Drug-susceptibility testing for TB: poised to take a turn?

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They studied resistance and susceptibility to the same four first-line drugs that the international consortium studied. “The value we think is most important is the susceptibility predictive value,” says Dr. Musser, whose laboratory wasn’t part of the large study. With the whole genome sequencing test, these values were rifampin, 100 percent; isoniazid, 99 percent; ethambutol, 99 percent; and pyrazinamide, 98 percent. Overall they saw a 99 percent predictive value for susceptibility to the four first-line drugs based on high-confidence mutations. Specificities were also between 99 percent and 100 percent (Shea J, et al. J Clin Microbiol. 2017;55[6]:1871–1882).

Dr. Musser

Dr. Musser and her colleagues added a third group of mutations that are currently unknowns. They were from archival strains that are sometimes resistant and sometimes susceptible. “Usually they have low-level resistance,” she says. “In our newest update to our pipeline, we added in those unknown mutations. At the same time, on Oct. 1, 2018, we went live using whole genome sequencing as our first line of testing. For any strains that test susceptible, we don’t do any additional testing. We report those out as susceptible with high confidence.”

For strains that test resistant or unknown, the Wadsworth laboratory continues to perform culture-based susceptibility testing. “Since strains in New York and the U.S. generally have a low level of resistance, we reduced the amount of culture-based susceptibility testing by about 70 percent,” Dr. Musser says. “Now we can focus more on drug resistance.”

There is work to be done to make the new technology accessible in resource-poor countries, Dr. Walker says, adding that Cepheid’s Gene­Xpert has the advantage of literally being a black box. “You put in a sample and, with minimal preparation, it gives you a result. To get DNA from clinical samples requires technical expertise. The new method won’t be rolled out around the world until someone completely automates it. In the meantime,” he says, “I suspect it will be restricted to labs with trained staff.” That’s what happened even with Gene­Xpert, he adds. “It was envisioned as a bedside test, but that didn’t happen.”

In Dr. Musser’s laboratory, all whole genome sequencing data are used to answer other questions. Epidemiological tracking is one application. To which member of the tuberculosis complex is a particular isolate related? Dr. Musser and colleagues relate each new organism to past strains and send a report including the strain or strains most closely related (by assessing the entire 4.4 million nucleotide genome) to the tuberculosis control epidemiologist. “It helps with those investigations,” Dr. Musser says. Testing with whole genome sequencing is faster than culture-based susceptibility testing and thus allows tuberculosis controllers and physicians to use the lab’s information more quickly, which is especially helpful for multidrug-resistant TB.

Thus, one whole genome sequencing test provides rapid, accurate, comprehensive drug prediction and replaces tests used for other purposes. WGS is an additional cost but one that is partially offset by reduced costs for conventional testing, she says, and ultimately the more rapid results save health care dollars. Dr. Musser’s conclusion: WGS susceptibility testing “pays for itself.” In New York there are about 800 cases per year, of which about 20 percent are drug resistant. “For states with fewer cases and a lower percentage of drug-resistant cases, it may not make sense to implement sequencing,” she says.

Cost for a whole genome sequencing assay is about $200 start to finish. “If we moved to a larger sequencing instrument, that might bring the cost down to $100,” Dr. Musser says. They are now using an Illumina MiSeq. “It fits our volume very well. We run about 15 specimens per week. We are using a NextSeq for some foodborne bacterial testing,” which she says can do 80 specimens per run. “We are thinking about also going to that for bacteria on which we do whole genome sequencing.”
Like Wadsworth, Public Health England decided to stop phenotyping isolates predicted to be susceptible to all first-line drugs, and “similar decisions have been made in the Netherlands,” according to the published international study.

Can the WGS assay be performed on DNA extracted directly from sputum, rather than wait for bacterial growth in culture? Says Dr. Walker: “We did all of our testing on culture isolates. The goal for many people in this field is to replicate those results without culture. The obvious thing is to sequence directly from clinical samples; that way you could get same-day results, which would transform the way we manage cases. We and others are working on isolating DNA from sputum.” They are making progress, he says: “I don’t think we are far from being able to isolate DNA from clinical samples.” Though considerable work is yet to be done, “it’s where we have to go,” he adds.

Dr. Musser’s group is exploring methods to enrich for M. tuberculosis DNA and to do target-based sample amplification. Their goal is to amplify only drug resistance prediction genes. “This is something we think about and work on every day,” she says.

William Check is a writer in Ft. Lauderdale, Fla.