Scientists at San Diego State University say they have identified rare genetic markers in Mycobacterium tuberculosis that could improve early detection of drug-resistant strains of the disease, helping prevent their spread.

To test whether someone has a strain of Tuberculosis (TB) that will no longer respond to standard treatment, clinicians culture samples of mucus from the respiratory tract and barrage them with antibiotics.

“But because TB grows so slowly, that takes weeks,” said San Diego State University professor of public health Faramarz Valafar, PhD. “In those weeks that patient is going around spreading TB that might be antibiotic-resistant.”

He says molecular diagnostic tools are much faster. These test for common genetic markers of drug-resistance and allow for more timely treatment. But TB strains with rare mechanisms of resistance still elude molecular detection.

“They don’t have the common genetic markers, but they are resistant,” continued Valafar. This leads clinicians to incorrectly conclude that standard TB drugs will kill the bacteria. “And so the patient is given the wrong medications and continues to infect others for weeks—sometimes months—before they realize that these drugs aren’t working. So we really want to prevent that.”

Derek Conkle-Gutierrez, a doctoral student in Valafar’s lab, led the search for rare genetic mutations associated with resistance. The researchers obtained samples of M. tuberculosis from seven different countries where antibiotic resistance is common. Culturing the samples revealed that some were indeed drug-resistant, even though molecular diagnostics had failed to catch them.

“First we confirmed that they didn’t have the known markers and then we started looking for what other mutations are showing up exclusively in these unexplained resistant isolates,” explained Conkle-Gutierrez.

The researchers identified one set of rare genetic mutations that may help block the common TB drug kanamycin from interfering with the pathogen’s ability to synthesize the proteins it needs, rendering it harmless to the pathogen. Another set of mutations may do the same for the TB drug capreomycin.

The study “Distribution of common and rare genetic markers of second-line-injectable-drug resistance in mycobacterium tuberculosis revealed by a genome-wide association study” appears in Antimicrobial Agents and Chemotherapy.

Anna Scavo, Sam Modlin, Derek Conkle Gutierrez, and Faramarz Valafar, members of the SDSU Laboratory for Pathogenesis of Clinical Drug Resistance and Persistence. Susanne Clara Bard, San Diego State University]

“Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics,” write the investigators.

“In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates.

“A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5′-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome.

“Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.”

“This manuscript identifies potential markers; confirmatory work for the selection of markers for the next generation of more comprehensive molecular diagnostic platforms lies ahead,” noted Valafar, adding that given the evolution of antibiotic resistance, molecular diagnostics will need to be updated frequently and be tailored to different regions of the world where antibiotic resistance in TB is common.

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