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.