Ndivhuho A Makhado1, Edith Matabane2, Mauro Faccin3, Claire Pinçon4, Agathe Jouet5, Fairouz Boutachkourt6, Léonie Goeminne6, Cyril Gaudin5, Gugu Maphalala7, Patrick Beckert8, Stefan Niemann8, Jean-Charles Delvenne9, Michel Delmée6, Lufuno Razwiedani10, Maphoshane Nchabeleng11, Philip Supply12, Bouke C de Jong13, Emmanuel André14. 1. Department of Medical Microbiology, National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, Pretoria, South Africa; Department of Microbiological Pathology, Sefako Makgatho Health Sciences University, Pretoria, South Africa; Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Global Health Institute, University of Antwerp, Belgium. 2. Department of Microbiological Pathology, Sefako Makgatho Health Sciences University, Pretoria, South Africa. 3. Institute of Information and Communication Technologies, Electronics and Applied Mathematics, University of Louvain, Brussels, Belgium. 4. Centre Hospitalier Universitaire, Lille, EA 2694-Santé Publique: Épidémiologie et Qualité des Soins, University of Lille, Lille, France. 5. Genoscreen, Lille, France. 6. Medical Microbiology Pole, Institute of Clinical and Experimental Medicine, UC Louvain, Brussels, Belgium. 7. National Reference Laboratory, Ministry of Health, Mbabane, eSwatini. 8. Molecular and Experimental Mycobacteriology, Research Centre Borstel, Leibniz Lung Centre, Borstel, Germany; German Centre for Infection Research, Borstel Site, Borstel, Germany. 9. Institute of Information and Communication Technologies, Electronics and Applied Mathematics, University of Louvain, Brussels, Belgium; Centre for Operations Research and Econometrics, UC Louvain, Louvain-La-Neuve, Belgium, Belgium. 10. Department of Community Medicine, Sefako Makgatho Health Sciences University, Pretoria, South Africa; Gauteng Department of Health, Hatfield, South Africa. 11. Department of Medical Microbiology, National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, Pretoria, South Africa; Department of Microbiological Pathology, Sefako Makgatho Health Sciences University, Pretoria, South Africa. 12. Centre National de la Recherche Scientifique, Lille, France; Institut National de la Santé et de la Recherche Médicale, Lille, France; Centre Hospitalier Universitaire de Lille, Lille, France; University of Lille, Lille, France; U1019, UMR 8204, Centre for Infection and Immunity of Lille, Institut Pasteur de Lille, Lille, France. 13. Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium. 14. Laboratory of Clinical Bacteriology and Mycology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium. Electronic address: emmanuel.andre@uzleuven.be.
Abstract
BACKGROUND: Global roll-out of rapid molecular assays is revolutionising the diagnosis of rifampicin resistance, predictive of multidrug-resistance, in tuberculosis. However, 30% of the multidrug-resistant (MDR) strains in an eSwatini study harboured the Ile491Phe mutation in the rpoB gene, which is associated with poor rifampicin-based treatment outcomes but is missed by commercial molecular assays or scored as susceptible by phenotypic drug-susceptibility testing deployed in South Africa. We evaluated the presence of Ile491Phe among South African tuberculosis isolates reported as isoniazid-monoresistant according to current national testing algorithms. METHODS: We screened records of 37 644 Mycobacterium tuberculosis positive cultures from four South African provinces, diagnosed at the National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, to identify isolates with rifampicin sensitivity and isoniazid resistance according to Xpert MTB/RIF, GenoType MTBDRplus, and BACTEC MGIT 960. Of 1823 isolates that met these criteria, 277 were randomly selected and screened for Ile491Phe with multiplex allele-specific PCR and Sanger sequencing of rpoB. Ile491Phe-positive strains (as well as 17 Ile491Phe-bearing isolates from the eSwatini study) were then tested by Deeplex-MycTB deep sequencing and whole-genome sequencing to evaluate their patterns of extensive resistance, transmission, and evolution. FINDINGS: Ile491Phe was identified in 37 (15%) of 249 samples with valid multiplex allele-specific PCR and sequencing results, thus reclassifying them as MDR. All 37 isolates were additionally identified as genotypically resistant to all first-line drugs by Deeplex-MycTB. Six of the South African isolates harboured four distinct mutations potentially associated with decreased bedaquiline sensitivity. Consistent with Deeplex-MycTB genotypic profiles, whole-genome sequencing revealed concurrent silent spread in South Africa of a MDR tuberculosis strain lineage extending from the eSwatini outbreak and at least another independently emerged Ile491Phe-bearing lineage. Whole-genome sequencing further suggested acquisition of mechanisms compensating for the Ile491Phe fitness cost, and of additional bedaquiline resistance following the introduction of this drug in South Africa. INTERPRETATION: A substantial number of MDR tuberculosis cases harbouring the Ile491Phe mutation in the rpoB gene in South Africa are missed by current diagnostic strategies, resulting in ineffective first-line treatment, continued amplification of drug resistance, and concurrent silent spread in the community. FUNDING: VLIR-UOS, National Research Foundation (South Africa), and INNOVIRIS.
BACKGROUND: Global roll-out of rapid molecular assays is revolutionising the diagnosis of rifampicin resistance, predictive of multidrug-resistance, in tuberculosis. However, 30% of the multidrug-resistant (MDR) strains in an eSwatini study harboured the Ile491Phe mutation in the rpoB gene, which is associated with poor rifampicin-based treatment outcomes but is missed by commercial molecular assays or scored as susceptible by phenotypic drug-susceptibility testing deployed in South Africa. We evaluated the presence of Ile491Phe among South African tuberculosis isolates reported as isoniazid-monoresistant according to current national testing algorithms. METHODS: We screened records of 37 644 Mycobacterium tuberculosis positive cultures from four South African provinces, diagnosed at the National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, to identify isolates with rifampicin sensitivity and isoniazid resistance according to Xpert MTB/RIF, GenoType MTBDRplus, and BACTEC MGIT 960. Of 1823 isolates that met these criteria, 277 were randomly selected and screened for Ile491Phe with multiplex allele-specific PCR and Sanger sequencing of rpoB. Ile491Phe-positive strains (as well as 17 Ile491Phe-bearing isolates from the eSwatini study) were then tested by Deeplex-MycTB deep sequencing and whole-genome sequencing to evaluate their patterns of extensive resistance, transmission, and evolution. FINDINGS:Ile491Phe was identified in 37 (15%) of 249 samples with valid multiplex allele-specific PCR and sequencing results, thus reclassifying them as MDR. All 37 isolates were additionally identified as genotypically resistant to all first-line drugs by Deeplex-MycTB. Six of the South African isolates harboured four distinct mutations potentially associated with decreased bedaquiline sensitivity. Consistent with Deeplex-MycTB genotypic profiles, whole-genome sequencing revealed concurrent silent spread in South Africa of a MDR tuberculosis strain lineage extending from the eSwatini outbreak and at least another independently emerged Ile491Phe-bearing lineage. Whole-genome sequencing further suggested acquisition of mechanisms compensating for the Ile491Phe fitness cost, and of additional bedaquiline resistance following the introduction of this drug in South Africa. INTERPRETATION: A substantial number of MDR tuberculosis cases harbouring the Ile491Phe mutation in the rpoB gene in South Africa are missed by current diagnostic strategies, resulting in ineffective first-line treatment, continued amplification of drug resistance, and concurrent silent spread in the community. FUNDING: VLIR-UOS, National Research Foundation (South Africa), and INNOVIRIS.
Authors: Armand Van Deun; Tom Decroo; Aung Kya Jai Maug; Mohamed Anwar Hossain; Murid Gumusboga; Wim Mulders; Nimer Ortuño-Gutiérrez; Lutgarde Lynen; Bouke C de Jong; Hans L Rieder Journal: PLoS One Date: 2020-05-18 Impact factor: 3.240
Authors: Nomonde R Mvelase; Melendhran Pillay; Wilbert Sibanda; Jacqueline N Ngozo; James C M Brust; Koleka P Mlisana Journal: Open Forum Infect Dis Date: 2019-02-12 Impact factor: 3.835