Surveillance on drug resistance in tuberculosis C N Paramasivan Tuberculosis Research centre (ICMR)

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

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

MDR TB in SEAR Thailand (2000 – 02) Nepal (2000 – 02) Myanmar (2003 – 04) Percentage

Prevalence of primary DR TRC Studies

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

DRS sites of India ( ) North Arcot – (2%), (1.7%), 1999 (3%) Pondicherry (0.9%) Tamil Nadu (3.3%) AFMS – (2.7%) Bangalore (2.2%) Mysore (1.2%) Raichur (3.2%), 1999 (2.5%) Wardha (0.5%) Jabalpur (1%) Mayurbhanj (0.7%) Hoogli (3%) Population covered = 8.1%

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

DRS in MDP area

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

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

Drug resistance trend in MDP area New Re Rx

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

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

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

Drug Resistance pattern of referred samples (n 2816 patients)

N

Few earlier studies on ADR in India

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

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

TRC Studies on Newer drugs and defining resistance

 Studies carried out at TRC DEFINITION OF RESISITANCE TO RIFAMPICIN MIC : 128µg/ml. PST (1% or more) : 40 µg/ml BACTEC : 2 µg/ml Indian J. Med. Res. 2001, 114,

Studies carried out at TRC  In vitro activity of capreomycin and ciprofloxacin against S.Indian isolates of M.tb Indian J Tuberculosis 1993; 40:  In vitro activity of ciprofloxacin and ofloxacin against S.Indian isolates of M.tb Indian J Tuberculosis 1994; 41:  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:

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

Recent TRC studies on newer Quinolones 1.Bactericidal action of Gatifloxacin, Rifamicin and isoniazid on Logarithmic – and Stationary – Phase Cultures of Mycobacterium tuberculosis. Antimicrob. Agents Chemother.2005, 49:627 – Moxifloxacin and Gatifloxacin in a new acid model of persistent M.tuberculosis. Antimicrob. Agents Chemother 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 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)

In vitro definition of resistance to gatifloxacin & Moxifloxacin  No. of strains : 50 (Sens. 30; Res. 20)  Methods used : Abs.conc. - LJ PST - LJ, 7H11 & BACTECRESULTS  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

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 Determination of MIC & Cross resistance in M.tb

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

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

Definition of resistance to Quinolones OFLO : NCCLS2.0 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)

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

Type Geometric mean LJ 7H11 Sensitive(46) (SHR) Resistant (46) (SHR/HR) Total (92) MIC of S/A against sensitive and resistant isolates of M.tuberculosis Microbios TRC study

Suggestions

Role of IQC and EQAP

Res. Pattern of strainResults obtained DrugPatternNo. of tests Agreement No. % S R S H RSRS R RSRS E RSRS K R S Ofl RSRS TOTAL TRC:IQC IN DST (June’98–Dec 2001)

Res. Pattern of strainResults obtained DrugPatternNo. of tests Agreement No. % S R S H RSRS R RSRS E RSRS K R S Ofl RSRS TOTAL TRC:IQC IN DST (Jan’2003 – Dec 2004)

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

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 / NO 3 reduction test  DST Indirect economic variant of PST Other methods

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

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

DEFINITION OF RESISTANCE ON LJ SIMPLIFIED VARIANT – PST* DRUGS CONC.(µg/ml) PR (%) INH0.21 Strep.410 Thioacetazone210 ETH2010 Kana 2010 Cyclo3010 Vio3010 Capreo2010 PZA Emb210 Rif Only one conc. of drug Canetti et al. Bull. WHO. 1969

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 is observed

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 2 nd 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 2 nd line drugs by different test systems

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 Determination of MIC & Cross resistance in M.tb

cumulative percentage inhibition

Cumulative percentage inhibition

Laboratory validation of second line drugs RECOMMENDED CONC. BACTEC PST (7H10) Capreomycin Cycloserine* * Ethionamide Kanamycin Amikacin Clofazimine Ofloxacin Rifabutin * No recommendation due to inconsistent result + No error between 2 method (excellent correlation) Pfyffer et al JCM 1999.

Multicenter evaluation of BACTEC & MGIT DRUGS : SHRE GOLD STANDARD : BACTEC MGIT: SENSITIVITY : 100% FOR ALL 4 DAYS SPECIFICITY : RANGE- 89.8% for S to 100% for Rif. MEAN TURN AROUND TIME : 6.5 DAYS (BACTEC) 7 DAYS (MGIT) Beamer et al JCM 2002.

Multicentre evaluation of MB / BACT Gold Standard :BACTEC 460 TB No. Strains tested: 166 Over all agreement: 98% for RHZE Sensitivity / Specificity: 100% for RHZ Sensitivity: 92.3% for Emb. Turn around time: SHRE: 6.6 d.;Z 7.8 d(MB/BACT) : SHRE 5 d ; Z 6.7d (BACTEC) BEMER et al JCM 2004.

Laboratory validation of second line drugs RECOMMENDED CONC. BACTEC PST (7H10) Capreomycin Cycloserine* * Ethionamide Kanamycin Amikacin Clofazimine Ofloxacin Rifabutin * No recommendation due to inconsistent result + No error between 2 method (excellent correlation) Pfyffer et al JCM 1999.

Multicenter evaluation of BACTEC & MGIT DRUGS : SHRE GOLD STANDARD : BACTEC MGIT: SENSITIVITY : 100% FOR ALL 4 DAYS SPECIFICITY : RANGE- 89.8% for S to 100% for Rif. MEAN TURN AROUND TIME : 6.5 DAYS (BACTEC) 7 DAYS (MGIT) Beamer et al JCM 2002.

Multicentre evaluation of MB / BACT Gold Standard :BACTEC 460 TB No. Strains tested: 166 Over all agreement: 98% for RHZE Sensitivity / Specificity: 100% for RHZ Sensitivity: 92.3% for Emb. Turn around time: SHRE: 6.6 d.;Z 7.8 d(MB/BACT) : SHRE 5 d ; Z 6.7d (BACTEC) BEMER et al JCM 2004.

Definition of resistance on LJ simplified variant – PST* DRUGS CONC.(µg/ml) PR (%) INH0.21 Strep.410 Thioacetazone210 ETH2010 Kana 2010 Cyclo3010 Vio3010 Capreo2010 PZA Emb210 Rif Only one conc. of drug Canetti et al. Bull. WHO. 1969

 Non-Inhibition of M.tb with high conc. of penicillin. Abraham et al 1941  ? Penicillinase Activity of M.tb Hand & Bains 1949 Penicillinase as ß lactamase Kasik 1964  Action of ß lactamase stable Oxacillin plus penicillin on M.tb Kasik et al Penicillin – Sparing effect  ß lactamase Inhibitors: eg: Sulbactam Clavulanate Carbapenems (imipenem and meropenem) Earlier studies on ß lactamase

 Clavulanic acid / sulbactam as specific inhibitors of ß lactamase currently used to protect ß lactam susc. Antibiotics.  Clavulanic acid - Natural Product  Sulbactam - Developed later  Augmentin (O): Clavulanic acid + amoxicillin  Timentin (P): Clavulanic acid + ticarcillin  Sulbacin (P): Sulbactam + ampicillin  Sultamicillin(O): Sulbactam + ampicillin Further details on ß lactams

Kasik et al 1966  Murine model  Penicillin + ß lactamase inhibitor combination Prabhakaran et al 1999  Bactericidal to M.tb H37Rv in vitro  M.leprae in mouse foot pad  Mycobacteria (M.simiae, M.haemophilum, M.avium & M.microti) 100 mg/L S/A killed 58-97% as etermined by cfu estimation Earlier studies on ß lactamase