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

William Check, PhD

January 2019—In a large international study, whole genome sequencing with next-generation sequencing technology has proved its ability to accurately assess susceptibility of Mycobacterium tuberculosis isolates to four first-line drugs. The data are convincing enough that the new technique has replaced phenotypic drug-susceptibility testing in some public health laboratories in the United States and Europe.

“What is remarkable is the lack of progress [in this area] in the last 100 years. It is only recently that we have made any advance at all,” says Timothy M. Walker, DPhil, of the Department of Microbiology, John Radcliffe Hospital, Oxford, United Kingdom. “Until the late 1990s we were still dependent on phenotypic testing [to determine resistance in M. tuberculosis isolates]. Now things appear to be moving.”

Dr. Walker led the study in 16 countries across six continents that analyzed 10,209 clinical specimens. It demonstrated the power of whole genome sequencing to correctly predict resistance and susceptibility of M. tuberculosis isolates (N Engl J Med. 2018;379[15]:1403–1415). “There is still a lot of work to be done,” he tells CAP TODAY, before this method can become standard in the clinical microbiology laboratory.

In the United States, whole genome sequencing to determine susceptibility of M. tuberculosis isolates is being performed at Wads­worth Center, the laboratory of the New York State Department of Health, in Albany. “We developed and validated a test based on whole genome sequencing that provides comprehensive resistance detection for this organism,” says Kimberlee A. Musser, PhD, Wads­worth’s chief of bacterial diseases. It was brought online in February 2016. “We performed that test side by side with culture-based susceptibility testing for more than two and a half years,” Dr. Musser says. Agreement was excellent, so whole genome sequencing was implemented as Wads­worth’s first-line clinical test.

Dr. Musser and Dr. Walker believe that direct detection of M. tuberculosis resistance in clinical samples by whole genome sequencing is feasible. Their laboratories are among several working now to achieve it.

Dr. Walker, who is an academic clinical lecturer in infectious diseases and microbiology, says there are two important conclusions from the international study, conducted by Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) and the 100,000 Genomes Project. First, “We are now at the point where our understanding of the molecular causes of resistance to first-line [antituberculosis] drugs is at a sufficiently high level that we can replace routine resistance testing by phenotypic methods with whole genome sequencing.” WGS can provide a result within 10 days rather than weeks to months, he notes.

Second, Dr. Walker says, “We argue that for the first time we can use a molecular method to predict susceptibility rather than just resistance. This is a slightly trickier concept to get our heads around. It is not complicated, but it is not the way people typically think about it.”

Cepheid’s GeneXpert MTB/RIF assay, for detection of rifampin resistance, lacks sufficient sensitivity, Dr. Walker says. As a result, “In the absence of a positive result we haven’t been able reliably to predict susceptibility.” In contrast, with a negative resistance result with whole genome sequencing, “we can now confidently say you can give this drug and avoid these others.”

In the study published Oct. 11, 2018 in the New England Journal of Medicine, there was a strong correlation between WGS-based predictions of susceptibility to four first-line tuberculosis drugs—isoniazid, rifampin, pyrazinamide, and ethambutol—and phenotypic susceptibility as determined by culture-based testing. Sequencing correctly predicted resistance, with sensitivities ranging from 91 percent to 98 percent. As a result, whole genome sequencing correctly predicted susceptibility to the four drugs, with specificities ranging from 93 percent (ethambutol) to 99 percent (isoniazid).

“[W]hole-genome sequencing can now characterize profiles of susceptibility to first-line antituberculosis drugs with a degree of accuracy sufficient for clinical use,” Dr. Walker and coauthors write, adding that the importance is twofold. “First, it shows that the genomic approach could be used to guide the choice of which drugs to prescribe and not just which drugs to avoid, in a way similar to phenotyping. Second, the data can be used to support plans to reduce the workload associated with culture and susceptibility analysis in places where routine whole-genome sequencing is performed.”

Dr. Musser’s laboratory started to work on a whole genome sequencing resistance test about five years ago. “We piloted a whole genome sequencing test to detect in a comprehensive way mutations that were known to cause resistance in M. tuberculosis strains,” she says. “In New York we have a mechanism to validate clinical tests of any kind.” Following this protocol produced reassuring results. “We found that whole genome sequencing predicted susceptibility and resistance with high sensitivity and specificity. Each year we refined our bioinformatic pipeline”—developed by a Wadsworth bioinformatician—“to a point where we felt we could detect all known indicators of resistance in M. tuberculosis as well as identify other mutations that never cause resistance.”