Sin t

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External quality assurance for drug
susceptibility testing of M. tuberculosis
against second line anti-tuberculosis drugs
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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 dHebron, 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

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OBJECTIVES

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

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Strains
Two laboratories collaborated 30 strains with
resistance to SLD Mycobacteriology Unit,
Institute of Tropical Medicine, Belgium Korean
Institute of Tuberculosis, Korea
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OBJECTIVES

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

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Reference methods

• Proportion method on Löwenstein-Jensen
• Proportion method on agar
• MGIT medium for Bactec 960
• Bactec 460

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Minimal inhibitory concentrations
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
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Kanamycin MIC (n 72)
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Capreomycin MIC (n 72)
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Ofloxacin MIC (n 72)
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PAS MIC (n 70)
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Ethionamide MIC (n 70)
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Cycloserine MIC (n 71)
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Reference methods vs MICs of the strains
evaluated (n 72)
Drug Peack serum level (1) Breackpoint MIC Agreement
KM 35-45 µg/ml ? 16 µg/ml 69
CPR 35-45 µg/ml ? 16 µg/ml 96
OFX 8-10 µg/ml ? 2 µg/ml nd
PAS 70 µg/ml ? 16 µg/ml nd
ETH 4 µg/ml ? 4 µg/ml 65
CS 20 µg/ml ? 8 µg/ml ?
(1) I. Bastian, F. Portaels. Multidrug-resistant
tuberculosis. 2000 Kluwer Academic Publishers.
The Netherlands.
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OBJECTIVES

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

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Selected strains (duplicate)
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Reference methods vs MICs 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
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Parameters evaluated
Sensitivity Specificity Predictive value
resistance Predictive value susceptibility Efficie
ncy Reproducibility
Drug Laboratories Serum level
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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 40 2 0.5 (5 labs) 1 (1 lab) 16 20 40 40
Proportion agar 5 (2 lab) 6 (1lab) 10 2 (2 lab) 1 (1lab) 2 (2 lab) 4 (1lab) 5 (1lab) 10 (2 lab)
6 laboratories used L-J
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Strains evaluated
Previous
Judicial Result
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All laboratories
KM CPR OFX PAS ETH CS
Sensitivity 100 100 100 67 79 -
Specificity 90 96 97 98 92 90
PV resistance 40 65 71 87 78 -
PV susceptib. 100 100 100 95 92 100
Efficiency 90 96 97 94 88 90
Reproductibility 94 93 99 95 87 -
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Efficiency of the different laboratories
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Efficiency of the different laboratories
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Efficiency of the different laboratories
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Efficiency of the different laboratories
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Global efficiency of the 10 laboratories
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Global efficiency / drugs
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All drug All laboratories
KM CPR OFX PAS ETH
True resistant 17 17 20 27 63
False resistant 25 9 8 4 18
True susceptible 224 240 269 253 198
False susceptible 0 0 0 13 16
TOTAL 266 266 297 297 295
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Gold standard
If the gold standard is the Judicial
Result Conclusion Excess of resistances!!
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Judicial Results vs MICs Kanamycin
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Judicial results vs MICs Capreomycin
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Judicial results vs MICs Ofloxacin
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Judicial results vs MICs PAS
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Judicial results vs MICs Ethionamide
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Judicial results vs MICs Cycloserine
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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
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Facts and questions

• A pool of strains with resistant to SLD but not
  with associate resistance to INHRP

Drug concentration/medium Reading time Inoculum?

• False resistant strains

• Cycloserine no correlation with MICs and severe
  psychiatric alterations

To continue testing?

• Other drugs for testing

Urgent
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Facts and questions

• 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?
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Thank you for your attention
Barcelona