Mycobacterium tuberculosis (Mtb) is transmitted in small aerosol droplets expectorated by individuals with active pulmonary tuberculosis (TB). There is still no established method for sampling these infectious aerosols and measuring the bacterial concentration in them. Little is known about the relationship between tuberculous aerosol output and infectiousness of TB cases; medical practice currently relies on the acid-fast smear status to gauge this and determine when it is safe to discharge TB patients. This study involved the development and evaluation of a rapid molecular assay and a mask sampling approach to increase our knowledge relating to TB transmission. A molecular assay targeting the mycobacterial 16S rDNA and differentiating Mtb complex from non-tuberculous mycobacteria was developed to facilitate recruitment of TB patients for the mask sampling study. Although the assay did not achieve this outcome during the study, we demonstrated its clinical utility on mycobacterial isolates. A second molecular assay targeting the Mtb global lineagedefining and locally prevalent RD750 polymorphism was also developed for preliminary genotyping of Mtb strains to assess for potential recent transmission events. Both the 16S and RD assays were performed in single thermocycler runs but in separate reaction tubes and utilised a combination of TaqMan and SYBR Green technologies to simultaneously differentiate two products. The 16S-RD assay shows promising potential for direct specimen analysis and detection of mixed infections. A novel method of mask aerosol sampling coupled to a mycobacteriophage amplification assay developed in this study was able to detect and to some extent quantify the amount of respiratory-borne Mtb. This approach could potentially be more reliable than smear microscopy in assessing TB infectivity. With further optimisation and improvement, both the 16S-RD assay and the mask sampling method have potential to facilitate better understanding of TB transmission and subsequently contribute to public and clinical management of this disease
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