Surveillance on drug resistance in tuberculosis

1
Surveillance on drug resistance in tuberculosis

• C N Paramasivan
• Tuberculosis Research centre (ICMR)

2
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

3
Earlier reports on combined resistance from India
and their limitations

• Case selection
• Sample size
• Methodology
• Source of drugs
• Definition of resistance

4
MDR TB in SEAR
Thailand (2000 02) 172 1505 Nepal (2000
02) 177 755 Myanmar (2003 04) 166 733
Percentage
5
Prevalence of primary DR TRC Studies 1956 - 2001
6
Prevalence of Primary Drug Resistance TRC
Studies (1974 -2001)

After the introduction of rifampicin in
Controlled Clinical Trail at TRC
7
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)
8
Level of MDR in New in different sites in India
(population covered 8.1)
TRC
NTI
9
DRS in MDP area
10
Drug resistance among newly diagnosed cases
MDP area N1603
Bangalore city N271
11
Drug susceptibility among previously treated cases
Bangalore city N226
MDP area N443
12
Drug resistance trend in MDP area
New 144 326
367
389 371 Re Rx.
46 98
100 103
93
13
MDR TB (Gujarat, India Jan 2000 Aug 2001)
N482
N822
12.4
3.9
Shah AR et al. Int J tuberc Lung Dis, 2003
6(12) 1098.
14
Drug Resistance in Patients With HIV / TB in
South India
New cases-167
Treated cases-37
Swaminathan S et al IJTLD 2004
15
Drug Resistance pattern of referred samples
2001-04
(n 2816 patients)
Susc. 32.5
Res. 1 or more 67.5
16
Drug Resistance pattern of referred samples
2001-04 (n 2816 patients)
17
Drug Resistance pattern of referred samples
2001-04 (n 2816 patients)
N 355
385
176
582
1498
18

• Few earlier studies on ADR in India

19
Level of MDR in New in different sites in India
(population covered 8.1)
TRC
Others
20
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
21
TRC Studies on Newer drugs and defining resistance
22
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,
187-191.
23
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

24
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

25
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, 49627 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)

26
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

27
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 gt SPFX gt OFLX gt CFLX gt LMFX

TRC Study J.Chemother,2005
28
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

29
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

30
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)
31
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 countrys requirement

32
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
33
Suggestions

34
Role of IQC and EQAP
35
TRCIQC IN DST (June98Dec 2001)
36
TRCIQC IN DST (Jan2003 Dec 2004)
37
The role of DST in DEC

• Failures of category II cases under DOT
• Tests should be very simple rapid for
• Primary culture
• Identification
• DST

38
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

39
Review of simple rapid tests DST for DEC

• Direct
• Primary culture
• Sputum swab method
• Sputum deposit after processing by Petroffs
• Identification
• Growth in 500 micrograms of PNB medium
• DST
• Standardization of direct PST only for H R
• Absolute concentration method
• Resistance ratio method

40
Role of speedier pheneotypic methods
? RIF. Resistance as an indicator of MDR TB
Direct methods MABA Nitrate reductase
assay MTT Assay MODS PhaB Others
41
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
42
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

43
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

44
Thank You
45
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 gt SPFX gt OFLX gt CFLX gt LMFX

TRC Study J.Chemother,2005
46
cumulative percentage inhibition
47
Cumulative percentage inhibition
48
Laboratory validation of second line drugs

RECOMMENDED CONC.
BACTEC PST
(7H10) Capreomycin 1.25
10.0 Cycloserine Ethionamide
1.25 5.0 Kanamycin 5.0 5.0
Amikacin 1.0 4.0 Clofazimine
0.5 1.0 Ofloxacin
2.0 2.0 Rifabutin
0.5
1.0 ----------------------------------------------
-----------------------No recommendation due to
inconsistent result No error between 2 method
(excellent correlation)
Pfyffer et al
JCM 1999.
49
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.
50
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.
51
Laboratory validation of second line drugs

RECOMMENDED CONC.
BACTEC PST
(7H10) Capreomycin 1.25
10.0 Cycloserine Ethionamide
1.25 5.0 Kanamycin 5.0 5.0
Amikacin 1.0 4.0 Clofazimine
0.5 1.0 Ofloxacin
2.0 2.0 Rifabutin
0.5
1.0 ----------------------------------------------
-----------------------No recommendation due to
inconsistent result No error between 2 method
(excellent correlation)
Pfyffer et al
JCM 1999.
52
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.
53
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.
54
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
55
Earlier studies on ß lactamase

• 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 1967
• - Penicillin Sparing effect
• ß lactamase Inhibitors
• eg Sulbactam
• Clavulanate
• Carbapenems (imipenem and
  meropenem)

56
Further details on ß lactams

• 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

57
Earlier studies on ß lactamase

• 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