Subtracting the background by reducing cell-free DNA's confounding effects on Mycobacterium tuberculosis quantitation and the sputum microbiome.
Charissa C Naidoo, Rouxjeane Venter, Francesc Codony, Gemma Agustí, Natasha Kitchin, Selisha Naidoo, Hilary Monaco, Hridesh Mishra, Yonghua Li, Jose C Clemente, Robin M Warren, Leopoldo N Segal, Grant Theron
Author Information
Charissa C Naidoo: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000, South Africa.
Rouxjeane Venter: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000, South Africa.
Francesc Codony: Municipal Laboratory - Aigües de Mataró, Mataró, Spain.
Gemma Agustí: Reactivos para Diagnóstico, Setmenat, Spain.
Natasha Kitchin: Department of Psychiatry, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
Selisha Naidoo: Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Hilary Monaco: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Hridesh Mishra: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000, South Africa.
Yonghua Li: Division of Pulmonary, Critical Care, and Sleep Medicine, New York University School of Medicine, New York, USA.
Jose C Clemente: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Robin M Warren: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000, South Africa.
Leopoldo N Segal: Division of Pulmonary, Critical Care, and Sleep Medicine, New York University School of Medicine, New York, USA.
Grant Theron: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town, 8000, South Africa. gtheron@sun.ac.za.
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.
