Developing a novel molecular bacterial load assay to improve clinical management of Mycobacterium abscessus infections

Abstract

Mycobacterium abscessus (M. abscessus) is one of the most common rapid growing non- tuberculous mycobacteria (NTM) causing pulmonary disease (PD). Treatment involves long and toxic multi-drug regimens with uncertain benefit. It is difficult to assess antibiotic efficacy as current treatment monitoring depends on semi-quantitative culture of serial clinical samples that takes weeks to provide results.

This thesis describes the development of the first molecular, quantitative treatment monitoring tool for NTM-PD. The M. abscessus molecular bacterial load assay (MBLA) assay which we developed provides results within a working day. The M. abscessus MBLA targets the hypervariable portions of 16S and pre-16S rRNA using real-time quantitative PCR to solely quantify the viable organisms from patients’ sputum samples. Both RNA targets showed a strong correlation with cell viability, having >90% degradation within four weeks following cell death.

The M. abscessus MBLA uses a standard curve to provide bacterial load quantification. An investigation into the impact of antibiotics used for M. abscessus- PD treatment on the standard curve revealed a correlation between the 16S rRNA quantification and bacterial load in the absence and presence of antibiotics.

The potential of using pre-16S rRNA to 16S rRNA ratio as a measure of metabolic activity within a bacterial population was explored, showing that the ratio followed the growth trends of M. abscessus in the absence and presence of antibiotics.

This thesis resulted in a treatment monitoring tool selective for M. abscessus-chelonae group species with an efficiency of 94% and limit of detection of log 10¹ CFU/ml. Preliminary clinical validation was limited by a small number of available samples from patients with M. abscessus- PD, but the assay accurately reported 6/8 positive results and 36/36 negative results against a gold standard of sputum culture. Larger clinical studies will be undertaken to fully evaluate the clinical utility of the test.

Date of Award	17 Jun 2022
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Derek James Sloan (Supervisor) & Stephen Henry Gillespie (Supervisor)