Technical report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis

WHO collaborating centre for the evaluation of new diagnostic technologies Technical Report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis 2018

WHO collaborating centre for the evaluation of new diagnostic technologies Technical Report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis © World Health Organization 2018 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 organisation, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. 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Printed in Switzerland WHO/CDS/TB/2018.5 Contents iii Contents Acknowledgements ............................................................................................................... xiv Members of the WHO Technical Expert Consultation Group ............................................ xiv Technical resource persons ................................................................................................. xiv Additional literature reviewers ............................................................................................. xiv Major data contributors ........................................................................................................ xiv Acknowledgement of financial support ............................................................................... xv Declaration and management of conflict of interest .......................................................... xv Abbreviations .........................................................................................................................xvii Glossary of terms .................................................................................................................. xix Executive summary ............................................................................................................... xxi 1. Introduction ........................................................................................................................... 3 1.0 Background ................................................................................................................ 3 1.1 Scope of the Technical Expert Consultation Meeting ................................................... 3 1.2 Systematic review ....................................................................................................... 4 1.2.1 Search methodology .......................................................................................... 4 1.2.2 Inclusion criteria ................................................................................................. 5 1.2.3 Studies identified through the systematic review ................................................ 5 1.3 Data presentation ........................................................................................................ 6 1.3.1 Format of this report .......................................................................................... 6 1.3.2 Format of MIC tables ......................................................................................... 6 2. Second-line injectable agents ............................................................................................. 8 2.0 SLI MIC data stratification and current breakpoints ...................................................... 8 2.A.1 KAN MIC data on LJ ................................................................................................ 8 2.A.1.1 KAN MICs for pWT isolates on LJ ................................................................... 8 2.A.1.2 KAN MICs for mutated isolates on LJ ............................................................. 9 2.A.1.3 Conclusion for KAN CC for LJ ........................................................................ 9 2.A.2 KAN MIC data on 7H10 ........................................................................................... 9 2.A.2.1 KAN MICs for pWT isolates on 7H10 .............................................................. 9 2.A.2.2 KAN MICs for mutated isolates on 7H10. ..................................................... 11 2.A.2.3 Conclusion for KAN CC for 7H10 ................................................................. 14 2.A.3 KAN MIC data on 7H11 ......................................................................................... 14 2.A.3.1 KAN MICs for pWT isolates on 7H11 ............................................................ 14 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS iv 2.A.3.2 KAN MICs for mutated isolates on 7H11 ...................................................... 14 2.A.3.3 Conclusion for KAN CC for 7H11 ................................................................. 14 2.A.4 KAN MIC data in MGIT ........................................................................................... 14 2.A.4.1 KAN MICs for pWT isolates in MGIT ............................................................. 14 2.A.4.2 KAN MICs for mutated isolates in MGIT ........................................................ 16 2.A.4.3 Conclusion for KAN CC in MGIT ................................................................... 17 2.A.5 References for KAN MIC studies ............................................................................ 17 2.B.1 AMK MIC data on LJ .............................................................................................. 19 2.B.1.1 AMK MICs for pWT isolates on LJ ................................................................ 19 2.B.1.2 AMK MICs for mutated isolates on LJ ........................................................... 19 2.B.1.3 Conclusion for AMK CC on LJ ...................................................................... 20 2.B.2 AMK MIC data on 7H10 ......................................................................................... 20 2.B.2.1 AMK MICs for pWT isolates on 7H10 ........................................................... 20 2.B.2.2 AMK MICs for mutated isolates on 7H10 ...................................................... 20 2.B.2.3 Conclusion for AMK CC on 7H10 ................................................................. 24 2.B.3 AMK MIC data on 7H11 ......................................................................................... 24 2.B.3.1 AMK MICs for pWT isolates on 7H11 ........................................................... 24 2.B.3.2 AMK MICs for mutated isolates on 7H11 ...................................................... 25 2.B.3.3 Conclusion for AMK CC on 7H11 ................................................................. 25 2.B.4 AMK MIC data in MGIT .......................................................................................... 25 2.B.4.1 AMK MICs for pWT isolates in MGIT ............................................................. 25 2.B.4.2 AMK MICs for mutated isolates in MGIT ....................................................... 25 2.B.5 References for AMK MIC studies ............................................................................ 28 2.C.1 CAP MIC data on LJ .............................................................................................. 30 2.C.1.1 CAP MICs for pWT isolates on LJ ................................................................ 30 2.C.1.2 CAP MICs for mutated isolates on LJ ........................................................... 30 2.C.1.3 Conclusion for CAP CC on LJ ...................................................................... 31 2.C.2 CAP MIC data on 7H10 ......................................................................................... 31 2.C.2.1 CAP MICs for pWT isolates on 7H10 ............................................................ 31 2.C.2.2 CAP MICs for mutated isolates on 7H10 ...................................................... 31 2.C.2.3 Conclusion for CAP CC on 7H10 ................................................................. 35 2.C.3 CAP MIC data on 7H11 ......................................................................................... 36 2.C.3.1 CAP MICs for pWT isolates on 7H11 ............................................................ 36 2.C.3.2 CAP MICs for mutated isolates on 7H11 ...................................................... 36 2.C.3.3 Conclusion for CAP CC on 7H11 ................................................................. 36 2.C.4 CAP MIC data in MGIT ........................................................................................... 36 2.C.4.1 CAP MICs for pWT isolates in MGIT ............................................................. 36 2.C.4.2 CAP MICs for mutated isolates in MGIT ........................................................ 36 2.C.5 References for CAP MIC studies ............................................................................ 39 Contents v 3. Clofazimine and bedaquiline ............................................................................................. 42 3.0 CFZ and BDQ MIC data stratification and current breakpoints ................................... 42 3.A.1 CFZ MIC data on LJ ............................................................................................... 42 3.A.1.1 CFZ MICs for pWT isolates on LJ ................................................................. 42 3.A.1.2 CFZ MICs for mutated isolates on LJ ............................................................ 42 3.A.1.3 Conclusion for CFZ CC for LJ ....................................................................... 42 3.A.2 CFZ MIC data on 7H10 .......................................................................................... 42 3.A.2.1 CFZ MICs for pWT isolates on 7H10 ............................................................ 42 3.A.2.2 CFZ MICs for mutated isolates on 7H10 ....................................................... 42 3.A.2.3 Conclusion for CFZ CC for 7H10 .................................................................. 42 3.A.3 CFZ MIC data on 7H11 .......................................................................................... 43 3.A.3.1 CFZ MICs for pWT isolates on 7H11 ............................................................ 43 3.A.3.2 CFZ MICs for mutated isolates on 7H11 ....................................................... 43 3.A.3.3 Conclusion for CFZ CC for 7H11 .................................................................. 43 3.A.4 CFZ MIC data in MGIT ........................................................................................... 43 3.A.4.1 CFZ MICs for pWT isolates in MGIT .............................................................. 43 3.A.4.2 CFZ MICs for mutated isolates in MGIT ........................................................ 44 3.A.4.3 Conclusion for CFZ CC for MGIT .................................................................. 44 3.A.5 References for CFZ MIC studies ............................................................................. 44 3.B.1 BDQ MIC data on LJ .............................................................................................. 45 3.B.1.1 BDQ MICs for pWT isolates on LJ ................................................................ 45 3.B.1.2 BDQ MICs for mutated isolates on LJ ........................................................... 45 3.B.1.3 Conclusion for BDQ CC for LJ ...................................................................... 45 3.B.2 BDQ MIC data on 7H10 ......................................................................................... 45 3.B.2.1 BDQ MICs for pWT isolates on 7H10 ........................................................... 45 3.B.2.2 BDQ MICs for mutated isolates on 7H10 ...................................................... 46 3.B.2.3 Conclusion for BDQ CC for 7H10 ................................................................. 46 3.B.3 BDQ MIC data on 7H11 ......................................................................................... 46 3.B.3.1 BDQ MICs for pWT isolates on 7H11 ........................................................... 46 3.B.3.2 BDQ MICs for mutated isolates on 7H11 ...................................................... 47 3.B.3.3 Conclusion for BDQ CC for 7H11 ................................................................. 48 3.B.4 BDQ MIC data in MGIT .......................................................................................... 49 3.B.4.1 BDQ MICs for pWT isolates in MGIT ............................................................. 49 3.B.4.2 BDQ MICs for mutated isolates in MGIT ....................................................... 49 3.B.4.3 Conclusion for BDQ CC for MGIT ................................................................. 50 3.B.5 References for BDQ MIC studies ............................................................................ 50 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS vi 4. Cycloserine and terizidone ................................................................................................ 52 4.0 DCS and TRD MIC data stratification and current breakpoints ................................... 52 4.1 DCS MIC data on LJ ................................................................................................. 52 4.1.1 DCS MICs for pWT isolates on LJ .................................................................... 52 4.1.2 DCS MICs for mutated isolates on LJ .............................................................. 52 4.1.3 Conclusion for DCS CC for LJ ......................................................................... 53 4.2 DCS MIC data on 7H10 ............................................................................................ 53 4.2.1 DCS MICs for pWT isolates on 7H10 ............................................................... 53 4.2.2 DCS MICs for mutated isolates on 7H10.......................................................... 53 4.2.3 Conclusion for DCS CC for 7H10..................................................................... 53 4.3 DCS MIC data on 7H11 ............................................................................................ 53 4.3.1 DCS MICs for pWT isolates on 7H11 ............................................................... 53 4.3.2 DCS MICs for mutated isolates on 7H11.......................................................... 53 4.3.3 Conclusion for DCS CC for 7H11..................................................................... 53 4.4 DCS MIC data in MGIT .............................................................................................. 53 4.4.1 DCS MICs for pWT isolates in MGIT ................................................................ 53 4.4.2 DCS MICs for mutated isolates in MGIT ........................................................... 54 4.4.3 Conclusion for DCS CC for MGIT ..................................................................... 54 4.5 References for DCS MIC studies ............................................................................... 54 5. Linezolid .............................................................................................................................. 55 5.0 LZD MIC data stratification and current breakpoints .................................................. 55 5.1 LZD MIC data on LJ .................................................................................................. 55 5.1.1 LZD MICs for pWT isolates on LJ ..................................................................... 55 5.1.2 LZD MICs for mutated isolates on LJ ............................................................... 55 5.1.3 Conclusion for LZD CC for LJ .......................................................................... 55 5.2 LZD MIC data on 7H10 ............................................................................................. 55 5.2.1 LZD MICs for pWT isolates on 7H10 ................................................................ 55 5.2.2 LZD MICs for mutated isolates on 7H10 .......................................................... 56 5.2.3 Conclusion for LZD CC for 7H10 ..................................................................... 56 5.3 LZD MIC data on 7H11 ............................................................................................. 56 5.3.1 LZD MICs for pWT isolates on 7H11 ................................................................ 56 5.3.2 LZD MICs for mutated isolates on 7H11 .......................................................... 57 5.3.3 Conclusion for LZD CC for 7H11 ..................................................................... 57 5.4 LZD MIC data in MGIT ............................................................................................... 57 5.4.1 LZD MICs for pWT isolates in MGIT ................................................................. 57 5.4.2 LZD MICs for mutated isolates in MGIT ............................................................ 57 5.3.3 Conclusion for LZD CC for MGIT ..................................................................... 58 5.5 References for LZD MIC studies ................................................................................ 58 Contents vii 6. Delamanid ........................................................................................................................... 61 6.0 DLM MIC data stratification and current breakpoints ................................................. 61 6.1 DLM MIC data on LJ ................................................................................................. 61 6.1.1 DLM MICs for pWT isolates on LJ .................................................................... 61 6.1.2 DLM MICs for mutated isolates on LJ .............................................................. 61 6.1.3 Conclusion for DLM CC for LJ ......................................................................... 61 6.2 DLM MIC data on 7H10 ............................................................................................ 61 6.2.1 DLM MICs for pWT isolates on 7H10 ............................................................... 61 6.2.2 DLM MICs for mutated isolates on 7H10 ......................................................... 62 6.2.3 Conclusion for DLM CC for 7H10 .................................................................... 62 6.3 DLM MIC data on 7H11 ............................................................................................ 62 6.3.1 DLM MICs for pWT isolates on 7H11 ............................................................... 62 6.3.2 DLM MICs for mutated isolates on 7H11 ......................................................... 62 6.3.3 Conclusion for DLM CC for 7H11 .................................................................... 63 6.4 DLM MIC data in MGIT .............................................................................................. 63 6.4.1 DLM MICs for pWT isolates in MGIT ................................................................ 63 6.4.2 DLM MICs for mutated isolates in MGIT ........................................................... 63 6.4.3 Conclusion for DLM CC for MGIT..................................................................... 64 6.5 References for DLM MIC studies ............................................................................... 64 7. Fluoroquinolones ................................................................................................................ 65 7.0 FQ MIC data stratification and current breakpoints .................................................... 65 7.A.1 OFX MIC data on LJ ............................................................................................... 66 7.A.1.1 OFX MICs for pWT isolates on LJ ................................................................. 66 7.A.1.2 OFX MICs for mutated isolates on LJ ............................................................ 66 7.A.1.3 Conclusion for OFX CC for LJ ....................................................................... 67 7.A.2 OFX MIC data on 7H10 .......................................................................................... 67 7.A.2.1 OFX MICs for pWT isolates on 7H10 ............................................................ 67 7.A.2.2 OFX MICs for mutated isolates on 7H10 ....................................................... 67 7.A.2.3 Conclusion for OFX CC for 7H10 .................................................................. 71 7.A.3 OFX MIC data on 7H11 .......................................................................................... 71 7.A.3.1 OFX MICs for pWT isolates on 7H11 ............................................................ 71 7.A.3.2 OFX MICs for mutated isolates on 7H11 ....................................................... 72 7.A.3.3 Conclusion for OFX CC for 7H11 .................................................................. 73 7.A.4 OFX MIC data in MGIT ........................................................................................... 73 7.A.4.1 OFX MICs for pWT isolates in MGIT .............................................................. 73 7.A.4.2 OFX MICs for mutated isolates in MGIT ........................................................ 73 7.A.5 References for OFX MIC studies ............................................................................. 75 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS viii 7.B.1 LFX MIC data on LJ ............................................................................................... 77 7.B.1.1 LFX MICs for pWT isolates on LJ .................................................................. 77 7.B.1.2 LFX MICs for mutated isolates on LJ ............................................................ 77 7.B.1.3 Conclusion for LFX CC for LJ ....................................................................... 78 7.B.2 LFX MIC data on 7H10 .......................................................................................... 78 7.B.2.1 LFX MICs for pWT isolates on 7H10 ............................................................. 78 7.B.2.2 LFX MICs for mutated isolates on 7H10 ....................................................... 79 7.B.2.3 Conclusion for LFX CC for 7H10 .................................................................. 82 7.B.3 LFX MIC data on 7H11 .......................................................................................... 82 7.B.3.1 LFX MICs for pWT isolates on 7H11 ............................................................. 82 7.B.3.2 LFX MICs for mutated isolates on 7H11 ....................................................... 83 7.B.4 LFX MIC data in MGIT ............................................................................................ 84 7.B.4.1 LFX MICs for pWT isolates in MGIT .............................................................. 84 7.B.4.2 LFX MICs for mutated isolates in MGIT ......................................................... 85 7.B.4.3 Conclusion for LFX CC for MGIT ................................................................... 86 7.B.5 References for LFX MIC studies ............................................................................. 86 7.C.1 GFX MIC data on LJ .............................................................................................. 88 7.C.1.1 GFX MICs for pWT isolates on LJ ................................................................. 88 7.C.1.2 GFX MICs for mutated isolates on LJ ........................................................... 88 7.C.1.3 Conclusion for GFX CC for LJ ...................................................................... 89 7.C.2 GFX MIC data on 7H10.......................................................................................... 89 7.C.2.1 GFX MICs for pWT isolates on 7H10 ............................................................ 89 7.C.2.2 GFX MICs for mutated isolates on 7H10 ....................................................... 89 7.C.2.3 Conclusion for GFX CC for 7H10 .................................................................. 89 7.C.3 GFX MIC data on 7H11.......................................................................................... 90 7.C.3.1 GFX MICs for pWT isolates on 7H11 ............................................................ 90 7.C.3.2 GFX MICs for mutated isolates on 7H11 ....................................................... 90 7.C.3.3 Conclusion for GFX CC for 7H11 .................................................................. 90 7.C.4 GFX MIC data in MGIT ........................................................................................... 91 7.C.4.1 GFX MICs for pWT isolates in MGIT .............................................................. 91 7.C.4.2 GFX MICs for mutated isolates in MGIT ........................................................ 91 7.C.4.3 Conclusion for GFX CC for MGIT .................................................................. 92 7.C.5 References for GFX MIC studies ............................................................................ 92 7.D.1 MFX MIC data on LJ .............................................................................................. 93 7.D.1.1 MFX MICs for pWT isolates on LJ ................................................................. 93 7.D.1.2 MFX MICs for mutated isolates on LJ ........................................................... 93 7.D.1.3 Conclusion for MFX CC and CB for LJ ......................................................... 94 7.D.2 MFX MIC data on 7H10 ......................................................................................... 94 Contents ix 7.D.2.1 MFX MICs for pWT isolates on 7H10 ............................................................ 94 7.D.2.2 MFX MICs for mutated isolates on 7H10 ...................................................... 95 7.D.2.3 Conclusion for MFX CC and CB for 7H10 ..................................................... 98 7.D.3 MFX MIC data on 7H11 ......................................................................................... 99 7.D.3.1 MFX MICs for pWT isolates on 7H11 ............................................................ 99 7.D.3.2 MFX MICs for mutated isolates on 7H11 ...................................................... 99 7.D.3.3 Conclusion for MFX CC and CB for 7H11 ................................................... 100 7.D.4 MFX MIC data in MGIT ......................................................................................... 101 7.D.4.1 MFX MICs for pWT isolates in MGIT ........................................................... 101 7.D.4.2 MFX MICs for mutated isolates in MGIT ...................................................... 101 7.D.4.3 Conclusion for MFX CC and CB for MGIT ................................................... 103 7.D.5 Rationale for MFX CB ........................................................................................... 103 7.D.6 References for MFX MIC studies .......................................................................... 104 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS x List of figures Figure 1. PRISMA diagram for overall search results and exclusion criteria......................... 5 List of tables Table 1. Critical concentrations and clinical breakpoints for medicines recommended for the treatment of rifampicin-resistant and multidrug-resistant TB. ................................... 2 Table 2. Overview of MIC data presentation. ...................................................................... 6 Table 3. Overview of current SLI CCs. ............................................................................... 8 Table 4. KAN MICs for pWT isolates on LJ. ....................................................................... 9 Table 5. KAN MICs for clinical rrs mutants on LJ. ............................................................... 9 Table 6. KAN MICs for pWT isolates on 7H10. ................................................................. 10 Table 7. KAN MICs for rrs allelic exchange mutants on 7H10. .......................................... 11 Table 8. KAN MICs for in vitro and clinical rrs mutants on 7H10. ...................................... 11 Table 9. KAN MICs for eis allelic exchange mutants on 7H10. ......................................... 12 Table 10. KAN MICs for in vitro and clinical eis mutants on 7H10. .................................... 13 Table 11. KAN MICs for whiB7 allelic exchange, in vitro and clinical mutants on 7H10. .... 13 Table 12. KAN MICs for in vitro and clinical tlyA mutants on 7H10. .................................. 13 Table 13. KAN MICs for pWT isolates on 7H11. ............................................................... 14 Table 14. KAN MICs for pWT isolates in MGIT.................................................................. 15 Table 15. KAN MICs for clinical rrs mutants in MGIT. ........................................................ 16 Table 16. KAN MICs for clinical eis mutants in MGIT. ....................................................... 16 Table 17. KAN MICs for clinical whiB7 mutant in MGIT. .................................................... 17 Table 18. AMK MICs for pWT isolates on LJ. ................................................................... 19 Table 19. AMK MICs for clinical rrs mutants on LJ. .......................................................... 19 Table 21. AMK MICs for rrs allelic exchange mutants on 7H10. ....................................... 20 Table 20. AMK MICs for pWT isolates on 7H10. .............................................................. 21 Table 22. AMK MICs for in vitro and clinical rrs mutants on 7H10. ................................... 22 Table 23. AMK MICs for eis allelic exchange mutants on 7H10. ....................................... 23 Table 24. AMK MICs for in vitro and clinical eis mutants on 7H10. ................................... 23 Table 25. AMK MICs for in vitro whiB7 mutant on 7H10. .................................................. 24 Table 26. AMK MICs for in vitro and clinical tlyA mutants on 7H10. .................................. 24 Table 27. AMK MICs for pWT isolates on 7H11. .............................................................. 24 Table 29. AMK MICs for clinical rrs mutants in MGIT. ....................................................... 25 Table 28. AMK MICs for pWT isolates in MGIT. ................................................................ 26 Table 30. AMK MICs for clinical eis mutants in MGIT. ....................................................... 27 Table 31. AMK MICs for clinical whiB7 mutant in MGIT. ................................................... 27 List of tables xi Table 32. AMK MICs for clinical tlyA mutants in MGIT. ...................................................... 27 Table 33. CAP MICs for pWT isolates on LJ. .................................................................... 30 Table 34. CAP MICs for clinical rrs mutants on LJ. ........................................................... 31 Table 36. CAP MICs for rrs allelic exchange mutants on 7H10. ........................................ 31 Table 35. CAP MICs for pWT isolates on 7H10. ............................................................... 32 Table 38. CAP MICs for in vitro and clinical eis mutants on 7H10. .................................... 33 Table 39. CAP MICs for in vitro whiB7 mutant on 7H10. .................................................. 33 Table 37. CAP MICs for in vitro and clinical rrs mutants on 7H10. .................................... 34 Table 40. CAP MICs for in vitro and clinical tlyA mutants on 7H10. .................................. 35 Table 41. CAP MICs for in vitro rrl mutant on 7H10. ......................................................... 35 Table 42. CAP MICs for pWT isolates on 7H11. ............................................................... 36 Table 44. CAP MICs for clinical rrs mutants in MGIT. ........................................................ 36 Table 43. CAP MICs for pWT isolates in MGIT. ................................................................. 37 Table 45. CAP MICs for clinical eis mutants in MGIT. ....................................................... 38 Table 46. CAP MICs for clinical whiB7 mutant in MGIT. .................................................... 38 Table 47. CAP MICs for clinical tlyA mutants in MGIT. ...................................................... 38 Table 48. CFZ MICs for pWT isolates on 7H10. ............................................................... 42 Table 49. CFZ MICs for pWT isolates on 7H11. ............................................................... 43 Table 50. CFZ MICs for pWT isolates in MGIT. ................................................................. 43 Table 51. CFZ MICs for in vitro and clinical atpE and mmpR mutants in MGIT. ................. 44 Table 52. BDQ MICs for pWT isolates on 7H10. .............................................................. 46 Table 53. BDQ MICs for pWT isolates on 7H11. .............................................................. 47 Table 54. BDQ MICs for in vitro and clinical atpE and mmpR mutants on 7H11. .............. 48 Table 55. BDQ MICs for pWT isolates in MGIT. ................................................................ 49 Table 56. BDQ MICs for in vitro and clinical atpE and mmpR mutants in MGIT. ................ 50 Table 57. Overview of current DCS and TRD CCs............................................................ 52 Table 58. DCS MICs for pWT and mutated isolates on LJ. ............................................... 52 Table 59. DCS MICs for pWT isolates on 7H10. ............................................................... 53 Table 60. DCS MICs for pWT isolates on 7H11. ............................................................... 53 Table 61. DCS MICs for pWT and mutated isolates in MGIT. ............................................ 54 Table 62. Overview of current LZD CCs. .......................................................................... 55 Table 63. LZD MICs for pWT isolates on 7H10. ............................................................... 56 Table 64. LZD MICs for pWT isolates on 7H11. ............................................................... 56 Table 65. LZD MICs for pWT isolates in MGIT. ................................................................. 57 Table 66. LZD MICs for in vitro mutants in MGIT. ............................................................. 58 Table 67. LZD MICs for mutated clinical isolates in MGIT. ................................................ 58 Table 68. DLM MICs for pWT isolates on 7H10. .............................................................. 61 Table 69. DLM MICs for pWT isolates on 7H11. .............................................................. 62 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS xii Table 70. DLM MICs for mutated clinical isolates on 7H11. .............................................. 63 Table 71. DLM MICs for pWT isolates in MGIT. ................................................................ 63 Table 72. DLM MICs for mutated clinical isolates in MGIT. ................................................ 64 Table 73. Overview of current FQ CCs and CBs. ............................................................. 65 Table 74. OFX MICs for pWT isolates on LJ. .................................................................... 66 Table 75. OFX MICs for clinical gyrA mutants on LJ. ........................................................ 66 Table 76. OFX MICs for clinical gyrB mutant on LJ. .......................................................... 67 Table 78. OFX MICs for gyrA allelic exchange mutants on 7H10. ..................................... 67 Table 77. OFX MICs for pWT isolates on 7H10. ............................................................... 68 Table 80. OFX MICs for gyrB allelic exchange mutants on 7H10. ..................................... 69 Table 79. OFX MICs for clinical gyrA mutants on 7H10. ................................................... 70 Table 81. OFX MICs for clinical gyrB mutants on 7H10. ................................................... 71 Table 82. OFX MICs for pWT isolates on 7H11. ............................................................... 71 Table 83. OFX MICs for mouse and clinical gyrA mutants on 7H11. ................................. 72 Table 84. OFX MICs for mouse gyrB mutants on 7H11. ................................................... 72 Table 85. OFX MICs for pWT isolates in MGIT. ................................................................. 73 Table 86. OFX MICs for clinical gyrA mutants in MGIT. ..................................................... 74 Table 87. OFX MICs for clinical gyrB mutants in MGIT. ..................................................... 74 Table 88. LFX MICs for pWT isolates on LJ. ..................................................................... 77 Table 89. LFX MICs for clinical gyrA mutants on LJ. ......................................................... 77 Table 90. LFX MICs for clinical gyrB mutants on LJ. ......................................................... 78 Table 91. LFX MICs for pWT isolates on 7H10. ................................................................ 78 Table 92. LFX MICs for gyrA allelic exchange mutants on 7H10. ...................................... 79 Table 93. LFX MICs for clinical gyrA mutants on 7H10. .................................................... 80 Table 94. LFX MICs for gyrB allelic exchange mutants on 7H10. ...................................... 81 Table 95. LFX MICs for clinical gyrB mutants on 7H10. .................................................... 82 Table 96. LFX MICs for pWT isolates on 7H11. ................................................................ 82 Table 97. LFX MICs for mouse and clinical gyrA mutants on 7H11. .................................. 83 Table 98. LFX MICs for mouse gyrB mutants on 7H11. ................................................... 83 Table 99. LFX MICs for pWT isolates in MGIT. .................................................................. 84 Table 100. LFX MICs for clinical gyrA mutants in MGIT. .................................................... 85 Table 101. LFX MICs for clinical gyrB mutants in MGIT. .................................................... 86 Table 102. GFX MICs for pWT isolates on LJ. .................................................................. 88 Table 103. GFX MICs for clinical gyrA mutants on LJ. ...................................................... 88 Table 104. GFX MICs for clinical gyrB mutants on LJ. ...................................................... 89 Table 105. GFX MICs for pWT isolates on 7H10. ............................................................. 89 Table 106. GFX MICs for pWT isolates on 7H11. ............................................................. 90 Table 107. GFX MICs for clinical gyrA mutants on 7H11. ................................................. 90 List of tables xiii Table 108. GFX MICs for pWT isolates in MGIT. ............................................................... 91 Table 109. GFX MICs for clinical gyrA mutants in MGIT. ................................................... 91 Table 110. GFX MICs for clinical gyrB mutants in MGIT. ................................................... 92 Table 111. MFX MIC distributions for the pWT population on LJ. ..................................... 93 Table 112. MFX MICs for clinical gyrA mutants on LJ. ...................................................... 93 Table 113. MFX MICs for clinical gyrB mutants on LJ. ...................................................... 94 Table 114. MFX MIC distributions for the pWT population on 7H10. ................................ 94 Table 115. MFX MICs for gyrA allelic exchange mutants on 7H10. ................................... 95 Table 116. MFX MICs for clinical gyrA mutants on 7H10. ................................................. 96 Table 117. MFX MICs for gyrB allelic exchange mutants on 7H10. ................................... 97 Table 118. MFX MICs for clinical gyrB mutants on 7H10. ................................................. 98 Table 119. MFX MICs for pWT isolates on 7H11. ............................................................. 99 Table 120. MFX MICs for mouse and clinical gyrA mutants on 7H11. ............................... 99 Table 121. MFX MICs for mouse gyrB mutants on 7H11. .............................................. 100 Table 122. MFX MICs for pWT isolates in MGIT. ............................................................. 101 Table 123. MFX MICs for clinical gyrA mutants in MGIT. ................................................. 102 Table 124. MFX MICs for clinical gyrB mutants in MGIT. ................................................. 103 TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS xiv Acknowledgements The development of this document was led by Christopher Gilpin and Alexei Korobitsyn with input from Karin Weyer (WHO Global TB Programme), on the basis of a systematic review of critical concentrations written by Claudio Köser (University of Cambridge, United Kingdom of Great Britain and Northern Ireland), Thomas Schön (Linköping University & Kalmar County Hospital, Sweden) and Sophia Georghiou (FIND, Switzerland) with additional input from Birte Vester (University of Southern Denmark, Denmark) and consensus agreed at a Technical Expert Group meeting convened by WHO on 24-26 April 2017, in Versoix, Switzerland. Members of the WHO Technical Expert Consultation Group Lynette Berkeley (US Food and Drug Administration, United States of America), Daniela Cirillo (San Raffaele Scientific Institute Milan, Italy), Chris Coulter (Queensland Mycobacterium Reference Laboratory, Australia), Armand Van Deun (Prince Leopold Institute of Tropical Medicine, Belgium), Francis Drobniewski (Imperial College London, the United Kingdom), Bernard Fourie (University of Pretoria, South Africa), Nazir Ismail (National Institute of Communicable Diseases, South Africa), Jacques Grosset (Johns Hopkins University, United States of America), Moses Joloba (National Reference Laboratory of the National TB and Leprosy Programme, Uganda), Leen Rigouts (Prince Leopold Institute of Tropical Medicine, Belgium), Max Salfinger (National Jewish Health, United States of America), Thomas Shinnick (Independent consultant, United States of America), Samuel Schumacher (FIND, Switzerland) and Sabira Tahseen (National TB Control Programme, Pakistan). Technical resource persons Claudio Köser, Sophia Georghiou and Timothy Rodwell (FIND, Switzerland). Additional literature reviewers Alexandra Aubry (Sorbonne Universités, France), Iñaki Comas (Institute of Biomedicine of Valencia, Spain), Hairong Huang (Beijing Tuberculosis and Thoracic Tumor Institute, China), Alexei Korobitsyn, Matthias Merker (Research Centre Borstel, Germany), Paolo Miotto (San Raffaele Scientific Institute, Italy), Vlad Nikolayevskyy (National Mycobacterium Reference Laboratory, the United Kingdom) and Nicolas Veziris (Sorbonne Universités, France). Major data contributors Koen Andries (Janssen Research and Development, Belgium), Alexandra Aubry, Ivan Bastian (SA Pathology, Australia), Erik Böttger (University of Zurich, Switzerland), Emmanuelle Cambau (National Reference Center for Mycobacteria and Antimycobacterial Resistance, France), Daniela Cirillo, Pauline Claxton (Scottish Mycobacteria Reference Laboratory, the United Kingdom), Edward Acknowledgement of financial support xv Desmond (California Department of Public Health, United States of America), Jurriaan de Steenwinkel (Erasmus University Medical Center, Netherlands), Keertan Dheda (University of Cape Town, South Africa), Andreas Diacon (MRC Centre for Tuberculosis Research, South Africa), David Dolinger (FIND, Switzerland), Anna Engström (Stockholm University, Sweden), Maha Farhat (Harvard Medical School, United States of America), Lanfranco Fattorini (Villa Marelli Institute, Italy), Scott Heysell (University of Virginia, United States of America), Doris Hillemann (National Reference Laboratory for Mycobacteria, Germany), Harald Hoffmann (WHO Supranational Reference Laboratory Munich- Gauting, Germany), Eric Houpt (University of Virginia, United States of America), Po-Ren Hsueh (National Taiwan University Hospital, Taiwan, China), Yi Hu (Fudan University, China), Nazir Ismail, Jasmine Jani (Becton Dickinson, India), Koné Kaniga (Janssen Research and Development, Belgium), Peter Keller (University of Zurich, Switzerland), Ian Laurenson (Scottish Mycobacteria Reference Laboratory, the United Kingdom), Jason Limberis (University of Cape Town, South Africa), Grace Lin (California Department of Public Health, United States of America), Yongge Liu (Otsuka Pharmaceutical Development & Commercialization, United States of America), Alexandre López- Gavín (Universitat de Barcelona, Spain), Harald Mauch (HELIOS Kliniken, Germany), Shaheed Omar (National Institute of Communicable Diseases, South Africa), Moisés Palaci (Universidade Federal do Espírito Santo, Brazil), Sharon Peacock (University of Cambridge, the United Kingdom), Therdsak Prammananan (National Center for Genetic Engineering and Biotechnology, Thailand), James Posey (Centers for Disease Control and Prevention, United States of America), Leen Rigouts, Jaime Robledo (Corporación Para Investigaciones Biológicas, Colombia), Camilla Rodrigues (P. D. Hinduja Hospital & Medical Research Centre, India), Thomas Schön, Meenu Sharma (National Reference Centre for Mycobacteriology, Canada), Thomas Shinnick, Frederick Sirgel (Stellenbosch University, South Africa), Sulochana Somasundaram (Sri Venkateswara College of Engineering, India), Timothy Sterling (Vanderbilt University School of Medicine, United States of America), Jakko van Ingen (Radboud University Medical Center, Netherlands), Dick van Soolingen (National Institute for Public Health and the Environment, Netherlands), Amour Venter (Stellenbosch University, South Africa), Nicolas Veziris, Cristina Villellas (Janssen Research and Development, Belgium), Rob Warren (Stellenbosch University, South Africa), Jim Werngren (Public Health Agency of Sweden, Sweden), Caie Yang (309th Hospital of the Chinese People’s Liberation Army, China), Wing Wai Yew (Chinese University of Hong Kong, Hong Kong Special Administrative Region, China) and Danila Zimenkov (Russian Academy of Sciences, Russian Federation). Acknowledgement of financial support FIND provided funding for performing the systematic review and preparing the draft report that was presented to the Technical Expert Group. Funding from the United States Agency for International Development through USAID-WHO Consolidated Grant No. GHA-G-00-09-00003 / US 2014-741 for convening the meeting is gratefully acknowledged. Claudio Köser is a research associate at Wolfson College and visiting scientist at the Department of Genetics (University of Cambridge). Declaration and management of conflict of interest All the contributors completed a WHO Declaration of Interest form. All stated declarations of interest were evaluated by members of the Steering Group for the existence of any possible financial conflict of interest which might warrant exclusion from membership of the Technical Expert Consultation Group or from the discussions as part of the consensus process. Intellectual conflict of interest was not considered for exclusion from membership of the Group, as broader expertise on DST TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS xvi methods for MTBC was considered as criteria for the selection. In addition, the diversity and representation in the Groups was large enough to balance and overcome any potential intellectual conflict of interest. During the consensus development process and the meeting, any emergence of intellectual conflict of interest was monitored by the Chair and there was no perceived intellectual conflict of interest identified during the meeting. The following interests were declared: None declared Christopher Coulter, Moses Joloba, Jacques Grosset, Lynette Berkeley, Sabira Tahseen, Armand Van Deun, Daniela Cirillo and Max Salfinger. Declared, insignificant Thomas Shinnick has declared that he was a former employee of the CDC until January 2016. As an employee, he had often represented CDC’s positions on laboratory services needed for tuberculosis diagnosis, treatment and control. Francis Drobniewski has declared that he was involved into the following activities: Grant to develop and implement a coordinated EU network of TB reference laboratories, ECDC, EUR 1.2 million; UK NIHR Health Technology Assessment and Innovate UK grants new TB diagnostic, systematic review and economic analysis modern TB diagnostics, £550,000; Unrestricted educational grant to develop and deliver training on clinical TB and MDR-TB management to medical doctors internationally, Otsuka, $75,000; EU FP7 grant PANNET; Research relating to MDR-TB diagnosis and management, EU FP7 grant, (approx. share of grant, Euro 1,000,000); Grant NIHR Imperial- PHE develop joint research between Imperial and PHE on DR, including TB, UK NIHR grants, £6,000; Grant UK Medical Research Council-evaluation of better physiological models for TB DR, UK MRC, £250,000; Activities of a consulting-training company providing training and supporting research in accord with WHO and international standards, total value £25,000. Bernard Fourie has declared that he holds the position of Non-Executive Director of the South African National Bioproducts Institute (core business fractionation, manufacturing and supply of blood/plasma-related products), $10,000 per annum. Nazir Ismail has declared that his unit received partial funding support for consumables for bedaquiline MIC surveillance and validation from J&J but no personal remuneration. Leen Rigouts has declared that she was involved Laboratory support to clinical trial C208-C209, phase II (J&J, no personal remuneration, funding for Mycobacteriology unit), supervising PhD research on clofazimine-bedaquiline cross resistance (with partial financing of J&J no personal remuneration, funding for Mycobacteriology unit). Abbreviations xvii Abbreviations 7H10 Middlebrook 7H10 medium 7H11 Middlebrook 7H11 medium AMK amikacin BDQ bedaquiline CAP capreomycin CB clinical breakpoint CC critical concentration CDC United States Centers for Disease Control and Prevention CFZ clofazimine CI exact binomial confidence interval CLSI Clinical & Laboratory Standards Institute DCS D-cycloserine DLM delamanid DMSO dimethyl sulfoxide DST drug-susceptibility testing ECOFF epidemiological cut-off value EUCAST European Committee on Antimicrobial Susceptibility Testing FDA United States Food & Drug Administration FIND Foundation for Innovative New Diagnostics FQ fluoroquinolone gDST genotypic drug-susceptibility testing GFX gatifloxacin gNWT genotypically non-wild type GTB Global TB Program gWT genotypically wild type KAN kanamycin LFX levofloxacin LPA line probe assay LJ Löwenstein-Jensen medium LZD linezolid LOF loss-of-function mutation (e.g. insertion, deletion or nonsense mutation) MDR multidrug-resistant TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS xviii MFX moxifloxacin MGIT BACTEC™ Mycobacterial Growth Indicator Tube™ 960 MIC minimum inhibitory concentration MTBC Mycobacterium tuberculosis complex OFX ofloxacin pDST phenotypic drug-susceptibility testing pNWT phenotypically non-wild type PMID PubMed ID pWT phenotypically wild type QC quality control QRDR quinolone resistance-determining region R resistance/resistant S susceptible/susceptibility SLI second-line injectable (drug) SOP standard operating procedure TB tuberculosis TEG technical expert group TRD terizidone v1 version 1 v2 version 2 WGS whole genome sequencing WHO World Health Organization XDR extensively drug-resistant Glossary of terms xix Glossary of terms Antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of an organism to an antimicrobial agent. For mycobacteria, two different categories, “critical concentration” and “minimum inhibitory concentration”, have been used to categorise the in vitro results. For strains of Mycobacterium tuberculosis complex, when tested against the lower concentration of some agents, the “critical concentration” category is applied. Testing of an additional higher concentration (a clinical breakpoint concentration) may also be recommended for some agents. However, there is no “intermediate” interpretive category, even when testing is performed both at the critical concentration and the clinical breakpoint concentration. Critical concentration of an anti-tuberculous agent has been adopted and modified from international convention. The critical concentration is defined as the lowest concentration of an anti-TB agent in vitro that will inhibit the growth of 99% of phenotypically wild type strains of M. tuberculosis complex. Clinical breakpoint – is the concentration or concentrations of an antimicrobial agent which defines an MIC above the critical concentration that separates strains that will likely respond to treatment from those which will likely not respond to treatment. This concentration is determined by correlation with available clinical outcome data, MIC distributions, genetic markers, and PK/PD data including drug dose. A dose increase can be used to overcome resistance observed at lower dosing, up until the maximum tolerated dose, and therefore a higher clinical breakpoint above which the particular drug is not recommended for use. The clinical breakpoint is used to guide individual clinical decisions in patient treatment. The clinical breakpoint is not applicable for drug resistance surveillance purposes. Critical proportion – is the proportion of resistant organisms within a particular cultured isolate that is used to determine resistance to a particular drug. A 1% critical proportion is used to differentiate susceptible and resistant strains. Any culture that shows less than 1% growth on a medium containing a critical concentration of the agent being tested when compared with the growth on a control without the agent is considered to be susceptible; a culture that has 1% or more growth on the medium containing the critical concentration of the agent is considered to be resistant, and the patient whose sample is being tested may not respond to the agent. The critical concentration and proportion criteria are used for testing most first-line and second-line anti-TB agents. Cross-resistance is resistance to multiple anti-tuberculosis agents caused by a single genetic change (or multiple changes, in case the given resistance mechanisms requires several genetic alterations), although in practice, such mutations may not be known. Epidemiological cut-off value (ECOFF), phenotypically wild type (pWT) and non-wild type (pNWT) strains • Typically, when MICs that are tested using a standardised method are aggregated for one species, a single Gaussian-shaped MIC distribution is formed, which corresponds to the pWT distribution for that species (i.e. the distribution for organisms that lack phenotypically detectable resistance mechanisms). Additional distributions with higher overall MICs are sometimes identified, even prior to the clinical use of the particular drug in question (or prior to the clinical use of another, related drug that shares the same resistance mechanism), that correspond to intrinsically or naturally resistant organisms. In this case, the distribution with the lowest MICs corresponds to the pWT distribution and the other distributions correspond to one or more pNWT distributions. • The ECOFF corresponds to the upper end of the pWT distribution (i.e. it typically encompasses 99% of pWT strains). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS xx • Excluding the scenario where it is difficult to distinguish pWT and pNWT strains because of methodological variation in MIC testing (i.e. where both distributions overlap), pWT strains are, by definition, genotypically WT (gWT). However, this does not mean that gWT strains are identical genotypically since they may harbour mutations in genes associated with resistance that do not change the MIC (e.g. the gyrA S95T mutation does not affect the MICs of fluoroquinolones). • Conversely, organisms with MICs above the ECOFF are by definition pNWT. Again, excluding the possibility of methodological testing variation close to the ECOFF, there should be a genetic basis for this phenotype (i.e. the strains should be genotypically NWT (gNWT)). Yet in practice, these gNWT strains may appear to be gWT if: – The gene conferring the phenotype was not interrogated. – The gene was interrogated, but the genetic change conferring the phenotype was not detected, as it occurred at a frequency below the level of detection of the molecular test (i.e. heteroresistance). – The genetic change was detected but could not be interpreted because of an incomplete understanding of the genotype-phenotype relationship. Indirect susceptibility test – a procedure based on inoculation of drug-containing media using organisms grown in culture. Minimum inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that prevents growth of more than 99% a microorganism in a solid medium or broth dilution susceptibility test. Potency – All antimicrobial agents are assayed for standard units of activity or potency. The assay units may differ widely from the actual weight of the powder and often may differ between drug production lots. Thus, a laboratory must standardise its antimicrobial solutions based on assays of the antimicrobial powder lots that are being used. The value for potency supplied by the manufacturer should include consideration for: • Purity measures (usually by high-performance liquid chromatography assay) • Water content (e.g. by Karl Fischer analysis or by weight loss on drying) • Salt/counter-ion fraction (if the compound is supplied as a salt instead of free acid or base) The potency may be expressed as a percentage, or in units of micrograms per milligrams (w/w). Proportion method: The proportion method was originally proposed by Canetti and colleagues, and modified later; it is the most common method used for testing the susceptibility of M. tuberculosis complex isolates. In this method, the inoculum used is monitored by testing two dilutions of a culture suspension, and the growth (that is, the number of colonies) on a control medium without an anti-TB agent is compared with the growth (the number of colonies) present on a medium containing the critical concentration of the anti-TB agent being tested; the ratio of the number of colonies on the medium containing the anti-TB agent to the number of colonies on the medium without the anti-TB agent is calculated, and the proportion is expressed as a percentage. A 1% critical proportion is used differentiate the proportion of resistant organisms within a particular strain that is used to determine resistance to a particular drug. Executive summary xxi Executive summary The End TB Strategy calls for early diagnosis and prompt treatment of all persons of all ages with any form of drug-susceptible or drug-resistant TB. The effective management of multi-drug and extensively-drug resistant TB (M/XDR-TB) relies upon the rapid diagnosis and treatment of resistant infections. Culture-based phenotypic drug susceptibility testing (DST) methods are currently the gold standard for drug resistance detection although these methods are time-consuming; require sophisticated laboratory infrastructure, qualified staff and strict quality assurance mechanisms. DST uses critical concentrations of anti-TB agents to determine the susceptibility or resistance of a culture of Mycobacterium tuberculosis complex. The critical concentration of an anti-TB agent has been adopted and modified from an international standard.1 The critical concentration is defined as the lowest concentration of an anti-TB agent in vitro that will inhibit the growth of 99% of phenotypically wild type strains of M. tuberculosis complex. Laboratory tests of the sensitivity of tubercle bacilli to anti-tuberculosis agents serve three main purposes. Firstly, they can be used as guidance in the choice of chemotherapy to be given to a patient. Secondly, they are of value in confirming that drug resistance has emerged when a patient failed to show a satisfactory response to treatment and thirdly can be used for the surveillance of emerging drug resistance. WHO Global TB Programme commissioned FIND to perform a systematic review of available minimum inhibitory concentration (MIC) data for phenotypically wild type as well as phenotypically non-wild type strains, including associated sequencing data for relevant resistance genes. The medicines included in the review were the second-line injectable agents (kanamycin, amikacin and capreomycin), clofazimine and bedaquiline, cycloserine and terizidone, linezolid, delamanid, and the fluoroquinolones (ofloxacin, levofloxacin, gatifloxacin and moxifloxacin). The following media were considered: Löwenstein Jensen, Middlebrook 7H10/7H11 and BACTEC™ Mycobacterial Growth Indicator Tube™ 960. In April 2017, WHO Global TB programme convened a Technical Expert Group to review the evidence for different critical concentrations and clinical breakpoints used for DST for the above- mentioned drug-media combinations. The revised or newly established breakpoints can be found in Table 1. A clinical breakpoint for the higher dose of moxifloxacin (800 mg/day) was established for the first time. Rifampicin and isoniazid critical concentrations were not evaluated as part of this review but should be re-evaluated as a priority. Finally, the Technical Expert Group highlighted the need for greater standardisation of DST protocols to minimise inter-laboratory differences. Supplementary data. The supplementary document and data files for this report can be downloaded at https://www.finddx.org/publication/supplement-critical-concentrations-for-dst-for- tb-drugs. 1 Canetti, G. et al. Mycobacteria: laboratory methods for testing drug sensitivity and resistance. Bull World Health Organ 29, 565-78 (1963). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 2 Table 1. Critical concentrations and clinical breakpoints for medicines recommended for the treatment of rifampicin-resistant and multidrug-resistant TB. Drug groups Drug LJ 7H10 7H11 MGIT (1) A. Fluoroquinolones (2) Levofloxacin (CC) (3) Moxifloxacin (CC) (3) Moxifloxacin (CB) (4) Gatifloxacin (CC) (3, 5) 2.0 1.0 – 0.5 1.0 0.5 2.0 – – 0.5 – – 1.0 0.25 1.0 0.25 B. Second-line injectable agents Amikacin Capreomycin Kanamycin (6) (Streptomycin) (7) 30.0 40.0 30.0 4.0 2.0 4.0 4.0 2.0 – – – 2.0 1.0 2.5 2.5 1.0 C. Other second-line agents Ethionamide (7) Prothionamide (7) Cycloserine / terizidone (8) Linezolid Clofazimine (9) 40.0 40.0 – – – 5.0 – – 1.0 – 10.0 – – 1.0 – 5.0 2.5 – 1.0 1.0 D. Add-on agents (not part of the core MDR-TB regimen) D1 Pyrazinamide (7) Ethambutol (7) – 2.0 – 5.0 – 7.5 100.0 5.0 D2 Bedaquiline (9) Delamanid (9) – – – – 0.25 0.016 1.0 0.06 D3 (10) p-aminosalicylic acid (7) Imipenem-cilastatin (7) Meropenem (7) Amoxicillin-clavulanate (7) (Thioacetazone) (7) – – – – – – – – – – – – – – – – – – – – All concentrations are in mg/L and apply to the proportion method with 1% as the critical proportion. Unless otherwise stated, they are critical concentrations (CCs), as opposed to clinical breakpoints (CBs). Changes to the previous version of the table are highlighted in red.(11) (1) MGIT is proposed as the reference method for performing DST for second-line anti-TB medicines. (2) Testing of ofloxacin is not recommended as it is no longer used to treat drug resistant-TB and laboratories should transition to testing the specific fluoroquinolones used in treatment regimens. During this transition, testing of ofloxacin at the CCs (i.e. 4.0 mg/L on LJ, 2.0 mg/L on 7H10, 2.0 mg/L on 7H11 and 2.0 mg/L in MGIT) may be performed instead of testing at the CCs for levofloxacin, moxifloxacin and gatifloxacin, but not for the CBs for moxifloxacin. (3) Levofloxacin, moxifloxacin and gatifloxacin interim CCs for LJ and gatifloxacin CC for MGIT established despite very limited data. (4) CBs for 7H10 and MGIT apply to high-dose moxifloxacin (i.e. 800 mg daily). (5) Gatifloxacin CC on 7H10 withdrawn due to limited evidence. (6) Kanamycin CC on 7H11 withdrawn due to limited evidence. (7) Drugs not reviewed as part of this report. (8) Cycloserine CC on LJ withdrawn due to limited evidence. (9) Interim CCs established. (10) Routine DST is not recommended for Group D3 anti-TB medicines as these agents are only to be used when a MDR-TB treatment regimen with five effective medicines cannot otherwise be composed. (11) World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). 1. Introduction 3 2 Global tuberculosis report 2017. Geneva: World Health Organization; 2017 (WHO/HTM/TB/2017.23; http://www.who.int/tb/publications/global_report/en/, accessed 1 November 2017). 3 The End TB Strategy: global strategy and targets for tuberculosis prevention, care and control after 2015. Geneva: World Health Organization; 2014 (http://www.who.int/tb/strategy/End_TB_Strategy.pdf, accessed 1 June 2017). 1. Introduction 1.0 Background Tuberculosis (TB) causes 10 million cases and 1.3 million deaths annually and it is estimated that 4 million cases go undiagnosed by public health services each year.2 Ending the global TB epidemic will be achievable over the next 20 years only if there is intensive action by all countries that have endorsed the End TB Strategy and its ambitious targets. It requires a paradigm shift from focused actions that gradually reduce the incidence of TB to enhanced, multisectoral actions that have been shown to drive down the epidemic at a rapid pace. MDR-TB and XDR-TB are major global public health problems and threaten progress made in TB care and prevention in recent decades. Drug resistance in Mycobacterium tuberculosis complex (MTBC) is caused by selection of naturally occurring genomic mutants. There are two ways that people get drug-resistant TB (DR-TB). Firstly, acquired DR-TB occurs when TB treatment is suboptimal due to inadequate policies and failures of health systems and care provision, poor quality of TB drugs, poor prescription practices, patient non-adherence, or a combination of the above. Secondly, primary DR-TB results from the direct transmission of DR-TB from one person to another. Globally, 4.1% of new and 19.0% of previously treated TB cases were estimated to have had MDR-TB or rifampicin-resistant TB in 2016.2 Each year MDR-TB or rifampicin-resistant TB leads to about 600,000 new cases and 240,000 deaths worldwide. Extensively drug-resistant TB (XDR- TB) has been reported by 123 countries. About 6.2% of patients with MDR-TB have XDR-TB globally. However, XDR-TB is more common among MDR-TB patients in some countries in Central Asia and Eastern Europe. The End TB Strategy calls for early diagnosis and prompt treatment of all persons of all ages with any form of drug-susceptible or drug- resistant TB. This requires ensuring access to WHO-recommended rapid diagnostics and universal access to DST for all patients with signs and symptoms of TB and no longer only prioritised for persons at greater risk of multidrug-resistant TB (MDR-TB) and-or HIV- associated TB. WHO defines universal access to DST as rapid DST for at least rifampicin, and further DST for at least fluoroquinolones and second-line injectable agents among all TB patients with rifampicin resistance.3 The effective management of M/XDR-TB relies upon the rapid diagnosis and treatment of resistant infections. Culture-based phenotypic drug susceptibility testing (DST) methods are currently the gold standard for drug resistance detection, but these methods are time-consuming, require sophisticated laboratory infrastructure, qualified staff and strict quality control. Traditionally, DST for MTBC has relied on the testing of a single, critical concentration (CC), which is used to differentiate resistant from susceptible strains of MTBC, and is specific for each anti-TB agent and test method. However, the definitions of CC for MTBC DST have evolved over time as have the definition of phenotypically wild type (pWT) vs. phenotypically non-wild type (pNWT) strains of MTBC. Laboratory tests of the sensitivity of tubercle bacilli to anti-tuberculosis agents serve three main purposes. Firstly, they can be used as guidance in the choice of chemotherapy to be given to a patient. Secondly, they are of value in confirming that drug resistance has emerged when a patient failed to show a satisfactory response to treatment and thirdly can be used for the surveillance of emerging drug resistance. 1.1 Scope of the Technical Expert Consultation Meeting The WHO GTB initiated and provided oversight to the process of evidence retrieval and analysis, was responsible for selection of members TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 4 for the TEG and External Review Group, for management of declarations of interest, organising the preparatory TEG meetings via webinar, and, finally, conducting the face-to- face TEG meeting. As a part of evidence retrieval and analysis, the WHO GTB commissioned the systematic review, which was performed by FIND in 2016- 17. The aim of the review was to collect the available data on MICs of pWT and pNWT isolates, including associated sequencing data for relevant gene regions, for the following anti- TB drugs: • SLIs (KAN, AMK and CAP) • CFZ and BDQ • DCS and TRD • LZD • DLM • FQs (OFX, LFX, GFX and MFX) The following media were considered: • LJ • 7H10 • 7H11 • MGIT A series of the webinars took place between November 2016 and April 2017 as part of the preparatory process for the TEG meeting. During these webinars, a consensus was achieved for revised definitions for performing DST. These revised definitions are included in the glossary of the current report. The objectives of the TEG were: • To revise and update the CCs and CBs for performing culture-based DST for SLIs, FQs, DCS and TRD. • To establish the CC for performing culture- based drug DST for the new anti-TB drugs (BDQ and DLM) and re-purposed agents (CFZ/TRD and LZD). The TEG meeting was convened by the Global TB Programme, WHO on 24-26 April 2017 in Versoix, Switzerland. During that meeting, the group assessed the MIC and sequencing data for each drug-medium combination, with a particular focus on potential sources of bias, as outlined in the supplement. Depending on the quality and quantity of the data, CCs and/ or CBs were either established, maintained or revised. Owing to the lack of data, the CCs were withdrawn in some cases. The decisions on the breakpoints for all anti-TB drugs in the review were based on consensus, which was defined as unanimous agreement among all TEG members. Consensus was achieved for all CCs and CBs both during the meeting and in consultations following the meeting. A CB for the higher dose of moxifloxacin (800 mg/day) was established for the first time. The outcome of the TEG was an updated table of the CCs and CBs for the anti-TB agents recommended for the treatment of the rifampicin- resistant and/or MDR forms of TB, formatted in accordance with the recent guidance for the management of M/XDR-TB (Table 1). Supplementary data. The supplementary document and data files for this report can be downloaded at https://www.finddx.org/ publication/supplement-critical-concentrations- for-dst-for-tb-drugs. 1.2 Systematic review 1.2.1 Search methodology A MEDLINE/PubMed search without date restrictions was conducted of all publications reporting quantitative DST results for the selected antibiotics. The search terms for each drug or group of drugs, which can be found in the supplement of this report, were intentionally broad since the titles or abstracts of papers do not necessarily mention MIC data. Moreover, MIC data were also solicited from the WHO Supranational Reference Laboratory Network and directly from key researchers, as identified through the literature search and laboratory network. Rifampicin and isoniazid were beyond the scope of the current review, but is envisaged to be assessed in phase 2 of the CC work and is planned for 2018. Studies in the following languages were reviewed independently by two people, with the exception of studies published in Chinese or Russian, where each study was screened by a single person: 1. Chinese: Hairong Huang 2. English: Sophia Georghiou and Claudio Köser 3. French (a partial review of the literature): 1. Introduction 5 Alexandra Aubry and Nicolas Veziris 4. German: Claudio Köser and Matthias Merker 5. Italian: Claudio Köser and Paolo Miotto 6. Russian: Alexei Korobitsyn or Vlad Nikolayevskyy 7. Spanish: Iñaki Comas and Sophia Georghiou 1.2.2 Inclusion criteria Studies identified as containing any MIC data through the full-text screening were further reviewed in detail by Sophia Georghiou or Claudio Köser. Studies that met the following criteria were included in the review: 1. The MICs for at least one of the anti-TB compounds of interest (with at least three concentrations tested per drug) were determined using the proportion method with a critical proportion of 1%, using LJ, 7H10, 7H11 or MGIT. 2. The drug concentrations tested were clearly defined (i.e. to assess potential truncations of the MIC results). 3. The number of isolates tested at each concentration was given (i.e. to evaluate the shape of the MIC distributions and determine the mode of the distributions). 4. The MIC data were available for at least 10 isolates per drug. For studies that reported only MIC ranges (i.e. did not meet the third criterion), raw study data were solicited directly from the corresponding authors and/or their co-authors. These studies were excluded if detailed MIC data could not be obtained. In exceptional circumstances, studies that did not meet all of these criteria were still included if they presented data that were particularly valuable, such as studies with sequencing data for new anti-TB drugs. 1.2.3 Studies identified through the systematic review A total of 6,096 unique records were identified for possible inclusion, along with further 47 additional datasets from other sources. As shown in Figure 1, 122 of these studies were included in the review, which were stratified further by medium (NB: the sum of the studies for individual media does not correspond to 122 as some studies featured MICs for multiple media). The exclusion criteria in this diagram were not stratified in detail as a study may have been excluded for one drug but contained relevant data for another. This information can be found in the supplement, which contains PRISMA diagrams for each drug or group of drugs. Figure 1. PRISMA diagram for overall search results and exclusion criteria TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 6 1.3 Data presentation 1.3.1 Format of this report Each chapter in the report covers a single drug or group of drugs that share at least one resistance mechanism (e.g. the SLIs). The results in each chapter are grouped by different media (LJ, 7H10, 7H11 and MGIT). For each medium, data are organised into three sections: (1) MICs for pWT isolates, including laboratory control strains (e.g. H37Rv), (2) MICs for isolates with mutations in relevant resistance genes (i.e. MICs from in vitro, animal or clinical isolates as well as allelic exchange experiments, where available), (3) conclusion for CC and CB for each combination of drug and medium, including the rationale for revising and establishing breakpoints. 1.3.2 Format of MIC tables This report contains abridged versions of the full Excel MIC data files, which are included in the supplement (an explanation of how to relate each table from this report with the raw data files can also be found in the supplement). Details for the information provided in each column of these files can be found below. However, only essential columns were included in this report. For example, the column with the ‘total [number of] MICs’ performed was included only if these numbers differed from the numbers of unique isolates tested (i.e. when isolates were tested repeatedly, as was the often the case for H37Rv). The following points are relevant for the interpretation of the data: • If a cell is empty, no information regarding the particular category were available (i.e. in the case of the ‘genotypic results’ column, blank cells are not equivalent to gWT (where sequencing or another genotypic method was carried out but no relevant genetic changes were found)). • MICs from different studies cannot be compared unless the concentrations and ranges of concentrations tested are considered. Shaded cells therefore designate the concentrations tested for each group of isolates (NB: some studies tested a wide range of concentrations. The columns that correspond to the concentrations at the lower and upper end of these ranges were included but hidden in the Excel datasheets for simplicity. These columns can be displayed by highlighting the columns on either side of these hidden columns and using the ‘unhide’ command after a right-click). Table 2 provides an overview of how MIC data are displayed. Table 2. Overview of MIC data presentation. Shaded cells correspond to the concentrations tested in a particular study (e.g. concentrations of 1, 2 and 4 mg/L were tested for study A, whereas 1 was not tested in study B, which means that MICs of 2 mg/L in both studies are not equivalent). Truncated MIC values were highlighted in red. If red was used in a shaded cell, the MIC was either ≤ or ≥ the concentration in question. For example, the lowest MIC value for study B was ≤0.5 mg/L, whereas the highest MICs were ≥8 mg/L. If red was used in an unshaded cell, the MIC was > the last concentration tested (for study A, the highest MICs were >4 mg/L, as opposed to 6 mg/L). The mode of the putative pWT MIC distribution was indicated by highlighting the corresponding number of MICs in bolded text (e.g. 2 mg/L for study A). In the case of study B, the truncation of the MIC values meant that a mode could not be identified (e.g. it was possible that the MICs of all 20 isolates with MICs ≤0.5 mg/L were actually 0.5 mg/L, in which case 0.5 mg/L would be the mode of the MIC distribution). The following information are provided in each data column. ‘Studies’ column: • The names of the studies with notable limitations were highlighted in red (e.g. if the same laboratory participated in multiple studies that used the same medium or a method other than sequencing was used for genotypic DST). The corresponding limitations were detailed below the tables in the footnotes in this report and in the ‘comment’ column in red in the supplementary MIC file. 1. Introduction 7 ‘Lab’ column: • The laboratories that participated in multiple studies using the same medium were highlighted in red. ‘Unique isolates’ & ‘total MICs’ columns: • Red entries correspond to isolates that were tested multiple times. ‘Genotypic results’ column: • In light of the recent endorsement of the Hain GenoType MTBDRsl v2 assay by WHO, the mutations that should be detected by this assay were noted.4 Mutations in bold represent mutations targeted by specific mutant probes (i.e. they are specifically identified), whereas underlined mutations can only be inferred by the absence of binding of a wild type probe. For this purpose, it was assumed that any mutation in an area covered by a wild type probe would prevent binding of the probe, even if the mutation in question was not explicitly listed on the package insert of the assay. It should also be noted that the actual performance of this assay may differ from these simulations (e.g. the mutant probes may not always identify their respective mutations and the frequency of the mutation in the sample in question may affect the results). ‘Comment’ column: • Additional remarks regarding the study in question were included in this column. Important limitations were highlighted in red. 4 Hain Lifescience. GenoType MTBDRsl VER 2.0. Instructions for use 06/2015. IFU-317A-02. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 8 2. Second-line injectable agents 2.0 SLI MIC data stratification and current breakpoints KAN, AMK and CAP MIC data were stratified by mutations in rrs (MTB000019), eis (Rv2416c), tlyA (Rv1694), whiB7 (Rv3197A) and rrl (MTB000020) (details regarding these resistance mechanisms can be found in the supplement). The M. tuberculosis numbering system was used for the nucleotide changes in rrs, which were numbered relative to the start of the gene, and the data were stratified by the mutations A1401G, C1402T and G1484T. Only mutations that were within 50 nucleotides upstream of the eis start codon were reported. The inter-genic whiB7 mutations were shown relative to the transcription start site of this gene (see supplement for more details). No assumptions were made about cross-resistance between KAN, AMK and CAP for this report, and therefore mutations in all resistance genes were analysed in relation to MIC data to all three drugs. Isolates with mutations in more than one resistance gene were excluded from the discussion, although these data are available in the supplementary MIC files. Version 2 of the Hain GenoType MTBDRsl assay interrogates mutations in rrs and the eis promotor region, whereas version 1 only covers rrs (the mutations that are specifically targeted by mutation probes by this assay are heighted in bold, whereas mutations that merely inferred by lack of binding of a wild type probe are underlined both in the supplementary MIC files and in the tables of this report).5 Table 3 provides an overview of the current WHO and CLSI CCs for KAN, AMK and CAP.6, 7 5 Tagliani, E. et al. Diagnostic performance of the new version of GenoType MTBDRsl (V2.0) assay for detection of resistance to fluoroquinolones and second line injectable drugs: a multicenter study. J Clin Microbiol 53, 2961-9 (2015). 6 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). 7 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). http://apps.who.int/iris/bitstream/10665/130918/1/9789241548809_eng. pdf?ua=1&ua=1 (accessed 13.8.2015). Table 3. Overview of current SLI CCs. LJ 7H10 7H11 MGIT Drug WHO CLSI WHO CLSI WHO CLSI WHO CLSI KAN 30.0 – 5.0 6.0 2.5 AMK 30.0 – 4.0 – – 1.0 CAP 40.0 – 4.0 10.0 – 10.0 2.5 Green CCs were set by both the WHO and CLSI; red CCs were set by WHO; blue CCs were set by CLSI. All concentrations are in mg/L. 2.A.1 KAN MIC data on LJ 2.A.1.1 KAN MICs for pWT isolates on LJ Three studies from the same laboratory were identified that reported KAN MIC data for the pWT population on LJ (Table 4). Most MICs were truncated, which meant that little insight about the shape of the pWT MIC distribution could be gained. 2. Second-line injectable agents 9 Table 4. KAN MICs for pWT isolates on LJ. The red line denotes the current WHO CC for KAN DST on LJ (30 mg/L). Notable limitation: All studies were performed in the same laboratory. 2.A.1.2 KAN MICs for mutated isolates on LJ rrs mutants Clinical isolates Three studies from the same laboratory were identified that reported KAN MIC data for rrs mutants on LJ (Table 5). All 74 rrs A1401G mutants (100% (95% CI, 95-100%)) tested resistant at the current CC, whereas the four rrs C1402T mutants were susceptible (100% (95% CI, 40-100%)), which was not the case on 7H10 (Section 2.A.2.2). Table 5. KAN MICs for clinical rrs mutants on LJ. The red line denotes the current WHO CC for KAN DST on LJ (30 mg/L). Notable limitation: All studies were performed in the same laboratory. 2.A.1.3 Conclusion for KAN CC for LJ The identified KAN MIC data on LJ were limited and provided little insight into the shape of the pWT distribution, which precluded a re- assessment of the CC. Nevertheless, the current CC of 30 mg/L was maintained given that the LJ proportion method is used widely and provides the only diagnostic option in some settings. 2.A.2 KAN MIC data on 7H10 2.A.2.1 KAN MICs for pWT isolates on 7H10 13 studies were identified that reported KAN MIC data for the pWT population on 7H10 (Table 6). Most distributions were not truncated and therefore provided a good understanding of the shape of the pWT MIC distributions. Some variation in testing was apparent, as some datasets had modes at 1 mg/L (e.g. Krüüner et al. & Engström et al. (Study 6)), for which a CC of 2 mg/L would be optimal. Others had pWT distributions that were slightly elevated (i.e. with a mode at 2 mg/L) and consequently supported a CC of 4 mg/L. In contrast to all other studies, the mode of Sowajassatakul et al. (Study 14) at 4 mg/L was high, with no MIC variation observed for the 28 pWT isolates that were tested in triplicate. Consequently, this study was excluded from all further analyses. Overall, these pWT MIC data supported lowering the current CC from 5 to 4 mg/L. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 10 Ta bl e 6. K AN M IC s f or p W T iso la te s o n 7H 10 . Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r K AN D ST o n 7H 10 (5 m g/ L) . N ot ab le lim ita tio ns : S tu di es 1 0- 12 a nd 1 6 w er e pe rfo rm ed in th e sa m e lab or at or y. St ud y 14 s ho w ed a h ig h m od e w ith ou t a ny v ar iab ilit y in th e M IC d ist rib ut io n. 2. Second-line injectable agents 11 2.A.2.2 KAN MICs for mutated isolates on 7H10. rrs mutants Allelic exchange results Reeves et al. (Study 10) conducted allelic exchange experiments in H37Rv, CDC1551 and Beijing strain backgrounds (Table 7). Compared to parental MICs of 2 mg/L, rrs A1401G conferred the largest MIC increase (>160 mg/L), followed by rrs G1484T (80-160 mg/L) and by rrs C1402T (10-15 mg/L). Table 7. KAN MICs for rrs allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CCs for KAN DST on 7H10 (5 mg/L). In vitro and clinical isolates All 199 in vitro or clinical rrs mutants tested resistant (100% (95% CI, 98-100%)), including the 11 rrs C1402T mutants (100% (95% CI, 72-100%)), which had the lowest KAN MICs (8-20 mg/L) (Table 8). Table 8. KAN MICs for in vitro and clinical rrs mutants on 7H10. The green line denotes the current WHO and CLSI CCs for KAN DST on 7H10 (5 mg/L). Notable limitation: Studies 16 and 17 were conducted in the same laboratory. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 12 eis mutants Allelic exchange results Zaunbrecher et al. (Study 11) were the first to study eis mutations (Table 9). The introduction of the eis C-14T promoter mutation into an H37Rv background increased the KAN MIC from 2 mg/L to 20-25 mg/L. Conversely, the restoration of the eis wild type sequence in an eis C-14T in vitro mutant lowered the MIC from 25 back to 2 mg/L. Zaunbrecher et al. further investigated this KAN resistance mechanism by complementing an unmarked eis deletion mutant of H37Rv with five different promoter mutations. For eis G-10A, C-12T, C-14T, and G-37T this resulted in KAN MICs of 10-20 mg/L. The MIC of the A-13G mutant was 5 mg/L, which is equal to the current WHO CC for this medium, but above the lowered CC of 4 mg/L. Pholwat et al. (Study 13) also conducted allelic exchange experiments in H37Rv, which confirmed that the aforementioned eis G-10A, C-12T, and C-14T mutations resulted in a KAN MIC increase above the current CC of 5 mg/L on 7H10 (i.e. from 2.5 mg/L to 10-40 mg/L). Notably, the C-14T mutation resulted in the largest KAN MIC increase (i.e. to 40 mg/L), as seen by Zaunbrecher et al. In addition, Pholwat et al. investigated two eis promoter mutations that were not evaluated by Zaunbrecher et al. Of these, the eis C-14G mutation did not change the KAN MIC compared to the wild type parent (2.5 mg/L), whereas the MIC of the eis C-15G mutation increased by one doubling dilution, which was within the normal variation of MIC testing. Pholwat et al. therefore concluded that these two mutations did not confer KAN resistance. Table 9. KAN MICs for eis allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for KAN DST on 7H10 (5 mg/L). In vitro and clinical isolates Four studies reported MIC data for 83 in vitro or clinical eis mutants without known mutations in other resistance genes (Table 10). 36 of these isolates (43% (95%CI, 33-55%)) were susceptible at the current CC. This was mainly due to eis G-10A mutants from Krüüner et al. & Engström et al. (Study 6). Notably, most of these isolates would have tested resistant at 2 mg/L, the optimal CC for this particular dataset (Section 2.A.2.1). However, even at 2 mg/L some eis G-10A mutants would have been misclassified as susceptible given that the lower end of the MIC distribution of this mutation was also 2 mg/L. By contrast, the variation in testing between datasets had less of an impact on the eis C-14T mutation (i.e. only 1 of the 25 mutants (4% (95% CI, 0-20%)) tested susceptible), as this mutation conferred larger MIC increases, which was in agreement with the allelic exchange data. 2. Second-line injectable agents 13 Table 10. KAN MICs for in vitro and clinical eis mutants on 7H10. The green line denotes the current WHO and CLSI CCs for KAN DST on 7H10 (5 mg/L). whiB7 mutants Allelic exchange results Reeves et al. (Study 12) conducted allelic exchange experiments to restore the wild type whiB7 sequence in three in vitro mutants that had KAN MICs of 10-20 mg/L (Table 11). This resulted in KAN MICs in the susceptible range (2-4 mg/L). Table 11. KAN MICs for whiB7 allelic exchange, in vitro and clinical mutants on 7H10. The green line denotes the current WHO and CLSI CCs for KAN DST on 7H10 (5 mg/L). In vitro and clinical isolates 10 additional whiB7 in vitro mutants obtained during the selection experiments had MICs of 10-20 mg/L, compared with 2 mg/L for the two parental strains (Table 11). The sole clinical mutant had an MIC of 20 mg/L. tlyA mutants In vitro and clinical isolates 38 in vitro and clinical isolates with tlyA mutations were reported by three studies from two laboratories (Table 12). Of those, 34 (89% (95% CI, 75-97%)) were susceptible at the current CC for KAN. Table 12. KAN MICs for in vitro and clinical tlyA mutants on 7H10. The green line denotes the current WHO and CLSI CC for KAN DST on 7H10 (5 mg/L). Notable limitation: Studies 16 and 17 were conducted in the same laboratory. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 14 2.A.2.3 Conclusion for KAN CC for 7H10 Few studies included 5 mg/L, the current KAN CC for 7H10, in their dilution series. Instead, 4 mg/L was adopted as the new CC to maximise the detection of eis mutants as well as yet unknown resistance mechanisms. This change should not affect the detection of rrs mutants, which reliably tested resistant even at 5 mg/L. Lowering the CC should also not result in the misclassification of tlyA mutants as resistant. Because of the variation between laboratories and/or different datasets and the fact that the MIC distributions for pWT isolates and some eis mutants overlapped (even when tested in the same laboratory), these eis mutants cannot be detected reliably using pDST. The implications of this finding for the interpretation of gDST results will be addressed in a later report. Based on the limited MIC data for whiB7 mutants and the fact that these mutants confer resistance through the over-expression of eis, it is possible that the ability of pDST to detect this class of mutants is also compromised. 2.A.3 KAN MIC data on 7H11 2.A.3.1 KAN MICs for pWT isolates on 7H11 Only one study was identified with KAN MIC data for 7H11 (Table 13). Table 13. KAN MICs for pWT isolates on 7H11. The green line denotes the current WHO and CLSI CC for KAN DST on 7H11 (6 mg/L) 2.A.3.2 KAN MICs for mutated isolates on 7H11 No studies presenting MIC data for mutants were identified. 2.A.3.3 Conclusion for KAN CC for 7H11 Given that KAN MIC data were only identified for a single study (which precluded an analysis of the inter-laboratory reproducibility) and that CCs for KAN for other media are supported by more evidence, the current CC of 6 mg/L was withdrawn. 2.A.4 KAN MIC data in MGIT 2.A.4.1 KAN MICs for pWT isolates in MGIT 10 studies were identified that reported KAN MIC data for the pWT population in MGIT (Table 14). Most studies used non-standard MIC dilution series as the current CC is 2.5 mg/L. Several studies were severely truncated at the lower end, including Rodrigues et al. (Study 26), which has been cited by the current CLSI document to support the current CC of 2.5 mg/L.8 Nevertheless, variation in testing was identifiable given that some studies had modes at 0.62 mg/L (e.g. Study 21) whereas others had modes that were one dilution higher, at 1.25 mg/L (e.g. Study 19). The current CC appeared to balance this variation appropriately, although more genotypic data for isolates with MICs just above this concentration is desirable to investigate the true upper end of the pWT distribution (e.g. in Study 22). 8 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). 2. Second-line injectable agents 15 Ta bl e 14 . K AN M IC s f or p W T iso la te s i n M GI T. Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r K AN D ST in M G IT (2 .5 m g/ L) . N ot ab le lim ita tio ns : S tu di es 1 9 an d 20 , a nd S tu di es 2 4 an d 26 w er e pe rfo rm ed in th e sa m e lab or at or y, re sp ec tiv ely . T he g en ot yp ic re su lts fo r S tu dy 2 3 w er e ba se d on a c om bi na tio n of s eq ue nc ing a nd a m icr oa rra y, w he re as S tu dy 2 4 re lie d on a c om bi na tio n of s eq ue nc ing a nd th e M TB DR sl v1 . TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 16 whiB7 mutant Clinical isolate One clinical mutant that tested KAN susceptible in MGIT (with an MIC equal to the CC of 2.5 mg/L) harboured a whiB7 A+238G mutation (Table 17). Reeves et al. (Study 12) had previously demonstrated that the removal of this mutation in an in vitro mutant restored KAN susceptibility on 7H10 (Section 2.A.2.2). Consequently, the MIC of this mutant might be close to the CC and might become resistant upon retesting as a result of the normal variation in pDST. Table 16. KAN MICs for clinical eis mutants in MGIT. The green line denotes the current WHO and CLSI CCs for KAN DST in MGIT (2.5 mg/L). Notable limitations: Studies 19 and 20 were performed in the same laboratory. The genotypic results for Study 23 were based on a combination of sequencing and a microarray, whereas Study 24 relied on a combination of sequencing and the MTBDRsl v1. 2.A.4.2 KAN MICs for mutated isolates in MGIT rrs mutants Clinical isolates Five studies conducted in four laboratories interrogated 105 clinical isolates harbouring the rrs A1401G mutation (Table 15). 100% (95% 97-100%) tested resistant at the current CC with MICs of >20 mg/L. Table 15. KAN MICs for clinical rrs mutants in MGIT. The green line denotes the current WHO and CLSI CCs for KAN DST in MGIT (2.5 mg/L). Notable limitations: Studies 19 and 20 were performed in the same laboratory. The genotypic results for Study 23 were based on a combination of sequencing and a microarray, whereas Study 24 relied on a combination of sequencing and the MTBDRsl v1. eis mutants Clinical isolates Four studies from three laboratories reported MIC data for 42 eis mutants that would be interpreted as resistant by MTBDRsl v2 assay (Table 16). Of these, seven (17% (95% CI, 7-31%)) were susceptible at the current CC. Notably, two of the ‘susceptible’ isolates (one eis G-10A and C-10C mutant) from Kambli et al. (Study 24) were tested in a laboratory with a relatively low pWT MIC distribution (Section 2.A.4.1) and still had clearly elevated MICs. 2. Second-line injectable agents 17 Table 17. KAN MICs for clinical whiB7 mutant in MGIT. The green line denotes the current WHO and CLSI CCs for KAN DST in MGIT (2.5 mg/L). 2.A.4.3 Conclusion for KAN CC in MGIT The current CC of 2.5 mg/L is likely appropriate to balance the variation in MIC testing and was consequently reaffirmed. As was the case for 7H10, the detection of eis mutants, but not rrs mutants, was likely affected by variation in MIC testing. 2.A.5 References for KAN MIC studies 1. Jugheli, L. et al. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob Agents Chemother 53, 5064-8 (2009). 2. Barletta, F., Zamudio, C., Rigouts, L. & Seas, C. Resistance to second-line anti-tuberculosis drugs among Peruvian multidrug resistant Mycobacterium tuberculosis strains. Rev Peru Med Exp Salud Publica 31, 676-82 (2014). 3. Vincent, V. et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int J Tuberc Lung Dis 16, 24-31 (2012). 4. Juréen, P. et al. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections. J Clin Microbiol 48, 1853-8 (2010). 5. Böttger, unpublished data. 6. (a) Krüüner, A., Juréen, P., Levina, K., Ghebremichael, S. & Hoffner, S. Discordant resistance to kanamycin and amikacin in drug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 47, 2971-3 (2003). (b) Engström, A., Perskvist, N., Werngren, J., Hoffner, S.E. & Juréen, P. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 66, 1247-54 (2011). 7. Udou, T. Comparative evaluation of the in vitro antimycobacterial activities of six aminoglycoside antibiotics using an agar dilution method. J Chemother 18, 610-6 (2006). 8. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 9. Hu, Y. et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in rural China. Antimicrob Agents Chemother 57, 3857-63 (2013). 10. Reeves, A.Z., Campbell, P.J., Willby, M.J. & Posey, J.E. Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 59, 444-9 (2015). 11. Zaunbrecher, M.A., Sikes, R.D., Jr., Metchock, B., Shinnick, T.M. & Posey, J.E. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 18 kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 106, 20004-9 (2009). 12. Reeves, A.Z. et al. Aminoglycoside cross- resistance in Mycobacterium tuberculosis due to mutations in the 5’ untranslated region of whiB7. Antimicrob Agents Chemother 57, 1857-1865 (2013). 13. Pholwat, S. et al. eis promoter C14G and C15G mutations do not confer kanamycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 60, 7522-7523 (2016). 14. Sowajassatakul, A., Prammananan, T., Chaiprasert, A. & Phunpruch, S. Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand. BMC Microbiol 14, 165 (2014). 15. Via, L. et al. Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean patients with drug-resistant tuberculosis. J Clin Microbiol 48, 402-11 (2010). 16. Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 3192-7 (2005). 17. Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 571-7 (2005). 18. Fattorini, L. et al. Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis. Microb Drug Resist 5, 265-70 (1999). 19. Tessema, B. et al. FIND tuberculosis strain bank: a resource for researchers and developers working on tests to detect Mycobacterium tuberculosis and related drug resistance. J Clin Microbiol 55, 1066-1073 (2017). 20. Heyckendorf, J. et al. What is resistance? Impact of phenotypic versus molecular drug resistance testing on multi- and extensively drug-resistant tuberculosis therapy. Antimicrob Agents Chemother, ahead of print (2017). 21. Stepanshina, V.N. et al. Determination of critical kanamycin concentration for Mycobacterium tuberculosis in the MGIT Bactec 960 system. Tuberk Biolezni Legkih, 71-8 (2009). 22. Zheng, X. et al. Determination of MIC breakpoints for second-line drugs associated with clinical outcomes in multidrug-resistant tuberculosis treatment in China. Antimicrob Agents Chemother 60, 4786-92 (2016). 23. (a) Gikalo, M.B., Nosova, E.Y., Krylova, L.Y. & Moroz, A.M. The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 67, 2107-9 (2012). (b) Zimenkov, D.V. et al. Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infect Dis 13, 240 (2013). 24. (a) Kambli, P. et al. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol 5, 1-6 (2016). (b) Kambli, P. et al. Corrigendum to “Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second- line injectables” [Int. J. Mycobacteriol. 5(1) 2016 1-6]. Int J Mycobacteriol 5, 370-372 (2016). 5. Bastian, I., Rigouts, L., Palomino, J.C. & Portaels, F. Kanamycin susceptibility testing of Mycobacterium tuberculosis using Mycobacterium Growth Indicator Tube and a colorimetric method. Antimicrob Agents Chemother 45, 1934-6 (2001). 26. Rodrigues, C. et al. Drug susceptibility testing of Mycobacterium tuberculosis against second- line drugs using the Bactec MGIT 960 System. Int J Tuberc Lung Dis 12, 1449-55 (2008). 27. Lin, unpublished data. 28. Sharma, M. et al. Canadian multicenter laboratory study for standardized second- line antimicrobial susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 49, 4112-6 (2011). 2. Second-line injectable agents 19 2.B.1 AMK MIC data on LJ 2.B.1.1 AMK MICs for pWT isolates on LJ Four studies from two laboratories were identified that reported AMK MIC data for the pWT population on LJ (Table 18). These data provided little insight into the shape of the pWT distribution due to truncations at the lower end of the distributions. Table 18. AMK MICs for pWT isolates on LJ. The red line denotes the current WHO CC for AMK DST on LJ (30 mg/L). Notable limitation: Studies 2, 3 and 4 were performed in the same laboratory. 2.B.1.2 AMK MICs for mutated isolates on LJ rrs mutants Clinical isolates Based on data from a single laboratory (Table 19), most rrs A1401G mutants (99% (95% CI, 93-100%)) were resistant at the current CC, whereas all four rrs C1402T mutants were susceptible (100% (95% CI, 40-100%)). Table 19. AMK MICs for clinical rrs mutants on LJ. The red line denotes the current WHO CC for AMK DST on LJ (30 mg/L). Notable limitation: All studies were performed in the same laboratory. 2.B.1.3 Conclusion for AMK CC on LJ The identified AMK MIC data for LJ were limited and provided little insight into the shape of the pWT distribution, which precluded a re-assessment of the CC of 30 mg/L. Nevertheless, this CC was maintained as LJ represents the only medium available for pDST in many settings. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 20 2.B.2 AMK MIC data on 7H10 2.B.2.1 AMK MICs for pWT isolates on 7H10 14 studies were identified that reported AMK MIC data for the pWT population on 7H10 (Table 20). Where the MIC distributions were not truncated, the modes varied between 0.5 to 2 mg/L. The distribution of Sowajassatakul et al. (Study 16) was particularly high, as also seen for KAN on 7H10 (Section 2.A.2.1). This study was therefore excluded from additional analyses. The remaining studies, apart from van Ingen et al. (Study 14), suggested that the CC should be lowered from 4 to 2 mg/L. 2.B.2.2 AMK MICs for mutated isolates on 7H10 rrs mutants Allelic exchange results Reeves et al. (Study 11) conducted allelic exchange experiments in H37Rv, CDC1551 and Beijing strain backgrounds (Table 21). The MICs of the three parent strains were 0.5-1 mg/L, compared to 2-4 mg/L for rrs C1402T, 100 mg/L for rrs G1484T and >128 mg/L for rrs A1401G. Lowering the CC from 4 to 2 mg/L meant that the interpretation of rrs C1402T, which is currently not regarded as a resistance mutation for AMK, should be reviewed. Table 21. AMK MICs for rrs allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for AMK DST on 7H10 (4 mg/L). In vitro and clinical isolates Seven studies from six laboratories presented MIC data for in vitro or clinical rrs mutants (Table 22). All 193 rrs A1401G or G1484T mutants (100% (95% CI, 98-100%)) were resistant at the current CC. The three studies that tested wide concentration ranges for the rrs C1402T mutations showed more varied results. The two mutants from Hu et al. (Study 13) and three mutants from van Ingen et al. (Study 14) had significantly higher MICs (32 mg/L and >20 mg/L, respectively) than the five MICs from Krüüner et al. & Engström et al. (Study 7), which ranged from 1-4 mg/L (with a mode at 2 mg/L). It is therefore possible that an unidentified mutation was responsible for the high MIC in Study 13. Similarly, it is possible that the isolates from van Ingen et al. actually harboured rrs A1401G mutations, given that the rrs C1402T was only inferred using the Hain Genotype MTBDRsl v1 assay (the authors noted that the hybridisation of the A1401G probe was generally weak in their study). By contrast, the MICs from Study 7 confirmed the allelic exchange results for rrs C1402T given that the corresponding distribution (1-4 mg/L, with a mode at 2 mg/L) was clearly elevated compared to the pWT MIC distribution in this dataset, which ranged from 0.12-1 mg/L (Section 2.B.2.1). 2. Second-line injectable agents 21 Ta bl e 20 . A M K M IC s f or p W T iso la te s o n 7H 10 . Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r A M K DS T on 7 H1 0 (4 m g/ L) . N ot ab le lim ita tio ns : S tu di es 1 1, 1 2 an d 18 w er e pe rfo rm ed in th e sa m e lab or at or y. Th e ge no ty pi c re su lts fo r S tu dy 1 4 w er e ba se d on th e M TB DR sl v1 . T he M IC d ist rib ut io n fo r S tu dy 1 6 w as u nu su all y hig h. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 22 Ta bl e 22 . A M K M IC s f or in vi tro a nd c lin ic al rr s m ut an ts o n 7H 10 . Th e gr ee n lin e de no te s t he c ur re nt W HO a nd C LS I C C fo r A M K DS T on 7 H1 0 (4 m g/ L) . N ot ab le lim ita tio ns : S tu di es 1 8 an d 19 w er e pe rfo rm ed in th e sa m e lab or at or y. Th e ge no ty pi c re su lts fo r S tu dy 1 4 w er e ba se d on th e M TB DR sl v1 . 2. Second-line injectable agents 23 eis mutants Allelic exchange results Zaunbrecher et al. (Study 12) tested all of the strains from their allelic exchange experiments (Section 2.A.2.2) against AMK (Table 23). Compared with the MIC of 0.5 mg/L for the parent strain, the introduction of eis G-10A, C-12T, A-13G, and G-37T mutations did not significantly affect the MICs. By contrast, the MIC of the C-14T mutant was increased by two doubling dilutions and was consequently susceptible at the current CC, but had an MIC above the lowered CC of 2 mg/L. This finding was confirmed by Pholwat et al. (Study 15), who observed an MIC of 4 mg/L for the C-14T mutation whereas the remaining eis mutants (i.e. G-10A, C-12T, C-14G, and C-15G) had MICs ≤2 mg/L. Table 23. AMK MICs for eis allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for AMK DST on 7H10 (4 mg/L). In vitro and clinical isolates Three studies reported MIC data for 71 in vitro or clinical isolates which only had eis promoter mutations (Table 24). The MIC results were varied. At 32-64 mg/L, the MICs of five mutants from Hu et al. (Study 13) were unusually high, which may be due to the presence of other, un-identified resistance mutations. The sole C-14T mutant tested in Pholwat et al. (Study 15) had an MIC of 4 mg/L, comparable to the findings of the allelic exchange experiments. Similarly, the MIC distribution of 1-4 mg/L (with a mode at 2 mg/L) for this mutation was clearly elevated in Krüüner et al. & Engström et al. (Study 7) compared with the corresponding pWT distribution, with some overlap at the dataset-specific CC of 1 mg/L (i.e. eis C-14T behaved similarly to the rrs C1402T mutation). By contrast, the eis C-14T distribution from Hu et al. (Study 13) was systematically lower by one dilution (i.e. 0.5-1 mg/L), although it should be noted that the shape of the pWT MIC distribution was unclear in this study. There was no clear indication that any of the remaining eis mutations conferred elevated MICs. Table 24. AMK MICs for in vitro and clinical eis mutants on 7H10. The green line denotes the current WHO and CLSI CC for AMK DST on 7H10 (4 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 24 whiB7 mutants In vitro isolate A single C+134 whiB7 in vitro mutant, which was resistant to KAN on 7H10 (Section 2.A.2.2), was susceptible to AMK on 7H10 at the current CC (Table 25). Table 27. AMK MICs for pWT isolates on 7H11. Table 25. AMK MICs for in vitro whiB7 mutant on 7H10. The green line denotes the current WHO and CLSI CC for AMK DST on 7H10 (4 mg/L). tlyA mutants In vitro and clinical isolates All 38 in vitro or clinical tlyA mutants (100% (95% 91-100%)) tested in two laboratories were susceptible at the current CC (Table 26). Looking at the non-truncated data from Krüüner et al. & Engström et al. (Study 7), lowering the CC to 2 mg/L would not change this conclusion. Table 26. AMK MICs for in vitro and clinical tlyA mutants on 7H10. The green line denotes the current WHO and CLSI CC for AMK DST on 7H10 (4 mg/L). Notable limitation: Studies 18 and 19 were performed in the same laboratory. 2.B.2.3 Conclusion for AMK CC on 7H10 On balance, the pWT MIC data supported 2 mg/L as the CC for AMK on 7H10. The current CC of 4 mg/L was consequently lowered accordingly. The implications of this change for the interpretation of the rrs C1402T and eis C-14T mutations will be addressed at a later date. 2.B.3 AMK MIC data on 7H11 2.B.3.1 AMK MICs for pWT isolates on 7H11 Two studies were identified that reported AMK MIC data for the pWT population on 7H11, which were determined to be insufficient to propose a CC (Table 27). 2. Second-line injectable agents 25 2.B.3.2 AMK MICs for mutated isolates on 7H11 No studies presenting MIC for the mutants were identified. 2.B.3.3 Conclusion for AMK CC on 7H11 A CC for AMK was not set for 7H11 given that only two studies were identified for this medium, and more evidence was available to set CCs for other media. 2.B.4 AMK MIC data in MGIT 2.B.4.1 AMK MICs for pWT isolates in MGIT 15 studies from 20 laboratories were identified that reported AMK MIC data for the pWT population in MGIT (Table 28). Most studies had a severely truncated pWT MIC distribution, including Rüsch-Gerdes et al. (Study 30) and Rodrigues et al. (Study 33), which have both been cited in the CLSI guidelines to support the current CC of 1 mg/L.9 Where modes were identifiable, they varied between 0.25 to 1 mg/L, which supported the current CC. 2.B.4.2 AMK MICs for mutated isolates in MGIT rrs mutants Clinical isolates Eight studies conducted in 13 laboratories were identified that interrogated 142 clinical isolates harbouring the rrs A1401G mutation (Table 29). All were resistant (100% (95% CI, 97-100%)) at the current CC. 9 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 29. AMK MICs for clinical rrs mutants in MGIT. The green lines denote the current WHO and CLSI CCs for AMK DST in MGIT (1 mg/L). Notable limitations: Studies 23 and 24 and Studies 34 and 36 had data from the same laboratories. The genotypic results for Study 26 were based on a combination of sequencing and a microarray, whereas Study 27 relied on a combination of sequencing and the MTBDRsl v1. eis mutants Clinical isolates Four studies from three laboratories presented data for clinical eis mutants (Table 30). All 34 mutants with eis mutations other than C-14T (100% (95% CI, 90-100%)) had MICs of ≤0.25-1 mg/L, and there was consequently no evidence that these mutations conferred elevated MICs to AMK. By contrast, there was a trend towards higher MICs for the C-14T mutants with 13 of the 17 MICs (77% (95% CI, 50-93%)) at 1-2 mg/L. In line with the 7H10 data (Section 2.B.2.2), this supported the hypothesis that this particular eis mutation conferred elevated AMK MICs. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 26 Ta bl e 28 . A M K M IC s f or p W T iso la te s i n M GI T. Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r A M K DS T in M G IT (1 m g/ L) . N ot ab le lim ita tio ns : O nly S tu di es 2 2, 2 5, 2 6, 2 9, 3 1, 3 2 an d 35 h ad d at a fro m u niq ue la bo ra to rie s. T he g en ot yp ic re su lts fo r S tu dy 2 6 w er e ba se d on a c om bi na tio n of s eq ue nc ing a nd a m icr oa rra y, w he re as St ud y 27 re lie d on a c om bi na tio n of s eq ue nc ing a nd th e M TB DR sl v1 . 2. Second-line injectable agents 27 Table 30. AMK MICs for clinical eis mutants in MGIT. The green line denotes the current WHO and CLSI CCs for AMK DST in MGIT (1 mg/L). Notable limitations: Studies 23 and 24 had data from the same laboratory. The genotypic results for Study 26 were based on a combination of sequencing and a microarray, whereas Study 27 relied on a combination of sequencing and the MTBDRsl v1. whiB7 mutants Clinical isolate The aforementioned whiB7 A+238G mutation, which correlated with resistance to KAN on 7H10 but not MGIT (Sections 2.A.2.2 and 2.A.4.2), was susceptible to AMK (Table 31). Table 31. AMK MICs for clinical whiB7 mutant in MGIT. The green line denotes the current WHO and CLSI CCs for AMK DST in MGIT (1 mg/L). tlyA mutants Clinical isolates Two of the five clinical tlyA mutants (40% (95% CI, 5-85%)) from Cambau et al. (Study 34) were resistant at the current CC (Table 32). The two isolates that tested resistant had MICs one dilution above the CC, which may be due to the presence of other resistance mutations. Table 32. AMK MICs for clinical tlyA mutants in MGIT. The green line denotes the current WHO and CLSI CCs for AMK DST in MGIT (1 mg/L). 2.B.4.3 Conclusion for AMK CC in MGIT The CC of 1 mg/L for AMK was reaffirmed. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 28 2.B.5 References for AMK MIC studies 1. Fabry, W., Schmid, E.N. & Ansorg, R. Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium tuberculosis. Zentralbl Bakteriol 282, 394-401 (1995). 2. Jugheli, L. et al. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob Agents Chemother 53, 5064-8 (2009). 3. Barletta, F., Zamudio, C., Rigouts, L. & Seas, C. Resistance to second-line anti-tuberculosis drugs among Peruvian multidrug resistant Mycobacterium tuberculosis strains. Rev Peru Med Exp Salud Publica 31, 676-82 (2014). 4. Vincent, V. et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int J Tuberc Lung Dis 16, 24-31 (2012). 5. Juréen, P. et al. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections. J Clin Microbiol 48, 1853-8 (2010). 6. Böttger, unpublished data. 7. (a) Krüüner, A., Juréen, P., Levina, K., Ghebremichael, S. & Hoffner, S. Discordant resistance to kanamycin and amikacin in drug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 47, 2971-3 (2003). (b) Engström, A., Perskvist, N., Werngren, J., Hoffner, S.E. & Juréen, P. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 66, 1247-54 (2011). 8. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 9. Udou, T. Comparative evaluation of the in vitro antimycobacterial activities of six aminoglycoside antibiotics using an agar dilution method. J Chemother 18, 610-6 (2006). 10. de Steenwinkel, J.E. et al. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR TB. Emerg Infect Dis 18, 660-3 (2012). 11. Reeves, A.Z., Campbell, P.J., Willby, M.J. & Posey, J.E. Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 59, 444-9 (2015). 12. Zaunbrecher, M.A., Sikes, R.D., Jr., Metchock, B., Shinnick, T.M. & Posey, J.E. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 106, 20004-9 (2009). 13. Hu, Y. et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in rural China. Antimicrob Agents Chemother 57, 3857-63 (2013). 14. van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 15. Pholwat, S. et al. eis promoter C14G and C15G mutations do not confer kanamycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 60, 7522-7523 (2016). 2. Second-line injectable agents 29 16. Sowajassatakul, A., Prammananan, T., Chaiprasert, A. & Phunpruch, S. Molecular char- acterization of amikacin, kanamycin and capreo- mycin resistance in M/XDR-TB strains isolated in Thailand. BMC Microbiol 14, 165 (2014). 17. Via, L. et al. Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean patients with drug-resistant tuberculosis. J Clin Microbiol 48, 402-11 (2010). 18. (a) Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 3192-7 (2005). (b) Reeves, A.Z. et al. Aminoglycoside cross- resistance in Mycobacterium tuberculosis due to mutations in the 5’ untranslated region of whiB7. Antimicrob Agents Chemother 57, 1857-1865 (2013). 19. Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 571-7 (2005). 20. Fattorini, L. et al. Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis. Microb Drug Resist 5, 265-70 (1999). 21. (a) Rey-Jurado, E., Tudó, G., de la Bellacasa, J.P., Espasa, M. & González-Martín, J. In vitro effect of three-drug combinations of antituberculous agents against multidrug- resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents 41, 278-80 (2013). (b) Rey-Jurado, E., Tudo, G., Soy, D. & Gonzalez-Martin, J. Activity and interactions of levofloxacin, linezolid, ethambutol and amikacin in three-drug combinations against Mycobacterium tuberculosis isolates in a human macrophage model. Int J Antimicrob Agents 42, 524-30 (2013). (c) López-Gavín, A. et al. In vitro time-kill curves study of three antituberculous combinations against Mycobacterium tuberculosis clinical isolates. Int J Antimicrob Agents 47, 97-100 (2016). 22. Gonzalo, X., Casali, N., Broda, A., Pardieu, C. & Drobniewski, F. Combination of amikacin and doxycycline against multidrug-resistant and extensively drug-resistant tuberculosis. Int J Antimicrob Agents 45, 406-12 (2015). 23. Tessema, B. et al. FIND tuberculosis strain bank: a resource for researchers and developers working on tests to detect Mycobacterium tuberculosis and related drug resistance. J Clin Microbiol 55, 1066-1073 (2017). 24. Heyckendorf, J. et al. What is resistance? Impact of phenotypic versus molecular drug resistance testing on multi- and extensively drug-resistant tuberculosis therapy. Antimicrob Agents Chemother, ahead of print (2017). 25. Sturegård, E. et al. Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10. Clin Microbiol Infect 21, 148 e5-7 (2015). 26. Zimenkov, D.V. et al. Detection of second- line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infect Dis 13, 240 (2013). 27. (a) Kambli, P. et al. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol 5, 1-6 (2016). (b) Kambli, P. et al. Corrigendum to “Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second- line injectables” [Int. J. Mycobacteriol. 5(1) 2016 1-6]. Int J Mycobacteriol 5, 370-372 (2016). 28. Matt, T. et al. Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin. Proc Natl Acad Sci U S A 109, 10984-9 (2012). 29. Lin, S.Y., Desmond, E., Bonato, D., Gross, W. & Siddiqi, S. Multicenter evaluation of Bactec MGIT 960 system for second-line drug susceptibility testing of Mycobacterium tuberculosis complex. J Clin Microbiol 47, 3630-4 (2009). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 30 30. Rüsch-Gerdes, S., Pfyffer, G.E., Casal, M., Chadwick, M. & Siddiqi, S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuber- culosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 44, 688-92 (2006). 31. Zheng, X. et al. Determination of MIC breakpoints for second-line drugs associated with clinical outcomes in multidrug-resistant tuberculosis treatment in China. Antimicrob Agents Chemother 60, 4786-92 (2016). 32. Sharma, M. et al. Canadian multicenter laboratory study for standardized second- line antimicrobial susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 49, 4112-6 (2011). 33. Rodrigues, C. et al. Drug susceptibility testing of Mycobacterium tuberculosis against second- line drugs using the Bactec MGIT 960 System. Int J Tuberc Lung Dis 12, 1449-55 (2008). 34. Cambau, E. et al. Revisiting susceptibility testing in MDR-TB by a standardized quanti- tative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 70, 686-96 (2015). 35. Sirgel, F.A. et al. Mutations in the rrs A1401G gene and phenotypic resistance to amikacin and capreomycin in Mycobacterium tuberculosis. Microb Drug Resist 18, 193-7 (2012). 36. Springer, B., Lucke, K., Calligaris-Maibach, R., Ritter, C. & Böttger, E.C. Quantitative drug susceptibility testing of Mycobacterium tubercu- losis by use of MGIT 960 and EpiCenter instru- mentation. J Clin Microbiol 47, 1773-80 (2009). Table 33. CAP MICs for pWT isolates on LJ. The red line denotes the current WHO CC for CAP DST on LJ (40 mg/L). Notable limitation: All studies were performed in the same laboratory. 2.C.1.2 CAP MICs for mutated isolates on LJ rrs mutants Clinical isolates Three studies from the same laboratory reported MICs for clinical rrs mutants (Table 34). All four rrs C1402T mutants (100% (95% 40-100%)) were resistant, with MICs of >160 mg/L. Six rrs A1401G mutants (8% (95% CI, 3-17%)) tested susceptible, which was mostly driven by the apparent overlap between the pWT distribution and the lower end of the MIC distribution of isolates with this mutation (i.e. it is likely that the five isolates with MICs of 40 mg/L would become resistant upon re-testing, whereas the isolate with an MIC of ≤10 mg/L might be the result of experimental error with either sequencing or pDST). 2.C.1 CAP MIC data on LJ 2.C.1.1 CAP MICs for pWT isolates on LJ Three studies from the same laboratory were identified that reported CAP MIC data for the pWT population on LJ (Table 33). Although the inter-laboratory reproducibility of testing could not be assessed, a mode at 20 mg/L was observed despite the truncation at the lower end of the pWT distribution. 2. Second-line injectable agents 31 Table 34. CAP MICs for clinical rrs mutants on LJ. The red line denotes the current WHO CC for CAP DST on LJ (40 mg/L). Notable limitation: All studies were performed in the same laboratory. 2.C.1.3 Conclusion for CAP CC on LJ The identified CAP MIC data for LJ were limited and provided little insight into the shape of the pWT distribution, which precluded a re- assessment of the current CC. Nevertheless, 40 mg/L was maintained as the CC, given the importance of LJ as a testing medium in many settings. An overlap between the MIC distributions of pWT isolates and rrs A1401G mutants was nonetheless noted. 2.C.2 CAP MIC data on 7H10 2.C.2.1 CAP MICs for pWT isolates on 7H10 11 studies from nine laboratories were identified that reported CAP MIC data for the pWT population on 7H10 (Table 35). Most distributions had modes at 1.25 or 2 mg/L, for which the WHO CC of 4 mg/L would be the optimal CC (e.g. for Krüüner et al. & Engström et al. (Study 6)). By contrast, Studies 9-11 had modes at either 4 or 5 mg/L. Among these, Reeves et al. (Study 9) was notable given that it presented data from the CDC, which uses 7H10 for routine pDST. The MIC distribution for the 16 isolates in this study was symmetrically centred around 4 mg/L, which meant that the use of the WHO CC would result in a systematic false-resistance rate of 13% (95% CI, 2-38%), assuming that these isolates are pWT. This would not be the case at the current CLSI CC of 10 mg/L or the equivalent concentration of 8 mg/L. For the pWT distribution in van Ingen et al. (Study 11), which consisted of 20 isolates, a false-resistance rate of 5% (95% CI, 0-25%) would occur at the WHO CC. However, it should be noted that relatively few isolates were tested as part of Studies 7 and 11 and it was therefore unclear whether these results are reproducible. 2.C.2.2 CAP MICs for mutated isolates on 7H10 rrs mutants Allelic exchange results Reeves et al. (Study 9) conducted allelic exchange experiments in H37Rv, CDC1551 and Beijing strain backgrounds (Table 36). The MICs of the three parent strains were 2-4 mg/L on 7H10, compared to 40 mg/L for rrs A1401G, 80 mg/L for rrs C1402T, and 160 to >320 mg/L for rrs G1484T. Table 36. CAP MICs for rrs allelic exchange mutants on 7H10. The red line denotes the current WHO CC for CAP DST on 7H10 (4 mg/L). The blue line denotes the current CLSI CC for CAP DST on 7H10 (10 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 32 Ta bl e 35 . C AP M IC s f or p W T iso la te s o n 7H 10 . Th e re d lin e de no te s t he c ur re nt W HO C C fo r C AP D ST o n 7H 10 (4 m g/ L) . T he b lue lin e de no te s t he c ur re nt C LS I C C fo r C AP D ST o n 7H 10 (1 0 m g/ L) . No ta bl e lim ita tio ns : S tu di es 9 , 1 3 an d 14 w er e do ne in th e sa m e lab or at or y. Th e ge no ty pi c re su lts fo r S tu dy 1 1 we re b as ed o n th e M TB DR sl v1 . 2. Second-line injectable agents 33 In vitro and clinical isolates Nine studies from seven laboratories reported MICs for in vitro or clinical rrs mutants (Table 37). All 41 rrs C1402T and G1484T mutants were resistant (100% (95% CI, 91-100%)) at the current WHO CC. By contrast, the detection of rrs A1401G was compromised by the variation between different laboratories and/or different datasets and the fact that the distributions of these isolates overlapped with the upper end of the pWT distributions. For example, Krüüner et al. & Engström et al. (Study 6) had systematically lower pWT MIC distributions and, consequently, a dataset-specific CC of 4 mg/L, which also represented the lower end of the rrs A1401G distribution (Section 2.C.2.1). By contrast, Reeves et al. (Study 9) and van Ingen et al. (Study 10) both had higher pWT MIC distributions and dataset-specific CCs of 8 and 10 mg/L, which were also the lower ends of the respective rrs A1401G distributions of these studies. Although all 242 rrs A1401G mutants likely had elevated MICs (100% (95% CI, 98-100%)), different percentages of these mutants would be misclassified depending on which CC was used. Using the WHO CC of 4 mg/L, only four isolates (2% (95% CI, 0-4%)) were misclassified as susceptible, whereas this increased to 56 (23% (95% CI, 18-29%)) with the CLSI CC of 10 mg/L. eis mutants In vitro and clinical isolates CAP MICs were reported for 73 eis mutants in four studies (Table 38). Excluding one isolate from Study 13, for which the lowest concentration tested was 10 mg/L, six isolates (8% (95% CI, 3-17%)) were resistant at the WHO CC, which decreased to five (7% (95% CI, 2-15%)) at the CLSI CC. The remaining five isolates had unusually high MICs (32-64 mg/L), likely due to the presence of another resistance mutation. Table 38. CAP MICs for in vitro and clinical eis mutants on 7H10. The red lines denote the current WHO CCs for CAP DST on 7H10 (4 mg/L). The blue line denotes the current CLSI CC for CAP DST on 7H10 (10 mg/L). whiB7 mutants In vitro isolate One in vitro whiB7 mutant, which was resistant to KAN on 7H10 (Section 2.A.2.2), was susceptible to CAP at the CLSI CC of 10 mg/L (Table 39). Table 39. CAP MICs for in vitro whiB7 mutant on 7H10. The red line denotes the current WHO CC for CAP DST on 7H10 (4 mg/L). The blue line denotes the current CLSI CC for CAP DST on 7H10 (10 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 34 Ta bl e 37 . C AP M IC s f or in vi tro a nd c lin ic al rr s m ut an ts o n 7H 10 . Th e re d lin e de no te s t he c ur re nt W HO C C fo r C AP D ST o n 7H 10 (4 m g/ L) . T he b lue lin e de no te s t he c ur re nt C LS I C C fo r C AP D ST o n 7H 10 (1 0 m g/ L) . No ta bl e lim ita tio ns : S tu di es 9 , 1 3 an d 14 w er e do ne in th e sa m e lab or at or y. Th e ge no ty pi c re su lts fo r S tu dy 1 1 we re b as ed o n th e M TB DR sl v1 . 2. Second-line injectable agents 35 tlyA mutants In vitro and clinical isolates Three studies from two laboratories reported MICs for 48 in vitro and clinical tlyA mutants, of which 92% (95% CI, 80-98%) were resistant at both CCs (Table 40). Two clinical mutants had very low MICs (i.e. 0.5 and 1 mg/L), which means that the mutations in question could be genuine polymorphisms that do not confer resistance. Two in vitro mutants from Krüüner et al. & Engström et al. (Study 6) had MICs of 8 mg/L, which were elevated compared with the dataset-specific CC of 4 mg/L for this study. Table 40. CAP MICs for in vitro and clinical tlyA mutants on 7H10. The red line denotes the current WHO CC for CAP DST on 7H10 (4 mg/L). The blue line denotes the current CLSI CC for CAP DST on 7H10 (10 mg/L). Notable limitation: Studies 13 and 14 were done in the same laboratory. rrl mutant In vitro isolate Maus et al. & Johansen et al. (Study 14) reported a CAP MIC of >160 mg/L for an in vitro rrl mutant (Table 41). This is the only rrl mutation implicated in CAP resistance to date. The authors reported that this mutant remained susceptible to both KAN and AMK. Table 41. CAP MICs for in vitro rrl mutant on 7H10. The red line denotes the current WHO CC for CAP DST on 7H10 (4 mg/L). The blue line denotes the current CLSI CC for CAP DST on 7H10 (10 mg/L). 2.C.2.3 Conclusion for CAP CC on 7H10 The available data supported 4 mg/L as the current CC for CAP DST on 7H10. However, it should be noted that the modes of two of the pWT distributions were 4 mg/L, which may mean that the gain in sensitivity in detecting pNWT iso- lates may come at the expense of a higher rate of false-resistance detection. However, if the CC was raised to 8 mg/L, there would be a greater risk that strains with the rrs A1401G mutation may be misclassified as susceptible. As more laboratories adopt 4 mg/L as the CC for pDST, both these possibilities should be monitored. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 36 2.C.3 CAP MIC data on 7H11 2.C.3.1 CAP MICs for pWT isolates on 7H11 Only Fattorini et al. (Study 14) reported CAP MIC data for 7H11 (Table 42). Table 42. CAP MICs for pWT isolates on 7H11. The blue line denotes the current CLSI CC for CAP DST on 7H11 (10 mg/L). 2.C.3.2 CAP MICs for mutated isolates on 7H11 No studies presenting MIC data for isolates with mutations in resistance genes were identified. 2.C.3.3 Conclusion for CAP CC on 7H11 Given that only a single study was identified for 7H11, which precluded an assessment of the inter-laboratory reproducibility, a CC for this medium could not be set. 2.C.4 CAP MIC data in MGIT 2.C.4.1 CAP MICs for pWT isolates in MGIT 13 studies from 19 laboratories featured CAP MIC data for the pWT population in MGIT (Table 43). Several of the distributions were severely truncated, including those for Rüsch-Gerdes et al. (Study 22) and Rodrigues et al. (Study 24), which were cited by CLSI in support of the current CC of 2.5 mg/L.10 Moreover, different dilution series were used by most of the studies, which complicated the direct comparison of the MICs. Nevertheless, the current CC appeared to be appropriate for the majority of studies, although there is at least one exception (i.e. Sturegård et al. (Study 16) would be better served by a CC of 4 mg/L, but additional untruncated MIC distributions, generated using standard dilution series, are required to investigate this possibility). 2.C.4.2 CAP MICs for mutated isolates in MGIT rrs mutants Clinical isolates Seven studies reported MIC data from 153 clinical rrs A1401G mutants from 12 laboratories (Table 44). Unlike LJ and 7H10, there did not appear to be an overlap between the pWT MIC distribution and the mutant MIC distribution in MGIT, as the lower end of the distribution of these mutants was 5 mg/L, so none of the isolates were misclassified (0% (95% CI, 0-2%)). 10 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 44. CAP MICs for clinical rrs mutants in MGIT. The green line denotes the current WHO and CLSI CC for CAP DST in MGIT (2.5 mg/L). Notable limitations: Studies 17, 18 and 28, and Studies 26 and 29 reported data from the same laboratory, respectively. The genotypic results for Study 19 were based on a combination of sequencing and a microarray, whereas Study 23 relied on a combination of sequencing and the MTBDRsl v1. 2. Second-line injectable agents 37 Ta bl e 43 . C AP M IC s f or p W T iso la te s i n M GI T. Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r C AP D ST in M G IT (2 .5 m g/ L) . N ot ab le lim ita tio ns : O nly s tu di es 1 6, 1 9- 21 , 2 5 an d 29 h ad da ta fr om u niq ue la bo ra to rie s. T he g en ot yp ic re su lts fo r S tu dy 1 9 w er e ba se d on a c om bi na tio n of s eq ue nc ing a nd a m icr oa rra y, w he re as S tu dy 2 3 re lie d on a c om bi na tio n of s eq ue nc ing a nd th e M TB DR sl v1 . TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 38 eis mutants Clinical isolates All 43 eis mutants tested (100% (95% CI, 92- 100%)) in three different laboratories were susceptible at the current CC (Table 45). Table 45. CAP MICs for clinical eis mutants in MGIT. The green line denotes the current WHO and CLSI CC for CAP DST in MGIT (2.5 mg/L). Notable limitations: Studies 17 and 18 reported data from the same laboratory. The genotypic results for Study 19 were based on a combination of sequencing and a microarray, whereas Study 23 relied on a combination of sequencing and the MTBDRsl v1. whiB7 mutant Clinical isolates The aforementioned whiB7 A+238G mutation, which was correlated with KAN resistance on 7H10 but not MGIT (Sections 2.A.2.2 and 2.A.4.2), was susceptible to CAP (Table 46). Table 46. CAP MICs for clinical whiB7 mutant in MGIT. The green line denotes the current WHO and CLSI CC for CAP DST in MGIT (2.5 mg/L). tlyA mutant Clinical isolates Five clinical tlyA mutations all tested CAP- resistant (100% (95% CI, 48-100%)) at the current CC (Table 47). Table 47. CAP MICs for clinical tlyA mutants in MGIT. The green line denotes the current WHO and CLSI CC for CAP DST in MGIT (2.5 mg/L). 2. Second-line injectable agents 39 2.C.4.3 Conclusion for CAP CC in MGIT The CC of 2.5 mg/L for CAP was reaffirmed. Unlike LJ and 7H10, no overlap of the MIC distributions of pWT isolates and rrs A1401G mutants was observed. 2.C.5 References for CAP MIC studies 1. Jugheli, L. et al. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob Agents Chemother 53, 5064-8 (2009). 2. Barletta, F., Zamudio, C., Rigouts, L. & Seas, C. Resistance to second-line anti-tuberculosis drugs among Peruvian multidrug resistant Mycobacterium tuberculosis strains. Rev Peru Med Exp Salud Publica 31, 676-82 (2014). 3. Vincent, V. et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int J Tuberc Lung Dis 16, 24-31 (2012). 4. Juréen, P. et al. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections. J Clin Microbiol 48, 1853-8 (2010). 5. Böttger, unpublished data. 6. (a) Krüüner, A., Juréen, P., Levina, K., Ghe- bremichael, S. & Hoffner, S. Discordant resis- tance to kanamycin and amikacin in drug-re- sistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 47, 2971-3 (2003). (b) Engström, A., Perskvist, N., Werngren, J., Hoffner, S.E. & Juréen, P. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 66, 1247-54 (2011). 7. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 8. Hu, Y. et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in rural China. Antimicrob Agents Chemother 57, 3857-63 (2013). 9. Reeves, A.Z., Campbell, P.J., Willby, M.J. & Posey, J.E. Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 59, 444-9 (2015). 10. Sowajassatakul, A., Prammananan, T., Chaiprasert, A. & Phunpruch, S. Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand. BMC Microbiol 14, 165 (2014). 11. Van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 12. Via, L. et al. Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean patients with drug-resistant tuberculosis. J Clin Microbiol 48, 402-11 (2010). 13. (a) Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 3192-7 (2005). (b) Zaunbrecher, M.A., Sikes, R.D., Jr., Metchock, B., Shinnick, T.M. & Posey, J.E. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 40 kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 106, 20004-9 (2009). (c) Reeves, A.Z. et al. Aminoglycoside cross- resistance in Mycobacterium tuberculosis due to mutations in the 5’ untranslated region of whiB7. Antimicrob Agents Chemother 57, 1857-1865 (2013). 14. (a) Maus, C.E., Plikaytis, B.B. & Shinnick, T.M. Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 571-7 (2005). (b) Johansen, S.K., Maus, C.E., Plikaytis, B.B. & Douthwaite, S. Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2’-O-methylations in 16S and 23S rRNAs. Mol Cell 23, 173-82 (2006). 15. Fattorini, L. et al. Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis. Microb Drug Resist 5, 265-70 (1999). 16. Sturegård, E. et al. Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10. Clin Microbiol Infect 21, 148 e5-7 (2015). 17. Tessema, B. et al. FIND tuberculosis strain bank: a resource for researchers and developers working on tests to detect Mycobacterium tuberculosis and related drug resistance. J Clin Microbiol 55, 1066-1073 (2017). 18. Heyckendorf, J. et al. What is resistance? Impact of phenotypic versus molecular drug resistance testing on multi- and extensively drug-resistant tuberculosis therapy. Antimicrob Agents Chemother, ahead of print (2017). 19. (a) Gikalo, M.B., Nosova, E.Y., Krylova, L.Y. & Moroz, A.M. The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 67, 2107-9 (2012). (b) Zimenkov, D.V. et al. Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infect Dis 13, 240 (2013). 20. Zheng, X. et al. Determination of MIC breakpoints for second-line drugs associated with clinical outcomes in multidrug-resistant tuberculosis treatment in China. Antimicrob Agents Chemother 60, 4786-92 (2016). 21. Lin, S.Y., Desmond, E., Bonato, D., Gross, W. & Siddiqi, S. Multicenter evaluation of Bactec MGIT 960 system for second-line drug susceptibility testing of Mycobacterium tuberculosis complex. J Clin Microbiol 47, 3630-4 (2009). 22. Rüsch-Gerdes, S., Pfyffer, G.E., Casal, M., Chadwick, M. & Siddiqi, S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 44, 688- 92 (2006). 23. (a) Kambli, P. et al. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol 5, 1-6 (2016). (b) Kambli, P. et al. Corrigendum to “Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second- line injectables” [Int. J. Mycobacteriol. 5(1) 2016 1-6]. Int J Mycobacteriol 5, 370-372 (2016). 24. Rodrigues, C. et al. Drug susceptibility testing of Mycobacterium tuberculosis against second-line drugs using the Bactec MGIT 960 System. Int J Tuberc Lung Dis 12, 1449-55 (2008). 25. Sharma, M. et al. Canadian multicenter laboratory study for standardized second- line antimicrobial susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 49, 4112-6 (2011). 26. Pietersen, E. et al. High frequency of resistance, lack of clinical benefit, and poor outcomes in capreomycin treated South African patients with extensively drug-resistant tuberculosis. PLoS One 10, e0123655 (2015). 27. Springer, B., Lucke, K., Calligaris-Maibach, R., Ritter, C. & Böttger, E.C. Quantitative drug susceptibility testing of Mycobacterium 2. Second-line injectable agents 41 tuberculosis by use of MGIT 960 and EpiCenter instrumentation. J Clin Microbiol 47, 1773-80 (2009). 28. Cambau, E. et al. Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 70, 686-96 (2015). 29. Sirgel, F.A. et al. Mutations in the rrs A1401G gene and phenotypic resistance to amikacin and capreomycin in Mycobacterium tuberculosis. Microb Drug Resist 18, 193-7 (2012). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 42 3. Clofazimine and bedaquiline 3.0 CFZ and BDQ MIC data stratification and current breakpoints MIC data were stratified by mutations in atpE (Rv1305), pepQ (Rv2535c) and mmpR (Rv0678) but not Rv1979c, given the substantial mutation variability observed for this gene (details regarding these resistance mechanisms can be found in the supplement). mmpR promoter mutations were assumed not to increase BDQ MICs, and so were shown as gWT in tables and data files.11 For BDQ, LOF mutations in mmpR were highlighted whenever possible. Additionally, some studies tested BDQ concentrations of 0.24, 0.48 and 0.96 mg/L, which were normalised to 0.25, 0.5 and 1 mg/L in order to best collate the data. No breakpoints for BDQ testing have been defined by CLSI, FDA or WHO to date. In parallel with the marketing authorisation by European Medicines Agency, EUCAST has designated 0.25 mg/L as a medium-independent CB for BDQ testing.12 No CC for CFZ currently exists. 3.A.1 CFZ MIC data on LJ 3.A.1.1 CFZ MICs for pWT isolates on LJ No studies with MICs for pWT isolates were identified. 3.A.1.2 CFZ MICs for mutated isolates on LJ No studies were found. 3.A.1.3 Conclusion for CFZ CC for LJ Given that no MIC data were identified for LJ, a CC could not be defined. 3.A.2 CFZ MIC data on 7H10 3.A.2.1 CFZ MICs for pWT isolates on 7H10 Two studies from two laboratories were identified that reported CFZ MIC data for the pWT population on 7H10 (Table 48). van Ingen et al. (Study 2) had a severely truncated MIC distribution, which precluded an assessment of its shape. 11 Villellas, C. et al. Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline. J Antimicrob Chemother 72, 684-690 (2017). 12 European Committee for Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 7.1, valid from 2017-03-10. http://www. eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_7.1_Breakpoint_Tables. xls (accessed 9.4.2017). Table 48. CFZ MICs for pWT isolates on 7H10. 3.A.2.2 CFZ MICs for mutated isolates on 7H10 No studies presenting MIC distributions for mutated isolates were found. 3.A.2.3 Conclusion for CFZ CC for 7H10 Only two studies were identified that reported CFZ MICs for 7H10. These data were insufficient to establish a CC. 3. Clofazimine and bedaquiline 43 3.A.3 CFZ MIC data on 7H11 3.A.3.1 CFZ MICs for pWT isolates on 7H11 A single study was identified with CFZ MIC data for 7H11, which did not include H37Rv (Table 49). Table 49. CFZ MICs for pWT isolates on 7H11. 3.A.3.2 CFZ MICs for mutated isolates on 7H11 No studies with MIC distributions for gNWT isolates on 7H11 were found. 3.A.3.3 Conclusion for CFZ CC for 7H11 Only a single study was identified that reported CFZ MICs on 7H11. Therefore, a CC was not set for this medium. 3.A.4 CFZ MIC data in MGIT 3.A.4.1 CFZ MICs for pWT isolates in MGIT Six studies from nine laboratories reported CFZ MIC data for the pWT population in MGIT (Table 50). Despite the fact that most distributions were truncated, variation in testing was apparent, as the modes of the pWT MIC distribution varied between 0.25 and 1 mg/L. These data therefore suggested a CC of 1 mg/L, although yet unknown resistance mechanisms for this drug may have confounded this analysis. Table 50. CFZ MICs for pWT isolates in MGIT. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 44 3.A.4.2 CFZ MICs for mutated isolates in MGIT In vitro and clinical isolates Five studies from eight laboratories presented MICs for in vitro and clinical mutants (Table 51). Köser et al. (Study 4) reported a low CFZ MIC of 0.12 mg/L for a BDQ-resistant, in vitro atpE mutant. The same set of mmpR in vitro mutants were tested in four laboratories by Ismail et al. (Study 7). Some of these mutants were also tested in a fifth laboratory by Köser et al. (Study 4), along with further mmpR in vitro mutants. The 62 MICs from these five laboratories varied between 1 and >4 mg/L, of which 32 were >1 mg/L (52% (95% CI, 39-65%)). By contrast, the MICs for the 17 clinical mmpR mutants ranged from ≤0.25 to >4 mg/L. This was likely due to the fact that some of these mutations negatively affected the function of MmpR and thus conferred elevated MICs, whereas others did not. For example, the mmpR V3I mutation, which was observed in seven isolates and was tested in two laboratories, always had MICs ≤0.5 mg/L and was therefore likely a neutral polymorphism. By contrast, the mmpR V1A mutation, which evolved during treatment with CFZ and correlated with cross-resistance to BDQ (Section 3.B.4.2), was significant, as it correlated with a MIC of ≥4 mg/L compared with 1 mg/L for the gWT parent. Table 51. CFZ MICs for in vitro and clinical atpE and mmpR mutants in MGIT. 3.A.4.3 Conclusion for CFZ CC for MGIT Because of the variation in testing, an interim CC of 1 mg/L was adopted. However, this concentration also corresponded to the lower end of the MIC distribution for mmpR in vitro isolates, which meant that the reproducibility of mmpR mutants that have truly elevated MICs would likely be poor (i.e. a large proportion would be misclassified as susceptible). In addition, Ismail et al. (Study 7) reported that the use of DMSO was crucial to prepare the inoculum and the dilutions for CFZ testing to prevent the precipitation of CFZ and, consequently, artificially high MICs (i.e. false-resistance). Therefore, more data for untruncated MIC distributions for pan-susceptible isolates are needed to better define the upper end of the pWT MIC distribution and the inter-laboratory reproducibility. Finally, additional studies of the various CFZ resistance mechanisms and their clinical relevance are needed. 3.A.5 References for CFZ MIC studies 1. Schön, T. et al. Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis. Int J Tuberc Lung Dis 15, 502-9 (2011). 2. Van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 3. López-Gavín, A., Tudó, G., Vergara, A., Hurtado, J.C. & Gonzalez-Martín, J. In vitro activity against Mycobacterium tuberculosis of levofloxacin, moxifloxacin and UB-8902 in combination with clofazimine and pretomanid. 3. Clofazimine and bedaquiline 45 Int J Antimicrob Agents 46, 582-5 (2015). 4. Köser, unpublished data. 5. Van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 6. Dheda, K. et al. Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study. Lancet Respir Med 5, 269-281 (2017). 7. Ismail, unpublished data. 8. (a) Bloemberg, G.V. et al. Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 373, 1986-8 (2015). (b) Somoskovi, A., Bruderer, V., Hömke, R., Bloemberg, G.V. & Böttger, E.C. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J 45, 554-7 (2015). (c) Böttger, unpublished data. 9. Werngren, unpublished data. 3.B.1 BDQ MIC data on LJ 3.B.1.1 BDQ MICs for pWT isolates on LJ No studies presented MIC distributions for pWT isolates on LJ. 3.B.1.2 BDQ MICs for mutated isolates on LJ No studies were found. 3.B.1.3 Conclusion for BDQ CC for LJ Given that no MIC data were identified for LJ, a CC could not be defined. 3.B.2 BDQ MIC data on 7H10 3.B.2.1 BDQ MICs for pWT isolates on 7H10 Three studies reported BDQ MIC data for the pWT population on 7H10 (Table 52): 1. Kaniga et al. (Study 1) conducted a study to determine the QC range for H37Rv ATCC 27294. This strain was tested 30 times in eight different centres. The results from laboratory 5 were excluded from the analysis by Kaniga et al., as the MICs from this laboratory were found to have unusually high MICs for one medium lot, which resulted in a bimodal MIC distribution. Excluding these data left 213 replicates of H37Rv, with MICs that ranged from ≤0.008- 0.25 mg/L with a mode at 0.06 mg/L. Based on these findings, 0.12 mg/L was defined as the upper end of the H37Rv QC range, which included 211 of the MICs (99% (95% CI, 97- 100%)). 2. Ismail et al. (Study 2) conducted a large, retrospective study of South African MTBC clinical isolates. WGS was conducted on all isolates. The MIC range for the 37 replicates of H37Rv ATCC 27294 tested in this study was 0.03-0.06 mg/L (with a mode at 0.06 mg/L), which was in agreement with the aforementioned QC range from Study 1, in which this laboratory participated. In contrast, the 371 gWT clinical isolates that had not been exposed to BDQ showed an unusually wide MIC range, with MICs spanning eight dilutions (0.015 to >2 mg/L).13 Notably, the mode of 0.25 mg/L of this distribution was two dilutions higher than the mode of the QC range from Study 1. When this same set of clinical isolates was tested in MGIT (as part of Study 12 in Section 3.B.4.1), the resulting mode of the pWT MIC distribution was within one dilution of the mode of the H37Rv MIC distribution in MGIT. Moreover, the MIC distribution for these clinical isolates (as well as the H37Rv MIC distribution from Study 12) was comparable 13 European Committee for Antimicrobial Susceptibility Testing. Standard Operating Procedure. MIC distributions and the setting of epidemiological cutoff (ECOFF) values. 17 November 2017. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 46 to the H37Rv and clinical MIC data in MGIT from another laboratory (Study 11). Together, these observations suggest a methodological problem with Ismail et al. (Study 2), which was therefore excluded from further analysis in this report. This observation underscored the need for careful standardisation of BDQ testing, which is more prone to variability than other drugs given that a variety of factors can affect MIC results (e.g. the type of plastic used, or protein (albumin) binding).14 3. Diacon et al. (Study 3) presented MICs for 44 clinical isolates from an early BDQ bactericidal activity trial. 33 MICs were truncated at the lower end of the distribution, which precluded an assessment of the mode of the distribution. 14 Lounis, N., Vranckx, L., Gevers, T., Kaniga, K. & Andries, K. In vitro culture conditions affecting minimal inhibitory concentration of bedaquiline against M. tuberculosis. Med Mal Infect 46, 220-5 (2016). Table 52. BDQ MICs for pWT isolates on 7H10. The purple line denotes the current EUCAST CB for BDQ testing (0.25 mg/L). Notable limitations: The laboratory in which Study 2 was conducted also participated in Study 1 (because this was a blinded study, it was unclear what the corresponding laboratory number was). Study 2 had an unusually wide pWT MIC distribution. 3.B.2.2 BDQ MICs for mutated isolates on 7H10 The data from Ismail et al. (Study 2), which featured mutated clinical isolates, were excluded (Section 3.B.2.1). However, these data can be found in the supplementary MIC file. 3.B.2.3 Conclusion for BDQ CC for 7H10 Because data from Ismail et al. (Study 2) were excluded for methodological reasons, only H37Rv QC data from and a truncated pWT MIC distribution were available for consideration. These data were insufficient to propose a CC. 3.B.3 BDQ MIC data on 7H11 3.B.3.1 BDQ MICs for pWT isolates on 7H11 Six studies reported BDQ MIC data for the pWT population on 7H11 (Table 53). Five of these studies either reported MICs for more than 10 pWT isolates or did extensive repeat MIC resting: 1. Kaniga et al. (Study 4) conducted a study to determine the QC range for H37Rv ATCC 27294. This strain was tested 30-32 times in eight different centres. The pooled MIC distribution of the total 242 replicates ranged from ≤0.008 to 0.25 mg/L (with a mode at 0.06 mg/L). Notably, the nine MICs at the higher end of the distribution were all from laboratory 5, which was excluded by Kaniga et al. from the evaluation of MIC data for 7H10 (Section 3.B.2.1). Based on these findings, 0.12 mg/L was defined as the upper end of the QC range, which included 233 of the total MICs (96% (95% CI, 93-98%)). 2. Diacon et al., Pym et al. and Villellas et al. (Study 5) presented MIC data for 325 gWT baseline clinical isolates from trials C208 and C209. The corresponding pWT MIC distribution 3. Clofazimine and bedaquiline 47 ranged from ≤0.008 to 0.25 mg/L (with a mode at 0.06 mg/L). This study also included data for four post-baseline isolates from trial C209 that were either gWT (n=3) or for which no genotypic information was available (n=1). The MICs of these isolates were 0.03-0.06 mg/L. The relationship between baseline BDQ MIC and culture conversion rates can be found in Supplementary Table 2. 3. Torrea et al. (Study 6) presented additional data from the laboratory that did the testing for Study 5. The pWT MIC distribution was ≤0.008- 0.25 mg/L (with a mode at 0.06 mg/L) for 77 clinical isolates. 4. Andries et al. (Study 7) reported a pWT MIC distribution of ≤0.008-0.12 mg/L for 22 clinical isolates. The distribution was bimodal with one mode at 0.015 and another at 0.06 mg/L. 5. Zimenkov et al. (Study 8) reported a pWT MIC distribution of ≤0.03-0.12 mg/L for 21 clinical isolates. However, the MIC distribution of 15 isolates was truncated at the lower end of the distribution, which precluded an assessment of its shape. The MIC of H37Rv ATCC 25618 was also truncated at ≤0.03 mg/L. Table 53. BDQ MICs for pWT isolates on 7H11. The purple line denotes the current EUCAST CB for BDQ testing (0.25 mg/L). Notable limitation: Studies 5 and 6 were conducted in the same laboratory. 3.B.3.2 BDQ MICs for mutated isolates on 7H11 In vitro and clinical isolates Torrea et al. (Study 6) reported MICs of 1-2 mg/L for 15 in vitro mmpR mutants. One mutant with an unknown resistance mechanism had an MIC of 1 mg/L, whereas an in vitro atpE mutant had an MIC >2 mg/L (Table 54). Four studies featured MIC data for clinical mmpR mutants (Table 54): 1. Torrea et al. (Study 6) reported one naturally resistant MDR mmpR mutant with an MIC of 0.5 mg/L. 2. Diacon et al., Pym et al. and Villellas et al. (Study 5) reported MICs for mmpR mutants from two groups of isolates: a. Eight baseline isolates from trials 208 and 209 had mmpR LOF mutations. Two of the isolates had MICs ≤0.008 mg/L, one had an MIC of 0.06 mg/L and the remaining five had MICs of at least 0.25 mg/L. 14 additional baseline isolates had other mmpR mutations, with MICs of 0.015-0.5 mg/L. 3. Seven mmpR LOF mutants from post- baseline isolates from trial C209 had MICs of 0.12 to >0.5 mg/L, whereas eight other mmpR mutants had MICs of 0.06 to >0.5 mg/L TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 48 (Supplementary Table 3 shows the MICs of all post-baseline isolate from this trial compared to their respective baseline isolates). Veziris et al. (Study 8) described two isolates with MICs of 0.25 and 0.5 mg/L that had different mmpR LOF mutations. Moreover, a mmpR mutant that was isolated from a patient failing therapy (with a regimen that included BDQ) had an MIC of 0.25 mg/L compared with ≤0.015 mg/L for the gWT parental isolate at baseline. 4. Zimenkov et al. (Study 9) reported 22 mmpR mutants with MICs of 0.06-0.25 mg/L. Moreover, they reported that one isolate with a baseline mmpR L142R mutation acquired an atpE A63V mutation during BDQ treatment, which resulted in a BDQ MIC increase from 0.25 mg/L to 1 mg/L (Supplementary Table 4 shows the isolates that acquired either mmpR or atpE mutations during the course of treatment in this study). Finally, the acquisition of an atpE D28N mutation resulted in an MIC increase from 0.03 mg/L to 0.12 mg/L. Notably, different mutations in both of these atpE codons had been previously described in BDQ-resistant in vitro mutants.15, 16 Even if 0.12 mg/L was used as the concentration to define resistance, 50% (95% CI, 29-71%) of these 24 mutants tested, including the atpE D28N mutant, would be deemed BDQ-susceptible despite the trend towards higher MICs for isolates that developed mutations during treatment. In light of the severe truncation of the pWT MIC distribution in this study, including the MIC for H37Rv, it was not possible to assess whether this was due to a methodological difference in MIC testing. As this is the first study to feature clinical atpE mutants, retesting of these isolates using standardised protocols and multiple media would be valuable. 15 Huitric, E. et al. Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother 54, 1022-8 (2010). 16 Preiss, L. et al. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci Adv 1, e1500106 (2015) Table 54. BDQ MICs for in vitro and clinical atpE and mmpR mutants on 7H11. The purple line denotes the current EUCAST CB for BDQ testing (0.25 mg/L). Notable limitation: Studies 5 and 6 were conducted in the same laboratory. 3.B.3.3 Conclusion for BDQ CC for 7H11 An interim CC of 0.25 mg/L was adopted based on the H37Rv QC and pWT MICs, even though most clinical MIC data were from a single laboratory. However, this CC did not necessarily allow for the adequate differentiation of clinical mmpR mutants from the pWT population. Some mmpR mutants might have had genuinely low MICs if the mutations in question were genetic polymorphisms that did not affect the action of the repressor. Other mmpR mutations may only have minor functional consequences and may therefore only result in slight MIC increases, leading to a gNWT MIC distribution that overlaps with the pWT MIC distribution. 3. Clofazimine and bedaquiline 49 3.B.4 BDQ MIC data in MGIT 3.B.4.1 BDQ MICs for pWT isolates in MGIT Three studies presented BDQ MIC data for the pWT population with MGIT (Table 55): 1. Bloemberg et al., Keller et al. and Somoskovi et al. (Study 10) used a different dilution series than the other studies and used crushed BDQ tablets rather than pure compound for MIC testing. The pWT MIC distribution for the total 22 clinical isolates tested was 0.2-1.6 mg/L (with a mode at 0.4 mg/L). 2. Torrea et al. (Study 11) reported an MIC distribution of 0.12-1 mg/L (with a mode at 0.25 mg/L) for 37 replicates of H37Rv ATCC 27294. 72 clinical isolates had a pWT MIC distribution of ≤0.03-1 mg/L (with a mode at 0.25 mg/L). Moreover, two additional clinical isolates were retested for a total of 17 times, which resulted in a MIC distribution of 0.12-1 mg/L (with two modes: at 0.25 mg/L and 1 mg/L). 3. Ismail et al. (Study 12) retested the same collection that had been tested on 7H10 (the data on that medium had to be excluded for methodological reasons (Section 3.B.2.1)). 31 replicates of H37Rv ATCC 27294 had an MIC distribution of ≤0.12-1 mg/L (with a mode at 0.25 mg/L). Moreover, 382 gWT clinical isolates (mostly from patients without prior exposure to BDQ) were tested. A pWT MIC distribution of approximately ≤0.12-2 mg/L (with a mode at 0.5 mg/L) was reported. The MICs of a proportion of these isolates were repeated (Supplementary Table 5). Table 55. BDQ MICs for pWT isolates in MGIT. The purple line denotes the current EUCAST CB for BDQ testing (0.25 mg/L). Notable limitation: Study 10 did not use pure BDQ for MIC testing. 3.B.4.2 BDQ MICs for mutated isolates in MGIT In vitro and clinical isolates Torrea et al. (Study 11) reported MICs of ≥2 mg/L for 14 in vitro mmpR mutants (Table 56). One additional in vitro mmpR mutant was tested nine times, which resulted in MICs of >2 mg/L in all cases. In the same study, one mutant with an unknown resistance mechanism also had an MIC of >2 mg/L, as did the nine replicates of an in vitro atpE mutant. Three studies reported MIC data for clinical mmpR or pepQ mutants (Table 56). 1. Torrea et al. (Study 11) reported an MIC of >2 mg/L for a naturally resistant mmpR clinical mutant. 2. Bloemberg et al., Keller et al. and Somoskovi et al. (Study 10) reported an MIC of 6.4 mg/L for two replicates of an mmpR mutant that developed in response to treatment with CFZ, compared with 0.8 mg/L for the gWT parental isolate (this mutant was also cross-resistant to CFZ (Section 3.A.4.2)). It should be noted that a major limitation of this study was that crushed, rather than pure, BDQ compound was used for testing.Ismail et al. (Study 12) observed BDQ MICs of 2-8 mg/L for nine mmpR mutants, which included six isolates from XDR patients who were failing BDQ therapy. Of the remaining three mmpR mutants, only one was from a patient with a prior history of BDQ treatment (repeat testing of these mutants yielded MICs of 1-2 mg/L (Supplementary Table 5)). Four isolates harboured three different pepQ mutations with MICs of 0.5-1 mg/L. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 50 Table 56. BDQ MICs for in vitro and clinical atpE and mmpR mutants in MGIT. The purple line denotes the current EUCAST CB for BDQ testing (0.25 mg/L). Notable limitation: Study 10 did not use pure BDQ for MIC testing. 3.B.4.3 Conclusion for BDQ CC for MGIT Based on the pWT and H37Rv MIC data from Studies 11 and 12, an interim CC of 1 mg/L for MGIT was adopted. The findings of Study 10 supported this value, despite having used crushed BDQ and a different dilution series. All 16 mmpR or atpE in vitro mutants tested (100% (95% CI, 79-100%)) and all 10 clinical mmpR mutants tested (100% (95% CI, 69- 100%)) would be interpreted as BDQ-resistant at this concentration. However, even in MGIT there appeared to be some overlap between the mmpR MIC distributions and the pWT population, as demonstrated by repeat MIC testing (i.e. the three mmpR mutants from Ismail et al. (Study 11) with initial BDQ MICs of 2-4 mg/L had MICs of 1-2 mg/L upon retesting (Supplementary Table 5)). 3.B.5 References for BDQ MIC studies 1. Kaniga, K. et al. A multilaboratory, multicoun- try study to determine bedaquiline MIC quality control ranges for phenotypic drug suscepti- bility testing. J Clin Microbiol 54, 2956-2962 (2016). 2. Ismail, unpublished data. 3. Diacon, A.H. et al. 14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial. Lancet 380, 986-93 (2012). 4. Kaniga, K. et al. A multilaboratory, multicountry study to determine bedaquiline MIC quality control ranges for phenotypic drug susceptibility testing. J Clin Microbiol 54, 2956- 2962 (2016). 5. (a) Diacon, A.H. et al. Multidrug-resistant tuberculosis and culture conversion with bedaquiline. N Engl J Med 371, 723-32 (2014). (b) Diacon, A.H., Lounis, N. & Dannemann, B. Multidrug-resistant tuberculosis and bedaquiline. N Engl J Med 371, 2436 (2014). (c) Pym, A.S. et al. Bedaquiline in the treatment of multidrug- and extensively drug-resistant tuberculosis. Eur Respir J 47, 564-74 (2016). (d) Villellas, C. et al. Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline. J Antimicrob Chemother 72, 684-690 (2017). 6. Torrea, G. et al. Bedaquiline susceptibility testing of Mycobacterium tuberculosis in an automated liquid culture system. J Antimicrob Chemother 70, 2300-5 (2015). 7. Andries, K. et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307, 223-7 (2005). 8. Veziris, N. et al. Rapid emergence of Mycobacterium tuberculosis bedaquiline resistance: lessons to avoid repeating past errors. Eur Respir J 49, 1601719 (2017). 9. Zimenkov, D.V. et al. Examination of beda- quiline- and linezolid-resistant Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 72, 1901-1906 (2017) 10. (a) Bloemberg, G.V. et al. Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 373, 1986-8 (2015). (b) Keller, P.M. et al. Determination of MIC distribution and epidemiological cutoff values for 3. Clofazimine and bedaquiline 51 bedaquiline and delamanid in Mycobacterium tuberculosis using the MGIT 960 System equipped with TB eXiST. Antimicrob Agents Chemother 59, 4352-5 (2015). (c) Somoskovi, A., Bruderer, V., Hömke, R., Bloemberg, G.V. & Böttger, E.C. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J 45, 554-7 (2015). 11. Torrea, G. et al. Bedaquiline susceptibility testing of Mycobacterium tuberculosis in an automated liquid culture system. J Antimicrob Chemother 70, 2300-5 (2015). 12. Ismail, unpublished data. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 52 4. Cycloserine and terizidone 4.0 DCS and TRD MIC data stratification and current breakpoints No MIC data were identified for TRD. DCS MIC data were stratified based on ald (Rv2780) and alr (Rv3423c) mutations (a detailed discussion of the known resistance mechanisms for this drug can be found in the supplement). The start codon of alr has been wrongly annotated in the H37Rv reference genome (GenBank accession number AL123456.3), and therefore the experimentally confirmed start codon was used to report mutations in this gene.17 The only CC for DCS was set by WHO (Table 57).18 No CC currently exists for TRD DST. 17 Strych, U., Penland, R.L., Jimenez, M., Krause, K.L. & Benedik, M.J. Characterization of the alanine racemases from two mycobacteria. FEMS Microbiol Lett 196, 93-8 (2001). 18 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). http://apps.who.int/iris/ bitstream/10665/130918/1/9789241548809_eng.pdf?ua=1&ua=1 (accessed 13.8.2015). Table 57. Overview of current DCS and TRD CCs. LJ 7H10 7H11 MGIT WHO CLSI WHO CLSI WHO CLSI WHO CLSI 30.0 – – – – Red CCs (in mg/L) were set by only WHO. 4.1 DCS MIC data on LJ 4.1.1 DCS MICs for pWT isolates on LJ Nakatani et al. (Study 1) only reported MICs for H37Rv tested in two laboratories and thus provided little insight into the pWT MIC distribution (Table 58). Table 58. DCS MICs for pWT and mutated isolates on LJ. The red line denotes the current WHO CC for DCS on LJ (30 mg/L). 4.1.2 DCS MICs for mutated isolates on LJ Clinical isolates Nakatani et al. (Study 1) demonstrated that the acquisition of an alr mutation (C to T at position -8 relative to the start of the gene) during MDR treatment correlated with a DCS MIC increase from 15 to 60 mg/L (Table 58). Three additional alr mutations, one of which coincided with an ald mutation, also correlated with MICs above the current CC. Nakatani et al. provided additional evidence by molecular modelling and direct measurements of enzymatic activity that these alr mutations are likely responsible for DCS resistance. 4. Cycloserine and terizidone 53 4.1.3 Conclusion for DCS CC for LJ As the data quantity was insufficient to propose even a tentative CC, the current CC of 30 mg/L was withdrawn. 4.2 DCS MIC data on 7H10 4.2.1 DCS MICs for pWT isolates on 7H10 Two studies were identified that reported DCS MIC data for the pWT population on 7H10 (Table 59). Schön et al. (Study 2) tested 110 clinical isolates that had a pWT MIC distribution of 8-32 mg/L (with a mode at 32 mg/L). Pholwat et al. (Study 3) reported a pWT MIC distribution of 3.75-15 mg/L (with a mode at 15 mg/L) for 21 clinical isolates. Table 59. DCS MICs for pWT isolates on 7H10. 4.2.2 DCS MICs for mutated isolates on 7H10 No studies presenting MICs for mutated isolates were identified. 4.2.3 Conclusion for DCS CC for 7H10 Only two studies were identified that reported DCS MICs for 7H10. These data were deemed insufficient to propose a CC. 4.3 DCS MIC data on 7H11 4.3.1 DCS MICs for pWT isolates on 7H11 Only one study was identified that reported DCS MIC data for the pWT population on 7H11 (Table 60). Fattorini et al. (Study 4) tested 46 clinical isolates, which were enriched for resistance to other drugs, and found an MIC distribution of 7.5-60 mg/L (with a mode at 15 mg/L). Table 60. DCS MICs for pWT isolates on 7H11. 4.3.2 DCS MICs for mutated isolates on 7H11 No studies presenting MIC distributions for mutated isolates were identified. 4.3.3 Conclusion for DCS CC for 7H11 A single study was identified, which was insufficient to set a CC. 4.4 DCS MIC data in MGIT 4.4.1 DCS MICs for pWT isolates in MGIT Only one study was identified that used MGIT to determine DCS MICs (Table 61). Nakatani et al. (Study 5) tested four closely-related clinical isolates, which all had a MIC of 16 mg/L compared to an MIC of 4 mg/L for H37Rv ATCC 27294. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 54 Table 61. DCS MICs for pWT and mutated isolates in MGIT. 19 Köser, C.U. et al. Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 369, 290-2 (2013). 4.4.2 DCS MICs for mutated isolates in MGIT Clinical isolates The alr mutation M319T, which has been predicted to result in DCS resistance through molecular modelling,19 was shown to correlate with a DCS MIC of 64 mg/L in MGIT compared to an MIC of 16 mg/L for closely-related alr wild type control isolates (Table 61). 4.4.3 Conclusion for DCS CC for MGIT The data from the single study identified were insufficient to set a CC. 4.5 References for DCS MIC studies 1. Nakatani, Y. et al. Role of alanine racemase mutations in Mycobacterium tuberculosis D-cycloserine resistance. Antimicrob Agents Chemother 61, e01575-17 (2017). 2. Schön, T. et al. Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis. Int J Tuberc Lung Dis 15, 502-9 (2011). 3. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 4. Fattorini, L. et al. Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis. Microb Drug Resist 5, 265-70 (1999). 5. Nakatani, Y. et al. Role of alanine racemase mutations in Mycobacterium tuberculosis D-cycloserine resistance. Antimicrob Agents Chemother 61, e01575-17 (2017). 5. Linezolid 55 5. Linezolid 5.0 LZD MIC data stratification and current breakpoints LZD MIC data were stratified based on mutations in the rrl (MTB000020) and rplC (Rv0701) genes (a detailed discussion of the known resistance mechanisms for this drug can be found in the supplement). In presenting the nucleotide changes for rrl mutants, two numbers were given: the first number represents the nucleotide position in M. tuberculosis, whereas the second number represents the corresponding nucleotide position in Escherichia coli. The latter numbering system is more commonly used to report mutations in this gene. To date, CLSI and WHO have only set a CC for LZD DST in MGIT (Table 62).20, 21 20 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). 21 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). http://apps.who.int/iris/ bitstream/10665/130918/1/9789241548809_eng.pdf?ua=1&ua=1 (accessed 13.8.2015). 22 European Committee for Antimicrobial Susceptibility Testing. Standard Operating Procedure. MIC distributions and the setting of epidemiological cutoff (ECOFF) values. 17 November 2017. Table 62. Overview of current LZD CCs. LJ 7H10 7H11 MGIT WHO CLSI WHO CLSI WHO CLSI WHO CLSI – – – 1.0 Green CCs (in mg/L) were set by both WHO and CLSI. 5.1 LZD MIC data on LJ 5.1.1 LZD MICs for pWT isolates on LJ No studies presenting MIC distributions for pWT isolates on LJ were identified. 5.1.2 LZD MICs for mutated isolates on LJ No studies with MIC distributions for mutated isolates on LJ were found. 5.1.3 Conclusion for LZD CC for LJ Given that no MIC data were identified for LJ, a CC could not be defined. 5.2 LZD MIC data on 7H10 5.2.1 LZD MICs for pWT isolates on 7H10 10 studies were identified that reported LZD MIC data for the pWT population on 7H10 (Table 63). The distribution in the study by Wang et al. (Study 1) was bimodal and consequently had to be excluded based on EUCAST rules for aggregating MIC data.22 The remaining nine studies featured more than 650 MICs for pWT isolates with modes at either 0.25 or 0.5 mg/L. These data suggested a CC of 1 mg/L for DST on 7H10. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 56 Table 63. LZD MICs for pWT isolates on 7H10. Notable limitation: The distribution in Study 1 was bimodal. 5.2.2 LZD MICs for mutated isolates on 7H10 No studies presenting MIC distributions for gNWT strains on 7H10 were identified. 5.2.3 Conclusion for LZD CC for 7H10 A CC of 1 mg/L was adopted for DST on 7H10. 5.3 LZD MIC data on 7H11 5.3.1 LZD MICs for pWT isolates on 7H11 Three studies were identified that reported LZD MIC data for the pWT population on 7H11 (Table 64). These studies featured more than 300 isolates, and only two MICs were truncated at the lower end of the pWT MIC distributions, which had modes of either 0.25 or 0.5 mg/L. These data suggest a CC of 1 mg/L for DST on 7H11. Table 64. LZD MICs for pWT isolates on 7H11. 5. Linezolid 57 5.3.2 LZD MICs for mutated isolates on 7H11 No studies presenting MIC distributions for gNWT strains on 7H11 were identified. 5.3.3 Conclusion for LZD CC for 7H11 A CC of 1 mg/L for DST on 7H11 was adopted. 5.4 LZD MIC data in MGIT 5.4.1 LZD MICs for pWT isolates in MGIT 12 studies were identified that reported LZD MIC data for the pWT population in MGIT (Table 65). Their distributions, including in Rüsch- Gerdes et al. (Study 23), which has been cited by the current CLSI guidelines to support a CC of 1 mg/L for MGIT, were often truncated at the lower or upper ends.23 Therefore little information regarding the shape of the distributions could be obtained. Where the modes could be defined, they varied between 0.25 to 1 mg/L, suggesting more inter-laboratory variation than on 7H10 and 7H11 (Sections 5.2.1 and 5.3.1). 23 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 65. LZD MICs for pWT isolates in MGIT. The green line denotes the current WHO and CLSI CC for LZD DST in MGIT (1 mg/L). Notable limitation: Some laboratories are in common to Studies 15, 18-20 and 23-25. 5.4.2 LZD MICs for mutated isolates in MGIT In vitro and clinical isolates Hillemann et al. and Beckert et al. (study 25) conducted in vitro selection experiments using six different parental strains with MICs of ≤1 mg/L in MGIT (Table 66). Four isolates with the rplC C154R mutation were obtained, as well as five isolates with two distinct rrl mutations (G2299/2061T and G2814/2576T). The LZD MICs of these mutants, including one isolate for which the resistance mechanism could not be elucidated, were all 4-32 mg/L. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 58 Table 66. LZD MICs for in vitro mutants in MGIT. The green line denotes the current WHO and CLSI CC for LZD DST in MGIT (1 mg/L). Three studies with LZD MICs reported sequencing data for pNWT clinical isolates (Table 67). Richter et al. and Beckert et al. (Study 15) reported MICs of 4-8 mg/L for four resistant isolates that arose during treatment, compared to MICs of 0.5-1 mg/L for their respective parental strains. The three isolates with MICs of 8 mg/L were rplC C154R mutants. The fourth mutant did not have mutations in rrl (rplC was not sequenced). The MIC for the rplC C154R mutant from Perdigão et al. (Study 26) could have been either 2 or 4 mg/L, as 2 mg/L was not tested. Similarly, the MIC for the rrl A2810/2572C and G2814/2576T double mutant from Bloemberg et al. and Somoskovi et al. (Study 27) could have been either 8 or 16 mg/L, as 8 mg/L was not tested. Table 67. LZD MICs for mutated clinical isolates in MGIT. The green line denotes the current WHO and CLSI CC for LZD DST in MGIT (1 mg/L). 5.3.3 Conclusion for LZD CC for MGIT Additional, untruncated MIC distributions for pWT isolates with sequence data are needed to better define the degree of overlap between pWT and pNWT distributions and the variation between different laboratories and/or different datasets. In the absence of these data, 1 mg/L was reaffirmed as the CC. 5.5 References for LZD MIC studies 1. Wang, J.Y. et al. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 59, 860-5 (2007). 2. Weiss, T. et al. Low minimal inhibitory concentrations of linezolid against multidrug- resistant tuberculosis strains. Eur Respir J 45, 285-7 (2015). 3. Schön, T. et al. Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis. Int J Tuberc Lung Dis 15, 502-9 (2011). 4. Ahmed, I., Jabeen, K., Inayat, R. & Hasan, R. Susceptibility testing of extensively drug-resistant and pre-extensively drug- resistant Mycobacterium tuberculosis against levofloxacin, linezolid, and amoxicillin- clavulanate. Antimicrob Agents Chemother 57, 2522-5 (2013). 5. McGee, B. et al. Population pharmacokinetics of linezolid in adults with pulmonary tuberculosis. Antimicrob Agents Chemother 53, 3981-4 (2009). 5. Linezolid 59 6. Koh, W.J. et al. Daily 300 mg dose of linezolid for the treatment of intractable multidrug- resistant and extensively drug-resistant tuberculosis. J Antimicrob Chemother 64, 388- 91 (2009). 7. Alcalá, L. et al. In vitro activities of linezolid against clinical isolates of Mycobacterium tuberculosis that are susceptible or resistant to first-line antituberculous drugs. Antimicrob Agents Chemother 47, 416-7 (2003). 8. Yang, C. et al. In vitro activity of linezolid against clinical isolates of Mycobacterium tuberculosis, including multidrug-resistant and extensively drug-resistant strains from Beijing, China. Jpn J Infect Dis 65, 240-2 (2012). 9. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 10. Van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 11. Rodríguez, J.C., Ruiz, M., López, M. & Royo, G. In vitro activity of moxifloxacin, levofloxacin, gatifloxacin and linezolid against Mycobacterium tuberculosis. Int J Antimicrob Agents 20, 464-7 (2002). 12. Kazemian, H. et al. Antimycobacterial activity of linezolid against multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis in Iran. Int J Antimicrob Agents 45, 668-70 (2015). 13. (a) Rey-Jurado, E., Tudó, G., de la Bellacasa, J.P., Espasa, M. & González-Martín, J. In vitro effect of three-drug combinations of antituberculous agents against multidrug- resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents 41, 278-80 (2013). (b) Rey-Jurado, E., Tudo, G., Soy, D. & Gonzalez-Martin, J. Activity and interactions of levofloxacin, linezolid, ethambutol and amikacin in three-drug combinations against Mycobacterium tuberculosis isolates in a human macrophage model. Int J Antimicrob Agents 42, 524-30 (2013). (c) López-Gavín, A. et al. In vitro time-kill curves study of three antituberculous combinations against Mycobacterium tuberculosis clinical isolates. Int J Antimicrob Agents 47, 97-100 (2016). 14. Yip, P.C., Kam, K.M., Lam, E.T., Chan, R.C. & Yew, W.W. In vitro activities of PNU-100480 and linezolid against drug-susceptible and drug- resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents 42, 96-7 (2013). 15. (a) Richter, E., Rüsch-Gerdes, S. & Hillemann, D. First linezolid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 51, 1534-6 (2007). (b) Beckert, P. et al. rplC T460C identified as a dominant mutation in linezolid-resistant Mycobacterium tuberculosis strains. Antimicrob Agents Chemother 56, 2743-5 (2012). 16. Dheda, K. et al. Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study. Lancet Respir Med 5, 269-281 (2017). 17. Ismail, unpublished data. 18. Van Ingen, J. et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 48, 2749-53 (2010). 19. Alffenaar, J.W. et al. Susceptibility of clinical Mycobacterium tuberculosis isolates to a potentially less toxic derivate of linezolid, PNU- 100480. Antimicrob Agents Chemother 55, 1287-9 (2011). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 60 20. Werngren, unpublished data. 21. Sharma, M. et al. Canadian multicenter laboratory study for standardized second- line antimicrobial susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 49, 4112-6 (2011). 22. Mirza, I.A., Satti, L., Khan, F.A. & Khan, K.A. In vitro evaluation of linezolid and meropenem against clinical isolates of multi drug resistant tuberculosis by Mycobacterial Growth Indicator Tube (MGIT) 960. J Coll Physicians Surg Pak 25, 427-30 (2015). 23. Rüsch-Gerdes, S., Pfyffer, G.E., Casal, M., Chadwick, M. & Siddiqi, S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 44, 688- 92 (2006). 24. Cambau, E. et al. Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 70, 686-96 (2015). 25. (a) Hillemann, D., Rüsch-Gerdes, S. & Richter, E. In vitro-selected linezolid-resistant Mycobacterium tuberculosis mutants. Antimicrob Agents Chemother 52, 800-1 (2008). (b) Beckert, P. et al. rplC T460C identified as a dominant mutation in linezolid-resistant Mycobacterium tuberculosis strains. Antimicrob Agents Chemother 56, 2743-5 (2012). 26. Perdigão, J. et al. Beyond extensively drug- resistant tuberculosis in Lisbon, Portugal: a case of linezolid resistance acquisition presenting as an iliopsoas abscess. Int J Antimicrob Agents (2016). 27. (a) Bloemberg, G.V. et al. Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 373, 1986-8 (2015). (b) Somoskovi, A., Bruderer, V., Hömke, R., Bloemberg, G.V. & Böttger, E.C. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J 45, 554-7 (2015). 6. Delamanid 61 6. Delamanid 6.0 DLM MIC data stratification and current breakpoints DLM MIC data were stratified based on mutations in the five known resistance genes: ddn (Rv3547), fgd1 (Rv0407), fbiA (Rv3261), fbiB (Rv3262), and fbiC (Rv1173) (a detailed discussion of the known resistance mechanisms for this drug can be found in the supplement). Since many isolates had mutations in resistance genes, only those mutations that correlated with elevated MICs were shown in the tables (i.e. all other mutations were regarded as gWT). No breakpoints for DLM testing have been defined by CLSI, FDA or WHO to date. In parallel with the marketing authorization by European Medicines Agency, EUCAST has designed 0.06 mg/L as a medium-independent CB for DLM testing.24 Otsuka, meanwhile, has proposed 0.05 mg/L as the ECOFF and 0.2 mg/L as the CC for DLM testing on both 7H10 and 7H11.25 6.1 DLM MIC data on LJ 6.1.1 DLM MICs for pWT isolates on LJ No studies presenting MIC distributions for pWT isolates on LJ were identified. 6.1.2 DLM MICs for mutated isolates on LJ No studies with MIC distributions for mutated isolates on LJ were found. 6.1.3 Conclusion for DLM CC for LJ Given that no MIC data were identified for LJ, a CC could not be defined. 6.2 DLM MIC data on 7H10 6.2.1 DLM MICs for pWT isolates on 7H10 One study was identified that reported DLM MIC data for 99 clinical isolates on 7H10 (Table 68). Dataset 1 included 24 baseline isolates from trial 102 and Dataset 2 consisted of 52 baseline isolates from trial 101. Dataset 3 featured 23 additional clinical isolates. With the exception of Dataset 1, which had a pWT MIC distribution of 0.006-0.025 mg/L (with a mode at 0.012 mg/L), modes could not be defined because the distributions were truncated at the lower end. 24 European Committee for Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 7.1, valid from 2017-03-10. http://www. eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_7.1_Breakpoint_Tables. xls (accessed 9.4.2017). 25 Stinson, K. et al. MIC of delamanid (OPC-67683) against Mycobacterium tuberculosis clinical isolates and a proposed critical concentration. Antimicrob Agents Chemother 60, 3316-22 (2016). Table 68. DLM MICs for pWT isolates on 7H10. The purple line denotes the current EUCAST CB for DLM testing (0.06 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 62 6.2.2 DLM MICs for mutated isolates on 7H10 No studies presenting MICs for mutated isolates on 7H10 were identified. 6.2.3 Conclusion for DLM CC for 7H10 All 7H10 MICs for clinical isolates were from a single laboratory, which precluded an assessment of the inter-laboratory reproducibility of testing. Moreover, many MICs were truncated. Consequently, these data were deemed insufficient to define a CC. 6.3 DLM MIC data on 7H11 6.3.1 DLM MICs for pWT isolates on 7H11 Two studies were identified that reported DLM MIC data for the pWT population on 7H11 (Table 69). Gler et al. and Stinson et al. (Study 2) presented data from two laboratories. Dataset 4 featured 45 clinical isolates from Japan, whereas Dataset 5 contained 314 pWT baseline isolates from trial 204. Only one MIC was truncated and the modes of the distributions in both laboratories were 0.004 mg/L. Schena et al. (Study 3) retested 13 baseline isolates from trial 204, which were also part of Dataset 5, and a 14th isolate provided by Otsuka that was not part of that trial. This is consequently not an independent dataset and could only provide information regarding the inter-laboratory reproducibility of MIC testing. The isolate that was not part of trial 204 had an unusually high MIC of 0.12 mg/L. The 13 isolates from trial 204 had MICs within the range of Study 2 (i.e. 0.002- 0.016 mg/L), but had two modes (one at 0.004 and a second at 0.016 mg/L). However, the sample size of this study was small. Table 69. DLM MICs for pWT isolates on 7H11. The purple line denotes the current EUCAST CB for DLM testing (0.06 mg/L). Notable limitation: 13 isolates from Dataset 5a represent a subset of Dataset 5. 6.3.2 DLM MICs for mutated isolates on 7H11 Clinical isolates Two studies reported MICs for mutated isolates on 7H11 (Table 70). Gler et al. and Stinson et al. (Study 2) described two naturally resistant clinical isolates from Egypt and the Republic of Korea from trial 204. DLM resistance in these isolates must have evolved independently, as they harboured unique ddn mutations (L107P and a 43bp deletion, respectively). Schena et al. (Study 3) retested the naturally re- sistant isolate with the ddn L107P mutation from Study 2 and confirmed an MIC of 1 mg/L. Four ddn mutants with three unique mutations that arose during treatment in trials NCT02573350 and NCT01131351 were also tested. 6. Delamanid 63 Table 70. DLM MICs for mutated clinical isolates on 7H11. The purple line denotes the current EUCAST CB for DLM testing (0.06 mg/L). Notable limitation: The same ddn L107P mutant was tested in both studies. 6.3.3 Conclusion for DLM CC for 7H11 Most 7H11 MICs came from just two laboratories. Nevertheless, more than 350 unique pWT isolates were tested, and so an interim CC of 0.016 mg/L was defined for this medium. All mutant isolates were detected at this CC. 6.4 DLM MIC data in MGIT 6.4.1 DLM MICs for pWT isolates in MGIT Two studies were identified that reported DLM MIC data for the pWT population in MGIT (Table 71). Schena et al. (Study 4) included data from two laboratories. Laboratory 4 tested a total of 149 gWT isolates, which formed a pWT MIC distribution of 0.002-0.06 mg/L (with a mode at 0.016 mg/L). This included the 13 baseline iso- lates from trial 204 and a 14th isolate provided by Otsuka (Dataset 5a) that had also been tested by Schena et al. on 7H11 (Study 3). The remain- ing 135 isolates were clinical isolates from other sources (Dataset 6). 51 isolates from the latter set were retested in laboratory 5 using two different dilution series (Dataset 6a). The distribution of the 20 isolates that were not truncated had a mode at 0.008 mg/L. 95 additional, unique pWT iso- lates were tested using a limited dilution series, which resulted in a truncated distribution (Data- set 7). Bloemberg et al. and Keller et al. (Study 5) used crushed DLM tablets rather than pure compound for MIC testing. They reported a pWT MIC distribution of 0.005-0.04 mg/L (with a mode at 0.01 mg/L) for 20 clinical isolates. Table 71. DLM MICs for pWT isolates in MGIT. The purple line denotes the current EUCAST CB for DLM testing (0.06 mg/L). Notable limitations: Dataset 6a represents a subset of Dataset 6. Study 5 did not use pure DLM for MIC testing. 6.4.2 DLM MICs for mutated isolates in MGIT Clinical isolates The same two studies mentioned in Section 6.4.1 (Studies 4 and 5) included MICs for mutated isolates in MGIT (Table 72). In Dataset 5a, Schena et al. (Study 4) tested a naturally resistant ddn L107P mutant, as well as four ddn mutants that arose during the course of trials NCT02573350 and NCT01131351 (these had also been tested on 7H11 in Studies 2 and 3). Moreover, three additional naturally resistant MDR Beijing isolates, which all harboured a nonsense mutation at codon 88 of ddn, and a fourth naturally resistant XDR Beijing isolate with a mutation at codon 250 of fbiA were tested in laboratories 4 and 5 (Datasets 6 and 6a). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 64 Bloemberg et al. and Keller et al. (Study 5), which did not use pure DLM for MIC testing, reported on the acquisition of DLM resistance during treatment. The identified fbiA D49T mutant had an MIC of >0.32 mg/L, compared to 0.01 mg/L for the gWT parent strain. Table 72. DLM MICs for mutated clinical isolates in MGIT. The purple line denotes the current EUCAST CB for DLM testing (0.06 mg/L). Notable limitations: The isolates in Datasets 6 and 6a are identical. Study 5 did not use pure DLM for MIC testing. 6.4.3 Conclusion for DLM CC for MGIT MGIT MICs were available from two laboratories that used pure compound and a third laboratory that relied on crushed DLM. Despite these methodological differences, the pWT MIC distributions were comparable and supported an interim CC of 0.06 mg/L. As was the case for 7H11, more data are required to strengthen this conclusion for MGIT. 6.5 References for DLM MIC studies 1. (a) Diacon, A.H. et al. Early bactericidal activity of delamanid (OPC-67683) in smear- positive pulmonary tuberculosis patients. Int J Tuberc Lung Dis 15, 949-54 (2011). (b) Stinson, K. et al. MIC of delamanid (OPC- 67683) against Mycobacterium tuberculosis clinical isolates and a proposed critical concentration. Antimicrob Agents Chemother 60, 3316-22 (2016). 2. (a) Gler, M.T. et al. Delamanid for multidrug- resistant pulmonary tuberculosis. N Engl J Med 366, 2151-2160 (2012) (b) Stinson, K. et al. MIC of delamanid (OPC- 67683) against Mycobacterium tuberculosis clinical isolates and a proposed critical concentration. Antimicrob Agents Chemother 60, 3316-22 (2016). 3. Schena, E. et al. Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC MGIT 960 system. J Antimicrob Chemother 71, 1532-9 (2016). 4. Schena, E. et al. Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC MGIT 960 system. J Antimicrob Chemother 71, 1532-9 (2016). 5. (a) Bloemberg, G.V. et al. Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 373, 1986-8 (2015). (b) Keller, P.M. et al. Determination of MIC distribution and epidemiological cutoff values for bedaquiline and delamanid in Mycobacterium tuberculosis using the MGIT 960 System equipped with TB eXiST. Antimicrob Agents Chemother 59, 4352-5 (2015). 7. Fluoroquinolones 65 7. Fluoroquinolones 7.0 FQ MIC data stratification and current breakpoints FQ MIC data were stratified based on mutations in gyrA (Rv0006) and gyrB (Rv0005) (details regarding these resistance mechanisms can be found in the supplement). For gyrA, only mutations within the QRDR spanning codons 74-109 were noted for this report, with the exception of T80A, A90G and S95T, which were not included in this report as these are considered natural polymorphisms.26, 27, 28 The 1998 numbering system, as described by Maruri et al., was used for gyrB mutations, as this is the same nomenclature employed by version 2 of the Hain Lifescience Genotype MTBDRsl assay. For gyrB, only mutations in codons 485- 543 were reported (codons 500-540 represent the QRDR according to Pantel et al. 2012, and the MTBDRsl v2 assay interrogates codons 536-541).29, 30 For Malik et al., all mutations were reported, as this study generated allelic exchange mutants to specifically study the role of putative polymorphisms and mutations within and outside of the gyrase QRDR regions.31 We excluded isolates with mutations in more than one resistance gene from the discussion, although these data are available in the supplementary MIC files. Version 2 of the Hain GenoType MTBDRsl assay interrogates mutations in both genes, whereas version 1 includes only gyrA (the mutations that are specifically targeted by mutation probes by this assay are highlighted in bold, whereas mutations that merely inferred by lack of binding of a wild type probe are underlined in the tables of this report). CLSI has set only CCs for the FQs, whereas WHO has also set CBs for MFX (Table 73).32, 33 26 Feuerriegel, S., Köser, C.U. & Niemann, S. Phylogenetic polymorphisms in antibiotic resistance genes of the Mycobacterium tuberculosis complex. J Antimicrob Chemother 69, 1205-10 (2014). 27 Ajileye, A. et al. Some synonymous and nonsynonymous gyrA mutations in Mycobacterium tuberculosis lead to systematic false-positive fluoroquinolone resistance results with the Hain GenoType MTBDRsl assays. Antimicrob Agents Chemother 61, e02169-16 (2017). 28 Maruri, F. et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 67, 819-31 (2012). 29 Pantel, A. et al. Extending the definition of the GyrB quinolone resistance-determining region in Mycobacterium tuberculosis DNA gyrase for assessing fluoroquinolone resistance in M. tuberculosis. Antimicrob Agents Chemother 56, 1990-6 (2012). 30 Tagliani, E. et al. Diagnostic performance of the new version of GenoType MTBDRsl (V2.0) assay for detection of resistance to fluoroquinolones and second line injectable drugs: a multicenter study. J Clin Microbiol 53, 2961-9 (2015). 31 Malik, S., Willby, M., Sikes, D., Tsodikov, O.V. & Posey, J.E. New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations. PLoS One 7, e39754 (2012). 32 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). 33 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). http://apps.who.int/iris/bitstream/10665/130918/1/9789241548809_eng. pdf?ua=1&ua=1 (accessed 13.8.2015). Table 73. Overview of current FQ CCs and CBs. LJ 7H10 7H11 MGIT Drug WHO CLSI WHO CLSI WHO CLSI WHO CLSI OFX 4.0 – 2.0 2.0 2.0 LFX – – 1.0 – 1.5 GFX – – 1.0 – – – MFX – – 0.5 & 2.0 0.5 – 0.5 0.5 & 2.0 0.25 Green CCs were set by both the WHO and CLSI; red CCs or CBs were set by WHO; blue CCs were set by CLSI. All concentrations are in mg/L. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 66 7.A.1 OFX MIC data on LJ 7.A.1.1 OFX MICs for pWT isolates on LJ Six studies from three laboratories were identified that reported OFX MIC data for the pWT population on LJ (Table 74). Most distributions were truncated at the lower end and Fabry et al. (Study 5) tested a non-standard dilution range. The insight into the shape of the pWT MIC distribution was therefore limited, but the current CC of 4 mg/L appeared to be appropriate. Table 74. OFX MICs for pWT isolates on LJ. The red line denotes the current WHO CC for OFX on LJ (4 mg/L). Notable limitations: Studies 1-4 were conducted in the same laboratory and Study 6 was enriched for isolates resistant to OFX. 7.A.1.2 OFX MICs for mutated isolates on LJ gyrA mutants Clinical isolates 67 clinical isolates from the same laboratory, har- bouring gyrA mutations that are targeted by the MTBDRsl assays, were tested on LJ medium (Table 75). Based on the current OFX CC for this medium, only 3 of these isolates (4% (95% CI, 1-13%) were phenotypically OFX-susceptible. 100% (95% CI, 29-100%) of these OFX-suscep- tible mutants had gyrA A90V or D94A mutations. Table 75. OFX MICs for clinical gyrA mutants on LJ. The red line denotes the current WHO CC for OFX DST on LJ (4 mg/L). Notable limitation: All studies were conducted in the same laboratory. gyrB mutants Clinical isolate Only a single gyrB mutant was tested on LJ and found to be resistant to OFX, which would be interpreted as conferring resistance with the MTBDRsl v2 assay (Table 76). 7. Fluoroquinolones 67 Table 76. OFX MICs for clinical gyrB mutant on LJ. The red line denotes the current WHO CC for OFX DST on LJ (4 mg/L). 7.A.1.3 Conclusion for OFX CC for LJ Although the identified MIC data were limited, the current OFX CC of 4 mg/L was maintained given the importance of LJ testing in some settings. Low-level gyrA mutants can be misclassified at this concentration because of the inherent variation in testing. However, it should be noted that testing of OFX is not recommended as it is no longer used to treat drug resistant-TB and laboratories should transition to testing the specific FQs used in treatment regimens. 7.A.2 OFX MIC data on 7H10 7.A.2.1 OFX MICs for pWT isolates on 7H10 10 studies from nine laboratories were identified that reported OFX MIC data for the pWT popula- tion on 7H10 (Table 77). Some variation in testing was apparent, as the modes of the distributions varied between 0.25 and 1 mg/L. The current CC of 2 mg/L therefore appeared to be appropriate. 7.A.2.2 OFX MICs for mutated isolates on 7H10 gyrA mutants Allelic exchange results Malik et al. (Study 13) generated allelic exchange mutants using either H37Rv, Erdman or CDC1551 (Table 78). The gyrA A74S, T80A, G247S, and A384V mutations (or combinations thereof) did not change the OFX MICs significantly compared to the parent strains. This also applied to the A90G mutation, which has been noted to cause a systematic- false positive result with the MTBDRsl assays.34 In contrast, the increase for the low-level A90V mutants were significant (i.e. >2 doubling dilutions), but spanned the CC (with reported MICs of 2-8 mg/L). This suggested that the MIC of A90V was close to the CC, which likely results in poor DST reproducibility for isolates with this mutation due to the normal technical variation inherent in MIC testing. This was not the case for the high-level D94G mutants, which had OFX MICs of between 8 and >16 mg/L. 34 Ajileye, A. et al. Some synonymous and nonsynonymous gyrA mutations in Mycobacterium tuberculosis lead to systematic false-positive fluoroquinolone resistance results with the Hain GenoType MTBDRsl assays. Antimicrob Agents Chemother 61, e02169-16 (2017). Table 78. OFX MICs for gyrA allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for OFX DST on 7H10 (2 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 68 Ta bl e 77 . O FX M IC s f or p W T iso la te s o n 7H 10 . Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r O FX D ST o n 7H 10 (2 m g/ L) . N ot ab le lim ita tio n: S tu di es 8 a nd 9 w er e co nd uc te d in th e sa m e lab or at or y. 7. Fluoroquinolones 69 Clinical isolates Six studies were identified with 166 MICs for isolates with gyrA mutations that are targeted by the MTBDRsl assays (Table 79). Based on the current OFX CC for this medium, 34 of these MICs were below the current CC (20% (95% CI, 15-27%)). 17 of these ‘susceptible’ results (50% (95% CI, 32-68%) were due to the gyrA A90V and D94A mutations. Methodological variation in MIC testing likely accounted for these results, in line the allelic exchange data for gyrA A90V. gyrB mutants Allelic exchange results Malik et al. (Study 13) generated allelic exchange mutants using H37Rv and Erdman (Table 80). The OFX MICs of some of these gyrB mutants were elevated above the current OFX CC (e.g. D500H mutants had MICs 4-8 mg/L in both genetic backgrounds). However, the majority of OFX MICs for gyrB mutants were 0.5-2 mg/L, and so these isolates were consequently OFX- susceptible based on the current OFX CC of 2 mg/L, as were the gWT control strains (MICs of 0.5 mg/L). These isolates included several mu- tants that would be interpreted as OFX-resistant by the MTBDRsl v2 assay (e.g. N538K). Howev- er, the OFX MICs for several of these ‘LPA-resis- tant’ isolates were equal to the CC, suggesting that these mutations only result in modest MIC increases. Table 80. OFX MICs for gyrB allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for OFX DST on 7H10 (2 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 70 Ta bl e 79 . O FX M IC s f or c lin ic al g yr A m ut an ts o n 7H 10 . Th e gr ee n lin e de no te s th e cu rre nt W HO a nd C LS I C C fo r O FX D ST o n 7H 10 (2 m g/ L) . 7. Fluoroquinolones 71 Clinical isolates Excluding the gyrB V340L mutation, which does not confer FQ resistance based on the aforementioned allelic exchange results, 12 isolates from four studies had gyrB mutations (Table 81). These included eight isolates that would be considered resistant by the MTBDRsl v2 assay, of which two were susceptible (25% (95% CI, 3-65%)). Table 81. OFX MICs for clinical gyrB mutants on 7H10. The green line denotes the current WHO and CLSI CC for OFX DST on 7H10 (2 mg/L). 7.A.2.3 Conclusion for OFX CC for 7H10 The current CC of 2 mg/L was reaffirmed based upon the identified MIC data, but it was apparent that because of the variation in testing known gyrA resistance mutations, particularly those that confer low-level resistance, can be misclassified as susceptible. However, it should be noted that testing of OFX is not recommended as it is no longer used to treat drug resistant-TB and laboratories should transition to testing the specific FQs used in treatment regimens. 7.A.3 OFX MIC data on 7H11 7.A.3.1 OFX MICs for pWT isolates on 7H11 Four studies were identified that reported OFX MIC data for the pWT population on 7H11 (Table 82). Given the truncations and lack of genotypic data for some studies, insights into the shape of the pWT MIC distributions were limited. Table 82. OFX MICs for pWT isolates on 7H11. The green line denotes the current WHO and CLSI CC for OFX DST on 7H11 (2 mg/L). Notable limitation: Study 20 was enriched for OFX-resistant isolates. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 72 7.A.3.2 OFX MICs for mutated isolates on 7H11 gyrA mutants Clinical isolates 44 isolates from two studies tested on 7H10 harboured gyrA mutations that are targeted by the MTBDRsl assays (Table 83). Based on the current OFX CC, two of these mutants (5% (95% CI, 1-15%) were phenotypically OFX-sus- ceptible. Table 83. OFX MICs for mouse and clinical gyrA mutants on 7H11. The green line denotes the current WHO and CLSI CC for OFX DST on 7H11 (2 mg/L). gyrB mutants Mouse isolates 12 gyrB mutants that arose during the treatment of mice with LFX were tested on 7H11, of which two had mutations that are targeted by the MTBDRsl v2 assay (Table 84). One of these isolates was susceptible at the current CC (50% (95% CI, 1-99%)). Table 84. OFX MICs for mouse gyrB mutants on 7H11. The green line denotes the current WHO and CLSI CC for OFX DST on 7H11 (2 mg/L). 7. Fluoroquinolones 73 7.A.3.3 Conclusion for OFX CC for 7H11 It was difficult to define the upper end of the pWT MIC distribution due to the truncations of MICs and a lack of supporting genetic data for some studies. Nevertheless, the current CC of 2 mg/L was maintained. However, it should be noted that testing of OFX is not recommended as it is no longer used to treat drug resistant-TB and laboratories should transition to testing the specific FQs used in treatment regimens. 7.A.4 OFX MIC data in MGIT 7.A.4.1 OFX MICs for pWT isolates in MGIT 12 studies were identified with OFX MIC data for pWT isolates tested in MGIT (Table 85). Based on their results, Cambau et al. (Study 30) suggested that the current CC should be lowered from 2 to 1 mg/L. Assessing this proposal was complicated by the fact that the pWT MIC distributions from most of the remaining studies, including Rüsch-Gerdes et al. (Study 28) and Rodrigues et al. (Study 29), which are cited in the CLSI guidelines in support of the current CC of 2 mg/L, were truncated.35 This was compounded by the lack of genotypic data for several studies. 35 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 85. OFX MICs for pWT isolates in MGIT. The green line denotes the current WHO and CLSI CC for OFX DST in MGIT (2 mg/L). Notable limitations: Studies 25 and 29 were conducted in the same laboratory. Some laboratories were in common to Studies 28, 30 and 32. The genotypic results in Study 25 were based on the MTBDRsl v1. 7.A.4.2 OFX MICs for mutated isolates in MGIT gyrA mutants Clinical isolates 262 isolates from six studies had gyrA mutations that are targeted by the MTBDRsl assays (Table 86). Of these, only four mutants (2% (95% CI, 0-4%) were susceptible to OFX. Given that these four isolates, of which two (50% (95% CI, 7-93%)) were gyrA A90V or D94A mutants, had MICs equal to the CC (2 mg/L), methodological variation in MIC testing likely accounted for these results. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 74 Table 86. OFX MICs for clinical gyrA mutants in MGIT. The green line denotes the current WHO and CLSI CC for OFX DST in MGIT (2 mg/L). Notable limitation: The genotypic results in Study 25 were based on the MTBDRsl v1. gyrB mutants Clinical isolates Three isolates from one study had gyrB mutations that are targeted by the MTBDRsl v2 assay (Table 87). Two of these mutants were susceptible, with an MIC of 1.5 mg/L (67% (95% CI, 9-99%)). Table 87. OFX MICs for clinical gyrB mutants in MGIT. The green line denotes the current WHO and CLSI CC for OFX DST in MGIT (2 mg/L). 7. Fluoroquinolones 75 7.A.4.3 Conclusion for OFX CC for MGIT Based on the data from their multi-centre investigation, Cambau et al. (Study 30) proposed that the current CC for MGIT should be lowered from 2 mg/L to 1 mg/L.36 As almost all identified studies reported truncated MIC distributions, little is known about the shape of the pWT MIC distribution in MGIT. This finding was compounded by the fact that some key studies lacked sequencing data, which would have helped to define the upper end of the pWT MIC distribution. In the end, the current CC of 2 mg/L was maintained, both based on the pWT MIC data and the fact that only 2% (95% CI, 0-4%) of gyrA mutants were misclassified as susceptible at this concentration. However, it should be noted that testing of OFX is not recommended as it is no longer used to treat drug resistant-TB and laboratories should transition to testing the specific FQs used in treatment regimens. 7.A.5 References for OFX MIC studies 1. Rigouts, L. et al. Specific gyrA gene mutations predict poor treatment outcome in MDR-TB. J Antimicrob Chemother 71, 314-23 (2016). 2. Coeck, N. et al. Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis. J Antimicrob Chemother 71, 1233-40 (2016). 3. Barletta, F., Zamudio, C., Rigouts, L. & Seas, C. Resistance to second-line anti-tuberculosis drugs among Peruvian multidrug resistant Mycobacterium tuberculosis strains. Rev Peru Med Exp Salud Publica 31, 676-82 (2014). 4. Vincent, V. et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int J Tuberc Lung Dis 16, 24-31 (2012). 5. Fabry, W., Schmid, E.N. & Ansorg, R. Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium tuberculosis. Zentralbl Bakteriol 282, 394-401 (1995). 6. Sulochana, S., Venkataraman, P. & Paramasivan, C.N. Evaluation of various methods of susceptibility to ofloxacin in strains of Mycobacterium tuberculosis. Indian J Med Res 110, 186-9 (1999). 7. Wang, J.Y. et al. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 59, 860-5 (2007). 8. Ängeby, K.A. et al. Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data. J Antimicrob Chemother 65, 946-52 (2010). 9. Niward, K. et al. Susceptibility testing breakpoints for Mycobacterium tuberculosis categorize isolates with resistance mutations in gyrA as susceptible to fluoroquinolones: implications for MDR-TB treatment and the definition of XDR-TB. J Antimicrob Chemother 71, 333-8 (2016). 10. (a) Pholwat, S., Heysell, S., Stroup, S., Foongladda, S. & Houpt, E. Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR. J Clin Microbiol 49, 69-75 (2011). (b) Pholwat, S., Ehdaie, B., Foongladda, S., Kelly, K. & Houpt, E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50, 754-61 (2012). (c) Pholwat, S. et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6, e02273 (2015). 11. Rey-Jurado, E., Tudó, G., Martínez, J.A. & González-Martín, J. Synergistic effect of two combinations of antituberculous drugs against Mycobacterium tuberculosis. Tuberculosis (Edinb) 92, 260-3 (2012). 36 Cambau, E. et al. Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 70, 686-96 (2015). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 76 12. Karak, K. & De, P.K. Comparative in vitro activity of fluoroquinolones against Mycobacterium tuberculosis. Indian J Med Res 101, 147-9 (1995). 13. Malik, S., Willby, M., Sikes, D., Tsodikov, O.V. & Posey, J.E. New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations. PLoS One 7, e39754 (2012). 14. (a) Blackman, A. et al. Microcolonies in fluoroquinolone agar proportion susceptibility testing of Mycobacterium tuberculosis: an indicator of drug resistance. Eur J Clin Microbiol Infect Dis 31, 2177-82 (2012). (b) Devasia, R. et al. High proportion of fluoroquinolone-resistant Mycobacterium tuberculosis isolates with novel gyrase polymorphisms and a gyrA region associated with fluoroquinolone susceptibility. J Clin Microbiol 50, 1390-6 (2012). (c) Van der Heijden, Y.F. et al. Fluoroquinolone susceptibility in Mycobacterium tuberculosis after pre-diagnosis exposure to older- versus newer-generation fluoroquinolones. Int J Antimicrob Agents 42, 232-7 (2013). (d) Eilertson, B. et al. High proportion of heteroresistance in gyrA and gyrB in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother 58, 3270-3275 (2014). 15. (a) Farhat, M.R. et al. Concordance of Mycobacterium tuberculosis fluoroquinolone resistance testing: implications for treatment. Int J Tuberc Lung Dis 19, 339-41 (2015). (b) Farhat, M.R. et al. Gyrase mutations are associated with variable levels of fluoroquinolone resistance in Mycobacterium tuberculosis. J Clin Microbiol 54, 727-33 (2016). 16. Hu, Y. et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in rural China. Antimicrob Agents Chemother 57, 3857-63 (2013). 17. (a) Fattorini, L. et al. Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis. Microb Drug Resist 5, 265-70 (1999). (b) Giannoni, F. et al. Evaluation of a new line probe assay for rapid identification of gyrA mu- tations in Mycobacterium tuberculosis. Antimi- crob Agents Chemother 49, 2928-33 (2005). 18. Rodríguez, J.C., Ruiz, M., Climent, A. & Royo, G. In vitro activity of four fluoroquinolones against Mycobacterium tuberculosis. Int J Antimicrob Agents 17, 229-31 (2001). 19. Bernard, C. et al. In vivo Mycobacterium tuberculosis fluoroquinolone resistance emergence: a complex phenomenon poorly detected by current diagnostic tests. J Antimicrob Chemother 71, 3465-3472 (2016). 20. Sulochana, S., Venkataraman, P. & Paramasivan, C.N. Evaluation of various methods of susceptibility to ofloxacin in strains of Mycobacterium tuberculosis. Indian J Med Res 110, 186-9 (1999). 21. Sturegård, E. et al. Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10. Clin Microbiol Infect 21, 148 e5-7 (2015). 22. Alvarez, N. et al. The structural modeling of the interaction between levofloxacin and the Mycobacterium tuberculosis gyrase catalytic site sheds light on the mechanisms of fluoroquinolones resistant tuberculosis in Colombian clinical isolates. Biomed Res Int 2014, 367268 (2014). 23. Sirgel, F.A. et al. gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis. J Antimicrob Chemother 67, 1088-93 (2012). 24. Kam, K.M. et al. Stepwise decrease in moxi- floxacin susceptibility amongst clinical isolates of multidrug-resistant Mycobacterium tuberculosis: correlation with ofloxacin susceptibility. Microb Drug Resist 12, 7-11 (2006). 25. Kambli, P. et al. Correlating minimum inhibitory concentrations of ofloxacin and moxifloxacin with gyrA mutations using the Genotype MTBDRsl assay. Tuberculosis (Edinb) 95, 137-41 (2015). 26. Rigouts, unpublished data. 7. Fluoroquinolones 77 27. Zheng, X. et al. Determination of MIC breakpoints for second-line drugs associated with clinical outcomes in multidrug-resistant tuberculosis treatment in China. Antimicrob Agents Chemother 60, 4786-92 (2016). 28. Rüsch-Gerdes, S., Pfyffer, G.E., Casal, M., Chadwick, M. & Siddiqi, S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 44, 688- 92 (2006). 29. Rodrigues, C. et al. Drug susceptibility testing of Mycobacterium tuberculosis against second- line drugs using the Bactec MGIT 960 System. Int J Tuberc Lung Dis 12, 1449-55 (2008). 30. Cambau, E. et al. Revisiting susceptibility testing in MDR-TB by a standardized quanti- tative phenotypic assessment in a European multicentre study. J Antimicrob Chemother 70, 686-96 (2015). 31. Sharma, M. et al. Canadian multicenter laboratory study for standardized second- line antimicrobial susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 49, 4112-6 (2011). 32. Springer, B., Lucke, K., Calligaris-Maibach, R., Ritter, C. & Böttger, E.C. Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter instrumentation. J Clin Microbiol 47, 1773-80 (2009). Table 88. LFX MICs for pWT isolates on LJ. 7.B.1.2 LFX MICs for mutated isolates on LJ gyrA mutants Clinical isolates 34 isolates from a single study by Coeck et al. (Study 1) harboured gyrA mutations that are targeted by the MTBDRsl assays and were tested on LJ medium (Table 89). Only a single mutant with a gyrA D94A mutation has MICs of ≤2 mg/L. Table 89. LFX MICs for clinical gyrA mutants on LJ. 7.B.1 LFX MIC data on LJ 7.B.1.1 LFX MICs for pWT isolates on LJ Only one study was identified that reported LFX MIC data for the pWT MIC distribution on LJ (Table 88). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 78 gyrB mutants Clinical isolates MIC data was found for just one clinical gyrB mutant, which would be interpreted as resistant using the MTBDRsl v2 assay (Table 90). Table 90. LFX MICs for clinical gyrB mutants on LJ. 7.B.1.3 Conclusion for LFX CC for LJ LJ represents the only medium available for DST in many high-TB burden settings. Therefore, an interim CC of 2 mg/L was set even though only a single study was identified for this drug- medium combination. This concentration was chosen as it corresponds to half the OFX CC on LJ (OFX consists of equal amounts of the active L-isomer of OFX (i.e. LFX) and the largely inactive D-isomer, which means that LFX is about twice as potent as OFX).37 This concentration was also in line with the limited pWT and pNWT MIC data presented by Coeck et al. (Study 1). 7.B.2 LFX MIC data on 7H10 7.B.2.1 LFX MICs for pWT isolates on 7H10 Eight studies were identified, including Sanders et al. (Study 7), which has been cited in the CLSI guidelines in support of the current CC of 1 mg/L, that reported LFX MIC data for the pWT population on 7H10 (Table 91).38 The modes of these distributions varied between 0.12 and 0.5 mg/L and thus supported the current CC. 37 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis (2014). http://apps.who.int/iris/bitstream/10665/130918/1/9789241548809_eng. pdf?ua=1&ua=1 (accessed 13.8.2015). 38 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 91. LFX MICs for pWT isolates on 7H10. The green line denotes the current WHO and CLSI CC for LFX DST on 7H10 (1 mg/L). Notable limitations: Studies 3 and 4, and Studies 6 and 8 were conducted in the same laboratory, respectively. 7. Fluoroquinolones 79 7.B.2.2 LFX MICs for mutated isolates on 7H10 gyrA mutants Allelic exchange results Malik et al. (Study 8) generated allelic exchange mutants using H37Rv, Erdman and CDC1551 (Table 92). The gyrA A74S, T80A, G247S, and A384V mutations (or combinations thereof) did not change the LFX MICs significantly (i.e. no MICs for these mutants were above the current LFX CC of 1 mg/L). This also applied to isolates with the A90G mutation, which has been noted to cause a systematic-false positive result with the MTBDRsl assays.39 As seen for OFX, the LFX MICs of the A90V mutants spanned the CC (with reported MICs of 0.5-4 mg/L), whereas the D94G mutants had LFX MICs of 8-16 mg/L. 39 Ajileye, A. et al. Some synonymous and nonsynonymous gyrA mutations in Mycobacterium tuberculosis lead to systematic false-positive fluoroquinolone resistance results with the Hain GenoType MTBDRsl assays. Antimicrob Agents Chemother 61, e02169-16 (2017). Table 92. LFX MICs for gyrA allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for LFX DST on 7H10 (1 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 80 Table 93. LFX MICs for clinical gyrA mutants on 7H10. The green line denotes the current WHO and CLSI CC for LFX DST on 7H10 (1 mg/L). Notable limitation: Studies 6 and 8 were conducted in the same laboratory. Clinical isolates 150 isolates from four studies that were tested in three laboratories had gyrA mutations that are targeted by the MTBDRsl assays (Table 93). Based on the current CC, 18 (12% (95% CI, 7-18%) of these mutants were phenotypically LFX-susceptible. Eight of these ‘susceptible’ isolates (44% (95% CI, 22-69%) harboured gyrA A90V or D94A mutations. 7. Fluoroquinolones 81 gyrB mutants Allelic exchange results Malik et al. (Study 8) generated allelic exchange mutants using H37Rv and Erdman (Table 94). The LFX MICs of some of these gyrB mutants were elevated above the current LFX CC (e.g. D500H mutants had MICs 2-4 mg/L in both ge- netic backgrounds tested). However, the major- ity of LFX MICs were ≤0.25-1 mg/L, and were consequently LFX-susceptible based on the current LFX CC of 1 mg/L, as were the gWT control strains (MICs of ≤0.25 mg/L). These iso- lates included several mutants that would be in- terpreted as resistant by the MTBDRsl v2 assay (e.g. N538K). However, the LFX MICs for sev- eral of these ‘LPA-resistant’ isolates were equal to the CC, suggesting that these mutations may only result in modest MIC increases. Table 94. LFX MICs for gyrB allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for LFX DST on 7H10 (1 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 82 Clinical isolates 11 isolates from two studies featured gyrB mutants that were tested on 7H10 (Table 95). One of the six isolates with mutations that are targeted by the MTBDRsl v2 assay tested susceptible on two occasions (17% (95% CI, 0-64%). Table 95. LFX MICs for clinical gyrB mutants on 7H10. The green lines denote the current WHO and CLSI CC for LFX DST on 7H10 (1 mg/L). Notable limitation: Studies 6 and 8 were conducted in the same laboratory. 7.B.2.3 Conclusion for LFX CC for 7H10 The current CC of 1 mg/L was reaffirmed, but it was noted that because of variation in testing certain gyrA mutants can be misclassified as susceptible at this concentration. 7.B.3 LFX MIC data on 7H11 7.B.3.1 LFX MICs for pWT isolates on 7H11 Four studies reported LFX MIC data for the pWT population on 7H11 (Table 96). Two of these studies reported MICs for at least 10 isolates. Table 96. LFX MICs for pWT isolates on 7H11. 7. Fluoroquinolones 83 7.B.3.2 LFX MICs for mutated isolates on 7H11 gyrA mutants Clinical isolates Two studies featured MICs on 7H11 for 44 isolates with gyrA mutations that are targeted by the MTBDRsl assays (Table 97). 42 of these isolates (96% (95% CI, 85-99%) had MICs >1 mg/L. Table 97. LFX MICs for mouse and clinical gyrA mutants on 7H11. gyrB mutants Mouse isolates 12 gyrB mutants that arose during the treatment of mice with LFX were tested on 7H11. Two of these isolates had mutations that are targeted by the MTBDRsl v2 assay (Table 98), with MICs that ranged from 0.5 to 2 mg/L. Table 98. LFX MICs for mouse gyrB mutants on 7H11. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 84 7.B.3.3 Conclusion for LFX CC for 7H11 Given that only two studies were identified with more than 10 pWT isolates tested on 7H11, no CC was set for this medium. 7.B.4 LFX MIC data in MGIT 7.B.4.1 LFX MICs for pWT isolates in MGIT 12 studies from 10 laboratories reported LFX MICs for the pWT population in MGIT (Table 99). The shape of most pWT distributions, including for Lin et al. (Study 22) which has been cited in the CLSI guidelines in support of the current CC, could not be assessed because of truncations.40 Where modes were identifiable, they ranged from 0.25 to 0.5 mg/L, which consequently pointed to 1 mg/L as the CC as opposed to the current concentration of 1.5 mg/L. 40 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 99. LFX MICs for pWT isolates in MGIT. The green line denotes the current WHO and CLSI CC for LFX DST in MGIT (1.5 mg/L). Notable limitations: Studies 16, 22 and 25, and Studies 17 and 18 were conducted in the same laboratory, respectively. The genotypic results for Study 14 were based on a combination of sequencing and a microarray, whereas Study 23 relied on the MTBDRsl v1. 7. Fluoroquinolones 85 7.B.4.2 LFX MICs for mutated isolates in MGIT gyrA mutants Clinical isolates Five studies featured MICs for 241 gyrA mutants that would be interpreted as resistant by the MTBDRsl assays (Table 100). Using the current CC of 1.5 mg/L, 29 of these mutants would be misclassified as susceptible (12% (95% CI, 8-17%). Lowering the CC to 1 mg/L would likely improve the detection of at least some of these ‘susceptible’ mutants, given that 28 (97% (95% CI, 82-100%) had MICs of 1.12-1.5 mg/L. Table 100. LFX MICs for clinical gyrA mutants in MGIT. The green line denotes the current WHO and CLSI CC for LFX DST in MGIT (1.5 mg/L). Notable limitations: The genotypic results for Study 14 were based on a combination of sequencing and a microarray, whereas Study 23 relied on the MTBDRsl v1. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 86 gyrB mutants Clinical isolates Four studies from three laboratories reported MGIT MICs for 13 gyrB mutants (Table 101). This included five isolates that would be considered resistant using the MTBDRsl v2 assay, yet four of the resulting MICs (67% (95% CI, 22-96%)) were susceptible at the current CC. The two MICs for the gyrB N538D mutant were 1 and 2 mg/L, which suggested that this mutation conferred MICs close to the CC, resulting in poor reproducibility of testing. Table 101. LFX MICs for clinical gyrB mutants in MGIT. The green line denotes the current WHO and CLSI CC for LFX DST in MGIT (1.5 mg/L). Notable limitations: Studies 17 and 18 were conducted in the same laboratory. The genotypic results for Study 14 were based on a combination of sequencing and a microarray. 7.B.4.3 Conclusion for LFX CC for MGIT The combined MIC data supported 1 mg/L as the CC, as opposed to the current breakpoint of 1.5 mg/L. The CC was lowered accordingly. 7.B.5 References for LFX MIC studies 1. Coeck, N. et al. Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis. J Antimicrob Chemother 71, 1233-40 (2016). 2. Wang, J.Y. et al. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 59, 860-5 (2007). 3. Ängeby, K.A. et al. Wild-type MIC distribu- tions of four fluoroquinolones active against My- cobacterium tuberculosis in relation to current critical concentrations and available pharmaco- kinetic and pharmacodynamic data. J Antimi- crob Chemother 65, 946-52 (2010). 4. Niward, K. et al. Susceptibility testing breakpoints for Mycobacterium tuberculosis categorize isolates with resistance mutations in gyrA as susceptible to fluoroquinolones: implications for MDR-TB treatment and the definition of XDR-TB. J Antimicrob Chemother 71, 333-8 (2016). 5. Peloquin, C.A. et al. Population pharmaco- kinetics of levofloxacin, gatifloxacin, and moxi- floxacin in adults with pulmonary tuberculosis. Antimicrob Agents Chemother 52, 852-7 (2008). 6. Willby, M., Sikes, R.D., Malik, S., Metchock, B. & Posey, J.E. Correlation between gyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 59, 5427-34 (2015). 7. Sanders, C.A., Nieda, R.R. & Desmond, E.P. Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol 42, 5225-8 (2004). 7. Fluoroquinolones 87 8. Malik, S., Willby, M., Sikes, D., Tsodikov, O.V. & Posey, J.E. New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations. PLoS One 7, e39754 (2012). 9. Hu, Y. et al. Prevalence and genetic char- acterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tu- berculosis in rural China. Antimicrob Agents Chemother 57, 3857-63 (2013). 10. Rodríguez, J.C., Ruiz, M., López, M. & Royo, G. In vitro activity of moxifloxacin, levofloxacin, gatifloxacin and linezolid against Mycobacterium tuberculosis. Int J Antimicrob Agents 20, 464-7 (2002). 11. Giannoni, F. et al. Evaluation of a new line probe assay for rapid identification of gyrA mu- tations in Mycobacterium tuberculosis. Antimi- crob Agents Chemother 49, 2928-33 (2005). 12. (a) Rey-Jurado, E., Tudó, G., de la Bellacasa, J.P., Espasa, M. & González-Martín, J. In vitro effect of three-drug combinations of antituberculous agents against multidrug- resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents 41, 278-80 (2013). (b) Rey-Jurado, E., Tudo, G., Soy, D. & Gonzalez-Martin, J. Activity and interactions of levofloxacin, linezolid, ethambutol and amikacin in three-drug combinations against Mycobacterium tuberculosis isolates in a human macrophage model. Int J Antimicrob Agents 42, 524-30 (2013). (c) López-Gavín, A., Tudó, G., Vergara, A., Hurtado, J.C. & Gonzalez-Martín, J. In vitro activity against Mycobacterium tuberculosis of levofloxacin, moxifloxacin and UB-8902 in combination with clofazimine and pretomanid. Int J Antimicrob Agents 46, 582-5 (2015). (d) López-Gavín, A. et al. In vitro time-kill curves study of three antituberculous combinations against Mycobacterium tuberculosis clinical isolates. Int J Antimicrob Agents 47, 97-100 (2016). 13. Bernard, C. et al. In vivo Mycobacterium tuberculosis fluoroquinolone resistance emer- gence: a complex phenomenon poorly de- tected by current diagnostic tests. J Antimicrob Chemother 71, 3465-3472 (2016). (a) Nosova, E.Y. et al. Analysis of mutations in the gyrA and gyrB genes and their association with the resistance of Mycobacterium tuberculosis to levofloxacin, moxifloxacin and gatifloxacin. J Med Microbiol 62, 108-13 (2013). (b) Zimenkov, D.V. et al. Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infect Dis 13, 240 (2013). 15. Sturegård, E. et al. Little difference between minimum inhibitory concentrations of Mycobac- terium tuberculosis wild-type organisms deter- mined with BACTEC MGIT 960 and Middlebrook 7H10. Clin Microbiol Infect 21, 148 e5-7 (2015). 16. Sanders, C.A., Nieda, R.R. & Desmond, E.P. Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol 42, 5225-8 (2004). 17. Tessema, B. et al. FIND tuberculosis strain bank: a resource for researchers and developers working on tests to detect Mycobacterium tuberculosis and related drug resistance. J Clin Microbiol 55, 1066-1073 (2017). 18. Heyckendorf, J. et al. What is resistance? Impact of phenotypic versus molecular drug resistance testing on multi- and extensively drug-resistant tuberculosis therapy. Antimicrob Agents Chemother, ahead of print (2017). 19. Zheng, X. et al. Determination of MIC breakpoints for second-line drugs associated with clinical outcomes in multidrug-resistant tuberculosis treatment in China. Antimicrob Agents Chemother 60, 4786-92 (2016). 20. Werngren, unpublished data. 21. Alvarez, N. et al. The structural modeling of the interaction between levofloxacin and the Mycobacterium tuberculosis gyrase catalytic site sheds light on the mechanisms of fluoroquinolones resistant tuberculosis in Colombian clinical isolates. Biomed Res Int 2014, 367268 (2014). 22. Lin, S.Y., Desmond, E., Bonato, D., Gross, W. & Siddiqi, S. Multicenter evaluation of Bactec MGIT 960 system for second-line drug susceptibility testing of Mycobacterium TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 88 tuberculosis complex. J Clin Microbiol 47, 3630-4 (2009). 23. Kambli, P. et al. Determination of MICs of levofloxacin for Mycobacterium tuberculosis with gyrA mutations. Int J Tuberc Lung Dis 19, 1227-9 (2015). 24. Rigouts, unpublished data. 25. Lin, unpublished data. 7.C.1 GFX MIC data on LJ 7.C.1.1 GFX MICs for pWT isolates on LJ Five studies were identified with GFX MIC data for the pWT population on LJ (Table 102). The MIC distributions were truncated at the lower end and only came from two laboratories. Somasundaram et al. (Study 1) was enriched for OFX-resistant isolates. Table 102. GFX MICs for pWT isolates on LJ. Notable limitations: Studies 2-5 were conducted in the same laboratory. Study 1 was enriched for resistant isolates. 7.C.1.2 GFX MICs for mutated isolates on LJ gyrA mutants Clinical isolates 137 isolates from the same laboratory harboured gyrA mutations that are targeted by the MTBDRsl assays (Table 103). Six of these mutants (5% (95% CI, 2-9%)) had MICs ≤0.5 mg/L. 100% (95% CI, 54-100%) of these isolates had gyrA A90V or D94A mutations. Table 103. GFX MICs for clinical gyrA mutants on LJ. Notable limitation: All studies were conducted in the same laboratory. 7. Fluoroquinolones 89 gyrB mutants Clinical isolates Seven clinical isolates from a single study had gyrB mutations (Table 104). Of the five isolates with mutations that are targeted by the MTBDRsl v2 assay, one (20% (95% CI, 1-72%)) had MICs ≤0.5 mg/L. Table 104. GFX MICs for clinical gyrB mutants on LJ. 7.C.1.3 Conclusion for GFX CC for LJ The pWT MICs distributions on LJ were severely truncated and only came from two laboratories. Despite these limitations, the consensus was to set 0.5 mg/L as an interim CC given that many high-TB burden settings use LJ for DST. Additional data from well-designed studies will be necessary to re-evaluate this CC. It was noted that because of the inherent variation in testing some gyrA mutants were misclassified at this concentration. 7.C.2 GFX MIC data on 7H10 7.C.2.1 GFX MICs for pWT isolates on 7H10 A single study with just 10 clinical isolates was identified with GFX MICs on 7H10 (Table 105). Table 105. GFX MICs for pWT isolates on 7H10. The red line denotes the current WHO CC for OFX DST on 7H10 (1 mg/L). 7.C.2.2 GFX MICs for mutated isolates on 7H10 No studies were found with MICs for mutants on 7H10. 7.C.2.3 Conclusion for GFX CC for 7H10 A single study with just 10 clinical isolates was identified by this review and no data were available for gNWT populations. The current GFX CC of 1 mg/L was therefore withdrawn. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 90 7.C.3 GFX MIC data on 7H11 7.C.3.1 GFX MICs for pWT isolates on 7H11 Three studies were identified that reported GFX MIC data for pWT isolates on 7H11 (Table 106). Rodriguez et al. (Study 7) featured a substantial number of MICs that were not truncated. Somasundaram et al. (Study 8) was enriched for OFX-resistant isolates (20 of the 50 isolates tested were resistant to OFX using 2 mg/L as the CC with the absolute concentration method on LJ). Table 106. GFX MICs for pWT isolates on 7H11. Notable limitation: Study 8 was enriched for resistant isolates. 7.C.3.2 GFX MICs for mutated isolates on 7H11 gyrA mutants Clinical isolates 17 clinical isolates from a single study Giannoni et al. (Study 9) harboured gyrA mutations that are targeted by the MTBDRsl assays (Table 107). The MICs were 0.5-2 mg/L. Table 107. GFX MICs for clinical gyrA mutants on 7H11. 7.C.3.3 Conclusion for GFX CC for 7H11 Rodriguez et al. (Study 7) was the only study that featured a substantial number of MICs that were not truncated. The available data were considered insufficient to set a CC. 7. Fluoroquinolones 91 7.C.4 GFX MIC data in MGIT 7.C.4.1 GFX MICs for pWT isolates in MGIT Two studies used MGIT to measure the GFX MICs for pWT isolates in MGIT (Table 108). The untruncated MIC distribution in Isaeva et al., Nosova et al. & Zimenkov et al. (Study 10), which included MICs for 32 unique isolates, was 0.06-0.25 mg/L (with a mode at 0.12 mg/L). Table 108. GFX MICs for pWT isolates in MGIT. Notable limitation: Isolates in Study 10 were interrogated using a combination of sequencing and microarray. 7.C.4.2 GFX MICs for mutated isolates in MGIT gyrA mutants Clinical isolates Two studies from two laboratories featured 98 clinical isolates with gyrA mutations that are targeted by the MTBDRsl assays (Table 109). 10 of these isolates (10% (95% CI, 5-18%)) had MICs ≤0.25 mg/L. 100% (95% CI, 69-100%) of these had gyrA A90V or D94A mutations. Table 109. GFX MICs for clinical gyrA mutants in MGIT. Notable limitation: Isolates in Study 10 were interrogated using a combination of sequencing and microarray. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 92 gyrB mutants Clinical isolates Two studies tested a total of six clinical gyrB mutants in MGIT (Table 110). Of the four mutants that would be regarded as GFX-resistant using the MTBDRsl v2 assay, two (50% (95% CI, 7-93%) had MICs ≤0.25 mg/L. Table 110. GFX MICs for clinical gyrB mutants in MGIT. Notable limitation: Isolates in Study 10 were interrogated using a combination of sequencing and microarray. 7.C.4.3 Conclusion for GFX CC for MGIT Only two studies were identified that reported GFX MICs for pWT and mutated isolates in MGIT. Owing to the fact that MGIT is widely used, an interim CC of 0.25 mg/L was, nevertheless, set. Additional data from well-designed studies will be necessary to re-evaluate this CC. As was the case for other media and FQs, gyrA mutants can be misclassified as susceptible at this CC due to the overlap between the MIC distributions of pWT and mutated isolates. 7.C.5 References for GFX MIC studies 1. Somasundaram, S. & Paramasivan, N.C. Susceptibility of Mycobacterium tuberculosis strains to gatifloxacin and moxifloxacin by different methods. Chemotherapy 52, 190-5 (2006). 2. (a) Aung, K.J. et al. Successful ‘9-month Bangladesh regimen’ for multidrug-resistant tuberculosis among over 500 consecutive pa- tients. Int J Tuberc Lung Dis 18, 1180-7 (2014). (b) Rigouts, L. et al. Specific gyrA gene muta- tions predict poor treatment outcome in MDR- TB. J Antimicrob Chemother 71, 314-23 (2016). 3. Coeck, N. et al. Correlation of different pheno- typic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis. J Antimicrob Chemother 71, 1233-40 (2016). 4. Barletta, F., Zamudio, C., Rigouts, L. & Seas, C. Resistance to second-line anti-tuberculosis drugs among Peruvian multidrug resistant Mycobacterium tuberculosis strains. Rev Peru Med Exp Salud Publica 31, 676-82 (2014). 5. Vincent, V. et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int J Tuberc Lung Dis 16, 24-31 (2012). 6. Peloquin, C.A. et al. Population pharmacokinetics of levofloxacin, gatifloxacin, and moxifloxacin in adults with pulmonary tuberculosis. Antimicrob Agents Chemother 52, 852-7 (2008). 7. Rodríguez, J.C., Ruiz, M., López, M. & Royo, G. In vitro activity of moxifloxacin, levofloxacin, gatifloxacin and linezolid against Mycobacterium tuberculosis. Int J Antimicrob Agents 20, 464-7 (2002). 8. Somasundaram, S. & Paramasivan, N.C. Susceptibility of Mycobacterium tuberculosis strains to gatifloxacin and moxifloxacin by different methods. Chemotherapy 52, 190-5 (2006). 9. Giannoni, F. et al. Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 2928-33 (2005). 10. (a) Isaeva, Y. et al. Determination of critical concentrations of moxifloxacin and gatifloxacin 7. Fluoroquinolones 93 for drug susceptibility testing of Mycobacterium tuberculosis in the BACTEC MGIT 960 system. J Antimicrob Chemother 68, 2274-81 (2013). (b) Nosova, E.Y. et al. Analysis of mutations in the gyrA and gyrB genes and their association with the resistance of Mycobacterium tuberculosis to levofloxacin, moxifloxacin and gatifloxacin. J Med Microbiol 62, 108-13 (2013). (c) Zimenkov, D.V. et al. Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infect Dis 13, 240 (2013). 11. Rigouts, unpublished data. Table 111. MFX MIC distributions for the pWT population on LJ. Notable limitations: Studies 2-5 were conducted in the same laboratory. Study 1 was enriched for resistant isolates. 7.D.1.2 MFX MICs for mutated isolates on LJ gyrA mutants Clinical isolates 67 clinical isolates that were tested in the same laboratory harboured gyrA mutations that would be interpreted as conferring resistance using the MTBDRsl assays (Table 112). Nine of these mutants (13% (95% CI, 6-24%) had MICs ≤1 mg/L. Eight of these nine isolates (89% (95% CI, 52-100%)) had A90V or D94A mutations. Table 112. MFX MICs for clinical gyrA mutants on LJ. Notable limitation: All studies were conducted in the same laboratory. 7.D.1 MFX MIC data on LJ 7.D.1.1 MFX MICs for pWT isolates on LJ Five studies from two laboratories were identified that reported MFX MIC data for the pWT population on LJ (Table 111). Most distributions were truncated. Somasundaram et al. (Study 1) was enriched for OFX-resistant isolates (20 of the 50 isolates tested were resistant to OFX using 2 mg/L as the CC with the absolute concentration method on LJ). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 94 gyrB mutant Clinical isolate The only gyrB mutant identified, which would have been interpreted as FQ-resistant using the MTBDRsl v2 assay, had an MIC of >8 mg/L (Table 113). Table 113. MFX MICs for clinical gyrB mutants on LJ. 7.D.1.3 Conclusion for MFX CC and CB for LJ The quantity and quality of data identified for MFX on LJ were similar to GFX on LJ, and con- sequently the same limitations applied. Taking into account the diagnostic importance of LJ in many settings, an interim CC of 1 mg/L for LJ was set. It was noted that gyrA mutants can be misclassified as susceptible at this concentration due to variations in testing. More MICs for pWT isolates are needed to re-evaluate this CC. A bet- ter understanding of the distributions of low- and high-level gyrA mutants is needed before a CB can be defined on this medium (Section 7.D.5). 7.D.2 MFX MIC data on 7H10 7.D.2.1 MFX MICs for pWT isolates on 7H10 13 studies from 11 laboratories were identified that reported MFX MIC data for the pWT population on 7H10 (Table 114). Table 114. MFX MIC distributions for the pWT population on 7H10. The green line denotes the current WHO and CLSI CC for MFX DST on 7H10 (0.5 mg/L). The red line denotes the CB recommended by the WHO for MFX DST on 7H10 (2 mg/L). Notable limitations: Studies 11 and 15, and Studies 17 and 18 were conducted in the same laboratory, respectively. In Study 14, the genotypic results were based upon MTBDRsl v1 testing. 41 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). 7. Fluoroquinolones 95 Despite truncations in some studies, the modes of five studies could be assessed, including for Ängeby et al. (Study 6), which has been cited in the CLSI guidelines in support of the current CC.41 The modes of the various distributions varied between 0.06 and 0.25 mg/L, and therefore supported the current CC of 0.5 mg/L. 7.D.2.2 MFX MICs for mutated isolates on 7H10 gyrA mutants Allelic exchange results Malik et al. (Study 18) generated allelic exchange mutants using H37Rv, Erdman and CDC1551 (Table 115). The gyrA T80A, G247S, and A384V mutations (or combinations thereof) did not sig- nificantly change the MFX MICs (i.e. no MICs for these mutants were above the current MFX CC of 0.5 mg/L). This also applied to the A90G mutation, which has been noted to cause a systematic-false positive result by MTBDRsl assays.42 The MFX MICs of the low-level A90V mutants spanned the CC (with reported MICs of 0.5-1 mg/L), which was also the case for the single A74S mutant. By contrast, the D94G mutants had more highly elevated MFX MICs of 2-16 mg/L. 42 Ajileye, A. et al. Some synonymous and nonsynonymous gyrA mutations in Mycobacterium tuberculosis lead to systematic false-positive fluoroquinolone resistance results with the Hain GenoType MTBDRsl assays. Antimicrob Agents Chemother 61, e02169-16 (2017) Table 115. MFX MICs for gyrA allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for MFX DST on 7H10 (0.5 mg/L). The red line denotes the additional WHO CB for MFX DST on 7H10 (2 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 96 Clinical isolates MICs on 7H10 were identified for 177 isolates with gyrA mutations that are targeted by the MTBDRsl assays (Table 116). 23 of these mutants (13% (95% CI, 8-19%)) were phenotypically MFX-susceptible at the current CC. The gyrA A90V and D94G mutations accounted for 14 (61% (95% CI, 39-80%)) of these ‘susceptible’ results. Table 116. MFX MICs for clinical gyrA mutants on 7H10. The green line denotes the current WHO and CLSI CC for MFX DST on 7H10 (0.5 mg/L). The red line denotes the additional WHO CB for MFX DST on 7H10 (2 mg/L). Notable limitations: Studies 17 and 18 were conducted in the same laboratory. In Study 14, the genotypic results were based upon MTBDRsl v1 testing. 7. Fluoroquinolones 97 gyrB mutants Allelic exchange results Malik et al. (Study 18) generated allelic exchange mutants using H37Rv and Erdman (Table 117). The MFX MICs of some of the gyrB mutants were elevated above the current MFX CC (e.g. N538K mutants had MICs 1-2 mg/L in both genetic backgrounds tested). However, the majority of MFX MICs were ≤0.25-0.5 mg/L, and were consequently susceptible based on the current MFX CC of 0.5 mg/L, as were the gWT control strains (MICs of ≤0.25 mg/L). These isolates included several mutants that would be interpreted as resistant by the MTBDRsl v2 assay (e.g. T539N). However, the MFX MICs for several of these ‘LPA-resistant’ isolates were equal to the CC, suggesting that these mutations may only result in subtle MIC increases. Table 117. MFX MICs for gyrB allelic exchange mutants on 7H10. The green line denotes the current WHO and CLSI CC for MFX DST on 7H10 (0.5 mg/L). The red line denotes the additional WHO CB for MFX DST on 7H10 (2 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 98 Clinical isolates Excluding the gyrB V340L mutation, which does not cause FQ resistance, 16 clinical isolates from four studies harboured gyrB mutations (Table 118). These included nine isolates that would be considered resistant by the MTBDRsl v2 assay, of which two were susceptible at the CC (22%, 95% CI, 3-60%). Table 118. MFX MICs for clinical gyrB mutants on 7H10. The green line denotes the current WHO and CLSI CC for MFX DST on 7H10 (0.5 mg/L). The red line denotes the additional WHO CB for MFX DST on 7H10 (2 mg/L). Notable limitation: Studies 17 and 18 were conducted in the same laboratory. 7.D.2.3 Conclusion for MFX CC and CB for 7H10 Based upon the identified MIC data, the current MFX CC of 0.5 mg/L was reaffirmed, although there was some overlap between the pWT MIC distribution and gyrA mutants, which meant that some mutants were misclassified as susceptible. The CB was designed to distinguish low-level gyrA mutants (e.g. A90V and D94A) from high- level mutants (the rationale for this decision can be found in Section 7.D.5). However, it should also be noted that the lower end of the MIC distribution of high-level resistance mutations (e.g. gyrA D94G) appears to be 2 mg/L, meaning that a proportion of these mutants will be misclassified as low-level resistant using the current CB due to the inherent variation in testing. This could be avoided by lowering the CB to 1 mg/L at the expense of increasing substantially the proportion of low-level resistant isolates that are misclassified as high-level resistant. Faced with this trade-off, 2 mg/L was reaffirmed as the CB. However, it was suggested that in order to minimise misclassifying high- level resistant strains as low-level resistant, the identification of high-level resistance mutations using a molecular assay (e.g. LPA identification of a gyrA D94G mutation) might overrule a susceptible phenotypic DST result at 2 mg/L. This proposal will be addressed in a later report. 7. Fluoroquinolones 99 7.D.3 MFX MIC data on 7H11 7.D.3.1 MFX MICs for pWT isolates on 7H11 Five studies were identified that reported MFX MIC data for the pWT population on 7H11 (Table 119). This included Somasundaram et al. (Study 22), which has been cited in the CLSI guidelines in support of a CC.43 This study was enriched for OFX-resistant isolates (20 of the 50 isolates tested were resistant to OFX using 2 mg/L as the CC with the absolute concentration method on LJ). Given the modes of these MIC distributions, which varied between 0.12-0.5 mg/L, the current CLSI CC of 0.5 mg/L appeared to be appropriate. Table 119. MFX MICs for pWT isolates on 7H11. The blue line denotes the current CLSI CC for MFX DST on 7H11 (0.5 mg/L). Notable limitation: Study 22 was enriched for resistant isolates. 7.D.3.2 MFX MICs for mutated isolates on 7H11 gyrA mutants Clinical isolates 44 isolates from two studies were identified with gyrA mutations that are targeted by the MTBDRsl assays (Table 120). Only one mutant (2% (95% CI, 0-12%)) was susceptible at the CLSI CC. 43 Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, 2nd edition Approved standard. CLSI document M24-A2. (2011). Table 120. MFX MICs for mouse and clinical gyrA mutants on 7H11. The blue line denotes the current CLSI CC for MFX DST on 7H11 (0.5 mg/L). TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 100 gyrB mutants Clinical isolates 12 gyrB mutants that arose during the treatment of mice with LFX were tested on 7H11, of which two had mutations targeted by the MTBDRsl v2 assay (Table 121). One of these was MFX- susceptible at the current CC (50% (95% CI, 1-99%)). Table 121. MFX MICs for mouse gyrB mutants on 7H11. The blue line denotes the CLSI CC for MFX DST on 7H11 media. 7.D.3.3 Conclusion for MFX CC and CB for 7H11 Based on the data from the five studies identified in this review, 0.5 mg/L was adopted as the CC, but a better understanding of the distributions of low- and high-level gyrA mutants was needed before a CB could be defined on this medium (Section 7.D.5). 7. Fluoroquinolones 101 Table 122. MFX MICs for pWT isolates in MGIT. The blue line denotes the current CLSI CC for MFX DST in MGIT (0.25 mg/L). The red lines denote the current WHO CC and CB for MFX DST in MGIT (0.5 and 2 mg/L). Notable limitations: Studies 25 and 39 were conducted in the same laboratory and some laboratories were in common to Studies 27, 28, 31 and 35. In Study 24, the genotypic results were based upon a combination of sequencing and microarray, whereas Study 25 used the MTBDRsl v1. 7.D.4.2 MFX MICs for mutated isolates in MGIT gyrA mutants Clinical isolates 416 isolates from 12 studies had gyrA mutations that are targeted by the MTBDRsl assays (Table 123). Using the CLSI CC of 0.25 mg/L for MGIT, only 19 (5% (95% CI, 3-7%)) of these gyrA mutants were phenotypically MFX-susceptible. Notably, 100% (95% CI, 82-100%) of these ‘susceptible’ results, which were likely due to variation in testing, were for isolates with A90V and D94A mutations. 7.D.4 MFX MIC data in MGIT 7.D.4.1 MFX MICs for pWT isolates in MGIT 16 studies were identified that reported MFX MIC data for the pWT population in MGIT (Table 122). Despite the truncation of most MIC distributions, the CLSI CC of 0.25 mg/L, rather than the current WHO CC of 0.5 mg/L, appeared to correspond to the upper end of the pWT MIC distribution. TECHNICAL REPORT ON CRITICAL CONCENTRATIONS FOR DRUG SUSCEPTIBILITY TESTING OF MEDICINES USED IN THE TREATMENT OF DRUG-RESISTANT TUBERCULOSIS 102 Table 123. MFX MICs for clinical gyrA mutants in MGIT. The blue line denotes the current CLSI CC for MFX DST in MGIT (0.25 mg/L). The red lines denote the current WHO CC and CB for MFX DST in MGIT (0.5 and 2 mg/L). Notable limitations: Some laboratories were in common to Studies 27, 31 and 35. In Study 24, the genotypic results were based upon a combination of sequencing and microarray, whereas Study 25 used the MTBDRsl v1. 7. Fluoroquinolones 103 gyrB mutants Clinical isolates 13 isolates from three laboratories had gyrB mutations (Table 124). These included five isolates that would be considered resistant by the MTBDRsl v2 assay, of which one (20% (95% CI, 1-72%)) was susceptible at 0.25 mg/L. Table 124. MFX MICs for clinical gyrB mutants in MGIT. The blue line denotes the current CLSI CC for MFX DST in MGIT (0.25 mg/L). The red lines denote the current WHO CC and CB for MFX DST in MGIT (0.5 and 2 mg/L). Notable limitations: Studies 27 and 28 were conducted in the same laboratory. In Study 24, the genotypic results were based upon a combination of sequencing and microarray. 7.D.4.3 Conclusion for MFX CC and CB for MGIT The CC was lowered from 0.5 to 0.25 mg/L for MGIT. There was some overlap between the MIC distributions of low-level gyrA mutants and pWT isolates, resulting in false-susceptible results. The CB was also lowered from 2 to 1 mg/L, as this captured most low-level resistant mutants (e.g. gyrA A90V and D94A). As was the case on 7H10, the distributions of low-level and high-level mutants overlapped, which meant that some high-level mutants were misclassified even at the lower CB (the rationale for the CB can be found in Section 7.D.5). 7.D.5 Rationale for MFX CB This CC is relevant for the daily dose 400 mg of MFX. One study conducted by Rigouts et al. showed that despite the higher MIC values for non-wild type isolates the outcome was still better with a high dose of GFX than without a FQ being included in the regimen.44 This is likely caused by synergy between GFX and other drug compounds in the regimen. Therefore, given treatment with a combination regimen, a high dose of MFX is of additional value (extrapolated from treatment with a high dose of the GFX). At MIC values >1 mg/L of GFX on LJ, however, the success rate dropped dramatically.44, 45 Of note, however, the CBs (1 mg/L in MGIT and 2 mg/L in 7H10) are for a daily dose of 800 mg of MFX. For any strain that is MFX-resistant at the CB of 1 mg/L in MGIT or 2 mg/L in 7H10 (i.e. with MICs above these values), MFX should not be considered as an effective drug in the regimen. Strains that have elevated MICs above the CC but below or equal to the CB may be effectively treated with the higher daily dose of 800 mg MFX. 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