Identifying and addressing the gaps impeding fast and accurate phenotypic drug-susceptibility testing of Mycobacterium tuberculosis

The gap in tuberculosis diagnostics has been a persistent challenge, spanning decades of efforts to improve the detection of Mycobacterium tuberculosis complex (MTBc) and its drug resistance. Roll-out of new drugs without access to drug-susceptibility testing (DST) and surveillance data risks repeating errors of the past, and rapidly losing these drugs to resistance when patients are treated ‘blindly’ with already compromised regimens. For the majority of anti-tuberculosis drugs, phenotypic DST (pDST) is still the gold standard. Thus, expanding research efforts to understand the current gaps in pDST in MTBc is crucial for gaining a more comprehensive understanding of the drug-resistance landscape and improving patient-centered TB management strategies.

This thesis sought to describe the factors impeding rapid and accurate detection of phenotypic drug resistance, as well as to determine whether faster minimum inhibitory concentration (MIC) testing offers advantages over traditional pDST, which relies on growth inhibition of MTBc in drug-containing medium at a single drug concentration known as the critical concentration (CC), yielding a binary classification as susceptible or resistant.

The main gaps identified in traditional pDST for MTBc during this PhD research include: (1) lack of a standardised culture medium that provides optimal nutrition to all MTBc variants, including fastidious strains, and (2) artefacts related to binary classification of drug susceptibility using the CCs, such as overlapping MICs of drug-susceptible and drug-resistant strains, leading to misclassification of low-level/borderline resistant mutants and (3) the lineage effect on in-vitro susceptibility to anti-TB drugs. These gaps underscore the need for a standardized quantitative pDST method, applicable for all MTBc variants.

As a potential solution, this PhD research assessed the recently WHO-endorsed microtiter-based broth microdilution (BMD) method, which represents a significant step towards standardization in pDST for MTBc. We semi-automated the method and successfully validated its use to assess new and repurposed anti-TB drugs. In addition, we demonstrated that freshly grown liquid cultures can be used as a basis for BMD inoculation, as opposed to standard inoculum preparation from cultures grown on solid medium, hence reducing the turnaround time (TAT) and increased field-friendliness. Finally, we addressed some of the challenges associated with traditional pDST methods.

Continuous improvements such as further reducing the TAT, standardize reading and inoculum preparation, as well as cost-effective automation to promote widespread adoption of MIC testing by the BMD method, will be critical for improving the quality and reliability of pDST for MTBc and, ultimately, TB treatment outcomes globally.