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Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
External quality assurance for drug susceptibility testing
of M. tuberculosis against
second line anti-tuberculosis drugs
S-J. KimS-J. Kim M. ZignyolM. EspinalM. ZignyolM. Espinal
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Participant laboratoriesParticipant 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 d’Hebron, 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
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 d’Hebron, 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
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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
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
OBJECTIVESOBJECTIVES
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Two laboratories collaborated 30 strains with resistance to SLD:
Mycobacteriology Unit,
Institute of Tropical Medicine, Belgium
Korean Institute of Tuberculosis,
Korea
Two laboratories collaborated 30 strains with resistance to SLD:
Mycobacteriology Unit,
Institute of Tropical Medicine, Belgium
Korean Institute of Tuberculosis,
Korea
StrainsStrains
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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
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
OBJECTIVESOBJECTIVES
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Reference methods
Reference methods
Proportion method on Löwenstein-Jensen
Proportion method on agar
MGIT medium for Bactec 960
Bactec 460
Proportion method on Löwenstein-Jensen
Proportion method on agar
MGIT medium for Bactec 960
Bactec 460
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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
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
Minimal inhibitory concentrations
Minimal inhibitory concentrations
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Kanamycin MIC(n = 72)
Kanamycin MIC(n = 72)
9 22 264
1
1
19
M I C µg/mlM I C µg/ml
128128
6464
3232
1616
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Capreomycin MIC(n = 72)
Capreomycin MIC(n = 72)
2 24 24 4
10
6
2
M I C µg/mlM I C µg/ml
128128
6464
3232
1616
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Ofloxacin MIC(n = 72)
Ofloxacin MIC(n = 72)
1 23 345
5
4
M I C µg/mlM I C µg/ml
3232
1616
44
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
PAS MIC(n = 70)
PAS MIC(n = 70)
1820 9 4 22
2
4
9
M I C µg/mlM I C µg/ml
128128
6464
3232
1616
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Ethionamide MIC(n = 70)
Ethionamide MIC(n = 70)
8 16 11 97
10
7
2
M I C µg/mlM I C µg/ml
128128
6464
3232
1616
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Cycloserine MIC(n = 71)
Cycloserine MIC(n = 71)
15
9 32
83
29
2
M I C µg/mlM I C µg/ml
128128
6464
3232
1616
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Reference methods vs MICs of the strains evaluated
(n = 72)
Reference methods vs MICs of the strains evaluated
(n = 72)
Drug Peack serum level (1)
BreackpointMIC 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.(1) I. Bastian, F. Portaels. Multidrug-resistant tuberculosis. 2000 Kluwer Academic Publishers. The Netherlands.
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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
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
OBJECTIVESOBJECTIVES
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Selected strains(duplicate)
Selected strains(duplicate)
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Reference methods vs MICs of the strains evaluated
(n = 15)
Reference methods vs MICs of the strains evaluated
(n = 15)
DrugBreackpoint
MICAgreement
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 %
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Drugs proposed by IUATLD/ WHODrugs proposed by IUATLD/ WHO Lot (Sigma-Aldrich)
Kanamycin (KM) 054K1169
Capreomycin (CPM) 083K1373
Ofloxacin (OFX) 123K1138
Para-aminosalicilic acid (PAS) 121K2522
Ethionamide (ETH) 044K1541
Cycloserine (CS) 083K0702
Lot (Sigma-Aldrich)
Kanamycin (KM) 054K1169
Capreomycin (CPM) 083K1373
Ofloxacin (OFX) 123K1138
Para-aminosalicilic acid (PAS) 121K2522
Ethionamide (ETH) 044K1541
Cycloserine (CS) 083K0702
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia Parameters evaluatedParameters evaluated
Sensitivity
Specificity
Predictive value resistance
Predictive value susceptibility
Efficiency
Reproducibility
Sensitivity
Specificity
Predictive value resistance
Predictive value susceptibility
Efficiency
Reproducibility
Drug
Laboratories
Serum level
Drug
Laboratories
Serum level
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Laboratories
Methods and drug concentrations
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
20.5 (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)
•Prop. on: Löw-Jensen 4, Agar Middlebrook 3•Bactec 460 2•Absolute concentration 1
•Prop. on: Löw-Jensen 4, Agar Middlebrook 3•Bactec 460 2•Absolute concentration 1
* 6 laboratories used L-J * 6 laboratories used L-J
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Strains evaluatedStrains evaluated
PreviousPrevious Judicial ResultJudicial Result
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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 -
All laboratoriesAll laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
KM CPR
Efficiency of the different laboratories
Efficiency of the different laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
OFX
Efficiency of the different laboratories
Efficiency of the different laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
PAS
Efficiency of the different laboratories
Efficiency of the different laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
ETH
Efficiency of the different laboratories
Efficiency of the different laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
Global efficiency of the 10 laboratories
Global efficiency of the 10 laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
50
60
70
80
90
100
%
KM CPR OFX PAS ETH
Global efficiency / drugsGlobal efficiency / drugs
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
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
All drug
All laboratories
All drug
All laboratories
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
“Gold standard”
If the gold standard is the Judicial Result
Conclusion
Excess of resistances!!
If the gold standard is the Judicial Result
Conclusion
Excess of resistances!!
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial Results vs MICs
Kanamycin
Judicial Results vs MICs
Kanamycin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1281283232
1616
88
44
Previous
JUDICIAL
Previous
JUDICIAL
R
S
R
S
R
R
R
R
M I C µg/mlM I C µg/ml
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial results vs MICs
Capreomycin
Judicial results vs MICs
Capreomycin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15R
S
R
S
3232
1616
88
44
6464
Previous
JUDICIAL
Previous
JUDICIAL
N
S
N
S
R
R
R
R
M I C µg/mlM I C µg/ml
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial results vs MICs
Ofloxacin
Judicial results vs MICs
Ofloxacin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1616
88
44
22
Previous
JUDICIAL
Previous
JUDICIAL
R
S
R
S
R
S
R
S
M I C µg/mlM I C µg/ml
R
S
R
S
R
R
R
R
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial results vs MICs
PAS
Judicial results vs MICs
PAS
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
128128
6464
3232
1616
Previous
JUDICIAL
Previous
JUDICIAL
R
S
R
S
R
R
R
R
M I C µg/mlM I C µg/ml
R
S
R
S
R
R
R
R
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial results vs MICs
Ethionamide
Judicial results vs MICs
Ethionamide
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
3232
1616
88
44
Previous
JUDICIAL
Previous
JUDICIAL
S
R
S
R
R
S
R
S
M I C µg/mlM I C µg/ml
R
S
R
S
R
R
R
R
R
R
R
R
R
R
R
R
S
S
S
S
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial results vs MICs
Cycloserine
Judicial results vs MICs
Cycloserine
0
4
8
12
16
20
24
28
32
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Previous
JUDICIAL
Previous
JUDICIAL
R
S
R
S
R
S
R
S
R
S
R
S
R
S
R
S
MIC µg/mlMIC µg/ml
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Judicial Results vs MICsAgreement of the round
Judicial Results vs MICsAgreement of the round
DrugBreackpoint
MICAgreement
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%
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
Facts and questionsFacts and questions
•Cycloserine: no correlation with MICs and severe psychiatric alterations
•Cycloserine: no correlation with MICs and severe psychiatric alterations To continue testing?To continue testing?
•False resistant strains•False resistant strainsDrug concentration/mediumReading timeInoculum?
Drug concentration/mediumReading timeInoculum?
•A pool of strains with resistant to SLD but not with associate resistance to INH+RP•A pool of strains with resistant to SLD but not with associate resistance to INH+RP
•Other drugs for testing•Other drugs for testing UrgentUrgent
Vall d’Hebron Vall d’Hebron
Servei de Servei de MicrobiologiaMicrobiologia
•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.
•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?Must to review the drug concentrations?
Could be useful to continue the proficiency testing for SLD?
Could be useful to continue the proficiency testing for SLD?
Are we increasing the difficulties of treating TB resistant patients?
Are we increasing the difficulties of treating TB resistant patients?
Facts and questionsFacts and questions