers of the new assay.

"For example, there are parts of Africa where M. canettii and M. africanum are considered to be the major infectious disease agent," Barry said. "And there are parts of South America where other … members of the complex may be more prevalent than M. tuberculosis." There is also concern in Europe, due to its relative proximity to Asia and Africa, of unusual Mycobacterium species popping up and causing problems in immunocompromised individuals, such as those infected with HIV, he noted.

Barry's group at NUI has, over the years, conducted a significant amount of molecular diagnostic development for industry, and during that time he identified tuberculosis as a relatively underserved disease area.

"It's not necessarily high on the radar of multinational companies [in the] developed world, but I thought there was a need to develop novel diagnostics for TB," he said. "When we set out to look at TB about three or four years ago, we looked to see [where] the gaps were in terms of TB diagnostic capability, and we identified PCR and in particular real-time PCR, and pushing it to its limits in terms of multiplex capability."

The group has previously published a pair of papers describing the stepwise multiplexing of an assay for the MTC, work that culminated in the development of a two-step, five-plex real-time PCR assay and algorithm that enables the accurate identification and differentiation of all eight members of the MTC, including the M. africanum West African 1 and M. africanum West African 2 strains; and that includes internal calibrator genes.

The researchers describe their assay, called SeekTB, and its performance in a validation study in a paper published this month in the Journal of Clinical Microbiology.

The NUI scientists identified primers and probes using in silico comparative genomics approaches, and designed their assay using TaqMan hydrolysis probes for use on a Roche LightCycler 480. The two-step assay is designed to be performed sequentially, with the first five-plex assay having the ability to detect the presence of the MTC while differentiating four of the member species; and the second five-plex assay designed to identify the remaining members of the complex if the first step yielded a positive MTC result but did not identify any of the species targeted in the first step.

The entire sequential assay takes about 3.5 hours to perform, the researchers said. The two-step process was necessary because "we're using hydrolysis-based probes, or TaqMan probes, so you're very limited with the spectra of fluorophores you can use," Kate Reddington, an NUI researcher and co-author on the paper, told PCR Insider. "The current limitation of real-time PCR machines for hydrolysis-based fluorescent or TaqMan probes is a five-plex."

In their study, the NUI team tested SeekTB against a panel of well-characterized bacterial strains and determined that the assay was 100 percent specific for members of the MTC. In addition, they blindly tested 125 Mycobacterium growth indicator tube-positive cultures using SeekTB, as well as Hain Lifesciences' Genotype MTBC kit, an amplification/hybridization-based assay that has been shown to be limited in its ability to accurately identify certain MTC members; and BD Diagnostics' MGIT TBc ID test, a lateral-flow immunochromatographic assay that detects the MPB64a protein in liquid cultures to identify only the general presence of MTC.

The researchers found that SeekTB and GenoType MTBC results were 100 percent concordant in identifying 84 of the 125 isolates as Mycobacterium tuberculosis, and 41 as non-MTC. Comparing the molecular methods and the MGIT TBc ID test produced nine discordant results, which the researchers resolved using sequencing, thus confirming the results of the molecular tests.

Barry said that their test represents the "absolute limit" of real-time PCR testing with commonly used fluorescent probes; while Reddington commented that SeekTB, in its current format, would "likely be something predominantly used in reference laboratories … and is conducive to epidemiological studies."

"Ideally I'd like to see this in the developing world, but the cost is just beyond the means of most countries in the developing world," Barry said. "You could see it being configured onto some handheld machine, maybe in a simpler format. Ideally you'd like to see this used directly on sputum, and used in the field," he added, but conceded that it would likely take years of development work to achieve such a goal.

Nevertheless, the NUI group hopes that a company will be interested in commercializing SeekTB because it could have a huge impact on understanding how the MTC complex contributes to human disease.

"I thought this technology was worth putting in the public domain, because … it could easily be used, if a commercial entity was interested, on a commercial platform," Barry said. "There might be one or two things we might tweak to make it better for them."