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Screening for non-fixed/mixed single nucleotide polymorphisms (SNPs) in serial Mycobacterium tuberculosis isolates during treatment for MDR-TB, the emergence of sugI mutations in patients receiving d-cycloserine.

R M Anthony(1) M Molemans(2) J Alffenaar(3,4,5) O Akkerman(6) M GG Sturkenboom(10) A Mulder(1) R de Zwaan(1) F CM van Leth(7) S Ghimire(10) N Yatskevich(8) A Skrahina(8) V Crudu(9) N Ciobanu(9) N Turcan(9)

1:National Tuberculosis Reference Laboratory, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), 3721BA Bilthoven, the Netherlands; 2:AIGHD Amsterdam Public Health Research Institute, Amsterdam, Netherlands; 3:Sydney Institute for Infectious Diseases, The University of Sydney, Sydney, NSW, Australia.; 4:School of Pharmacy, The University of Sydney Faculty of Medicine and Health, Sydney, NSW, Australia.; 5:Westmead Hospital, Sydney, NSW, Australia.; 6:Department of Pulmonary Diseases and Tuberculosis, University Medical Center Groningen, University of Groningen, Haren, The Netherlands, Tuberculosis Center Beatrixoord, University Medical Center Groningen, University of Groningen, Haren, The Netherlands; 7:Department of Health Sciences, Vrije Universiteit Amsterdam; 8:TB-NET; 9:Phthisiopneumology Institute, Chi?inau, Republic of Moldova; 10:Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

This study aimed to investigate the adaptation  of multi-drug resistant Mycobacterium tuberculosis (MDR-TB) during treatment.

Multiple M. tuberculosis DNA samples were collected from 54 patients at diagnosis and between 7 days and two months of MDR-TB treatment, shortly after the publication of the 2019 WHO consolidated guidelines on drug-resistant tuberculosis treatment in two sites.

Whole Genome Sequencing (WGS) data from 120 isolates was obtained and screened for unfixed single nucleotide polymorphisms (SNPs / mixed loci). Confidently mixed SNPs were identified in samples from multiple patients in only five genes (gyrA, pncA, Rv1129c [prpR], Rv1148c, and sugI), all other  genes with confidently mixed SNPs were identified in isolates from only a single patient. Three different mixed SNPs were identified in the sugI gene from follow up isolates from three different patients (sugI - P7A, P7T, and Q6stop).

Mutations in sugI have been previously reported in spontaneous in vitro d-cycloserine resistant mutants. Alterations in sugI may indicate a sub-optimal treatment regimen containing d-cycloserine and potentially be of clinical significance with respect to adaptation to d-cycloserine. The majority of patients (52 of 54 patients) received d-cycloserine as part of their initial treatment. D-cycloserine was used within a new bedaquiline containing regimen at one site but as mainly within of regimens not containing bedaquiline at the second site.

Monitoring the accumulation of low frequency escape mutants has the potential to identify weaknesses in regimens and identify key drugs at risk of resistance selection.

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