NEW YORK (GenomeWeb) – The first report of 10-color detection capabilities deployed in a molecular diagnostic test cartridge for extensively drug resistant (XDR) tuberculosis shows the test can distinguish 32 mutant sequences with a sensitivity about equivalent to Cepheid's widely adopted Xpert MTB/RIF test.
Published last week in the Journal of Clinical Microbiology, the work is part of an ongoing collaboration between Cepheid and researchers in the lab of David Alland at the New Jersey Medical School at Rutgers University.
"Cepheid and my laboratory collaborate very closely; we have weekly phone calls where we share data and we really are one team," Alland told GenomeWeb in an interview.
Alland is PI on the project and his team developed the concept and the assay, and did most of the testing of it, he said. Cepheid, meanwhile, developed the new fluorophore dyes at its Bothell, Washington facility, and also devised a new cartridge that permits more DNA to be tested.
Most PCR systems have four-, five-, or six-color detection capabilities. "Going from six to 10 has been a first in the PCR world," David Persing, Cepheid's chief medical and technology officer, said in an interview.
"We had to create some new dyes that could work with our existing six-color excitation spectrum," Persing added.
Dyes typically are excited by one wavelength and then emit fluorescence at another wavelength. "You have an excitation channel and a detection channel, and those channels are at different wavelengths," Persing explained. Cepheid has now created four large Stokes shift, or "off axis," dyes, to allow two dyes that absorb the same wavelength to emit at two different wavelengths. In this way, the firm essentially created new detection channels using the existing six excitation channels in the system.
The JCM study describes using the new 10-color method in a three-phase, double-nested PCR approach followed by melting temperature analysis using a standard GeneXpert platform. The newly developed dyes allow the six-channel system to detect the four additional dyes, while a technology called "sloppy molecular beacons" identifies many different mutations using a small number of probes.
The sloppy beacon technology enables the detection of a number of different mutations within a given region. Alland noted that, unlike regular molecular beacons, which are designed to be pretty much either "all on" or "all off" their target, sloppy molecular beacons are more tolerant of mismatches. "[They] are pretty sticky, and so they melt off their target at different temperatures [and] we can use one sloppy molecular beacon to detect and distinguish among several mutations in a target region based on the melting temperature," Alland said.
The use of melt thus allows specificity in identification of individual mutations, "like doing a virtual sequencing reaction in the cartridge," Persing said, because individual SNPs can be resolved.
For the TB test, "This is important because many of the regions we were targeting had mutations, some of which were significant and others of which were not," he said.
The XDR test can distinguish 32 different mutations that can confer resistance to isoniazid, rifampin, aminoglycosides, and fluoroquinolones.
But the 10-color capability combined with sloppy beacons and melt analysis also lend themselves to other types of testing.
"It allows for resolution of up to five individual targets per color channel, so now with a 10-color system you've got 50-target resolution capability in a single reaction," Persing said.
Persing noted in a previous interview that the firm had been planning to release a TB Ultra test in 2016 and the XDR about a year and half later.
However, since acquisition by Danaher the firm is emphasizing commercial products with a good return on investment and there is currently no launch date estimated for the XDR cartridge nor is it on the firm's five-year plan. That plan now highlights oncology and virology products as well as the creation of rapid "Xpress" chemistry versions of many of the firm's currently-available tests, a strategy outlined by Cepheid last year. Persing also noted that Cepheid is planning to use the new 10-color technology to develop respiratory and gastrointestinal panel-based tests, which could expand the firm's versatility and help leverage the GeneXpert into new diagnostic spaces.
But it still might be possible to develop the XDR test. "We are talking with several outside partners to see if we can make that happen," Persing said.
In the meantime, Alland pointed out that the XDR test has the benefit of improving treatment and saving patients, and it could also be expected to reduce transmission of resistant strains within institutions.
He noted that the World Health Organization has recommended a short-course multi-drug therapeutic regimen for all patients with rifampin-resistant TB, but that the protocol should not be used if a patient might be resistant to any of the drugs in the cocktail.
"Of course there is really no way to know whether someone would have one of those additional forms of drug resistance and whether that would be the right regimen to put people on, but our test will really nail it," Alland said.
As such, he sees the test being used as a reflex test. "Sometimes it should be used on everyone with TB, in situations where drug resistance is very high, and other times we would expect it to be used only when patients have rifampin-resistant TB."
And until it is available, Alland will continue to field calls from stakeholders in the TB community. "It is still an experimental cartridge and it is not yet in production; that's all I can say at this point," he said, adding, "there is certainly a lot of excitement from people that are in the trenches."