Speeding up drug susceptibility testing in Mycobacterium tuberculosis using RNA biomarkers

Abstract

Background: Efficient management of drug-resistant tuberculosis relies on fast diagnostics. To accelerate phenotypic drug susceptibility testing [pDST] for Mycobacterium tuberculosis [TB], we introduce TRACeR-TB, a test that infers drug resistance from antibiotic-specific mRNA biomarkers.

Methods: To develop TRACeR-TB, target genes were first identified through RNA sequencing experiments conducted on two drug-exposed, susceptible strains for four antitubercular drugs. Based on these findings, we designed drug-specific multiplex Quantigene panels to quantify mRNA levels of 8-9 biomarkers per drug (class), directly from crude cell lysates. The performance of TRACeR-TB was compared to the widely used Mycobacteria Growth Indicator Tube [MGIT] pDST by subjecting 238 strains with diverse drug resistance profiles to both methods, and aligning results to genotypic data. Furthermore, we explored TRACeR-TB's potential for evaluating molecules that enhance antibiotic efficacy, and investigated its applicability in macrophage models to assess Mtb's intracellular stress responses to drugs.

Findings: Antituberculosis drugs trigger distinct transcriptional stress responses in susceptible, but not resistant bacilli, enabling a differentiation of the antibiotic phenotype in only 6 h. Validation on 238 strains showed TRACeR-TB had 100% (95% CI: 93·1-100%) sensitivity and 89·5% (95% CI: 74·7-97·2%) specificity compared to, respectively, 82·3% (95% CI: 69·2%-91·5%) and 94·8% (95% CI: 81·9%-99·4%) for MGIT pDST. TRACeR-TB specificity is likely underestimated due to the inclusion of isolates harbouring uncharacterised mutations. TRACeR-TB demonstrated 100% concordance with MGIT for drugs with reliable MGIT outcomes (moxifloxacin and isoniazid). Additionally, its sensitivity outperformed current rifampicin testing, detecting resistance in all borderline-resistant strains that MGIT missed, and bedaquiline testing. Furthermore, the assay detected the predicted effect of a novel drug booster and the intracellular drug-induced stress in macrophage models, highlighting its potential for drug optimisation.

Interpretation: TRACeR-TB is a complementary addition to current DSTs and can have a substantial impact on the TB diagnostics field. This tool can also play a vital role in identifying resistance mutations, thereby closing gaps in genotypic knowledge, and contribute to drug discovery and development.

Funding: Institut Pasteur, Agence Nationale de la Recherche.

Keywords: Antimicrobial resistance; Biomarkers; Diagnosis; RNA; Tuberculosis.

Fig. 1

Overview of the TRACeR-TB assay for rapid DST of M. tuberculosis based on RNA quantification. To distinguish susceptible from resistant strains, we expose aliquots from culture positive Mycobacteria Growth Indicator Tube [MGIT] broths in exponential growth to an antibiotic or its solvent only for six hours at 37 °C. Following transcriptional arrest and a simple cell lysis step, we add a drug-specific oligonucleotide probe and magnetic bead set (QuantiGene technology, ThermoFisher Scientific) to the crude lysate, and the xMAP technology (Luminex Corporation) quantifies biomarker expression. A three-step normalization/transformation approach is used to convert raw fluorescent gene signals into fold-change values, employing housekeeping genes and solvent-exposed controls. We determine transcript induction or repression by comparing drug-exposed to solvent-exposed samples. Finally, the expression values are condensed into a single numeric value: the squared projected distance [SPD], calculated from a drug-specific core reference set. Strains that are susceptible to a certain drug exhibit small SPDs close to 0, as a result of their distinctive, pronounced transcriptional responses, whereas antibiotic-resistant strains display higher SPDs near 1, indicative of little to no change in biomarkers expression. A threshold SPD at 0·26 allows the distinction between susceptible and resistant isolates. Created with Biorender.com.

Fig. 2

TRACeR-TB biomarker selection and outcome in relation to MGIT and genotype. Left panel. Transcriptional response of selected biomarkers of two pan-susceptible M. tuberculosis strains to four antitubercular drugs. The heatmap depicts their log2 FoldChange after 2, 3, 6, or 24 h of incubation with moxifloxacin (0·5 μg ml⁻¹), rifampicin (2 μg ml⁻¹), bedaquiline (3·2 μg ml⁻¹), and isoniazid (0·2 μg ml⁻¹). Right panels. The TRACeR-TB validation for moxifloxacin, rifampicin, bedaquiline, and isoniazid, with SPDs obtained after 6 h of exposure at the critical concentration [CC] (left side) and a higher concentration (right side) with the number of isolates tested [n]. MGIT-DST outcomes for the tested strains are presented in different colours: susceptible [MGIT-S] in green and resistant [MGIT-R] in orange. Isoniazid MGIT-DST was performed with two concentrations (0·1 and 0·4 μg ml⁻¹) to distinguish low/high-level of resistance [MGIT low/high-level R] in, respectively, light and dark orange in the figure. Genotypic outcomes for the tested strains are presented with different patterns. Drug resistance mutations are classified according to the latest WHO catalogue. Mutations with a final confidence grading of “1) Assoc w R” and “2) Assoc w R – Interim” are labelled as (low/high) resistant [R]. Mutations with a final confidence grading of “3) Uncertain significance”, or mutations not described in the catalogue are marked as “uncertain significance” [unc. sign.] and are only depicted as such if a resistant outcome was obtained in MGIT DST and/or TRACeR-TB. There is no separate labelling for mutations with a final confidence grading of “4) Not assoc w R - Interim” and “5) Not assoc w R”. Borderline rifampicin resistance-conferring mutations are reported separately among the resistant strains (Appendix S2).

Fig. 3

Applications of TRACeR-TB in drug development. (a) SMARt751 effect detection in TRACeR-TB. SPDs obtained for five ethionamide MGIT-susceptible strains (strains 1–5) and seven ethionamide MGIT-resistant strains carrying the inhA c-15t mutation (strains 6 and 7) or an ethA mutation (strains 8–12). We tested each strain in five conditions: exposure to SMARt751, exposure to two ethionamide concentrations and exposure to two ethionamide concentrations combined with SMARt751 (green border). Triangles replace dots for values above 1 or below −1. Strain 1 is H37Rv, strains 2–7 are clinical strains from the Belgian National Reference Center for tuberculosis and Mycobacteria-Sciensano (2: S16BD02813, 3: S16BD05456, 4: S16BD05457, 5: S17BD00453, 6: 13MY0376- SRR13180422, and 7: 11MY0210- SRR13180328) and strains 8–12 are in vitro selected with ethA mutations provided by Sciensano (8: Y84C, 9: C253Stop, 10: G343A, 11: Y386Stop, and 12: W256Stop) (b) Susceptibility testing in macrophages. SPDs obtained for uninfected macrophages (no signal), H37Rv and macrophages infected by H37Rv exposed to 0·1 and 1 μg ml⁻¹ of isoniazid.