vall d’hebron servei de microbiologia external quality assurance for drug susceptibility testing...

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



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



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



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



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



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



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



Capreomycin MIC(n = 72)

Capreomycin MIC(n = 72)

2 24 24 4

10

6

2


128128

6464

3232

1616



Ofloxacin MIC(n = 72)

Ofloxacin MIC(n = 72)

1 23 345

5

4


3232

1616

44



PAS MIC(n = 70)

PAS MIC(n = 70)

1820 9 4 22

2

4

9


128128

6464

3232

1616



Ethionamide MIC(n = 70)

Ethionamide MIC(n = 70)

8 16 11 97

10

7

2


128128

6464

3232

1616



Cycloserine MIC(n = 71)

Cycloserine MIC(n = 71)

15

9 32

83

29

2


128128

6464

3232

1616



Reference methods vs MICs of the strains evaluated

(n = 72)


(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.



Selected strains(duplicate)

Selected strains(duplicate)




(n = 15)


(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 %



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


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



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



Strains evaluatedStrains evaluated

PreviousPrevious Judicial ResultJudicial Result



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



50

60

70

80

90

100

%

KM CPR

Efficiency of the different laboratories




50

60

70

80

90

100

%

OFX





50

60

70

80

90

100

%

PAS





50

60

70

80

90

100

%

ETH





50

60

70

80

90

100

%

Global efficiency of the 10 laboratories

Global efficiency of the 10 laboratories



50

60

70

80

90

100

%

KM CPR OFX PAS ETH

Global efficiency / drugsGlobal efficiency / drugs



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



“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!!



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




Judicial results vs MICs

Capreomycin


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





Ofloxacin


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


R

S

R

S

R

R

R

R




PAS


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


R

S

R

S

R

R

R

R




Ethionamide


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


R

S

R

S

R

R

R

R

R

R

R

R

R

R

R

R

S

S

S

S




Cycloserine


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



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%



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



•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



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vall d’hebron servei de microbiologia external quality assurance for drug susceptibility testing...

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