Role of TRC in DRS for India & SEAR

1. Role of TRC in DRS for India & SEAR • SNRL and ref. Lab of the WHO • NRL for India Renders assistance for the following: • Preparation of generic protocol • Developing laboratories for culture & DST • Preparation of manuals and SOPs • Training of laboratory personnel • Instituting uniform methods for DST • Ensuring quality thro’ QAP • Supply of standard strains, drugs & reagents • Periodic site visits

2. Earlier reports on combined resistance from India and their limitations • Case selection • Sample size • Methodology • Source of drugs • Definition of resistance

3. MDR TB in SEAR Thailand (2000 – 02) 172 1505 Nepal (2000 – 02) 177 755 Myanmar (2003 – 04) 166 733 Percentage

4. Prevalence of primary DRTRC Studies 1956 - 2001

5. Prevalence of Primary Drug Resistance TRC Studies – (1974 -2001) * After the introduction of rifampicin in Controlled Clinical Trail at TRC

6. DRS sites of India (1985-2003) Population covered = 8.1% Jabalpur - 2002 (1%) Wardha - 2001 (0.5%) Hoogli - 2003 (3%) AFMS – 1995 -1999 (2.7%) Mayurbhanj - 2002 (0.7%) Raichur - 1989(3.2%), 1999 (2.5%) North Arcot – 1985-89 (2%), 1989-90 (1.7%), 1999 (3%) Pondicherry - 1985 (0.9%) Mysore - 2001 (1.2%) Tamil Nadu - 1995 (3.3%) Bangalore - 2002 (2.2%)

7. Level of MDR in ‘New’ in different sites in India (population covered 8.1%) TRC NTI

8. DRS in MDP area

9. Drug resistance among newly diagnosed cases MDP area N=1603 Bangalore city N=271

10. Drug susceptibility among previously treated cases Bangalore city N=226 MDP area N=443

11. Drug resistance trend in MDP area New 144 326 367 389 371 Re Rx. 46 98 100 103 93

12. MDR TB (Gujarat, India Jan 2000 – Aug 2001) N=482 N=822 12.4 % 3.9 % Shah AR et al. Int J tuberc Lung Dis, 2003; 6(12): 1098.

13. Drug Resistance in Patients With HIV / TB in South India New cases-167 Treated cases-37 Swaminathan S et al IJTLD 2004

14. Drug Resistance pattern of referred samples 2001-04 (n 2816 patients) Susc. 32.5% Res. 1 or more 67.5%

15. Drug Resistance pattern of referred samples 2001-04 (n 2816 patients)

16. Drug Resistance pattern of referred samples 2001-04(n 2816 patients) N 355 385 176 582 1498

17. Few earlier studies on ADR in India

18. Level of MDR in ‘New’ in different sites in India (population covered 8.1%) TRC Others

19. Year 2005 DRS sites of India Population being covered in 2005 = 25% Gujarat, 53.8 millions (4.9%), I Qrt 2005 Orissa, 38.2 millions (3.5%), II Qrt 2005 Resurvey – Tamilnadu DRS-Sikkim,2005-06 Maharashtra, 102.8 millions (9.4%), I Qrt 2005 Andhra Pradesh, 78.7 millions (7.2%), II Qrt 2005

20. TRC Studies on Newer drugs and defining resistance

21. Studies carried out at TRCDEFINITION OF RESISITANCE TO RIFAMPICINMIC : 128µg/ml. PST (1% or more) : 40 µg/ml BACTEC : 2 µg/mlIndian J. Med. Res. 2001, 114, 187-191.

22. Studies carried out at TRC In vitro activity of capreomycin and ciprofloxacin against S.Indian isolates of M.tb Indian J Tuberculosis 1993; 40: 21-25 In vitro activity of ciprofloxacin and ofloxacin against S.Indian isolates of M.tb Indian J Tuberculosis 1994; 41: 87-90 MIC of Lomefloxacin and Minocycline Against Drug- Sensitive & resistant Isolates of M.tuberculosis Compared on L-J and 7H11 Media Int J Leprosy 1997; 65: 375-378

23. Studies carried out at TRC • Evaluation of various methods of susceptibility to ofloxacin in strains of M.tb Indian J Med Res 1999; 110: 186-189 Evaluation of bactericidal action of ofloxacin and sulbactam/ampicillin alone & in combination with R & H on M.tb invitro Antimicrob Agents Chemother 1996; 40: 2296-2299 • A multi centre study of the early bactericidal activity of anti- tuberculosis drugs J Antimicrobial Chemother 2000; 45: 859-870

24. Recent TRC studies on newer Quinolones • Bactericidal action of Gatifloxacin, Rifamicin and isoniazid on Logarithmic – and Stationary – Phase Cultures of Mycobacterium tuberculosis. Antimicrob. Agents Chemother.2005, 49:627 – 631 • Moxifloxacin and Gatifloxacin in a new acid model of persistent M.tuberculosis. Antimicrob. Agents Chemother.2005 • In vitro activity of fluoroquinlones against M.tuberculosis. J. Chemotherapy.April,2005 (Accepted) 4. In vitro definitions of MIC of gati and moxifloxacin by different test methods. FEMS Microbiology.2005 5. Bactericidal action of Moxifloxacin, Rifampicin and Isoniazid on Logarithmic – and Stationary phase cultures of M.tuberculosis. J Antimicro.Agents and Chemother. (2005 Communicated) 6. Analysis of Fluoroquinolone resistance in clinical isolates of M.tuberculosis from India. J. Clinical Microbiology,2005 ( Communicated)

25. In vitro definition of resistance to gatifloxacin & Moxifloxacin • No. of strains : 50 (Sens. 30; Res. 20) • Methods used : Abs.conc. - LJ PST - LJ, 7H11 & BACTEC RESULTS • MIC of GATI LJ : 1 μg ml • Critical conc. LJ & 7H11 : 0.5 μg/ml BACTEC : 0.25 μg/ml • MIC of MOXI LJ : 1 μg /ml • Critical conc. LJ : 1 μg/ml 7H11 & BACTEC : 0. 5 μg/ml

26. Determination of MIC & Cross resistance in M.tb No of strains : 55 (oflox-Res 33; Susc 22) Method of testing : MIC Drugs tested : Spar, Oflo, Cipro, Lome,moxi & Gati Media used : LJ & 7H11 RESULT : Fluoroquinolones exhibited cross resistance at different levels. MIC of quinolones were in the order of GTFX = MOXI > SPFX > OFLX > CFLX > LMFX TRC Study J.Chemother,2005

27. TRC study findings In vitro MIC studies • Quinolones showed low and similar MIC on both drug sens & resist. population of M.tb • Cipro showed higher mean MIC than Ofloxacin • Almost 100% cross resistance was seen • Ofloxacin MICs were lower than other quinolones tested • PST on LJ showed 2mg/l as a criterion of resistance for Ofloxacin • Absolute Concentration Method (Ofloxacin) :8mg/l

28. TRC study findings In vitro simulation experiment with ofloxacin • Showed high EBA either alone or in combinations on exponential growth • Expect high bactericidal activity in the early phase of the Rx • Comparatively low level of SA against stationary phase growth • However, it enhanced activity in combination with H, R & HR

29. Definition of resistance to Quinolones OFLO : NCCLS 2.0 µg/ml (7H10 & 7H11) TRC 8 µg/ml ACM (LJ) 2 µg/ml PST (LJ) GATI : TRC 1 µg/ml – LJ; 0.5 & 0.25 (7H11 & BACTEC) MOXI : TRC 1 µg/ml – (LJ); 0.5 (7H 11 & BACTEC)

30. Standardisation of DST to newer drugs • NCCLS (2002) Guidelines : • 7H10 & 7H 11: Capreo, Eth, Kan, Oflo, PAS, RBU & Strep • BACTEC : PZA • Canetti etal (1969)& Various TRC Publications • LJ : INH, Capreo, Amikacin,Rif,RBU, Kan, Eth, Cyclo • TRC: Lomi, Cipro,Oflo,Gati & Moxi • Developing SOP for country’s requirement

31. MIC of S/A against sensitive and resistant isolates of M.tuberculosis Type Geometric mean LJ 7H11 Sensitive(46) 63.97 26.73 (SHR) Resistant (46) 65.92 23.82 (SHR/HR) Total (92) 65.01 25.23 Microbios 89 135-141 1997 TRC study

32. Suggestions

33. Role of IQC and EQAP

34. Res. Pattern of strain Results obtained R Agreement Drug Pattern No. oftests S No. % S 232 369 217 367 93.5 99.5 H R S 381 299 376 286 98.7 95.7 R R S 384 307 382 305 99.5 99.3 E R S 282 384 272 383 96.5 99.7 K R S 46 169 44 168 95.7 99.4 Ofl R S 38 219 36 219 94.7 100.0 TOTAL TRC:IQC IN DST (June’98–Dec 2001)

35. Res. Pattern of strain Results obtained R Agreement Drug Pattern No. of tests S No. % S 334 329 334 323 100 98.2 H R S 567 138 567 137 100 99.3 R R S 417 294 416 292 99.8 99.3 E R S 268 458 258 457 96.3 99.8 K R S 28 266 27 261 96.4 98.1 Ofl R S 67 296 63 292 94.0 99.8 TOTAL TRC:IQC IN DST (Jan’2003 – Dec 2004)

36. The role of DST in DEC Failures of category II cases under DOT • Tests should be very simple & rapid for • Primary culture • Identification & • DST

37. DST in DEC • Drug resistance surveillance • The tests should be as per global DRS guidelines • Identification • Growth rate. • Growth in 500 micrograms of PNB medium • Niacin test / NO3 reduction test • DST • Indirect economic variant of PST • Other methods

38. Review of simple & rapid tests DST for DEC Direct • Primary culture • Sputum swab method • Sputum deposit after processing by Petroff’s • Identification • Growth in 500 micrograms of PNB medium • DST • Standardization of direct PST only for H & R • Absolute concentration method • Resistance ratio method

39. Role of speedier pheneotypic methods ? RIF. Resistance as an indicator of MDR TB Direct methods MABA Nitrate reductase assay MTT Assay MODS PhaB & Others

40. DEFINITION OF RESISTANCE ON LJSIMPLIFIED VARIANT – PST* ----------------------------------------------------------- DRUGS CONC.(µg/ml) PR (%) ------------------------------------------------------------------------ INH 0.2 1 Strep. 4 10 Thioacetazone 2 10 ETH 20 10 Kana 20 10 Cyclo 30 10 Vio 30 10 Capreo 20 10 PZA 100 10 Emb 2 10 Rif 40 1 ----------------------------------------------------------------------- Only one conc. of drug Canetti et al. Bull. WHO. 1969

41. DRS – Salient Observations • Among new cases : No evidence of an increase in the prevalence of resistance • Reports on higher prevalence of ADR ( TRC findings, Gujarat, N.Arcot, N.Delhi, Tamilnadu ,Bombay, UP.) • TRC studies :Low level prevalence of MDR TB • TRC studies : Paediatric & Extra-pulmonary cases • low level resistance to H (5-10%) • low level resistance to S (2-14%) • absence of MDR TB • Compared to global situation • a lesser prevalence of primary resistance • a much higher level of acquired resistance isobserved

42. Issues to be considered • Steps and Time Tables • Preparation of SOP • Culture system and methodology • Training • Organizing EQAP for second line drugs • IQC Measures (Drugs, Techniques, Periodicity, Monitoring) • Role of Speedier methods for DST of 2nd line drugs • Role of simpler phenotypic methods for detecting MDR TB • Rif. Resistance as an indicator for detecting MDR TB • DST for PZA – Its relevance • Multi - centric approach for defining resistance to various 2nd line drugs by different test systems

43. Thank You