Drug Resistance Surveillance

1
Drug Resistance Surveillance
C.N. PARAMASIVAN Tuberculosis Research Centre
Indian Council of Medical Research Chennai
2
Anti-TB Drug Resistance in the World
Definition of resistance A decrease in
sensitivity to sufficient degree to be
reasonably certain that the strain concerned is
different from a sample of wild stains of human
type that have never come in contact with the
drugs
Prof. Mitchison
3
Anti-TB Drug Resistance in the World

• The Probability of Resistance
• Mutation per reproductive unit 10-6
• No of bacilli in a cavity 107 -
  109
• Chances of dev. res. to H R 10-14( 106
  x 108)
• Probability of dev. res. to 3 drugs 10-18
• Possibility of dev. res. with
• multi drug therapy Nil

4
Earlier studies from India
5
Reports on combined resistance and their
limitations

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

6
Studies Reported From NTI AFMC
7
Studies on Primary Drug Resistance Among
M.Tuberculosis Isolates NTI Reports
8
Drug Resistant TB in Armed Forces Chest Disease
Hospital, Pune (1995 99)

• No of pts. 2562

2.7
14.4
83.9
Kailash Chand et al. Med J Armed Forces India
2000 56 130.
9
TRC Studies
10
Drug Resistance on Admission in Pondicherry
Paramasivan CN et al Tubercle and Lung Dis.1993,
74 23-27
11
Drug Resistance on Admission in N Arcot Dt.
Paramasivan CN et al Tubercle and Lung Dis.1993,
74 23-27
12
Drug Resistance Survey in N Arcot Dt.
13
Drug Resistance Survey in N Arcot Dt.
14
Drug Resistance Survey in Raichur Dt.
15
Drug Resistance Survey in Raichur Dt.
P.G.Gopi et al. Ind J Tub. 1997 44 171
16
Prevalence of Primary Drug Resistance TRC
Studies - 1956 to 2001
17
Prevalence of Primary Drug Resistance TRC
Studies (1974 -2001)

After the introduction of rifampicin in
Controlled Clinical Trail at TRC
18
Recent Reports From TRCOn DRS based
onWHO/IUATLD Guidelines
19
WHO / IUATLD Global Project on Anti TB Drug
Resistance

• Objectives
• To collect data on the Worldwide extent on the
  severity of anti-TB drug resistance in a
  standardized manner at a country level
• To monitor drug resistance levels in countries
  identified as a priority for assistance or in
  countries where, for various reasons, knowledge
  of drug resistance levels may be important for
  global policy decisions

20
Summary of Patterns of Resistance
History of previous treatment in 384 patients
WHO sponsored study on surveillance of drug
resistance in TB in Tamil Nadu, India.
Tuberculosis Research Centre (ICMR)
21
Summary of Patterns of Resistance
N 384
N 16
22
Surveillance of drug resistance in TB in N.Arcot
Dt, Tamil Nadu,1999
Total No of specimens 635 No of Patients
320 Patients with culture Neg/Cont.
20 Sensitivity results not available
2 No of patients with DS results 298
23
Surveillance of drug resistance in N.Arcot Dt,
TN,1999
24
Surveillance of primary drug resistance N.Arcot
dt,TN,1999
25
Primary Drug resistance - NArcot . Dt, TN
26
Surveillance in Drug Resistance, Raichur Dt,
Karnataka,1999 -2000
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Surveillance in Drug Resistance Raichur Dt.
Karnataka 1999 -2000
28
PRIMARY DRUG RESISTANCE-RAICHUR
29
Surveillance in Drug Resistance Wardha District
Maharashtra 2000 -2001
30
Primary drug resistance Wardha district
N197
N323
H
HR
H
HR
2000-01
1982-89
31
Primary drug resistance Jabalpur district (n273)
32
Primary resistance in the Model DOTS area (1999-
2003)
N1893
33
Drug Resistance in HIV/TB (2000-02)
New -168
Treated -37
34
Recent reports from NTI
35
DRS STUDIES OF NTI, BANGLAORE
36
Primary Drug Resistance NTI studies 2003
37
Other Reports on ADR
38
ADR- MDR TB - INDIAN STUDIES
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ADR-MDR TB-TRC RESULTS
HIV/TB
40
Anti-TB Drug Resistance in the World
Prof. Jacques grosset foreword The
WHO/IUATLD global project on anti-tb
drs WHO/CDS/TB/2000.278
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EVOLUTION OF THE GLOBAL PROJECT ON DRS
(WHO/IUATLD OTHER PARTNERS)
THREE MAIN PRINCIPLES 1) SAMPLES TO BE
REPRESENTATIVE OF ALL CASES IN DEFINED
SETTINGS 2) DRUG RESISTANCE SHOULD BE CLEARLY
DISTINGUISHED AMONG
- NEVER TREATED
- PREVIOUSLY
TREATED 3) ENSURE OPTIMAL LAB PERFORMANCE
THRO. QAP
42
DEVELOPMENT OF SRLN
TO ENSURE DST PERFORMANCE THROUGH QAP LOCATION
OF 20 LABS IN 5 REGIONS COORDINATING CENTRE
THE PRINCE LEOPOLD INST. OF TROP.MEDICINE
ANTWERP, BELGIUM (F.PORTAELS/ARMOND)
COMPLETION OF 10 ROUNDS OF PT EACH SRLN
COMPLETED SEVERAL ROUNDS OF PT IN THEIR
RESPECTIVE NRLS OTHERS.


43
PROGRESS OF THE GLOBAL PROJECT ON DRS

• -------------------------------------------------
  ------------------------------
• PHASE No. of MAJOR FINDINGS
• SITES
• -------------------------------------------------
  -----------------------------
• 1994-97 35 MDR TB WAS A PROBLEM
• PREVALENCE
  OF DR
• CORRELATED
  WITH GOOD
• TB CONTROL
• 2. 1997-99 58 PRESENCE OF DR IN ALL
  SETTINGS
• (51 COUNTRIES) HIGH RATES OF MDR TB
  IN SEVERAL
• SETTINGS .
  No.INCREASE IN MDR
• TB IN PLACES
  WITH SOUND TB
• CONTROL
  PROGRAMME.

44
PROGRESS OF THE GLOBAL PROJECT ON DRS
THIRD PHASE 1999
2002 DATA FROM 77 SETINGS - NEWS CASES 75
- PREVIOUSLY Rx. 65 - COMBINED
CASES 70 THE BURDEN OF MDR TB ESTIMATION 69
COUNTRIES


45
THIRD PHASE GLOBAL DRS 1999-02

• ANALYSIS BASED ON 0.25 MILLION STRAINS
• 105 GEOGRAPHICAL SETTINGS (90 COUNTRIES)
• ISSUES ADDRESSED
• - THE DYNAMICS OF ANTI TB DR OVER TIME,
  TRENDS
• - THE MAJOR DETERMINANTS OF DR
• - EVALUATION OF HOT SPOTS AND HOW TO DEFINE
• MDR TB BURDEN
• - USE OF Rif. AS A SERROGATE MARKER FOR MDR
• - ANALYSIS OF SRLN PT RESULTS OVER TIME.

46
THIRD PHASE GLOBAL DRS 1999-02
CONCLUSIONS
DR WAS PRESENT IN
ALMOST ALL SETTINGS SURVEYED. HIGH PREVALENCE OF
MDR- TB RUSSIA, AREAS OF FORMER SU,
ISRAEL, LIAONING AND HENAN PROVINCES IN
CHINA, EQUADOR. REPRESENTATIVE DR DATA URGENTLY
NEEDED FROM RUSSIA, FORMER SU, INDIA,
CHINA, PAKISTAN, AFGHANISTAN, INDONESIA,
NIGERIA, DR CONGO AND TANZANIA. GAP IN
SURVEILLANCE INADEQUATE LABORATORYCAPACITRY
(OR) -
COMPLETE LACK OF FUNCTIONAL NRL. CONTINUOUS
MONITORING OF TRENDS IN HIGH PREVALENCE MDR
SETTINGS.

47
WHO/IUATLD GLOBAL PROJECT 1999-2002
COUNTRYWIDE DRS NEPAL 2001 RESISTANCE
PATTERN NEW (755)
PREVIOUSLY TREATED (177)

Susc. to all 89 59.1 Res. to
H 5.4 33.3 R 1.7
20.5 Emb 0.9
9.9 S 8.9
31.0 MDR TB
1.3
20.5 ---------------------------------------------
----------------------------------------- Cluster
sampling
48
WHO/IUATLD GLOBAL PROJECT 1999-2002
COUNTRYWIDE DRS THAILAND 2001 RESISTANCE
PATTERN NEW (N1505)
PREVIOUSLY TREATED (N172)

Susc. to all 85.2 61.0 Res. to
H 9.5 30.8 R 1.4
22.7 Emb 1.1
15.1 S 8.2
24.4 MDR TB
0.9
20.3 ---------------------------------------------
----------------------------------------- Proporti
onal Cluster
49
DRS IndonesiaPrimary Resistance

• Year H R S E MDR
• 1996 1.3 5.2 2.9 - 5.8
• 1997 1.6 5.9 2.1 - 5.0
• 1998 5.4 1.7 5.4 0.9 6.6
• PERSHABATAN HOSPITAL - JAKARTA

50
DRS IndonesiaSecondary Resistance

• Year H R S E MDR
• 1996 6.4 6.4 1.6 - 24.5
• 1997 6.0 5.8 0.7 - 41.8
• 1998 6.8 5.8 4.3 - 24.7
• PERSHABATAN HOSPITAL - JAKARTA

51
DRS Few comments

• No evidence of an increase in the prevalence of
  primary resistance
• Reports on higher prevalence of ADR
• (Gujarat, N.Arcot, N.Delhi, Tamilnadu
  ,Bombay, UP.)
• Low level prevalence of MDR TB in TRC studies
• Paediatric 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

52
Anti-TB Drug Resistance in the World
At more serious examination.. the prevalence
of MDR strain is worrysome not only because it
is high but also because it was high in the first
resurvey and still high in the second resurvey,
indicating that more preventive and curative
interventions are needed in these settings.
Although ,on a median basis, the present
prevalence of drug resistance is not alarming,
several indicators are quite frightening and
should be understood as telling us that the DOTS
strategy should be implemented every where at
any cost, and surveillance of drug resistance
should not only continue but ever extended.
Prof. Jacques Grosset
53
DRUGS USED FOR THE THERAPY OF MYCOBACTERIAL
INFECTION
54
Drug susceptibility testing

• Direct method
• Indirect method
• MIC
• RR
• Proportion method
• BACTEC 460 (first second line )
• E- test
• Micro well alamar blue assay
• Microscopic observation broth Drug
• susc. Assay (MODS)

55
Antimicrobial susceptibility tests

• Phenotypic methods
• Microcolony detection method
• MGIT
• The PhaB assay, LRP assay
• E Test
• Rapid metobolic tests
• Alamar Blue assay
• Bioluminescence assay

56
MOLECULAR BASIS OF DRUG RESISTANCE
Drug Target Molecules Molecular Basis R RNA
polymerase Point mutation in ?
subunit rpoB prevent drug binding H NAD
reductase(InhA) Point mutation in
catalase-peroxidase InhA(A94S) Point
mutation in Kat G gene S S12 ribosomal
protein Mutations in rpsl gene K 16S rRNA
gene Alteration in 16S rRNA(rrs) Z
Amidase Mutations in pnc A gene Quinol DNA
gyrase-DNA complex Single mutations in gyr A or
gyrB Emb Action on Arabinan over expression
Emb protein biosynthesis (Emb R, Emb A, Emb
B Emb C
57
FACTORS ASSOCIATED WITH MDR TB

• RESISTANCE IS DUE TO CHROMOSOMAL MUTATION
• MUTATIONUNLINKEDOCCUR AT LOW FREQ 1X106 -
  108
• MDR STRAINS ARISE BY SEQUENTIAL ACCUMULATION
  OF RES. MUTATIONS FOR INDIV.
  DRUGS.
• ADR COMMONLY SEEN IN CAVITARY LUNG DISEASE
• MULTIPLE MEDICATIOIN PROTECTS AGAINST ADR
• MUTANTS TO AN INDIV. DRUG GET KILLED BY
• ANOTHER DRUG.
• HIGHLY IMPROBABLE FOR A SINGLE ORGANISM TO
• EVOLVE SPONTANEOUS MUTATION

58
RAPID DETECTION OF DRUG RESISTANT STRAINS

• AMONG MANY METHODS EMPLOYED, AUTOMATED
• DNA SEQUENCING OF PCR PRODUCTS IS WIDELY
• APPLIED
• ADVANTAGE
• SEQUENCE- BASED APPROACH RESULTANT DATA
• ARE VIRTUALLY UNAMBIGUOUS BECAUSE
• RESISTANCE ASSOCIATED MUTATION IS EITHER
• PRESENT OR ABSENT

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RAPID DETECTION OF DRUG RESISTANT STRAIN

• PRINCIPLE DETECTION OF SPECIFIC POLYMORPHISM
• SEEN IN RESISANT STRAIN
• DIRECT SEQUENCING OF PCR PRODUCTS
• SSCP ANALYSIS
• HETERODUPLEX ANALYSIS
• DIDEOXY FINGER PRINTING
• RNA/RNA DUPLEX, BASE PAIR MISMATCHING ASSAY
• LUCIFERASE MYCOBACTERIOPHAGE STRATEGY
• rRNA / DNA BIOLUMINESCENCE LABELLED
• PROBE METHOD
• REVERSE HYBRIDISATION - LIPA
• OTHER STRATEGIES

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Cost MODS versus other methods
Caviedes.L. et al. J.Clin. Microbiol. 2000, 38,
1203
61
Comparisons of turnaround-time
Sequencing Methods
DNA Micro array
PCR-SSCP
Hybridization Assays
Cost/Sophistication
BACTEC
MGIT
Metabolic Methods
Micro colonies
Pha B Assay
Conventional Method
Time to Result
Palamino JC, MDR TB, p.145-162, 2000. Ed
Bastian,I Portaels,F.
Palomin
62
What is feasible in DEC?

• Sputum smear microsocopy
  
• Direct
• ? Conc. by NaOCl sedimendation
• diagnosis / monitoring
• a) SWAB ? Direct (LJ/ LJPNB/ LJ 40 mg/l RIF)
• b) Deposit ? Direct (LJ /LJPNB /LJ 40 mg/l
  RIF)
• One plate strategy (7H 10 /7H 11)
• Smear negative Agar biplate
• Smear pos. Agar quadrant plate
• (Control, H 0.2 1.0, Rif. 1.0)
• A similar approach in LJ