Subtracting the background by reducing cell-free DNA’s confounding effects on Mycobacterium tuberculosis quantitation and the sputum microbiome

Abstract

DNA characterisation in people with tuberculosis (TB) is critical for diagnostic and microbiome evaluations. However, extracellular DNA, more frequent in people on chemotherapy, confounds results. We evaluated whether nucleic acid dyes [propidium monoazide (PMA), PEMAX] and DNaseI could reduce this. PCR [16S Mycobacterium tuberculosis complex (Mtb) qPCR, Xpert MTB/RIF] was done on dilution series of untreated and treated (PMA, PEMAX, DNaseI) Mtb. Separately, 16S rRNA gene qPCR and sequencing were done on untreated and treated sputa before (Cohort A: 11 TB-negatives, 9 TB-positives; Cohort B: 19 TB-positives, PEMAX only) and 24-weeks after chemotherapy (Cohort B). PMA and PEMAX reduced PCR-detected Mtb DNA for dilution series and Cohort A sputum versus untreated controls, suggesting non-intact Mtb is present before treatment-start. PEMAX enabled sequencing-based Mycobacterium-detection in 7/12 (58%) TB-positive sputa where no such reads otherwise occurred. In Cohort A, PMA- and PEMAX-treated versus untreated sputa had decreased α- and increased β-diversities. In Cohort B, β-diversity differences between timepoints were only detected with PEMAX. DNaseI had negligible effects. PMA and PEMAX (but not DNaseI) reduced extracellular DNA in PCR and improved pathogen detection by sequencing. PEMAX additionally detected chemotherapy-associated taxonomic changes that would otherwise be missed. Dyes enhance microbiome evaluations especially during chemotherapy.