To counter the long turnaround time of standard phenotypic Drug Susceptibility Testing (DST) of M. tuberculosis (Mtb), multiple DNA-based methods were successfully introduced over the last years. Although these are fast and sensitive, they (a) are based on the knowledge on resistance mutations which is limited for especially 2nd-line and new drugs, (b) do not distinguish living from death cells, (c) ignore all intrinsic resistance mechanisms like efflux pump overexpression, and (d) ignore the multifactorial influence of compensatory mutations. Here, we introduce a next-generation diagnostic test based on quantification of antibiotic-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within minutes to a few hours. A major advantage of this method is that it avoids a long culture-dependent step, yet detects the resistance phenotype, independent of the specific cause of resistance.
First, the global transcriptional response of H37Rv and clinical Mtb strains on ten anti-TB drugs including Bedaquiline, Pyrazinamide and Delamanid was determined, to identify RNA Biomarkers. In a next phase, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGIT culture is spiked with a specific concentration of a drug, while a control sample is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysate using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fischer) technology. Normalized, relative genes expression levels (control vs drug) are combined to one numeric value which determines the drug susceptibility of the investigated strain.
The assay was optimized for parameters as cell density, incubation time and lysis method. I will present the results, showing that following a biomarker set of 5 responsive genes and 3 normalizing genes enables to distinguish low- and high resistant Mtb strains after an incubation step of 6 hours. With a total of 8 biomarker sets under optimization, the phenotypic drug resistance profile of up to 14 drugs can be determined for any combination of antibiotics in 96 well format.