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In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible <jats:italic>M. tuberculosis<\/jats:italic> mother strain (S) and its <jats:italic>in vitro<\/jats:italic> selected resistant daughter strain harbouring the D94G mutation in <jats:italic>gyrA<\/jats:italic> (R) were mixed at different ratio\u2019s. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDR<jats:italic>sl<\/jats:italic> and GenoScholar-FQ\u2009+\u2009KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25\u2009mg\/L allows the phenotypic detection of \u22651% resistant bacilli, whereas at 0.5\u2009mg\/L\u2009\u2265\u20095% resistant bacilli are detected. Line probe assays detected \u22655% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected \u22641% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.<\/jats:p>","DOI":"10.1038\/s41598-019-48289-9","type":"journal-article","created":{"date-parts":[[2019,8,13]],"date-time":"2019-08-13T10:02:28Z","timestamp":1565690548000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":59,"title":["Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3301-9480","authenticated-orcid":false,"given":"L.","family":"Rigouts","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4610-2427","authenticated-orcid":false,"given":"P.","family":"Miotto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Schats","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P.","family":"Lempens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9727-6465","authenticated-orcid":false,"given":"A. 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