Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance

For further information, please contact: Global Tuberculosis Programme World Health Organization 20 Avenue Appia CH-1211 Geneva 27 Switzerland Web site: https://www.who.int/teams/ global-tuberculosis-programme/overview Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance Second edition

Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance Second edition Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, second edition ISBN 978-92-4-008241-0 (electronic version) ISBN 978-92-4-008242-7 (print version) © World Health Organization 2023 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. 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Design by Inis Communication Contents Acknowledgements v Abbreviations and acronyms vii 1 Introduction 1 2 Overview of the second edition of the catalogue 5 2.1 Understanding and using the catalogue 5 2.2 Main changes from the first edition of the catalogue 7 2.3 Overview of clinical sample sources and types of pDST data 9 2.4 Performance of mutations in the catalogue for predicting phenotypic resistance 12 3 Results for individual drugs 15 3.1 Instructions for reading the mutation tables 15 3.2 Rifampicin and rifapentine 19 3.3 Isoniazid 26 3.4 Ethambutol 33 3.5 Pyrazinamide 36 3.6 Levofloxacin and moxifloxacin 48 3.7 Bedaquiline and clofazimine 52 3.8 Linezolid 60 3.9 Delamanid and pretomanid 62 3.10 Amikacin 64 3.11 Streptomycin 69 3.12 Ethionamide and prothionamide 72 iii iv Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 4 Future research priorities 77 Types of data to be analysed 77 Grading criteria 78 Bioinformatics pipeline 78 5 Methods 81 5.1 Overview 81 5.2 Revised computational architecture 82 5.3 Data sources 83 5.4 Curation of pDST data 83 5.5 Prioritization of pDST results 85 5.6 Variant analysis 87 5.7 Association studies 92 5.8 Confidence grading 97 Criteria for initial confidence grading 100 Additional grading rules applied for final confidence grading 101 6 Data contributors 103 References 109 Annex 1. Further information 121 Annex 2. Conflict of interest assessment 127 vAcknowledgements Development of this document was led by Nazir Ahmed Ismail, Carl-Michael Nathanson and Alexei Korobitsyn, with support from Matteo Zignol and under the overall direction of Tereza Kasaeva, Director of the WHO Global TB Programme. The document is based on systematic analysis of a large WHO database of whole-genome sequencing and phenotypic drug-susceptibility testing data from analysis of clinical isolates of Mycobacterium tuberculosis complex collected from global collaborators. The primary team responsible for data curation, analysis, developing and running the algorithms for the catalogue and preparing the report were: Timothy Rodwell, FIND, Geneva, Switzerland, and University of California, San Diego (CA), United States of America (USA); Paolo Miotto, San Raffaele Scientific Institute, Milan, Italy; Claudio Köser, University of Cambridge, United Kingdom of Great Britain and Northern Ireland (United Kingdom); Timothy Walker, University of Oxford, United Kingdom; Leonid Chindelevitch, Imperial College, London, United Kingdom; Sacha Laurent, FIND, Geneva, Switzerland; and Maha Farhat, Harvard Medical School, Boston (MA), USA. Further support was provided by: Daniela M. Cirillo, San Raffaele Scientific Institute, Milan, Italy; Iñaki Comas, Biomedicine Institute of Valencia, Spain; Jamie Posey, Centers for Disease Control and Prevention, Atlanta (GA), USA; Shaheed V. Omar, National Institute for Communicable Diseases, Johannesburg, South Africa; and Leen Rigouts, Institute of Tropical Medicine, Antwerp, Belgium. Reviewing and editing of drafts was provided by Anita Suresh, Swapna Uplekar, Rebecca Colman and Sophia Georghiou, FIND, Geneva, Switzerland. This work was funded by grants from Unitaid and the US Agency for International Development. WHO thanks the following people for contributing to selection experiments, reviewing the results them or providing feedback on resistance genes: Emmanuel André, KU Leuven, Belgium; Sönke Andres, Research Center Borstel, Germany; Richard Anthony, National Institute for Public Health and the Environment, Bilthoven, Netherlands (Kingdom of the); Ivan Barilar, Research Center Borstel, Germany; Patrick Beckert, Research Center Borstel, Germany; Chen-Yi Cheung, University of Otago, Dunedin, New Zealand; Daniela M. Cirillo, San Raffaele Scientific Institute, Milan, Italy; Gregory Cook, University of Otago, Dunedin, New Zealand; Teresa Cortes, Biomedicine Institute of Valencia, Spain; Federico Di Marco, San Raffaele Scientific Institute, Milan, Italy; Sebastien Gagneux, Swiss Tropical and Public Health Institute, Allschwil, Switzerland; Qian Gao, Fudan University, Shanghai, China; Doris Hillemann, Research Center Borstel, Germany; Hairong Huang, Beijing Chest Hospital, China; Nabila Ismail, South African Medical Research Council Centre for Tuberculosis Research, Stellenbosch University, Cape Town, South Africa; Jongseok Lee, International Tuberculosis Research Center, Changwon, Republic of Korea; Jie Lu, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, China; Marisa Klopper, South African Medical Research Council Centre for Tuberculosis Research, Stellenbosch University, Cape Town, South Africa; Matthew McNeil, vi Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition University of Otago, Dunedin, New Zealand; Matthias Merker, Research Center Borstel, Germany; Satoshi Mitarai, Research Institute of Tuberculosis, Kiyose, Japan; Stefan Niemann, Research Center Borstel, Germany; Shaheed V. Omar, National Institute for Communicable Diseases, Johannesburg, South Africa; Christian Otum, South African Medical Research Council Centre for Tuberculosis Research, Stellenbosch University, Cape Town, South Africa; Rui Pi, Fudan University, Shanghai, China; Paola M.V. Rancoita, Vita-Salute San Raffaele University, Milan, Italy; Leen Rigouts, Institute of Tropical Medicine, Antwerp, Belgium; Emmanuel Rivière, University of Antwerp, Belgium; Camilla Rodrigues, P.D. Hinduja Hospital and Medical Research Centre, Mumbai, India; Max Salfinger, University of South Florida, Tampa (FL), USA; Jin Shi, Beijing Chest Hospital, Capital Medical University, China; Jihad Snobre, Institute of Tropical Medicine, Antwerp, Belgium; Lindsay Sonnenkalb, Research Center Borstel, Germany; Philip Supply, Institut Pasteur de Lille, France; Akiko Takaki, Research Institute of Tuberculosis, Kiyose, Japan; Annelies Van Rie, University of Antwerp, Belgium; Natalie Waller, University of Otago, Dunedin, New Zealand. WHO is grateful to the following people for further technical input: Heidi Albert, FIND, Cape Town, South Africa; Uladzimir Antonenka, IML red GmbH, Gauting, Germany; Arnold Bainomugisa, Queensland Department of Health, Brisbane, Australia; Francesc Coll, London School of Hygiene & Tropical Medicine, United Kingdom; Sarah Cook-Scalise, US Centers for Disease Control and Prevention, Atlanta (GA), USA; Chris Coulter, Queensland Department of Health, Brisbane, Australia; Alan Cristoffels, University of the Western Cape, Bellville, South Africa; James Dawson, US Centers for Disease Control and Prevention, Atlanta (GA), USA; Anna Dean, WHO, Geneva, Switzerland; Dennis Falzon, WHO, Geneva, Switzerland; Philip Fowler, University of Oxford, United Kingdom; Patricia Hall, US Centers for Disease Control and Prevention, Atlanta (GA), USA; Zahra Hasan, The Aga Khan University, Karachi, Pakistan; Harald Hoffmann, Institute of Microbiology and Laboratory Medicine, Gauting, Germany; Zamin Iqbal, European Bioinformatics Institute, Hinxton, United Kingdom; Moses Joloba, National Reference Laboratory of the National TB and Leprosy Programme, Kampala, Uganda; George Kasule, National Reference Laboratory of the National TB and Leprosy Programme, Kampala, Uganda; Alexei Korobitsyn, WHO, Geneva, Switzerland; Sanjana Kulkarni, Harvard Medical School, Boston (MA), USA; Marguerite Massinga Loembé, African Society for Laboratory Medicine, Libreville, Gabon; Alberto Mendoza, Ministry of Health, Lima, Peru; Matthias Merker, Research Center Borstel, Germany; Cecily Miller, WHO, Geneva, Switzerland; Satoshi Mitarai, Research Institute of Tuberculosis, Tokyo, Japan; Stefan Niemann, Research Center Borstel, Germany; Amy Piatek, US Agency for International Development, Washington DC, USA; Camilla Rodrigues, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; Samuel Schumacher, WHO, Geneva, Switzerland; Wayne van Gemert, Stop TB Partnership, Geneva Switzerland; Zhao Yanlin, National Tuberculosis Reference Laboratory, China Center for Disease Control and Prevention, Beijing, China; Matteo Zignol, WHO, Geneva, Switzerland; Danila Zimenkov, Engelhardt Institute of Molecular Biology, Moscow, Russian Federation. All individuals who provided technical input were required to disclose any potential conflicts of interest, encompassing both financial and non-financial interests. A “significant” conflict of interest included: “intellectual bias”, involvement in research or publication of materials related to issues under review; and a financial interest above US$ 5000. Upon review no significant conflict of interest were identified. vii Abbreviations and acronyms Additional abbreviations and acronyms used are listed in Tables 4–5. 7H10 Middlebrook 7H10 7H11 Middlebrook 7H11 ALL data set data set with all acceptable phenotypic drug-susceptibility testing results AMK amikacin Assoc w R associated with resistance Assoc w R–interim associated with resistance–interim aR algorithmically resistant aS algorithmically susceptible aU algorithmically uncertain BDQ bedaquiline BMD broth microdilution bp base pairs CAP capreomycin CC critical concentration CFZ clofazimine CI exact binomial confidence interval DLM delamanid DST drug-susceptibility testing gDST genotypic drug-susceptibility testing pDST phenotypic drug-susceptibility testing EMB ethambutol ETO ethionamide FE-sig significant with Fisher exact test FQ fluoroquinolone HGVS Human Genome Variation Society indel insertion/deletion INH isoniazid KAN kanamycin lb lower bound viii Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition LFX levofloxacin LJ Löwenstein-Jensen LoF loss-of-function LZD linezolid MGIT BACTEC™ Mycobacterial Growth Indicator Tube™ 960 MIC minimum inhibitory concentration MODS microscopic observation drug-susceptibility MTBC Mycobacterium tuberculosis complex MFX moxifloxacin Not assoc w R not associated with resistance Not assoc w R–interim not associated with resistance–interim OFX ofloxacin OR odds ratio pDST phenotypic drug susceptibility testing PMD pretomanid PPV positive predictive value PTO prothionamide PZA pyrazinamide R resistant or resistance RIF rifampicin RPT rifapentine RRDR rifampicin resistance-determining region S susceptible or susceptibility SOLO lone STM streptomycin TB tuberculosis ub upper bound WGS whole-genome sequencing WHO data set data set with WHO-endorsed phenotypic drug-susceptibility testing results 11 Introduction A total of 1.6 million people died of tuberculosis (TB) in 2021, and approximately 10.6 million people developed active TB disease due to Mycobacterium tuberculosis complex (MTBC). Of the 10.6 million new cases, an estimated 450 000 had TB resistant to rifampicin (RIF), which requires rapid, accurate detection and characterization to initiate appropriate treatments (1). Detection of RIF resistance has improved significantly with the introduction of rapid molecular diagnostic tools that require less complex infrastructure and are simpler to perform than conventional phenotypic methods (2). In 2012, globally, only 7% of individuals with bacteriologically confirmed TB were tested for RIF resistance (3); by 2021, that proportion was 71% (1). Over the same period, the number of individuals started on treatment for multi-drug- or RIF-resistant TB more than doubled, from 77 321 to 161 746, highlighting the central role of diagnostics in the TB response (1,3). The molecular basis of RIF resistance in MTBC isolates is almost exclusively mutations in the RIF resistance-determining region (RRDR), an 81-base-pair fragment of the rpoB gene (4). This knowledge and development of molecular tools that target RRDR have been critical to the delivery of new diagnostic solutions in the past decade (5). WHO recommends routine testing of all TB patients for resistance to RIF and isoniazid (INH), while resistance to the fluoroquinolones (FQs) is tested only when isolates are known to be RIF- or INH-resistant TB (5). MTBC resistance mechanisms to INH and FQs are well understood, and molecular tools are commercially available for detecting mutations associated with phenotypic resistance to these drugs at a WHO-endorsed critical concentration (CC) (6). After a long period of stagnation in innovation for TB treatment, the introduction of new drugs and repurposing of existing antimicrobial agents for the treatment of TB have significantly increased the potential for improved TB treatment. As resistance to new and repurposed drugs gradually increases in the community, however, concern has been raised about the lack of options for rapid detection of resistance to these new and repurposed drugs (7–9). The recent WHO recommendation of several targeted next-generation sequencing assays for culture-free diagnosis of drug-resistant TB represents an important step towards improving the diagnostic landscape (10). These assays provide sequences directly from sputum and reveal much more of the resistance-associated MTBC genome than traditional genotypic drug-susceptibility testing (gDST) assays (2). They have been recommended for detecting resistance to up to 10 antibiotics simultaneously directly from clinical samples, but their sensitivity for predicting resistance to some drugs remains limited by incomplete understanding of the molecular basis of resistance (10). While allelic exchange experiments are the reference standard for demonstrating that a specific mutation is both necessary and sufficient to confer phenotypic resistance, these approaches are expensive, slow and technically demanding (11). Therefore, association studies based on Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 2 whole-genome sequencing (WGS) and standardized phenotypic drug-susceptibility testing (pDST) data from large numbers of globally diverse MTBC isolates are indispensable for comprehensive investigations of the genetic basis of resistance, particularly in non-essential genes, where hundreds of loss-of-function (LoF) mutations can result in phenotypic resistance at a clinically relevant WHO CC (12,13). A major obstacle to the development and diagnostic utility of sequence-based technologies and next-generation molecular diagnostics for gDST has been the lack of a standardized, comprehensive catalogue of mutations and their association with phenotypic drug resistance. In 2021, WHO published the first mutation catalogue, consisting of a high-quality, comprehensive list of confidence-graded MTBC genetic markers of phenotypic resistance (14). The aim was to provide a resource that could be used to distinguish clinically relevant resistant variants (i.e. variants statistically associated with a resistant pDST result at a WHO CC) from those not associated with resistance and from those for which there are insufficient data to establish a meaningful association. While that catalogue helped to reduce continuing technical uncertainty about the number, identity and clinical interpretation of genomic resistance-determining regions for legacy drugs, data on graded mutations associated with new and repurposed drugs was very limited and so too was the representation of some geographical regions (14). The primary reason for revising the Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, published in 2021 (the first edition (14)) was to add sufficient new data on clinical MTBC isolates from global sources to the WHO database of genotypes and phenotypes to identify new genomic variants associated with phenotypic resistance to the new and repurposed TB therapeutics, while also improving detection of variants associated with resistance to all TB therapeutics and improving the geographical representation of data in the catalogue. WHO held an expert consultation on 28 February, 1 March and 9 March 2023. All individuals who provided technical input were required to disclose any potential conflicts of interest, encompassing both financial and non-financial interests. Upon review no significant conflict of interests were identified. Participants’ statements were summarized by the WHO at the start of the meeting and can be found in Annex 1. For the analysis presented here, WGS and pDST data on the largest collection of multinational MTBC isolates to date (~52 000, up from ~38 000 analysed in 2021) were assembled to produce the Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, second edition. The second edition is intended to be a common, standardized reference for interpreting resistance to all first-line drugs (RIF, INH, ethambutol [EMB] and pyrazinamide [PZA]) and also to second-line drugs in group A (levofloxacin [LFX], moxifloxacin [MFX], bedaquiline [BDQ] and linezolid [LZD]), group B (clofazimine [CFZ]) and group C (delamanid [DLM], amikacin [AMK], streptomycin [STM], ethionamide [ETO] and prothionamide [PTO]). While kanamycin (KAN) and capreomycin (CAP) are no longer recommended for MDR-TB treatment by WHO, they are included in this analysis for historical context and because mutations associated with KAN resistance provide useful insights for interpreting some mutations that confer resistance to AMK (15,16). Although WHO has not yet set a CC for pretomanid (PMD), this second edition provides some early guidance for interpretation of LoF mutations in the six genes required for activation 1 Introduction 3 of this prodrug (17,18). Guidance is also provided for rifapentine (RPT) (4). This report on the second edition describes the revisions to the methods used to create the catalogue, the mutations identified and summaries of key findings for each drug. New areas for future research are also outlined. The report is intended to inform the development of new and improved molecular assays, based on sequencing or other methods, for comprehensive detection of resistance to TB drugs.

52 Overview of the second edition of the catalogue 2.1 Understanding and using the catalogue The second edition consists of three related elements: • a list of graded variants (the catalogue master file and a file with the “genomic coordinates in VCF”); • evidence-based “additional grading rules”, of which some are general and others drug-specific (Table 1); and • evidence-based “other interpretation criteria” (Table 1). These related elements can be used to interpret MTBC mutations detected with NGS or other gDST methods using the workflow demonstrated below (Fig. 1). Fig. 1. Instructions for using the catalogue Is the individual mutation graded in the catalogue? Group 2: Assoc w R–interim Group 1: Assoc w R Marker of resistance Group 3: Uncertain signifi cance Uncertain signifi cance Group 4: Not assoc w R–interim Group 5: Not assoc w R Not marker of resistance Assign Group Interpretationb Is the mutation a silent varianta? Yes Yes Yes No No No Does a drug-specifi c “additional grading rule” apply? a General “additional grading rule” (not shown in Table 1). b “Other interpretation criteria”, such as the level of resistance, must be considered for some Groups 1/2 variants (Table 1). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 6 Table 1. Summary of “additional grading rules” and “other interpretation criteria” used in the second edition Drug Drug-specific additional grading rulesa Other interpretation criteria for Groups 1 and 2 mutations RIF & RPT Non-silent variants in RRDR of rpoBb • Groups 1–5 classifications for RIF also apply to RPT. INH LoF in katG • katG mutations assumed to confer high-level INH resistance. • fabG1-inhA mutations assumed to confer low-level INH resistance. • Multiple, genetically linked low-level resistance mutations have additive effects and should be considered to confer high-level INH resistance. • fabG1-inhA mutations confer cross-resistance to ETO and INH. PZA LoF in pncA • If isolate identified as M. canettii, infer intrinsic PZA resistance. LFX & MFX • gyrA and gyrB mutations confer cross-resistance to LFX and MFX, but the level of LFX resistance is not stratified. • gyrA Gly88Cys, Asp94Asn, Asp94Gly, Asp94His and Asp94Tyr mutations assumed to confer high-level MFX resistance. • Remaining gyrA and gyrB mutations assumed to confer low-level MFX resistance. • Multiple, genetically linked low-level MFX resistance mutations have additive effects and should be considered to confer high-level MFX resistance. BDQ & CFZ LoF in Rv0678 and pepQ • Rv0678 and pepQ mutations confer cross-resistance to BDQ and CFZ. • Rv0678 mutations cannot confer resistance if genetically linked with LoF variants in mmpL5 (epistasis). DLM & PMD LoF in ddn, fbiA, fbiB, fbiC, fgd1, and Rv2983 AMK & KANc • eis promoter mutations cannot confer resistance if genetically linked with LoF variants in eis coding region (epistasis). STM LoF in gid ETO & PTO LoF in ethA • Groups 1–5 classifications for ETO also apply to PTO. • fabG1-inhA mutations confer cross-resistance to ETO and INH. CAPc LoF in tlyA For simplicity, the general “additional grading rule” whereby any novel silent variant is classified in Group 4 (Fig. 1) was not included in this table but applies to all genes for all drugs. a When these drug-specific rules apply, the mutation in question is classified in Group 2 (Fig. 1). b Changes at position 1346 in codon 449 of rpoB detected with Illumina sequencing, particularly if unfixed, may represent artefacts. c No longer recommended for TB treatment. Variants were graded if they occurred at an allele frequency of at least 75% in at least one isolate and if the pDST result was valid (see Section 5 for details). Specifically, these variants were stratified into one of five groups according to the amount and quality of evidence available to support the association statistically. • Group 1: Associated with resistance (Assoc w R) • Group 2: Associated with resistance–interim (Assoc w R–interim) • Group 3: Uncertain significance • Group 4: Not associated with resistance–interim (Not assoc w R–interim) • Group 5: Not associated with resistance (Not assoc w R) Groups 1 and 2 variants should be interpreted as markers of clinically relevant phenotypic resistance (i.e. mutations associated with phenotypic resistance at a WHO-endorsed CC), whereas Groups 2 Overview of the second edition of the catalogue 7 4 and 5 variants are not markers of resistance (Fig. 1). The role of Group 3 mutations remains uncertain from the available evidence. Although the grading process for the second edition remained largely unchanged from that for the first edition, some new methods were introduced (Section 2.2), and considerably more isolates were analysed (Section 2.3). The performance of the resulting mutations listed as predictors of phenotypic resistance is summarized in Section 2.4. Fig. 1 and Table 1 indicate how the three elements of the catalogue can be used to grade variants that were previously graded and those that were not. Some examples are given below to demonstrate the logic of the workflow: • katG Ser315Thr and Ser315Ile mutations are graded as Group 1 and Group 2 mutations, respectively, and would, therefore, be interpreted as markers of INH resistance. In accordance with the “other interpretation criteria” (Table 1), they would be reported as conferring high- level INH resistance. • The katG Gly33fs mutation is not listed in the catalogue but would be classified in Group 2 according to the katG LoF “additional grading rule” (Table 23) and considered to confer high- level INH resistance according to the “other interpretation criteria”. • The G>A mutation at position of 1350 of rpoB, resulting in a silent change at codon 450, is not included in the catalogue, but would be classified in Group 4 according to the general “additional grading rule” for RIF and RPT. • The ddn Trp20* is in Group 2 for DLM and would also be reported as being in Group 2 for PMD by the LoF “additional grading rule” (Table 23). • A sample with gyrA Ala90Val and Asp94Ala mutations would be reported as resistant to LFX. In contrast, the level of MFX resistance is stratified as low and high. Both variants are low-level resistance mutations. If they are not genetically linked (not phased according to the sequencing reads), or it is not clear whether they are genetically linked, they would be reported as “at least low-level resistant to MFX” (i.e. the sample is definitely low-level resistant, but high-level resistance due to another low-frequency mutation cannot be excluded, particularly at low sequencing coverage (19)). If the two mutations are genetically linked, their effects would be additive, and the sample would be reported as high-level resistant. 2.2 Main changes from the first edition of the catalogue A detailed description of the methods used to create the second edition is provided in Section 5. The main improvements over the first edition are as follows. • Data: – increased number and geographical diversity of MTBC isolates, including increased absolute number and prevalence of resistant isolates for most of the drugs considered (see Section 2.3); and – improved curation and prioritization of pDST results to increase the diversity of testing methods that could be included in analyses (see Sections 5.3 and 5.4). • Bioinformatics pipeline: – improved pipeline for more appropriate handling of null calls and large deletions (20) (see Section 5.6); and Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 8 – variants classified if they occurred at an allele frequency of ≥ 75% (previously ≥ 90%), with additional estimates of the impact of lowering the threshold of calling variants to ≥ 25% (see Table A.1 in the Annex). • Association studies: – updated the list of target genes associated with resistance to all the drugs considered (see Table 21, p. 89) and revised the corresponding upstream regions (see Table 22 , p. 89); – revised the algorithm for Group 4/5 to reduce the risk of misclassifying mutations as not relevant for resistance and, consequently, of very major errors (Sections 5.7 and 5.8); – revised definition of LoF mutations (excluding inframe insertion/deletion [indel] mutations) to reduce the risk of overestimating resistance (Sections 3.4 and 5.6); – translated the Stata association algorithms into R in order to validate the association workflow code independently (quality control) and improve public access to the code (Section 5.2); – included new grading criteria: added new cross-resistance “additional grading rules” and added new evidence from laboratory mutation selection studies to improve genotype– phenotype associations (Sections 5.7 and 5.8). Notably, the catalogue now contains some mutations that confer resistance to BDQ and LZD that were identified only in laboratory- based selection experiments from at least two independent sites. These were clearly labelled as not occurring in our collection of clinical isolate data and were classified into Group 2 to reflect this; and – assessed the impact of epistasis for BDQ/CFZ and AMK/KAN (Sections 3.7 and 3.10). A summary of the changes in the total number of variants by group and the justifications for those changes are provided in Fig. 2. A total of 226 mutations (green) were classified with higher certainty in the second edition; while group changes to 38 mutations (yellow) did not alter the final interpretation; and 22 mutations (red) were moved to Group 3 from other more certain groups. As relevant, these changes are discussed in more detail for each drug in Section 3. 2 Overview of the second edition of the catalogue 9 Fig. 2. Changes (with explanations) in the classification of variants in the second edition as compared with the first edition Group 1: Assoc w R Group 2: Assoc w R–interim Group 3: Uncertain significance Group 4: Not assoc w R–interim Group 5: Not assoc w R N/A Group 1 167 53 18 8 Group 2 9 880 107 126 Group 3 4 7 (98)a 12581 3 8 21178 Group 4 (1)a 7 20 29 1 (12358)b Group 5 36 5 136 182 (126)b Previous catalogue Cu rre nt ca ta lo gu e • Association now supported by WHO-endorsed assays • Association now supported by ALL dataset only • Association now supported by “relaxed” pncA thresholds • Association from WHO dataset • Algorithm pass 2 association no longer supported by data • Inframe now excluded from LoF rule • Association no longer supported by data • Association no longer supported by “relaxed” pncA thresholds • Association no longer supported by “relaxed” pncA thresholds • Due to the switch from PPV|SOLO to PPV SOLO in neutral algorithm • Now Group 4 by literature/ previous evidence • Association no longer supported by data First edition Se co nd ed iti on Yellow boxes denote number of variants downgraded within category (e.g. from Group 1 to Group 2); red boxes denote a downgrade across categories (e.g. from Group 1/2 to Group 3/4). a Number of inframe mutations reported in brackets. b Number of silent mutations reported in brackets. 2.3 Overview of clinical sample sources and types of pDST data A total of 556 918 individual pDST results were collated for 64 622 MTBC isolates. Matching WGS data were available for 61 986 isolates. After prioritization and curation of the individual pDST data and quality control of processed sequencing data, data on 9 419 isolates were dropped from further consideration, leaving a total of 52 567 isolates to be used in the analyses for the second edition (up from ~38 000 for the first edition (14)). Sixty-seven countries contributed data for ≥ 5 MTBC isolates, 54 countries for ≥ 50 isolates, 21 for ≥ 500 isolates, and India and the United Kingdom contributed data for ≥ 5000 isolates (Fig. 3). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 10 Fig. 3. Global origin and regional numbers of MTBC isolates included in the first edition (panel A) and the second edition (panel B) A) B) 50-499 >5000 0-4 5-49 500-4999 No data Not applicable 50-499 >5000 No data Not applicable 0-4 5-49 500-4999 The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of WHO concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement. 2 Overview of the second edition of the catalogue 11 Approximately 52 000 isolates were considered for the second edition (Table 2). The number of isolates for which pDST results were available varied widely by drug, with as few as 11 803 isolates with pDST results for DLM in the WHO data set, which comprises pDST results obtained according to current or previous WHO-endorsed methods, to as many as 48 706 for INH in the ALL data set, which consists of the WHO data set plus other methods that are not WHO-endorsed but were deemed acceptable for inclusion (Sections 5.4 and 5.5). Of most significance to the analysis of genotypic–phenotypic associations for new and repurposed drugs, the second edition includes > 5800 new isolates with BDQ pDST and almost 7000 new isolates with LZD pDST in the ALL data set. There were substantial increases in the proportions of resistant isolates to every drug in the catalogue, except for ETO, for which more susceptible than resistant isolates with WHO phenotypes were included in the second edition, reducing the overall prevalence of resistance to ETO. Table 2. Summary of pDST results included in the first and second edition, stratified by drug and data set Drug Dataset First edition Second edition Variation Total % R (95% CI) Total % R (95% CI) Change % R RIF WHO 27063 24.9 (24.4–25.4) 35401 32.6 (32.1–33.1) 31 ALL 34375 28.7 (28.2–29.2) 47730 35.3 (34.9–35.8) 23 INH WHO 26727 31.6 (31.0–32.1) 34881 38.5 (38.0–39.0) 22 ALL 34437 35.4 (34.9–35.9) 48706 43.0 (42.6–43.5) 21 EMB WHO 23706 15.2 (14.8–15.7) 33240 19.8 (19.4–20.3) 30 ALL 30708 16.0 (15.5–16.4) 45515 21.0 (20.6–21.3) 31 PZA WHO 15903 14.6 (14.1–15.2) 19889 19.1 (18.6–19.7) 30 ALL 15902 14.6 (14.1–15.2) 21319 20.8 (20.2–21.3) 42 LFX WHO 10305 19.6 (18.8–20.4) 12441 22.0 (21.3–22.7) 12 ALL 18277 17.0 (16.5–17.6) 27576 21.3 (20.8–21.8) 25 MFX WHO 6904 15.8 (15.0–16.7) 8439 20.8 (19.9–21.7) 31 ALL 13351 14.0 (13.4–14.6) 22783 17.7 (17.2–18.2) 26 BDQ WHO 88 3.4 (0.7–9.6) 2165 41.7 (39.6–43.7) 1122 ALL 8321 0.9 (0.7–1.1) 14135 7.3 (6.9–7.8) 736 LZD WHO 1131 0.8 (0.4–1.5) 6825 2.0 (1.7–2.3) 152 ALL 11018 1.1 (0.9–1.3) 18010 2.1 (1.9–2.3) 86 CFZ WHO 3635 0.6 (0.4–0.9) 5027 4.3 (3.7–4.8) 576 ALL 10179 1.2 (1.0–1.5) 14904 4.5 (4.2–4.9) 270 DLM WHO 89 2.2 (0.3–7.9) 575 9.4 (7–11.8) 318 ALL 7778 1.1 (0.8–1.3) 11803 2.1 (1.9–2.4) 103 AMK WHO 8040 8.3 (7.7–8.9) 8958 12.5 (11.9–13.2) 52 ALL 16978 7.6 (7.2–8.0) 24710 10.0 (9.7–10.4) 32 STM WHO 9043 28.3 (27.4–29.3) 19747 39.3 (38.7–40.0) 39 ALL 13984 33.1 (32.4–33.9) 26166 39.8 (39.2–40.4) 20 ETO WHO 2184 40.5 (38.4–42.6) 5999 36.4 (35.2–37.6) -10 ALL 13918 21.3 (20.6–22.0) 20936 25.0 (24.4–25.6) 18 KANa WHO 7381 9.3 (8.7–10.0) 8014 20.1 (19.3–21.0) 116 ALL 16154 9.2 (8.7–9.6) 24582 14.5 (14.1–15.0) 58 CAPa WHO 9103 7.7 (7.2–8.3) 10025 13.1 (12.5–13.8) 70 ALL 11526 8.4 (7.9–8.9) 17716 11.7 (11.2–12.1) 39 a No longer recommended for TB treatment. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 12 2.4 Performance of mutations in the catalogue for predicting phenotypic resistance One of the simplest ways to determine whether the mutation catalogue has captured most of the important genetic predictors of phenotypic resistance to a specific drug is to calculate the sensitivity, specificity and positive predictive value (PPV) of all of the Groups 1 and 2 mutations listed in the catalogue relative to the total number of phenotypically resistant isolates in the catalogue (Table 3). For the reasons given below, this is an oversimplification and the results should not be considered to represent the theoretical maximum sensitivity, specificity and PPV that could be achieved in clinical settings, where the prevalence of resistance depends on the location. • Although we prioritized pDST results, the phenotypic reference standard was imperfect for some isolates. For example, some WHO-endorsed CCs had been set too high, and the CCs for interpreting broth microdilution (BMD) minimum inhibitory concentrations (MICs) have not been reviewed by WHO (16,21–25). Moreover, use of data collected from many different laboratories is inherently more prone to error than use of data from smaller, well-controlled multi-centre studies specifically designed to assess the performance of gDST, and the specificity would probably be higher under the latter circumstances for at least some drugs. • As we had no independent data set against which to validate results, we calculated the performance of the catalogue by making predictions for the same data set from which it was derived; consequently, the data may have been overfitted (26,27). Independent validation against different data sets will still be necessary to better understand the performance of mutations that occur in both WHO and outside data sets; however, validating rare mutations will remain a challenge. • Owing to transmission of a limited number of clones globally, particularly for drug-resistant TB, the prevalence of any specific mutation will impact sensitivity estimates. Clones with unclassified mutations that are globally rare but locally frequent can result in much lower sensitivity of the catalogue estimates in some settings. In contrast, in environments where transmission of clones with classified mutations is common, the sensitivity would be higher. Understanding local epidemiology is therefore important for using this catalogue and any WHO-endorsed gDST assays (28–31). The pre-test probability of resistance to second-line drugs is one of the factors to be considered in this context. For the reasons outlined above, we explored several alternative analytical scenarios with different assumptions to determine how sensitivity and specificity estimates are affected in our data set. Tables A.1–A.3 in the Annex show the results of those analyses. A more detailed discussion of the performance of the catalogue mutations for each drug is provided in Section 3. Table 3 provides an overview of Groups 1–5 mutations in the second edition and their performance in predicting phenotypic resistance. 2 O verview of the second ed ition of the catalogue 13 Table 3. Groups 1–5 mutations for the second edition and their performance for predicting phenotypic resistance Group 1: Assoc w R Group 2: Assoc w R–interim Group 3: Uncertain significance Group 4: Not assoc w R–interim Group 5: Not assoc w R RIF No. of variants identified 26 110 4484 2 (2568) 52 (32) Sens, spec, PPV (% [95% CI]) 92.1 (91.7–92.5), 97.1 (96.9–97.3), 94.5 (94.2–94.9) 1.1 (1.0–1.3), 99.8 (99.8–99.9), 76.5 (70.8–81.6) Combined performance INH No. of variants identified 7 135 5404 11 (1671) 41 (16) Sens, spec, PPV (% [95% CI]) 89.6 (89.2–90.0), 98.2 (98.1–98.4), 97.5 (97.2–97.7) 2.0 (1.8–2.2), 99.7 (99.6–99.7), 82.5 (78.9–85.7) Combined performance EMB No. of variants identified 13 0 4943 10 (2068) 50 (34) Sens, spec, PPV (% [95% CI]) 81.1 (80.3–81.9), 91.6 (91.3–91.9), 71.9 (71.0–72.8) 0 (0–0), 100.0 (100.0–100.0), 0 (0–0) Combined performance PZA No. of variants identified 139 202 1465 20 (720) 17 (8) Sens, spec, PPV (% [95% CI]) 63.5 (62.0–64.9), 98.6 (98.5–98.8), 92.4 (91.4–93.4) 14.6 (13.5–15.6), 99.2 (99.1–99.3), 82.9 (80.1–85.5) Combined performance LFX No. of variants identified 12 6 2016 2 (983) 19 (9) Sens, spec, PPV (% [95% CI]) 83.6 (82.6–84.5), 97.3 (97.0–97.5), 89.2 (88.4–90.0) 1.2 (1.0–1.6), 99.6 (99.6–99.7), 48.3 (40.1–56.6) Combined performance MFX No. of variants identified 10 8 1775 2 (904) 15 (9) Sens, spec, PPV (% [95% CI]) 84.8 (83.7–85.9), 94.0 (93.6–94.3), 75.2 (73.9–76.4) 0.9 (0.6–1.2), 99.5 (99.4–99.6), 29.5 (21.6–38.4) Combined performance BDQ No. of variants identified 5 81 947 5 (424) 1 Sens, spec, PPV (% [95% CI]) 26.3 (23.7–29.1), 99.4 (99.3–99.5), 78.2 (73.5–82.4) 23.0 (20.5–25.7), 99.3 (99.1–99.4), 72.0 (66.8–76.8) Combined performance LZD No. of variants identified 1 7 844 0 (71) 4 (1) Sens, spec, PPV (% [95% CI]) 27.3 (22.8–32.1), 99.8 (99.8–99.9), 78.5 (70.4–85.2) 6.7 (4.4–9.7), 100.0 (99.9–100.0), 78.1 (60.0–90.7) Combined performance 85.7 (84.6–86.8), 93.5 (93.2–93.9), 74.0 (72.7–75.2) 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 91.6 (91.2–92.0), 97.9 (97.8–98.1), 97.1 (96.8–97.3) 81.1 (80.3–81.9), 91.6 (91.3–91.9), 71.9 (71.0–72.8) 78.0 (76.8–79.2), 97.9 (97.6–98.1), 90.5 (89.5–91.4) 84.8 (83.9–85.7), 96.9 (96.7–97.1), 88.1 (87.3–89.0) 49.4 (46.3–52.5), 98.7 (98.5–98.9), 75.2 (71.8–78.4) 34.0 (29.2–39.0), 99.8 (99.7–99.9), 78.4 (71.3–84.5) Group 1: As oc w R -interi r : i i fi -i C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 14 Group 1: Assoc w R Group 2: Assoc w R–interim Group 3: Uncertain significance Group 4: Not assoc w R–interim Group 5: Not assoc w R CFZ No. of variants identified 2 56 1256 0 (576) 11 (1) Sens, spec, PPV (% [95% CI]) 4.3 (2.9–6.1), 99.8 (99.7–99.8), 46.0 (33.4–59.1) 12.7 (10.3–15.4), 98.9 (98.7–99.1), 36.0 (29.9–42.4) Combined performance DLM No. of variants identified 0 24 579 0 (334) 0 Sens, spec, PPV (% [95% CI]) 0 (0–1.5), 100.0 (100.0–100.0), 0 (0–0) 14.7 (10.6–19.7), 99.9 (99.8–99.9), 72.5 (58.3–84.1) Combined performance AMK No. of variants identified 2 2 1772 1 (343) 68 (2) Sens, spec, PPV (% [95% CI]) 68.9 (67.0–70.7), 99.2 (99.0–99.3), 90.1 (88.7–91.4) 4.0 (3.2–4.8), 99.2 (99.0–99.3), 34.3 (28.8–40.1) Combined performance STM No. of variants identified 14 144 2342 1 (538) 15 (6) Sens, spec, PPV (% [95% CI]) 72.1 (71.2–72.9), 97.6 (97.4–97.8), 95.2 (94.7–95.7) 7.6 (7.1–8.1), 96.5 (96.2–96.8), 58.8 (56.1–61.4) Combined performance ETO No. of variants identified 5 281 1944 0 (515) 2 Sens, spec, PPV (% [95% CI]) 45.8 (44.4–47.1), 94.0 (93.6–94.3), 71.7 (70.1–73.2) 29.1 (27.8–30.3), 91.9 (91.5–92.3), 54.6 (52.7–56.4) Combined performance KANa No. of variants identified 6 2 1862 3 (353) 12 (2) Sens, spec, PPV (% [95% CI]) 74.4 (73.0–75.9), 96.7 (96.5–97.0), 79.4 (78.0–80.8) 0.4 (0.3–0.7), 100.0 (99.9–100.0), 66.7 (44.7–84.4) Combined performance CAPa No. of variants identified 5 64 2273 1 (253) 52 (5) Sens, spec, PPV (% [95% CI]) 61.2 (59.1–63.3), 98.0 (97.8–98.2), 80.4 (78.4–82.3) 4.9 (4.0–6.0), 99.8 (99.7–99.9), 76.1 (68.0–83.1) Combined performance 74.8 (73.6–76.0), 85.9 (85.3–86.4), 63.9 (62.7–65.1) 74.9 (73.4–76.3), 96.7 (96.4–96.9), 79.3 (77.9–80.7) 66.2 (64.1–68.2), 97.8 (97.6–98.1), 80.1 (78.1–81.9) 17.0 (14.2–20.0), 98.7 (98.5–98.9), 38.1 (32.6–43.8) 14.7 (10.6–19.7), 99.9 (99.8–99.9), 72.5 (58.3–84.1) 72.8 (71.0–74.6), 98.3 (98.1–98.5), 82.8 (81.2–84.4) 79.7 (78.9–80.5), 94.1 (93.7–94.4), 89.9 (89.3–90.5) Group 1: As oc w R -interi r : i i fi -i CI, exact binomial confidence interval; sens, sensitivity; spec, specificity. Only majority variants (allele frequency ≥ 75%) are listed and used for calculations with the ALL data set. The number of silent mutations in each group is shown in parentheses. a Drugs no longer recommended for TB treatment. 15 3 Results for individual drugs 3.1 Instructions for reading the mutation tables As in the first edition, this section includes abridged mutation tables by drug. The searchable tables are available as supplementary material (WHO-UCN-TB-2023.5-eng.xlsx). The terms and abbreviations used in the mutation tables are listed in Tables 4–5. Below are the criteria that were used for grading mutations into different groups that relied on specific lone (SOLO) mutations associated with resistance (see Sections 5.7 and 5.8 for more details). The colour coding shown in parentheses was used in the tables to show whether the criteria for the initial confidence grading were met. Group 1: Assoc w R Mutations that met five criteria: 1. sum of resistant and susceptible isolates with the SOLO mutation (Present_SOLO_SR) ≥ 5 (red); 2. lower bound (lb) of 95% CI of PPV conditional on being SOLO (PPV|SOLO_lb) ≥ 25% (red); 3. odds ratio (OR) > 1, which always applies if criterion 4 is met (red); 4. OR SOLO > 1 (red); and 5. statistical significance of OR SOLO (OR SOLO_FE-sig) with Fisher exact false discovery rate- corrected (red). Criteria 4 and 5 are merged in the “OR SOLO” column of the simplified tables in this report and are shown in red if both criteria were met. Group 2: Assoc w R–interim Mutations that met “relaxed” criteria for pncA: 1. resistant isolates with the SOLO mutation (Present_SOLO_R) ≥ 2 (yellow) 2. PPV ≥ 50% (yellow) Group 3: Uncertain significance Mutations that did not meet the criteria for inclusion in Group 1, 2, 4 or 5 Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 16 Group 4: Not assoc with R– interim Silent mutations that do not fulfil the requirements for other groups Mutations that meet “relaxed” criteria for pncA: 1. PPV SOLO < 40% (blue) 2. upper bound (ub) of 95% CI of PPV SOLO (PPV_SOLO_ub) < 75% (blue) Group 5: Not assoc with R Neutral mutations that were masked before use of the algorithm Fig. 4. Example of an abridged variant classification table Dr ug Va ria nt MU T Pr es en t_ ph en o S MU T Ab se nt _p he no S MU T Pr es en t_ ph en o R MU T Ab se nt _p he no R SE NS IT IV IT Y SP EC IF IC IT Y PP V PP V SO LO PP V SO LO _u b PP V| SO LO _lb OR SO LO INITIAL CONFIDENCE GRADING SU PP OR TI NG DA TA SE T Additional grading criteria applied FINAL CONFIDENCE GRADING Fo ot no te s CH AN GE S vs ve r.1 RIF rpoB_p.Ser450Leu 226 30643 10859 6002 64.4% 99.3% 98.0% 97.9% 98.2% 97.1% 234.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Val 17 30515 1154 15656 6.9% 99.9% 98.5% 98.8% 99.4% 97.5% 162.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asp 10 30522 608 16202 3.6% 100.0% 98.4% 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Tyr 11 30521 593 16217 3.5% 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Tyr 93 30439 341 16469 2.0% 99.7% 78.6% 70.9% 76.5% 59.7% 4.5 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu452Pro 102 30430 281 16529 1.7% 99.7% 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Trp 5 30527 238 16572 1.4% 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Leu 20 30512 217 16593 1.3% 99.9% 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu430Pro 185 30347 215 16595 1.3% 99.4% 53.8% 25.5% 32.0% 17.1% 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ser450Phe 1 30531 206 16604 1.2% 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Dr ug Va ria nt MU T Pr es en t_ ph en o S MU T Ab se nt _p he no S MU T Pr es en t_ ph en o R MU T Ab se nt _p he no R S en si tiv ity S pe ci fic ity PP V PP V SO LO PP V SO LO _u b PP V| SO LO _lb OR S OL O Initial confidence grading S up po rt in g da ta se t Additional grading criteria applied Final confidence grading N o te s C ha ng es v s pr ev . v er . Ser 50 226 643 10859 6002 64.4 3 8 0 7 9 8 2 97 1 234.4 Asp 5Val 17 515 1154 5656 6 9 9 98 5 98 8 99 4 9 5 162.4 1) AwR His 45Asp 10 22 6 8 2 2 3 6 8 4 98.4 99.3 6 8 118.3 Tyr 11 1 593 217 3 5 8 2 6 4 6 0 128.2 Asp435Tyr 9 3 341 469 2 0 78 6 70 9 76 5 59 7 4 5 1) AwR L u452Pro 102 30 281 529 1 73 4 64 9 70 7 56 1 3 4 ALL+WHO I r .Ser45 Trp 5 27 238 572 1.4 . .9 .4 .1 94.0 68.9 ) ) I r . i Leu 20 12 217 593 1.3 99.9 91.6 .4 4. 85.8 .6 ) ) I r .Leu 0 r 185 347 215 595 1.3 99.4 53.8 25.5 32.0 17.1 0.6 3) Uncertain Borderline ) I r .Ser 50 1 31 206 604 1.2 . 9.5 100.0 100.0 9 .2 Inf ) ) ss c I rpo _p.His445Arg 3 30529 136 16674 0.8 100.0 97.8 98.1 99.8 92.1 96.1 1) LL 1) ssoc IF rpo _p.His445Asn 83 30449 126 16684 0.7 99.7 60.3 33.7 43.6 21.6 0.9 3) Uncertain LL+ Borderline 1) ssoc IF rpoB_p.Ile491Phe 88 30444 116 16694 0.7 99.7 56.9 55.5 63.0 44.7 2.3 1) A ALL Borderline 1) Assoc RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5 100.0 97.8 97.6 99.9 83.8 73.0 1) AwR ALL+ H 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4 100.0 89.7 91.5 96.8 79.5 19.8 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4 100.0 98.6 98.1 100.0 89.7 93.0 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Additional grading criteria applied when relevant to reach the Final confidence grading Mutatio named as described in Section 5.6 Final confidence grading of a mutation Drug in focus In the first xample in Fig. 4, the drug considered is RIF. The variant is in the rpoB gene, the amino acid change is at codon 445 (MTBC codon numbering), and th change is from serine to leucine (this corresponds to codon 526 in the previous Escherichia coli nomenclature (4,32)). This variant was found in 20 phenotypically susceptible isolates and in 217 resistant isolates. The mutation was not found in 30 51 susceptible isolates or 16 593 resistant isolates. The sensitivity, specificity and PPV represent the performance of this mutation in predicting a resistant phenotype in the data set. The next four columns indic te the statistical perf rmance of this mutation when it occurs as a SOLO mutation in the genomic regions selected when assessing RIF resistance. The values given are the mid-point PPV and the corresponding lb and ub and the odds ratio for the SOLO mutation (OR SOLO). The initial confidence grading for rpoB His445Leu was Group 1 because: • present_SOLO_SR (see catalogue master file) was 210 and, consequently, ≥ 5; • the PPV|SOLO_lb of 85.8% was ≥ 25%; and • OR SOLO of 19.6 was > 1 and statistically significant. As the initial confidence grading of the WHO and the ALL data sets was concordant for this mutation, the figures shown are for the ALL data set. Additional grading criteria were not applied to this mutation; therefore, the final confidence grading was unchanged. In contrast, the initial confidence grading for rpoB Leu430Pro was revised according to the additional grading rule 3 Results for individual drugs 17 related to borderline RIF resistance mutations, which are shown in purple (4, 33). More details can be found in the Section 5. Table 4. Terms used in the mutation tables Term used in the report Description ALL only information only from the ALL data set AwR associated with resistance AwRI associated with resistance–interim FQ X-R fluoroquinolone cross-resistance BDQ-CFZ X-R bedaquiline-clofazimine cross-resistance INH-ETO X-R isoniazid-ethionamide cross-resistance Inf infinity Lit. information from the literature NotAwR not associated with resistance NotAwRI not associated with resistance–interim Prev. WHO previous WHO guidance Uncert. Sig. uncertain significance WHO-end. gDST WHO-endorsed genotypic drug susceptibility testing assay Drug name of drug Interim on WHO initial Group 2 classification by WHO was used Pot. infl. PPV potentially inflated positive predictive value Selection information from selection studies Present_S number of susceptible isolates with the mutation Absent_S number of susceptible isolates without the mutation Present_R number of resistant isolates with the mutation Absent_R number of resistant isolates without the mutation Sensitivity true positive rate of mutation Specificity true negative rate of mutation PPV positive predictive value of mutation PPV|SOLOa positive predictive value conditional on being SOLO PPV SOLOa positive predictive value of SOLO mutation OR SOLO odds ratio as SOLO mutation Initial confidence grading initial grouping of mutation Supporting data set data set(s) used to derive the initial confidence grading Additional grading criteria criteria used to change the initial confidence grading (e.g. previous WHO guidance or WHO- endorsed genotypic DST assays) to yield the final confidence grading Final confidence grading final grouping of mutation after relevant additional grading criteria were applied Footnotes additional details provided for specific variants Changes vs ver.1 changes from first edition (Table 5) Additional variables shown in the master file Description Tier a-priori grouping of genomic regions; tiers 1 and 2 Algorithm pass algorithm pass during which mutation was classified; 0, before algorithm (i.e. neutral mutation); 1, first pass; 2, second pass Genome position genomic position in H37Rv for indels, inter-genetic and ribosomal mutations Present_SOLO_R number of resistant isolates with the SOLO mutation Present_SOLO_SR total number of resistant and susceptible isolates with the SOLO mutation Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 18 Additional variables shown in the master file Description Sensitivitya true positive rate of mutation Specificitya true negative rate of mutation PPVa positive predictive value of mutation ORa odds ratio of mutation OR SOLOa odds ratio of SOLO mutation Sentivity SOLOa true positive rate of SOLO mutation Specificity SOLOa true negative rate of SOLO mutation OR SOLO_FE-sig Fisher exact test for the false discovery rate-corrected P for the OR SOLO; TRUE = false discovery rate-corrected P ≤ 0.05; FALSE = false discovery rate-corrected P > 0.05 Neutral masked 0 = not masked; 1 = masked Previous WHO guidance NGS Guide 2018, Level of resistance to INH or MFX, RIF CC guide 2021, Miotto et al. (PubMed 29284687) (4, 6, 15, 19, 34) WHO-recommended genotypic DST assays Abbott RealTime MTB RIF/INH (35), BD MAX™ MDR-TB (36), Cepheid Xpert® MTB/RIF (37), Cepheid Xpert® MTB/RIF Ultra (38), Cepheid Xpert® MTB/XDR (39), Hain FluoroType® MTBDR VER 2.0 (40), Hain GenoType MTBDRplus VER 2.0 (41), Hain GenoType MTBDRsl VER 2.0 (42), Molbio Truenat® MTB-RIF Dx (43), Nipro Genoscholar™ NTM+MDRTB II (44), Nipro Genoscholar™ PZA-TB II (45), Roche Cobas® MTB-RIF/INH (46). a The lb and ub of the 95% CI for these figures are provided in additional columns. Table 5. Symbols and abbreviations used in the mutation tables Icon Changes Summary from first edition to second editionSummary from previous catalogue to current catalogue DOWN Assoc w R Uncertain significance DOWN Assoc w R–interim Uncertain significance DOWN Not assoc w R Uncertain significance DOWN Not assoc w R–interim Uncertain significance SWITCH Assoc w R–interim Not assoc w R–interim DOWN Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim UP Assoc w R–interim Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance Assoc w R UP Uncertain significance Assoc w R–interim UP Uncertain significance Not assoc w R UP Uncertain significance Not assoc w R–interim NEW Not present Not assoc w R NEW Not present Assoc w R NEW Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification DOWN Assoc w R Uncertain si ificanceSummary from previous catalogue to current catalogue DO N Assoc w R Uncertain significance DO N Assoc w R–interim Uncertain significance DO N Not assoc w R Uncertain significance DOWN Not assoc w R–interim Uncertain significance SWITCH Assoc w R–interim Not assoc w R–interim DO N Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim UP Assoc w R–interim Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance Assoc w R UP Uncertain significance Assoc w R–interim UP Uncertain significance Not assoc w R UP Uncertain significance Not assoc w R–interim NE Not present Not assoc w R NE Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification DOWN Assoc w R–interi Uncertain si ificanceSummary from previous catalogue to current catalogue DOWN Assoc w R Uncertain significance DOWN Assoc w R–interim Uncertain significance DO N Not assoc w R Uncertain significance DOWN Not assoc w R–interim Uncertain significance SWITCH Assoc w R–interim Not assoc w R–interim DO N Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim UP Assoc w R–interim Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance Assoc w R UP Uncertain significance Assoc w R–interim UP Uncertain significance Not assoc w R UP Uncertain significance Not assoc w R–interim NEW Not present Not assoc w R NEW Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification DOWN N t assoc w R Uncertain si ificanceSummary from previous catalogue to current catalogue DO N Assoc w R Uncertain significance DOWN Assoc w R–interim Uncertain significance DO N Not assoc w R Uncertain significance DOWN Not assoc w R–interim Uncertain significance SWITCH Assoc w R–interim Not assoc w R–interim DO N Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim UP Assoc w R–interim Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance Assoc w R UP Uncertain significance Assoc w R–interim UP Uncertain significance Not assoc w R UP Uncertain significance Not assoc w R–interim NE Not present Not assoc w R NEW Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification DOWN N t assoc w R–interim Uncertain si ificance Summary from previous catalogue to u re t catalogu ss rt i si ifi Assoc w R–interim rt i si ifi t ss rt i si ifi DOWN Not assoc w R–interim Uncertain significance SWITCH ss –interim Not assoc w R–interim Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim Assoc w R–interim Assoc w R Not assoc w R–interim Not assoc w R rt i si ifi ss rt i si ifi Assoc w R–interim rt i si ifi t ss UP Uncertain significance t ss –interim t r s t Not assoc w R t r s t ss t r s t Assoc w R–interim EW Not present Not assoc w R–interim No change Icon Ch nges Same classification SWITCH Assoc w R–interi Not assoc w R–interim W W W DOWN Assoc w R A soc –interim WDOWN N t assoc w R Not assoc R–interim Summary from previous catalogue to current catalogue DOWN Assoc w R Uncertain significance DOWN Assoc w R–interim Uncertain significance DO N N t assoc w R Un e tai significance Not assoc w R–i teri SWIT Not as oc –interim Assoc w R Assoc w R–interim Not assoc w R–interim UP Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance Assoc w R Uncertain significance –interim Uncertain significance Not assoc w R–interim NEW Not present Not assoc w R NEW Not present Assoc w R NE Not present Assoc w R–interim –interim o change Icon Changes Same classification UP Assoc w R–interim A soc Summary from previous catalogue to current catalogue DO N ss c Uncertain significance DO N Assoc w R–interim Uncertain significance t ass c certai si ifica ce DO N Not assoc w R–interim Uncertain significance SWITCH ss c –interim Not assoc w R–interim Assoc w R Assoc w R–interim DOWN Not assoc w R Not assoc w R–interim UP Assoc w R–interim Assoc w R UP Not assoc w R–interim Not assoc w R UP Uncertain significance ss c UP Uncertain significance Assoc w R–interim certai si ifica ce Not assoc w R UP Uncertain significance t ass c –interim NE Not present Not assoc w R NE Not present ss c N t rese t Assoc w R–interim E Not present Not assoc w R–interim No change Icon Ch nges Same classification UP Not assoc w R–i teri Not a soc Summary fr m previous catalogue to u re t catalogue Assoc w R t i i i i Not assoc w R Uncertain significance –interim Uncertain significance SWITCH t i t i DO N –interim DOWN t t –interim Assoc w R–interim Not assoc w R–interim Assoc w R t i i i i Assoc w R–interim UP Uncertain significance t UP Uncertain significance Not assoc w R–interim t t Assoc w R EW Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification UP Uncertain significanc A soc Summary fr m previous catalogue to u re t catalogue ssoc Uncertain significance ot assoc Uncertain s gnificance –interim Uncertain significance SWITCH ot assoc –interim DO N Assoc w R ssoc –interim DOWN Not assoc w R Not assoc w R–interim Assoc w R–interim Not assoc w R–interim Assoc w R UP Uncertain significance ssoc –interim UP Uncertain significance Not assoc w R UP Uncertain significance ot assoc –interi E Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification UP Uncertain significanc Assoc R–interim Summary fr m previous catalogue to u re t catalogue ssoc Uncertain significance ot assoc Uncertain significance –interim Uncertain significance SWITCH ot assoc –interim DO N Assoc w R ssoc –interim DOWN Not assoc w R Not assoc w R–interim Assoc w R–interim Not assoc w R–interim Assoc w R UP Uncertain significance ssoc –interim UP Uncertain significance Not assoc w R UP Uncertain significance ot assoc –interi E Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification UP Uncertain significanc Not assoc Summary fr m previous catalogue to u re t catalogue ssoc Uncertain significance Uncertain significance –interim Uncertain significance SWITCH ot assoc –interim DO N Assoc w R ssoc –interim DOWN Not assoc w R Assoc w R–interim assoc w R–interim UP Uncertain significance ssoc –interim UP Uncertain signific nce Not assoc w R UP Uncertain significance ot assoc –interi EW Not present Assoc w R NE Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification UP Uncertain significanc Not assoc –interim f i s t l t t t l i t ri t i t ri t i i i i I i t ri t ss i t ri ss ss i t ri t ss t ss i t ri ss i t ri ss t ss i t ri t ss i t ri t i i i i i t ri t i t ri t t t ss i t ri I s l ssifi ti NEW Not pr sent Not assoc ary fro revio s catalog e to re t catalog ssoc –interi ot assoc –interi t i i i i I –interi ot as oc –interi ssoc ssoc –interi ot assoc t i t i –interi t –interi t ssoc –interi t i i i i –interi t ssoc –interi t t ot assoc –interi o change Ico ges a e classification NE Not pr sent Assoc w R Summary fr m previous catalogue to u re t catalogue –inte i Uncertain significance Uncertain significance SWITCH oc R–inte i DO N DOWN –interim Assoc w R–interim assoc w R–interim UP Uncertain significance UP Uncertain signific nce ot a oc R Assoc w R E Not present Assoc w R–interim NEW Not present Not assoc w R–interim No change Icon Changes Same classification NEW Not present Assoc R–interim t t i t ri t NE Not pr sent Not assoc –interim c r ss c r m t ass c W ss r m ass c t ass c i teri N ss w No change Same classification The new symbols were added to the second edition to denote mutations that were new (NEW) or downgraded (DOWN), upgraded (UP), changed from being associated with resistance to not being associated (SWITCH) or remained the same in both catalogues. Because a new method of annotating indels was adopted for the second edition, comparison with the first edition was not possible for these variants. 3 Results for ind ivid ual d rugs 19 3.2 Rifampicin and rifapentine The rpoB Val170Phe and Ile491Phe remained the only mutations outside RRDR, spanning the rpoB codons 426–452 that are markers of RIF resistance (Table 6). Although some RRDR mutations (e.g. Ser450Tyr) were upgraded from Group 2 in the first edition to Group 1 in the second edition, this did not change final interpretation of the mutations, which means that the combined sensitivity of Groups 1 and 2 mutations in the two catalogues was identical at 93.3% (95% CI: 92.9–93.7) (Table A.1). The added sensitivity of the 110 Group 2 mutations that were almost exclusively classified according to the WHO-endorsed “additional grading rule” (any non-silent RRDR mutation was assumed to confer RIF resistance in the absence of evidence to the contrary (Table 1) (4,33)) increased the sensitivity by only 1.1% (95% CI: 1.0–1.3) from that of the 26 Group 1 mutations alone (Table 3). Lowering the variant frequency cut-off for calling Groups 1 and 2 mutations from 75% to 25% increased the combined sensitivity by 1.1% (Table A.1). It should be noted that the WHO-endorsed GenoScreen Deeplex® Myc-TB assay based on Illumina sequencing does not call minority variants at a frequency below 10% at nucleotide 1346 of rpoB (genomic position 761152), because this position is considered to be “noisy” (47). Moreover, an external quality assessment scheme for WGS questioned the validity of observed T to A changes at this position (rpoB Leu449Gln) (48). Specifically, two laboratories reported this change in the “2018 FASTQ14” sequence file, and one of the two laboratories reported the same change in “2019 FASTQ14” (in all three cases, the allele frequency was reported to be < 10%) (48).1 Work is under way to investigate whether this represents a potential Illumina-specific artefact (e.g. linked to library preparation or sequencing) or whether alternative explanations, such as low-level contamination with DNA from other species, apply (48). Any rpoB changes at this position identified by Illumina sequencing, particularly if unfixed, should be analysed carefully to avoid overcalling RIF resistance, as codon 449 falls within RRDR and is therefore subject to the associated “additional grading rule”. It is not known whether this potential limitation extends to other sequencing technologies. WHO has previously endorsed use of RIF as a surrogate for RPT, meaning that the Groups 1–5 classifications for RIF also apply to RPT (4). 1 Anthony R, personal communication, 2023. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 20 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . RIF rpoB_p.Ser450Leu 226 30643 10859 6002 64.4% 99.3% 98.0% 97.9% 98.2% 97.1% 234.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Val 17 30515 1154 15656 6.9% 99.9% 98.5% 98.8% 99.4% 97.5% 162.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asp 10 30522 608 16202 3.6% 100.0% 98.4% 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Tyr 11 30521 593 16217 3.5% 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Tyr 93 30439 341 16469 2.0% 99.7% 78.6% 70.9% 76.5% 59.7% 4.5 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu452Pro 102 30430 281 16529 1.7% 99.7% 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Trp 5 30527 238 16572 1.4% 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Leu 20 30512 217 16593 1.3% 99.9% 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu430Pro 185 30347 215 16595 1.3% 99.4% 53.8% 25.5% 32.0% 17.1% 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ser450Phe 1 30531 206 16604 1.2% 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Table 6. Abridged variant classification for RIF D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.His445Gln 2 30530 21 16789 0.1% 100.0% 91.3% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Asp 6 30526 15 16795 0.1% 100.0% 71.4% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn438del 2 30530 13 16797 0.1% 100.0% 86.7% 83.3% 97.9% 51.6% 9.1 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln429His 1 30531 13 16797 0.1% 100.0% 92.9% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Gln 0 30532 12 16798 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim RIF rpoB_p.Asp435Ala 6 30526 12 16798 0.1% 100.0% 66.7% 0.0% 70.8% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Val 2 30530 12 16798 0.1% 100.0% 85.7% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Gly 3 30529 11 16799 0.1% 100.0% 78.6% 70.0% 93.3% 34.8% 4.2 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Arg448Gln 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln429Leu 0 30532 10 16800 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu449Met 0 30532 10 16800 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Gly 1 30531 10 16800 0.1% 100.0% 90.9% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Pro 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ala451Val 6 30526 8 16802 0.0% 100.0% 57.1% 0.0% 70.8% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432Glu 0 30532 8 16802 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Met 1 30531 8 16802 0.0% 100.0% 88.9% 88.9% 99.7% 51.8% 14.5 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Arg 1 30531 7 16803 0.0% 100.0% 87.5% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437His 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Asn 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Arg 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Gln 1 30531 5 16805 0.0% 100.0% 83.3% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Thr 0 30532 5 16805 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Met 0 30532 5 16805 0.0% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Val 4 30528 5 16805 0.0% 100.0% 55.6% 0.0% 60.2% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ile 0 30532 5 16805 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Arg448Lys 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435His 0 30532 4 16806 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Thr 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430_Ser431insArg 1 30531 4 16806 0.0% 100.0% 80.0% 66.7% 99.2% 9.4% 3.6 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434_Asp435del 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T INAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 I . . . . . . . . I r . r r . . . . . . . ) ) I r . i . . . . . . . ) ) I r . r . . . . . . . ) c rt i r rli ) ss c I rpo _p. er450 he 1 30531 206 16604 1.2 100.0 99.5 100.0 100.0 97.2 Inf 1) LL 1) ssoc IF rpo _p. is445 rg 3 30529 136 16674 0.8 100.0 97.8 98.1 99.8 92.1 96.1 1) LL+ 1) ssoc IF rpoB_p. is445Asn 83 30449 126 16684 0.7 99.7 60.3 33.7 43.6 21.6 0.9 3) ncertain ALL+ Borderline 1) Assoc RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7 99.7 56.9 55.5 63.0 44.7 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5 100.0 97.8 97.6 99.9 83.8 73.0 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4 100.0 89.7 91.5 96.8 79.5 19.8 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+ HO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim 3 Results for ind ivid ual d rugs 21 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . RIF rpoB_p.Ser450Leu 226 30643 10859 6002 64.4% 99.3% 98.0% 97.9% 98.2% 97.1% 234.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Val 17 30515 1154 15656 6.9% 99.9% 98.5% 98.8% 99.4% 97.5% 162.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asp 10 30522 608 16202 3.6% 100.0% 98.4% 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Tyr 11 30521 593 16217 3.5% 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Tyr 93 30439 341 16469 2.0% 99.7% 78.6% 70.9% 76.5% 59.7% 4.5 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu452Pro 102 30430 281 16529 1.7% 99.7% 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Trp 5 30527 238 16572 1.4% 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Leu 20 30512 217 16593 1.3% 99.9% 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu430Pro 185 30347 215 16595 1.3% 99.4% 53.8% 25.5% 32.0% 17.1% 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ser450Phe 1 30531 206 16604 1.2% 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.His445Gln 2 30530 21 16789 0.1% 100.0% 91.3% 0.0% 84.2% 0.0% 0.0 3) Unc rtain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Asp 6 30526 15 16795 0.1% 100.0% 71.4% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn438del 2 30530 13 16797 0.1% 100.0% 86.7% 83.3% 97.9% 51.6% 9.1 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln429His 1 30531 13 16797 0.1% 100.0% 92.9% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim I r . r Gln 0 532 12 16798 0.1 100.0 100.0 100.0 100.0 66.4 Inf ) ALL ALL only 2) Assoc w R - Interim I r . Ala 6 26 2 6798 0.1 100.0 66.7 0.0 70.8 0.0 0.0 3) Uncertain RRDR 2) Assoc w R - Interim I r .Met434Val 2 30 12 798 0.1 . 85.7 NA NA 0.0 NA 3) Uncertain RRDR 2) Assoc w R - Interim I r . is Gly 3 9 11 799 0.1 . 7 .6 70.0 3.3 34.8 4.2 3) Uncertain RRDR 2) Assoc w R - Interim I rpo _p. rg448Gln 0 30532 10 16800 0.1 100.0 100.0 100.0 100.0 15.8 Inf 3) Uncertain LL+ RRDR 2) Assoc w R - Interim IF rpo _p.Gln429Leu 0 30532 10 16800 0.1 100.0 100.0 NA NA NA NA 3) Uncertain LL+ RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu449Met 0 30532 10 16800 0.1 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ H RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Gly 1 30531 10 16800 0.1 100.0 90.9 0.0 97.5 0.0 0.0 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Pro 0 30532 9 16801 0.1 100.0 100.0 100.0 100.0 54.1 Inf 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ala451Val 6 30526 8 16802 0.0% 100.0% 57.1% 0.0% 70.8% 0.0% 0.0 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432Glu 0 30532 8 16802 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Met 1 30531 8 16802 0.0% 100.0% 88.9% 88.9% 99.7% 51.8% 14.5 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Arg 1 30531 7 16803 0.0% 100.0% 87.5% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437His 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Asn 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Arg 0 30532 6 16804 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Gln 1 30531 5 16805 0.0% 100.0% 83.3% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Thr 0 30532 5 16805 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Met 0 30532 5 16805 0.0% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Val 4 30528 5 16805 0.0% 100.0% 55.6% 0.0% 60.2% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ile 0 30532 5 16805 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Arg448Lys 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435His 0 30532 4 16806 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Thr 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430_Ser431insArg 1 30531 4 16806 0.0% 100.0% 80.0% 66.7% 99.2% 9.4% 3.6 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434_Asp435del 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Ad itional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Met434Arg 0 30532 4 16806 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Gly 1 30531 4 16806 0.0% 100.0% 80.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Gln432insArg 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Gln432insHis 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Ala 1 30531 4 16806 0.0% 100.0% 80.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427_Ser428del 1 30531 4 16806 0.0% 100.0% 80.0% 80.0% 99.5% 28.4% 7.3 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ala 1 30531 4 16806 0.0% 100.0% 80.0% 50.0% 98.7% 1.3% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432His 0 30532 3 16807 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436del 1 30531 3 16807 0.0% 100.0% 75.0% 100.0% 100.0% 19.4% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445Phe 0 30532 3 16807 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Phe433_Asp435del 0 30532 3 16807 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Ile 1 30531 3 16807 0.0% 100.0% 75.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Asn 0 30532 3 16807 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Val 0 30532 3 16807 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Cys 5 30527 3 16807 0.0% 100.0% 37.5% 33.3% 77.7% 3.7% 0.9 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Gly 2 30530 3 16807 0.0% 100.0% 60.0% 50.0% 93.2% 6.8% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ser 0 30532 3 16807 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Ile 0 30532 3 16807 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ala451Gly 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435_Gln436delinsGlu 1 30531 2 16808 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 3.6 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln429Pro 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Met434delinsLeu 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432del 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly442Glu 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430_Gln432del 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Arg 1 30531 2 16808 0.0% 100.0% 66.7% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Glu 1 30531 2 16808 0.0% 100.0% 66.7% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Thr 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Phe433_Gln436del 1 30531 2 16808 0.0% 100.0% 66.7% 100.0% 100.0% 1.3% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Thr 1 30531 2 16808 0.0% 100.0% 66.7% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 22 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . RIF rpoB_p.Ser450Leu 226 30643 10859 6002 64.4% 99.3% 98.0% 97.9% 98.2% 97.1% 234.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Val 17 30515 1154 15656 6.9% 99.9% 98.5% 98.8% 99.4% 97.5% 162.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asp 10 30522 608 16202 3.6% 100.0% 98.4% 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Tyr 11 30521 593 16217 3.5% 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Tyr 93 30439 341 16469 2.0% 99.7% 78.6% 70.9% 76.5% 59.7% 4.5 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu452Pro 102 30430 281 16529 1.7% 99.7% 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Trp 5 30527 238 16572 1.4% 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Leu 20 30512 217 16593 1.3% 99.9% 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu430Pro 185 30347 215 16595 1.3% 99.4% 53.8% 25.5% 32.0% 17.1% 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ser450Phe 1 30531 206 16604 1.2% 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Met434Arg 0 30532 4 16806 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser428Gly 1 30531 4 16806 0.0% 100.0% 80.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Gln432insArg 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Gln432insHis 0 30532 4 16806 0.0% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Ala 1 30531 4 16806 0.0% 100.0% 80.0% 0.0% 97.5% 0.0% 0.0 3) Unc rtain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427_Ser428del 1 30531 4 16806 0.0% 100.0% 80.0% 80.0% 99.5% 28.4% 7.3 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ala 1 30531 4 16806 0.0% 100.0% 80.0% 50.0% 98.7% 1.3% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432His 0 30532 3 16807 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Gln 36d l 1 531 3 16807 0.0 100.0 75 100.0 100.0 19 4 Inf 3) Uncertain RRDR 2) Assoc w R - Interim I .His445Phe 0 32 3 6807 0.0 100.0 100.0 100.0 100.0 15.8 Inf 3) Uncertain RRDR 2) Assoc w R - Interim I r .Phe433_Asp435del 0 3 3 8 7 0.0 . 100.0 100.0 100.0 29.2 Inf 3) Uncertain RRDR 2) Assoc w R - Interim I r .Ser 28Ile 1 3 3 80 0.0 . 75.0 NA NA 0.0 NA 3) Uncertain RRDR 2) Assoc w R - Interim I r .Ser 1Asn 0 5 2 3 807 0. 100.0 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim I rpo _p.Ser441Val 0 30532 3 16807 0.0 100.0 100.0 100.0 100.0 29.2 Inf 3) Uncertain LL RRDR 2) Assoc w R - Interim IF rpo _p. er450Cys 5 30527 3 16807 0.0 100.0 37.5 33.3 77.7 3.7 0.9 3) Uncertain LL+ RRDR 2) Assoc w R - Interim IF rpoB_p.Ser450Gly 2 30530 3 16807 0.0 100.0 60.0 50.0 93.2 6.8 1.8 3) Uncertain ALL+ RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Ser 0 30532 3 16807 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ H RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Ile 0 30532 3 16807 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ala451Gly 0 30532 2 16808 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435_Gln436delinsGlu 1 30531 2 16808 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 3.6 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln429Pro 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Met434delinsLeu 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432del 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly442Glu 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430_Gln432del 0 30532 2 16808 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Arg 1 30531 2 16808 0.0% 100.0% 66.7% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Glu 1 30531 2 16808 0.0% 100.0% 66.7% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Thr 0 30532 2 16808 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Phe433_Gln436del 1 30531 2 16808 0.0% 100.0% 66.7% 100.0% 100.0% 1.3% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431Thr 1 30531 2 16808 0.0% 100.0% 66.7% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Arg448Leu 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437_Asn438del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Ile 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Ser 3 30529 1 16809 0.0% 100.0% 25.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn438His 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435_Gln436del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435del 1 30531 1 16809 0.0% 100.0% 50.0% 100.0% 100.0% 1.3% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Leu 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Phe433del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432Asn 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436_Asn437del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436Arg 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436Pro 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445_Lys446del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.His445_Lys446delinsGln 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu443_Lys446delinsProGln 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu443Phe 1 30531 1 16809 0.0% 100.0% 50.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu443Ser 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu443Trp 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu452Val 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Lys446Thr 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434_Asn437delinsIle 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434_Asp435insVal 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Phe425_Gly426del 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Ala 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Ser 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Met434del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Trp 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450_Leu452del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Ala 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim 3 Results for ind ivid ual d rugs 23 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . RIF rpoB_p.Ser450Leu 226 30643 10859 6002 64.4% 99.3% 98.0% 97.9% 98.2% 97.1% 234.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Val 17 30515 1154 15656 6.9% 99.9% 98.5% 98.8% 99.4% 97.5% 162.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asp 10 30522 608 16202 3.6% 100.0% 98.4% 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Tyr 11 30521 593 16217 3.5% 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Tyr 93 30439 341 16469 2.0% 99.7% 78.6% 70.9% 76.5% 59.7% 4.5 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu452Pro 102 30430 281 16529 1.7% 99.7% 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Trp 5 30527 238 16572 1.4% 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Leu 20 30512 217 16593 1.3% 99.9% 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Leu430Pro 185 30347 215 16595 1.3% 99.4% 53.8% 25.5% 32.0% 17.1% 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ser450Phe 1 30531 206 16604 1.2% 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Arg448Leu 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437_Asn438del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Ile 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Ser 3 30529 1 16809 0.0% 100.0% 25.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn438His 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435_Gln436del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435del 1 30531 1 16809 0.0% 100.0% 50.0% 100.0% 100.0% 1.3% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Leu 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Phe433del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432Asn 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436_Asn437del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436Arg 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Gln 36Pro 0 532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim His445_Lys446del 0 32 1 6809 0 0 100.0 100.0 100.0 100.0 2.5 Inf 3) Uncertain RRDR 2) Assoc w R - Interim i _Lys446delinsGln 0 3 1 8 9 0 0 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim I .Leu443_Lys446delinsProGln 0 32 1 809 0.0 . 100.0 100.0 100.0 2.5 Inf 3) Uncertain RRDR 2) Assoc w R - Interim I r .Leu 43Phe 1 5 1 1 80 0. 100.0 50.0 0.0 97. 0.0 0.0 3) Uncertain RRDR 2) Assoc w R - Interim I r . 43Ser 5 2 1 80 0.0 100.0 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim I r .L u 43 r 0 32 1 809 0.0 . 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim I r .Leu452Val 0 3 809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain RRDR 2) Assoc w R - Interim IF rpo _p.Lys446Thr 0 30532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) ncertain LL+ RRDR 2) Assoc w R - Interim IF rpoB_p.Met434_Asn437delinsIle 0 30532 1 16809 0.0 100.0 100.0 100.0 100.0 2.5 Inf 3) Uncertain ALL+ RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434_Asp435insVal 0 30532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ H RRDR 2) Assoc w R - Interim RIF rpoB_p.Phe425_Gly426del 0 30532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Ala 0 30532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Ser 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser431_Met434del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Trp 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450_Leu452del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser450Ala 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Thr427_Gln429del 0 30532 1 16809 0.0 100.0 100.0 100.0 100.0 2.5 Inf 3) Uncertain ALL+ H RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427_Gln429delinsLys 0 30532 1 16809 0.0 100.0 100.0 100.0 100.0 2.5 Inf 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Pro 0 30532 1 16809 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+ HO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Pro 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Ser 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Tyr 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Met434del 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436Asn 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly426_Thr427del 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly426Ser 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu452Met 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Leu 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Leu 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Lys 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Asn 2 30530 0 16810 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Gly 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Val695Leu 71 30461 126 16684 0.7% 99.8% 64.0% 9.5% 18.5% 3.7% 0.2 3) Uncertain ALL+WHO 3) Uncertain significance RIF Rv2752c_p.Lys435Glu 33 23674 11 11491 0.1% 99.9% 25.0% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim RIF rpoB_p.Glu250Gly 49 23658 5 11497 0.0% 99.8% 9.3% 2.0% 10.9% 0.1% 0.0 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim RIF mtrB_p.Met517Leu 19355 4352 9872 1630 85.8% 18.4% 33.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-28T>C 11233 12474 6324 5178 55.0% 52.6% 36.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF mtrB_p.Pro18Ser 4186 19521 4916 6586 42.7% 82.3% 54.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Glu1092Asp 1076 22631 1887 9615 16.4% 95.5% 63.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Gly594Glu 3984 19723 1064 10438 9.3% 83.2% 21.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_c.-61C>T 3347 20360 640 10862 5.6% 85.9% 16.1% 1.2% 1.7% 0.8% 0.0 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Val460Ala 3310 20397 404 11098 3.5% 86.0% 10.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF lpqB_p.Asp142Gly 3284 20423 403 11099 3.5% 86.1% 10.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_c.-138T>C 2918 20789 371 11131 3.2% 87.7% 11.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Ala172Val 2777 20930 366 11136 3.2% 88.3% 11.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Val192fs 115 23592 173 11329 1.5% 99.5% 60.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 24 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoB_p.Thr427_Gln429del 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427_Gln429delinsLys 0 30532 1 16809 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Pro 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Pro 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr444Ser 0 30532 1 16809 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asn437Tyr 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln432_Met434del 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gln436Asn 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly426_Thr427del 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Gly426Ser 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu452Met 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Leu 1 30531 0 16810 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Pro439Leu 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Ser441Lys 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Asn 2 30530 0 16810 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Thr427Gly 1 30531 0 16810 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Val695Leu 71 30461 126 16684 0.7% 99.8% 64.0% 9.5% 18.5% 3.7% 0.2 3) Uncertain ALL+WHO 3) Uncertain significance RIF Rv2752c_p.Lys435Glu 33 23674 11 11491 0.1% 99.9% 25.0% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim RIF rpoB_p.Glu250Gly 49 23658 5 11497 0.0% 99.8% 9.3% 2.0% 10.9% 0.1% 0.0 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim RIF mtrB_p.Met517Leu 19355 4352 9872 1630 85.8% 18.4% 33.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-28T>C 11233 12474 6324 5178 55.0% 52.6% 36.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF mtrB_p.Pro18Ser 4186 19521 4916 6586 42.7% 82.3% 54.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Glu1092Asp 1076 22631 1887 9615 16.4% 95.5% 63.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Gly594Glu 3984 19723 1064 10438 9.3% 83.2% 21.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_c.-61C>T 3347 20360 640 10862 5.6% 85.9% 16.1% 1.2% 1.7% 0.8% 0.0 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Val460Ala 3310 20397 404 11098 3.5% 86.0% 10.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF lpqB_p.Asp142Gly 3284 20423 403 11099 3.5% 86.1% 10.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_c.-138T>C 2918 20789 371 11131 3.2% 87.7% 11.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Ala172Val 2777 20930 366 11136 3.2% 88.3% 11.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Val192fs 115 23592 173 11329 1.5% 99.5% 60.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoC_p.Pro601Leu 1634 22073 172 11330 1.5% 93.1% 9.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Pro906Ala 249 23458 135 11367 1.2% 98.9% 35.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2477c_p.Gly41Glu 83 23624 127 11375 1.1% 99.6% 60.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Val300Ala 76 23631 101 11401 0.9% 99.7% 57.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Pro123Leu 206 23501 93 11409 0.8% 99.1% 31.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_p.Glu319Lys 412 23295 75 11427 0.7% 98.3% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Ala621Thr 379 23328 66 11436 0.6% 98.4% 14.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-48A>C 310 23397 64 11438 0.6% 98.7% 17.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-49A>C 241 23466 44 11458 0.4% 99.0% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_c.-261A>G 435 23272 44 11458 0.4% 98.2% 9.2% 1.3% 2.9% 0.4% 0.0 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Met31Ile 127 23580 37 11465 0.3% 99.5% 22.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Gly161Ser 100 23607 36 11466 0.3% 99.6% 26.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-48A>G 266 23441 35 11467 0.3% 98.9% 11.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF mtrA_c.-162C>G 56 23651 28 11474 0.2% 99.8% 33.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-9T>G 601 23106 28 11474 0.2% 97.5% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-68C>T 83 23624 27 11475 0.2% 99.6% 24.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Glu209Lys 49 23658 24 11478 0.2% 99.8% 32.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_p.Pro34Leu 230 23477 24 11478 0.2% 99.0% 9.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Asp271Gly 226 23481 23 11479 0.2% 99.0% 9.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_p.Thr167Met 135 23572 17 11485 0.1% 99.4% 11.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-46C>G 49 23658 14 11488 0.1% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-310T>A 71 23636 13 11489 0.1% 99.7% 15.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Leu228Val 345 23362 11 11491 0.1% 98.5% 3.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-124delC 141 23566 8 11494 0.1% 99.4% 5.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Ile925Val 73 23634 8 11494 0.1% 99.7% 9.9% 1.4% 7.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Ala296Val 112 23595 6 11496 0.1% 99.5% 5.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF lpqB_p.Ser394Leu 206 23501 5 11497 0.0% 99.1% 2.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Glu639Asp 55 23652 4 11498 0.0% 99.8% 6.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Ser388Leu 56 23651 4 11498 0.0% 99.8% 6.7% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Gly191dup 30 23677 2 11500 0.0% 99.9% 6.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confid nce grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . Ser450 226 643 10859 6002 64.4 3 98 97.9 9 2 97.1 234.4 1) AwR 1) Assoc w R rpoB_p.As 435Val 17 30515 1 54 5656 6 9 98 5 98.8% 99.4% 97.5 162.4 1) AwR ALL+WHO 1) Assoc w R I .His445Asp 10 30522 608 6202 3.6 100.0 98.4 98.4 99.3 96.8 118.3 1) AwR ALL+WHO 1) Assoc w R I rpo .His445Tyr 11 30521 593 16217 3.5 100.0 98.2 98.6% 99.4% 96.0 128.2 1) AwR ALL+WHO 1) Assoc w R I rpoB_p.Asp435 yr 93 30 39 341 16469 2.0 99.7 78.6 70.9% 76.5% 59.7 4.5 1) AwR ALL+WHO 1) Assoc w R I rpo .Leu452Pro 102 30430 281 16529 1.7 99.7 73.4 64.9% 70.7% 56.1 3.4 1) AwR ALL+WHO 1) Assoc w R I r B .Ser450Trp 5 30527 238 16572 1.4 100.0 97.9 97.4% 99.1% 94.0 68.9 1) AwR ALL+WHO 1) Assoc w R I rpoB_p.His445Leu 20 30512 217 16593 1.3 99.9 91.6 91.4% 94.8% 85.8 19.6 1) AwR ALL+WHO 1) Assoc w R IF rpo _p.Leu430Pro 185 30347 215 16595 1.3 99.4 53.8 25.5 32.0 17.1 0.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R IF rpoB_p.Ser450Phe 1 30531 206 16604 1.2 100.0 99.5 100.0% 100.0% 97.2 Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8 100.0 97.8 98.1% 99.8% 92.1 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7 99.7 60.3 33.7% 43.6% 21.6 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R IF rpoB_p.His445Cys 8 30524 70 16740 0.4 100.0 89.7 91.5% 96.8% 79.5 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4 100.0 98.6 98.1% 100.0% 89.7 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3 100.0 92.3 90.7% 97.4% 77.9 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3 100.0 100.0 100.0% 100.0% 83.9 Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim 3 Results for ind ivid ual d rugs 25 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 RIF rpoC_p.Pro601Leu 1634 22073 172 11330 1.5% 93.1% 9.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Pro906Ala 249 23458 135 11367 1.2% 98.9% 35.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2477c_p.Gly41Glu 83 23624 127 11375 1.1% 99.6% 60.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Val300Ala 76 23631 101 11401 0.9% 99.7% 57.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Pro123Leu 206 23501 93 11409 0.8% 99.1% 31.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_p.Glu319Lys 412 23295 75 11427 0.7% 98.3% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Ala621Thr 379 23328 66 11436 0.6% 98.4% 14.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-48A>C 310 23397 64 11438 0.6% 98.7% 17.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-49A>C 241 23466 44 11458 0.4% 99.0% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_c.-261A>G 435 23272 44 11458 0.4% 98.2% 9.2% 1.3% 2.9% 0.4% 0.0 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Met31Ile 127 23580 37 11465 0.3% 99.5% 22.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Gly161Ser 100 23607 36 11466 0.3% 99.6% 26.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-48A>G 266 23441 35 11467 0.3% 98.9% 11.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF mtrA_c.-162C>G 56 23651 28 11474 0.2% 99.8% 33.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-9T>G 601 23106 28 11474 0.2% 97.5% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-68C>T 83 23624 27 11475 0.2% 99.6% 24.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Glu209Lys 49 23658 24 11478 0.2% 99.8% 32.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_p.Pro34Leu 230 23477 24 11478 0.2% 99.0% 9.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Asp271Gly 226 23481 23 11479 0.2% 99.0% 9.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF nusG_p.Thr167Met 135 23572 17 11485 0.1% 99.4% 11.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv1129c_c.-46C>G 49 23658 14 11488 0.1% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-310T>A 71 23636 13 11489 0.1% 99.7% 15.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Leu228Val 345 23362 11 11491 0.1% 98.5% 3.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoA_c.-124delC 141 23566 8 11494 0.1% 99.4% 5.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Ile925Val 73 23634 8 11494 0.1% 99.7% 9.9% 1.4% 7.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Ala296Val 112 23595 6 11496 0.1% 99.5% 5.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF lpqB_p.Ser394Leu 206 23501 5 11497 0.0% 99.1% 2.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Glu639Asp 55 23652 4 11498 0.0% 99.8% 6.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF rpoB_p.Ser388Leu 56 23651 4 11498 0.0% 99.8% 6.7% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R RIF glpK_p.Gly191dup 30 23677 2 11500 0.0% 99.9% 6.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S e n s i t i v i t y S p e c i f i c i t y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S u p p o r t i n g d a t a s e t Additional grading criteria applied Final confidence grading N o t e s C h a n g e s v s p r e v . v e r . rpoB_p.Ser450Leu 226 30 43 10859 6002 64.4 3 98 0 97.9% 98.2% 97.1 234.4 1) AwR ALL+WHO 1) Assoc w R B p Asp435Val 17 30515 1 54 5656 6 9 9 98 98.8% 99.4% 97.5 162.4 1) AwR ALL+WHO 1) Assoc w R rpoB .His445Asp 10 3052 608 6202 3 6 100.0 98.4 98.4% 99.3% 96.8% 118.3 1) AwR ALL+WHO 1) Assoc w R B p His 45Tyr 11 30 21 593 6217 3 5 100.0% 98.2% 98.6% 99.4% 96.0% 128.2 1) AwR ALL+WHO 1) Assoc w R Asp435Tyr 9 304 9 341 6 69 2 0 78.6% 70.9% 76.5% 59.7% 4 5 1) AwR ALL+WHO 1) Assoc w R rpoB_p.Leu452Pro 0 30430 281 6529 1 7 7 73.4% 64.9% 70.7% 56.1% 3.4 1) AwR ALL+WHO 1) Assoc w R r o . r450Trp 5 30 27 238 6572 1 4 100.0% 97.9% 97.4% 99.1% 94.0% 68.9 1) AwR ALL+WHO 1) Assoc w R I .His445Leu 20 3051 217 65 3 1.3 .9 91.6% 91.4% 94.8% 85.8% 19.6 1) AwR ALL+WHO 1) Assoc w R I r .L u430Pro 185 30347 215 65 5 1.3 .4 53.8% 25.5% 32.0 17.1% .6 3) Uncertain ALL+WHO Borderline 1) Assoc w R I rpoB .Ser450Phe 1 30531 206 66 4 1.2 100.0% 99.5% 100.0% 100.0% 97.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Arg 3 30529 136 16674 0.8% 100.0% 97.8% 98.1% 99.8% 92.1% 96.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Asn 83 30449 126 16684 0.7% 99.7% 60.3% 33.7% 43.6% 21.6% 0.9 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Ile491Phe 88 30444 116 16694 0.7% 99.7% 56.9% 55.5% 63.0% 44.7% 2.3 1) AwR ALL Borderline 1) Assoc w R RIF rpoB_p.Val170Phe 2 30530 89 16721 0.5% 100.0% 97.8% 97.6% 99.9% 83.8% 73.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Cys 8 30524 70 16740 0.4% 100.0% 89.7% 91.5% 96.8% 79.5% 19.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Pro 1 30531 69 16741 0.4% 100.0% 98.6% 98.1% 100.0% 89.7% 93.0 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Phe 4 30528 48 16762 0.3% 100.0% 92.3% 90.7% 97.4% 77.9% 17.8 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Lys 0 30532 46 16764 0.3% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Leu 1 30531 38 16772 0.2% 100.0% 97.4% 94.4% 99.9% 72.7% 30.9 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser441Gln 2 30530 30 16780 0.2% 100.0% 93.8% 93.8% 99.2% 79.2% 27.3 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432Leu 1 30531 28 16782 0.2% 100.0% 96.6% 94.1% 99.9% 71.3% 29.1 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Phe433dup 0 30532 26 16784 0.2% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.His445Ser 7 30525 19 16791 0.1% 100.0% 73.1% 66.7% 86.7% 38.4% 3.6 3) Uncertain ALL+WHO Borderline 1) Assoc w R RIF rpoB_p.Thr444dup 0 30532 10 16800 0.1% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Gln432_Asp435delinsHis 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Ser450Tyr 0 30532 9 16801 0.1% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R RIF rpoB_p.Asp435Gly 8 30524 148 16662 0.9% 100.0% 94.9% 50.0% 84.3% 9.9% 1.8 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Met434Ile 2 30530 33 16777 0.2% 100.0% 94.3% 100.0% 100.0% 0.8% Inf 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Leu430Arg 3 30529 32 16778 0.2% 100.0% 91.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim RIF rpoB_p.Asp435Glu 0 30532 21 16789 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO RRDR 2) Assoc w R - Interim Mutations shown in purple correspond to the seven WHO-endorsed borderline resistance mutations (4,33). Silent mutations are not listed in the table but can be found in the catalogue master file. D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 Ly 9 4Glu 49 23658 2 1500 0 99.8% 3.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R mtr Met260Val 107 23600 1 1501 0 5 0.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R I Rv2477c_p.Thr372Lys 70 23637 1501 .0 . 1.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R I r C .Arg69Pro 72 236 5 1 150 .0 99.7 1.4 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R I glpK .Gly260Ala 42 23665 0 1502 .0 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R I nusG_p.Thr3Asn 46 2366 0 1502 .0 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R I Rv1129c_c.-29A>G 65 23642 0 150 .0 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R I rpo _p.Asn381His 76 23631 0 11502 0.0 99.7% 0.0% 0.0% 4.7% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R IF rpoB_p.Asp853_Glu854dup 33 23674 0 11502 0.0 99.9% 0.0% 0.0% 10.6 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R RIF rpoC_p.Glu784 ln 58 23649 0 11502 0.0 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R RIF Rv2752c_p.Ala273Val 108 23599 0 11502 0.0 99.5% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 26 3.3 Isoniazid The combined sensitivity of Groups 1 and 2 resistance mutations for predicting phenotypic INH resistance was 91.6% (95% CI: 91.2–92.0), representing a gain of 0.6% over the first edition (Table A.1). Lowering the cut-off for calling Groups 1 and 2 mutations from 75% to 25% increased the combined sensitivity by only 0.5% (Table A.1). Two new Group 2 promoter mutations upstream of the fabG1-inhA operon (i.e. -17G>T and -8T>G, Table 7) were recognized by additional grading rules because they are detected with the WHO-endorsed FluoroType® MTBDR VER 2.0 assay (40) (see Section 5.8 for details of all additional grading rules applied). The remaining five mutations that confer resistance by over-expression of inhA (the shared target of INH and ETO) were -16A>G, -15C>T, -8T>A and -8T>C, which are upstream of fabG1; and the fabG1 609G>A Leu203Leu mutation that creates an alternative inhA promoter (49,50). In light of this detailed mechanistic understanding, a new “additional grading rule” for cross-resistance was endorsed for the second edition (Table 7), whereby any inhA resistance mutation for INH was also recognized as a resistance mutation for ETO and vice versa (see Section 5.8) (19). With this rule, inhA Ser94Ala, which was in Group 2 for ETO (Table 19), was also classified in Group 2 for INH. This is supported by transduction experiments for this mutation (50). Only six katG mutations (Met1?, Ser315Arg, Ser315Asn, Ser315Ile, Ser315Thr and Trp328Leu) were sufficiently frequent in this data set to be classified into Group 1 or 2 (Table 7). The WHO-endorsed “additional grading” rule was also used to classify 128 of the 135 Group 2 mutations, whereby any katG LoF mutation was assumed to result in an LoF phenotype and, consequently, INH resistance (Tables 1 and 23). In the second edition, inframe mutations were excluded from the additional grading rule because they are less likely to abolish katG function. All katG mutations were assumed to confer high-level INH resistance, whereas inhA mutations were considered to confer low-level resistance if they occurred in isolation. Genetically linked low-level inhA mutations (e.g. an upstream mutation with a coding mutation) have an additive effect, however, and confer high-level INH resistance (4,19,51,52). Potential role of rare variants It is well recognized that various rare katG mutations do not meet the standard criteria used to classify resistance mutations in our analyses (Fig. 8) but which can be inferred by using mutations upstream of ahpC with the Xpert® MTB/XDR assay (39,53). We explored their role in this data set by applying the “relaxed” criteria endorsed for pncA (Fig. 9) to katG. This increased the combined sensitivity of INH mutations by 1.2% while reducing the specificity and PPV by 0.2% (Table A.3). If it is assumed that any non-silent Group 3 coding mutation in katG (in RIF-resistant isolates) confers INH resistance, the sensitivity is increased by 1.7% at the cost of lowering the specificity by 0.4% and the PPV by 0.4% (Table A.3). These calculations indicate that only a small proportion of the lack of sensitivity in this data set was probably due to katG resistance mutations. 3 Results for ind ivid ual d rugs 27 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . INH katG_p.Ser315Thr 250 27505 16302 4649 77.8% 99.1% 98.5% 98.3% 98.5% 97.5% 336.7 1) AwR ALL+WHO 1) Assoc w R G INH inhA_c.-777C>T 161 27307 4449 16397 21.3% 99.4% 96.5% 87.8% 89.6% 83.7% 12.0 1) AwR ALL+WHO 1) Assoc w R F, L INH inhA_c.-154G>A 64 27404 745 20101 3.6% 99.8% 92.1% 70.1% 76.5% 61.1% 3.2 1) AwR ALL+WHO 1) Assoc w R F, H INH katG_LoF 11 27457 254 20592 1.2% 100.0% 95.8% 94.0% 98.0% 79.2% 21.1 1) AwR ALL+WHO 1) Assoc w R G INH katG_p.Ser315Asn 5 27463 206 20640 1.0% 100.0% 97.6% 99.2% 100.0% 91.4% 170.3 1) AwR ALL+WHO 1) Assoc w R G INH katG_p.Met1? 0 27468 29 20817 0.1% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R G INH katG_p.Ser315Arg 1 27467 14 20832 0.1% 100.0% 93.3% 100.0% 100.0% 64.0% Inf 1) AwR ALL+WHO 1) Assoc w R G INH katG_p.Trp328Leu 0 27468 12 20834 0.1% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO 1) Assoc w R G INH inhA_c.-770T>C 17 27451 388 20458 1.9% 99.9% 95.8% 41.7% 63.4% 19.4% 1.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, J INH inhA_c.-779G>T 26 27442 339 20507 1.6% 99.9% 92.9% 58.9% 71.9% 42.4% 1.9 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, N INH inhA_p.Ser94Ala 27 27441 213 20633 1.0% 99.9% 88.8% 58.3% 72.4% 37.1% 1.9 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim F INH inhA_c.-770T>A 8 27460 166 20680 0.8% 100.0% 95.4% 70.6% 89.7% 36.1% 3.2 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, I INH katG_p.Ser315Ile 0 27468 20 20826 0.1% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim G INH inhA_c.-770T>G 0 27468 19 20827 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, K INH inhA_c.-778A>G 1 27467 0 20846 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, M INH katG_p.Ala480del 1 27467 5 20841 0.0% 100.0% 83.3% 50.0% 98.7% 1.3% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Ala359_Gly362del 0 27468 3 20843 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gly121del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gly124del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Pro429del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Thr380_Gly494delinsSer 0 27468 2 20844 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Ala480dup 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Asn133_Glu233delinsLys 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gln50_Asn51insThr 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu195_Asn236del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu233_Pro239del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu67del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Leu343_Ser346delinsArg 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Lys154dup 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Pro288_Glu289del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Table 7. Abridged variant classification for INH C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 28 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 INH katG_p.Pro29_Val30delinsLeu 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Thr380del 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Tyr353_Thr354insAsn 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Tyr390dup 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Val151_Tyr155delinsAsp 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Val23_Val30del 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Asp513_Leu514insHis 1 27467 0 20846 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Leu641_Gly644del 1 27467 0 20846 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance INH Rv2752c_p.Val300Ala 68 21245 108 13266 0.8% 99.7% 61.4% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH Rv2752c_p.Pro123Leu 175 21138 100 13274 0.7% 99.2% 36.4% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH ndh_p.Arg268His 99 21214 84 13290 0.6% 99.5% 45.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim INH Rv2752c_p.Met31Ile 140 21173 52 13322 0.4% 99.3% 27.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH mshA_p.Asp218Ala 157 21156 27 13347 0.2% 99.3% 14.7% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim INH ndh_c.-70G>T 44 21269 27 13347 0.2% 99.8% 38.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim INH Rv2752c_p.Ala296Val 101 21212 14 13360 0.1% 99.5% 12.2% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH Rv2752c_p.Lys435Glu 32 21281 12 13362 0.1% 99.8% 27.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH mshA_p.Arg443His 35 21278 8 13366 0.1% 99.8% 18.6% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim INH katG_c.-354C>T 67 21246 3 13371 0.0% 99.7% 4.3% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim INH ndh_p.Gly313Arg 65 21248 2 13372 0.0% 99.7% 3.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim INH Rv1129c_c.-28T>C 9795 11518 7551 5823 56.5% 54.0% 43.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH katG_p.Arg463Leu 9704 11609 7507 5867 56.1% 54.5% 43.6% 4.2% 4.7% 1.9% 0.1 5) NotAwR WHO 5) Not assoc w R INH Rv1258c_p.Glu194fs 3517 17796 5469 7905 40.9% 83.5% 60.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH mshA_p.Ala187Val 3361 17952 5236 8138 39.2% 84.2% 60.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH ahpC_c.-88G>A 3214 18099 974 12400 7.3% 84.9% 23.3% 17.3% 30.3% 0.1% 0.3 5) NotAwR WHO 5) Not assoc w R INH glpK_p.Val460Ala 2919 18394 753 12621 5.6% 86.3% 20.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Ile87Met 2537 18776 703 12671 5.3% 88.1% 21.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH mshA_p.Asn111Ser 2163 19150 702 12672 5.2% 89.9% 24.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH katG_c.-85C>T 494 20819 510 12864 3.8% 97.7% 50.8% 2.5% 4.3% 1.2% 0.0 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Ala26Val 844 20469 272 13102 2.0% 96.0% 24.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH ndh_p.Val18Ala 695 20618 235 13139 1.8% 96.7% 25.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . Ser315Thr 250 505 16302 4649 77.8 99.1 98.5 98.3% 98.5% 97.5% 336.7 1) AwR 1) Assoc w R G inhA c.-777C>T 161 307 4449 16397 21.3 99.4 96.5 87.8% 89.6% 83.7% 12.0 1) AwR 1) Assoc w R F, L inhA c.-154G>A 64 04 745 101 3 6 99.8 92.1 70.1% 76.5% 61.1% 3.2 1) AwR 1) Assoc w R F, H LoF 11 57 254 592 1 2 95.8 94.0% 98.0% 79.2% 21.1 1) AwR 1) Assoc w R G Ser315Asn 5 3 206 6 0 1 97.6 99.2 91.4 170.3 1) AwR 1) Assoc w R G Met1? 29 17 1 100.0% 100.0% 85.8% Inf 1) AwR 1) Assoc w R G Ser3 5Arg 14 32 1 93.3 1 0.0% 100.0 64.0 Inf 1) AwR 1) Assoc w R G Trp328Leu 0 8 12 34 1 1 0.0% 1 0.0% 100.0 73.5 Inf 1) AwR 1) Assoc w R G inhA_c.-770T>C 17 7451 38 20458 1 9 9 95 8 41.7% 63.4% 19.4 1.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, J inhA_c.-779G>T 26 7442 339 20507 1 6 9 92 9 58.9% 71.9% 42.4 1.9 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, N inhA_p.Ser94Ala 27 7441 213 20633 1 0 9 88 8 58.3% 72.4% 37.1 1.9 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim F inhA_c.-770T>A 8 7460 166 20680 8 100.0 95 4 70.6% 89.7% 36.1 3.2 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, I katG_p.Ser315Ile 0 7468 0 20826 1 100.0 100.0 100.0% 100.0% 71.5 Inf 1) AwR ALL ALL only 2) Assoc w R - Interim G inhA_c.-770T>G 0 74 8 19 2082 1 100.0 100.0 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, K inhA_c.-778 >G 1 7467 0 20846 0 100.0 0.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, M katG_p.Ala480del 1 7467 5 20841 0 100.0 83 50.0% 98.7% 1 3 1.3 3) Uncertain ALL+WHO 3) Uncertain significance katG_p.Ala359_Gly362del 0 746 3 20843 0 100.0 100.0 NA 3) Uncertain ALL+WHO 3) Uncertain significance p Gly121del 0 7468 2 20844 100.0 100.0% 1 0.0% 100.0 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain significance katG_p.Gly 24del 0 746 20844 100.0 100.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain significance katG_p.Pro4 9del 0 2746 2 20844 0.0 100.0 100.0 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance Thr380_Gly494delinsSer 0 27468 2 20844 0.0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Ala480dup 0 2 468 1 20845 0.0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Asn 33_G u233delinsLys 0 2 468 1 20845 0.0 100.0 100.0 NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Gln50_ sn51insThr 0 27468 1 20845 0 0 100.0 100.0 NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG .Glu195_Asn236del 0 27468 1 20845 0 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Glu233_Pro239del 0 27468 1 20845 0 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Glu67del 0 27468 1 20845 0 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance p Leu343_Ser346delinsArg 0 7468 20 45 0 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Lys154dup 0 7 8 1 20845 0 100.0 100.0 NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Pro288_Glu289del 0 746 1 20845 0 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance 3 Results for ind ivid ual d rugs 29 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 INH Rv0010c_c.-141A>G 317 20996 185 13189 1.4% 98.5% 36.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH dnaA_p.Pro124Leu 474 20839 169 13205 1.3% 97.8% 26.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH inhA_c.-40C>T 604 20709 136 13238 1.0% 97.2% 18.4% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R INH dnaA_c.-133G>T 572 20741 116 13258 0.9% 97.3% 16.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1129c_c.-9T>G 511 20802 109 13265 0.8% 97.6% 17.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Tyr95Cys 494 20819 107 13267 0.8% 97.7% 17.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH dnaA_c.-32C>T 479 20834 100 13274 0.7% 97.8% 17.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1129c_c.-48A>C 280 21033 94 13280 0.7% 98.7% 25.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH dnaA_c.-58G>A 227 21086 66 13308 0.5% 98.9% 22.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH glpK_p.Leu228Val 291 21022 62 13312 0.5% 98.6% 17.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH katG_c.-278G>C 258 21055 58 13316 0.4% 98.8% 18.4% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R INH Rv1129c_c.-49A>C 229 21084 51 13323 0.4% 98.9% 18.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1129c_c.-48A>G 236 21077 43 13331 0.3% 98.9% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv2752c_p.Gly161Ser 96 21217 41 13333 0.3% 99.5% 29.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Leu111fs 188 21125 32 13342 0.2% 99.1% 14.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1258c_p.Pro414Ser 117 21196 25 13349 0.2% 99.5% 17.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Thr4Ile 56 21257 19 13355 0.1% 99.7% 25.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1258c_p.Gly88fs 49 21264 17 13357 0.1% 99.8% 25.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH dnaA_p.His156Arg 184 21129 10 13364 0.1% 99.1% 5.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Thr121Ser 182 21131 10 13364 0.1% 99.1% 5.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH inhA_p.Val78Ala 55 21258 6 13368 0.0% 99.7% 9.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH glpK_p.Gly191dup 15 21298 3 13371 0.0% 99.9% 16.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH dnaA_c.-48G>A 53 21260 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH inhA_c.-522C>G 45 21268 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH katG_p.Val469Leu 46 21267 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Ser82Pro 47 21266 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv0010c_p.Thr40Ala 48 21265 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1129c_c.-29A>G 65 21248 0 13374 0.0% 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv1258c_p.Glu243Ala 48 21265 0 13374 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R INH Rv2752c_p.Ala273Val 108 21205 0 13374 0.0% 99.5% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . katG_p.Ser3 5Thr 250 7505 16302 4649 77.8 9 1 98 5 98.3% 98.5% 97.5 336.7 1) AwR ALL+WHO 1) Assoc w R G inhA_c.-777C>T 161 7307 4449 6397 21.3 9 4 9 5 87.8% 89.6% 83.7 12.0 1) AwR ALL+WHO 1) Assoc w R F, L 154G>A 64 74 4 745 20101 3 6 9 8 92 1 70.1 76 61.1 3 2 1) AwR ALL+WHO 1) Assoc w R F, H katG_LoF 11 7457 254 20592 1 2 100.0 95 8 94.0% 98.0% 79.2 21.1 1) AwR ALL+WHO 1) Assoc w R G katG_p.Ser315Asn 5 7463 2 6 20640 1 0 100.0 9 99.2% 100.0% 91.4 170.3 1) AwR ALL+WHO 1) Assoc w R G katG_p.Met1? 0 7468 29 2081 1 100.0 100.0 100.0% 100.0% 85.8 Inf 1) AwR ALL+WHO 1) Assoc w R G katG_p.Ser315Arg 1 7467 14 20832 1 100.0 93 100.0% 100.0% 64.0 Inf 1) AwR ALL+WHO 1) Assoc w R G katG_p.Trp32 Leu 0 7468 12 20834 1 100.0 100.0 100.0% 100.0% 73.5 Inf 1) AwR ALL+WHO 1) Assoc w R G inhA_c.-770T C 17 7451 388 2045 1 9 9 95 8 41.7% 63.4% 19.4 1.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Ass c w R - Interim F, J inhA c -779G>T 26 744 339 20507 1 6 9 9 92 9 58.9% 71.9% 42.4 1.9 3) Uncertain ALL+WHO WHO-end. gDST 2) Ass c w R - Interim F, N inhA p Ser94Ala 27 7441 213 20633 1 0 9 9 8 8 58.3 72 4 37.1 1 9 3) Uncertain ALL+WHO INH-ETO X-R 2) Ass c w R - Interim F inhA_c.-770T> 8 7460 166 20680 8 100.0 95 4 70.6% 89.7% 36.1 3.2 3) Uncertain ALL+WHO WHO-end. gDST 2) Ass c w R - Interim F, I katG_p.Ser315Ile 0 7468 20 20826 1 100.0 100.0 100.0% 100.0% 71.5 Inf 1) AwR ALL ALL only 2) Ass c w R - Interim G inhA_c.-770T>G 0 7468 19 20827 1 100.0 100.0 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO WHO-end. gDST 2) Ass c w R - Interim F, K inhA_c.-778A>G 1 7467 0 208 6 0 100.0 0.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Ass c w R - Interim F, M katG_p.Ala480del 7467 5 208 1 0 100.0 83 3 50.0% 98.7% 1 3 1.3 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Ala359_Gly362del 0 7468 3 20843 0 100.0 100.0 NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Gly121del 0 74 8 2 20844 0 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Gly 24del 0 7468 2 2084 0 100.0 100.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Pro429del 0 7468 2 2084 0 100.0 100.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG .Thr3 0_Gly494delinsSer 0 746 2 20844 100.0 100.0% NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG .A a480 0 746 1 20845 100.0 100.0 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Asn133_Glu233delinsLys 0 74 8 1 20845 100.0 1 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG p.Gln50_Asn51insThr 0 74 1 20845 100.0 1 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain ignificance Glu195_Asn236del 0 74 8 1 20845 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Glu233_Pro239del 0 74 8 1 20845 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Glu67del 0 74 8 1 20845 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Leu343_Ser346delinsArg 0 746 1 20845 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Lys154dup 0 74 8 1 20845 100.0 1 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain ignificance katG_p.Pro288_Glu289del 7468 1 20845 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 30 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 INH katG_p.Ser315Gly 1 27467 29 20817 0.1% 100.0% 96.7% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gln127Pro 4 27464 23 20823 0.1% 100.0% 85.2% 50.0% 93.2% 4.3% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Pro232Ser 1 27467 13 20833 0.1% 100.0% 92.9% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_c.-10A>C 1 27467 10 20836 0.0% 100.0% 90.9% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp142Gly 2 27466 10 20836 0.0% 100.0% 83.3% 87.5% 99.7% 40.0% 9.2 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly699Glu 1 27467 9 20837 0.0% 100.0% 90.0% 87.5% 99.7% 47.3% 9.2 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Tyr337Cys 1 27467 9 20837 0.0% 100.0% 90.0% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Ala109Val 0 27468 8 20838 0.0% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asn138Ser 0 27468 8 20838 0.0% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp189Gly 2 27466 8 20838 0.0% 100.0% 80.0% 66.7% 95.7% 22.3% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly279Asp 0 27468 8 20838 0.0% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Val423Ile 7 27461 8 20838 0.0% 100.0% 53.3% 22.2% 60.0% 2.8% 0.4 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asn138His 0 27468 7 20839 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asn138Asp 2 27466 6 20840 0.0% 100.0% 75.0% 66.7% 95.7% 22.3% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gln461Pro 3 27465 6 20840 0.0% 100.0% 66.7% 50.0% 93.2% 5.3% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gln525Pro 1 27467 6 20840 0.0% 100.0% 85.7% 80.0% 99.5% 28.4% 5.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly297Val 1 27467 6 20840 0.0% 100.0% 85.7% 80.0% 99.5% 28.4% 5.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly299Ser 0 27468 6 20840 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Pro232Ala 0 27468 5 20841 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Thr344Pro 1 27467 5 20841 0.0% 100.0% 83.3% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Trp161Arg 2 27466 5 20841 0.0% 100.0% 71.4% 100.0% 100.0% 29.0% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_c.-185T>C 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Ala172Val 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp419His 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp94Ala 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp94Gly 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly118Ser 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Leu141Phe 1 27467 4 20842 0.0% 100.0% 80.0% 80.0% 99.5% 28.4% 5.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Thr271Ile 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Thr394Ala 1 27467 4 20842 0.0% 100.0% 80.0% 100.0% 100.0% 19.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . Thr 250 505 16302 4649 77.8 99.1 8 5 98 3 8 5 97.5 336.7 1) AwR 1) Assoc w R G inhA c.-777C>T 161 307 4449 16397 21.3 99.4 96 5 87 8 89 6 83.7 12.0 1) AwR 1) Assoc w R F, L inhA c.-154G>A 64 04 745 101 3 6 99.8 1 70 1 76 5 61.1 3 2 1) AwR 1) Assoc w R F, H LoF 11 5 254 592 1 2 5 8 94.0 98.0 79.2 21.1 1) AwR 1) Assoc w R G Ser315Asn 5 3 206 640 1 97 6 99 2 100.0 91 4 170.3 1) AwR 1) Assoc w R G Met1? 0 8 29 1 1 100.0 100.0 100.0 85 8 Inf 1) AwR 1) Assoc w R G Ser 15Arg 14 2 1 3 3 64.0 1) AwR 1) Assoc w R G Trp328Leu 12 4 1 73 5 1) AwR 1) Assoc w R G inhA c.-770T>C 17 51 3 8 45 1 9 99.9 95.8 41.7 63.4 1 4 1.0 WHO-end. gDST 2) Assoc w R - Interim F, J inhA c.-779G>T 26 42 339 507 1 6 99.9 92 9 58 9 71 9 4 4 1 9 WHO-end. gDST 2) Assoc w R - Interim F, N inhA .Ser94Ala 27 41 213 6 3 1 99.9 88.8 58.3 72.4 37 1.9 INH-ETO X-R 2) Assoc w R - Interim F inhA c.-770T>A 8 0 166 680 8 95 4 70 6 89 7 36.1 3 2 WHO-end. gDST 2) Assoc w R - Interim F, I Ser315Ile 20 26 1 71 5 1) AwR ALL ALL only 2) Assoc w R - Interim G inhA c.-770T>G 0 8 19 27 1 100.0 100.0 100.0 9 2 Inf WHO-end. gDST 2) Assoc w R - Interim F, K inhA c.-778A>G 1 7 0 6 0.0 NA NA 0 0 NA WHO-end. gDST 2) Assoc w R - Interim F, M Ala480del 5 1 3 3 5 8 7 1.3 1 Ala359_G y362del 0 8 3 3 100.0 NA NA NA NA 121d l 2 4 2.5 Gly124del 2 4 2.5 Pro429del 0 8 2 4 100.0 100.0 100.0 15.8 Inf hr380_Gly494delinsSer 0 8 2 4 100.0 NA NA NA NA p Ala480dup 1 5 2.5 sn133_Glu233delinsLys 1 5 NA NA NA NA Gln50_Asn51insThr 1 5 NA NA NA NA Glu195_Asn236del 1 5 2.5 Glu233_Pro239del 1 5 2.5 u67del 1 5 2.5 3 3_S r346delinsArg 0 8 1 5 100.0 100.0 100.0 2.5 Inf Lys154dup 1 5 NA NA NA NA Pro288_Glu289del 0 8 1 5 100.0 2.5 3 Results for ind ivid ual d rugs 31 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 INH katG_p.Trp149Arg 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Trp300Gly 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Tyr98Cys 0 27468 4 20842 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_c.-485_-484insTGTCT 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_c.-8G>A 1 27467 3 20843 0.0% 100.0% 75.0% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Arg104Gln 1 27467 3 20843 0.0% 100.0% 75.0% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Arg484His 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Arg571His 1 27467 3 20843 0.0% 100.0% 75.0% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp189Asn 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp311Gly 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp487Asn 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp735Ala 1 27467 3 20843 0.0% 100.0% 75.0% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gln439Arg 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly125Asp 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly285Val 2 27466 3 20843 0.0% 100.0% 60.0% 50.0% 93.2% 6.8% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly491Ser 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Leu48Pro 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Thr271Pro 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Trp300Arg 0 27468 3 20843 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Ala476Val 2 27466 2 20844 0.0% 100.0% 50.0% 50.0% 93.2% 6.8% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Arg128Trp 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Arg146Gly 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asn493Lys 1 27467 2 20844 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asn637Lys 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp189Ala 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Asp381Ala 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Glu588Gly 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly121Asp 2 27466 2 20844 0.0% 100.0% 50.0% 100.0% 100.0% 6.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly156Asp 1 27467 2 20844 0.0% 100.0% 66.7% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Gly234Arg 1 27467 2 20844 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 2.6 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . Ser315Thr 250 505 16302 4649 77.8 99.1 98.5 98.3 98.5 97 5 336.7 1) AwR 1) Assoc w R G inhA c.-777C>T 161 307 4449 16397 21.3 99.4 96.5 87.8 89.6 83 7 12.0 1) AwR 1) Assoc w R F, L inhA c.-154G>A 64 04 7 5 101 3 6 99.8 92.1 70.1 76.5 61 1 3.2 1) AwR 1) Assoc w R F, H LoF 11 57 254 592 1 2 95.8 94.0 98.0 7 21.1 1) AwR 1) Assoc w R G p Ser315Asn 5 3 206 6 0 1 97 6 99.2 91.4 170.3 1) AwR 1) Assoc w R G Met ? 0 8 29 17 1 100.0 100.0 100.0 85.8 Inf 1) AwR 1) Assoc w R G Ser315Arg 1 7 14 32 1 93.3 64 0 1) AwR 1) Assoc w R G T p328Leu 0 8 12 34 1 100.0 100.0 100.0 73.5 Inf 1) AwR 1) Assoc w R G inhA c.-770T>C 17 51 388 458 1 9 99.9 95.8 41.7 63.4 9 4 1.0 WHO-end. gDST 2) Assoc w R - Interim F, J inhA c.-779G>T 26 42 3 9 507 1 6 99.9 92.9 58.9 71.9 42 4 1.9 WHO-end. gDST 2) Assoc w R - Interim F, N inhA .Ser94Ala 27 41 213 63 1 99.9 88.8 58.3 72.4 37 1 1.9 INH-ETO X-R 2) Assoc w R - Interim F inhA c.-770T>A 8 0 166 680 8 9 4 70.6 89.7 36.1 3.2 WHO-end. gDST 2) Assoc w R - Interim F, I Ser315Ile 20 26 1 71 5 1) AwR ALL ALL only 2) Assoc w R - Interim G inhA c.-770T>G 19 27 1 29 2 WHO-end. gDST 2) Assoc w R - Interim F, K inhA c.-778A>G 1 7 0 6 0.0 NA NA 0 0 NA WHO-end. gDST 2) Assoc w R - Interim F, M Ala 80d l 1 7 5 1 83.3 50.0 98.7 1.3 1.3 Ala359_Gly362del NA NA NA NA Gly12 del 2 4 2.5 Gly124del 2 4 2.5 Pro 29de 0 8 100.0 100.0 100.0 15.8 Inf Thr380_Gly494delinsSer NA NA NA NA la480dup 1 5 2.5 13 _Glu233delinsLys 0 8 1 5 100.0 NA NA NA NA Gl 50_Asn51insThr 1 5 NA NA NA NA Glu 95_Asn236del 1 5 2.5 Glu233_Pro239del 1 5 2.5 67del 1 5 2.5 Leu343_Ser346delinsArg 0 8 1 5 100.0 2 5 Lys 4du 0 8 1 5 100.0 NA NA NA NA Pro 88_Glu289del 0 8 1 5 100.0 100.0 100.0 2 5 Inf C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 32 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 INH katG_p.Gly699Arg 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Leu333Pro 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Leu427Pro 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Pro288His 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Ser160Pro 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Ser700Pro 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Thr322Ala 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O INH katG_p.Tyr229Cys 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . Ser315Thr 250 505 16302 4649 77.8 99.1 98.5 98.3 98.5 97 5 336.7 1) AwR 1) Assoc w R G inhA c.-777C>T 161 307 4449 16397 21.3 99.4 96.5 87.8 89.6 83 7 12.0 1) AwR 1) Assoc w R F, L inhA c.-154G>A 64 04 745 101 3 6 99.8 92.1 70.1 76.5 61 1 3.2 1) AwR 1) Assoc w R F, H LoF 11 57 254 592 1 2 95.8 94.0 98.0 79 2 21.1 1) AwR 1) Assoc w R G 315Asn 5 3 206 6 0 1 97.6 99.2 91 4 170.3 1) AwR 1) Assoc w R G M t1? 29 17 1 8 1) AwR 1) Assoc w R G Se 15 rg 1 7 14 32 1 93.3 64 0 1) AwR 1) Assoc w R G rp3 8Leu 12 3 1 73 5 1) AwR 1) Assoc w R G INH inhA_c.-770T>C 17 27451 388 20458 1.9% 99.9% 95.8% 41.7% 63.4% 19.4% 1.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, J INH inhA_c.-779G>T 26 27442 339 20507 1.6% 99.9% 92.9% 58.9% 71.9% 42.4% 1.9 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, N INH inhA_p.Ser94Ala 27 27441 213 20633 1.0% 99.9% 88.8% 58.3% 72.4% 37.1% 1.9 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim F INH inhA_c.-770T>A 8 27460 166 20680 0.8% 100.0% 95.4% 70.6% 89.7% 36.1% 3.2 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, I INH katG_p.Ser315Ile 0 27468 20 20826 0.1% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim G INH inhA_c.-770T>G 0 27468 19 20827 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, K INH inhA_c.-778A>G 1 27467 0 20846 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F, M INH katG_p.Ala480del 1 27467 5 20841 0.0% 100.0% 83.3% 50.0% 98.7% 1.3% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Ala359_Gly362del 0 27468 3 20843 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gly121del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gly124del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Pro429del 0 27468 2 20844 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Thr380_Gly494delinsSer 0 27468 2 20844 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Ala480dup 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Asn133_Glu233delinsLys 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Gln50_Asn51insThr 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu195_Asn236del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu233_Pro239del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Glu67del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Leu343_Ser346delinsArg 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Lys154dup 0 27468 1 20845 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance INH katG_p.Pro288_Glu289del 0 27468 1 20845 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Individual LoF mutations in the coding regions of katG that are classified in Group 2 because of the associated additional gradi g rule (see Table 23 and Section 5.8) and silent mutatio s are not listed in this table but can be found in the catalogue master file. F Low-level resistance (multiple, genetically linked low-level resistance mutations are additive and confer high-level resistance). G High-level resistance. H Alias fabG1_p.Leu203Leu. I Alias fabG1_c.-8T>A. J Alias fabG1_c.-8T>C. K Alias f bG1_c.-8T>G. L Alias fabG1_c.-15C>T. M Alias fabG1_c.-16A>G. N Alias fabG1_c.-17G>T. O Group 2 by “relaxed” threshold (not endorsed). 3 Results for ind ivid ual d rugs 33 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 34.2% 98.1% 82.6% 79.9% 81.5% 74.0% 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6% 96.8% 62.8% 54.5% 56.7% 45.3% 5.5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8482 10.5% 99.4% 81.7% 73.0% 76.8% 58.8% 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3.5% 99.4% 62.2% 53.0% 59.0% 37.4% 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3.2% 99.6% 68.7% 64.2% 69.9% 49.9% 6.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 9207 2.9% 99.4% 56.4% 46.9% 53.2% 31.5% 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2.2% 99.9% 81.3% 80.7% 85.7% 73.0% 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2.0% 99.8% 71.1% 61.1% 70.9% 34.8% 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1.5% 99.9% 74.0% 53.0% 65.4% 30.2% 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1.5% 99.9% 74.4% 70.4% 79.2% 48.6% 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 0.5% 100.0% 90.2% 84.0% 95.5% 60.6% 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7% 99.8% 48.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7% 99.8% 47.8% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0% 99.9% 6.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R 3.4 Ethambutol Thirteen non-synonymous mutations in embB and one intergenic mutation upstream of embA met the criteria for Group 1 mutations; no Group 2 mutations were identified (Table 8). The combined sensitivity of Group 1 mutations for predicting phenotypic EMB resistance was 81.1% (95% CI: 80.3–81.9), while the specificity was 91.6% (95% CI: 91.3–91.9) and the PPV 71.9% (95% CI: 71.0–72.8) (Table 3). The reduced specificity is probably due to the fact that many embB mutations confer MICs close to the CC, resulting in poor categorical agreement with pDST (11,22,54–56). Indeed, the Clinical and Laboratory Standards Institute has an “inconclusive” category for EMB MICs equal to the CC on Sensititre MYCOTB plates (21,57). Additionally, it is not clear whether the currently endorsed CCs are identical to the epidemiological cut-off values for EMB. Inappropriately high breakpoints may be responsible for high rates of misclassification of embB phenotypes and consequent discordance with gDST results, as was the case with borderline RIF resistance mutations (21, 33). Notably, two markers of resistance in the first edition (embA -12C>T as Group 1 and embB Leu74Arg as Group 2) were downgraded to Group 3 in the second edition. As a result, EMB is one of only four drugs the sensitivity of which decreased slightly from that in the first edition (i.e. by 2.7%, as shown in Table A.1). Inclusion of mutations with a variant allele frequency below 75% increased the sensitivity by only 1% (Table A.1) suggesting a minimal role of heteroresistance. Table 8. Abridged variant classification for EMB C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 34 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 34.2% 98.1% 82.6% 79.9% 81.5% 74.0% 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6% 96.8% 62.8% 54.5% 56.7% 45.3% 5.5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8482 10.5% 99.4% 81.7% 73.0% 76.8% 58.8% 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3.5% 99.4% 62.2% 53.0% 59.0% 37.4% 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3.2% 99.6% 68.7% 64.2% 69.9% 49.9% 6.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 9207 2.9% 99.4% 56.4% 46.9% 53.2% 31.5% 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2.2% 99.9% 81.3% 80.7% 85.7% 73.0% 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2.0% 99.8% 71.1% 61.1% 70.9% 34.8% 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1.5% 99.9% 74.0% 53.0% 65.4% 30.2% 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1.5% 99.9% 74.4% 70.4% 79.2% 48.6% 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 0.5% 100.0% 90.2% 84.0% 95.5% 60.6% 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7% 99.8% 48.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7% 99.8% 47.8% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0% 99.9% 6.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 EMB glpK_p.Val460Ala 3427 23078 248 6312 3.8% 87.1% 6.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB ubiA_p.Glu149Asp 3404 23101 247 6313 3.8% 87.2% 6.8% 0.0% 60.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Thr270Ile 3246 23259 243 6317 3.7% 87.8% 7.0% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embA_p.Pro913Ser 3014 23491 226 6334 3.4% 88.6% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Asn394Asp 3000 23505 225 6335 3.4% 88.7% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embR_p.Cys110Tyr 2931 23574 222 6338 3.4% 88.9% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-900C>T 945 25560 132 6428 2.0% 96.4% 12.3% 1.2% 2.4% 0.3% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embA_p.Val206Met 1689 24816 117 6443 1.8% 93.6% 6.5% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-1743G>A 473 26032 99 6461 1.5% 98.2% 17.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Thr1082Ala 108 26397 96 6464 1.5% 99.6% 47.1% 1.2% 6.7% 0.0% 0.1 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-100C>T 255 26250 93 6467 1.4% 99.0% 26.7% 0.0% 2.1% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-529T>C 107 26398 83 6477 1.3% 99.6% 43.7% 0.0% 4.6% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Pro123Leu 210 26295 68 6492 1.0% 99.2% 24.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Ala774Ser 141 26364 65 6495 1.0% 99.5% 31.6% 1.8% 6.3% 0.2% 0.1 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Val300Ala 114 26391 63 6497 1.0% 99.6% 35.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-1193C>T 103 26402 36 6524 0.5% 99.6% 25.9% 1.2% 6.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB aftB_p.Lys522Arg 306 26199 29 6531 0.4% 98.8% 8.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Gly161Ser 113 26392 24 6536 0.4% 99.6% 17.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Met31Ile 147 26358 19 6541 0.3% 99.4% 11.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embA_p.Glu951Asp 125 26380 15 6545 0.2% 99.5% 10.7% 1.8% 6.3% 0.2% 0.1 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-1419G>A 143 26362 15 6545 0.2% 99.5% 9.5% 1.7% 6.0% 0.2% 0.1 5) NotAwR WHO 5) Not assoc w R EMB ubiA_p.Gly268Asp 143 26362 15 6545 0.2% 99.5% 9.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB Rv2477c_p.Val85Ile 84 26421 13 6547 0.2% 99.7% 13.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-589C>G 88 26417 11 6549 0.2% 99.7% 11.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val104Met 299 26206 10 6550 0.2% 98.9% 3.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg567His 375 26130 9 6551 0.1% 98.6% 2.3% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB ubiA_c.-32delG 713 25792 8 6552 0.1% 97.3% 1.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB glpK_p.Leu228Val 349 26156 7 6553 0.1% 98.7% 2.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB aftB_p.Ile327Val 194 26311 6 6554 0.1% 99.3% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Asn13Ser 192 26313 6 6554 0.1% 99.3% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . Met3 Val 685 020 3245 6236 34.2 8 1 82 6 79 9 81 5 74 0 22.4 B p Met306Ile 1159 4 46 1953 7528 20.6 6 8 62 8 54 5 56 7 45 3 5 5 1) AwR 1) Assoc w R Gln497Arg 224 481 999 8 82 10.5 99.4 81 7 73.0 76 8 58.8 11.3 1) AwR 1) Assoc w R B p Gly406Ala 199 3 5 6 328 9 3 3.5 9 4 62 2 53.0 59.0 37.4 4 4 1) AwR ALL+WHO 1) Assoc w R Asp354Ala 137 3556 301 9180 3 2 6 6 7 64.2% 69.9% 49.9 6.9 1) AwR ALL+WHO 1) Assoc w R B Gly406Asp 212 35 93 274 920 2 9 4 56 4 46.9% 53.2% 31.5 3.4 1) AwR ALL+WHO 1) Assoc w R B Tyr31 4 3565 204 9277 2 9 8 3 80.7% 85.7% 73.0 16.1 1) AwR ALL+WHO 1) Assoc w R B p Gly406Ser 7 3562 187 929 2 0 71 1 61.1% 70.9% 34.8 6.0 1) AwR ALL+WHO 1) Assoc w R B Gln 97Lys 1 35654 145 9336 1 5 9 74 0 53.0% 65.4% 30.2 4.3 1) AwR ALL+WHO 1) Assoc w R B p Met306Leu 50 35655 1 5 933 1 5 9 74.4 70.4% 79.2% 48.6 9.1 1) AwR ALL+WHO 1) Assoc w R embB_p.Asp328Tyr 5 35700 46 9435 5 100.0 90.2 84.0% 95.5% 60.6 19.9 1) AwR ALL+WHO 1) Assoc w R Ty 319Cys 12 93 27 33 4 100.0 69.2 59.3% 77.6% 37.2 5.9 1) AwR 1) Assoc w R Gly406Cys 8 35677 36 9445 4 56.3 58.1% 73.0% 33.3 5.2 1) AwR ALL+WHO 1) Assoc w R embA_c.-12C>T 173 35532 635 8846 6.7 99 5 78 6 30.9% 41.1% 10.2 1.8 3) Uncertain ALL+WHO 3) Uncertain ignificance B Leu74Arg 8 35697 21 9460 0 2 100.0 72 4 0 97.5 0 3) Uncertain ALL+WHO 3) Uncertain ignificance 1188C>T 1498 5007 1403 5157 21.4 4 3 48 4 5 4 7.0 2 7 3 Lit. (PMID 32143680) 4) Not assoc w R - Interim B Gly156Cys 47 64 8 45 515 0 7 99 8 48.9 NA NA 0 NA Lit. (PMID 32143680) 4) Not assoc w R - Interim A Ala813G y 47 6458 43 517 0 7 99 8 47.8 Lit. (PMID 32143680) 4) Not assoc w R - Interim embA_p.Pro 39Ser 57 644 16 544 0.2 99.8 21.9 . ) t Lit. (PMID 32143680) 4) Not assoc w R - Interim embC_c.-20A>C 56 6449 16 544 0.2 99.8 22.2 NA NA . NA ) t Lit. (PMID 32143680) 4) Not assoc w R - Interim A .Val468Ala 57 6448 16 544 0.2 99. 21.9 NA NA . NA ) t Prev. WHO 4) Not assoc w R - Interim C c.-1520C>A 7 6 34 4 556 0.1 99.7 5.3 . ) t Prev. WHO 4) Not assoc w R - Interim ubiA_p.Val49Ile 72 6433 4 556 0.1 99. 5.3 . ) t Prev. WHO 4) Not assoc w R - Interim B .Ser1054Pro 31 64 55 0.0 99. 6.1 . ) t Prev. WHO 4) Not assoc w R - Interim B p.Val668Ile 23 6482 2 55 0. 9.9 8.0 NA NA .0 NA ) t Lit. (PMID 32143680) 4) Not assoc w R - Interim aftB_p.Asp397Gly 5235 1270 3363 3197 51.3 80.2 39.1 NA NA . NA ) t ) t p.Val981Leu 4076 2429 516 044 7.9 84.6 1.2 1.0% 1.7% .2 0.0 ) t ) t C c.-565C>T 2287 4218 332 228 5.1 1.4 12.7 0.0 70.8 . .0 ) t ) t p.Arg738Gln 3853 26 2 327 233 5.0 85.5 7.8 1.1 1.6 .5 . ) t ) t B p.Glu378Ala 3386 3119 251 309 3.8 87.2 6.9 NA NA . NA ) t ) t 3 Results for ind ivid ual d rugs 35 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 34.2% 98.1% 82.6% 79.9% 81.5% 74.0% 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6% 96.8% 62.8% 54.5% 56.7% 45.3% 5.5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8482 10.5% 99.4% 81.7% 73.0% 76.8% 58.8% 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3.5% 99.4% 62.2% 53.0% 59.0% 37.4% 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3.2% 99.6% 68.7% 64.2% 69.9% 49.9% 6.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 9207 2.9% 99.4% 56.4% 46.9% 53.2% 31.5% 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2.2% 99.9% 81.3% 80.7% 85.7% 73.0% 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2.0% 99.8% 71.1% 61.1% 70.9% 34.8% 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1.5% 99.9% 74.0% 53.0% 65.4% 30.2% 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1.5% 99.9% 74.4% 70.4% 79.2% 48.6% 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 0.5% 100.0% 90.2% 84.0% 95.5% 60.6% 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7% 99.8% 48.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7% 99.8% 47.8% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0% 99.9% 6.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 E B e bC_c.-270G>A 64 26441 5 6555 0.1 99.8 7.2 0.0 97.5 0.0 0.0 5) NotAwR WHO 5) Not assoc w R E B e bR_p.Ala70Ser 120 26385 3 6557 0.0 99.5 2.4 NA NA 0.0 NA 5) otA 5) Not assoc w R E B Rv2752c_p.Ala296Val 113 26392 2 6558 0.0 99.6 1.7 A A 0.0 A 5) otA 5) Not assoc w R E B Rv2477c_p.Arg86His 54 26451 1 6559 0.0 99.8 1.8 A A 0.0 A 5) otA 5) Not assoc w R E B e bA_p.Pro383Ser 58 26447 1 6559 0.0 99.8 1.7 A A 0.0 A 5) otA 5) Not assoc w R E B e bB_p.Arg213Gln 207 26298 1 6559 0.0 99.2 0.5 0.0% 1.8% 0.0 0.0 5) otA 5) Not assoc w R E B e bB_p.Gln139His 138 26367 1 6559 0.0 99.5 0.7 A A 0.0 A 5) otA 5) Not assoc w R E B e b _p.Leu661Ile 58 26447 1 6559 0.0 99.8 1.7 A A 0.0 A 5) otA 5) Not assoc w R E B embR_p.Cys372Gly 4 26501 0 6560 0.0 100.0 0.0 A A 0.0 A 5) otA 5) Not assoc w R E B e bR_p.Phe376Leu 4 26501 0 6560 0.0 100.0 0.0 A A 0.0 A 5) otA 5) Not assoc w R E B glpK_p.Gly260Ala 42 26463 0 6560 0.0 99.8 0.0 A A 0.0 A 5) otA 5) Not assoc w R E B Rv2752c_p.Ala273Val 108 26397 0 6560 0.0 99.6 0.0 A A 0.0 A 5) otA 5) ot assoc E B e b _c.-1803G>C 58 26447 0 6560 0.0 99.8 0.0 0.0 7.0 0.0 0.0 5) otA 5) ot assoc E B e bA_p.Ala201Thr 136 26369 0 6560 0.0 99.5 0.0 NA NA 0.0 NA 5) otA 5) ot assoc E B e bA_p.Thr308Ala 46 26459 0 6560 0.0 99.8 0.0 NA NA 0.0 NA 5) otA 5) ot assoc Silent mutations are not listed in this table but can be found in the catalogue master file. D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 EMB glpK_p.Val460Ala 3427 23078 248 6312 3.8% 87.1% 6.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB ubiA_p.Glu149Asp 3404 23101 247 6313 3.8% 87.2% 6.8% 0.0% 60.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Thr270Ile 3246 23259 243 6317 3.7% 87.8% 7.0% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embA_p.Pro913Ser 3014 23491 226 6334 3.4% 88.6% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Asn394Asp 3000 23505 225 6335 3.4% 88.7% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embR_p.Cys110Tyr 2931 23574 222 6338 3.4% 88.9% 7.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-900C>T 945 25560 132 6428 2.0% 96.4% 12.3% 1.2% 2.4% 0.3% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embA_p.Val206Met 1689 24816 117 6443 1.8% 93.6% 6.5% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-1743G>A 473 26032 99 6461 1.5% 98.2% 17.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Thr1082Ala 108 26397 96 6464 1.5% 99.6% 47.1% 1.2% 6.7% 0.0% 0.1 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-100C>T 255 26250 93 6467 1.4% 99.0% 26.7% 0.0% 2.1% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-529T>C 107 26398 83 6477 1.3% 99.6% 43.7% 0.0% 4.6% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Pro123Leu 210 26295 68 6492 1.0% 99.2% 24.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Ala774Ser 141 26364 65 6495 1.0% 99.5% 31.6% 1.8% 6.3% 0.2% 0.1 5) NotAwR WHO 5) Not assoc w R EMB Rv2752c_p.Val300Ala 114 26391 63 6497 1.0% 99.6% 35.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-1193C>T 103 26402 36 6524 0.5% 99.6% 25.9% 1.2% 6.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R aftB_p.Lys522Arg 306 26199 9 5 1 0.4 8 8.7 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R Rv2752c_p.Gly161Ser 113 26392 24 6 36 0.4 9 6 17 5 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R Rv2752c_p.Met31Ile 147 26358 19 6541 0.3 1 4 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R A u951 sp 25 26380 15 6545 0 2 5 10 7 1.8 6.3 0.2 0 1 5) NotAwR WHO 5) Not assoc w R C c -1419G>A 43 263 2 15 6545 0 5 9.5 1.7 6.0 0.2 0 1 5) NotAwR WHO 5) Not assoc w R ubiA_p.Gly268Asp 143 26362 15 6545 0 2 5 9.5 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R Rv2477c_p.Val85Ile 84 26421 13 654 0 7 13 4 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C c -589C>G 88 26417 11 6549 0 2 7 1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C Val104Met 299 26206 10 6550 0 2 8 3.2 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C Arg567His 375 26130 9 6551 0 1 8 6 2.3 0.0 97 5 0.0 0 0 5) Not wR WHO 5) Not assoc w R ubiA_c.-32delG 713 2 92 8 6552 1 97.3 1.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R glpK_p.Leu228Val 349 156 7 5 1 98.7 2.0 NA NA 0.0 NA 5) NotAwR 5) Not assoc w R aftB_p.Ile327Val 194 26311 6 6554 1 3 3.0 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R B p Asn13Ser 92 26313 6 6554 0 1 3 3.0 NA NA 0.0 NA 5) NotAwR WHO 5) Not as oc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 36 3.5 Pyrazinamide As in the first edition, only pncA mutations were classified as markers of PZA resistance in this analysis (Table 9). While only 139 pncA mutations were sufficiently frequent to meet the grading criteria for Group 1, resulting in a combined sensitivity of 63.5% (95% CI: 62.0–64.9) (Table 3), 49 of the 202 identified Group 2 mutations were classified according to the WHO-endorsed LoF additional grading rule (Tables 1 and 23), increasing the sensitivity to 78.0% (95% CI: 76.8–79.2). This represents a 4.3% increase in sensitivity over that in the first edition, with a negligible decrease in specificity and PPV (Table A.1). Notably, the inframe pncA Gly113_Leu116delinsVal indel, which was subject to the LoF grading rule in the first edition but was excluded when we changed the definition of LoF mutations (see Table 23, p. 91), was not associated with resistance on an interim basis (Group 4), confirming that inframe indels are less likely to be markers of resistance. Inclusion of mutations with variant allele frequencies below 75% increased the sensitivity by 2% (Table A.1), suggesting that mixed populations may play a role in observed PZA resistant phenotypes. Most M. bovis isolates are intrinsically resistant to PZA because of the Group 1 pncA His57Asp mutation (58). M. canettii, which is rarely found outside the Horn of Africa, is also intrinsically PZA-resistant (59,60). The genetic basis of this phenotype is unclear, however, and most of the isolates described to date do not have a plausible pncA resistance mutation (61,62). This intrinsic resistance is therefore best inferred by identification of M. canettii (Table 1). Ideally, identification should rely on multiple phylogenetically informative mutations in genes that are not under selection; in practice, however, the choice of markers depends on which parts of the MTBC genome are interrogated by an assay. The 138A>G synonymous mutation at codon 46 of pncA, which has been proposed as a marker for M. canettii in the literature (62), was also observed in some lineage 4 MTBC isolates in this data set. As a result, it was not endorsed as a marker to infer intrinsic PZA resistance for M. canettii (i.e. it was classified as a Group 4 mutation instead). Nevertheless, a footnote was included for this synonymous mutation to indicate that M. canettii may be present, in order to minimize the possibility that intrinsic PZA resistance is missed by assays that analyse pncA but not other loci for differentiating MTBC (Table 9). Potential role of rare variants The sensitivity estimates for pncA mutations are based on “relaxed” grading criteria (Fig. 9) rather than the standard criteria used for all the other drugs (Fig. 8). In the first edition, an additional grading criterion was proposed, but not endorsed, whereby the sensitivity could be increased further by assuming that any coding pncA mutation, except silent and Group 4 or 5 mutations, in pncA was a valid marker of resistance if it occurred in genotypically RIF-resistant isolates. Inferring resistance is the principal approach used for the WHO-endorsed Genoscholar PZA-TB II assay, as it cannot differentiate most pncA mutations (45,63,64). Use of this approach would increase the overall sensitivity by 2.6%, with an accompanying decrease in specificity of 0.7% and a decrease in the PPV of 2.4% (Table A.3). In settings where non-silent but still neutral pncA mutations are more frequent, the decrease in specificity and PPV might be considerably greater (28). This assumption was therefore explored only for the analyses presented in Table A.3 and was not used to generate the master file results. 3 Results for ind ivid ual d rugs 37 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_LoF 42 16780 573 3836 13.0% 99.8% 93.2% 93.6% 95.5% 89.8% 63.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His57Asp 2 16820 166 4243 3.8% 100.0% 98.8% 98.8% 99.9% 95.6% 319.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_c.-11A>G 10 16812 141 4268 3.2% 99.9% 93.4% 93.3% 96.8% 88.1% 55.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His51Arg 5 16817 82 4327 1.9% 100.0% 94.3% 94.3% 98.1% 87.1% 63.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln141Pro 5 16817 75 4334 1.7% 100.0% 93.8% 93.8% 97.9% 86.0% 58.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln10Pro 5 16817 70 4339 1.6% 100.0% 93.3% 93.2% 97.8% 84.9% 53.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val139Ala 5 16817 59 4350 1.3% 100.0% 92.2% 91.9% 97.3% 82.2% 44.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln10Arg 3 16819 57 4352 1.3% 100.0% 95.0% 94.6% 98.9% 85.1% 68.3 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly132Ala 2 16820 57 4352 1.3% 100.0% 96.6% 96.6% 99.6% 88.1% 108.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His57Arg 5 16817 56 4353 1.3% 100.0% 91.8% 91.8% 97.3% 81.9% 43.3 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val131fs 0 16822 54 4355 1.2% 100.0% 100.0% 100.0% 100.0% 93.3% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr76Pro 3 16819 52 4357 1.2% 100.0% 94.5% 94.5% 98.9% 84.9% 66.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His51Asp 1 16821 48 4361 1.1% 100.0% 98.0% 98.0% 99.9% 89.1% 185.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly97Asp 5 16817 46 4363 1.0% 100.0% 90.2% 95.7% 99.9% 61.9% 84.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu27Pro 0 16822 44 4365 1.0% 100.0% 100.0% 100.0% 100.0% 91.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp49Gly 2 16820 43 4366 1.0% 100.0% 95.6% 95.6% 99.5% 84.9% 82.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr135Pro 1 16821 43 4366 1.0% 100.0% 97.7% 97.7% 99.9% 88.0% 165.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Cys14Arg 1 16821 42 4367 1.0% 100.0% 97.7% 97.7% 99.9% 87.7% 161.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu4Ser 3 16819 42 4367 1.0% 100.0% 93.3% 93.0% 98.5% 80.9% 51.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His71Arg 2 16820 38 4371 0.9% 100.0% 95.0% 94.9% 99.4% 82.7% 71.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Glu173fs 0 16822 37 4372 0.8% 100.0% 100.0% 100.0% 100.0% 90.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr153fs 2 16820 35 4374 0.8% 100.0% 94.6% 94.6% 99.3% 81.8% 67.3 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His71Tyr 2 16820 34 4375 0.8% 100.0% 94.4% 94.4% 99.3% 81.3% 65.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val7Gly 2 16820 34 4375 0.8% 100.0% 94.4% 94.1% 99.3% 80.3% 61.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Pro54Leu 2 16820 32 4377 0.7% 100.0% 94.1% 93.8% 99.2% 79.2% 57.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Trp68Gly 2 16820 32 4377 0.7% 100.0% 94.1% 94.1% 99.3% 80.3% 61.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu172Pro 0 16822 31 4378 0.7% 100.0% 100.0% 100.0% 100.0% 88.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ile133Thr 18 16804 29 4380 0.7% 99.9% 61.7% 61.7% 75.5% 46.4% 6.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu120Pro 0 16822 28 4381 0.6% 100.0% 100.0% 100.0% 100.0% 87.7% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ile5Ser 0 16822 27 4382 0.6% 100.0% 100.0% 100.0% 100.0% 87.2% Inf 1) AwR ALL+WHO 1) Assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 3502 3245 62 34 2 8 1 82 6 79 9 81 74 0 22 4 EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62 54 5 56 7 4 3 5.5 EMB embB_p.Gln497Arg 224 35481 999 8482 10.5 4 81 7 7 0 7 5 8 11 3 EMB embB_p.Gly406Ala 199 35506 328 9153 3 5 99.4 62 2 53 0 59 0 3 4 4.4 EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 68 7 64 2 69 49 9 6.9 EMB embB_p.Gly406Asp 212 35493 274 9207 2 9 99.4 56 4 46 9 53 2 31 5 3.4 EMB embB_p.Tyr319Ser 47 35658 204 9277 2 2 99.9 81 3 80 7 85 7 73 0 16 EMB embB_p.Gly406Ser 76 3562 187 9294 2 0 99.8 71 1 61 1 70 34 8 6.0 EMB embB_p.Gln497Lys 51 35654 145 9 36 5 99.9 74 0 53 0 65 4 30 2 4.3 EMB embB_p.Met306Leu 50 35655 145 9 36 5 99.9 74 4 70 4 79 2 48 6 9.1 EMB embB_p.Asp 28Tyr 5 35700 46 943 0 5 90.2 84.0 95.5 60 6 19.9 EMB embB_p.Tyr319Cys 12 2 493 27 6533 0 4 69 2 59 3 77 6 37 2 5.9 WHO EMB embB_p.Gly406Cys 28 35677 36 9445 0 4 99.9 56 3 5 1 73 0 33 3 5.2 EMB embA_c.-12C>T 173 35532 635 8846 6 7 99.5 78 6 30 9 41 1 10 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 94 0 0 2 72.4 .0% 97.5 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1 03 5157 21.4 94.3 48 4 5.4 7.0 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 5 6515 0 7 99.8 48 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 3 651 0 7 99.8 4 8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 16 6544 0 2 99.8 21 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 6544 2 99.8 22 2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 6556 1 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 1 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 482 2 6558 0 99.9 8.0 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 2127 3 63 319 51.3% 80.2 39 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 224 9 516 6044 7 9 84.6 11.2 1.0% 1.7% 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 22 7 24218 332 6228 5 1 1 4 12 0.0 0 8 0.0 0 0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 2265 327 6233 5 0 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6 09 3 8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R Table 9. Abridged variant classification for PZA C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 38 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Asp63Ala 5 16817 26 4383 0.6% 100.0% 83.9% 83.3% 94.4% 65.3% 19.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Tyr103* 0 16822 26 4383 0.6% 100.0% 100.0% 100.0% 100.0% 86.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_c.-11A>C 0 16822 24 4385 0.5% 100.0% 100.0% 100.0% 100.0% 85.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser59Pro 0 16822 24 4385 0.5% 100.0% 100.0% 100.0% 100.0% 85.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ile31Ser 4 16818 23 4386 0.5% 100.0% 85.2% 85.2% 95.8% 66.3% 22.0 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp8Gly 1 16821 22 4387 0.5% 100.0% 95.7% 95.7% 99.9% 78.1% 84.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser67Pro 0 16822 22 4387 0.5% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ala146Val 0 16822 21 4388 0.5% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His71Pro 0 16822 20 4389 0.5% 100.0% 100.0% 100.0% 100.0% 83.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Trp68Cys 0 16822 20 4389 0.5% 100.0% 100.0% 100.0% 100.0% 83.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly97Cys 1 16821 19 4390 0.4% 100.0% 95.0% 94.7% 99.9% 74.0% 69.0 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu151Ser 6 16816 19 4390 0.4% 100.0% 76.0% 75.0% 90.2% 53.3% 11.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Lys96Thr 1 16821 19 4390 0.4% 100.0% 95.0% 95.0% 99.9% 75.1% 72.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val125Gly 0 16822 19 4390 0.4% 100.0% 100.0% 100.0% 100.0% 82.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp12Ala 7 16815 18 4391 0.4% 100.0% 72.0% 72.0% 87.9% 50.6% 9.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp136Gly 6 16816 18 4391 0.4% 100.0% 75.0% 75.0% 90.2% 53.3% 11.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Trp68Arg 1 16821 18 4391 0.4% 100.0% 94.7% 94.7% 99.9% 74.0% 69.0 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Tyr34Asp 0 16822 18 4391 0.4% 100.0% 100.0% 100.0% 100.0% 81.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val180Phe 2 16820 18 4391 0.4% 100.0% 90.0% 90.0% 98.8% 68.3% 34.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Arg154Gly 7 16815 17 4392 0.4% 100.0% 70.8% 70.8% 87.4% 48.9% 9.3 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly108Arg 1 16821 17 4392 0.4% 100.0% 94.4% 94.4% 99.9% 72.7% 65.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr47Ala 11 16811 17 4392 0.4% 99.9% 60.7% 60.7% 78.5% 40.6% 5.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ala146Thr 4 16818 15 4394 0.3% 100.0% 78.9% 78.9% 93.9% 54.4% 14.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu120Arg 0 16822 15 4394 0.3% 100.0% 100.0% 100.0% 100.0% 78.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu182Ser 4 16818 15 4394 0.3% 100.0% 78.9% 77.8% 93.6% 52.4% 13.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu4Trp 1 16821 15 4394 0.3% 100.0% 93.8% 93.8% 99.8% 69.8% 57.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe106fs 2 16820 15 4394 0.3% 100.0% 88.2% 88.2% 98.5% 63.6% 28.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val139Gly 0 16822 15 4394 0.3% 100.0% 100.0% 100.0% 100.0% 78.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp8Asn 0 16822 14 4395 0.3% 100.0% 100.0% 100.0% 100.0% 76.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Cys72Arg 0 16822 14 4395 0.3% 100.0% 100.0% 100.0% 100.0% 76.8% Inf 1) AwR ALL+WHO 1) Assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3 45 6236 34.2 98.1 2 6 79 9 81 5 74 0 22 4 EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62.8 54.5 56.7 45 3 5.5 EMB embB_p.Gln497Arg 224 35481 999 84 2 10.5 99.4 81.7 73.0 76.8 58 8 11.3 EMB embB_p.Gly406Ala 199 35506 328 9153 3 99.4 62.2 53.0 59.0 37 4 4.4 EMB embB_p.Asp354Ala 137 3556 301 91 0 3 2 99.6 68 7 64 69 9 49 9 6.9 EMB embB_p.Gly406Asp 2 2 35493 274 920 2 9 99.4 56 4 46 9 53 2 31 5 3.4 EMB embB_p.Tyr319Ser 47 35658 204 927 2 2 99.9 81.3 80.7 85.7 7 0 16.1 EMB embB_p.Gly 06Ser 76 356 9 187 9294 2 0 99.8 71.1 61.1 70.9 34 8 6.0 EMB embB_p.Gln497Lys 51 35654 145 9 36 1 99.9 74.0 53.0 65.4 30 4.3 EMB embB_p.Met306Leu 50 35655 145 9 36 1 99.9 74.4 70.4 79.2 48 6 9.1 EMB embB_p.Asp328Tyr 5 35700 46 9435 5 0 2 8 0 5 5 60 6 1 9 EMB embB_p.Tyr319Cys 12 2 493 27 6533 69 2 59 3 77 6 37 2 5.9 WHO EMB embB_p.Gly406Cys 28 35677 36 9445 99.9 56 3 58 1 73 0 33 3 5.2 EMB embA_c.-12C>T 173 3553 635 8846 6 7 99.5 78.6 30.9 41.1 10 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 2 4 0.0 9 5 0.0 0 0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3 48 4 5.4 7.0 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 6515 7 99.8 48 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47.8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639S r 57 2 448 6 6544 2 99.8 21 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 6 6544 2 99.8 22 2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 6 6544 2 99.8 21 9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 7 2 434 4 6556 1 7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 1 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Il 23 2 482 2 6558 0 99.9 8.0 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 31 7 51.3% 80.2 39 1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 224 9 516 604 7 9 84.6 11 1.0 1.7 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5 1 91.4 12.7 .0% 70.8 0.0 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 2265 327 6233 5 0 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6 09 3 8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R 3 Results for ind ivid ual d rugs 39 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Gln10His 0 16822 14 4395 0.3% 100.0% 100.0% 100.0% 100.0% 75.3% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Pro69Leu 5 16817 14 4395 0.3% 100.0% 73.7% 73.7% 90.9% 48.8% 10.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr142Ala 2 16820 14 4395 0.3% 100.0% 87.5% 86.7% 98.3% 59.5% 24.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val128Gly 0 16822 14 4395 0.3% 100.0% 100.0% 100.0% 100.0% 76.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ala134Val 1 16821 13 4396 0.3% 100.0% 92.9% 92.9% 99.8% 66.1% 49.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp12Gly 3 16819 13 4396 0.3% 100.0% 81.3% 81.3% 96.0% 54.4% 16.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly97Ser 2 16820 13 4396 0.3% 100.0% 86.7% 86.7% 98.3% 59.5% 24.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe58Leu 4 16818 13 4396 0.3% 100.0% 76.5% 76.5% 93.2% 50.1% 12.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr142Met 1 16821 13 4396 0.3% 100.0% 92.9% 92.3% 99.8% 64.0% 45.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp129_Val131del 0 16822 12 4397 0.3% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His57Tyr 0 16822 12 4397 0.3% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ile6Thr 0 16822 12 4397 0.3% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Met175Val 3 16819 12 4397 0.3% 100.0% 80.0% 78.6% 95.3% 49.2% 14.0 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser164Pro 1 16821 12 4397 0.3% 100.0% 92.3% 92.3% 99.8% 64.0% 45.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr160Pro 0 16822 12 4397 0.3% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ala152fs 0 16822 11 4398 0.2% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln122* 0 16822 11 4398 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Glu127_Asp129del 0 16822 11 4398 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_c.-5delG 3 16819 10 4399 0.2% 100.0% 76.9% 76.9% 95.0% 46.2% 12.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp49Ala 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly97Arg 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu19Pro 1 16821 10 4399 0.2% 100.0% 90.9% 90.9% 99.8% 58.7% 38.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu85Pro 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Lys96Arg 2 16820 10 4399 0.2% 100.0% 83.3% 83.3% 97.9% 51.6% 19.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Lys96Glu 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Met175Arg 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe94Leu 3 16819 10 4399 0.2% 100.0% 76.9% 75.0% 94.5% 42.8% 11.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Pro62Leu 1 16821 10 4399 0.2% 100.0% 90.9% 90.9% 99.8% 58.7% 38.2 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val180Ala 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val9Gly 0 16822 10 4399 0.2% 100.0% 100.0% 100.0% 100.0% 69.2% Inf 1) AwR ALL+WHO 1) Assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t iti l i it i li Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 350 0 32 5 6236 34.2 98.1 82.6 79.9 81.5 4.0 22.4 ) ) EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62.8 54.5 56.7 5.3 5.5 ) ) EMB embB_p.Gln 97Arg 2 4 35481 999 8482 10.5 99.4 1.7 73.0 76.8 8.8 11.3 ) ) EMB embB_p.Gly406Ala 199 35506 328 9153 3.5 99.4 62.2 53.0 59.0 37.4 4.4 ) ) EMB embB_p.Asp 54Ala 137 35568 301 9180 3.2 99.6 68.7 64.2 6 .9 49.9 6.9 ) ) EMB embB_p.Gly406Asp 212 35493 274 9207 2.9 99.4 56.4 46.9 53.2 31.5 3.4 ) ) EMB embB_p.T r319Ser 47 35658 204 9277 2.2 99.9 1.3 0. 85.7 73.0 16.1 ) ) EMB embB_p.Gly406Ser 76 35629 187 92 4 2.0 99.8 1.1 61.1 70.9 34.8 6.0 ) ) EMB embB_p.Gln 97Lys 51 35654 145 9 3 1.5 99.9 74.0 53.0 65.4 30.2 4.3 ) ) EMB embB_p.Met306Leu 50 35655 145 9 36 1.5 99.9 74.4 70.4 79.2 48.6 9.1 ) ) EMB embB_p.A p328Tyr 5 35700 46 9435 .5 . 90.2 84.0 95.5 60.6 19.9 ) ) EMB embB_p.Tyr319Cys 12 2 493 27 6533 .4 . 69.2 59.3 77.6 37.2 5.9 ) WHO ) EMB embB_p.Gly406Cys 28 35677 36 9445 .4 99.9 56.3 5 .1 73.0 33.3 5.2 ) ) EMB embA_c.-12C>T 173 35532 635 8846 6.7 99.5 78.6 30.9 41.1 10.2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 .2 . 72.4 .0% 97.5 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3 48.4 5.4% 7.0% 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 6515 .7 99.8 48.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 .7 99.8 47.8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 6 6544 . 99.8 21. NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 6 6544 . 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 6 6544 . 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 6556 .1 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 .1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 .0 99.9 6.1 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 48 2 6558 .0 99.9 8.0% NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp39 Gly 5235 21270 3363 31 7 51.3% 80.2 39.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val 81Leu 4076 2242 516 6044 7.9 84.6 11.2 1.0 1.7 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1 91.4 12.7 0.0 70. 0.0 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 2265 327 6233 5.0 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6 0 3.8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 40 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Asp129fs 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp136fs 1 16821 9 4400 0.2% 100.0% 90.0% 90.0% 99.7% 55.5% 34.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp8Glu 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Cys138Arg 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Glu144fs 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly132Ser 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu85Arg 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Met1? 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe94Cys 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser84fs 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Tyr103Cys 4 16818 9 4400 0.2% 100.0% 69.2% 69.2% 90.9% 38.6% 8.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val155Gly 1 16821 9 4400 0.2% 100.0% 90.0% 90.0% 99.7% 55.5% 34.4 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val180Gly 0 16822 9 4400 0.2% 100.0% 100.0% 100.0% 100.0% 66.4% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ala102Pro 1 16821 8 4401 0.2% 100.0% 88.9% 87.5% 99.7% 47.3% 26.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His51Gln 1 16821 8 4401 0.2% 100.0% 88.9% 88.9% 99.7% 51.8% 30.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His51Pro 0 16822 8 4401 0.2% 100.0% 100.0% 100.0% 100.0% 63.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu159Arg 2 16820 8 4401 0.2% 100.0% 80.0% 80.0% 97.5% 44.4% 15.3 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr177Pro 1 16821 8 4401 0.2% 100.0% 88.9% 88.9% 99.7% 51.8% 30.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr76Ile 2 16820 8 4401 0.2% 100.0% 80.0% 87.5% 99.7% 40.0% 26.8 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val128fs 0 16822 8 4401 0.2% 100.0% 100.0% 100.0% 100.0% 63.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val139Met 0 16822 8 4401 0.2% 100.0% 100.0% 100.0% 100.0% 63.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val155Met 1 16821 8 4401 0.2% 100.0% 88.9% 88.9% 99.7% 51.8% 30.6 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val7Leu 0 16822 8 4401 0.2% 100.0% 100.0% 100.0% 100.0% 63.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_c.-12T>C 1 16821 7 4402 0.2% 100.0% 87.5% 87.5% 99.7% 47.3% 26.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp86fs 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln10* 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gln141* 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ile90Ser 3 16819 7 4402 0.2% 100.0% 70.0% 70.0% 93.3% 34.8% 8.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu156fs 1 16821 7 4402 0.2% 100.0% 87.5% 87.5% 99.7% 47.3% 26.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser104Arg 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 350 0 3245 6236 34.2 98.1 82.6 79.9 81.5 74 0 22.4 EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62 8 54 5 56 4 3 5.5 EMB embB_p.Gln497Arg 224 35481 9 9 8 82 10.5 99.4 81.7 73.0 76.8 58 8 11.3 EMB embB_p.Gly406 la 199 35506 328 9153 3 5 99.4 62.2 53.0 59.0 37 4.4 EMB embB_p.Asp354Ala 137 35568 301 918 3 99.6 68.7 64.2 69.9 49 9 6.9 EMB embB_p.Gly406Asp 212 35493 274 92 7 2 9 99.4 56.4 46.9 53.2 31 5 3.4 EMB embB_p.Tyr319Ser 47 35658 204 9277 2 99.9 81.3 80.7 85.7 73 0 16.1 EMB embB_p.Gly406Ser 76 356 9 187 9294 2 0 99.8 71.1 61.1 70.9 34 8 6.0 EMB embB_p.Gln497Lys 51 35654 145 9336 1 5 99.9 74.0 53.0 65.4 30 2 4.3 EMB embB_p.Met306Leu 50 35655 145 9336 1 5 99.9 74.4 70.4 79.2 48 6 9.1 EMB embB_p.Asp328Tyr 5 35700 46 9 35 5 90 84 0 5 5 60 19.9 EMB embB_p.Tyr319C s 12 2 493 27 6533 4 69 2 59 3 77 6 37 2 5.9 WHO EMB embB_p.Gly4 6C s 28 35677 36 9 45 4 99.9 56.3 58.1 73.0 33 3 5.2 EMB embA_c.-12C>T 173 35532 635 8846 6 7 99.5 7 6 30 9 41 1 10 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9 60 72 4 0.0 7 5 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3 48.4 5.4% 7.0% 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 6515 7 99.8 48 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47 8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro 39Ser 57 2 448 16 6544 99.8 21 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 6544 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 6556 1 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val6 8Ile 23 2 48 2 6558 0 99.9 8.0% NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2 39.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 224 9 516 6044 7 9 84.6 11.2 1.0% 1.7% 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 242 8 332 6228 5 1 91.4 12 7 0.0 70 8 0.0 0 0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5 0 85.5 7.8 1.1 1.6 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 63 9 3 8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R 3 Results for ind ivid ual d rugs 41 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Tyr103His 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Tyr64* 1 16821 7 4402 0.2% 100.0% 87.5% 87.5% 99.7% 47.3% 26.7 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Val130fs 0 16822 7 4402 0.2% 100.0% 100.0% 100.0% 100.0% 59.0% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp12Glu 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Asp63fs 1 16821 6 4403 0.1% 100.0% 85.7% 85.7% 99.6% 42.1% 22.9 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly105Asp 2 16820 6 4403 0.1% 100.0% 75.0% 75.0% 96.8% 34.9% 11.5 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Leu182Trp 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe13Ile 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Pro54Arg 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Ser66Pro 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Trp119* 0 16822 6 4403 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Arg154Met 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Gly24Asp 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His51Tyr 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.His82Arg 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Lys96Gln 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Phe13Ser 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr47Pro 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Thr61fs 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Trp68* 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL+WHO 1) Assoc w R PZA pncA_p.Lys48Thr 26 16796 20 4389 0.5% 99.8% 43.5% 43.5% 58.9% 28.9% 2.9 1) AwR ALL ALL only 2) Assoc w R - Interim PZA pncA_p.Asp126_Val130del 1 16821 12 4397 0.3% 100.0% 92.3% 66.7% 99.2% 9.4% 7.7 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Ala146Glu 0 16822 8 4401 0.2% 100.0% 100.0% 100.0% 100.0% 63.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim PZA pncA_p.Asp63Gly 5 16041 6 3780 0.2% 100.0% 54.5% 54.5% 83.3% 23.4% 5.1 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Leu116Pro 4 16818 6 4403 0.1% 100.0% 60.0% 42.9% 81.6% 9.9% 2.9 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val7Ala 3 16819 6 4403 0.1% 100.0% 66.7% 62.5% 91.5% 24.5% 6.4 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp12Asn 1 16045 5 3781 0.1% 100.0% 83.3% 80.0% 99.5% 28.4% 17.0 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Val125Phe 3 16043 5 3781 0.1% 100.0% 62.5% 62.5% 91.5% 24.5% 7.1 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Asp49Asn 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL ALL only 2) Assoc w R - Interim PZA pncA_p.His57Leu 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL ALL only 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 350 0 3245 6236 34.2 98.1 82.6 79.9 81.5 74 22.4 EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62 8 54 56 5 5.5 EMB embB_p.Gln497Arg 224 35481 999 8 8 10.5 99.4 81.7 73.0 76.8 8 8 11.3 EMB embB_p.Gly406Ala 199 35506 328 915 3 5 99.4 62.2 53.0 59.0 37 4 4.4 EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 68 64 2 6 9 9 9 6.9 EMB embB_p.Gly406Asp 212 35493 274 92 7 2 9 99.4 56 4 46 9 53 2 1 5 3.4 EMB embB_p.Tyr319Ser 47 35658 204 9277 2 2 99.9 81.3 80.7 85.7 73 0 16.1 EMB embB_p.Gly406Ser 76 356 9 187 9294 2 0 99.8 71.1 61.1 70.9 3 8 6.0 EMB embB_p.Gln497Lys 51 35654 145 9336 1 5 99.9 74.0 53.0 65.4 30 2 4.3 EMB embB_p.Met306Leu 50 35655 145 9336 1 5 99.9 74.4 70.4 79.2 48 6 9.1 EMB embB_p.Asp328Tyr 5 35700 46 9 35 5 90.2 84.0 95.5 60 6 19.9 EMB embB_p.Tyr319Cys 12 2 493 27 6533 4 69.2 59.3 77.6 3 2 5.9 WHO EMB embB_p.Gly406Cys 28 35677 36 9 45 4 99.9 56 58 1 73 0 3 3 5.2 EMB embA_c.-12C>T 173 3553 635 8846 6 7 99.5 78.6 30.9 41.1 10 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9 60 2 72.4 .0% 97.5 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3 48 4 5.4 7.0 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly 56Cys 47 2 458 45 6515 7 99.8 48 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47.8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 16 654 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 654 2 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 2 21 9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 6556 1 99.7 5.3 NA NA 0 0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1 NA NA 0.0 NA 5) NotAwR Pr v. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 482 2 6558 0 99.9 8.0 NA NA 0 0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2 39 1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7 9 84.6 11 2 1.0 1.7 0.2 0.0 5) NotAwR 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5 91.4 1 7 0.0 70 8 0.0 0 0 5) NotAwR 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5 0 85.5 7.8 1.1 1.6 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 2 1 63 9 3 8 87.2 6.9 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 42 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Val131Gly 1 16821 5 4404 0.1% 100.0% 83.3% 83.3% 99.6% 35.9% 19.1 1) AwR ALL ALL only 2) Assoc w R - Interim PZA pncA_p.Val93Leu 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim PZA pncA_p.Leu159Pro 0 16822 5 4404 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Leu35Pro 3 16819 5 4404 0.1% 100.0% 62.5% 62.5% 91.5% 24.5% 6.4 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Phe13Leu 1 16821 5 4404 0.1% 100.0% 83.3% 75.0% 99.4% 19.4% 11.5 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Trp119Cys 1 16821 5 4404 0.1% 100.0% 83.3% 100.0% 100.0% 19.4% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Leu116Arg 0 16046 4 3782 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Met175Thr 0 16046 4 3782 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Thr168Pro 0 16046 4 3782 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Trp119Arg 0 16046 4 3782 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Gly108Glu 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_c.-11A>T 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala171Glu 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala46Glu 1 16821 4 4405 0.1% 100.0% 80.0% 80.0% 99.5% 28.4% 15.3 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala46Val 1 16821 4 4405 0.1% 100.0% 80.0% 80.0% 99.5% 28.4% 15.3 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly105Val 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly162Asp 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly17Asp 1 16821 4 4405 0.1% 100.0% 80.0% 80.0% 99.5% 28.4% 15.3 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly78Asp 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly78Val 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.His137Pro 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Leu120Gln 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Lys96Asn 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Met175Ile 4 16818 4 4405 0.1% 100.0% 50.0% 50.0% 84.3% 15.7% 3.8 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro62Arg 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ter187Trpext*? 1 16821 4 4405 0.1% 100.0% 80.0% 80.0% 99.5% 28.4% 15.3 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Thr61Pro 2 16820 4 4405 0.1% 100.0% 66.7% 60.0% 94.7% 14.7% 5.7 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val131Phe 1 16821 4 4405 0.1% 100.0% 80.0% 80.0% 99.5% 28.4% 15.3 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val44Gly 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val7Phe 0 16822 4 4405 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met 06Val 685 350 0 3245 6236 34.2 98.1 2 6 79 9 81 5 74 0 22 4 ALL+WHO 1) Assoc w R EMB embB_p.Met 06Ile 1159 34546 1953 7528 20.6 96.8 62.8 54.5 56.7 5 3 5.5 ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8 82 10.5 99.4 81.7 73.0 76.8 58 11.3 1) 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3 5 99.4 2 53 0 59 0 37 4 4 1) 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 68 7 64 2 6 9 4 9 6.9 1) 1) Assoc w R EMB embB_p.Gly406Asp 2 2 35493 274 92 7 2 9 99.4 56 4 46.9 53.2 31 5 3.4 1) 1) Assoc w R EMB embB_p.Tyr3 9Ser 47 35658 204 9277 2 2 99.9 81.3 80.7 85.7 73 0 16.1 1) ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2 0 99.8 71.1 61.1 70.9 4 6.0 1) ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 1 5 9336 1 5 99.9 74.0 53.0 65.4 0 2 4.3 1) ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 1 5 9336 1 5 99.9 74.4 70.4 79.2 48 6 9.1 1) ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9 3 5 90.2 84.0 95.5 60 6 19.9 1) ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 2 493 27 6533 4 69.2 59.3 77.6 7 2 5.9 1) WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9 4 4 99.9 56.3 58.1 73.0 3 3 5.2 1) 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6 7 99.5 78 6 3 9 41 1 10 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9 60 2 72 4 0.0 7 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4 94.3 48.4 5.4% 7.0% 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 651 7 99.8 48.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47 8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 6544 2 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 6556 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val4 Ile 72 2 433 6556 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.S r1054Pro 31 2 474 2 6558 0 99.9 6.1 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 48 2 6558 0 99.9 8.0% NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3 80.2 39 1 NA NA 0.0 NA 5) NotAwR WHO 5) N t assoc w R EMB embC_p.Val981Leu 4076 224 9 516 6044 7 9 84.6 11 2 1.0 1.7 0.2 0 0 5) NotAwR WHO 5) N t assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5 91.4 12 7 0.0 70 8 0.0 0.0 5) NotAwR WHO 5) N t assoc w R EMB embC_p.Arg738Gln 3853 2265 327 6233 5 0 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) N t assoc w R EMB embB_p.Glu378Ala 3386 23119 251 63 9 3 8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) N t assoc w R 3 Results for ind ivid ual d rugs 43 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Trp119Gly 0 16046 3 3783 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Val21Gly 0 16046 3 3783 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.His43Pro 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Ile90Thr 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_c.-7T>C 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala143Gly 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala3Glu 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp129Asn 1 16821 3 4406 0.1% 100.0% 75.0% 75.0% 99.4% 19.4% 11.5 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp8Ala 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp8Tyr 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly78Ser 2 16820 3 4406 0.1% 100.0% 60.0% 60.0% 94.7% 14.7% 5.7 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly97Val 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Leu159Val 2 16820 3 4406 0.1% 100.0% 60.0% 60.0% 94.7% 14.7% 5.7 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Phe106Ser 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Phe94Ser 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro54Gln 1 16821 3 4406 0.1% 100.0% 75.0% 75.0% 99.4% 19.4% 11.5 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro54Ser 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro62Ser 2 16820 3 4406 0.1% 100.0% 60.0% 75.0% 99.4% 14.7% 11.5 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro62Thr 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Thr100Pro 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Trp119Leu 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Tyr64Asp 2 16820 3 4406 0.1% 100.0% 60.0% 60.0% 94.7% 14.7% 5.7 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val130Gly 0 16822 3 4406 0.1% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val163Ala 1 16821 3 4406 0.1% 100.0% 75.0% 75.0% 99.4% 19.4% 11.5 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Arg140Pro 0 16046 2 3784 0.1% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI WHO Interim on WHO 2) Assoc w R - Interim PZA pncA_p.Phe58Cys 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Thr142Pro 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Thr160Lys 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Trp68Leu 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim PZA pncA_p.Val130Met 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35 20 3245 6236 34.2 98.1 82.6 79.9 81.5 74 0 22.4 1) ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 345 1953 7528 20.6 96.8 62.8 54.5 56.7 45 3 5.5 1) ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8 82 10.5 99.4 81.7 73.0 76.8 58 11.3 1) ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3 5 99.4 62.2 53.0 59.0 37 4 4.4 1) ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 68.7 64.2 69.9 4 9 6.9 1) 1) Assoc w R EMB embB_p.Gly 06Asp 212 35493 274 92 7 2 9 99.4 56.4 46.9 53.2 31 5 3.4 1) 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2 2 99.9 81.3 80.7 85.7 73 0 16.1 1) 1) Assoc w R EMB embB_p.Gly406Ser 76 356 9 187 9294 2 0 99.8 1 1 61 1 70 9 34 8 6.0 1) 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 933 1 5 99.9 74.0 53.0 65.4 30 4.3 1) 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 933 1 5 99.9 74.4 70.4 79.2 48 6 9.1 1) 1) Assoc w R EMB embB_p.Asp328Tyr 5 3570 46 9 35 5 9 2 84 5 5 60 6 19.9 1) 1) Assoc w R EMB embB_p.T r319Cys 12 2 493 27 6533 4 69.2 59.3 77.6 37 5.9 1) WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9 45 4 99.9 56 3 58 1 73 0 33 3 2 1) 1) Assoc w R EMB embA_c.-12C>T 173 3553 6 5 884 6 7 99.5 78.6 30.9 41.1 10 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9 60 2 72.4 .0% 97.5 0.0 0.0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4 94.3 48 4 5.4 7.0 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 6515 7 99.8 48.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47 8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 6544 2 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 655 99.7 5.3 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 655 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 482 6558 0 99.9 8.0% NA NA 0.0 NA 5) NotAwR Li . (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 319 51.3 80.2 39.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 224 9 516 6044 7 9 84.6 11.2 1.0% 1.7% 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5 1 91.4 12.7 .0% 70.8 0.0 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 2265 3 7 6233 5 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu 78Ala 3386 23119 251 63 9 3 8 87.2 6.9% NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 44 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Ala102Thr 2 16820 2 4407 0.0% 100.0% 50.0% 50.0% 93.2% 6.8% 3.8 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala171Pro 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ala178_Ser179del 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Arg121Pro 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp136Val 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp49Glu 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp63_Ser67delinsGlu 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Asp8His 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Cys14Trp 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Cys72Tyr 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly132Asp 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Gly132Cys 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.His57Gln 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ile90Asn 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Lys48Glu 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Phe106Leu 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Phe58Ser 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Pro54Thr 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ser104_Gly108delinsArg 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ser104Gly 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ser104Ile 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ter187Argext*? 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Thr114Pro 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Thr135Asn 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Thr142Lys 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Tyr103Asp 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Tyr34Ser 0 16822 2 4407 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Tyr95Asp 2 16820 2 4407 0.0% 100.0% 50.0% 50.0% 93.2% 6.8% 3.8 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val139Leu 1 16821 2 4407 0.0% 100.0% 66.7% 66.7% 99.2% 9.4% 7.6 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Val155Ala 2 16820 2 4407 0.0% 100.0% 50.0% 50.0% 93.2% 6.8% 3.8 2) AwRI ALL+WHO 2) Assoc w R - Interim D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 350 3245 6236 34.2 98.1 82 6 79 9 81 5 74.0 22.4 1) 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62.8 54.5 56.7 4 3 5.5 1) 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8 82 10.5 99.4 81.7 73.0 76.8 58 11.3 1) 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 3 8 9153 3 5 99.4 62.2 53.0 59.0 37 4 4.4 1) 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 68.7 64.2 69.9 49 9 6.9 1) 1) Assoc w R EMB embB_p.Gly 06Asp 212 35493 274 92 2 9 99.4 56.4 46.9 53.2 31 5 3.4 1) 1) Assoc w R EMB embB_p.Tyr319 47 35658 204 927 2 2 99.9 81 3 80 85 7 73.0 16.1 1) 1) Assoc w R EMB embB_p.Gly406Ser 76 356 9 187 9294 2 99.8 71.1 61.1 70.9 34 6.0 1) 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1 5 99.9 74 0 53 0 65 4 30.2 4 3 1) 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1 5 99.9 74.4 70.4 79.2 48 6 9.1 1) 1) Assoc w R EMB embB_p.Asp 8Tyr 5 35700 46 9 35 5 90.2 84.0 95.5 60 6 19.9 1) 1) Assoc w R EMB embB_p.T r319Cys 12 2 493 27 6533 4 69.2 59.3 77.6 37 2 5.9 1) WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9 45 4 99.9 56.3 58.1 73.0 33 3 5.2 1) 1) Assoc w R EMB embA_c.-12C>T 173 3553 635 8846 6 7 99.5 78.6 30.9 41.1 0 2 1.8 3) Uncertain 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9 60 2 72 4 0.0 7 5 0 0 0 0 3) Uncertain 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 515 21.4 94.3 48.4 5.4% 7.0% 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 2 458 45 6515 7 99.8 48.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 2 458 43 651 7 99.8 47 8 NA NA 0 0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 2 449 16 6544 2 99.8 22 2 NA NA 0 0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 2 448 16 6544 2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 2 434 4 6556 1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 2 433 4 6556 1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 2 474 6558 99.9 6.1% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 2 48 6558 99.9 8.0% NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 319 51.3 80.2 39 1 NA NA 0 0 NA 5) NotAwR WHO 5) N t assoc w R EMB embC_p.Val981Leu 4076 224 9 516 6044 7 9 84.6 11.2 1.0% 1.7% 0.2 0.0 5) NotAwR WHO 5) N t assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5 1 91.4 12 7 0.0 70 8 0 0 0 0 5) NotAwR WHO 5) N t assoc w R EMB embC_p.Arg738Gln 3853 22652 3 7 6233 5 85.5 7.8 1.1 1.6 0 5 0 0 5) NotAwR WHO 5) N t assoc w R EMB embB_p.Glu378Ala 3386 23119 251 63 9 3 8 87.2 6.9 NA NA 0 0 NA 5) NotAwR WHO 5) N t assoc w R 3 Results for ind ivid ual d rugs 45 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Val93Ala 2 16820 2 4407 0.0% 100.0% 50.0% 50.0% 93.2% 6.8% 3.8 2) AwRI ALL+WHO 2) Assoc w R - Interim PZA pncA_p.Ile31Thr 2 16820 1 4408 0.0% 100.0% 33.3% 33.3% 90.6% 0.8% 1.9 3) Uncertain ALL+WHO Lit. (PMID 32571824) 2) Assoc w R - Interim PZA pncA_p.Ala171Val 6 16816 3 4406 0.1% 100.0% 33.3% 33.3% 70.1% 7.5% 1.9 3) Uncertain ALL+WHO 3) Uncertain significance PZA Rv1258c_p.Gly88fs 37 16785 3 4406 0.1% 99.8% 7.5% 0.0% 10.3% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Phe81Val 5 16817 2 4407 0.0% 100.0% 28.6% 28.6% 71.0% 3.7% 1.5 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ala165del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ala28_Leu172del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ala3_Ile5del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Asp110_Asn112del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Asp129del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Asp136_Leu156del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Asp136Asn 3 16819 1 4408 0.0% 100.0% 25.0% 25.0% 80.6% 0.6% 1.3 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Glu107del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Gly132_Thr135del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Gly150_Leu151insAspAla ValArgAsnGly 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Gly17_Ala25del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Gly55del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Gly60_Thr61del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ile6_Val7del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Leu172Arg 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Leu19_Val21del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Lys48_Val155delinsMet 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Phe81_His82del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Pro54_Asp56del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ser18_Val44del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ser66del 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Tyr103Ser 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Val155dup 0 16822 1 4408 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ala79Val 2 16820 0 4409 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Ser74_Pro77del 1 16821 0 4409 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met 06Val 685 350 3245 6236 34.2 98.1 82 6 79 9 81 5 74.0 22.4 1) 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 752 20.6 96.8 62 8 54 5 56 7 45.3 5 5 1) AwR 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8 82 10.5 99.4 81 7 7 0 6 8 58.8 11.3 1) AwR 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3 5 4 62.2 53.0 59 0 37.4 4 4 1) AwR 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 99.6 6 7 64 2 69 9 49.9 6 9 1) AwR 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 92 7 2 9 99.4 56.4 46.9 53.2 31.5 3.4 1) AwR 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2 2 99.9 81.3 80.7 85.7 73.0 16.1 1) AwR 1) Assoc w R EMB embB_p.Gly406Ser 76 356 9 187 9294 2 99.8 71.1 61.1 70.9 34.8 6.0 1) AwR 1) Assoc w R EMB embB_p.Gln497Ly 51 35654 145 9336 1 5 99.9 74.0 53.0 65.4 30.2 4.3 1) AwR 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1 5 99.9 74.4 70.4 79.2 48.6 9.1 1) AwR 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9 35 5 90.2 84.0 95.5 60.6 19.9 1) AwR 1) Assoc w R EMB embB_p.Tyr319Cys 12 2 493 27 6533 4 69 2 59 3 77 37.2 5 9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9 45 4 99.9 56.3 58.1 73.0 33.3 5.2 1) AwR 1) Assoc w R EMB embA_c.-12C>T 173 3553 635 8846 6 7 99.5 78.6 30.9 41.1 10.2 1.8 EMB embB_p.Leu74Arg 8 35697 21 9 60 2 72.4 .0% 97.5 0 0 0.0 EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7 99.8 48.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7 99.8 47.8 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2 99.8 22.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468 la 57 26448 16 6544 0.2 99.8 21.9 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1 99.7 5.3% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0 99.9 6.1% NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0 99.9 8.0% NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3 80.2 39.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9 84.6 11.2 1.0% 1.7% 0.2 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1 91.4 12.7 0.0% 70.8 0.0 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0 85.5 7.8% 1.1% 1.6% 0.5 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8 87.2 6.9 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 46 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA pncA_p.Thr160_Ala171del 1 16821 0 4409 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Thr168_Val169del 1 16821 0 4409 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance PZA pncA_p.Val7del 1 16821 0 4409 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance PZA Rv3236c_p.Thr102Ala 2934 13112 1976 1810 52.2% 81.7% 40.2% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim PZA clpC1_p.Val63Ala 1261 14785 241 3545 6.4% 92.1% 16.0% 11.1% 13.0% 8.4% 0.5 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim PZA rpsA_p.Met432Thr 545 15501 158 3628 4.2% 96.6% 22.5% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim PZA Rv1258c_p.Pro414Ser 113 15933 14 3772 0.4% 99.3% 11.0% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim PZA pncA_p.Ala102Val 11 16035 5 3781 0.1% 99.9% 31.3% 31.3% 58.7% 11.0% 1.9 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Leu35Arg 30 16016 4 3782 0.1% 99.8% 11.8% 9.1% 24.3% 1.9% 0.4 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Phe58Val 12 16034 3 3783 0.1% 99.9% 20.0% 14.3% 42.8% 1.8% 0.7 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Thr160Ala 3 16043 3 3783 0.1% 100.0% 50.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.His137Arg 5 16041 2 3784 0.1% 100.0% 28.6% 28.6% 71.0% 3.7% 1.7 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_c.-3_-2insC 10 16036 1 3785 0.0% 99.9% 9.1% 0.0% 30.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Ile6Leu 18 16028 1 3785 0.0% 99.9% 5.3% 0.0% 18.5% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Val21Ala 6 16040 1 3785 0.0% 100.0% 14.3% 0.0% 52.2% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_c.-33G>A 5 16041 0 3786 0.0% 100.0% 0.0% 0.0% 52.2% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Ala79Thr 3 16043 0 3786 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Glu15Gly 3 16043 0 3786 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Gly113_Leu116delinsVal 3 16043 0 3786 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Ser66Leu 8 16038 0 3786 0.0% 100.0% 0.0% 0.0% 36.9% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Thr114Met 3 16043 0 3786 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Thr168Ile 3 16043 0 3786 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_p.Thr87Met 17 16029 0 3786 0.0% 99.9% 0.0% 0.0% 19.5% 0.0% 0.0 4) NotAwRI WHO 4) Not assoc w R - Interim PZA pncA_c.138A>G 0 16822 3 4406 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 4) Not assoc w R - Interim Q PZA PPE35_p.Leu896Ser 7025 9021 2563 1223 67.7% 56.2% 26.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA panD_c.-1937C>T 82 15964 164 3622 4.3% 99.5% 66.7% 0.0% 5.9% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R PZA Rv3236c_p.Ala370Thr 516 15530 95 3691 2.5% 96.8% 15.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Pro822Ser 920 15126 92 3694 2.4% 94.3% 9.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Gly258Asp 470 15576 87 3699 2.3% 97.1% 15.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA Rv3236c_p.Val151Ala 425 15621 87 3699 2.3% 97.4% 17.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met3 6Val 685 350 0 3245 6236 34.2 98.1 82.6 79.9 81 74.0 22.4 1) AwR 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 96.8 62.8 54.5 56 7 45.3 5 5 1) AwR 1) Assoc w R EMB embB_p.Gln497Arg 224 3548 999 8 82 10.5 99.4 81.7 73.0% 76.8% 58.8 11.3 1) AwR 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3.5 99 4 62 53.0% 59.0% 37.4 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB Asp354Ala 137 35568 30 9180 3 2 9 6 68 7 64 2 69 9 49.9 6 9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 3 493 274 9207 2 9 9 4 56 4 46.9% 53.2% 31.5 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 3 658 204 92 7 2 2 9 8 3 80.7% 85.7% 73.0 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2 0 8 7 1 6 1 70 9 34 8 6 0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 1 5 9336 1 5 9 74 0 53.0 65 4 30.2 4 3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.M t306Leu 50 35655 145 9336 1 5 74 4 70 4 79 2 48.6 9 1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 5 9 2 84.0 95 5 60.6 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 2 493 27 6533 4 69 2 59 3 7 6 37.2 5 9 1) AwR 1) Assoc w R EMB embB_p.Gly406 ys 28 35677 36 944 4 56.3 58.1 73 0 33.3 5 2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6 7 5 78.6 30.9 41 1 10.2 1 8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 2 72 4 97 5 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25 07 1403 5157 21.4 94.3 48.4 5 4 7.0 2 7 3 5) t Lit. (PMID 32143680) EMB embB_p.Gly156Cys 47 2 458 45 6515 7 99.8 48.9 NA NA NA 5) t Lit. (PMID 32143680) EMB embA_p.Ala813Gly 47 2 458 43 6517 7 99.8 47.8 NA NA NA 5) t Lit. (PMID 32143680) EMB embA_p.Pro6 9S r 57 2 4 8 16 6544 2 99.8 21.9 NA NA NA 5) t Lit. (PMID 32143680) EMB embC_c.-20A>C 56 2 449 16 6544 2 99.8 22.2 NA NA NA 5) t Lit. (PMID 32143680) EMB embA_p.Val468Ala 57 2 4 8 16 6544 2 99.8 21.9 NA NA NA 5) t Prev. WHO EMB embC_c.-1520C>A 71 2 434 4 655 1 99.7 5 3 NA NA NA 5) t Prev. WHO EMB ubiA_p.Val49Ile 72 2 433 4 655 1 7 5 3 NA NA NA 5) t Prev. WHO EMB embB_p.Ser1054Pro 31 2 474 2 6558 0 99.9 6.1% 0.0% 5) NotAwR WHO Prev. WHO EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99 9 8.0% Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 197 51.3 80 2 39 1 NA NA NA EMB embC_p.Val981Leu 4076 22429 516 6044 7 9 84 6 1 2 1.0% 1.7% 2 0.0 EMB embC_c.-565C>T 2287 24218 332 6228 5 1 1 4 12.7 0.0% 70.8% 0.0 EMB embC_p.Arg738Gln 3853 22652 327 6233 5 0 85 5 7.8% 1.1% 1.6% 5 0.0 EMB embB_p.Glu378Ala 3386 23119 251 630 3 8 8 2 6.9 3 Results for ind ivid ual d rugs 47 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 PZA PPE35_p.Thr712Pro 55 15991 37 3749 1.0% 99.7% 40.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA clpC1_p.Asp326Asn 428 15618 32 3754 0.8% 97.3% 7.0% 1.6% 3.3% 0.6% 0.1 5) NotAwR WHO 5) Not assoc w R PZA Rv3236c_c.-520A>G 430 15616 32 3754 0.8% 97.3% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA sigE_p.Arg8Trp 126 15920 23 3763 0.6% 99.2% 15.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA clpC1_p.Pro796Leu 178 15868 17 3769 0.4% 98.9% 8.7% 2.8% 6.5% 0.9% 0.1 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Ile15Met 171 15875 17 3769 0.4% 98.9% 9.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Gly51Glu 317 15729 15 3771 0.4% 98.0% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Pro670Leu 286 15760 10 3776 0.3% 98.2% 3.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA PPE35_p.Ser948Ile 38 16008 0 3786 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA rpsA_p.Ile70Leu 47 15999 0 3786 0.0% 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R PZA Rv1258c_p.Glu243Ala 41 16005 0 3786 0.0% 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 3 020 3245 6236 34.2 8 1 82 6 79.9% 81.5% 74.0 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB Met 0 Ile 1159 34546 1953 7528 20.6 6 8 62.8 54.5 56.7 45.3 5 5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497 rg 224 3 481 999 8482 10.5 9 4 81.7 73.0% 76.8% 58.8 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 99 3 506 328 915 3 5 4 62 2 53.0% 59.0% 37.4 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB Asp354Ala 37 3 5 301 9180 3 2 9 6 68.7 64.2 69.9 49.9 6 9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.G y406Asp 212 3 493 274 9207 2 9 9 4 56.4 46.9% 53.2% 31.5 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 3 658 204 92 7 2 2 9 9 81.3 80.7% 85.7% 73.0 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 3 629 187 9294 2 0 9 8 71.1 61.1% 70.9% 34.8 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln4 7Lys 51 35654 145 933 1 5 9 74.0 53.0% 65.4% 30.2 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 3 655 145 933 1 5 9 74.4 70.4% 79.2% 48.6 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 357 0 46 9435 5 100.0 90.2 84.0% 95.5% 60.6 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7% 99.8% 48.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7% 99.8% 47.8% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0% 99.9% 6.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R Individual LoF mutations in the coding regions of pncA that are classified in Group 2 because of the associated additional grading rule (see Table 23 and Section 5.8) and silent mutations, except for pncA 138A>G, are not listed in this table but can be found in the catalogue master file. Q Consider that this variant might indicate that the isolate is M. canettii, which is intrinsically PZA-resistant. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 48 3.6 Levofloxacin and moxifloxacin The most significant change to fluoroquinolone (FQ) resistance mutations in the second edition is upgrading of gyrA Asp89Asn and three gyrB mutations (Ser447Phe, Asn499Thr and Asp461His; Tables 10 and 11). These mutations were Group 3 in the first edition but, with additional evidence, are now recognized as either Group 1 or 2 mutations for LFX and/or MFX, increasing the overall sensitivity of the combined Groups 1 and 2 mutations by 1.1% to 84.8% (95% CI: 83.9–85.7) for LFX and by 0.7% to 85.7% (95% CI: 84.6–86.8) for MFX when variants at allele frequencies ≥ 75% are called (Table A.1). Lowering the cut-off for calling variants to allele frequency of 25% increased the sensitivity of these mutations for predicting resistant phenotypes by approximately 4.5% for both drugs (Table A.1), which confirmed published evidence that heteroresistance plays an important role in FQ resistance (65,66). All gyrA and gyrB mutations were classified as conferring low-level resistance to MFX, except for the high-level resistance gyrA mutations Gly88Cys, Asp94Asn, Asp94Gly, Asp94His and Asp94Tyr (Table 1). Genetically linked low-level resistance mutations probably have additive effects and should be considered high-level resistant (Table 1). Table 10. Abridged variant classification for LFX D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 LFX gyrA_p.Asp94Gly 131 21332 2128 3715 36.4% 99.4% 94.2% 94.2% 95.2% 92.5% 93.4 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Ala90Val 157 21306 1311 4532 22.4% 99.3% 89.3% 88.7% 90.4% 85.1% 36.8 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Ala 81 21382 441 5402 7.5% 99.6% 84.5% 81.1% 84.9% 75.1% 17.0 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Asn 31 21432 375 5468 6.4% 99.9% 92.4% 92.0% 94.6% 88.2% 45.3 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Ser91Pro 51 21412 274 5569 4.7% 99.8% 84.3% 83.8% 87.9% 77.0% 19.8 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Tyr 19 21444 229 5614 3.9% 99.9% 92.3% 91.5% 94.8% 87.1% 41.2 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94His 7 21456 99 5744 1.7% 100.0% 93.4% 93.8% 97.7% 85.8% 56.7 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asp461Asn 18 21445 44 5799 0.8% 99.9% 71.0% 60.0% 74.3% 44.3% 5.5 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Gly88Cys 1 21462 43 5800 0.7% 100.0% 97.7% 97.4% 99.9% 86.5% 140.6 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asn499Thr 6 9633 12 2708 0.4% 99.9% 66.7% 62.5% 84.8% 35.4% 5.9 1) AwR WHO 1) Assoc w R LFX gyrA_p.Asp89Asn 9 21454 20 5823 0.3% 100.0% 69.0% 66.7% 83.5% 46.0% 7.4 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asn499Asp 7 21456 19 5824 0.3% 100.0% 73.1% 77.8% 93.6% 43.0% 12.9 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Glu501Asp 33 21430 45 5798 0.8% 99.8% 57.7% 29.5% 45.2% 16.0% 1.5 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim LFX gyrB_p.Ala504Val 10 21453 32 5811 0.5% 100.0% 76.2% 37.5% 64.6% 15.2% 2.2 3) Uncertain ALL+WHO Prev. WHO 2) Assoc w R - Interim LFX gyrB_p.Ser447Phe 19 21444 21 5822 0.4% 99.9% 52.5% 50.0% 68.1% 28.8% 3.7 1) AwR ALL ALL only 2) Assoc w R - Interim LFX gyrB_p.Asp461His 4 21459 19 5824 0.3% 100.0% 82.6% 77.8% 97.2% 30.8% 12.9 1) AwR ALL ALL only 2) Assoc w R - Interim LFX gyrA_p.Gly88Ala 12 21451 16 5827 0.3% 99.9% 57.1% 45.0% 68.5% 21.8% 3.0 3) Uncertain ALL+WHO Prev. WHO 2) Assoc w R - Interim LFX gyrB_p.Glu501Val 1 21462 0 5843 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim LFX gyrA_p.Ala90Gly 23 9616 1 2719 0.0% 99.8% 4.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28137812) 4) Not assoc w R - Interim LFX gyrA_p.Arg252Leu 26 9613 0 2720 0.0% 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim LFX gyrA_p.Glu21Gln 9541 98 2707 13 99.5% 1.0% 22.1% 3.1% 4.4% 0.2% 0.2 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Gly668Asp 8388 1251 2489 231 91.5% 13.0% 22.9% 9.1% 41.3% 0.0% 0.5 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Ser95Thr 8486 1153 2390 330 87.9% 12.0% 22.0% 0.0% 18.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R LFX Rv1129c_c.-28T>C 4932 4707 1831 889 67.3% 48.8% 27.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Gly247Ser 680 8959 136 2584 5.0% 92.9% 16.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX glpK_p.Val460Ala 589 9050 45 2675 1.7% 93.9% 7.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Ala384Val 534 9105 43 2677 1.6% 94.5% 7.5% 50.0% 98.7% 0.0% 3.4 5) NotAwR WHO 5) Not assoc w R LFX gyrB_p.Met291Ile 519 9120 42 2678 1.5% 94.6% 7.5% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R LFX gyrA_c.-34C>T 118 9521 14 2706 0.5% 98.8% 10.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Ala463Ser 58 9581 14 2706 0.5% 99.4% 19.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 4 2 8 1 82 6 79 9 81 5 74 0 22 EMB embB_p.Met306Ile 1159 3454 953 7 28 0 6 6 8 62 8 54 5 56 7 4 3 5.5 EMB embB_p.Gln 97Arg 224 354 1 999 8 8 10.5 4 1 7 73 0 76 8 58 8 1 3 EMB embB_p.Gly406Ala 199 35506 28 9153 3 5 4 6 2 53 59 0 37 4 4.4 EMB embB_p.Asp354Ala 137 35568 301 9180 3 2 6 68 7 64 2 69 49 9 6.9 EMB embB_p.Gly406Asp 212 35 93 74 9207 2 4 56 4 46 9 53 2 31 5 3.4 EMB embB_p.Tyr319Ser 47 356 8 204 9277 2 2 99.9 81 3 80 7 85 73 0 1 1 EMB embB_p.Gly406Ser 76 35629 187 92 4 2 0 8 1 1 1 0 9 3 8 6 0 EMB embB_p.Gln497Lys 51 35654 145 9336 1 5 99.9 74 0 53 0 65 4 30 2 .3 EMB embB_p.Met306Leu 50 35655 145 9336 1 5 74 4 70 4 79 2 48 6 9 1 ALL+WHO EMB embB_p.Asp328Tyr 5 35700 46 9435 5 90 2 84 0 95 60 6 19.9 EMB embB_p.Tyr319Cys 12 6 93 27 6533 4 69 2 59 3 77 37 2 5.9 WHO EMB embB_p.Gly406Cys 28 35677 36 9445 4 9 6 3 58 1 73 0 33 3 5 2 1) AwR 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8 46 6 7 99.5 8 6 0 9 41 1 0 1 8 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 9460 2 100.0 7 4 0.0 97 5 0.0 0 0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 5007 1403 157 21.4 94.3 48 4 5.4 7.0 2.7 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 6 8 45 6515 7 8 48 9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala8 3G y 47 6 58 43 6517 7 99.8 47.8 NA NA NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 2 21.9 32 43680 EMB embC_c.-20A>C 5 26449 16 6544 2 8 22.2 Lit. (PMID 32143680) EMB embA_p.Val468Ala 7 26448 16 6544 0.2 99.8 1 9 NA NA 0 NA Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1 99 7 5.3 NA NA NA Prev. WHO 4) Not assoc w R - Interim EMB ubi Val49Ile 72 26433 4 6556 0.1 99 7 5.3 NA NA NA Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0 99 9 6.1 Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6 58 0 9 8.0 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3 80 2 39.1 EMB embC_p.Val981Leu 4076 22429 516 6044 7 9 8 6 11.2 1.0 1.7 2 0 0 EMB embC_c.-565C>T 2287 24218 332 622 5 1 1 4 12.7 70 8 EMB embC_p.Arg738Gln 3853 22652 327 6233 5 0 85 5 7.8 1.1% 1.6% 5 0.0 EMB embB_p.Glu378Ala 33 6 23119 251 63 9 3 8 87 2 6.9 3 Results for ind ivid ual d rugs 49 Silent mutations are not listed in this table but can be found in the catalogue master file. D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 LFX Rv2752c_p.Pro123Leu 65 9574 13 2707 0.5% 99.3% 16.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX Rv1129c_c.-48A>C 126 9513 12 2708 0.4% 98.7% 8.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrB_p.Val301Leu 90 9549 11 2709 0.4% 99.1% 10.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Gln613Glu 92 9547 5 2715 0.2% 99.0% 5.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrB_p.Pro94Leu 206 9433 5 2715 0.2% 97.9% 2.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX glpK_p.Cys29Tyr 72 9567 3 2717 0.1% 99.3% 4.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Thr80Ala 72 9567 3 2717 0.1% 99.3% 4.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrB_p.Gly520Ala 36 9603 1 2719 0.0% 99.6% 2.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LFX gyrB_p.Ala403Ser 41 9598 1 2719 0.0% 99.6% 2.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 34.2% 98.1% 82.6% 79.9% 81.5% 74.0% 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6% 96.8% 62.8% 54.5% 56.7% 45.3% 5.5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8482 10.5% 99.4% 81.7% 73.0% 76.8% 58.8% 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ala 199 35506 328 9153 3.5% 99.4% 62.2% 53.0% 59.0% 37.4% 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 9180 3.2% 99.6% 68.7% 64.2% 69.9% 49.9% 6.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 9207 2.9% 99.4% 56.4% 46.9% 53.2% 31.5% 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2.2% 99.9% 81.3% 80.7% 85.7% 73.0% 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2.0% 99.8% 71.1% 61.1% 70.9% 34.8% 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1.5% 99.9% 74.0% 53.0% 65.4% 30.2% 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1.5% 99.9% 74.4% 70.4% 79.2% 48.6% 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 0.5% 100.0% 90.2% 84.0% 95.5% 60.6% 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 515 21.4 4 48 4 5.4% 7.0% 2 7 0.3 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 7 9 8 48.9 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.A a8 3Gly 47 26458 43 6517 7 8 47 8 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro6 9Ser 57 26448 16 6544 8 21.9 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 9 8 22.2 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 2 8 21.9 Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 1 26434 4 6556 7 5 3 Prev. WHO 4) Not assoc w R - Interim EMB ubiA Val49Ile 72 26433 4 6556 1 7 5 3 Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 3 26474 2 6558 9 6 1 Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 LFX gyrA_p.Asp94Gly 131 21332 2128 3715 36.4% 99.4% 94.2% 94.2% 95.2% 92.5% 93.4 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Ala90Val 157 21306 1311 4532 22.4% 99.3% 89.3% 88.7% 90.4% 85.1% 36.8 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Ala 81 21382 441 5402 7.5% 99.6% 84.5% 81.1% 84.9% 75.1% 17.0 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Asn 31 21432 375 5468 6.4% 99.9% 92.4% 92.0% 94.6% 88.2% 45.3 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Ser91Pro 51 21412 274 5569 4.7% 99.8% 84.3% 83.8% 87.9% 77.0% 19.8 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94Tyr 19 21444 229 5614 3.9% 99.9% 92.3% 91.5% 94.8% 87.1% 41.2 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Asp94His 7 21456 99 5744 1.7% 100.0% 93.4% 93.8% 97.7% 85.8% 56.7 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asp461Asn 18 21445 44 5799 0.8% 99.9% 71.0% 60.0% 74.3% 44.3% 5.5 1) AwR ALL+WHO 1) Assoc w R LFX gyrA_p.Gly88Cys 1 21462 43 5800 0.7% 100.0% 97.7% 97.4% 99.9% 86.5% 140.6 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asn499Thr 6 9633 12 2708 0.4% 99.9% 66.7% 62.5% 84.8% 35.4% 5.9 1) AwR WHO 1) Assoc w R LFX gyrA_p.Asp89Asn 9 21454 20 5823 0.3% 100.0% 69.0% 66.7% 83.5% 46.0% 7.4 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Asn499Asp 7 21456 19 5824 0.3% 100.0% 73.1% 77.8% 93.6% 43.0% 12.9 1) AwR ALL+WHO 1) Assoc w R LFX gyrB_p.Glu501Asp 33 21430 45 5798 0.8% 99.8% 57.7% 29.5% 45.2% 16.0% 1.5 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim LFX gyrB_p.Ala504Val 10 21453 32 5811 0.5% 100.0% 76.2% 37.5% 64.6% 15.2% 2.2 3) Uncertain ALL+WHO Prev. WHO 2) Assoc w R - Interim LFX gyrB_p.Ser447Phe 19 21444 21 5822 0.4% 99.9% 52.5% 50.0% 68.1% 28.8% 3.7 1) AwR ALL ALL only 2) Assoc w R - Interim LFX gyrB_p.Asp461His 4 21459 19 5824 0.3% 100.0 . 7.8 97.2 30.8 1 .9 ALL ALL only 2) Assoc w R - Interim LFX gyrA_p.Gly88Ala 2 21451 16 58 7 0.3 9.9 57.1 45.0 68.5 21.8 3.0 3) Uncertain Prev. WHO 2) Assoc w R - Interim LFX gyrB_p.Glu501Val 1 21 62 0 5843 0.0 100.0 0.0 0.0 97.5 0.0 0.0 3) Uncertain WHO-end. gDST 2) Assoc w R - Interim LFX gyrA_p.Ala90Gly 23 9616 1 2719 0.0 .8 4.2 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 28137812) 4) Not assoc w R - Interim LFX gyrA_p.Arg252Leu 26 9613 272 0.0 .7 0.0 NA NA 0.0 NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim LFX gyrA_p.Glu21Gln 9541 98 2707 13 99.5 1.0 22.1 3.1 4.4 0.2 0.2 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Gly668Asp 8388 1251 2489 231 91.5 13.0 22.9 9.1 41.3 0.0 0.5 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Ser95Thr 8486 1153 2390 330 87.9 12.0 22.0 0.0 18.5 0.0 0. 5) NotAwR WHO 5) Not assoc w R LFX Rv1129c_c.-28T>C 4932 4707 1831 889 67.3 48.8 27.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Gly247Ser 680 8959 36 2584 5.0 2. 16.7 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R LFX glpK_p.Val460Ala 589 9050 5 267 1.7 93.9 7.1 NA NA 0.0 NA 5) NotAwR WHO 5) Not assoc w R LFX gyrA_p.Ala384Val 534 9105 43 2677 1.6 94.5 7.5 0.0 98.7 0.0 3.4 5) NotAwR 5) Not assoc w R LFX gyrB_p.Met291Ile 519 9120 42 2678 1.5 4.6 7.5 0.0 84.2 0.0 0.0 5) NotAwR WHO 5) Not assoc w R LFX gyrA_c.-34C>T 18 9521 14 270 0.5 8.8 10. NA NA 0.0 NA 5) NotAwR WHO 5) Not as oc w R LFX gyrA_p.Ala463Ser 58 9581 14 2706 .5 99.4 19. NA NA . NA 5) NotAwR WHO 5) Not as oc w R C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 50 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 MFX gyrA_p.Asp94Gly 260 18275 1692 2310 42.3% 98.6% 86.7% 86.4% 88.0% 83.8% 50.4 1) AwR ALL+WHO 1) Assoc w R G MFX gyrA_p.Ala90Val 452 18083 720 3282 18.0% 97.6% 61.4% 56.7% 59.8% 51.1% 7.2 1) AwR ALL+WHO 1) Assoc w R F MFX gyrA_p.Asp94Asn 53 18482 296 3706 7.4% 99.7% 84.8% 83.5% 87.5% 78.7% 25.3 1) AwR ALL+WHO 1) Assoc w R G MFX gyrA_p.Asp94Ala 172 18363 261 3741 6.5% 99.1% 60.3% 52.5% 58.1% 44.0% 5.4 1) AwR ALL+WHO 1) Assoc w R F MFX gyrA_p.Ser91Pro 68 18467 175 3827 4.4% 99.6% 72.0% 72.1% 78.1% 62.2% 12.5 1) AwR ALL+WHO 1) Assoc w R F MFX gyrA_p.Asp94Tyr 36 18499 171 3831 4.3% 99.8% 82.6% 81.9% 87.1% 74.7% 21.9 1) AwR ALL+WHO 1) Assoc w R G MFX gyrA_p.Asp94His 14 18521 70 3932 1.7% 99.9% 83.3% 85.9% 92.7% 72.7% 28.7 1) AwR ALL+WHO 1) Assoc w R G MFX gyrB_p.Glu501Asp 10 18525 61 3941 1.5% 99.9% 85.9% 79.1% 90.0% 62.2% 17.8 1) AwR ALL+WHO 1) Assoc w R F MFX gyrA_p.Gly88Cys 5 18530 33 3969 0.8% 100.0% 86.8% 85.7% 95.2% 69.7% 28.0 1) AwR ALL+WHO 1) Assoc w R G MFX gyrA_p.Asp89Asn 6 18529 20 3982 0.5% 100.0% 76.9% 76.9% 91.0% 56.4% 15.5 1) AwR ALL+WHO 1) Assoc w R F MFX gyrB_p.Asn499Thr 15 18520 23 3979 0.6% 99.9% 60.5% 25.0% 52.4% 6.1% 1.6 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrB_p.Ala504Val 10 18525 14 3988 0.3% 99.9% 58.3% 28.6% 71.0% 2.1% 1.9 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrB_p.Asn499Asp 5 18530 12 3990 0.3% 100.0% 70.6% 81.8% 97.7% 35.1% 20.9 1) AwR ALL ALL only 2) Assoc w R - Interim F MFX gyrB_p.Asp461Asn 24 18511 12 3990 0.3% 99.9% 33.3% 12.0% 31.2% 2.4% 0.6 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrA_p.Gly88Ala 13 18522 11 3991 0.3% 99.9% 45.8% 41.2% 67.1% 15.4% 3.2 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrB_p.Ser447Phe 20 18515 7 3995 0.2% 99.9% 25.9% 13.6% 34.9% 2.8% 0.7 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrB_p.Asp461His 4 18531 3 3999 0.1% 100.0% 42.9% 25.0% 80.6% 0.5% 1.5 3) Uncertain ALL+WHO FQ X-R 2) Assoc w R - Interim F MFX gyrB_p.Glu501Val 1 18534 0 4002 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim F MFX gyrA_p.Thr80Ala 17 6623 1 1741 0.1% 99.7% 5.6% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28137812) 4) Not assoc w R - Interim MFX gyrA_p.Ala90Gly 2 6638 0 1742 0.0% 100.0% 0.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28137812) 4) Not assoc w R - Interim MFX gyrA_p.Glu21Gln 6570 70 1737 5 99.7% 1.1% 20.9% 1.8% 3.4% 0.1% 0.3 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Gly668Asp 5921 719 1654 88 94.9% 10.8% 21.8% 0.0% 33.6% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Ser95Thr 5987 653 1598 144 91.7% 9.8% 21.1% 0.0% 26.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R MFX Rv1129c_c.-28T>C 3897 2743 1399 343 80.3% 41.3% 26.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Gly247Ser 404 6236 59 1683 3.4% 93.9% 12.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX glpK_p.Val460Ala 762 5878 34 1708 2.0% 88.5% 4.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrB_p.Met291Ile 720 5920 34 1708 2.0% 89.2% 4.5% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Ala384Val 734 5906 32 1710 1.8% 88.9% 4.2% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Ala463Ser 46 6594 25 1717 1.4% 99.3% 35.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrA_c.-34C>T 171 6469 13 1729 0.7% 97.4% 7.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 6236 34 2 1 2 6 79 9 1 5 74 0 22 EMB embB_p.Met306Ile 1159 34546 1953 7528 20 6 6 8 2 8 4 5 6 7 45 3 5 5 EMB embB_p.Gln 97Arg 224 35 1 9 9 8482 10.5 4 1 7 7 0 76 8 5 8 11 EMB embB_p.Gly406Ala 99 35506 328 9153 3 4 2 2 3 0 9 0 37 4 4 EMB embB_p.Asp354Ala 137 355 8 301 9180 3 2 68 7 64 2 69 9 49 9 6.9 EMB embB_p.Gly406Asp 212 35 3 2 4 9207 2 9 4 56 4 46 53 2 31 5 3.4 EMB embB_p.Tyr319Ser 47 35658 204 9277 2 2 1 0 7 85 3 0 16 1 EMB embB_p.Gly406Ser 76 356 9 187 9294 2 0 8 71 1 61 7 9 34 8 6.0 EMB embB_p.Gln497Lys 51 35654 145 9336 1 5 99.9 74 0 53 0 6 4 30 2 4.3 EMB embB_p.Met306Leu 50 35655 145 9336 1 99.9 4 4 0 4 79 2 48 6 9.1 EMB embB_p.Asp328Tyr 5 3570 46 9435 5 100.0 9 2 84 95 5 60.6% 19.9 1) AwR 1) Assoc w R EMB embB_p.Tyr319Cys 2 26493 27 6533 4 100.0 69 2 59 3 7 6 37.2% 5 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 4 99.9 56 3 58 1 73 0 3 3 5.2 ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35 32 635 8846 6 7 5 78 6 30 9 4 1 10.2 1 8 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 2 9460 2 100.0 72 4 0.0 9 5 0.0 0 0 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4 4 3 48 4 5.4 7.0 7 3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 7 99.8 8 NA NA 0 NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala8 3G y 47 26458 43 6517 7 99.8 47.8 NA NA NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.P o639Ser 5 26448 16 65 4 2 8 21.9 32 43680 EMB embC_c.-20A>C 56 26449 16 65 4 2 99.8 22.2 32 43680 EMB embA_p.Val468Ala 26448 16 6544 0.2 99.8 1 NA NA 0 NA Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1 99 7 5.3 NA NA NA Prev. WHO 4) Not assoc w R - Interim EMB ubi Val49Ile 72 2 4 3 4 6556 0.1 99.7 5.3 NA NA NA Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0 99 9 6.1 Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0 0 9 8.0 Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3 63 3197 51.3 0 2 39.1 EMB embC_p.Val 81Leu 4076 22429 516 6044 7 9 4 6 11.2 1 1.7 2 EMB embC_c.-565C>T 2287 24218 332 6228 5 1 91 4 12.7 70 8 EMB embC_p.Arg7 8Gln 3853 22652 327 6233 5 0 85 5 7.8 1.1% 1.6% 5 0.0 EMB embB_p.Glu378Ala 3386 23119 251 630 3 8 8 2 6 9 Table 11. Abridged variant classification for MFX 3 Results for ind ivid ual d rugs 51 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 MFX Rv1129c_c.-48A>C 99 6541 8 1734 0.5% 98.5% 7.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrB_p.Val301Leu 94 6546 8 1734 0.5% 98.6% 7.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX Rv2752c_p.Gly161Ser 49 6591 3 1739 0.2% 99.3% 5.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrB_p.Pro94Leu 206 6434 3 1739 0.2% 96.9% 1.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R MFX gyrA_p.Gln613Glu 87 6553 2 1740 0.1% 98.7% 2.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . EMB embB_p.Met306Val 685 35020 3245 62 6 34.2 1 82.6 79.9% 81.5% 74.0 22.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Ile 1159 34546 1953 7528 20.6 6 8 62.8 54.5% 56.7% 45.3 5.5 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Arg 224 35481 999 8482 10.5 4 81.7 73.0% 76.8% 58.8 11.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly 06Ala 199 35506 328 9153 3 5 9 4 62.2 53.0% 59.0% 37.4 4.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp354Ala 137 35568 301 918 3 2 9 6 68.7 64.2% 69.9% 49.9 6.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Asp 212 35493 274 9207 2.9% 99.4% 56.4% 46.9% 53.2% 31.5% 3.4 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Ser 47 35658 204 9277 2.2% 99.9% 81.3% 80.7% 85.7% 73.0% 16.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gly406Ser 76 35629 187 9294 2.0% 99.8% 71.1% 61.1% 70.9% 34.8% 6.0 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Gln497Lys 51 35654 145 9336 1.5% 99.9% 74.0% 53.0% 65.4% 30.2% 4.3 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Met306Leu 50 35655 145 9336 1.5% 99.9% 74.4% 70.4% 79.2% 48.6% 9.1 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Asp328Tyr 5 35700 46 9435 0.5% 100.0% 90.2% 84.0% 95.5% 60.6% 19.9 1) AwR ALL+WHO 1) Assoc w R EMB embB_p.Tyr319Cys 12 26493 27 6533 0.4% 100.0% 69.2% 59.3% 77.6% 37.2% 5.9 1) AwR WHO 1) Assoc w R EMB embB_p.Gly406Cys 28 35677 36 9445 0.4% 99.9% 56.3% 58.1% 73.0% 33.3% 5.2 1) AwR ALL+WHO 1) Assoc w R EMB embA_c.-12C>T 173 35532 635 8846 6.7% 99.5% 78.6% 30.9% 41.1% 10.2% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance EMB embB_p.Leu74Arg 8 35697 21 9460 0.2% 100.0% 72.4% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance EMB embC_c.-1188C>T 1498 25007 1403 5157 21.4% 94.3% 48.4% 5.4% 7.0% 2.7% 0.3 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embB_p.Gly156Cys 47 26458 45 6515 0.7% 99.8% 48.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Ala813Gly 47 26458 43 6517 0.7% 99.8% 47.8% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Pro639Ser 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embC_c.-20A>C 56 26449 16 6544 0.2% 99.8% 22.2% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB embA_p.Val468Ala 57 26448 16 6544 0.2% 99.8% 21.9% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embC_c.-1520C>A 71 26434 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB ubiA_p.Val49Ile 72 26433 4 6556 0.1% 99.7% 5.3% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Ser1054Pro 31 26474 2 6558 0.0% 99.9% 6.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim EMB embB_p.Val668Ile 23 26482 2 6558 0.0% 99.9% 8.0% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 32143680) 4) Not assoc w R - Interim EMB aftB_p.Asp397Gly 5235 21270 3363 3197 51.3% 80.2% 39.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Val981Leu 4076 22429 516 6044 7.9% 84.6% 11.2% 1.0% 1.7% 0.2% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_c.-565C>T 2287 24218 332 6228 5.1% 91.4% 12.7% 0.0% 70.8% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embC_p.Arg738Gln 3853 22652 327 6233 5.0% 85.5% 7.8% 1.1% 1.6% 0.5% 0.0 5) NotAwR WHO 5) Not assoc w R EMB embB_p.Glu378Ala 3386 23119 251 6309 3.8% 87.2% 6.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R Silent mutations are not listed in this table but can be found in the catalogue master file. F Low-level resistance (multiple, genetically linked low-level resistance mutations are additive and confer high-level resistance). G High-level resistance. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 52 3.7 Bedaquiline and clofazimine In the first edition, no mutations met Group 1 or 2 grading criteria for predicting BDQ or CFZ resistance. In the second edition, because of the addition of substantial new data, mutations in Rv0678, atpE and pepQ met criteria for Group 1 or 2 grading (Table 12), for a combined sensitivity of 49.4% (95% CI: 46.3–52.5), a specificity of 98.7% (95% CI: 98.5–98.9) and a PPV of 75.2% (95% CI: 71.8–78.4) (Table 3). Mutations with an allele frequency below 75% were found to play a major role in predicting BDQ phenotypes, as their inclusion increased the combined sensitivity of Groups 1 and 2 mutations by 10.2% to 59.6% (95% CI: 56.5–62.6) (Table A.1). Six atpE mutations (atpE Asp28Ala, Asp28Gly, Asp28Val, Glu61Asp, Ile66Met and Ala63Pro) were classified as Group 2 resistance mutations. Notably, two of these mutations (Ala63Pro and Ile66Met) occurred as SOLO mutations at sufficient frequency to meet thresholds for association with phenotypic resistance, indicating that atpE mutations can arise independently of Rv0678 mutations. The remaining four atpE mutations met criteria based on in-vitro selection experiments, which also supported our additional grading rule that any LoF mutation in Rv0678 and pepQ was assumed to confer BDQ resistance (Tables 1 and 23) (67–74). It should be noted that the contributing laboratory that supplied most of the BDQ pDST results for the WHO data set (referred to as laboratory 1 below) contributed only categorical results for isolates that were resistant on pDST at two independent laboratories (isolates that tested pDST resistant to BDQ in peripheral laboratories were referred to a reference laboratory for confirmatory testing at the WHO CC and were sequenced and contributed to the catalogue only if the initial R result was confirmed at the reference laboratory). Therefore, it is possible that the PPV|SOLO of the mutations in these isolates was inflated, given that no BDQ susceptible were submitted from this collection. For example, the grading of mutation Rv0678 Met146Thr, which is frequent in Eswatini (29) but considered to be rare elsewhere, was potentially affected by this isolate collection. This mutation was assigned an initial confidence grading in Group 1 on the basis of a PPV|SOLO of 100% (95% CI: 72–100) in the WHO data set. This was calculated from 12 categorical pDST BACTEC™ Mycobacterial Growth Indicator Tube™ 960 (MGIT) results in the collection that included only resistant isolates. This laboratory also determined that the MGIT MIC range for 9 of the 12 Rv0678 Met146Thr mutants as 1–4 mg/L (see result from laboratory 1 in Fig. 5), which, while still mainly resistant, represents a lower PPV|SOLO of 67% (95% CI: 30–93) when calculated by MIC result. Because our rules for addressing pDST data prioritized categorical pDST results over MIC data (see Sections 5.4 and 5.5), however, these MIC results were not considered in the WHO data set. Given these results, we also examined the BDQ MICs for 21 isolates with the same SOLO Rv0678 Met146Thr mutation, but from a different laboratory. These isolates showed MGIT MICs of 0.25–1 mg/L in the susceptible range (see laboratory 2 in Fig. 5) but were not considered in the WHO data set, as MIC results were analysed only for the ALL data set. Because of potentially inflated PPVs for these isolates, the working group decided to downgrade all Group 1 BDQ mutations that met the Group 1 criteria only on the basis of results from laboratory 1 to Group 2, and a footnote (C) was added to the BDQ mutation table (Table 12) to identify all 18 mutations with potentially elevated PPVs. It should be emphasized, however, that only three Rv0678 mutations (i.e. Cys46Arg, Ile67Ser, Met146Thr) were ultimately downgraded from Group 1 to Group 2 to mitigate potential PPV inflation. The remaining 15 mutations would have had a 3 Results for individual drugs 53 final confidence grading of Group 2 even without the data from laboratory 1 because they were covered by the Rv0678 or pepQ LoF “additional grading rule” (10 mutations), their classification was based on the ALL data set only (three mutations) or was supported by in-vitro selection data (atpE Ala63Pro and Ile66Met). Fig. 5. BDQ MICs for MTBC isolates with Rv0678 Met146Thr 14 10 6 2 0 12 8 4 0.25 0.5 1 MGIT MIC (mg/L) Laboratory 1 Laboratory 2 St ra in s te st ed 2 4 MGIT CC Published evidence suggests that Rv0678 mutations confer resistance only if the corresponding efflux pump is functional. It appears that mmpL5 LoF mutations, which may be frequent in some settings, such as Lima, Peru (75), can confer a hyper-susceptible phenotype to BDQ and CFZ (76). We evaluated this in our data set by determining that the BDQ PPV for combined Groups 1 and 2 mutations was significantly higher in isolates with silent or only Group 4 or 5 mutations in mmpL5 (i.e. the corresponding protein was functional) as compared with isolates with LoF mutations in mmpL5 (75% [95% CI: 71–79] vs. 0% [95% CI: 0–16]). Consequently, for assays that interrogate only Rv0678, inclusion of a disclaimer should be considered, acknowledging the possibility that epistasis affects the predictive power of Rv0678 mutations (Table 1). Definitive identification of those isolates affected by epistasis is difficult, given the requirement to phase the Rv0678 mutation results with mmpL5 LoF mutations (see Section 3.10 for a discussion of this point for AMK). When epistasis could be identified, it was corrected for in our calculations of the predictive performance of Rv0678 mutations in the second edition (Section 2.4 and in the Annex). Because this data set did not feature any mmpS5 LoF mutants with BDQ pDST results, the potential role of those mutations in epistasis could not be investigated (75,77,78). Only pooled Rv0678 LoF mutations and two individual Rv0678 mutations (Glu49fs and Gly121Arg) could be classified as CZF resistance mutations in this data set (Table 13). As Rv0678 and pepQ mutations are known to confer cross-resistance to BDQ and CFZ in selection experiments from independent laboratories, however, the working group decided that any Group 1 or 2 BDQ mutation in these two genes should also be classified as a Group 2 mutation for CFZ (Table 1). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 54 In accordance with these findings for BDQ, epistasis caused by mmpL5 LoF was considered also relevant for CFZ and was corrected (Section 2.4 and the Annex). The sensitivity and PPV for CFZ were significantly lower than those for BDQ (Table 3). The greater technical variability of pDST for CFZ probably explains these differences, but other explanations should be explored (e.g. whether some CCs for CFZ are too high). Potential role of rare variants The assumption that rare Rv0678 promoter and coding mutations that meet the same “relaxed” grading criteria used for pncA (Fig. 9) confer resistance increased the sensitivity for BDQ by a further 9.2% while decreasing the specificity by only 0.1% (Table A.3). If it is assumed that all the coding mutations observed (except silent and Groups 4 and 5 mutations) confer BDQ resistance in RIF-resistant isolates, the combined sensitivity is increased by 15.5% to 75.1% and the specificity decreased by 0.7% (Table A.3). Taken together, these analyses suggest that detection of heteroresistance down to at least 25% is important for predicting BDQ resistance, that there is probably a wide variety of rare resistance-conferring mutations (13) in existing target genes and that revision of grading rules will be more informative than including new gene targets in the next version of the catalogue. 3 Results for ind ivid ual d rugs 55 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . BDQ Rv0678_LoF 134 12817 424 611 41.0% 99.0% 76.0% 79.5% 82.9% 71.3% 81.1 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Glu49fs 26 12925 142 893 13.7% 99.8% 84.5% 85.7% 90.7% 77.6% 86.8 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Asp47fs 25 12926 61 974 5.9% 99.8% 70.9% 71.1% 80.5% 59.3% 32.6 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Ile67fs 35 12916 52 983 5.0% 99.7% 59.8% 76.1% 85.7% 48.2% 41.9 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Gly121Arg 1 12950 9 1026 0.9% 100.0% 90.0% 90.0% 99.7% 55.5% 113.6 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Leu117Arg 4 12947 8 1027 0.8% 100.0% 66.7% 66.7% 90.1% 34.9% 25.2 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Met146Thr 0 1249 11 890 1.2% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Ile67Ser 0 12951 12 1023 1.2% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Cys46Arg 1 12950 9 1026 0.9% 100.0% 90.0% 90.0% 99.7% 55.5% 113.6 1) AwR ALL+WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ pepQ_LoF 4 12947 8 1027 0.8% 100.0% 66.7% 63.6% 89.1% 30.8% 22.1 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ atpE_p.Ala63Pro 0 12951 7 1028 0.7% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ atpE_p.Ile66Met 0 12951 7 1028 0.7% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ Rv0678_p.Ala36Val 0 12951 6 1029 0.6% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Asn70Asp 1 12950 6 1029 0.6% 100.0% 85.7% 85.7% 99.6% 42.1% 75.5 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Leu32Ser 1 12950 5 1030 0.5% 100.0% 83.3% 83.3% 99.6% 35.9% 62.9 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ atpE_p.Glu61Asp 3 12948 2 1033 0.2% 100.0% 40.0% 50.0% 93.2% 5.3% 12.5 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Ala NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Gly NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Val NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ mmpL5_LoF 247 12704 0 1035 0.0% 98.1% 0.0% 0.0% 1.7% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance D BDQ mmpL5_p.Ile948Val 1245 4 895 6 99.3% 0.3% 41.8% 7.7% 10.6% 2.0% 0.1 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ Rv1979c_c.-129A>G 1238 11 893 8 99.1% 0.9% 41.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpL5_p.Thr794Ile 760 489 635 266 70.5% 39.2% 45.5% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ Rv1979c_p.Asp286Gly 48 1201 71 830 7.9% 96.2% 59.7% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpS5_c.-74G>T 3 1246 36 865 4.0% 99.8% 92.3% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpL5_p.Asp767Asn 509 740 146 755 16.2% 59.2% 22.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R Table 12. Abridged variant classification for BDQ C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 56 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 BDQ Rv0678_p.Leu142Arg 2 12949 7 1028 0.7% 100.0% 77.8% 50.0% 93.2% 6.8% 12.6 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Gly41Asp 0 12951 4 1031 0.4% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser53Pro 0 12951 4 1031 0.4% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Thr91Ile 0 12951 4 1031 0.4% 100.0% 100.0% 100.0% 100.0% 39.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg94Trp 1 12950 3 1032 0.3% 100.0% 75.0% 75.0% 99.4% 19.4% 37.6 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Asp47dup 0 12951 3 1032 0.3% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Gly65Glu 0 12951 3 1032 0.3% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu60Pro 0 12951 3 1032 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Pro48Leu 1 12950 3 1032 0.3% 100.0% 75.0% 75.0% 99.4% 19.4% 37.6 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser2Ile 1 12950 3 1032 0.3% 100.0% 75.0% 75.0% 99.4% 19.4% 37.6 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser63Gly 0 12951 3 1032 0.3% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ala102Thr 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ala99Pro 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg107Cys 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg109Pro 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg34Gln 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg34Trp 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Arg50Gln 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Asn70Ile 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Cys46Trp 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Gln115Pro 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Gly78Arg 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ile67Leu 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu122Pro 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu35Trp 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu40Phe 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu60Gln 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Leu95Ser 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Met139Ile 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Phe79Leu 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . LoF 134 817 424 611 41.0 99.0 6 0 79 5 82 9 71.3 81 1 1) AwR 1) Assoc w R E u 9fs 26 25 1 2 893 13.7 99.8 84.5 85.7 90.7 77 6 86.8 1) AwR 1) Assoc w R E Asp47fs 25 26 61 974 5 9 99.8 70.9 71.1 80.5 5 3 32.6 1) AwR 1) Assoc w R E Ile67fs 35 16 52 983 5 0 99.7 59.8 76.1 85.7 48 2 41.9 1) AwR 1) Assoc w R E Gly121Arg 9 26 9 90 90 7 55 5 113.6 1) AwR 1) Assoc w R E Leu117Arg 4 47 8 27 8 66.7 66.7 90.1 34 9 25.2 1) AwR 1) Assoc w R E Met146Thr 1249 11 890 1 2 71 5 1) AwR WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O Il 67Se 12 23 1 2 73 5 1) AwR Pot. infl. PPV 2) Assoc w R - Interim C, E, O Cys 6Arg 9 26 9 90 90 7 55 5 113.6 1) AwR Pot. infl. PPV 2) Assoc w R - Interim C, E, O pepQ_LoF 4 47 8 27 8 66 7 63 6 8 1 30 8 22 1 1) AwR ALL ALL only 2) Assoc w R - Interim C atpE_p.Ala63Pro 7 28 7 47 8 1) AwR ALL ALL only 2) Assoc w R - Interim C atpE_p.Ile66Met 7 28 7 54 1 1) AwR ALL ALL only 2) Assoc w R - Interim C 36Val 6 29 6 54 1 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O sn7 Asp 6 29 6 85 85 6 42.1% 7 5 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O Leu32Ser 1 0 5 0 5 83.3 83.3 99.6 3 9 62.9 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O atpE_p.Glu61Asp 3 48 40.0 50.0 93.2 5.3 12.5 Selection 2) Assoc w R - Interim atpE_p.Asp28Ala NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim atpE_p.Asp28Gly NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim atpE_p.Asp28Val NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim mmpL5 LoF 247 704 0 5 0 98.1 .0% .0% 1.7% 0.0 0.0 D mmpL5 .Ile948Val 1245 4 895 6 99.3% .3% 41.8 7.7% 10.6 2.0 0.1 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim 19 9c_c.-129A>G 1238 11 893 8 99.1% .9% 41.9 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim mmpL5 .Thr794Ile 760 489 635 266 70.5% 39.2 45.5 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BD Rv1979c_p.Asp286Gly 48 1201 71 830 7.9 96.2 59.7 NA NA 0.0 NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpS5_c.-74G>T 3 1246 36 865 4.0% 99.8% 92.3% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpL5_p.Asp767Asn 509 740 146 755 16.2% 59.2% 22.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R 3 Results for ind ivid ual d rugs 57 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 BDQ Rv0678_p.Phe93Leu 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser151Pro 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser52Phe 0 12951 2 1033 0.2% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Ser63Arg 1 12950 2 1033 0.2% 100.0% 66.7% 66.7% 99.2% 9.4% 25.1 3) Uncertain ALL+WHO 3) Uncertain significance O BDQ Rv0678_p.Tyr157Ser 1 12950 2 1033 0.2% 100.0% 66.7% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . LoF 134 817 4 4 611 41.0 99.0 7 0 79 5 82 9 71.3 81 1) AwR 1) Assoc w R E Glu49fs 26 25 142 893 13.7 99.8 84.5 85.7 90.7 77 6 86.8 1) AwR 1) Assoc w R E Asp47fs 25 26 61 974 5 9 99.8 70.9 71.1 80.5 59 3 32.6 1) AwR 1) Assoc w R E Il 67fs 35 16 52 983 5 0 99.7 59 8 7 1 85 7 48.2 41 9 1) AwR 1) Assoc w R E Gly 21Arg 9 26 9 90 0 90.0 99.7 55.5 113.6 1) AwR 1) Assoc w R E BDQ Rv0678_p.Leu117Arg 4 12947 8 1027 0.8% 100.0% 66.7% 66.7% 90.1% 34.9% 25.2 1) AwR ALL+WHO 1) Assoc w R E BDQ Rv0678_p.Met146Thr 0 1249 11 890 1.2% 100.0% 100.0% 100.0% 100.0% 71.5% Inf 1) AwR WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Ile67Ser 0 12951 12 1023 1.2% 100.0% 100.0% 100.0% 100.0% 73.5% Inf 1) AwR ALL+WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Cys46Arg 1 12950 9 1026 0.9% 100.0% 90.0% 90.0% 99.7% 55.5% 113.6 1) AwR ALL+WHO Pot. infl. PPV 2) Assoc w R - Interim C, E, O BDQ pepQ_LoF 4 12947 8 1027 0.8% 100.0% 66.7% 63.6% 89.1% 30.8% 22.1 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ atpE_p.Ala63Pro 0 12951 7 1028 0.7% 100.0% 100.0% 100.0% 100.0% 47.8% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ atpE_p.Ile66Met 0 12951 7 1028 0.7% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C BDQ Rv0678_p.Ala36Val 0 12951 6 1029 0.6% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Asn70Asp 1 12950 6 1029 0.6% 100.0% 85.7% 85.7% 99.6% 42.1% 75.5 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ Rv0678_p.Leu32Ser 1 12950 5 1030 0.5% 100.0% 83.3% 83.3% 99.6% 35.9% 62.9 1) AwR ALL ALL only 2) Assoc w R - Interim C, E, O BDQ atpE_p.Glu61Asp 3 12948 2 1033 0.2% 100.0% 40.0% 50.0% 93.2% 5.3% 12.5 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Ala NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Gly NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ atpE_p.Asp28Val NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim BDQ mmpL5_LoF 247 12704 0 1035 0.0% 98.1% 0.0% 0.0% 1.7% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance D BDQ mmpL5_p.Ile948Val 1245 4 895 6 99.3% 0.3% 41.8% 7.7% 10.6% 2.0% 0.1 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ Rv1979c_c.-129A>G 1238 11 893 8 99.1% 0.9% 41.9% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpL5_p.Thr794Ile 760 489 635 266 70.5% 39.2% 45.5% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ Rv1979c_p.Asp286Gly 48 1201 71 830 7.9% 96.2% 59.7% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpS5_c.-74G>T 3 1246 36 865 4.0% 99.8% 92.3% NA NA 0.0% NA 5) NotAwR WHO Lit. (PMID 28031270; 34503982) 4) Not assoc w R - Interim BDQ mmpL5_p.Asp767Asn 509 740 146 755 16.2% 59.2% 22.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R Individual LoF mutations in the coding regions of pepQ and Rv0678 that are classified in Group 2 because of the associated additional grading rule (see Table 23 and Section 5.8) and silent mutations are not listed in this table but can be found in the catalogue master file. C Includes data from one site that only submitted resistant strains, which may have inflated the PPV. D Abrogates effect of genetically linked Groups 1 and 2 Rv0678 mutations. E Can confer resistance only if genetically linked to a functional MmpL5. O Group 2 by “relaxed” threshold (not endorsed). C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 58 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 4666 63 151 29.4% 98.6% 48.1% 46.6% 56.0% 35.3% 26.9 1) AwR WHO 1) Assoc w R E CFZ Rv0678_p.Glu49fs 30 14013 29 645 4.3% 99.8% 49.2% 50.0% 66.2% 26.4% 21.7 1) AwR ALL+WHO 1) Assoc w R E CFZ Rv0678_p.Gly121Arg 1 14042 4 670 0.6% 100.0% 80.0% 80.0% 99.5% 28.4% 83.8 1) AwR ALL ALL only 2) Assoc w R - Interim E CFZ Rv0678_p.Leu117Arg 9 14034 4 670 0.6% 99.9% 30.8% 30.8% 61.4% 9.1% 9.3 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Cys46Arg 0 14043 3 671 0.4% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ pepQ_LoF 3 14040 3 671 0.4% 100.0% 50.0% 33.3% 90.6% 0.6% 10.5 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim CFZ Rv0678_p.Ala36Val 1 14042 3 671 0.4% 100.0% 75.0% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Asn70Asp 0 14043 2 672 0.3% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Met146Thr 36 14007 2 672 0.3% 99.7% 5.3% 5.4% 18.2% 0.6% 1.2 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Leu32Ser 3 14040 1 673 0.1% 100.0% 25.0% 25.0% 80.6% 0.6% 7.0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 0 674 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ mmpL5_LoF 242 13801 7 667 1.0% 98.3% 2.8% 3.0% 6.4% 0.9% 0.6 3) Uncertain ALL+WHO 3) Uncertain significance D CFZ mmpL5_p.Ile948Val 4719 15 209 5 97.7% 0.3% 4.2% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7% 0.4% 4.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6% 20.1% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9% 67.1% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Val170Met 518 4216 10 204 4.7% 89.1% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2.8% 86.0% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1.9% 95.2% 1.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Lys270Met 108 4626 1 213 0.5% 97.7% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-389C>A 249 4485 1 213 0.5% 94.7% 0.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fbiA_p.Thr302Met 39 4695 0 214 0.0% 99.2% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-327C>A 42 4692 0 214 0.0% 99.1% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv0678_p.Ser53Pro 0 14043 4 670 0.6% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance P Table 13. Abridged variant classification for CFZ 3 Results for ind ivid ual d rugs 59 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 CFZ Rv0678_p.Asn70Ile 1 14042 1 673 0.1% 100.0% 50.0% 50.0% 98.7% 1.3% 20.9 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu40Phe 0 14043 1 673 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Pro48Leu 2 14041 1 673 0.1% 100.0% 33.3% 33.3% 90.6% 0.8% 10.4 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser52Phe 0 14043 1 673 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Tyr157Ser 0 14043 1 673 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg107Cys 1 14042 0 674 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg94Trp 1 14042 0 674 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ile67Leu 1 14042 0 674 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu60Gln 1 14042 0 674 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu95Ser 2 14041 0 674 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Met139Ile 2 14041 0 674 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Phe93Leu 2 14041 0 674 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser2Ile 1 14042 0 674 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance P D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . LoF 68 4666 63 151 29.4 98.6 48 1 46 6 56 0 35.3 6 1) AwR WHO 1) Assoc w R E Gl 9fs 30 1 29 45 4 3 99.8 49.2 50.0 66.2 26.4 21.7 1) AwR 1) Assoc w R E Gly121Arg 1 2 4 0 6 80 0 80 0 9 5 28.4 83 8 1) AwR ALL ALL only 2) Assoc w R - Interim E L u117Arg 9 34 4 0 6 99.9 30.8 30.8 61.4 9 1 9.3 BDQ-CFZ X-R 2) Assoc w R - Interim E C s46Arg 3 1 4 29.2 BDQ-CFZ X-R 2) Assoc w R - Interim E pepQ_LoF 3 0 3 1 4 50.0 33.3 0 6 6 10.5 BDQ-CFZ X-R 2) Assoc w R - Interim la36Val 3 1 4 75.0 1 0.0% 100.0 9 4 Inf BDQ-CFZ X-R 2) Assoc w R - Interim E Asn 0Asp 0 3 2 2 3 1 0.0% NA NA NA NA BDQ-CFZ X-R 2) Assoc w R - Interim E Met146Thr 36 07 2 2 3 99.7 5 3 5 4 18 2 6 1 2 BDQ-CFZ X-R 2) Assoc w R - Interim E 32 3 0 1 3 1 25.0 25.0 0 6 6 7 BDQ-CFZ X-R 2) Assoc w R - Interim E Ile67Ser 1 2 NA NA NA BDQ-CFZ X-R 2) Assoc w R - Interim E mmpL5 LoF 242 380 7 67 1 98.3 2 8 3 6.4 9 6 D mmpL5 .Ile948Val 4719 15 2 9 5 97.7 .3% 4 2 0.0% 84.2% 0.0 5) NotAwR WHO 5) Not as oc w R CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7% 0.4% 4.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6% 20.1% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9% 67.1% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Val170Met 518 4216 10 204 4.7% 89.1% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2.8% 86.0% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1.9% 95.2% 1.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Lys270Met 108 4626 1 213 0.5% 97.7% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-389C>A 249 4485 1 213 0.5% 94.7% 0.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fbiA_p.Thr302Met 39 4695 0 214 0.0% 99.2% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-327C>A 42 4692 0 214 0.0% 99.1% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv0678_p.Ser53Pro 0 14043 4 670 0.6% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance P Individual LoF mutations in the coding regions of pepQ and Rv0678 that are classified in Group 2 because of the associated additional grading rule (see Table 23 and Section 5.8) and silent mutations are not listed in this table but can be found in the catalogue master file. D Abrogates effect of genetically linked Groups 1 and 2 Rv0678 mutations. E Can only confer resistance if genetically linked to a functional MmpL5. P Group 2 by “relaxed” threshold (not endorsed) and BDQ cross-resistance. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 60 3.8 Linezolid The rplC Cys154Arg mutation was the only marker of resistance in the first edition and remained the only Group 1 mutation with an associated sensitivity of 27.3% (95% CI: 22.8–32.1) (Table 3). The seven newly endorsed Group 2 mutations (Table 14) were in rrl and increased the combined sensitivity by 6.7% (95% CI: 4.4–9.7) (Table A.1). Six of the rrl mutations, three of which were not found in our data set (2270G>C, 2689A>T and 2746G>A), met the criteria as interim resistance markers according to the new in-vitro selection grading rule based on data from at least two independent laboratories (79–84).2 The combined sensitivity of all Groups 1 and 2 mutations was, however, still only 34.0% (95% CI: 29.2–39.0). Sensitivity increased by only 1.0% when variant allele frequency variants ≥ 25% were considered (Table A.1). The observed sensitivity is probably an underestimate, as the PPV of the pDST reference standard itself is likely to be low, because the estimated overall prevalence of LZD resistance is only 2.1% (95% CI: 1.9–2.3), rising to 3.0% (95% CI: 2.7–3.4) among genotypically RIF-resistant isolates (Table A.2) (30). Alternatively, there may be unknown resistance mechanisms and novel gene targets for which we did not account in this analysis. 2 Lee J, personal communication, 2023; Takaki A, Mitarai S, personal communication, 2023; Andres S, personal communication, 2023. 3 Results for ind ivid ual d rugs 61 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 LZD rplC_p.Cys154Arg 24 17415 102 271 27.3% 99.9% 81.0% 81.0% 89.0% 62.2% 274.2 1) AwR ALL+WHO 1) Assoc w R LZD rrl_n.2814G>T 4 17435 19 354 5.1% 100.0% 82.6% 77.8% 97.2% 30.8% 172.4 1) AwR ALL ALL only 2) Assoc w R - Interim LZD rrl_n.2270G>T 4 17435 13 360 3.5% 100.0% 76.5% 66.7% 95.7% 15.7% 96.9 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim LZD rrl_n.2269_2270insT 1 17438 1 372 0.3% 100.0% 50.0% 50.0% 98.7% 1.3% 46.9 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim LZD rrl_n.2299G>T 0 17439 1 372 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim LZD rrl_n.2270G>C NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim LZD rrl_n.2689A>T NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim LZD rrl_n.2746G>A NA NA NA NA NA NA NA NA NA NA NA NA NA Selection 2) Assoc w R - Interim LZD tsnR_p.Leu232Pro 6238 386 120 17 87.6% 5.8% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R LZD rplC_c.-452C>A 731 5893 3 134 2.2% 89.0% 0.4% 0.2% 1.1% 0.0% 0.1 5) NotAwR WHO 5) Not assoc w R LZD rrl_n.344C>T 225 6399 3 134 2.2% 96.6% 1.3% 0.9% 3.3% 0.1% 0.4 5) NotAwR WHO 5) Not assoc w R LZD tsnR_p.Tyr147Cys 392 6232 2 135 1.5% 94.1% 0.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 4666 63 15 9 4 8 6 48 1 46 6 56 35 3 26.9 WHO E CFZ Rv067 _p.Glu49fs 30 4013 2 645 4 3 99.8 49 2 50 0 66 26 4 21.7 ALL+WHO 1) Assoc w R E CFZ Rv0678_p.Gly121Arg 1 4042 4 67 0 6 80 0 80 0 9 5 28 4 83 8 1) AwR ALL ALL only E CFZ Rv0678_p.Leu117Arg 9 40 4 4 6 0 6 99.9 3 8 3 8 61 4 9 1 9.3 BDQ-CFZ X-R E CFZ Rv0678_p.Cys46Arg 4043 3 6 1 4 29.2 BDQ-CFZ X-R E CFZ pepQ_LoF 3 14040 3 671 0.4% 100.0% 50.0% 33.3% 90.6% 0.6% 10.5 3) Uncertain ALL+WHO BDQ-CFZ X-R CFZ Rv0678_p.Ala36Val 1 14042 3 671 0.4% 100.0% 75.0% 100.0% 100.0% 9.4% Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R E CFZ Rv0678_p.Asn70Asp 0 14043 2 672 0.3% 100.0% 100.0% 3) Uncertain ALL+WHO BDQ-CFZ X-R E CFZ Rv0678_p.Met146Thr 36 14007 672 0.3 99.7 5 3 5.4% 18.2% 6 1.2 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Leu32Ser 14040 1 673 0 1 100.0 25.0 25.0 80.6 6 7 0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 0 67 0 0 100.0 0 0 NA NA 0 NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ mmpL5_LoF 24 13801 7 667 0 8 3 2 8 3.0% 6.4% 9 0.6 3) Uncertain ALL+WHO 3) Uncertain ignificance D CFZ mmpL5_p.Ile948Val 4719 15 209 5 97.7% 0.3% 4.2% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7% 0.4% 4.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6% 20.1% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9% 67.1% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Val170Met 518 4216 10 204 4.7% 89.1% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2.8% 86.0% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1.9% 95.2% 1.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Lys270Met 108 4626 1 213 0.5% 97.7% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-389C>A 249 4485 1 213 0.5% 94.7% 0.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fbiA_p.Thr302Met 39 4695 0 214 0.0% 99.2% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-327C>A 42 4692 0 214 0.0% 99.1% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv0678_p.Ser53Pro 0 14043 4 670 0.6% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance P Table 14. Abridged variant classification for LZD Silent mutations are not listed in this table but can be found in the catalogue master file. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 62 3.9 Delamanid and pretomanid The ddn Leu49Pro amino acid change was the only mutation that was sufficiently frequent in the first edition to be classified as a Group 2 mutation for DLM resistance, which remained the case for the second edition (Table 15). In contrast, pooled ddn LoF mutations (Table 23) met the criteria in the ALL data set for Group 2. Moreover, LoF mutations in the remaining five resistance genes considered to be associated with DLM resistance (fbiA, fbiB, fbiC, fgd1 and Rv2983) were recognized as Group 2 mutations for DLM on the basis of laboratory selection, complementation and knockdown experiments conducted for DLM and/or PMD in at least two independent laboratories (84–87). Despite addition of these new grading rules, the sensitivity of all Group 2 mutations for the second edition increased to only 14.7% (95% CI: 10.6–19.7) as compared with 4.4% (95% CI: 2.2–7.7) in the first edition (Table A.1). Heteroresistance did not appear to play a large role, as the sensitivity increased by only 0.4% when the cut- off for calling variants was reduced from an allele frequency of ≥ 75% to ≥ 25% (Table A.1). WHO has not yet set a CC for PMD because of the different intrinsic susceptibility of different MTBC lineages; however, the issue should be settled in 2023 (17,18). Standard analysis for PMD was therefore not possible for the second edition. Irrespective of whether lineage 1 isolates (which have intrinsically higher PMD MICs than lineages 2, 3, 4 and 7) should be considered susceptible or not (17), there was sufficient evidence from several laboratories that LoF mutations in ddn, fbiA, fbiB, fbiC, fgd1 and Rv2983 confer cross-resistance to DLM and PMD and should, therefore, be interpreted as Group 2 interim resistance mutations for both DLM and PMD (Tables 1 and 23) (84–87). LoF mutations have also been described in at least some of these genes in clinical isolates that have not been exposed to nitroimidazoles, indicating that intrinsic resistance to DLM and PMD is possible, although this appears to be rare globally (17,88,89). As the relative increase in MIC for each drug differs for the six genes, and at least some mutations do not appear to affect the MIC for both agents, a general cross-resistance rule was not used for the second edition (86,90). This question will be reviewed for the next version, once a full genotype/phenotype analysis is possible for PMD with a WHO-endorsed CC. Potential role of rare variants Application of “relaxed” thresholds to upstream and coding mutations in all tier-1 target genes for DLM increased the combined sensitivity for predicting DLM resistance by only 2.8%, without decreasing specificity, and increased PPV by 3.9% (Table A.3). The assumption that all Group 3 non-silent coding variants in the six tier-1 genes conferred resistance if they occurred in RIF-resistant isolates increased the combined sensitivity by 9.9% but reduced the specificity by 8.2% and the PPV by 56.2% (Table A.3). These results suggested the presence of some additional resistance mutations in tier 1 resistance genes but that they account for only a small portion of the reduced sensitivity and are less frequent than as-yet- unclassified neutral mutations in these genes. Most of the decrease in sensitivity as compared with pDST, which is similar among isolates that are susceptible rather than resistant to RIF, could be due either to unknown resistance genes or to random false-resistant pDST results, which may be 3 Results for ind ivid ual d rugs 63 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 DLM ddn_LoF 5 11389 19 230 7.6% 100.0% 79.2% 69.2% 90.9% 35.1% 111.4 1) AwR ALL ALL only 2) Assoc w R - Interim R DLM ddn_p.Leu49Pro 2 11392 11 238 4.4% 100.0% 84.6% 100.0% 100.0% 54.6% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim DLM fbiC_LoF 4 11390 3 246 1.2% 100.0% 42.9% 0.0% 60.2% 0.0% 0.0 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim R DLM fbiA_LoF 1 11393 2 247 0.8% 100.0% 66.7% NA NA 0.0% NA 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim R DLM fgd1_LoF 0 11394 2 247 0.8% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim R DLM fbiB_LoF 1 11393 0 249 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim R DLM Rv2983_LoF 1 11393 0 249 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO Selection 2) Assoc w R - Interim R DLM ddn_p.Tyr122_Met129del 0 11394 3 246 1.2% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O DLM ddn_p.Tyr29del 0 11394 2 247 0.8% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O DLM fbiA_p.Arg321Ser 0 11394 2 247 0.8% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance O D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 4666 63 151 29.4 98.6 48 1 46 6 56 0 3 26.9 WHO 1) Assoc w R E CFZ Rv0678_p.Glu49fs 30 4013 29 645 3 99.8 49 2 50.0 66.2 26 4 21.7 ALL+WHO 1) Assoc w R E CFZ Rv0678_p.Gly121Arg 1 4042 4 670 0 6 80 0 80.0 99 5 28.4 83.8 1) AwR ALL ALL only E CFZ Rv0678_p.Leu117Arg 9 4034 4 670 6 99.9 30 8 30.8% 61.4% 9 1 9.3 BDQ-CFZ X-R E CFZ Rv0678_p.Cys46Arg 4043 3 671 4 100.0% 100.0% 29.2% Inf BDQ-CFZ X-R E CFZ pepQ_LoF 3 4040 3 671 4 50.0 33.3% 90.6% 6 10.5 BDQ-CFZ X-R CFZ 0678 p.Ala36Val 4042 3 671 4 75.0 1 0.0% 100.0 9 4 Inf BDQ-CFZ X-R E CFZ Rv0678_p.Asn70Asp 4043 2 672 0 3 NA NA NA NA BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Met146Thr 36 4007 672 3 99.7 5.3% 5.4% 18.2 0.6 1.2 BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Leu32Ser 3 4040 1 673 1 25.0 25.0 80.6 0.6 7.0 BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 0 674 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ mmpL5_LoF 242 13801 7 667 1.0% 98.3% 2.8% 3.0% 6.4% 0.9% 0.6 3) Uncertain ALL+WHO 3) Uncertain significance D CFZ mmpL5_p.Ile948Val 4719 15 209 5 97.7% 0.3% 4.2% 0.0% 84.2% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7% 0.4% 4.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6% 20.1% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9% 67.1% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Val170Met 518 4216 10 204 4.7% 89.1% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2.8% 86.0% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1.9% 95.2% 1.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Lys270Met 108 4626 1 213 0.5% 97.7% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-389C>A 249 4485 1 213 0.5% 94.7% 0.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fbiA_p.Thr302Met 39 4695 0 214 0.0% 99.2% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-327C>A 42 4692 0 214 0.0% 99.1% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv0678_p.Ser53Pro 0 14043 4 670 0.6% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance P more frequent than the true prevalence of resistance for DLM. The latter possibility is plausible, as the approximate prevalence of resistance was only 2% by pDST, irrespective of whether the isolates were susceptible or resistant to RIF (Table A.2), probably resulting in an underestimate of the true sensitivity of gDST (as for LZD) (30). Table 15. Abridged variant classification for DLM Individual LoF mutations in the coding regions of ddn, fbiA, fbiB, fbiC, fgd1 and Rv2983, which are classified in Group 2 because of the associated additional grading rule (see Table 23 and Section 5.8), and silent mutations are not listed in this table but can be found in the catalogue master file. R Confer DLM-PMD cross-resistance. O Group 2 by “relaxed” threshold (not endorsed). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 64 3.10 Amikacin In contrast to the first edition, eis -14C>T had a PPV|SOLO with a lower 95% CI bound of 20% in the ALL data set (see Table 17) and 19% in the WHO data set, which was below the 25% threshold required for Group 1 or 2 grading (Fig. 8). Previous allelic exchange experiments with this mutation, however, clearly showed a modest effect on AMK resistance (15). To err on the side of caution and to remain consistent with other WHO-endorsed assays (Cepheid Xpert® MTB/XDR and Hain GenoType MTBDRsl VER 2.0 (19, 39)), it was decided to continue to classify eis -14C>T and rrs 1402C>T, which also did not meet the PPV|SOLO threshold, in Group 2 (see Table 17). Consequently, the same four mutations were considered resistance mutations in both catalogues. The potential effect of epistasis was evaluated formally (75). The AMK PPV for eis -14C>T was significantly higher when the eis coding region had only silent mutations rather than LoF mutations (Table 16). As expected, the difference in the equivalent KAN PPVs for eis -14C>T were more marked (16). We found only a single isolate with one of the remaining four eis KAN resistance mutations and an eis LoF mutation, although there were 1 212 such eis mutants and only 293 eis -14C>T mutants (Table 16). It has been hypothesized that overrepresentation of LoF mutations that have evolved repeatedly after acquisition of eis -14C>T might be due to the higher fitness cost of this promoter mutation, which results in the greatest over-expression of eis (75). Epistasis was, therefore, recognized for eis -14C>T in both AMK and KAN (Table 1). Assays to recognize this eis promoter mutation should include a disclaimer that this mutation could over-call resistance to AMK (KAN is no longer recommended for clinical use). Assays to recognize the eis promoter and coding region should, if possible, be refined to recognize samples that are potentially affected by epistasis (e.g. when all eis -14C>T reads are linked to an LoF mutation and no other AMK resistance mutation is present in rrs). This is difficult in practice (e.g. when sequencing reads are too short for phasing multiple eis mutations). In the absence of pDST results that prove definitively that an eis -14C>T mutant is susceptible to AMK because of epistasis, it is advisable to interpret eis -14C>T routinely as AMK resistant. Correction for epistasis in this data set decreased the sensitivity by < 0.05% and increased the specificity by 0.2% and the PPV by 1.5% (Table A.1), illustrating the minor overall effect, as other mutations confer most of the AMK resistance. Thus, as the AMK resistance mutations did not differ between both catalogues, the slight difference in performance was due to epistasis. As expected, the corresponding effects of epistasis were more marked for KAN (Table A.1) (16). Low- frequency resistance mutations increased the sensitivity to AMK by 2.6%, with negligible effects on the specificity and PPV (Table A.1). 3 Results for individual drugs 65 Table 16. Impact of LoF mutations on PPV of eis mutations Drug eis mutationa PPV (%, [95% CI]), n With eis silent mutations onlyb With eis LoF mutationc Total eis mutants AMK -14C>T 34 (28–40), 241 2 (0–13), 41 282 KANd -37G>T 82 (76–87), 209 na, 0 209 -14C>T 88 (83–92), 259 18 (7–35), 34 293 -12C>T 44 (40–48), 664 na, 0 664 -10G>A 69 (64–74), 331 0 (0–98), 1 332 -8delC 86 (42–100), 7 na, 0 7 a Promoter mutation at a frequency of at least 90% with no other Group 1 or 2 mutation for the relevant agent at any frequency. b No coding mutation at any frequency, except silent or Group 4 or 5 mutations. c LoF mutation at a frequency of at least 90% (Table 23). d Unlike for AMK (Table 17), an abridged variant table is not included in this report for KAN, as this drug is no longer recommended for TB treatment. The full list of KAN classifications can be found in the catalogue master file. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 66 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 AMK rrs_n.1401A>G 147 21831 1682 778 68.4% 99.3% 92.0% 92.6% 93.8% 89.8% 351.0 1) AwR ALL+WHO 1) Assoc w R AMK rrs_n.1484G>T 5 21973 17 2443 0.7% 100.0% 77.3% 70.6% 89.7% 44.0% 21.6 1) AwR ALL+WHO 1) Assoc w R AMK eis_c.-14C>T 213 21765 90 2370 3.7% 99.0% 29.7% 36.0% 43.1% 20.3% 5.2 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim A AMK rrs_n.1402C>T 14 21964 9 2451 0.4% 99.9% 39.1% 33.3% 59.0% 11.9% 4.5 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim AMK eis_LoF 240 21738 10 2450 0.4% 98.9% 4.0% 2.2% 5.4% 0.4% 0.2 3) Uncertain ALL+WHO 3) Uncertain significance B AMK whiB7_c.-178C>T 18 7755 0 1111 0.0% 99.8% 0.0% 0.0% 41.0% 0.0% 0.0 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim AMK whiB6_c.-75delG 7518 255 1092 19 98.3% 3.3% 12.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK ccsA_p.Ile245Met 3180 4593 617 494 55.5% 59.1% 16.2% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB6_p.Thr51Pro 655 7118 216 895 19.4% 91.6% 24.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.514A>C 241 7532 99 1012 8.9% 96.9% 29.1% 0.6% 3.6% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R AMK bacA_p.Ile273Thr 751 7022 68 1043 6.1% 90.3% 8.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK eis_c.-12C>T 620 7153 44 1067 4.0% 92.0% 6.6% 2.6% 4.4% 1.1% 0.2 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.517C>T 269 7504 43 1068 3.9% 96.5% 13.8% 2.9% 8.4% 0.2% 0.2 5) NotAwR WHO 5) Not assoc w R AMK ccsA_p.Val27Ile 445 7328 39 1072 3.5% 94.3% 8.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK eis_c.-10G>A 278 7495 15 1096 1.4% 96.4% 5.1% 4.5% 7.9% 1.9% 0.3 5) NotAwR WHO 5) Not assoc w R AMK bacA_p.Ile603Val 219 7554 8 1103 0.7% 97.2% 3.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB6_c.-82C>T 197 7576 8 1103 0.7% 97.5% 3.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK eis_p.Val163Ile 126 7647 7 1104 0.6% 98.4% 5.3% 0.9% 5.0% 0.0% 0.1 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.492C>T 229 7544 3 1108 0.3% 97.1% 1.3% 1.4% 4.0% 0.3% 0.1 5) NotAwR WHO 5) Not assoc w R AMK whiB6_c.-75_-73delGCTinsCC 52 7721 2 1109 0.2% 99.3% 3.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB6_c.-75_-73delGCTinsCG 48 7725 2 1109 0.2% 99.4% 4.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB6_p.Arg107Cys 68 7705 2 1109 0.2% 99.1% 2.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1050C>T 32 7741 1 1110 0.1% 99.6% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1208T>A 12 7761 1 1110 0.1% 99.8% 7.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1217T>A 15 7758 1 1110 0.1% 99.8% 6.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1223A>G 16 7757 1 1110 0.1% 99.8% 5.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1507C>T 10 7763 1 1110 0.1% 99.9% 9.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.292G>A 11 7762 1 1110 0.1% 99.9% 8.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.533G>T 7 7766 1 1110 0.1% 99.9% 12.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.534T>G 8 7765 1 1110 0.1% 99.9% 11.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 4666 3 151 29 8 6 48 1 46 56 0 35 3 26.9 WHO E CFZ Rv0678_p. lu49fs 30 1401 29 645 4 3 99.8 49 2 5 0 66 2 26 4 7 E CFZ Rv0678_p.Gly121Arg 14042 4 670 0 6 100.0 80 0 80 99 5 8 4 83.8 1) AwR ALL ALL only E CFZ Rv0678_p.Leu117Arg 9 1403 4 670 6 0 8 0 8 61 4 9.1 9 3 BDQ-CFZ X-R E CFZ Rv0678_p.Cys46Arg 0 14043 3 671 100.0 100.0% 100.0% 100.0% 29.2 Inf BDQ-CFZ X-R 2) Assoc w R - Interim E CFZ pepQ_LoF 3 14040 3 671 4 100.0 50.0 33.3 90 6 6 10.5 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Assoc w R - Interim CFZ Rv0678_p.Ala36Val 1 14042 3 671 0.4 100.0% 75 0 100.0% 100.0% 9 4 Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Asn70Asp 0 14043 2 672 0.3 100.0 100.0 NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Met146Thr 36 14007 2 672 0.3 9 7 5.3 5.4% 18.2% 6 1.2 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Leu32Ser 3 14040 1 673 0 1 100.0 5 0 25.0 80.6 6 7 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 0 674 0 0 100.0 0 0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ mmpL5_LoF 242 13801 7 667 1 8 3 2 8 3 0 6 0 9 6 3) Uncertain ALL+WHO 3) Uncertain ignificance D CFZ mmpL5_p.Ile948Val 4719 15 209 5 97.7 0.3 4.2 0 0 84.2 0 0 CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7 0.4 4 2 CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6 20 1 4 5 NA NA 0 0 NA CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9 6 1 0 CFZ fgd1_p.Val170Met 518 421 10 204 4 89 1 1 CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2 8 86 0 0 9 NA NA NA CFZ mmpL5_p.Phe696Leu 7 4 07 4 210 1 9 5 2 7 NA NA 0 NA CFZ fgd1_p.Lys270Met 108 46 6 1 213 5 7 7 0 9 CFZ Rv1979 _c.-389C>A 249 448 1 213 5 4 7 0 4 CFZ fbiA_p.Thr302Met 39 469 0 214 0 2 0 0 CFZ Rv1979c_c.-327C>A 4 4692 0 214 0 1 0 CFZ Rv0678_p.Ser53Pro 0 14043 4 670 6 100.0 100.0% 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 3 100.0 100.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p. rg50Gln 0 14043 2 672 3 100.0 100.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 3 100.0 100.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 3 100.0 100.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 3 100.0 100.0 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ala99Pro 0 14043 673 100.0 100.0 NA 3) Uncertain ALL+WHO 3) Uncertain ignificance P Table 17. Abridged variant classification for AMK 3 Results for ind ivid ual d rugs 67 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 AMK rrs_n.537G>A 8 7765 1 1110 0.1% 99.9% 11.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.555C>T 21 7752 1 1110 0.1% 99.7% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.685G>A 20 7753 1 1110 0.1% 99.7% 4.8% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.726G>C 18 7755 1 1110 0.1% 99.8% 5.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.868T>C 16 7757 1 1110 0.1% 99.8% 5.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.871C>T 15 7758 1 1110 0.1% 99.8% 6.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.899A>G 22 7751 1 1110 0.1% 99.7% 4.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.936C>T 27 7746 1 1110 0.1% 99.7% 3.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.948A>T 26 7747 1 1110 0.1% 99.7% 3.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.958T>A 24 7749 1 1110 0.1% 99.7% 4.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1190G>A 33 7740 1 1110 0.1% 99.6% 2.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1211A>T 19 7754 1 1110 0.1% 99.8% 5.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.305T>A 21 7752 1 1110 0.1% 99.7% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.306C>T 10 7763 1 1110 0.1% 99.9% 9.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.327T>C 33 7740 1 1110 0.1% 99.6% 2.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.358G>A 12 7761 1 1110 0.1% 99.8% 7.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.736A>T 17 7756 1 1110 0.1% 99.8% 5.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1277T>A 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1302G>C 8 7765 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1319C>G 8 7765 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1327T>C 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1332G>A 12 7761 0 1111 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1347A>G 12 7761 0 1111 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1407T>C 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.261G>A 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.499C>T 18 7755 0 1111 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.51T>C 8 7765 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.534T>C 4 7769 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.672T>A 8 7765 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.816A>G 5 7768 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 46 6 63 151 29.4 8 6 48 46.6% 56.0% 35.3 26.9 1) AwR 1) Assoc w R E CFZ Rv0678_p.Glu49fs 30 14013 29 645 4 3 8 49.2 50.0% 66.2% 26.4 21.7 1) AwR ALL+WHO 1) Assoc w R E CFZ Rv0678_p.Gly121Arg 1 14042 4 670 6 100.0 80.0 80.0% 99.5% 28.4 83.8 1) AwR ALL ALL only 2) Ass c w R - Interim E CFZ Rv0678_p.Leu117Arg 9 14034 4 670 6 9 30.8 30.8% 61.4% 9 1 9.3 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Cys46Arg 0 14043 3 671 4 100.0 100.0% 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ pepQ_LoF 3 14040 3 671 4 100.0 50.0 33.3% 90.6% 6 10.5 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim CFZ Rv0678_p.Ala36Val 1 14042 3 671 4 100.0 75.0 100.0% 100.0% 9 4 Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Asn70Asp 0 14043 2 672 3 100.0 100.0% NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Met146Thr 3 14007 2 672 3 5 3 5.4% 18.2% 6 1.2 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Leu32Ser 3 14040 673 100.0 25.0 25.0% 80.6% 6 7.0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 0 674 0 100.0 0 0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ mmpL5_LoF 242 13801 7 667 1 0 8 3 2 8 3.0% 6.4% 9 0.6 3) Uncertain ALL+WHO 3) Uncertain ignificance D CFZ mmpL5_p.Ile948Val 47 9 1 209 5 97.7 0.3 2 0.0% 84.2% 0.0 CFZ Rv1979c_c.-129A>G 4713 21 207 7 96.7 0.4 4 2 CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6 20 1 4 5 CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9 67 1 3 0 CFZ fgd1_p.Val170Met 518 421 10 204 4 7 8 1 1 9 CFZ Rv1979c_p.Asp286Gly 665 40 9 6 208 2 8 86 0 9 CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1 9 5 2 1 7 CFZ fgd1_p.Lys270Met 108 4626 1 213 5 7 7 9 CFZ Rv1979c_c.-389C>A 249 4485 1 213 5 4 7 4 CFZ fbiA_p.Thr302Met 39 4695 214 2 CFZ Rv1979c_c.-327C>A 4 4692 214 1 CFZ Rv0678_p.Ser53Pro 0 14043 4 670 6 100.0 1 0.0% 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 3 100.0 1 0.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 3 100.0 1 0.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 3 100.0 1 0.0% 100.0% 100.0% 2 5 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 3 100.0 1 0.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 3 100.0 1 0.0% 100.0% 100.0% 15.8 Inf 3) Uncertain ALL+WHO 3) Uncertain ignificance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 1 100.0 1 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain ignificance P C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 68 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 AMK rrs_n.852T>C 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.889C>T 11 7762 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.896G>A 11 7762 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.979T>A 3 7770 0 1111 0.0% 100.0% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.983T>C 3 7770 0 1111 0.0% 100.0% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB7_p.Gly64fs 48 7725 0 1111 0.0% 99.4% 0.0% 0.0% 9.3% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1145A>G 7 7766 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1276T>C 12 7761 0 1111 0.0% 99.8% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1328C>T 9 7764 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.1414C>T 4 7769 0 1111 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.406G>A 16 7757 0 1111 0.0% 99.8% 0.0% 0.0% 97.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R AMK rrs_n.906A>G 34 7739 0 1111 0.0% 99.6% 0.0% 0.0% 11.9% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R AMK whiB6_p.Arg54Gln 51 7722 0 1111 0.0% 99.3% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R AMK whiB7_c.-100T>C 86 7687 0 1111 0.0% 98.9% 0.0% 0.0% 4.6% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . CFZ Rv0678_LoF 68 46 63 151 29.4 8 6 48.1 46.6% 56.0% 35.3 26.9 1) AwR 1) Assoc w R E CFZ Rv0678_p.Glu49fs 30 14013 29 645 4 3 8 49.2 50.0% 66.2% 26.4 21.7 1) AwR ALL+WHO 1) Assoc w R E CFZ Rv0678_p.Gly121Arg 1 14042 4 670 6 100.0 80.0 80.0% 99.5% 28.4 83.8 1) AwR ALL ALL only 2) Ass c w R - Interim E CFZ Rv0678_p.Leu117Arg 9 14034 4 670 6 99.9 30.8 30.8% 61.4% 9 1 9.3 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Cys46Arg 0 14043 3 671 4 1 0.0% 100.0% 100.0% 29.2 Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ pepQ_LoF 3 14040 3 671 4 100.0 50.0 33.3 90.6 6 10.5 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim CFZ Rv0678_p. la36Val 1 14042 3 671 4 100.0 75.0 100.0% 100.0% 9 4 Inf 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Asn70Asp 0 14043 2 672 3 100.0 1 0.0% NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Met146Thr 36 14007 2 672 3 7 5 3 5.4% 18.2% 6 1.2 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Leu32Ser 3 14040 1 673 1 100.0 25.0 25.0% 80.6% 6 7.0 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ Rv0678_p.Ile67Ser 1 14042 674 100.0 NA NA NA 3) Uncertain ALL+WHO BDQ-CFZ X-R 2) Ass c w R - Interim E CFZ mmpL5_LoF 242 13801 7 667 1 8 3 2 8 3 6.4 9 6 3) Uncertain ALL+WHO 3) Uncertain ignificance D CFZ mmpL5_p.Ile9 8Val 47 9 15 2 9 5 97.7 0.3 4 2 0.0% 84.2% 0.0 CFZ Rv19 9c_c.-129A>G 4713 21 2 7 7 96.7 0.4 4 2 NA NA NA CFZ mmpL5_p.Thr794Ile 3782 952 179 35 83.6% 20.1% 4.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Asp767Asn 1559 3175 49 165 22.9% 67.1% 3.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Val170Met 518 4216 10 204 4.7% 89.1% 1.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_p.Asp286Gly 665 4069 6 208 2.8% 86.0% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ mmpL5_p.Phe696Leu 227 4507 4 210 1.9% 95.2% 1.7% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fgd1_p.Lys270Met 108 4626 1 213 0.5% 97.7% 0.9% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-389C>A 249 4485 1 213 0.5% 94.7% 0.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ fbiA_p.Thr302Met 39 4695 0 214 0.0% 99.2% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv1979c_c.-327C>A 42 4692 0 214 0.0% 99.1% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R CFZ Rv0678_p.Ser53Pro 0 14043 4 670 0.6% 100.0% 100.0% 100.0% 100.0% 29.2% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala102Thr 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Arg50Gln 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu142Arg 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Leu35Trp 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ser151Pro 0 14043 2 672 0.3% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance P CFZ Rv0678_p.Ala99Pro 0 14043 1 673 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance P Silent mutations are not listed in this table but can be found in the catalogue master file. A Can confer resistance only if genetically linked to a functional Eis. B Abrogates effect of genetically linked Groups 1 and 2 eis mutations. 3 Results for ind ivid ual d rugs 69 3.11 Streptomycin The combined sensitivity of the mutations from the second edition for predicting STM resistance increased by only 0.5%, to 79.7% (95% CI: 78.9– 80.5) over that in the first edition; a further 0.6% was gained by including variants with an allele frequency ≥ 25% (Table A.1). The latter marginal gain was expected, as the evolution of STM resistance often predates INH resistance, so that most STM resistance is transmitted (91). The rrs G878A mutation, first implicated in STM resistance in the first edition, remained an interim resistance mutation (Table 18), consistent with recently published experimental data from MTBC (92). Application of the WHO-endorsed LoF additional grading rule for gid resulted in classification of 137 of the 144 Group 2 mutations (Tables 1 and 23), which increased the sensitivity by 7.6% (95% CI: 7.1–8.1) as compared with Group 1 mutations alone (Table 3). The PPV of only 58.8% (95% CI: 56.1–61.4) was probably due to the small increases in MIC conferred by LoF gid mutations. The current CCs therefore divide the resulting MIC distributions at their lower end (55,56,93–95). As for EMB, it is not clear how well the currently used CCs correspond to the epidemiological cut-off values, which may exacerbate the very major pDST error rate for this resistance mechanism (30). C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 70 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading F F i i n n a a l l c c o o n n f f i i d d e e n n c c e e g g r r a a d d i i n n g g Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . STM rpsL_p.Lys43Arg 100 15508 4715 5653 45.5% 99.4% 97.9% 97.7% 98.2% 96.5% 116.9 1) AwR ALL+WHO 1) Assoc w R STM rpsL_p.Lys88Arg 29 15579 1280 9088 12.3% 99.8% 97.8% 97.8% 98.6% 95.9% 77.2 1) AwR ALL+WHO 1) Assoc w R STM gid_LoF 577 15031 938 9430 9.0% 96.3% 61.9% 60.5% 63.7% 45.0% 2.4 1) AwR ALL+WHO 1) Assoc w R STM rrs_n.517C>T 54 15554 600 9768 5.8% 99.7% 91.7% 90.3% 92.8% 86.7% 14.9 1) AwR ALL+WHO 1) Assoc w R STM rrs_n.514A>C 18 15590 550 9818 5.3% 99.9% 96.8% 96.5% 98.2% 91.3% 44.0 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Arg39fs 69 15539 130 10238 1.3% 99.6% 65.3% 60.2% 68.5% 45.3% 2.3 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Gln125* 20 15588 87 10281 0.8% 99.9% 81.3% 71.1% 83.6% 47.0% 3.7 1) AwR ALL+WHO 1) Assoc w R STM rpsL_p.Lys88Met 0 15608 31 10337 0.3% 100.0% 100.0% 100.0% 100.0% 85.2% Inf 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Glu99* 4 15604 23 10345 0.2% 100.0% 85.2% 88.0% 97.5% 65.1% 11.1 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Ala134Glu 0 15608 22 10346 0.2% 100.0% 100.0% 100.0% 100.0% 83.9% Inf 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Val105Glu 2 15606 21 10347 0.2% 100.0% 91.3% 90.0% 98.8% 68.3% 13.6 1) AwR ALL+WHO 1) Assoc w R STM gid_p.His48Gln 5 15603 20 10348 0.2% 100.0% 80.0% 81.8% 94.8% 56.3% 6.8 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Ser70Asn 3 15605 17 10351 0.2% 100.0% 85.0% 91.7% 99.8% 49.2% 16.6 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Gly73Glu 2 15606 14 10354 0.1% 100.0% 87.5% 91.7% 99.8% 54.6% 16.6 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Ala200Glu 2 15606 13 10355 0.1% 100.0% 86.7% 92.3% 99.8% 57.2% 18.1 1) AwR ALL+WHO 1) Assoc w R STM gid_p.Pro84Leu 36 15572 56 10312 0.5% 99.8% 60.9% 59.3% 70.1% 45.9% 2.2 1) AwR ALL ALL only 2) Assoc w R - Interim STM rrs_n.878G>A 5 15603 50 10318 0.5% 100.0% 90.9% 86.8% 95.6% 71.9% 10.0 1) AwR ALL ALL only 2) Assoc w R - Interim STM gid_p.Gly73Ala 14 15594 33 10335 0.3% 99.9% 70.2% 70.3% 84.1% 48.3% 3.6 1) AwR ALL ALL only 2) Assoc w R - Interim STM rpsL_p.Lys88Gln 4 15604 15 10353 0.1% 100.0% 78.9% 66.7% 92.5% 26.2% 3.0 3) Uncertain ALL+WHO Prev. WHO 2) Assoc w R - Interim STM gid_p.Asp67His 1 15607 9 10359 0.1% 100.0% 90.0% 100.0% 100.0% 51.8% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim STM gid_p.Gly69Asp 4 15604 13 10355 0.1% 100.0% 76.5% 71.4% 91.6% 41.9% 3.8 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Pro75Arg 2 15606 11 10357 0.1% 100.0% 84.6% 77.8% 97.2% 40.0% 5.3 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Arg83dup 0 15608 2 10366 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Asp67_Gly71del 0 15608 2 10366 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ala119_Glu120insAspGluIleV alArgGlyArgAla 0 15608 1 10367 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Gly30_Pro38del 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ile4_Pro38del 1 15607 1 10367 0.0% 100.0% 50.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ile55dup 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Leu152_Arg154dup 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Leu44_Asp46delinsHis 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Table 18. Abridged variant classification for STM 3 Results for ind ivid ual d rugs 71 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 STM gid_p.Pro14_Gly42del 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Pro84_Arg137del 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ser9_Ala25del 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Val89_Arg102delinsGly 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Arg118_Ser149del 3 15605 0 10368 0.0% 100.0% 0.0% 0.0% 70.8% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Arg83_Glu92del 1 15607 0 10368 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance STM rrs_n.492C>T 381 11493 43 7693 0.6% 96.8% 10.1% NA NA 0.0% NA 5) NotAwR WHO Prev. WHO 4) Not assoc w R - Interim STM rpsL_c.-165T>C 11673 201 7623 113 98.5% 1.7% 39.5% 3.9% 4.5% 1.3% 0.1 5) NotAwR WHO 5) Not assoc w R STM gid_p.Glu92Asp 2620 9254 4202 3534 54.3% 77.9% 61.6% 37.5% 75.5% 0.0% 1.6 5) NotAwR WHO 5) Not assoc w R STM Rv1258c_p.Glu194fs 2467 9407 4033 3703 52.1% 79.2% 62.0% 20.0% 71.6% 0.0% 0.6 5) NotAwR WHO 5) Not assoc w R STM gid_p.Leu16Arg 1630 10244 679 7057 8.8% 86.3% 29.4% 0.0% 28.5% 0.0% 0.0 5) NotAwR WHO 5) Not assoc w R STM glpK_p.Val460Ala 1732 10142 331 7405 4.3% 85.4% 16.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM bacA_p.Ile603Val 1673 10201 326 7410 4.2% 85.9% 16.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM gid_p.Tyr195His 152 11722 117 7619 1.5% 98.7% 43.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM whiB7_p.Gly64fs 421 11453 44 7692 0.6% 96.5% 9.5% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM whiB7_c.-100T>C 101 11773 36 7700 0.5% 99.1% 26.3% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM whiB7_c.-242G>C 76 11798 9 7727 0.1% 99.4% 10.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM rpsL_c.-125G>C 60 11814 7 7729 0.1% 99.5% 10.4% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM rrs_n.1328C>T 8 11866 1 7735 0.0% 99.9% 11.1% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM Rv1258c_p.Gly363Val 38 11836 1 7735 0.0% 99.7% 2.6% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM rrs_n.1327T>C 9 11865 0 7736 0.0% 99.9% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R STM Rv2477c_p.Thr372Lys 38 11836 0 7736 0.0% 99.7% 0.0% NA NA 0.0% NA 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading F F i i n n a a l l c c o o n n f f i i d d e e n n c c e e g g r r a a d d i i n n g g Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . rpsL_p.Lys 3Arg 1 0 5 4715 5653 45.5 99.4 97.9 97.7 98.2 96.5 116.9 1) AwR 1) Assoc w R rpsL_p.Lys88 29 579 1280 9088 12.3 99.8 97.8 97.8 98.6 95.9 77.2 1) AwR 1) Assoc w R LoF 577 031 938 9430 9 96.3 61.9 60.5 63.7 45.0 2.4 1) AwR 1) Assoc w R rrs n.517C>T 54 554 600 9768 5 8 99.7 91.7 90.3 92.8 86.7 14.9 1) AwR 1) Assoc w R rrs n.514A>C 18 590 550 9818 5 3 99.9 96.8 96.5 98 2 91.3 44.0 1) AwR 1) Assoc w R 39fs 69 539 130 23 1 3 99.6 65.3 60.2 68 45.3 2 3 1) AwR 1) Assoc w R gid p.Gln125* 20 5588 87 10281 8 9 9 81 3 71.1% 83.6% 47.0 3.7 1) AwR ALL+WHO 1) Assoc w R p Lys88Met 0 15608 31 103 7 0.3 100.0% 100.0 100.0% 100.0% 85.2 Inf 1) AwR ALL+WHO 1) Assoc w R 9* 4 15604 3 10345 0.2 100.0 85 2 88 0 97 65.1 11.1 1) AwR ALL+WHO 1) Assoc w R gid_p.Ala134Glu 0 15608 22 10346 0.2 100.0 100.0 100.0 100.0 83.9 Inf 1) AwR ALL+WHO 1) Assoc w R Val105Glu 2 5606 21 10347 0 2 100.0 91 3 90.0 9 8 68.3 13.6 1) AwR ALL+WHO 1) Assoc w R id_p.His48Gln 5 5603 20 10348 0 2 100.0 80 81.8% 94.8% 56.3 6.8 1) AwR ALL+WHO 1) Assoc w R gid_p.Ser70Asn 3 56 5 17 10351 0 100.0 85 0 91.7% 99.8% 49.2 16.6 1) AwR ALL+WHO 1) Assoc w R Gly73Glu 2 5606 14 10354 0 1 100.0 87 91.7% 99.8% 54.6 16.6 1) AwR ALL+WHO 1) Assoc w R gid_p.Ala200Glu 5606 13 10355 1 100.0 86.7 92.3% 99.8% 57.2 18.1 1) AwR ALL+WHO 1) Assoc w R gid_p.Pro84Leu 36 55 2 5 10312 8 60 9 59.3% 70.1% 45.9 2.2 1) AwR ALL ALL only 2) Ass c w R - Interim rrs_n.878G>A 5 5603 50 10318 5 100.0 9 9 86.8% 95.6% 71.9 10.0 1) AwR ALL ALL only 2) Ass c w R - Interim gid_p.Gly73Ala 14 559 33 10335 3 9 7 2 70.3% 84.1% 48.3 3.6 1) AwR ALL ALL only 2) Ass c w R - Interim psL_p.Lys88Gln 4 5604 15 10353 1 100.0 78 9 66.7% 92.5% 26.2 3.0 3) Uncertain ALL+WHO Prev. WHO 2) Ass c w R - Interim gid_p.Asp67His 1 5607 9 10359 1 100.0 90.0 100.0% 100.0% 51.8 Inf 1) AwR ALL ALL only 2) Ass c w R - Interim gid p.Gly69Asp 4 5604 13 10355 1 100.0 76.5 71.4% 91.6% 41.9 3.8 3) Uncertain ALL+WHO 3) Uncertain ignificance gid_p.Pro75Arg 2 560 11 10357 1 100.0 84.6 77.8% 97.2% 40.0 5.3 3) Uncertain ALL+WHO 3) Uncertain ignificance STM gid_p.Arg83dup 0 15608 2 10366 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Asp67_Gly71del 0 15608 2 10366 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ala119_Glu120insAspGluIleV alArgGlyArgAla 0 15608 1 10367 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Gly30_Pro38del 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ile4_Pro38del 1 15607 1 10367 0.0% 100.0% 50.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Ile55dup 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Leu152_Arg154dup 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance STM gid_p.Leu44_Asp46delinsHis 0 15608 1 10367 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Individual LoF mutations in the coding regions of gid, which are classified in Group 2 because of the associated additional grading rule (see Table 23 and Section 5.8), and silent mutations are not listed in this table but can be found in the catalogue master file. C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 72 3.12 Ethionamide and prothionamide ETO and PTO were considered to be equivalent for DST and are reported as ETO in this report. Thus, all Groups 1–5 classifications for ETO also apply to PTO (see Section 5.4). There are numerous mechanisms of resistance to ETO (Table 19), some of which include a wide diversity of potential resistance mutations distributed across gene targets, as they are non-essential genes (96,97). Five Group 1 and 281 Group 2 mutations yielded a combined sensitivity of 74.8% (95% CI: 73.6–76.0), a gain of 2.3% over that in the first edition (Table A.1), but with an associated specificity of only 85.9% (95% CI: 85.3–86.4) and a PPV of 63.9% (95% CI: 62.7–65.1) (Table 3). Of those mutations, 258 were classified in Group 2 according to the WHO-endorsed additional grading rule that any LoF mutation in ethA (Table 1) should be assumed to confer ETO resistance. (This rule now excludes indels, in contrast to the first edition [Table 23]). In this analysis, an additional grading rule was applied to ensure that any inhA mutation that met the criteria for ETO resistance was also considered to confer resistance to INH and vice versa (see Table 1 and Section 3.3). Notably, the –154G>A mutation upstream of inhA (i.e. 609G>A in codon 203 of fabG1) was classified as a Group 2 mutation in the ALL data set, which is consistent with published allelic exchange data (49). In contrast, this mutation is interpreted as a marker for INH resistance only in the Xpert® MTB/XDR assay, which should be updated (39). The low PPV (Table 3) observed is probably due mainly to the modest increases in MIC conferred by many ETO resistance mutations, resulting in a considerable overlap with the MIC distribution of susceptible isolates when these mutations occur alone (55,56,98). Development of ETO resistance may be similar to that to EMB, evolving in a stepwise manner, as it is not uncommon for isolates to have many mechanisms with presumably additive effects (54). For example, the most frequent Group 1 mutation, inhA -777C>T, commonly referred to as fabG1 -15C>T, upstream of the fabG1- inhA operon, can co-occur with ethA mutations or the Group 2 inhA S94A, which mutation confers ETO and INH cross-resistance in transduction experiments (50,52,55,99,100). 3 Results for ind ivid ual d rugs 73 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Additional grading criteria applied Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . ETO inhA_c.-777C>T 745 14742 2166 3046 41.6% 95.2% 74.4% 65.5% 67.9% 53.3% 9.2 1) AwR ALL+WHO 1) Assoc w R L ETO ethA_LoF 936 14551 1270 3942 24.4% 94.0% 57.6% 51.0% 53.7% 38.8% 3.8 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Lys37fs 86 15401 109 5103 2.1% 99.4% 55.9% 49.4% 57.3% 40.7% 2.9 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Met1? 49 15438 58 5154 1.1% 99.7% 54.2% 52.5% 62.7% 41.3% 3.3 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Arg207Gly 24 15463 36 5176 0.7% 99.8% 60.0% 63.2% 75.6% 46.5% 5.1 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Tyr235fs 11 15476 26 5186 0.5% 99.9% 70.3% 68.6% 83.1% 50.7% 6.5 1) AwR ALL+WHO 1) Assoc w R ETO inhA_c.-154G>A 138 15349 309 4903 5.9% 99.1% 69.1% 46.2% 54.7% 26.3% 2.7 1) AwR ALL ALL only 2) Assoc w R - Interim H ETO inhA_c.-770T>C 164 15323 186 5026 3.6% 98.9% 53.1% 34.6% 55.7% 2.4% 1.6 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim J ETO inhA_c.-779G>T 139 15348 122 5090 2.3% 99.1% 46.7% 11.5% 22.2% 1.9% 0.4 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim N ETO inhA_p.Ser94Ala 24 15463 118 5094 2.3% 99.8% 83.1% 60.7% 78.5% 26.3% 4.7 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-770T>A 20 15467 64 5148 1.2% 99.9% 76.2% 33.3% 70.1% 2.8% 1.5 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim I ETO ethA_p.Asn379Asp 84 15403 61 5151 1.2% 99.5% 42.1% 40.9% 49.8% 30.9% 2.1 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_c.-7T>C 67 15420 53 5159 1.0% 99.6% 44.2% 43.5% 53.4% 32.1% 2.3 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ser390Phe 5 15482 19 5193 0.4% 100.0% 79.2% 79.2% 92.9% 57.8% 11.3 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ala341Val 7 15480 17 5195 0.3% 100.0% 70.8% 71.4% 88.7% 45.1% 7.4 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Leu35Arg 2 15485 12 5200 0.2% 100.0% 85.7% 81.8% 97.7% 48.2% 13.4 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ser57Tyr 1 15486 11 5201 0.2% 100.0% 91.7% 90.9% 99.8% 58.7% 29.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-770T>G 1 15486 11 5201 0.2% 100.0% 91.7% 66.7% 99.2% 9.4% 6.0 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim K ETO ethA_p.Thr88Ile 1 15486 9 5203 0.2% 100.0% 90.0% 88.9% 99.7% 51.8% 23.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Val202Gly 1 15486 7 5205 0.1% 100.0% 87.5% 87.5% 99.7% 47.3% 20.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Cys403Tyr 0 15487 6 5206 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-778A>G 1 15486 0 5212 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim M ETO ethA_p.Val398dup 3 15484 36 5176 0.7% 100.0% 92.3% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Pro378Leu 60 15427 34 5178 0.7% 99.6% 36.2% 39.0% 50.8% 23.7% 1.9 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Arg150_Pro160del 3 15484 33 5179 0.6% 100.0% 91.7% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Tyr32Asp 14 15473 15 5197 0.3% 99.9% 51.7% 48.1% 68.1% 28.7% 2.8 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Gly11Val 1 15486 14 5198 0.3% 100.0% 93.3% 85.7% 99.6% 42.1% 17.9 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala222_Ile338del 0 15487 7 5205 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Asn287_Leu333del 8 15479 4 5208 0.1% 99.9% 33.3% 11.1% 48.2% 0.3% 0.4 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Gly184del 0 15487 3 5209 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Table 19. Abridged variant classification for ETO C atalogue of m utations in M ycob acterium tub erculosis com p lex and their association w ith d rug resistance - second ed ition 74 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 ETO ethA_p.Thr2_Met41del 0 15487 3 5209 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Thr323dup 5 15482 3 5209 0.1% 100.0% 37.5% 37.5% 75.5% 8.5% 1.8 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala248dup 0 15487 2 5210 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala252_Asp300del 0 15487 2 5210 0.0% 100.0% 100.0% 100.0% 100.0% 15.8% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala20dup 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala237dup 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Asp357del 0 15487 1 5211 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Glu318_Ile339del 1 15486 1 5211 0.0% 100.0% 50.0% 50.0% 98.7% 1.3% 3.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Gly299_Val310del 0 15487 1 5211 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ile339del 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ile339dup 0 15487 1 5211 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Leu129_Val312del 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Leu194_Ala195delinsPro 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Lys30_Ser31insArg 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Lys370_Tyr382del 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Met233_Thr236delinsIle 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Pro160del 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ser251_Ala252insGly 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Trp116_Cys137del 0 15487 1 5211 0.0% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Val85del 0 15487 1 5211 0.0% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Glu223_Lys224del 2 15485 0 5212 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.His201_Lys370del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ile81_His102del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Leu344del 1 15486 0 5212 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Lys224dup 1 15486 0 5212 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Met263_Phe320delinsIle 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Phe431_Thr435del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Pro284_Leu344del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Pro297_Asn388del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Thr366_Tyr369del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . in c.-777C>T 745 4742 2166 3046 41.6 95.2 74.4 65.5 67.9 53.3 9.2 1) AwR 1) Assoc w R L LoF 936 4551 1270 3942 24.4 94.0 5 6 51 0 53 7 38.8 3 1) AwR 1) Assoc w R Lys37fs 86 01 109 103 2 1 99.4 55.9 49.4% 57.3% 40.7% 2.9 1) AwR 1) Assoc w R Met1? 49 38 58 154 1 1 99.7 54.2 52.5 62.7 41 3 3.3 1) AwR 1) Assoc w R rg 7Gly 24 63 36 176 7 99.8 60.0 63.2% 75.6% 46.5% 5.1 1) AwR 1) Assoc w R Tyr 5fs 11 76 26 186 5 99.9 70.3 68.6% 83.1% 50.7% 6.5 1) AwR 1) Assoc w R in c.-154G>A 138 349 309 4903 5 9 99.1 69.1 46.2 54.7 26.3 2.7 1) AwR ALL ALL only 2) Assoc w R - Interim H in c.-770T>C 164 323 186 026 3 6 98.9 3 1 34 6 55 2 4 1 6 WHO-end. gDST 2) Assoc w R - Interim J in c.-779G>T 139 348 122 090 2 3 99.1 46.7 11.5 22.2 1 9 0.4 INH-ETO X-R 2) Assoc w R - Interim N in S r94Ala 24 63 1 8 094 2 3 99.8 83.1 60.7% 78.5% 26.3% 4.7 1) AwR ALL ALL only 2) Assoc w R - Interim in c.-770T>A 20 6 64 148 1 2 99.9 76.2 33.3 70.1 8 1.5 WHO-end. gDST 2) Assoc w R - Interim I Asn37 Asp 84 03 61 15 1 2 99.5 42.1 40.9% 49.8% 30.9% 2.1 1) AwR ALL ALL only 2) Assoc w R - Interim c -7T>C 67 20 53 159 1 99.6 44.2 43.5% 53.4% 32.1% 2.3 1) AwR ALL ALL only 2) Assoc w R - Interim Ser 90Phe 5 2 19 193 4 79.2 79.2% 92.9% 57.8% 11.3 1) AwR ALL ALL only 2) Assoc w R - Interim Ala 41Val 7 0 17 195 3 70.8 71.4% 88.7% 45.1% 7.4 1) AwR ALL ALL only 2) Assoc w R - Interim Leu35Arg 2 5 12 00 2 85.7 81.8% 97.7% 48.2% 13.4 1) AwR ALL ALL only 2) Assoc w R - Interim Ser57Tyr 1 6 11 0 2 91.7 90.9% 99.8% 58.7% 29.8 1) AwR ALL ALL only 2) Assoc w R - Interim in c.-770T>G 1 6 11 0 2 91.7 66.7% 99.2% 9.4% 6.0 INH-ETO X-R 2) Assoc w R - Interim K hr88Ile 1 6 9 03 2 90.0 88.9% 99.7% 51.8% 23.8 1) AwR ALL ALL only 2) Assoc w R - Interim 202Gly 1 6 7 05 1 87.5 87.5 99.7 47.3 20.8 1) AwR ALL ALL only 2) Assoc w R - Interim Cys40 Tyr 0 7 6 06 1 1 0.0% 100.0% 100.0% 54.1 Inf 1) AwR ALL ALL only 2) Assoc w R - Interim in c.-778A>G NA NA NA WHO-end. gDST 2) Assoc w R - Interim M Val398dup 3 4 36 176 7 92.3 NA NA NA Pro 78L u 60 27 34 178 7 99.6 36.2 39.0% 50.8% 23.7 1.9 Arg150_Pro160del 3 4 33 179 6 91.7 Tyr32Asp 14 73 15 197 3 99.9 51.7 48.1 68 1 28.7 2 8 Gly11Val 14 198 3 93.3 85.7 9 6 42.1 17.9 Ala 22 Il 3 8del 0 7 7 05 1 1 0.0% NA NA NA NA Asn 8 Leu 33 l 8 79 4 08 1 99.9 33.3 11.1 48 2 3 4 Gly184del 0 7 3 09 1 1 0.0% 1 0.0% 100.0 2 5 Inf 3 Results for ind ivid ual d rugs 75 D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S E N S I T I V I T Y S P E C I F I C I T Y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O INITIAL CONFIDENCE GRADING S U P P O R T I N G D A T A S E T Additional grading criteria applied FINAL CONFIDENCE GRADING F o o t n o t e s C H A N G E S v s v e r . 1 ETO ethA_p.Tyr32dup 2 15485 0 5212 0.0% 100.0% 0.0% 0.0% 84.2% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Val188_Ser251del 1 15486 0 5212 0.0% 100.0% 0.0% 0.0% 97.5% 0.0% 0.0 3) Uncertain ALL+WHO 3) Uncertain significance ETO mshA_p.Ala187Val 1563 2227 987 1183 45.5% 58.8% 38.7% 6.7% 8.6% 2.5% 0.1 5) NotAwR WHO 5) Not assoc w R ETO mshA_p.Asn111Ser 173 3617 62 2108 2.9% 95.4% 26.4% 3.7% 8.4% 0.9% 0.1 5) NotAwR WHO 5) Not assoc w R D r u g V a r i a n t M U T P r e s e n t _ p h e n o S M U T A b s e n t _ p h e n o S M U T P r e s e n t _ p h e n o R M U T A b s e n t _ p h e n o R S S e e n n s s i i t t i i v v i i t t y y S S p p e e c c i i f f i i c c i i t t y y P P V P P V S O L O P P V S O L O _ u b P P V | S O L O _ l b O R S O L O Initial confidence grading S S u u p p p p o o r r t t i i n n g g d d a a t t a a s s e e t t Final confidence grading N N o o t t e e s s C C h h a a n n g g e e s s v v s s p p r r e e v v . . v v e e r r . . in c.-777C>T 745 4742 2166 3046 41.6 95.2 74.4 65.5 67 9 53.3 9 2 1) AwR 1) Assoc w R L LoF 936 4551 1270 394 24.4 94.0 57.6 51.0 53 7 38.8 3 8 1) AwR 1) Assoc w R ethA_p.Lys37fs 8 15401 109 5 0 2.1 99 4 55 9 49.4 57.3 40.7 2 9 1) AwR ALL+WHO 1) Assoc w R ethA_p.Met1? 49 15438 58 5 54 1 1 9 7 54 2 52.5 62.7 41.3 3 3 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Arg207Gly 24 15463 36 5176 0.7% 99.8% 60.0% 63.2% 75.6% 46.5% 5.1 1) AwR ALL+WHO 1) Assoc w R ETO ethA_p.Tyr235fs 11 15476 26 5186 0.5% 99.9% 70.3% 68.6% 83.1% 50.7% 6.5 1) AwR ALL+WHO 1) Assoc w R ETO inhA_c.-154G>A 138 15349 309 4903 5.9% 99.1% 69.1% 46.2% 54.7% 26.3% 2.7 1) AwR ALL ALL only 2) Assoc w R - Interim H ETO inhA_c.-770T>C 164 15323 186 5026 3.6% 98.9% 53.1% 34.6% 55.7% 2.4% 1.6 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim J ETO inhA_c.-779G>T 139 15348 122 5090 2.3% 99.1% 46.7% 11.5% 22.2% 1.9% 0.4 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim N ETO inhA_p.Ser94Ala 24 15463 118 5094 2.3% 99.8% 83.1% 60.7% 78.5% 26.3% 4.7 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-770T>A 20 15467 64 5148 1.2% 99.9% 76.2% 33.3% 70.1% 2.8% 1.5 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim I ETO ethA_p.Asn379Asp 84 15403 61 5151 1.2% 99.5% 42.1% 40.9% 49.8% 30.9% 2.1 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_c.-7T>C 67 15420 53 5159 1.0% 99.6% 44.2% 43.5% 53.4% 32.1% 2.3 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ser390Phe 5 15482 19 5193 0.4% 100.0% 79.2% 79.2% 92.9% 57.8% 11.3 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ala341Val 7 15480 17 5195 0.3% 100.0% 70.8% 71.4% 88.7% 45.1% 7.4 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Leu35Arg 2 15485 12 5200 0.2% 100.0% 85.7% 81.8% 97.7% 48.2% 13.4 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Ser57Tyr 1 15486 11 5201 0.2% 100.0% 91.7% 90.9% 99.8% 58.7% 29.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-770T>G 1 15486 11 5201 0.2% 100.0% 91.7% 66.7% 99.2% 9.4% 6.0 3) Uncertain ALL+WHO INH-ETO X-R 2) Assoc w R - Interim K ETO ethA_p.Thr88Ile 1 15486 9 5203 0.2% 100.0% 90.0% 88.9% 99.7% 51.8% 23.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Val202Gly 1 15486 7 5205 0.1% 100.0% 87.5% 87.5% 99.7% 47.3% 20.8 1) AwR ALL ALL only 2) Assoc w R - Interim ETO ethA_p.Cys403Tyr 0 15487 6 5206 0.1% 100.0% 100.0% 100.0% 100.0% 54.1% Inf 1) AwR ALL ALL only 2) Assoc w R - Interim ETO inhA_c.-778A>G 1 15486 0 5212 0.0% 100.0% 0.0% NA NA 0.0% NA 3) Uncertain ALL+WHO WHO-end. gDST 2) Assoc w R - Interim M ETO ethA_p.Val398dup 3 15484 36 5176 0.7% 100.0% 92.3% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Pro378Leu 60 15427 34 5178 0.7% 99.6% 36.2% 39.0% 50.8% 23.7% 1.9 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Arg150_Pro160del 3 15484 33 5179 0.6% 100.0% 91.7% NA NA 0.0% NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Tyr32Asp 14 15473 15 5197 0.3% 99.9% 51.7% 48.1% 68.1% 28.7% 2.8 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Gly11Val 1 15486 14 5198 0.3% 100.0% 93.3% 85.7% 99.6% 42.1% 17.9 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Ala222_Ile338del 0 15487 7 5205 0.1% 100.0% 100.0% NA NA NA NA 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Asn287_Leu333del 8 15479 4 5208 0.1% 99.9% 33.3% 11.1% 48.2% 0.3% 0.4 3) Uncertain ALL+WHO 3) Uncertain significance ETO ethA_p.Gly184del 0 15487 3 5209 0.1% 100.0% 100.0% 100.0% 100.0% 2.5% Inf 3) Uncertain ALL+WHO 3) Uncertain significance Individual LoF mutations in the coding regions of ethA, which are classified in Group 2 by the associated additional grading rule (see Table 23 and Section 5.8), and silent mutations are not listed in this table but can be found in the catalogue master file. H Alias fabG1_p.Leu203Leu. I Alias fabG1_c.-8T>A. J Alias fabG1_c.-8T>C. K Alias fabG1_c.-8T>G. L Alias fabG1_c.-15C>T. M Alias fabG1_c.-16A>G. N Alias fabG1_c.-17G>T.

77 4 Future research priorities The mutation catalogue will be updated and revised regularly according to need and emerging evidence. The following research areas have been identified as priorities for future catalogues. Types of data to be analysed Allelic exchange, enzymatic, lineage and MIC data: • Current WHO treatment guidelines indicate that the levels of resistance to INH and MFX have notable implications for treatment. For example, high-dose INH may be useful for low-resistance isolates, and isolates with high-level resistance mutations to MFX cannot be treated with high- dose MFX, even as part of a longer, individualized regimen (5). The assumption that all katG mutations confer high-level INH resistance should be investigated. Moreover, more MICs are required on the rarer FQ resistance mutations. For example, the limited MIC data for gyrA Gly88Cys suggest that this mutation causes high-level resistance, which is not in agreement with enzymatic measurements (15,101). • Some mutations result in only modest increases in MIC that are difficult to classify from categorical pDST data (e.g. rrs 1402C>T and some inhA promoter mutations already included in WHO-endorsed gDST assays). Ideally, the shape and particularly the mode of the MIC distribution of individual mutations should be reviewed to identify potential borderline resistance mechanisms (30,33). Moreover, areas of technical uncertainty, as defined by the European Committee on Antimicrobial Susceptibility Testing, may be necessary to minimize very major pDST errors (4,21, 29,102). Inclusion of additional allelic exchange or selection data may be relevant in this context. • Lineage effects (e.g. lineage 3 for FQs and lineage 1 for PMD and PZA (16,17,103)) and homoplasy, which may be a signal for selection, should be explored systematically (64,104). A more strategic approach to collecting data, commissioning additional testing and interpreting the findings to maximize the usefulness of gDST: • To support this approach, a global TB sequencing knowledgebase has been established at WHO, in collaboration with FIND, as a repository of associated phenotypic–genotypic data sets, with user-friendly dashboards and features for browsing and querying. The portal will support gathering of data contributions by global partners for future updates of the catalogue. It is expected to be ready for use by the end of 2023 and available at TB sequencing knowledgebase. Potential data contributors who seek more information are invited to contact the WHO Global TB Programme (tbsequencing@who.int) . Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 78 • Susceptible and resistant pDST must be collected to avoid inflated PPVs and potential false associations with resistance. Given the frequency of Rv0678 Met146Thr in Eswatini, additional unbiased results for this mutation are particularly important (29). • Group 3 mutations that may result in the largest potential gain in sensitivity should be a priority, particularly if they are homoplasic. • Additional isolates are required from countries and MTBC lineages that are currently underrepresented. • Exceptions to the “additional grading rules” (Table 1) that may result in significant harm in some settings should be studied. For example, rpoB Thr427Ala may not confer RIF resistance despite being in the RRDR (33,105). Grading criteria • A comprehensive analysis of PMD is warranted as soon as a WHO-endorsed CC becomes available (17). • The validity of the breakpoints used should be assessed, particularly if dosing of antibiotics changes (e.g. if a higher dose of RIF is endorsed (4)). • Prioritization of categorical pDST results over MIC results obtained with the same method and exclusion of discordant pDST results may have to be reconsidered, as became apparent in the analysis of Rv0678 Met146Thr. • The calculations in Table A.3 show that rare resistance mutations in non-essential genes probably play an important role for some key drugs (e.g. in Rv0678 for BDQ). Less stringent grading criteria, akin to the “relaxed” thresholds already endorsed for pncA (Fig. 9), might have to be endorsed. • Alternative approaches, such as regression analyses, will be required to classify markers of resistance that, by definition, have a low PPV|SOLO (e.g. compensatory mechanisms, such as in ahpC, or resistance mutations that usually occur in combination). • Selection of genes and corresponding regulatory regions should be revised in the light of the latest scientific evidence. For example, the role of Rv1979c in BDQ and CFZ resistance has been questioned, whereas Rv1453 may be involved in CFZ resistance (13,106,107). Moreover, dprE2 (Rv3791) was recently found to be the shared target of DLM and PMD, fusA1 (Rv0684) may have to be considered for KAN/AMK, and mshC (Rv2130c) may be relevant for ETO/INH, and (84,108). Bioinformatics pipeline • Large insertions, such as an IS6110 insertion that can cause resistance to several drugs, including BDQ, cannot be detected reliably with the current pipeline (109,110). Moreover, some small inframe changes are sometimes reported as two frameshifts, resulting in inappropriate application of the LoF additional grading rule, e.g. katG changes in samples “2020 DNA6” and “2020 DNA10” from an external quality assessment scheme in 2020 (48). • So far, only aftA, fabG1 and furA have been analysed at nucleotide instead of amino acid level, because mutations in these genes could affect the expression of downstream resistance genes 4 Future research priorities 79 (49,54,111). Such analysis might have to be extended to other translated genes if there is evidence of selection of synonymous mutations (e.g. if they are homoplasic) (112–114). • The assumption that different nucleotide changes that result in the same amino acid substitution have the same effect may have to be reconsidered (64,115). • The pipeline should be adapted for analysis of next-generation sequencing data from genomic technologies other than Illumina, such as those from Oxford Nanopore Technologies. Guidance should be developed on what, if any, confirmatory testing should be conducted if a marker for resistance is found and how discordant results should be resolved if an isolate is found to be susceptible by pDST (i.e. the extent to which a composite reference standard should be endorsed for individual patient treatment (30,33)). The relative contributions of the following factors should be considered for each mechanism and/or mutation. • It is well understood that traditional gDST assays can yield false-resistant or false-susceptible results because of inherent limitations in the underlying technology (e.g. some mutations are missed, even though they are targeted, and low bacillary loads may affect the accuracy (30,116– 118)). Limitations specific to certain technologies for next-generation sequencing are also possible (Section 3.2). Manufacturers of sequencing technology, assay developers and users should notify WHO and other relevant parties of suspected problems (e.g. positions that are more prone to sequencing errors). The outcomes of the investigation of such issues should be shared with users in a timely manner (116,119). • The reproducibility of pDST, the accuracy of the breakpoint used and the prevalence of resistance should be evaluated. • Studies should be conducted on whether a mutation results in MICs close to the breakpoint. • The classification of some mutations according to additional grading rules or to previous WHO decisions may be incorrect. For example, a nonsense mutation one codon before the actual stop codon of katG is unlikely to confer INH resistance. Such exceptions could be excluded from the additional grading rules by adding them to Group 4 or 5, depending on the quality of the evidence. • Investigation should be made of whether epistasis can confound the interpretation of a mutation in genes other than Rv0678 and the eis promoter region. For example, if mymA (Rv3083) is naturally overexpressed in some isolates, it could counteract an LoF mutation in ethA, although this has not been described to date (97).

81 5 Methods 5.1 Overview Four primary components were essential for developing the second edition. 1. High-quality pDST DST data were curated manually to ensure that the best available phenotypic data were used as a reference standard for genotypic/phenotypic associations. As different DST methods were used during the period over which the data were collected and as WHO-endorsed CCs changed with time, the phenotypic methods were rank-ordered from WHO-endorsed methods (highest ranking) to non-endorsed methods (lowest); among WHO-endorsed methods, they were ranked from most recent (highest) to oldest (lowest). For data on isolates with multiple phenotypes, a hierarchy was used, in which the most recent WHO-endorsed DST methods were ranked highest, and older or non-WHO methods were ranked lower. A large data set on BMD DST was also included, which contributed substantial data on new and repurposed drugs. As the method and criteria for its interpretation have not been reviewed or endorsed by WHO, findings based on these data alone were classified as “interim” associations in the catalogue. 2. High-quality, standardized WGS for generating unbiased sequence data Only WGS data that were generated with Illumina instruments were included in the analysis. These next-generation sequencing platforms are currently the most widely used globally. This ensures that the raw sequencing output data are standardized to the greatest extent possible among the various Illumina next-generation sequencing instruments. The output file of raw sequencing reads was used as the starting-point for the bioinformatics analyses. 3. A comprehensive bioinformatics pipeline for variant detection and annotation To ensure that mutations were identified uniformly in all the raw sequencing data, a single bioinformatic pipeline based on the “clockwork” architecture (https://github.com/iqbal- lab-org/clockwork) (120) was used to process the data through quality checks, align the reads to the M. tuberculosis H37Rv reference genome and identify variants. The pipeline was designed to maximize variant detection of both single nucleotide polymorphisms and small insertions and deletions up to 15 bp in length. The pipeline also generated sequencing depth and coverage values for each sample, so that quality control filters could be applied further downstream. For the second edition, we also identified large-scale deletions of up to 100 kbp with an additional open access tool (121). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 82 4. A validated algorithmic approach for statistically associating variants with phenotypes The matched and curated phenotype and genotype data were then processed in a multistage algorithm that was first used to identify neutral mutations (not associated with resistance). Masking the neutral mutations from further analysis, it then identified MTBC isolates with SOLO mutations, defined as the single remaining mutation among candidate genes (selected by the working group as having the highest pre-test probability of being associated with drug resistance to a specific drug, Table 21). All mutations and corresponding phenotypes were then analysed statistically for confidence grading, including determining odds ratios (ORs) and PPVs for association of the variant with phenotypic resistance. As the data set was large and heterogeneous, strict criteria were applied, and, when applicable, the bounds of the 95% CI were considered with the relevant statistics. The bioinformatics pipeline for identifying variants and the algorithms for identifying variants “associated with” and “not associated with” resistant phenotypes was adapted from approaches developed by the CRyPTIC Consortium (22), and the confidence grading method was developed for the Seq & Treat project (6,122). All these elements were described in detail in the first edition (14). Methods were refined and optimized for the second edition during a series of meetings of an international panel of expert advisors in sequencing, bioinformatics, biostatistics and mycobacteriology. Methods were proposed, adapted and finalized in webinars and e-mail communication due to travel restrictions during the coronavirus disease 2019 pandemic. Details of the methods and changes are described below. 5.2 Revised computational architecture One of the secondary but still critical goals of the second edition of the mutation catalogue was to ensure a more centralized, efficient, sustainable workflow for future revisions of the catalogue, with minimal hands-on work. For the first edition (14), all the phenotypic and genotypic data and all the computational processes, including bioinformatics processing of the sequencing data and statistical grading of the variants, were based on a restricted-access, academic high- performance computing cluster. For the second edition, all the data and computational processes were transferred to a WHO-supervised cloud service and adapted to run on a public cloud service provider. A new relational database schema was developed to house the data in the WHO cloud. The schema was designed to include information ranging from samples and associated sequencing data, to genotype calls identified after bioinformatics processing. A complete relational model for the required variables for downstream bioinformatics and statistical analyses helped to form a reliable, accessible, transparent and reproducible service. PostgreSQL was chosen as the open- source relational database management system. We incorporated tables from the open-source BioSQL database format (https://github.com/biosql/biosql) and used the associated suite of tools to load the reference genome and annotation in the associated table. Then, we included additional tables for the specific study of genetic variants and their association with phenotypes. The full database table definition (which we called GenPhenSQL) is available for review and dissemination at https://github.com/GTB-tbsequencing/mutation-catalogue-2023/tree/main/ Final%20Result%20Files. For the second edition, the GenPhenSQL database was used to provide 5 Methods 83 all inputs for the downstream computational analyses, starting with bioinformatics processing of the sequencing data. 5.3 Data sources As for the first edition, data were aggregated from various sources: legacy data sets from publications and consortium initiatives and direct submissions in response to public calls for contributions by the WHO Global Tuberculosis Programme. The minimum acceptable phenotypic data included the pDST method and a categorical result (resistant, susceptible or intermediate), but MIC ranges were also collected in tabular or simple text formats when available. pDST and MIC data were collected in tabular format and were inserted into the GenPhenSQL database as specific extract-transform-load scripts built with Python/Pandas. We synchronized the GenPhenSQL daily with the International Nucleotide Sequence Database Collaboration databases to connect pDST and MIC submissions linked to publicly available sequencing data. We used the public National Center for Biotechnology Information ENTREZ application programming interface for BioSample and Sequence Read Archive databases for that purpose. Consequently, our extract–transform– load script could accept pDST or MIC submissions with sample identification labels from the National Center for Biotechnology Information or the European Nucleotide Archive directly. We also accepted direct private sequencing data submission. We recorded the origin of the sequencing data (i.e. private or publicly available) in GenPhenSQL so that the analytical workflow could correctly retrieve the raw data at runtime. Whenever possible, submissions linked directly to publicly available sequencing data were preferred. When contributors preferred private, direct submission of sequencing data, we accepted only raw FASTQ files, so that all samples could be processed with the same bioinformatics pipeline. In a few cases in which only BAM files were available, we converted the BAM back to FASTQ format with SAMtools. 5.4 Curation of pDST data All pDST results associated with MTBC isolates for which WGS data were also available were collated and analysed. Categorical (resistant, susceptible or intermediate) and/or MIC pDST data were considered. Intermediate categorical pDST results were converted to binary results (R or S) or excluded according to additional grading rules approved by the working group. MIC data were converted into categorical binary results (resistant or susceptible) according to CCs appropriate to the pDST method used, as described below. We then stratified the pDST data into eight categories according to the level of WHO endorsement of the method, as described in detail below and summarized in Fig. 6. Category 1. pDST methods currently endorsed by WHO (WHO CURRENT) Categorical pDST results for Löwenstein-Jensen (LJ), Middlebrook 7H10 (7H10), Middlebrook 7H11 (7H11) and MGIT were regarded as “current” if the CCs in the latest published WHO DST manual (123) were used, with the following exceptions. We used the updated RIF CCs from 2021 (4). Results for ofloxacin (OFX) and KAN were regarded as “current” if they were based on the Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 84 CCs from the 2018 WHO technical report (15). KAN was still included, even though it is no longer recommended for TB treatment, as the data provide useful insights into the effect of eis promoter mutations and rrs 1402C>T on AMK (15,16). Similarly, although WHO no longer recommends testing for OFX to ensure that it is not used clinically, pDST for OFX resistance at concentration x is equivalent to testing LFX at x/2, as OFX consists of equal amounts of the active L-isomer of OFX (i.e. LFX) and the largely inactive D-isomer, as reflected in the CCs for the two drugs (15). For this reason and because OFX was still widely tested in WHO-endorsed media, pDST results for OFX and LFX were pooled and reported as LFX results in the mutation tables in this report. Similarly, pDST results for ETO and PTO were pooled and reported as ETO. For MFX, we also considered non-WHO CCs that were lower than previous WHO CCs but above the current WHO CC, as these non_WHO_ CC_SR results were considered more accurate than results with previous WHO CCs (15). In some cases, we had to assume that pDST was conducted by the proportion method with the correct critical proportion, as this information was not collected systematically from all contributors. In practice, this assumption was probably correct for the majority of results (i.e. only a minority of testing on LJ may have been done with the resistance ratio or absolute concentration method). Microscopic observation drug-susceptibility (MODS) results with a CC of 1 mg/L RIF or 0.4 mg/L INH were also considered to be “current” (124,125). Category 2. pDST methods previously endorsed by WHO (WHO PAST) This category included pDST results for LJ, 7H10, 7H11, MGIT or BACTEC™ 460 obtained either with outdated WHO CCs or simply reported to have been based on WHO CCs without providing the drug concentration tested, in which case it was not clear which WHO CC was followed (126– 128). It was assumed that the proportion method with the correct critical proportion was used. Category 3. Other pDST methods This category consisted primarily of a very large genotypic and pDST data set from the CRyPTIC Consortium, which used novel BMD plates manufactured by Thermo Fisher for pDST. Although Clinical and Laboratory Standards Institute CCs exist for RIF, INH and EMB for the MYCOTB(I) BMD plate, which is also manufactured by Thermo Fisher, BMD plates and the corresponding CCs are not endorsed by WHO. Two different CRyPTIC plates were used to produce raw MIC data, namely the UKMYC5 and UKMYC6 plates. MIC data were translated into binary resistant or susceptible results with the CCs listed in Table 20, and the pDST results were given the code MYCOTB_MIC in the data set. The same CCs were also used to translate MICs from the MYCOTB(I) plates into categorical pDST (16). The rationale and derivation of these CCs can be found elsewhere (22). Category 3 also included pDST results derived with methods for which it was unclear whether they met either current or previous WHO guidelines, either because no information on the CC was provided, or, a CC was available, but it was not clear whether 7H10 or 7H11 had been used. In addition, resistance results from LJ, 7H10, 7H11, MGIT or BACTEC™ 460 from categorical or MIC data with a breakpoint above the latest WHO CC were considered and given the Non_WHO_CC_R code. Susceptible results from LJ, 7H10, 7H11, MGIT or BACTEC™ 460 from categorical or MIC data with a breakpoint below or equal to the latest WHO CC were included with the Non_WHO_CC_S code. 5 Methods 85 Table 20. Drugs tested with two CRyPTIC BMD plate designs and corresponding CC used to translate MIC data into binary resistant or susceptible categories Group Drug Concentration range (mg/L) CC used for interpretation as R or S (mg/L)a UKMYC5 UKMYC6 First-line RIF 0.06–4 0.03–8 0.5 INH 0.025–1.6 0.025–12.8 0.1b EMB 0.125–8 0.25–32 4c Group A LFX 0.125–8 0.12–8 1 MFX 0.6–4 0.06–4 1 BDQ 0.016–2 0.008–1 0.25 LZD 0.03–2 0.06–4 1 Group B CFZ 0.06–4 0.03–2 0.25 Group C DLM 0.016–1 0.008–0.5 0.125 AMK 0.25–8 0.25–16 1 ETO 0.25–8 0.25–8 4 Otherd KAN 1–16 1–16 4 a Neither method nor CC is endorsed by WHO, and some of the CCs have been questioned (16,22–25). b Equivalent to the Clinical and Laboratory Standards Institute CC of 0.12 mg/L for the MYCOTB plate (57). c Identical to the Clinical and Laboratory Standards Institute CC of 4 mg/L for the MYCOTB plate on the assumption that the “inconclusive” concentration of 4 mg/L corresponds to an area of technical uncertainty, as defined by the European Committee on Antimicrobial Susceptibility Testing (21,57). d Drug no longer endorsed for TB treatment. Category 4. Excluded pDST results This category included all pDST results that did not fit into categories 1–3 and were excluded from the analysis. 5.5 Prioritization of pDST results As the reference pDST methods and the CCs used to evaluate genotype/phenotype associations could significantly affect the outcomes of our statistical analyses, we completed two separate association analyses, first using only data with pDST results in categories 1 and 2 (WHO CURRENT and PAST) – which we considered to be more conservative but also less inclusive – and then using pDST data from categories 1, 2 and 3 (ALL) – which we considered less conservative and more inclusive. After completing independent association calculations, we compared and contrasted the resulting associations stratified by pDST category and reported both in the ALL and WHO (WHO CURRENT and PAST) columns of the master table of mutation associations. For many MTBC isolates, pDST results were obtained with more than one method. In these cases, we ordered them by priority to include only the pDST data most recently endorsed by WHO according to the following hierarchy: • category 1 > category 2 > category 3 Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 86 • Within the same category, solid media pDST results were given higher priority than those in liquid media, because the latter are more likely to miss key, clinically relevant rpoB mutations (129). • Liquid media methods within the same category were prioritized as follows: MGIT > MODS > BACTEC™ 460 > CRyPTIC, as BACTEC™ 460 are no longer used, and MGIT has undergone more validation than MODS. • Solid media methods in the same category were prioritized as follows: 7H10 > LJ > 7H11, because there is generally more supporting evidence for 7H10 CCs. This hierarchical organization enabled creation of standardized data sets that included only isolates that had been tested with the same methods, e.g. only currently WHO-endorsed methods (WHO) or any acceptable DST method (ALL) (Fig. 6). While there was probably more pDST variation and phenotype noise in the ALL data set, the number of isolates in that data set was significantly larger, which was at times critical for detecting a statistically significant relation between genotype and observed phenotype. If an isolate was tested several times with the same method (same media and same CC) but with discrepant results, all the discordant results were excluded, and concordant results were considered. The same rule was applied to binarized MIC data. Fig. 6. Summary of pDST stratification and hierarchy WHO current Categorical LJ, 7H10, 7H11 or MGIT results using latest WHO CCs. MODS results with a CC of 1 mg/L RIF or 0.4 mg/L INH were also considered to be “current” non_WHO_CC_R Resistant results from LJ, 7H10, 7H11, MGIT or BACTEC™ 460 from categorical or MIC data using a breakpoint above the latest WHO CC WHO past Categorical LJ, 7H10, 7H11, MGIT or BACTEC™ 460 results using outdated WHO CCs CRyPTIC_MIC MICs from custom BMD Thermo Fisher plates that were converted into categorical results using CRyPTIC CCs (not WHO-endorsed) non_WHO_CC_SR Categorical results that did not use WHO CCs but were more accurate than those using past WHO CCs (i.e. the breakpoints used were below the past WHO CC but above the current WHO CC). Applies to MFX only non_WHO_CC_S Susceptible results from LJ, 7H10, 7H11, MGIT or BACTEC™ 460 from categorical or MIC data using a breakpoint below or equal to the latest WHO CC WHO undefi ned Categorical LJ, 7H10, 7H11, MGIT or BACTEC™ 460 results reportedly obtained using WHO guidelines but where the CC or precise medium was not specifi ed MYCOTB_MIC MICs from commercial BMD Thermo Fisher plates that were converted into categorical results using CRyPTIC CCs (not WHO-endorsed) A LL d at as et W H O d at as et Stars denote changes from the approach used for the first edition. 5 Methods 87 5.6 Variant analysis Bioinformatics pipeline Although the bioinformatics pipeline for variant detection was adapted to run on a public cloud provider for the second edition, most of the steps in the analysis remained the same for the two catalogues. Paired reads were mapped with Burrows-Wheeler Aligner to the same reference (GenBank NC_000962.3), and PCR duplicates were removed with SAMtools. When more than one sequencing run was available for a single isolate, alignments for each pair were merged before genotyping. For genotyping, a standard caller (Freebayes) was used to detect single nucleotide polymorphisms and small indels, and another caller (delly), capable of detecting large deletions, was also used. For quality control, the taxonomy of aligned reads was assigned with kraken; global sequencing statistics metrics were collected with SAMtools, and gene-specific sequencing statistics were calculated with mosdepth. At the end of bioinformatics processing, four types of information were inserted into the GenPhenSQL database: all genotype calls, the global sequencing metrics, the per-gene sequencing metrics, and the taxonomy assignments of aligned reads. A scheme of the pipeline is provided in Annex Fig. A.1. Variant callers Genotype calls were generated with two different callers, which handle small-scale events separately: Freebayes for genotypes typically shorter than 15 bp and delly for large-scale deletions. All variants found in more than 20% of the reads (fraction of supporting reads) were considered and inserted into the GenPhenSQL. Freebayes is a haplotype-based Bayesian genotype caller and can therefore handle many consecutive nucleotide changes, ensuring that point mutations that affect the same codon are appropriately accounted for at the annotation step. Before insertion into GenPhenSQL, all variant coordinates were normalized with bcftools norm; variants that spanned several nucleotides were not decomposed into single variants in order to preserve the correctness of the annotation. Large-scale deletions were inserted next to the other genotype calls according to the coordinates of the deletion, as determined by delly. For overall quality control of per-sample sequencing, we recorded the median sequencing depth for the full genome as well as the percentage of the genome covered at 15×, 20×, 25× and 30×. We included samples in the analysis if their median depth was greater than 15× and at least 95% of the reference genome had been sequenced at least 20 times. We collected the same metrics for each gene and considered that a gene was missing from a sample if at least one of its nucleotides had been sequenced fewer than 10 times. Variant annotation One benefit of using a relational model for computation was that variant annotation (which relies on a given set of coordinates for a reference genome) could be performed independently from identification of genotypes in samples. In practice, this meant that only iterative extraction of new entries from the “variant” table of GenPhenSQL was necessary to perform the annotation. Annotation was performed with SnpEff, and the same reference genome and was configured with Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 88 the GFF annotation file for the entry GCF_000195955.2_ASM19595v2 and the bacterial genetic code (translation table 12 in GenBank). To correctly annotate dnaA (Rv0001), the first gene on the linearized sequence, and its promoter variants at the end of the linear sequence, we created a modified sequence whereby nucleotides located at the end of a reference were collated in front. Only variants on the dnaA promoter were annotated with that modified reference. From the raw output of the annotation returned by SnpEff, a custom Python script curated some of issues that have been identified, such as incorrect Human Genome Variation Society (HGVS) nomenclature, in order to improve compatibility with bioinformatic interpretation tools over that in the first edition of the catalogue (130), and normalized the information for insertion into GenPhenSQL. The relation modelled between variants and annotation was “many-to-many”, as one variant could have had different annotation values with respect to different genes, and an annotation could be associated with more than one variant because of redundancy of the genetic code, such as when different nucleotide changes lead to the same missense variant. After normalization and insertion into the database, the annotation table included the predicted effect of the detected variant (e.g. missense or synonymous) on the gene or protein considered, as well as the tentative HGVS nomenclature annotation as provided by SnpEff for that effect. With respect to the annotation of deletion variants detected by delly, one annotation entry was inserted for every gene that fell entirely within the range of the deletion. The “predicted_effect” value was set as “feature_ablation”. Data extraction The rules for setting the hierarchy and prioritization for categorizing pDST and MIC data were based on the large-scale data analysis, open-source engine, Apache Spark and its Python implementation (PySpark). Quality control per sample and per gene was also assured with PySpark, as was transformation of the raw information on variant annotation inserted into the GenPhenSQL. The aim of variant curation was to link genotypes identified in samples unambiguously to predicted changes in the tiered genes included in the resistance association list (Table 21). For each resistance gene, relevant promoter and/or upstream regions were defined according to the primary transcriptional start site (Table 22). Two pieces of information for each variant were determined from the inserted SnpEff output for statistical grading: the predicted effect and the variant category. Depending on the context, the final unit of study for the grading algorithm could be a protein encoded by the gene (for missense, frameshift or in-frame insertion or deletion included in a coding sequence) or relative to the gene or mRNA itself (for non-coding genes, synonymous or intergenic variants). All possible values and examples are listed in Table 23. 5 Methods 89 Table 21. Candidate resistance genes Drug Tier 1 Tier 2 References INH fabG1 (Rv1483), inhA (Rv1484), katG (Rv1908c), furA (Rv1909c), ahpC (Rv2428) dnaA (Rv0001), Rv0010c, mshA (Rv0486), hadA (Rv0635), Rv1129c, Rv1258c, ndh (Rv1854c), Rv2752c, glpK (Rv3696c) 49,111,131–141 RIF rpoB (Rv0667) nusG (Rv0639), rpoC (Rv0668), Rv1129c, Rv2477c, Rv2752c, lpqB (Rv3244c), mtrB (Rv3245c), mtrA (Rv3246c), rpoA (Rv3457c), glpK 6,76,135,136,139–142 EMB aftA (Rv3792), embC (Rv3793), embA (Rv3794), embB (Rv3795), ubiA (Rv3806c) embR (Rv1267c), Rv2477c, Rv2752c, glpK, aftB 54,76,135,13–141,143 PZA pncA (Rv2043c), clpC1 (Rv3596c), panD (Rv3601c) Rv1258c, rpsA (Rv1630), sigE (Rv1221), PPE35 (Rv1918c), Rv3236c 6,137,139,144–146 FQ gyrB (Rv0005), gyrA (Rv0006) Rv1129c, Rv2477c, Rv2752c, glpK 6,76,136,140,141 BDQ mmpL5 (Rv0676c), mmpS5 (Rv0677c), Rv0678, atpE (Rv1305), pepQ (Rv2535c) Rv1979c, lpqB, mtrB, mtrA 13,76 LZD rplC (Rv0701), rrl (MTB000020) tsnR (Rv1644) 13,76 CFZ mmpL5, mmpS5, Rv0678, Rv1979c, pepQ fgd1 (Rv0407), fbiC (Rv1173), Rv2983, fbiA (Rv3261), fbiB (Rv3262) 13,84,147 DLM fgd1, ddn (Rv3547), fbiC, Rv2983, fbiA, fbiB ndh 86,148 AMK rrs (MTB000019), eis (Rv2416c), whiB7 (Rv3197A) ccsA (Rv0529), bacA (Rv1819c), Rv2477c, whiB6 (Rv3862c) 15,76,139,149 STM rpsL (Rv0682), Rv1258c, rrs, whiB7, gid (Rv3919c) bacA, Rv2477c, glpK 6,76,137,149,150 ETO mshA, fabG1, inhA, ethA (Rv3854c) Rv0565c, ndh, Rv3083, ethR (Rv3855) 49,97,133,134,138,139,151, 152 KAN rrs, eis, whiB7 ccsA, bacA, Rv2477c, whiB6 6,76,139,149 CAP rrs, tlyA (Rv1694) ccsA, rrl, bacA, Rv2680, Rv2681, whiB6 6,76,135,139,153 Table 22. Upstream/promoter regions of candidate resistance genes Gene Upstream/ promoter regions Primary transcriptional start site Reference aftB 1–129 4268914 154 ahpC 1–93 2726151 131 atpE 1–51 1461045 154 bacA 1–81 2064758 155 ccsA 1–191 619751 155 clpC1 1–106 4040759 155 ddn 1–51 3986844 155 dnaA 1–314 4411270 155 eis 1–84 2715365 156 embA 1–86 4243233 155 embC 1–1982a embR 1–103 1417399 155 ethA 1–51 4327473 155 ethR 1–26 4327505 154 fbiA 1–138 3640456 155 fbiC 1–127 1302855 155 Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 90 Gene Upstream/ promoter regions Primary transcriptional start site Reference fgd1 1–51 490783 155 gid 1–79 4408230 157 glpK 1–52 4139756 155 gyrA 1–35 gyrB 1–108 5183 155 hadA 1–51 731930 155 inhA 1–813b 1673440 155 katG 1–532c 2156592 158 mmpS5 1–85d 778965 155 mshA 1–669 574730 155 mtrA 1–376 3627674 155 mtrB 1–50 3626821 155 ndh 1–96 2103087 155 nusG 1–201 734104 155 panD 1–51 & 1838–1949 4046179 155 pepQ 1–51 2860418 155 pncA 1–51 2289241 155 PPE35 1–122 2170683 155 rplC 1–51 & 323–503 800357 154 rpoA 1–536 3878992 154 rpoB 1–263 759595 155 rpoC 1–45 rpsA 1–100 1833493 155 rpsL 1–234 781377 155 rrl 1–51 rrs 1–151 1471746 3 Rv0010c 1–156 Rv0565c 1–78 657497 155 Rv1129c 1–51 1254510 154 Rv1258c 1–58 1407347 155 Rv1979c 1–470 2223583 154 Rv2477c 1–88 2784079 155 Rv2680 1–153 2996003 155 Rv2681 1–2 Rv2752c 1–51 & 934–984 3067124 155 Rv2983 1–51 3339118 155 Rv3083 1–51 3448504 155 Rv3236c 1–51 & 488–538 3613603 155 sigE 1–51 1364413 155 tlyA 1–51 & 185–236 1917755 155 tsnR 1–51 ubiA 1–51 whiB6 1–126 4338596e whiB7 1–404 3569032 159 3 Cortes T, personal communication, 2023. 5 Methods 91 a Includes aftA, which was analysed at the nucleotide level. b Includes fabG1, which was analysed at the nucleotide level. c Includes furA, which was analysed at the nucleotide level. d Includes the upstream region for Rv0678 and its transcriptional start site at 778990 (155). e Transcriptional start site unknown; 4338596 was used instead, as a G insertion at this position is known to affect whiB6 expression (160). Table 23. Predicted effects and variant categories used in the SOLO association algorithm Predicted effect Variant category format Example of variant category values feature_ablationa,b Constant value deletion frameshifta Proteic HGVS p.Arg158fs inframe_deletion Proteic HGVS p.Ala136_Ala139del p.Lys766_Asp770delinsAsn inframe_insertion Proteic HGVS p.Cys46_Asp47insGly initiator_codon_variantc Nucleotidic HGVS c.3G>A missense_variant Proteic HGVS p.Ala277Thr non_coding_transcript_exon_variant Nucleotidic HGVS n.1016G>T start_losta Proteic HGVS p.Met1? stop_gaineda Proteic HGVS p.Trp36* stop_lost Proteic HGVS p.Ter215Argext*? stop_retained_variantc Nucleotidic HGVS c.1044G>A synonymous_variantc Nucleotidic HGVS c.1212G>A upstream_gene_variant Nucleotidic HGVS c.-134G>T c.-30_-29insGCG a Assumed to be an LoF mutation. Unlike in the first edition, inframe indels were excluded from this category because they are less likely to cause a LoF phenotype. b Assigned when an entire gene was deleted. c Considered to be a silent variant. The PySpark extract-transform-load script for generating the input for the SOLO and grading algorithm was developed to abide by the following rules: • Mutations in promoter and upstream regions were analysed because of their potential to alter the expression of downstream resistance genes. Therefore, all mutations in these regions were interpreted with the nucleotidic HGVS system, even if they overlapped with upstream protein- coding genes that were not considered to be resistance genes. • Different genetic changes in resistance genes (Table 21) that had the same protein-altering effect were grouped and interpreted with the standard protein-based naming system, proteic HGVS. Three resistance genes (aftA, fabG1 and furA) were analysed with the nucleotidic HGVS system, because variants in these regions can potentially affect the expression of downstream resistance genes (i.e. they were considered part of the upstream/promoter regions of embC, inhA and katG respectively, Table 22) (49,54,111). • Genetic changes that cause a shift in the reading frame at the same position in the genetic code were also grouped and interpreted with the proteic HGVS system. • Synonymous changes, which do not alter the protein, were treated individually and interpreted with a nucleotide-based naming system (nucleotidic HGVS). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 92 • The predicted effects “synonymous_variant”, “initiator_codon_variant” (mutations that change a start codon to another start codon in bacteria) and “stop_retained_variant” were all treated as silent in the SOLO and grading algorithms (Table 23). • Consecutive point mutations that affect several nucleotides were analysed together only if they occurred in the same codon and resulted in a protein-altering effect. Point mutations on different codons were always treated individually within their respective codon. For instance, one rare, consecutive point change in rpoB (variant occurring at genomic position 761138 that leads to replacement of the nucleotide sequence CCACA by GTCCC) was interpreted as three different units: one synonymous change in codon 444, one missense variant in codon 445 (p.His445Ser) and one missense variant on codon 446 (p.Lys446Gln). • Consecutive nucleotide changes in non-coding sequences were evaluated separately. • Leading amino acids encoded by non-canonical start codons in bacteria were corrected to methionine. Although these non-canonical start codons encode amino acids other than methionine everywhere else in the gene sequence, translation is always initiated with a special tRNA bound to formylmethionine (161–163). The transformation scripts for these methods are available at https://github.com/ GTB-tbsequencing/mutation-catalogue-2023. 5.7 Association studies SOLO – an algorithmic method for identifying SOLO mutations The second edition of individual mutations associated independently with resistance was produced with a method that was applied to MTBC in 2015 (12) and adapted in 2021 to produce the first mutation catalogue (14). The approach resembles the “definite defectives” algorithm used in group testing, pioneered in 1943 (164,165). For MTBC genomic data, it is used to characterize the effects of specific mutations in various genes and promoters that are considered highly likely to be linked to expression of phenotypic resistance. Deviations from the published method (12) are described below. The algorithm, presented in Fig. 7, is used to characterize variants as resistance-determining (algorithmically resistant or “aR”) or as consistent with susceptibility (algorithmically susceptible or “aS”). Silent mutations were assumed to be neutral (“aS”) and were masked before step b. Any non-silent variant found to be the only variant in the relevant resistance regions (i.e. a “SOLO” variant) in a phenotypically resistant isolate was classified as “aR”. Any non-silent variant that was found, either overall or as a SOLO variant, only in phenotypically susceptible isolates was classified as “aS”. Any non-silent mutation that could not be characterized as either “aR” or “aS”, such as a mutation that never appeared as a SOLO mutation and was never seen in either phenotypically susceptible or phenotypically resistant isolates, was classified as algorithmically uncertain (“aU”). The resulting SOLO mutations were further classified with the grading algorithm (Section 5.8). 5 Methods 93 Fig. 7. Steps in the SOLO algorithm for determining neutral mutations (a and b) and for quantifying associations between genotype and phenotype Silent masked (after step a) a b c CONFIDENCE GRADING SYSTEM PPV_ub <10% PPV_ub <10% Remove strains with previously documented R mutations Literature neutral mutations (incl. silent) Data- Set C Data- Set C-D1 PPV SOLO_ub <10% PPV SOLO_ub <10% Neutral mutations masked (Set F) Neutral mutations: Set D1 Neutral mutations: Set C aR aU aS masking Data- Set F Neutral mutations: Set A Neutral mutations: Set B Algorithm Algorithm Neutral mutations: Set D2 aR aU aS WHO dataset only (including silent mut) Neutral mutations: WHO cat. ver. 1 Stars denote changes from the approach used in the first edition. Before running the algorithm, the raw data were prepared for analysis. 1. Selection of candidate genes and promoters The success of the algorithmic approach requires exploration of relatively short genetic sequences. In the method published in 2015, the range was 1–8 genes and up to an arbitrarily defined 100 bp upstream, which was curtailed if it ran into an adjacent coding sequence (12). The target genes (Table 21) and upstream and promoter regions were further refined from the first edition (Table 22). To minimize the effect of excessive genomic variation on the algorithm, we retained the division of candidate genomic regions into two tiers from the first edition, although the classification of some genes was adjusted (e.g. ubiA was upgraded to tier 1 from tier 2). Tier 1 comprised gene sequences and associated promoters that were considered most likely to contain resistance mutations. Tier 2 included the remaining candidate genes and associated promoters, considered to have a lower, but still reasonable pre-test probability of containing resistance mutations. 2. Sequencing defects and fraction of supporting reads When sequencing defects that could not be explained by deletion calls generated by delly or Freebayes were detected, the information was inserted into the grading algorithm, as were all variants identified in at least 25% of reads for a particular sample. This ensured that running the algorithm with ≥ 75% as the cut-off for calling mutations did not result in false SOLO mutations. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 94 For example, a gyrA mutation with a variant frequency of 80% could not be a SOLO if it co-occurred with another gyrA mutation with a frequency of 30% in the same sample. 3. Quality control Sample mislabelling is relatively common and can lead to spurious results in algorithm-based analyses. One way of mitigating such error is to exclude all isolates with a previously well- established resistance mutation but a susceptible phenotype in the data set, i.e. isolates in which a susceptible phenotype is not credible. With this logic, MTBC isolates that had a katG Ser315Thr mutation for INH resistance or a rpoB Ser450Leu mutation for RIF resistance but were recorded as having “susceptible” phenotypes for the corresponding drug in the data were excluded from further consideration, on the assumption that these mutations were best explained by sample mislabelling. A separate analysis was performed only for these two mutations, which included all the isolates, phenotypically susceptible and resistant alike, to ensure accurate associations for katG Ser315Thr and rpoB Ser450Leu. 4. Initial identification of neutral mutations As the aim of the algorithmic approach is to identify specific SOLO mutations associated with resistance, a preliminary step should be to identify as many “neutral” (i.e. not associated with resistance) mutations as possible and to mask them to downstream analyses. Isolates for which there were category 1 and 2 pDST results (forming the WHO data set) were analysed separately in four ways to identify neutral mutations. In each case, the quality control steps (see above) were implemented, and uncertain base calls were masked. Any mutation with a lower bound of 95% CI of PPV or PPV_SOLO < 10% was considered neutral. To ensure a conservative approach, phenotypes unique to category 3 (i.e. not WHO-endorsed) were not used to identify neutral mutations. Neutral mutations were identified in a stepwise fashion, described below and shown in Fig. 7a and 7b. (i) PPV = Present_RPresent_R + Present_S was computed for each mutation. (ii) The PPV was computed for each mutation after removal of isolates that contained one of a few previously documented resistance mutations, defined as follows: a. any mutation, excluding indels, classified as Group 1/2 in the first edition; b. all non-synonymous mutations and indels in RRDR (codons 426–452 rpoB-RIF); c. any LoF mutation in non-essential tier 1 genes (Table 21) in which at least some LoF mutations are known to confer resistance; and d. any mutation, except silent ones, in Rv0678 (for BDQ and CFZ). (iii) A list of mutations identified specifically as neutral from evidence in the literature for RIF, INH, EMB and PZA (107), and for BDQ (166, 167) was appended to the results of (i) and (ii), and neutral mutations were masked from all downstream analyses, with silent mutations not classified as neutral in (i) or (ii). 5 Methods 95 (iv) PPV_SOLO = !"#$#%&_*+,+_- !"#$#%&_*+,+_- ' !"#$#%&_*+,+_* was computed for each mutation that remained as the only (SOLO) mutation in a set of candidate genes after masking the neutral mutations identified in steps (i), (ii) and (iii). (v) PPV_SOLO was computed for each mutation that remained as the only SOLO mutation in a set of candidate genes after masking of the neutral mutations identified in steps (i)–(iv). A list of mutations identified specifically as neutral in the first edition was then appended to the results, and neutral mutations were masked from all downstream analyses. Running the algorithm to identify mutations associated with resistance The algorithm was run separately for each category of pDST data (WHO and ALL). In each instance, the quality control steps outlined above were first applied, then the neutral mutations identified in Fig. 7a and 7b were masked, with silent mutations, before the final algorithm was run in two iterations (Fig. 7c). Prioritizing gene targets As the algorithm approach used was hierarchical, tier-1 target gene sequences (with a higher probability of association with phenotypic resistance) were investigated first. For a resistant phenotype linked to an “aR” or “aU” mutation, no additional sequence was analysed. Tier-2 gene sequences were investigated for the remaining data. Two passes of the algorithm were performed for tier-1 sequences (Fig. 7c), whereby mutations characterized as “aS” in the first pass were masked in the second pass in order to characterize further the now solitary mutations as “aS” or “aR”. Tier-2 sequences were assessed only after the second pass of tier-1 sequences. Only one pass of the algorithm was performed for tier-2 sequences because of the lower probability of finding resistance mutations in those targets. Validation and optimization of the SOLO algorithm It is our long-term goal to produce iterative improvements to the WHO mutation catalogue as sufficient new data are accumulated, or at least annually. For this work to be sustainable, we must ensure that the methods used to create the catalogue are accurate and as simple, efficient and cost-effective as possible. The genotypic and phenotypic associations included in the first edition were produced with a SOLO algorithm that was programmed in Stata. For the second edition, we sought to reduce the dependence of future work on the catalogue on proprietary or licensed software (e.g. Stata) and therefore asked an independent expert to review the entire Stata code set and to recreate it in R. This provided independent coding of the SOLO algorithm to validate the revised Stata code outputs, all of which were cross-checked to ensure that both codes provided the same intermediate and final statistical outputs. In the final Stata and R versions of the SOLO algorithm, all outputs were effectively identical. Future catalogues will probably be run on the open-source R version of the SOLO algorithm. For the second edition, both the R and the Stata version are available online (https://github.com/GTB-tbsequencing/mutation-catalogue-2023). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 96 Strengths and limitations of the algorithmic approach The strength of the SOLO algorithmic approach is that it is known to perform well for building catalogues (12). Its theoretical basis in group testing provides additional confidence (164). By focusing on a set of candidate sequences with a high probability of being associated with phenotypic resistance, the chances of correctly identifying SOLO “aR” mutations are enhanced, although the risk of not addressing additional relevant sequences remains. In the original method, all phylogenetically deep-rooted mutations were masked; in both catalogues, however, they were masked only if there was sufficient evidence of their neutrality. This analysis may therefore be more vulnerable to confounding due to MTBC population structure but may be less prone to misclassification that leads to arbitrary masking of the wrong mutations. While it was assumed in the first edition that mutation calls with a fraction of supporting reads below a threshold (formerly 0.9, now 0.75) were wild-type, and thus effectively removed from the analysis, in the second edition we kept all such mutations for analysis. By propagating the uncertainty associated with their presence, we ensured that other mutations in the same isolate and tier were not spuriously classified as SOLO mutations. This helped to prevent misclassification of mutations as SOLO in the presence of potential other mutations, albeit with less strong support. Statistical support for resistance mutations The numbers of resistant and susceptible isolates with and without a mutation were collated into a 2 × 2 contingency table in order to assess genotypic/phenotypic associations, from which ORs were computed in a Fisher exact test, with corresponding P values according to the hypergeometric distribution and CIs computed with a normal approximation. To control for multiple testing, a Benjamini-Hochberg correction was used, with a false discovery rate of 5%. The set of hypotheses was assumed to contain all mutations except those determined to be neutral in the first edition, those determined to be neutral in the literature (107,166,167), and silent ones. This correction was applied because only variants that fit into one of the above categories could be classified as not associated with resistance before the data were seen, while the status of the remaining ones (including those that were not finally classified with the algorithm) was determined only after the analysis. The same statistical procedure was applied to the ORs for SOLO mutations (OR SOLO), whereby only isolates with a SOLO mutation were counted instead of all isolates with a mutation and compared with the corresponding numbers of isolates without the mutation: OR SOLO = Present_SOLO_R Absent_R 0 Present_SOLO_S Absent_S PPV|SOLO was calculated for all mutations as the number of times the mutation was observed as a SOLO with a resistant phenotype, divided by the sum of that number and the number of times the mutation was observed with a susceptible phenotype. PPV_SOLO was calculated for all mutations as the number of times the mutation was observed as a SOLO with a resistant phenotype divided by the sum of that number and the number of times the mutation was observed as a SOLO with a susceptible phenotype: 5 Methods 97 PPV|SOLO = Present_SOLO_R Present_SOLO_R + Present_S PPV SOLO = Present_SOLO_R Present_SOLO_R + Present_SOLO_S 95% CIs were obtained with the Clopper-Pearson method. These statistical metrics were then used to stratify and prioritize mutations in the confidence grading algorithm described below. Because of the quality control steps taken before these analyses, mutations at katG S315T and at rpoB S450L would necessarily have appeared to be perfectly associated with resistance to INH and RIF, respectively. To generate real-world data for these mutations, a separate analysis was performed in which mutations to INH and RIF were reanalysed without removing probably mislabelled isolates. Only the results for katG Ser315Thr and rpoB Ser450Leu were retained from that analysis and substituted for the results for those mutations in the main analysis. 5.8 Confidence grading Once variants that were and were not associated with resistant phenotypes were identified with the SOLO algorithm and relevant association statistics were generated, as described in “Association studies” above, a set of consensus statistical thresholds and additional grading rules for confidence grading and ranking the observed MTBC mutations were applied to stratify the associations into five Groups according to the strength of the evidence for a genotype–phenotype association and the prioritization (according to level of support) of the phenotypic method used (see “Prioritization of pDST results” above): Group 1: Assoc with R Group 2: Assoc with R–interim Group 3: Uncertain significance Group 4: Not assoc with R–interim Group 5: Not assoc with R Grading criteria (see specific criteria below) were applied equally to all mutations for all drugs, as shown in Fig. 8. As individual mutations associated with PZA resistance are both found less frequently and distributed more broadly among genes such as pncA than other resistance mutations (such as those in rpoB), they required special consideration. Therefore, we applied “relaxed” grading criteria with less stringent thresholds to identify additional, infrequent mutations associated and not associated with resistance to PZA in the pncA gene only (Fig. 9). Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 98 Fig. 8. Standard criteria for grading variants no no no no Neutral by literature • LoF in Rv0678 (BDQ, CFZ), pepQ (BDQ, CFZ), tlyA (CAP), ddn (DLM), fbiA (DLM), fbiB (DLM), fbiC (DLM), fgd1 (DLM), Rv2983 (DLM), katG (INH), ethA (ETO), gid (STM) • Any non-synonymous mutation or indel in the RRDR of rpoB (RIF) • Previous WHO guidance and mutations specifi cally interpreted as marker for R in WHO-endorsed gDST assay • Cross-resistance: gyrA and gyrB (LFX/MFX), inhA (ETO/INH), Rv0678 and pepQ (BDQ vs CFZ only) • Literature evidence • Selection evidence • Potentially infl ated PPV (BDQ) • Borderline rpoB mutations (RIF) • Previous WHO guidance • Literature evidence • Silent mutations not graded as Group 5 Group 1: Assoc w R Group 2: Assoc w R–interim Group 4: Not assoc w R–interim Group 3: Uncertain signifi cance Group 5: Not assoc w R yes yes yes yes N SOLO ≥5 OR SOLO >1 FDR SOLO sig WHO dataset ALL dataset only Algorithm pass 2 PPV|SOLO_ lb ≥25% WHO dataset only Neutral masked Stars denote changes from the approach used for the first edition. 5 Methods 99 Fig. 9. “Relaxed” criteria for grading PZA variants yes yesyes no no Neutral by literature yes yes yes no no no no OR SOLO >1 WHO dataset ALL dataset only Algorithm pass 2 PPV|SOLO_ lb ≥25% PPV ≥50% Present_ SOLO_R ≥2 yes • LoF in pncA (PZA) • Previous WHO guidance • Literature evidence • Previous WHO guidance • Literature evidence • Silent mutations not graded as Group 5 N SOLO ≥5 WHO dataset only Neutral masked yes no no PPV SOLO <40% PPV SOLO_ ub <75% FDR SOLO sig Group 1: Assoc w R Group 2: Assoc w R–interim Group 4: Not assoc w R–interim Group 3: Uncertain signifi cance Group 5: Not assoc w R Stars denote changes from the approach used for the first edition. General principles of the grading approach and presentation of mutation tables • While most mutations were identified from the data set that we analysed, some that were assigned to Group 1, 2 or 4 were identified from the literature and from published WHO documents, according to additional grading rules. Any mutations in the tables from these sources are clearly marked as such. All mutations not flagged as such were processed in the algorithm and then classified according to the grading criteria detailed below. The additional grading rules were based on the available evidence and will be revised as new evidence emerges. • The grading criteria used to stratify mutations into Group 2 or 4 were generally more permissive than those for Group 1 or 5 or identified solely with pDST methods that have not been endorsed by WHO. • Group 3 mutations could not be classified from the available data. They comprise all mutations that do not fall into Group 1, 2, 4 or 5. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 100 • While the MICs of the mutations analysed in this data set were not evaluated to determine whether the observed mutations were associated with high or low MICs to specific drugs, WHO previously endorsed specific mutations associated with “high” or “low” MICs to INH and MFX (4,15,19). These mutations are therefore flagged as “high” or “low” in the tables in the relevant report (see Table 1). Criteria for initial confidence grading Group 1: Assoc w R Mutations that met five criteria: 1. number of resistant and susceptible isolates with the SOLO mutation (Present_SOLO_SR) ≥ 5 2. lower bound of 95% CI of PPV conditional on being SOLO (PPV|SOLO_lb) ≥ 25% 3. OR > 1 (always applies if criterion 4 is met) 4. OR SOLO mutation > 1 5. statistical significance of OR as SOLO mutation (OR SOLO_FE-sig) with Fisher exact test for false discovery rate, false discovery rate-corrected P ≤ 0.05 Group 2: Assoc w R–interim Mutations that met “relaxed” criteria for pncA: 1. resistant isolates with the SOLO mutation (Present_SOLO_R) ≥ 2 2. PPV ≥ 50% Group 3: Uncertain significance All mutations that did not meet the criteria for inclusion in Group 1, 2, 4 or 5. Group 4: Not assoc with R–interim Silent mutations not classified as neutral in steps a and b of the neutral algorithm (Fig. 7a and 7b). Mutations that met “relaxed” criteria for pncA: 1. PPV SOLO < 40% 2. upper bound of 95% CI of PPV SOLO < 75% To ensure a conservative approach, phenotypes unique to category 3 (i.e. not WHO-endorsed) were not used to identify Group 4 or 5 mutations. Group 5: Not assoc with R Neutral mutations that were masked before use of the algorithm (see “Initial identification of neutral mutations”). 5 Methods 101 Additional grading rules applied for final confidence grading When the initial confidence grades of mutations selected according to statistical thresholds conflicted with strong evidence before the analysis, additional grading rules and established precedent were applied. As far as possible, the grading was based on the available data, and such changes were kept to a minimum. When additional criteria were used to override the initial confidence grading, the mutation in question was annotated with the specific criterion applied in the mutation tables. The basis for these changes and the abbreviations for each criterion are described below (the rules relevant for classifying mutations that are not graded in this catalogue are also included in Table 1). Group 1 (Assoc w R): 1. Four “borderline” rpoB resistance mutations (Leu430Pro, His445Asn, His445Ser and Ile491Phe) were assigned a final grading of Group 1 because previous WHO guidance explicitly states that these mutations are valid markers of resistance, do not require confirmation by pDST and that their detection overrules a susceptible pDST result (4). Without this reclassification, their final confidence grading would have been Group 3, except for Ile491Phe, which would have been Group 2. Group 2 (Assoc w R–interim): 1. All only: tier-1 mutations that met the criteria for Group 1 during initial confidence grading only because the ALL data set contained methods that were not endorsed by WHO. 2. Pass 2: tier-1 mutations that met the criteria for Group 1 only during initial confidence grading in pass 2 of the algorithmic method (i.e. after masking mutations classified as neutral during pass 1). 3. Tier 2: probably-2 mutations that met the criteria for Group 1 during the initial confidence grading (i.e. based on algorithmic decisions about tier 1-mutations). This did not apply to any of the mutations in this analysis. 4. WHO precedent (“prev. WHO”): Two LFX mutations (gyrA Gly88Ala and Ala504Val) and one STM mutation (rpsL Lys88Gln) were recognized as Group 2 in accordance with WHO precedents (6,168). 5. WHO-endorsed genotypic DST assays (“WHO-end. gDST”): any mutations specifically interpreted as markers of resistance in one or more of the WHO-endorsed assays listed in Table 4. 6. RRDR additional grading rule (“RRDR”): Any non-synonymous mutation or indel in the RRDR of rpoB (4). 7. FQ cross-resistance additional grading rule (“FQ X-R”): Any gyrA or gyrB mutation that was ultimately graded into Group 1 or 2 for LFX but initially graded into Group 3 for MFX was upgraded to Group 2 for MFX and vice versa. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 102 8. INH-ETO cross-resistance additional grading rule (“INH-ETO X-R”): Any inhA or fabG1 mutation that was ultimately graded into Group 1 or 2 for INH but initially graded into Group 3 for ETO was upgraded to Group 2 for ETO and vice versa (19,49,50). 9. BDQ-CFZ cross-resistance additional grading rule (“BDQ-CFZ X-R”): Any Rv0678 or pepQ mutation that was ultimately graded into Group 1 or 2 for BDQ but initially graded into Group 3 for CFZ was upgraded to Group 2 for CFZ (70,73,74). 10. Selection experiment additional grading rule (“Selection”): specific mutations in atpE (67– 69,71,72) or rrl (79–84) found to confer resistance during in-vitro or animal selection in MTBC in at least two laboratories.4 This two-laboratory threshold was also met for pooled LoF mutations in ddn, fbiA, fbiB, fbiC, fgd1 and Rv2983 for DLM and/or PMD (84–87). 11. LoF additional grading rule (“LoF”): Any premature stop codon (i.e. nonsense mutation), gene deletion (feature_ablation), frameshift or start-loss mutation in the coding regions of ethA (ETO), gid (STM), katG (INH), pncA (PZA), Rv0678 (BDQ, CFZ), pepQ (BDQ, CFZ), ddn (DLM), fbiA (DLM), fbiB (DLM), fbiC (DLM), fgd1 (DLM), Rv2983 (DLM) and tlyA (CAP) was considered a Group 2 mutation for that drug. The mechanisms by which LoF mutations in these genes confer resistance is well understood, and, except for Rv0678, no epistatic interaction is known that could render an isolate with a LoF mutation in one of these genes susceptible (64,93,94,97,145,169–171). 12. Literature evidence additional grading rule (“Lit.”): This rule was used to support classification of the pncA Ile31Thr mutation into drug susceptibility 2 for PZA (64). 13. Potentially inflated PPV (Pot. infl. PPV): This rule was used to downgrade all Group 1 BDQ SOLO mutations from laboratory 1 into Group 2. 14. Interim on WHO: If the initial confidence grading was Group 2 for the WHO data set but Group 1 for the ALL data set, the Group 2 classification was used. Group 4 (Not assoc w R-interim): 1. Silent mutations not classified as neutral in steps a and b of the neutral algorithm. 2. Previous WHO guidance: mutations previously documented as “not associated with R” (6, 34) that did not meet the criteria for Group 1, 2 or 5. 3. Literature evidence additional grading rule (“Lit.”): mutations previously documented as “Not associated with R” that are frequent in some settings were placed in Group 4: a. Mutations that could not be classified as neutral in the WHO data set but that were classified as neutral in the literature for RIF, INH, EMB and PZA (107), and for BDQ (166,167) according to pDST data and were consequently masked before use of the algorithmic method; and b. gyrA Thr80Ala and Ala90Gly, because these are frequent in the Uganda genotype (6,28). 4 Lee J, personal communication, 2023; Takaki A, Mitarai S, personal communication, 2023; Andres S, personal communication, 2023. 103 6 Data contributors The following people contributed additional isolates and data for the second edition: Yuliana Atanasova, National Centre of Infectious and Parasitic Diseases, National TB Reference Laboratory, Sofia, Bulgaria; Ewa Augustynowicz-Kopeć, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland; Elizabeta Bachiyska, National Centre of Infectious and Parasitic Diseases, National TB Reference Laboratory, Sofia, Bulgaria; Ágnes Bakos, National Koranyi Institute for Pulmonology, Budapest, Hungary; Anna Bateson, Centre for Clinical Microbiology, University College London; Virginia Batignani, San Raffaele Scientific Institute, Milan, Italy; Ana Baykova, National Centre of Infectious and Parasitic Diseases, National TB Reference Laboratory, Sofia, Bulgaria; Dorte Bek Folkvardsen, Statens Serum Institut, Copenhagen, Denmark; Riccardo Bollini, San Raffaele Scientific Institute, Milan, Italy; Emanuele Borroni, San Raffaele Scientific Institute, Milan, Italy; Chhavarath Dary, University of Health Sciences, Phnom Penh, Cambodia; Matus Dohal, Comenius University, Bratislava, Slovakia; Věra Dvořáková, National Reference Laboratory for Mycobacteria, National Institute of Public Health, Prague, Czechia; Konstantin Edokimov, Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, Singapore; Vegard Eldholm, National Reference Laboratory for Mycobacteria, Department of Bacteriology, Norwegian Institute of Public Health, Oslo, Norway; Margaret Fitzgibbon, St James’s Hospital, Dublin, Ireland; Arash Ghodousi, San Raffaele Scientific Institute, Milan, Italy; Agnieszka Głogowska, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland; Marjo Haanperä, Finnish Institute for Health and Welfare, Helsinki, Finland; Qing Hao Miow, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Dan Hartley, Critical Path Institute, Tucson (AZ), USA; Twee Hee Rick Ong, Saw Swee Hock School of Public Health, National University of Singapore, Singapore; Li-Yang Hsu, Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, Singapore; Alamdar Hussain, National TB Reference Laboratory, Islamabad, Pakistan; Kadri Klaos, Department of Mycobacteriology, United Laboratories, Tartu University Hospital, Tartu, Estonia; Lex Leong, University of South Australia, Adelaide, Australia; Federico Lorenzo, Microbacterial Service, Instituto Nacional de Enfermedades Infecciosas, Administración Nacional de Laboratorios e Institutos de Salud Malbrán, Argentina; Yelena Losev, Tuberculosis National Center, Tel Aviv, Israel; Austin March, Critical Path Institute, Tucson (AZ), USA; Faisal Masood, National TB Reference Laboratory, Islamabad, Pakistan; Timothy McHugh, Centre for Clinical Microbiology, University College London, United Kingdom; Roxana Mihaela Coriu, Marius Nasta Institute of Pneumology, Bucharest, Romania; Simone Mok, St James’ Hospital, Dublin, Ireland; Mihaela Obrovac, National Reference Laboratory for TB, Croatian Institute of Public Health, Zagreb, Croatia; Shaheed Vally Omar, National Institute for Communicable Diseases, Pretoria, South Africa; Janne Oseberg Rønning, National Reference Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 104 Laboratory for Mycobacteria, Department of Bacteriology, Norwegian Institute of Public Health, Oslo, Norway; Roxana Paul, Servicio Micobacterias, Microbacterial Service, Instituto Nacional de Enfermedades Infecciosas, Administración Nacional de Laboratorios e Institutos de Salud Malbrán, Argentina; Anna Ralph, Queensland Mycobacterium Reference Laboratory, Brisbane, Australia; Sylvia Rofael, University College London, United Kingdom; Emma Roycroft, St James’ Hospital, Dublin, Ireland; Vonthanak Saphonn, University of Health Sciences, Phnom Penh, Cambodia; Marco Schito, Critical Path Institute, Tucson (AZ), USA; Keith Scollick, Critical Path Institute, Tucson (AZ), USA; Caitlin Selway, South Australia Pathology, Adelaide, Australia; Giri Shan Rajahram, Clinical Research Centre, Queen Elizabeth Hospital, Kota Kinabalu, Malaysia; Lisa Shepard, South Australia Pathology, Adelaide, Australia; Norberto Simboli, Servicio Micobacterias, Servicio Micobacterias, Microbacterial Service, Instituto Nacional de Enfermedades Infecciosas, Administración Nacional de Laboratorios e Institutos de Salud Malbrán, Argentina; Dan Sivilli, Critical Path Institute, Tucson (AZ), USA; Neil Stoker, St George’s, University of London, United Kingdom; Elisa Tagliani, San Raffaele Scientific Institute, Milan, Italy; Sabira Tahseen, National Tuberculosis Control Program, Islamabad, Pakistan; Juliano Timm, TB Alliance, Washington DC, USA; Anne Torunn Mengshoel, National Reference Laboratory for Mycobacteria, Department of Bacteriology, Norwegian Institute of Public health, Oslo, Norway; Laima Vasiliauskaite, Centre of Laboratory Medicine, Tuberculosis Laboratory, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; Timothy William, Clinical Research Centre, Queen Elizabeth Hospital, Kota Kinabalu, Malaysia; Adam Witney, St George’s, University of London, United Kingdom; Yoshiyuki Yamada, Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, Singapore; Stanislava Yordanova, National Centre of Infectious and Parasitic Diseases, National TB Reference Laboratory, Sofia, Bulgaria; Danila Zimenkov, Engelhardt Institute of Molecular Biology, Moscow, Russian Federation; Ljiljana Žmak, National Reference Laboratory for Tuberculosis, Croatian Institute of Public Health, Zagreb, Croatia. The following people contributed isolates and data for the catalogue published in 2021: Solange Alves Vinhas, Federal University of Espírito Santo, Vitória, Brazil; Jason Andrews, Stanford University School of Medicine, Stanford (CA), USA; Simone Battaglia, San Raffaele Scientific Institute, Milan, Italy; Marcel Behr, McGill University Health Centre, Montreal, Canada; Emanuele Borroni, San Raffaele Scientific Institute, Milan, Italy; Andrea Cabibbe, San Raffaele Scientific Institute, Milan, Italy; Joshua Carter, University of Oxford, Oxford, United Kingdom; Angkana Chaiprasert, Mahidol University, Bangkok, Thailand; Alvaro Chiner-Oms, Biomedicine Institute of Valencia, Spain; Joconiah Chirenda, University of Zimbabwe, Harare, Zimbabwe; Daniela Cirillo, San Raffaele Scientific Institute, Milan, Italy; Pauline Claxton, Scottish Mycobacteria Reference Laboratory, Edinburgh, United Kingdom; David Clifton, University of Oxford, United Kingdom; Ted Cohen, Yale University, New Haven (CT), USA; Iñaki Comas, Biomedicine Institute of Valencia, Spain; Tom Connor, Public Health Wales, Cardiff, United Kingdom; Marcelo Cordeiro dos Santos, Doctor Heitor Vieira Dourado Foundation of Tropical Medicine, Manaus, Brazil; Jorge Coronel, London School of Hygiene & Tropical Medicine, London, United Kingdom, and Cayetano Heredia Peruvian University, Lima, Peru; Julio Croda, Federal University of Mato Grosso do Sul, Campo Grande, Brazil; Derrick Crook, University of Oxford, United Kingdom; Keertan Dheda, University of Cape Town, South Africa; Sarah Dunstan, University of Melbourne, Australia; Tatiana Dutra, Federal University of Rio de Janeiro, Brazil; Sarah Earle, University of Oxford, United 6 Data contributors 105 Kingdom; Vincent Escuyer, New York State Department of Health, Albany (NY), USA; Kiatichi Faksri, Khon Kaen University, Khon Kaen, Thailand; Maha Farhat, Harvard Medical School, Boston (MA), USA; Lucilaine Ferrazoli, Institute Adolfo Lutz, São Paulo, Brazil; Philip Fowler, University of Oxford, United Kingdom; Sebastien Gagneux, Swiss Tropical and Public Health Institute, Basel, Switzerland; Neel Gandhi, Emory University, Atlanta (GA), USA; George Gao, China Centers for Disease Control, Beijing, China; Jennifer Gardy, University of British Columbia, Vancouver, Canada; Amy Gaskin, Public Health Wales, Cardiff, United Kingdom; Arash Ghodousi, San Raffaele Scientific Institute, Milan, Italy; Ana Gibertoni Cruz, University of Oxford, United Kingdom; Simon Grandjean Lapierre, Institut Pasteur de Madagascar, Antananarivo, Madagascar; Jennifer Guthrie, Public Health Ontario, Toronto, Canada; Tanya Halse, New York State Department of Health, Albany (NY), USA; Guangxue He, China Center for Disease Control, Beijing, China; Harald Hoffmann, Institute of Microbiology and Laboratory Medicine, Gauting, Germany; Kathryn Holt, Monash University, Melbourne, Australia; Sarah Hoosdally, University of Oxford, United Kingdom; Martin Hunt, European Bioinformatics Institute, London, United Kingdom; Stella Huo, University of California at San Francisco (CA), USA; Zamin Iqbal, European Bioinformatics Institute, London, United Kingdom; Frances Jamieson, Public Health Ontario, Toronto, Canada (retired); Lisa Jarrett, Public Health England, Birmingham, United Kingdom; Bouke de Jong, Institute of Tropical Medicine, Antwerp, Belgium; Lavania Joseph, National Institute for Communicable Diseases, Pretoria, South Africa; Ruwen Jou, Center for Disease Control Taiwan, Taipei, Taiwan (China); Priti Kambli, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; Rukhsar Khot, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; HeeJin Kim, Korean Institute of Tuberculosis, Seoul, Republic of Korea; Jeff Knaggs, European Bioinformatics Institute, London, United Kingdom; Anastasia Koch, University of Cape Town, South Africa; Thomas Kohl, Research Center Borstel, Germany; Samaneh Kouchaki, University of Oxford, United Kingdom; Afrânio Kritski, Federal University of Rio de Janeiro, Brazil; Julianna Kus, Public Health Ontario, Toronto, Canada; Alexander Lachapelle, University of Oxford, United Kingdom; Ajit Lalvani, Imperial College London, United Kingdom; Pascal Lapierre, New York State Department of Health, Albany (NY), USA; Ian Laurenson, Scottish Mycobacteria Reference Laboratory, Edinburgh, United Kingdom; Brice Letcher, European Bioinformatics Institute, London, United Kingdom; Wan-Hsuan Lin, Center for Disease Control Taiwan, Taipei, Taiwan (China); Chunfa Liu, China Center for Disease Control, Beijing, China; Maria Lucia Rossetti, Centre for Scientific and Technological Development, Porto Alegre, Brazil; Maria Luiza Bazzo, Federal University of Santa Catarina, Florianópolis, Brazil; Aijing Ma, China Center for Disease Control, Beijing, China; Juliana Maira Watanabe Pinhata, Institute Adolfo Lutz, São Paulo, Brazil; Kerri Malone, European Bioinformatics Institute, London, United Kingdom; Ayan Mandal, Foundation for Medical Research, Mumbai, India; Flora Martinez, Federal University of Grande Dourados, Brazil; Neil Martinson, University of the Witwatersrand, Johannesburg, South Africa; Daniela Matias, Public Health England, Birmingham, United Kingdom; Vidya Mave, BJ Medical College, Pune, India; Conor Meehan, University of Bradford, United Kingdom; Fabrizio Menardo, Swiss Tropical and Public Health Institute, Basel, Switzerland; Carl Mendel, TB Alliance, London, United Kingdom (retired); Matthias Merker, Research Center Borstel, Germany; James Millard, Africa Health Research Institute, Durban, South Africa; Paolo Miotto, San Raffaele Scientific Institute, Milan, Italy; Nerges Mistry, Foundation for Medical Research, Mumbai, India; David Moore, London School of Hygiene & Tropical Medicine, London, United Kingdom, and Cayetano Heredia Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 106 Peruvian University, Lima, Peru; Kimberlee Musser, New York State Department of Health, Albany (NY), USA; Han de Neeling, National Institute for Public Health and the Environment, Bilthoven, Netherlands (Kingdom of the); Isabela Neves de Almeida, Federal University of Minas Gerais, Belo Horizonte, Brazil; Dumisani Ngcamu, National Institute for Communicable Diseases, Pretoria, South Africa; Hoang Ngoc Nhung, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Stefan Niemann, Research Center Borstel, Germany; Kayzad Nilgiriwala, Foundation for Medical Research, Mumbai, India; Camus Nimmo, University College London, United Kingdom; Nana Okozi, National Institute for Communicable Diseases, Pretoria, South Africa; Shaheed Vally Omar, National Institute for Communicable Diseases, Pretoria, South Africa; Rick Ong Twee Hee, National University of Singapore, Singapore; Moises Palaci, Federal University of Espírito Santo, Vitória, Brazil; Nicholas Paton, National University of Singapore, Singapore; Tim Peto, University of Oxford, United Kingdom; Sara Plesnik, Institute of Microbiology and Laboratory Medicine, Gauting, Germany; Jamie Posey, Centers for Disease Control and Prevention, Atlanta (GA), USA; Marie-Sylvianne Rabodoarivelo, Institut Pasteur de Madagascar, Antananarivo, Madagascar; Niaina Rakotosamimanana, Institut Pasteur de Madagascar, Antananarivo, Madagascar; Priti Rathod, Public Health England, Birmingham, United Kingdom; Elis Regina Dalla Costa, Centre for Scientific and Technological Development, Porto Alegre, Brazil; Camilla Rodrigues, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; Tom Rogers, Trinity College Dublin, Ireland; Alex Rosenthal, National Institutes of Health, Bethesda (MD), USA; Emma Roycroft, St James’ Hospital, Dublin, Ireland; Richard Salvato, Centre for Scientific and Technological Centre, Mumbai, India; Rukhsar Khot, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; HeeJin Kim, Korean Institute of Tuberculosis, Seoul, Republic of Korea; Jeff Knaggs, European Bioinformatics Institute, London, United Kingdom; Anastasia Koch, University of Cape Town, South Africa; Thomas Kohl, Research Center Borstel, Germany; Samaneh Kouchaki, University of Oxford, United Kingdom; Afrânio Kritski, Federal University of Rio de Janeiro, Brazil; Julianna Kus, Public Health Ontario, Toronto, Canada; Alexander Lachapelle, University of Oxford, United Kingdom; Ajit Lalvani, Imperial College London, United Kingdom; Pascal Lapierre, New York State Department of Health, Albany (NY), USA; Ian Laurenson, Scottish Mycobacteria Reference Laboratory, Edinburgh, United Kingdom; Brice Letcher, European Bioinformatics Institute, London, United Kingdom; Wan-Hsuan Lin, Center for Disease Control Taiwan, Taipei, Taiwan (China); Chunfa Liu, China Center for Disease Control, Beijing, China; Maria Lucia Rossetti, Centre for Scientific and Technological Development, Porto Alegre, Brazil; Maria Luiza Bazzo, Federal University of Santa Catarina, Florianópolis, Brazil; Aijing Ma, China Center for Disease Control, Beijing, China; Juliana Maira Watanabe Pinhata, Institute Adolfo Lutz, São Paulo, Brazil; Kerri Malone, European Bioinformatics Institute, London, United Kingdom; Ayan Mandal, Foundation for Medical Research, Mumbai, India; Flora Martinez, Federal University of Grande Dourados, Brazil; Neil Martinson, University of the Witwatersrand, Johannesburg, South Africa; Daniela Matias, Public Health England, Birmingham, United Kingdom; Vidya Mave, BJ Medical College, Pune, India; Conor Meehan, University of Bradford, United Kingdom; Fabrizio Menardo, Swiss Tropical and Public Health Institute, Basel, Switzerland; Carl Mendel, TB Alliance, London, United Kingdom (retired); Matthias Merker, Research Center Borstel, Germany; James Millard, Africa Health Research Institute, Durban, South Africa; Paolo Miotto, San Raffaele Scientific Institute, Milan, Italy; Nerges Mistry, Foundation for Medical Research, Mumbai, India; David Moore, London School of Hygiene & Tropical Medicine, London, 6 Data contributors 107 United Kingdom, and Cayetano Heredia Peruvian University, Lima, Peru; Kimberlee Musser, New York State Department of Health, Albany (NY), USA; Han de Neeling, National Institute for Public Health and the Environment, Bilthoven, Netherlands (Kingdom of the); Isabela Neves de Almeida, Federal University of Minas Gerais, Belo Horizonte, Brazil; Dumisani Ngcamu, National Institute for Communicable Diseases, Pretoria, South Africa; Hoang Ngoc Nhung, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Stefan Niemann, Research Center Borstel, Germany; Kayzad Nilgiriwala, Foundation for Medical Research, Mumbai, India; Camus Nimmo, University College London, United Kingdom; Nana Okozi, National Institute for Communicable Diseases, Pretoria, South Africa; Shaheed Vally Omar, National Institute for Communicable Diseases, Pretoria, South Africa; Rick Ong Twee Hee, National University of Singapore, Singapore; Moises Palaci, Federal University of Espírito Santo, Vitória, Brazil; Nicholas Paton, National University of Singapore, Singapore; Tim Peto, University of Oxford, United Kingdom; Sara Plesnik, Institute of Microbiology and Laboratory Medicine, Gauting, Germany; Jamie Posey, Centers for Disease Control and Prevention, Atlanta (GA), USA; Marie-Sylvianne Rabodoarivelo, Institut Pasteur de Madagascar, Antananarivo, Madagascar; Niaina Rakotosamimanana, Institut Pasteur de Madagascar, Antananarivo, Madagascar; Priti Rathod, Public Health England, Birmingham, United Kingdom; Elis Regina Dalla Costa, Centre for Scientific and Technological Development, Porto Alegre, Brazil; Camilla Rodrigues, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; Tom Rogers, Trinity College Dublin, Ireland; Alex Rosenthal, National Institutes of Health, Bethesda (MD), USA; Emma Roycroft, St James’ Hospital, Dublin, Ireland; Richard Salvato, Centre for Scientific and Technical Development, Porto Alegre, Brazil; Marco Schito, Critical Path Institute, Tucson (AZ), USA; Sanchi Shah, Foundation for Medical Research, Mumbai, India; Joseph Shea, New York State Department of Health, Albany (NY), USA; Soyoun Shin, Korean Institute of Tuberculosis, Seoul, Republic of Korea; Elisangela Silva, Federal University of Rio de Janeiro, Brazil; Vitali Sintchenko, University of Sydney, Australia; Rosangela Siqueira de Oliveira, Institute Adolfo Lutz, São Paulo, Brazil; Grace Smith, Public Health England, Birmingham, United Kingdom; Walter Solano, London School of Hygiene & Tropical Medicine, London, United Kingdom, and Cayetano Heredia Peruvian University, Lima, Peru; Dick van Soolingen, National Institute for Public Health and the Environment, Bilthoven, Netherlands (Kingdom of the); Mel Spigelman,TB Alliance, New York City (NY), USA; Silvana Spindola de Miranda, Federal University of Minas Gerais, Belo Horizonte, Brazil; Andrea Spitaleri, San Raffaele Scientific Institute, Milan, Italy; Prapaporn Srilohasin, Mahidol University, Bangkok, Thailand; Vijay Srinivasan, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Angela Starks, Centers for Disease Control and Prevention, Atlanta (GA), USA; Utkarsha Surve, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India; Sabira Tahseen, National TB Control Programme, Islamabad, Pakistan; Jill Taylor, Association of Public Health Laboratories, Silver Spring (MD), USA; Grant Theron, Stellenbosch University, Cape Town, South Africa; Nguyen Thuy Thuong Thuong, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Guy Thwaites, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Katharina Todt, Institute of Microbiology and Laboratory Medicine, Gauting, Germany; Alberto Trovato, San Raffaele Scientific Institute, Milan, Italy; Annelies Van Rie, University of Antwerp, Belgium; Mirela Verza, Federal University of Santa Catarina, Florianópolis, Brazil; Timothy Walker, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam; Sarah Walker, University of Oxford, United Kingdom; Rob Warren, University of Stellenbosch, Cape Town, South Africa; Jim Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 108 Werngren, Public Health Agency, Stockholm, Sweden; Robert Wilkinson, University of Cape Town, Imperial College London and Francis Crick Institute, South Africa; Daniel Wilson, University of Oxford, United Kingdom; Penelope Wintringer, European Bioinformatics Institute, London, United Kingdom; Yu-Xin Xiao, Center for Disease Control Taiwan, Taipei, Taiwan (China); Yang Yang, University of Oxford, United Kingdom; Zhao Yanlin, National Tuberculosis Reference Laboratory, China Center for Disease Control, China; Shen Yuan Yao, National Institute for Communicable Diseases, Pretoria, South Africa; Baoli Zhu, Institute of Microbiology, Chinese Academy of Science, Beijing, China. 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121 Annex 1. Further information The performance of the first edition was compared with that of the second edition for majority variants (allele frequency ≥ 75%) in the ALL data set. The latter figures correspond to the “combined performance” numbers in Table 3 of the main text. The role of minority variants (allele frequency ≥ 25%) was calculated for the second edition only. Table A1.1. Comparison of the performance of the first and second edition and impact of minority variants Drug Catalogue, threshold for variants (%) Sensitivity, specificity, PPV (% [95% CI]) Changes in sensitivity, specificity, PPV (%) RIF 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 0, 0, 0 94.4 (94.0–94.7), 96.8 (96.6–97.0), 94.2 (93.8–94.5) 1.1, -0.1, 0 INH First, 75 91.0 (90.6–91.4), 98.1 (97.9–98.2), 97.3 (97.1–97.5) Second, 75 91.6 (91.2–92.0), 97.9 (97.8–98.1), 97.1 (96.8–97.3) 0.6, -0.2, -0.2 Second, 25 First, 75 Second, 75 Second, 25 92.1 (91.7–92.4), 97.9 (97.7–98.0), 97.0 (96.8–97.2) 0.5, 0.0, -0.1 EMB First, 75 83.8 (83.1–84.6), 91.2 (90.9–91.5), 71.6 (70.7–72.4) Second, 75 81.1 (80.3–81.9), 91.6 (91.3–91.9), 71.9 (71.0–72.8) -2.7, 0.4, 0.3 Second, 25 82.1 (81.3–82.9), 91.4 (91.1–91.7), 71.7 (70.9–72.6) 1, -0.2, -0.2 PZA First, 75 73.7 (72.4–75.0), 98.0 (97.8–98.2), 90.8 (89.8–91.7) Second, 75 78.0 (76.8–79.2), 97.9 (97.6–98.1), 90.5 (89.5–91.4) 4.3, -0.1, -0.3 Second, 25 80.0 (78.8–81.2), 97.7 (97.5–98.0), 90.3 (89.4–91.2) 2, -0.2, -0.2 LFX First, 75 83.7 (82.7–84.6), 97.1 (96.9–97.3), 88.7 (87.8–89.5) Second, 75 84.8 (83.9–85.7), 96.9 (96.7–97.1), 88.1 (87.3–89.0) 1.1, -0.2, -0.6 Second, 25 89.2 (88.4–90.0), 96.7 (96.4–96.9), 87.9 (87.0–88.7) 4.4, -0.2, -0.2 MFX First, 75 85.0 (83.8–86.1), 93.8 (93.4–94.1), 74.5 (73.2–75.8) Second, 75 85.7 (84.6–86.8), 93.5 (93.2–93.9), 74.0 (72.7–75.2) 0.7, -0.3, -0.5 Second, 25 90.3 (89.4–91.2), 93.2 (92.8–93.5), 74.0 (72.7–75.2) 4.6, -0.3, 0 BDQ First, 75 0 (0–0.4), 100.0 (100.0–100.0), 0 (0–0) Second, 75 49.4 (46.3–52.5), 98.7 (98.5–98.9), 75.2 (71.8–78.4) 49.4, -1.3, 75.2 Second, 25 59.6 (56.5–62.6), 98.4 (98.2–98.6), 75.0 (71.9–77.9) 10.2, -0.3, -0.2 LZD First, 75 27.3 (22.8–32.1), 99.8 (99.8–99.9), 78.5 (70.4–85.2) Second, 75 34.0 (29.2–39.0), 99.8 (99.7–99.9), 78.4 (71.3–84.5) 6.7, 0, -0.1 Second, 25 35.0 (30.2–40.1), 99.8 (99.7–99.8), 75.7 (68.6–81.9) 1.0, 0, -2.7 CFZ First, 75 0 (0–0.5), 100.0 (100.0–100.0), 0 (0–0) Second, 75 17.0 (14.2–20.0), 98.7 (98.5–98.9), 38.1 (32.6–43.8) 17.0, -1.3, 38.1 Second, 25 21.3 (18.2–24.5), 98.4 (98.2–98.6), 38.9 (33.9–44.1) 4.3, -0.3, 0.8 DLM First, 75 4.4 (2.2–7.7), 100.0 (99.9–100.0), 84.6 (54.6–98.1) Second, 75 14.7 (10.6–19.7), 99.9 (99.8–99.9), 72.5 (58.3–84.1) 10.3, -0.1, -12.1 Second, 25 15.1 (10.9–20.1), 99.8 (99.7–99.9), 62.3 (49.0–74.4) 0.4, -0.1, -10.2 AMK First, 75 72.9 (71.1–74.6), 98.1 (97.9–98.3), 81.3 (79.6–82.9) Second, 75 72.8 (71.0–74.6), 98.3 (98.1–98.5), 82.8 (81.2–84.4) -0.1, 0.2, 1.5 Second, 25 75.4 (73.7–77.1), 98.2 (98.0–98.4), 82.2 (80.6–83.8) 2.6, -0.1, -0.6 STM First, 75 79.2 (78.4–80.0), 94.1 (93.7–94.5), 89.9 (89.2–90.5) Second, 75 79.7 (78.9–80.5), 94.1 (93.7–94.4), 89.9 (89.3–90.5) 0.5, 0, 0 Second, 25 80.3 (79.5–81.1), 94.0 (93.6–94.4), 89.8 (89.2–90.4) 0.6, -0.1, -0.1 ETO First, 75 72.5 (71.3–73.7), 86.6 (86.1–87.2), 64.4 (63.2–65.7) Second, 75 74.8 (73.6–76.0), 85.9 (85.3–86.4), 63.9 (62.7–65.1) 2.3, -0.7, -0.5 Second, 25 76.1 (74.9–77.2), 85.6 (85.0–86.1), 63.8 (62.6–65.0) 1.3, -0.3, -0.1 KANa First, 75 75.1 (73.6–76.5), 96.5 (96.3–96.8), 78.7 (77.2–80.0) Second, 75 74.9 (73.4–76.3), 96.7 (96.4–96.9), 79.3 (77.9–80.7) -0.2, 0.2, 0.6 Second, 25 77.2 (75.8–78.5), 96.6 (96.3–96.8), 79.2 (77.8–80.5) 2.3, -0.1, -0.1 CAPa First, 75 66.4 (64.3–68.4), 97.8 (97.6–98.1), 80.1 (78.2–82.0) Second, 75 66.2 (64.1–68.2), 97.8 (97.6–98.1), 80.1 (78.1–81.9) -0.2, 0, 0 Second, 25 69.3 (67.3–71.3), 97.6 (97.3–97.8), 79.2 (77.3–81.1) 3.1, -0.2, -0.9 Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 122 Drug Catalogue, threshold for variants (%) Sensitivity, specificity, PPV (% [95% CI]) Changes in sensitivity, specificity, PPV (%) RIF 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 0, 0, 0 94.4 (94.0–94.7), 96.8 (96.6–97.0), 94.2 (93.8–94.5) 1.1, -0.1, 0 INH First, 75 91.0 (90.6–91.4), 98.1 (97.9–98.2), 97.3 (97.1–97.5) Second, 75 91.6 (91.2–92.0), 97.9 (97.8–98.1), 97.1 (96.8–97.3) 0.6, -0.2, -0.2 Second, 25 First, 75 Second, 75 Second, 25 92.1 (91.7–92.4), 97.9 (97.7–98.0), 97.0 (96.8–97.2) 0.5, 0.0, -0.1 EMB First, 75 83.8 (83.1–84.6), 91.2 (90.9–91.5), 71.6 (70.7–72.4) Second, 75 81.1 (80.3–81.9), 91.6 (91.3–91.9), 71.9 (71.0–72.8) -2.7, 0.4, 0.3 Second, 25 82.1 (81.3–82.9), 91.4 (91.1–91.7), 71.7 (70.9–72.6) 1, -0.2, -0.2 PZA First, 75 73.7 (72.4–75.0), 98.0 (97.8–98.2), 90.8 (89.8–91.7) Second, 75 78.0 (76.8–79.2), 97.9 (97.6–98.1), 90.5 (89.5–91.4) 4.3, -0.1, -0.3 Second, 25 80.0 (78.8–81.2), 97.7 (97.5–98.0), 90.3 (89.4–91.2) 2, -0.2, -0.2 LFX First, 75 83.7 (82.7–84.6), 97.1 (96.9–97.3), 88.7 (87.8–89.5) Second, 75 84.8 (83.9–85.7), 96.9 (96.7–97.1), 88.1 (87.3–89.0) 1.1, -0.2, -0.6 Second, 25 89.2 (88.4–90.0), 96.7 (96.4–96.9), 87.9 (87.0–88.7) 4.4, -0.2, -0.2 MFX First, 75 85.0 (83.8–86.1), 93.8 (93.4–94.1), 74.5 (73.2–75.8) Second, 75 85.7 (84.6–86.8), 93.5 (93.2–93.9), 74.0 (72.7–75.2) 0.7, -0.3, -0.5 Second, 25 90.3 (89.4–91.2), 93.2 (92.8–93.5), 74.0 (72.7–75.2) 4.6, -0.3, 0 BDQ First, 75 0 (0–0.4), 100.0 (100.0–100.0), 0 (0–0) Second, 75 49.4 (46.3–52.5), 98.7 (98.5–98.9), 75.2 (71.8–78.4) 49.4, -1.3, 75.2 Second, 25 59.6 (56.5–62.6), 98.4 (98.2–98.6), 75.0 (71.9–77.9) 10.2, -0.3, -0.2 LZD First, 75 27.3 (22.8–32.1), 99.8 (99.8–99.9), 78.5 (70.4–85.2) Second, 75 34.0 (29.2–39.0), 99.8 (99.7–99.9), 78.4 (71.3–84.5) 6.7, 0, -0.1 Second, 25 35.0 (30.2–40.1), 99.8 (99.7–99.8), 75.7 (68.6–81.9) 1.0, 0, -2.7 CFZ First, 75 0 (0–0.5), 100.0 (100.0–100.0), 0 (0–0) Second, 75 17.0 (14.2–20.0), 98.7 (98.5–98.9), 38.1 (32.6–43.8) 17.0, -1.3, 38.1 Second, 25 21.3 (18.2–24.5), 98.4 (98.2–98.6), 38.9 (33.9–44.1) 4.3, -0.3, 0.8 DLM First, 75 4.4 (2.2–7.7), 100.0 (99.9–100.0), 84.6 (54.6–98.1) Second, 75 14.7 (10.6–19.7), 99.9 (99.8–99.9), 72.5 (58.3–84.1) 10.3, -0.1, -12.1 Second, 25 15.1 (10.9–20.1), 99.8 (99.7–99.9), 62.3 (49.0–74.4) 0.4, -0.1, -10.2 AMK First, 75 72.9 (71.1–74.6), 98.1 (97.9–98.3), 81.3 (79.6–82.9) Second, 75 72.8 (71.0–74.6), 98.3 (98.1–98.5), 82.8 (81.2–84.4) -0.1, 0.2, 1.5 Second, 25 75.4 (73.7–77.1), 98.2 (98.0–98.4), 82.2 (80.6–83.8) 2.6, -0.1, -0.6 STM First, 75 79.2 (78.4–80.0), 94.1 (93.7–94.5), 89.9 (89.2–90.5) Second, 75 79.7 (78.9–80.5), 94.1 (93.7–94.4), 89.9 (89.3–90.5) 0.5, 0, 0 Second, 25 80.3 (79.5–81.1), 94.0 (93.6–94.4), 89.8 (89.2–90.4) 0.6, -0.1, -0.1 ETO First, 75 72.5 (71.3–73.7), 86.6 (86.1–87.2), 64.4 (63.2–65.7) Second, 75 74.8 (73.6–76.0), 85.9 (85.3–86.4), 63.9 (62.7–65.1) 2.3, -0.7, -0.5 Second, 25 76.1 (74.9–77.2), 85.6 (85.0–86.1), 63.8 (62.6–65.0) 1.3, -0.3, -0.1 KANa First, 75 75.1 (73.6–76.5), 96.5 (96.3–96.8), 78.7 (77.2–80.0) Second, 75 74.9 (73.4–76.3), 96.7 (96.4–96.9), 79.3 (77.9–80.7) -0.2, 0.2, 0.6 Second, 25 77.2 (75.8–78.5), 96.6 (96.3–96.8), 79.2 (77.8–80.5) 2.3, -0.1, -0.1 CAPa First, 75 66.4 (64.3–68.4), 97.8 (97.6–98.1), 80.1 (78.2–82.0) Second, 75 66.2 (64.1–68.2), 97.8 (97.6–98.1), 80.1 (78.1–81.9) -0.2, 0, 0 Second, 25 69.3 (67.3–71.3), 97.6 (97.3–97.8), 79.2 (77.3–81.1) 3.1, -0.2, -0.9 Drug Catalogue, threshold for variants (%) Sensitivity, specificity, PPV (% [95% CI]) Changes in sensitivity, specificity, PPV (%) RIF 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 93.3 (92.9–93.7), 96.9 (96.7–97.1), 94.2 (93.9–94.6) 0, 0, 0 94.4 (94.0–94.7), 96.8 (96.6–97.0), 94.2 (93.8–94.5) 1.1, -0.1, 0 INH First, 75 91.0 (90.6–91.4), 98.1 (97.9–98.2), 97.3 (97.1–97.5) Second, 75 91.6 (91.2–92.0), 97.9 (97.8–98.1), 97.1 (96.8–97.3) 0.6, -0.2, -0.2 Second, 25 First, 75 Second, 75 Second, 25 92.1 (91.7–92.4), 97.9 (97.7–98.0), 97.0 (96.8–97.2) 0.5, 0.0, -0.1 EMB First, 75 83.8 (83.1–84.6), 91.2 (90.9–91.5), 71.6 (70.7–72.4) Second, 75 81.1 (80.3–81.9), 91.6 (91.3–91.9), 71.9 (71.0–72.8) -2.7, 0.4, 0.3 Second, 25 82.1 (81.3–82.9), 91.4 (91.1–91.7), 71.7 (70.9–72.6) 1, -0.2, -0.2 PZA First, 75 73.7 (72.4–75.0), 98.0 (97.8–98.2), 90.8 (89.8–91.7) Second, 75 78.0 (76.8–79.2), 97.9 (97.6–98.1), 90.5 (89.5–91.4) 4.3, -0.1, -0.3 Second, 25 80.0 (78.8–81.2), 97.7 (97.5–98.0), 90.3 (89.4–91.2) 2, -0.2, -0.2 LFX First, 75 83.7 (82.7–84.6), 97.1 (96.9–97.3), 88.7 (87.8–89.5) Second, 75 84.8 (83.9–85.7), 96.9 (96.7–97.1), 88.1 (87.3–89.0) 1.1, -0.2, -0.6 Second, 25 89.2 (88.4–90.0), 96.7 (96.4–96.9), 87.9 (87.0–88.7) 4.4, -0.2, -0.2 MFX First, 75 85.0 (83.8–86.1), 93.8 (93.4–94.1), 74.5 (73.2–75.8) Second, 75 85.7 (84.6–86.8), 93.5 (93.2–93.9), 74.0 (72.7–75.2) 0.7, -0.3, -0.5 Second, 25 90.3 (89.4–91.2), 93.2 (92.8–93.5), 74.0 (72.7–75.2) 4.6, -0.3, 0 BDQ First, 75 0 (0–0.4), 100.0 (100.0–100.0), 0 (0–0) Second, 75 49.4 (46.3–52.5), 98.7 (98.5–98.9), 75.2 (71.8–78.4) 49.4, -1.3, 75.2 Second, 25 59.6 (56.5–62.6), 98.4 (98.2–98.6), 75.0 (71.9–77.9) 10.2, -0.3, -0.2 LZD First, 75 27.3 (22.8–32.1), 99.8 (99.8–99.9), 78.5 (70.4–85.2) Second, 75 34.0 (29.2–39.0), 99.8 (99.7–99.9), 78.4 (71.3–84.5) 6.7, 0, -0.1 Second, 25 35.0 (30.2–40.1), 99.8 (99.7–99.8), 75.7 (68.6–81.9) 1.0, 0, -2.7 CFZ First, 75 0 (0–0.5), 100.0 (100.0–100.0), 0 (0–0) Second, 75 17.0 (14.2–20.0), 98.7 (98.5–98.9), 38.1 (32.6–43.8) 17.0, -1.3, 38.1 Second, 25 21.3 (18.2–24.5), 98.4 (98.2–98.6), 38.9 (33.9–44.1) 4.3, -0.3, 0.8 DLM 4.4 (2. –7.7), 100.0 (99.9–100.0), 84.6 (54.6–98.1) 14 7 10 6 19 9 9 8 9 9 72 5 58 3 8 1 10.3, -0.1, -12.1 15 1 10 9 20 1 9 9 7 9 9 62 3 49 0 7 4 0.4, -0.1, -1 .2 AMK 72 9 71 1 74 6 3 81 79 6 82 9 72 8 7 0 74 6 8 3 8 1 5 82 8 81 2 84 4 -0.1, . , 1.5 75 4 73 77 1 8 2 8 0 4 82 2 80 6 83 8 2.6, - .1, - .6 STM 79 2 78 4 0 0 4 1 3 7 4 89 9 89 2 90 5 79 7 78 9 0 5 4 1 3 7 4 4 89 89 3 90 5 0.5, 0, 0 3 79 5 1 1 4 0 3 6 4 4 89 8 89 2 90 4 0.6, -0.1, -0.1 ETO 2 5 1 3 3 7 86 6 86 1 87 64 4 63 2 65 4 8 3 6 6 0 85 85 3 86 4 63 9 62 7 65 1 2 7 5 76 1 4 9 77 85 6 85 0 86 1 63 8 62 6 65 0 1.3, -0.3, -0.1 KANa 75 1 73 6 76 5 6 5 3 6 8 7 7 2 0 0 7 9 7 4 76 3 7 4 6 9 79 3 7 9 0 7 -0.2, . , . 77 75 8 78 5 6 3 8 79 2 7 8 0 5 2 3 1 1 CAPa 66 4 64 3 68 4 7 7 6 8 80 1 8 82 0 66 2 6 1 68 2 7 8 7 6 8 1 80 1 8 1 81 9 -0.2, 0, 69 67 3 7 3 7 6 7 3 7 8 9 2 7 3 81 1 3.1, -0.2, -0.9 a Drugs no longer recommended for TB treatment. The performance of the second edition for minority variants in the ALL data set (these figures are also included in Table A.1) was stratified by the genotypic RIF result (pDST could not be used as it was not available for all isolates). Results for RIF are not shown, as stratifying them by genotypic RIF resistance would not have yielded meaningful results. Annex 1. Further information 123 Table A1.2. Impact of rifampicin resistance on the performance of the second edition Drug Genotypic RIF results % R (95% CI) Sensitivity, specificity, PPV (% [95% CI]) Changes in sensitivity, specificity, PPV (%) INH R+S 43.0 (42.6–43.5) 92.1 (91.7–92.4), 97.9 (97.7–98.0), 97.0 (96.8–97.2) R 93.5 (93.2–93.9) 96.7 (96.4–97.0), 73.1 (70.5–75.7), 98.1 (97.9–98.3) 4.6, -24.8, 1.1 S 14.2 (13.8–14.6) 74.8 (73.5–76.0), 98.9 (98.8–99.0), 92.0 (91.0–92.9) -17.3, 1.0, -5 EMB R+S 21.0 (20.6–21.3) 82.1 (81.3–82.9), 91.4 (91.1–91.7), 71.7 (70.9–72.6) R 56.8 (56.0–57.6) 86.0 (85.2–86.7), 56.9 (55.7–58.1), 72.4 (71.6–73.3) 3.9, -34.5, 0.7 S 2.3 (2.1–2.5) 32.1 (28.6–35.8), 99.4 (99.3–99.4), 54.1 (49.1–59.0) -50, 8.0, -17.6 PZA R+S 20.8 (20.2–21.3) 80.0 (78.8–81.2), 97.7 (97.5–98.0), 90.3 (89.4–91.2) R 58.4 (57.2–59.6) 88.7 (87.6–89.7), 86.3 (85.0–87.6), 90.1 (89.1–91.0) 8.7, -11.4, -0.2 S 4.9 (4.5–5.2) 36.0 (32.5–39.6), 99.9 (99.8–99.9), 93.2 (89.6–95.9) -44, 2.2, 2.9 LFX R+S 21.3 (20.8–21.8) 89.2 (88.4–90.0), 96.7 (96.4–96.9), 87.9 (87.0–88.7) R 36.0 (35.3–36.8) 92.6 (91.8–93.2), 93.0 (92.4–93.5), 88.1 (87.2–88.9) 3.4, -3.7, 0.2 S 3.6 (3.3–4.0) 49.6 (44.9–54.3), 99.6 (99.5–99.7), 83.0 (78.0–87.3) -39.6, 2.9, -4.9 MFX R+S 17.7 (17.2–18.2) 90.3 (89.4–91.2), 93.2 (92.8–93.5), 74.0 (72.7–75.2) R 35.1 (34.2–36.0) 93.3 (92.5–94.1), 82.6 (81.7–83.5), 74.4 (73.1–75.6) 3, -10.6, 0.4 S 2.6 (2.3–2.9) 54.9 (49.2–60.5), 99.3 (99.1–99.4), 66.3 (60.2–72.0) -35.4, 6.1, -7.7 BDQ R+S 7.3 (6.9–7.8) 59.6 (56.5–62.6), 98.4 (98.2–98.6), 75.0 (71.9–77.9) R 14.2 (13.3–15.0) 61.8 (58.7–64.9), 96.6 (96.1–97.1), 75.0 (71.8–78.0) 2.2, -1.8, 0 S 1.0 (0.8–1.3) 31.6 (21.4–43.3), 99.9 (99.8–100.0), 75.0 (56.6–88.5) -28, 1.5, 0.1 LZD R+S 2.1 (1.9–2.3) 35.0 (30.2–40.1), 99.8 (99.7–99.8), 75.7 (68.6–81.9) R 3.0 (2.7–3.4) 44.9 (39.0–50.9), 99.6 (99.4–99.7), 75.7 (68.6–82.0) 9.9, -0.2, 0 S 1.0 (0.8–1.3) 3.4 (0.7–9.5), 100.0 (99.9–100.0), 75.0 (19.4–99.4) -31.6, 0.2, -0.7 CFZ R+S 4.5 (4.2–4.9) 21.3 (18.2–24.5), 98.4 (98.2–98.6), 38.9 (33.9–44.1) R 6.3 (5.7–6.9) 31.5 (27.2–36.2), 96.8 (96.3–97.2), 39.6 (34.4–45.0) 10.2, -1.6, 0.7 S 3.1 (2.7–3.5) 3.6 (1.7–6.8), 99.7 (99.6–99.8), 31.0 (15.3–50.8) -17.7, 1.3, -7.9 DLM R+S 2.1 (1.9–2.4) 15.1 (10.9–20.1), 99.8 (99.7–99.9), 62.3 (49.0–74.4) R 2.4 (2.0–2.8) 16.0 (9.9–23.8), 99.8 (99.6–99.9), 63.3 (43.9–80.1) 0.9, 0, 1 S 2.0 (1.7–2.3) 14.3 (8.8–21.4), 99.8 (99.7–99.9), 61.3 (42.2–78.2) -0.8, 0, -1 AMK R+S 10.0 (9.7–10.4) 75.4 (73.7–77.1), 98.2 (98.0–98.4), 82.2 (80.6–83.8) R 17.0 (16.4–17.7) 80.2 (78.5–81.8), 96.5 (96.1–96.8), 82.4 (80.7–83.9) 4.8, -1.7, 0.2 S 1.7 (1.5–2.0) 19.8 (14.5–26.1), 99.9 (99.8–99.9), 76.5 (62.5–87.2) -55.6, 1.7, -5.7 STM R+S 39.8 (39.2–40.4) 80.3 (79.5–81.1), 94.0 (93.6–94.4), 89.8 (89.2–90.4) R 77.7 (76.8–78.5) 85.0 (84.2–85.8), 76.5 (74.7–78.2), 92.6 (92.0–93.2) 4.7, -17.5, 2.8 S 14.9 (14.4–15.5) 64.3 (62.4–66.3), 97.0 (96.7–97.3), 79.0 (77.1–80.8) -16, 3, -10.8 ETO R+S 25.0 (24.4–25.6) 76.1 (74.9–77.2), 85.6 (85.0–86.1), 63.8 (62.6–65.0) R 38.6 (37.7–39.5) 77.9 (76.6–79.1), 72.5 (71.5–73.5), 64.1 (62.8–65.3) 1.8, -13.1, 0.3 S 7.4 (6.8–7.9) 63.8 (60.0–67.4), 96.8 (96.4–97.2), 61.6 (57.9–65.2) -12.3, 11.2, -2.2 KANa R+S 14.5 (14.1–15.0) 77.2 (75.8–78.5), 96.6 (96.3–96.8), 79.2 (77.8–80.5) R 25.1 (24.4–25.9) 82.3 (81.0–83.6), 92.8 (92.3–93.3), 79.3 (77.9–80.7) 5.1, -3.8, 0.1 S 2.5 (2.2–2.8) 17.6 (13.4–22.5), 99.8 (99.7–99.9), 71.4 (59.4–81.6) -59.6, 3.2, -7.8 CAPa R+S 11.7 (11.2–12.1) 69.3 (67.3–71.3), 97.6 (97.3–97.8), 79.2 (77.3–81.1) R 16.6 (15.9–17.2) 72.8 (70.7–74.8), 96.3 (95.9–96.7), 79.7 (77.7–81.5) 3.5, -1.3, 0.5 S 2.4 (2.0–2.8) 23.6 (16.9–31.4), 99.7 (99.5–99.8), 64.2 (49.8–76.9) -45.7, 2.1, -15 a Drugs no longer recommended for TB treatment. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 124 We explored the extent to which rare Group 3 mutations in the following nonessential genes that did not meet the standard grading thresholds (Fig. 8) might account for the “missing” sensitivity as compared with pDST: INH (katG), PZA (pncA), BDQ and CFZ (Rv0678), and DLM (ddn, fbiA, fbiB, fbiC, fgd1, and Rv2983). We used two approaches. First, we extended the “relaxed” grading criteria that were WHO-endorsed in the first edition to classify rare pncA mutations into Group 2 (Fig. 9) to the remaining genes of interest (coding and upstream regions). The mutations that met these “relaxed” criteria were highlighted in footnotes in the catalogue master file. The resulting sensitivities, specificities and PPVs are shown in the “1+2+3 relaxed” entries in the table below. As the “relaxed” criteria were already approved for pncA, there is no “1+2+3 relaxed” entry for PZA (i.e. the mutations in question were already in Group 2 and therefore included under “Groups 1+2”). Secondly, we assumed that any coding mutation (upstream regions excluded) in the above genes, except silent and Groups 4/5 mutations, might also be valid markers of resistance in isolates that are genotypically resistant to RIF. The resulting figures are shown as “1+2+3 coding” entries. All calculations were conducted with a 25% allele frequency as the threshold for calling mutations in the ALL data set. These results are only for information available at this time, and the same limitations for e.g. sampling and overfitting discussed in Section 2.4 apply to these calculations. A more detailed discussion of these findings is provided in the relevant sections for drugs in Section 3. Annex 1. Further information 125 Table A1.3. Potential role of rare Group 3 mutations for predicting resistance to INH, PZA, BDQ and DLM Drug Genotypic RIF results % R (95% CI) Mutation Groups Sensitivity, specificity, PPV (% [95% CI]) Changes in sensitivity, specificity, PPV relative to Group 1+2 (%) INH 2+1S+R 92.1 (91.7–92.4), 97.9 (97.7–98.0), 97.0 (96.8–97.2) 1+2+3 relaxed 93.3 (93.0–93.6), 97.7 (97.5–97.9), 96.8 (96.6–97.1) 1.2, -0.2, -0.2 1+2+3 coding 93.8 (93.4–94.1), 97.5 (97.3–97.7), 96.6 (96.4–96.9) 1.7, -0.4, -0.4 2+1R 96.7 (96.4–97.0), 73.1 (70.5–75.7), 98.1 (97.9–98.3) 1+2+3 relaxed 97.6 (97.4–97.9), 71.8 (69.1–74.4), 98.0 (97.8–98.3) 0.9, -1.3, -0.1 1+2+3 coding 98.8 (98.7–99.0), 65.3 (62.4–68.0), 97.6 (97.4–97.9) 2.1, -7.8, -0.5 2+1S 74.8 (73.5–76.0), 98.9 (98.8–99.0), 92.0 (91.0–92.9) 1+2+3 relaxed 77.0 (75.7–78.2), 98.8 (98.7–98.9), 91.5 (90.5–92.4) 2.2, -0.1, -0.5 PZA 2+1S+R 80.0 (78.8–81.2), 97.7 (97.5–98.0), 90.3 (89.4–91.2) 1+2+3 coding 82.6 (81.5–83.7), 97.0 (96.7–97.3), 87.9 (86.8–88.9) 2.6, -0.7, -2.4 2+1R 88.7 (87.6–89.7), 86.3 (85.0–87.6), 90.1 (89.1–91.0) 1+2+3 coding 91.8 (90.8–92.6), 81.6 (80.1–83.0), 87.5 (86.4–88.5) 3.1, -4.7, -2.6 S 4.9 (4.5–5.2) 1+2 36.0 (32.5–39.6), 99.9 (99.8–99.9), 93.2 (89.6–95.9) BDQ 2+1S+R 59.6 (56.5–62.6), 98.4 (98.2–98.6), 75.0 (71.9–77.9) 1+2+3 relaxed 68.8 (65.8–71.6), 98.3 (98.1–98.5), 76.6 (73.7–79.3) 9.2, -0.1, 1.6 1+2+3 coding 75.1 (72.4–77.7), 97.7 (97.4–97.9), 71.9 (69.1–74.5) 15.5, -0.7, -3.1 2+1R 61.8 (58.7–64.9), 96.6 (96.1–97.1), 75.0 (71.8–78.0) 1+2+3 relaxed 71.0 (68.0–73.8), 96.4 (95.9–96.9), 76.6 (73.7–79.4) 9.2, -0.2, 1.6 1+2+3 coding 78.6 (75.8–81.1), 94.9 (94.3–95.4), 71.8 (68.9–74.5) 16.8, -1.7, -3.2 2+1S 31.6 (21.4–43.3), 99.9 (99.8–100.0), 75.0 (56.6–88.5) 1+2+3 relaxed 40.8 (29.6–52.7), 99.9 (99.7–99.9), 75.6 (59.7–87.6) 9.2, 0, 0.6 CFZ 2+1S+R 21.3 (18.2–24.5), 98.4 (98.2–98.6), 38.9 (33.9–44.1) 1+2+3 relaxed 24.2 (21.0–27.6), 98.3 (98.1–98.5), 40.6 (35.8–45.6) 2.9, -0.1, 1.7 1+2+3 coding 29.4 (26.0–33.0), 97.7 (97.4–97.9), 37.6 (33.5–41.9) 8.1, -0.7, -1.3 2+1R 31.5 (27.2–36.2), 96.8 (96.3–97.2), 39.6 (34.4–45.0) 1+2+3 relaxed 35.5 (31.0–40.3), 96.6 (96.2–97.1), 41.4 (36.3–46.6) 4, -0.2, 1.8 1+2+3 coding 44.4 (39.6, 49.2), 95.2 (94.6, 95.7), 38.0 (33.7, 42.4) 12.9, -1.6, -1.6 2+1S 3.6 (1.7–6.8), 99.7 (99.6–99.8), 31.0 (15.3–50.8) 1+2+3 relaxed 4.8 (2.5–8.3), 99.7 (99.5–99.8), 32.4 (18.0–49.8) 1.2, 0, 1.4 DLM 2+1S+R 15.1 (10.9–20.1), 99.8 (99.7–99.9), 62.3 (49.0–74.4) 1+2+3 relaxed 17.9 (13.3–23.2), 99.8 (99.7–99.9), 66.2 (53.7–77.2) 2.8, 0, 3.9 1+2+3 coding 25.0 (19.8–30.8), 91.6 (91.0–92.1), 6.1 (4.7–7.7) 9.9, -8.2, -56.2 2+1R 16.0 (9.9–23.8), 99.8 (99.6–99.9), 63.3 (43.9–80.1) 1+2+3 relaxed 17.6 (11.3–25.7), 99.8 (99.6–99.9), 65.6 (46.8–81.4) 1.6, 0, 2.3 1+2+3 coding 37.0 (28.3–46.3), 80.4 (79.3–81.5), 4.4 (3.2–5.8) 21, -19.4, -58.9 2+1S 14.3 (8.8–21.4), 99.8 (99.7–99.9), 61.3 (42.2–78.2) 1+2+3 relaxed 18.0 (11.9–25.6), 99.8 (99.7–99.9), 66.7 (49.0–81.4) 3.7, 0, 5.4 2.4 (2.0–2.8) 2.0 (1.7–2.3) 14.2 (13.3–15.0) 1.0 (0.8–1.3) 4.5 (4.2–4.9) 6.3 (5.7–6.9) 3.1 (2.7–3.5) 2.1 (1.9–2.4) 7.3 (6.9–7.8) 43.0 (42.6–43.5) 93.5 (93.2–93.9) 14.2 (13.8–14.6) 20.8 (20.2–21.3) 58.4 (57.2–59.6) Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 126 Fig. A1.1. Scheme of the bioinformatics pipeline for detecting variants in the second edition SRAPass S3Pass PrepareKraken FilterDeletions ChoiceDelly Delly NoDelly CalculateStats PrepareStats SamtoolsStats MosDepth BwaMem SamtoolsSort ChoiceLibraryNumber GATKRemoveDuplicates FastQC CleanUpFiles Initialize Start End RenameAlignment WGSPass tNGSPass MergeAlignments Bcftools Freebayes GATKHaplotypeCaller PrepareGenotypes CopyReadsFromS3 choiceS3orNGDCFTP ChoiceSampleOriginForDownload DownloadReadByFtp ChoiceSampleType DownloadReadsFromSRA NGDC-CNCBPass ConvertToCram Kraken 127 Annex 2. Conflict of interest assessment WHO held an expert consultation on 28 February, 1 March and 9 March 2023. All individuals who provided technical input were required to disclose any potential conflicts of interest, encompassing both financial and non-financial interests. The candidates’ DOI forms and information retrieved from the internet, were examined by WHO staff members Nazir Ismail and Carl-Michael Nathanson to assess whether there were, or might be, actual or perceived conflicts of interest and, if so, whether a management plan was required. This evaluation process, and resultant management plans, were based on the Guidelines for declaration of interests (WHO experts) and the WHO handbook for guideline development (2nd edition). Both financial and non-financial interests were considered. A “significant” conflict of interest would include: • “intellectual bias”, where an individual may have repeatedly and publicly taken a position on an issue under review, which may affect the individual’s objectivity and independence in the global policy development process; • involvement in research or publication of materials related to issues under review; and • a financial interest above US$ 5000. Developers of any assay are never involved in the process of policy development – such involvement is automatically considered a conflict of interest. Upon review no significant conflict of interest were identified. Participants’ statements were summarized by the WHO at the start of the meeting. The review findings are summarized in Table A2.1. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance - second edition 128 Table A2.1. Declarations of interests Participant Interest declared Conclusion Heidi Albert FIND has several clinical research projects to evaluate multiple new diagnostic tests against published Target Product Profiles that have been defined through consensus processes. Conflict of interest not significant Uladzimir Antoneka None declared No conflict of interest Arnold Bainomugisa None declared No conflict of interest Leonid Chindelevitch None declared No conflict of interest Daniela Cirillo None declared No conflict of interest Francesc Coll Consulting and employment, Next Gen Diagnostics in the past Conflict of interest not significant Rebecca Colman None declared No conflict of interest Iñaki Comas None declared No conflict of interest Sarah Cook-Scalise None declared No conflict of interest Chris Coulter None declared No conflict of interest James Dawson None declared No conflict of interest Maha Farhat None declared No conflict of interest Philip Fowler Consulting - Scientific and Technical consultant for GPAS Ltd Conflict of interest not significant Sophia Georghiou None declared No conflict of interest Patricia Hall-Eidson None declared No conflict of interest Zahra Hasan None declared No conflict of interest Harald Hoffmann None declared No conflict of interest Zamin Iqbal Grant to support development and pilot of a cloud-based global TB genomic surveillance tool, and testing in Buenos Aires. Conflict of interest not significant George Kasule None declared No conflict of interest Claudio Köser Consulting for BD and TB Alliance, remuneration not declared, advisor for Cepheid in 2022 pro bono, collaboration with Janssen, PZA Innocation and Thermo Fisher with no remuneration Conflict of interest not significant Sanjana Kulkarni Not declared, observer N/A Sacha Laurent None declared No conflict of interest Marguerite Massinga Loembé Grant (acting as project scientific lead for ASLM who is one of the grant recipient) from EDCTP Grant (acting as project scientific lead for ASLM who is the grant recipient) from FIND/BMGF Conflict of interest not significant Heather McLaughlin Not declared, observer N/A Alberto Mendoza None declared No conflict of interest Matthias Merker None declared No conflict of interest Paolo Miotto None declared No conflict of interest Satoshi Mitarai Research grant from Roche diagnostics KK, ceased in 2021 Conflict of interest not significant Stefan Niemann Consultation for Illumina, 3700 Euro reimbursement, ended 2022 Conflict of interest not significant Jamie Posey None declared No conflict of interest Leen Rigouts None declared No conflict of interest Camilla Rodrigues Research grant from FIND Conflict of interest not significant Annex 2. Conflict of interest assessment 129 Participant Interest declared Conclusion Timothy Rodwell Co-inventor on a patent involving the processing of sequencing data for the purposes of detecting drug resistant TB mutations. All rights to future exploitation of and potential income from the patent have been transferred to UCSD Conflict of interest not significant Anita Suresh None declared No conflict of interest Swapna Uplekar None declared No conflict of interest Shaheed Vally Omar Reserarch grant from Janssen Pharma to assess prevalence of BDQ resistance using WGS Conflict on interest not significant Wayne van Gemert None declared No conflict of interest Timothy Walker None declared No conflict of interest Zhao Yanlin None declared No conflict of interest Danila Zimenkov None declared No conflict of interest For further information, please contact: Global Tuberculosis Programme World Health Organization 20 Avenue Appia CH-1211 Geneva 27 Switzerland Web site: https://www.who.int/teams/ global-tuberculosis-programme/overview Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance For further info mation, please contact: Global Tuberculosis Programme World Health Organization 20 Avenue Appia CH-1211 Geneva 27 Switzerland Web sit : ht ps://www.who int/teams/ global-tuberculosis-programme/overview Catalogue of mutations in Mycobacterium tuberculosi complex and their association with drug resi tance C atalo g u e o f m u tatio n s in M yco b acteriu m tu b ercu lo sis co m p lex an d th eir asso ciatio n w ith d ru g resistan ce