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LABORATORY BASED DST FOR BEDAQUILINE AND INTRODUCTION IN COUNTRIES
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NDWG Annual meeting 2015, Cape Town, South Africa
Rigouts Leen, Institute of Tropical Medicine, Antwerp, Belgium
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Bedaquiline (BDQ) –Sirturo®
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Andries et al., Science 2005
- Diarylquinolonine
- Inhibits ATP synthase enzyme
- Molecular target = atpE gene
2012 – Food and Drug Administration (FDA)
• approved for adult MDR-TB • after Phase II clinical trial (C209)
under provisions of accelerated approval regulations for
"serious or life-threatening illnesses" (21CFR314.500)
Cross resistance BDQ - clofazimine (CLF) (Hartkoorn RC et al., AAC 2014; Andries et al., PloseOne 2014)
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Efflux-based resistance
Low level resistance
Mutations in Rv0678 gene
2- to 8-fold increase in MIC MICs 0.25 to 0.50 µg/mL
High level resistance Mutations in atpE gene
8- to 133-fold increase in MIC MICs 0.25 to 4.0 µg/mL
Target- based resistance
3 Cross-resistance BDQ-CFZ Slide from Koen Andries
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mmpL5 mmpS5 Rv0678
Cross-resistance BDQ-CFZ due to Rv0678 mutations
Palindromi
c
region
Intergenic
region
Rv0678 mmpL5 mmpS5
4 Slide from Koen Andries
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• MIC ranges M. tuberculosis : 0.03 – 0.12 µg/ml
– Reference strains on agar
– Clinical strains in BACTEC - radiometric
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Phenotypic drug-susceptibility testing (DST)
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• Data from Phase II clinical trial – Minimal inhibitory concentration (MIC)
– BDQ: Stock in DMSO (10.000 µg/ml stock)
Working solutions in sterile distilled water
– QC by H37Rv and BK12 (Andries et al., aptE mutant)
– Middlebrook 7H11 agar medium (Diacon et al., 2009 – 2012 – 2014)
• 21 days incubation at 37°C + 5% CO2
• Polystyrene tubes
– REMA (Palomino et al., 2012)
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Phenotypic drug-susceptibility testing (DST)
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EUCAST Clinical Breakpoint Table v 5.0 (valid from 2015-01-01)
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Objective: To evaluate performance of the MGIT960 for BDQ susceptibility in Mtb
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MGIT-BDQ drug-susceptibility testing
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QC range and ECOFF (Torrea et al., 2015)
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QC range H37Rv in MGIT
• 10 different days
• 3 different batches MGIT
ECOFF in MGIT and on 7H11
• Parallel testing
• 45 probable susceptible strains
H37Rv MICs and ECOFF in MGIT ~ 4-fold 7H11
ECOFF ~ EUCAST: the epidemiological cutoff
value is the wild type MIC value identifying
the upper limit for the wild-type population
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Accuracy / trueness (Torrea et al., 2015)
• 74 Probable-susceptible (PS) strains
• 18 Probable resistant (PR) strains (AtpE or Rv0678 mutants)
• Parallel testing
Bimodal curves
Breakpoints close to ECOFF
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Reproducibility – Repeatability (Torrea et al., 2015)
100% (39/39) replicates differed by ≤ 1 dilution
5 strains
2 operators
Different weeks
Same day testing
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• MGIT960 is reproducible and accurate vs. M7H11 agar – ECOFF ~ Breakpoint
– 1,00 µg/ml for MGIT960 and 0,25 µg/ml for M7H11
• MGIT960 and M7H11 ECOFFs being only a 2-fold concentration lower than the lowest MICs for mutant strains ~ risk to misclassify mutant strains as susceptible, if not performed meticulously.
• Further PK/PD studies and clinical data from a larger number of susceptible and MDR strains will be needed to finally set the clinical breakpoint.
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Conclusion MGIT-BDQ (Torrea et al., 2015)
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• MIC distribution and ECOFF
• 10 Pan-susceptible strains, BDQ-treatment naive
• 12 MDR and pre-XDR isolates
• BDQ tablets
• Drug stability test in MGIT (0-3 weeks pre-incubation)
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BDQ in MGIT using TB Exist software (Keller et al., 2015)
Non-significant difference between medians
pan-susceptible and MDR/XDR
ECOFF > Torrea: tablets?
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• Two studies peformed
• 8 different laboratories and locations
• 7H10 and 7H11 agar medium versus 7H9 broth medium
• QC data H37Rv (personal communication D. Cirillo)
– 0.015 to 0.12 μg/mL on 7H10 et 7H11 agar
– 0.015 to 0.06 μg/mL in 7H9 broth
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Multicenter evaluations
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• Löwenstein-Jensen: not suitable ~ protein binding
• Stability stock solution in DMSO
– Minimum 3 months at <-18°C
• Stability diluted solutions in broth (REMA plates)
– Maximum 2 weeks at 2-8°C
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Additional experience at ITM, Antwerp, Belgium
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Summary phenotypic DST
Medium ECOFF
(µg/ml)
Breakpoint
(µg/ml)
QC range H37Rv
(µg/ml)
7H10 agar 0.015 - 0.12
7H11 agar 0.25 0.25 0.015 - 0.12
7H9 broth (REMA) 0.06 – 0.015
MGIT960 1.0 1.0 0.12 – 0.5
LJ NA NA NA
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How to proceed if you want to test in your lab?
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Order BDQ powder: https://www.aidsreagent.org/register.cfm.
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• Not yet included in EQA proficiency panels
• Molecular testing – No atpE mutants in clinical isolates so far
– Broad variety in Rv0678 mutations
– Rv0678 mutations ~ MIC levels: to be determined
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Remaining challenges
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