A Molecular Approach to the Diagnosis, Assessment, Monitoring and Treatment of Pulmonary Non-Tuberculous Mycobacterial Disease

Abstract

Introduction: Non-Tuberculous Mycobacteria (NTM) can cause disease of the lungs and sinuses, lymph nodes, joints and central nervous system as well as disseminated infections in immunocompromised individuals. Efforts to tackle infections in NTM are hampered by a lack of reliable biomarkers for diagnosis, assessment of disease activity, and prognostication. Aims: The broad aims of this thesis are: 1. to develop molecular assays capable of quantifying the 6 most common pathogenic mycobacteria (M. abscessus, M. avium, M. intracellulare, M. malmoense, M. kansasii, M. xenopi) and calculate comparative sensitivities and specificities for each assay. 2. to assess patients’ clinical course over 12 – 18 months by performing the developed molecular assays against DNA extracted from sputum from patients with NTM infection. 3. to assess dynamic bacterial changes of the lung microbiome in patients on treatment for NTM disease and those who are treatment na ve. Methods: DNA was extracted from a total of 410 sputum samples obtained from 38 patients who were either: • commencing treatment for either M. abscessus or Mycobacterium avium complex. • considered colonised with M. abscessus or Mycobacterium avium complex (i.e. cultured NTM but were not deemed to have infection as they did not meet ATS or BTS criteria for disease). • Diagnosed with cystic fibrosis (CF) or non-CF bronchiectasis but had never cultured NTM. For the development of quantitative molecular assays, NTM hsp65 gene sequences were aligned and interrogated for areas of variability. These variable regions enabled the creation of species specific probes. In vitro sensitivity and specificity for each probe was determined by testing each probe against a panel of plasmids containing hsp65 gene inserts from different NTM species. Quantification accuracy was determined by using each assay against a mock community containing serial dilutions of target DNA. Each sample was tested with the probes targeting: M. abscessus, M. avium and M. intracellulare producing a longitudinal assessment of NTM copy number during each patient’s clinical course. In addition, a total of 64 samples from 16 patients underwent 16S rRNA gene sequencing to characterise longitudinal changes in the microbiome of both NTM disease and controls. Results: In vitro sensitivity for the custom assays were 100% and specificity ranged from 91.6% to 100%. In terms of quantification accuracy, there was no significant difference between the measured results of each assay and the expected values when performed in singleplex. The assays were able to accurately determine NTM copy number to a theoretical limit of 10 copies/μl. When used against samples derived from human sputum and using culture results as a gold standard, the sensitivity of the assay for M. abscessus was found to be 0.87 and 0.86 for MAC. The specificity of the assay for M. abscessus was 0.95 and 0.62 for MAC. The negative predictive value of the assay for M. abscessus was 0.98 and 0.95 for MAC. This resulted in an AUC of 0.92 for M. abscessus and 0.74 for MAC. Longitudinal analysis of the lung microbiome using 16SrRNA gene sequencing showed that bacterial burden initially decreases after initiation of antibiotic therapy but begins to return to normal levels over several months of antibiotic therapy. This effect is mirrored by changes in alpha diversity. The decrease in bacterial burden and loss of alpha diversity was found to be secondary to significant changes in specific genera such as Veillonella and Streptococcus. The abundance of other Proteobacteria such as Pseudomonas remain relatively constant. Conclusion: The molecular assay has shown high in vitro sensitivity and specificity for the detection and accurate quantification of the 6 most commonly pathogenic NTM species. The assays successfully identified NTM DNA from human sputum samples. A notable association between NTM copy number and the cessation of one or more antibiotics existed (i.e. when one antibiotic was stopped because of patient intolerance, NTM copy number increased, often having been unrecordable prior to this). The qPCR assays developed in this thesis provide an affordable, real time and rapid measurement of NTM burden allowing clinicians to act on problematic results sooner than currently possible. There was no significant difference between the microbiome in bronchiectasis and cystic fibrosis nor was there a significant difference between the microbiome in patients requiring treatment for NTM and those who did not. Patients receiving treatment experienced an initial decrease in bacterial burden over the first weeks of treatment followed by a gradual increase towards baseline over the next weeks to months. This change was mirrored in measures of alpha diversity. Changes in abundance and diversity were accounted for by decreases in specific bacteria whilst the abundance of other bacteria increased, occupying the microbial niche created. These bacteria (for example Pseudomonas spp) are often associated with morbidity.Open Acces