1. Vall d’Hebron Servei de Microbiologia External quality assurance for drug susceptibility testing of M. tuberculosis against second line anti-tuberculosis drugs S-J. Kim M. Zignyol M. Espinal M. Zignyol M. Espinal

2. Vall d’Hebron Servei de Microbiologia Participant laboratories 1.Bai G_H. Korean Institute of Tuberculosis Korea 2.Boulahbal F. Laboratoire de la Tuberculose Institut Pasteur Algeria 3.Fattorini L. Dipartimento di Malattie Infettive, Instituto Superiore di Sanità, Italy 4.Gilpin C. Queensland Mycobacterium Reference Laboratory, The Prince Charles Hospital, Australia 5.Hoffner S. Department of Bacteriology Swedish Institute for Infectious Disease Control Sweden 6.Kam KM. Tuberculosis Reference Laboratory Public Health Laboratory Centre, Hong Kong 7.Martín-Casabona N. Servicio de Microbiologia, Hospital Universitaris Vall d’Hebron, Spain 8.Mitarai S. Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Japan 9.Rigouts L. Mycobacteriology Unit, Institute of Tropical Medicine, Belgium 10.Rüsch- Gerdes S. National Reference Center for Mycobacteria Germany 1.Bai G_H. Korean Institute of Tuberculosis Korea 2.Boulahbal F. Laboratoire de la Tuberculose Institut Pasteur Algeria 3.Fattorini L. Dipartimento di Malattie Infettive, Instituto Superiore di Sanità, Italy 4.Gilpin C. Queensland Mycobacterium Reference Laboratory, The Prince Charles Hospital, Australia 5.Hoffner S. Department of Bacteriology Swedish Institute for Infectious Disease Control Sweden 6.Kam KM. Tuberculosis Reference Laboratory Public Health Laboratory Centre, Hong Kong 7.Martín-Casabona N. Servicio de Microbiologia, Hospital Universitaris Vall d’Hebron, Spain 8.Mitarai S. Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Japan 9.Rigouts L. Mycobacteriology Unit, Institute of Tropical Medicine, Belgium 10.Rüsch- Gerdes S. National Reference Center for Mycobacteria Germany

3. Vall d’Hebron Servei de Microbiologia 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3.To evaluate the proficiency of SLD susceptibility testing of the participating laboratories 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3.To evaluate the proficiency of SLD susceptibility testing of the participating laboratories OBJECTIVES

4. Vall d’Hebron Servei de Microbiologia Two laboratories collaborated 30 strains with resistance to SLD: Mycobacteriology Unit, Institute of Tropical Medicine, Belgium Korean Institute of Tuberculosis, Korea Two laboratories collaborated 30 strains with resistance to SLD: Mycobacteriology Unit, Institute of Tropical Medicine, Belgium Korean Institute of Tuberculosis, Korea Strains

5. Vall d’Hebron Servei de Microbiologia 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3. To evaluate the proficiency of SLD susceptibility testing of the participating laboratories 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3. To evaluate the proficiency of SLD susceptibility testing of the participating laboratories OBJECTIVES

6. Vall d’Hebron Servei de Microbiologia Reference methods  Proportion method on Löwenstein-Jensen  Proportion method on agar  MGIT medium for Bactec 960  Bactec 460  Proportion method on Löwenstein-Jensen  Proportion method on agar  MGIT medium for Bactec 960  Bactec 460

7. Vall d’Hebron Servei de Microbiologia Method: Agar dilution susceptibility testing Medium: Middlebrook 7H10 Antibiotics: Kanamycin, capreomycin, ofloxacin, PAS, ethiomamide, cycloserine Drug concentrations: Twofold dilutions from 0,5 µg/ml to 128 µg/ml Strains: Mycobacterium tuberculosis 69 and Mycobacterium bovis 3 Method: Agar dilution susceptibility testing Medium: Middlebrook 7H10 Antibiotics: Kanamycin, capreomycin, ofloxacin, PAS, ethiomamide, cycloserine Drug concentrations: Twofold dilutions from 0,5 µg/ml to 128 µg/ml Strains: Mycobacterium tuberculosis 69 and Mycobacterium bovis 3 Minimal inhibitory concentrations

8. Vall d’Hebron Servei de Microbiologia Kanamycin MIC (n = 72) Kanamycin MIC (n = 72) M I C µg/ml 128 64 32 16

9. Vall d’Hebron Servei de Microbiologia Capreomycin MIC (n = 72) Capreomycin MIC (n = 72) M I C µg/ml 128 64 32 16

10. Vall d’Hebron Servei de Microbiologia Ofloxacin MIC (n = 72) Ofloxacin MIC (n = 72) M I C µg/ml 32 16 4 4

11. Vall d’Hebron Servei de Microbiologia PAS MIC (n = 70) PAS MIC (n = 70) M I C µg/ml 128 64 32 16

12. Vall d’Hebron Servei de Microbiologia Ethionamide MIC (n = 70) Ethionamide MIC (n = 70) M I C µg/ml 128 64 32 16

13. Vall d’Hebron Servei de Microbiologia Cycloserine MIC (n = 71) Cycloserine MIC (n = 71) M I C µg/ml 128 64 32 16

14. Vall d’Hebron Servei de Microbiologia Reference methods vs MIC s of the strains evaluated (n = 72) Reference methods vs MIC s of the strains evaluated (n = 72) Drug Peack serum level (1) Breackpoint MIC Agreement KM35-45 µg/ml  16 µg/ml 69 % CPR35-45 µg/ml  16 µg/ml 96 % OFX8-10 µg/ml  2 µg/ml nd PAS70 µg/ml  16 µg/ml nd ETH4 µg/ml  4 µg/ml 65 % CS20 µg/ml  8 µg/ml ? (1) I. Bastian, F. Portaels. Multidrug-resistant tuberculosis. 2000 Kluwer Academic Publishers. The Netherlands.

15. Vall d’Hebron Servei de Microbiologia 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3. To evaluate the proficiency of SLD susceptibility testing of the participating laboratories 1.To collect strains from different laboratories 2.To determine the patterns of susceptibility or resistance of the strains against SLD 3. To evaluate the proficiency of SLD susceptibility testing of the participating laboratories OBJECTIVES

16. Vall d’Hebron Servei de Microbiologia Selected strains (duplicate) Selected strains (duplicate)

17. Vall d’Hebron Servei de Microbiologia Reference methods vs MIC s of the strains evaluated (n = 15) Reference methods vs MIC s of the strains evaluated (n = 15) Drug Breackpoint MIC Agreement Kanamycin  16 µg/ml 93 % Capreomycin  16 µg/ml 94 % Ofloxacin  2 µg/ml 80 % PAS  16 µg/ml 87 % Ethionamide  4 µg/ml 80 %

18. Vall d’Hebron Servei de Microbiologia Drugs proposed by IUATLD/ WHO Lot (Sigma-Aldrich) Kanamycin (KM)054K1169 Capreomycin (CPM)083K1373 Ofloxacin (OFX) 123K1138 Para-aminosalicilic acid (PAS)121K2522 Ethionamide (ETH) 044K1541 Cycloserine (CS)083K0702 Lot (Sigma-Aldrich) Kanamycin (KM)054K1169 Capreomycin (CPM)083K1373 Ofloxacin (OFX) 123K1138 Para-aminosalicilic acid (PAS)121K2522 Ethionamide (ETH) 044K1541 Cycloserine (CS)083K0702

19. Vall d’Hebron Servei de Microbiologia Parameters evaluated Sensitivity Specificity Predictive value resistance Predictive value susceptibility Efficiency Reproducibility Sensitivity Specificity Predictive value resistance Predictive value susceptibility Efficiency Reproducibility Drug Laboratories Serum level Drug Laboratories Serum level

20. Vall d’Hebron Servei de Microbiologia Laboratories Methods and drug concentrations Laboratories Methods and drug concentrations KM µg/ml CPR µg/ml OFX µg/ml PAS* µg/ml ETH µg/ml Proportion L-J 16 20 30 40 16 20 40 2 0.5 (5 labs) 1 (1 lab) 16 20 40 Proportion agar 5 (2 lab) 6 (1lab) 10 2 (2 lab) 1 (1lab) 2 (2 lab) 4 (1lab) 5 (1lab) 10 (2 lab) Prop. on: Löw-Jensen 4, Agar Middlebrook3 Bactec460 2 Absolute concentration1 Prop. on: Löw-Jensen 4, Agar Middlebrook3 Bactec460 2 Absolute concentration1 * 6 laboratories used L-J

21. Vall d’Hebron Servei de Microbiologia Strains evaluated Previous Judicial Result

22. Vall d’Hebron Servei de Microbiologia KMCPROFXPASETHCS Sensitivity100 6779- Specificity909697989290 PV resistance4065718778- PV susceptib.100 9592100 Efficiency909697948890 Reproductibility9493999587- All laboratories

23. Vall d’Hebron Servei de Microbiologia Efficiency of the different laboratories

24. Vall d’Hebron Servei de Microbiologia Efficiency of the different laboratories

25. Vall d’Hebron Servei de Microbiologia Efficiency of the different laboratories

26. Vall d’Hebron Servei de Microbiologia Efficiency of the different laboratories

27. Vall d’Hebron Servei de Microbiologia Global efficiency of the 10 laboratories

28. Vall d’Hebron Servei de Microbiologia Global efficiency / drugs

29. Vall d’Hebron Servei de Microbiologia KMCPROFXPASETH True resistant17 202763 False resistant2598418 True susceptible224240269253198 False susceptible0001316 TOTAL266 297 295 All drug All laboratories All drug All laboratories

30. Vall d’Hebron Servei de Microbiologia “Gold standard” If the gold standard is the Judicial Result Conclusion Excess of resistances!! If the gold standard is the Judicial Result Conclusion Excess of resistances!!

31. Vall d’Hebron Servei de Microbiologia Judicial Results vs MICs Kanamycin Judicial Results vs MICs Kanamycin 128 32 16 8 8 4 4 Previous JUDICIAL Previous JUDICIAL RSRS RSRS RRRR RRRR M I C µg/ml

32. Vall d’Hebron Servei de Microbiologia Judicial results vs MICs Capreomycin Judicial results vs MICs Capreomycin RSRS RSRS 32 16 8 8 4 4 64 Previous JUDICIAL Previous JUDICIAL NSNS NSNS RRRR RRRR M I C µg/ml

33. Vall d’Hebron Servei de Microbiologia Judicial results vs MICs Ofloxacin Judicial results vs MICs Ofloxacin 16 8 8 4 4 2 2 Previous JUDICIAL Previous JUDICIAL RSRS RSRS RSRS RSRS M I C µg/ml RSRS RSRS RRRR RRRR

34. Vall d’Hebron Servei de Microbiologia Judicial results vs MICs PAS Judicial results vs MICs PAS 128 64 32 16 Previous JUDICIAL Previous JUDICIAL RSRS RSRS RRRR RRRR M I C µg/ml RSRS RSRS RRRR RRRR

35. Vall d’Hebron Servei de Microbiologia Judicial results vs MICs Ethionamide Judicial results vs MICs Ethionamide 32 16 8 8 4 4 Previous JUDICIAL Previous JUDICIAL SRSR SRSR RSRS RSRS M I C µg/ml RSRS RSRS RRRR RRRR RRRR RRRR RRRR RRRR SSSS SSSS

36. Vall d’Hebron Servei de Microbiologia Judicial results vs MICs Cycloserine Judicial results vs MICs Cycloserine Previous JUDICIAL Previous JUDICIAL RSRS RSRS RSRS RSRS RSRS RSRS RSRS RSRS MIC µg/ml

37. Vall d’Hebron Servei de Microbiologia Judicial Results vs MICs Agreement of the round Judicial Results vs MICs Agreement of the round Drug Breackpoint MIC Agreement Kanamycin  16 µg/ml 100% Capreomycin  16 µg/ml 100% Ofloxacin  2 µg/ml 100% PAS  16 µg/ml 100% Ethionamide  4 µg/ml 100%

38. Vall d’Hebron Servei de Microbiologia Facts and questions Cycloserine: no correlation with MICs and severe psychiatric alterations To continue testing? False resistant strains Drug concentration/medium Reading time Inoculum? Drug concentration/medium Reading time Inoculum? A pool of strains with resistant to SLD but not with associate resistance to INH+RP Other drugs for testing Urgent

39. Vall d’Hebron Servei de Microbiologia Laboratories do not use the drug concentrations recommended. After this round, some laboratories have done modifications in method use to improve their results. There were an excess of resistant results. Laboratories do not use the drug concentrations recommended. After this round, some laboratories have done modifications in method use to improve their results. There were an excess of resistant results. Must to review the drug concentrations? Could be useful to continue the proficiency testing for SLD? Are we increasing the difficulties of treating TB resistant patients? Facts and questions

40. Vall d’Hebron Servei de Microbiologia Barcelona Thank you for your attention Thank you for your attention