DELAMANID SUSCEPTIBILITY TESTING IN AN AUTOMATED …

DELAMANID SUSCEPTIBILITY TESTING IN AN AUTOMATED LIQUID

CULTURE SYSTEM

Daniela Maria Cirillo

San Raffaele Scientific Institute, Milan

COI/CA

• OSR as signed MTA with Janssen and Otzuka as SRL and is involved in the development of MGIT test for delamanid

• OSR has received reimbursement for participation to the reproducibility study sponsored by Janssen

• OSR is leading the SRLN project partially financed by Otzuka for the development of DLM DST in MGIT

DELAMANID (DLM)

Delamanid is a nitromidoxazole compound that specifically impairs the bio-synthesis of methoxy- and keto-mycolic acids, by disrupting metabolism of the cell wall (Matsumoto M, 2006, Plos Med). Active on both replicating and not bacilli (Matsumoto M, 2006, Plos Med).

(Matsumoto M. et al., Plos One 2006)

Like other Nitroimidazoles, DLM require activation by mycobacterial F420-dependent deazaflavin-dependent nitroreductase (Rv3547 or Ddn)

Mechanism of resistance: Mutations in genes involved in coenzyme F420 biosynthesis and metabolism [fbiA (Rv3261), fbiB (Rv3262), fbiC (Rv1173), fgd1 (Rv0407)] has been proposed as possible mechanisms of resistance to DLM (Choi KP et al., J. Bacteriol. 2002) Spontaneous rate of resistance to delamanid in in he range of 6.44 x 10-6 - 4.19 x 10-5 (EMA, 2013)

• Well tolerated in TB patients, associated to higher favourable treatment outcomes and significantly lower mortality, not associated with clinically relevant drug-drug interactions (Blair HA, 2015,Drugs; Gler MT, 2012, Engl J Med).

• Conditional approval by WHO in 2014 for MDR-XDR TB treatment

• Deltyba has received marketing authorization by the European Medicine Agency and the Japanese Ministry of Health, Labor and Welfare in 2014 (Ryan NJ, 2014, Drugs).

• Delamanid is available in the UK, Germany and other European Countries for the management of MDR-TB patients and has received conditional approval by the world health organization (WHO).

DELAMANID - DELTYBA

AIMS

• To develop a standardized protocol for rapid Delamanid (DLM) susceptibility testing (DST) using the semi-automated BACTEC™ MGIT™ 960

• To define a breakpoint able to accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis (MTB) towards DLM.

EXPERIMENTAL PLAN

Validation of the resazurin microtiter assay (REMA) and MGIT MIC against the 7H11 agar based protocol (APM) on a panel of 19 Otsuka pre-characterized strains

Determination of MIC distribution in REMA and MGIT of clinical isolates never exposed to the drug. Results confirmed by APM

WGS of study strains to explore genetic polymorphisms in the five genes involved in the F420 mediated activation

RESISTANT STRAINS SUSCEPTIBLE STRAINS

8.0-4.0-2.0-1.0-0.5-0.25-0.125-0.06-0.03-0.016-0.008-0.004-0.002-0.001-≤ 0.0005 µg/ml

7H11/REMA/MGIT

• A break point of 0,2 µg/ml has been proposed by Otsuka based on previous work on a collection of wt and resistant mutant in vitro generated strains

• The break point has been discussed with the EUCAST committee (0,06 µg/ml)

DLM mic

Otsuka has independently established a 7H11-based DST method for testing strains and based on MIC performed on a collection of wt and resistant mutant in vitro generated strains defined a Breakpoint of 0,2 mg/L.

Determination of DLM MIC for a panel of 19 reference strains using REMA (from 0,0005 to 32 mg/L), MGIT and 7H11 (from 0,0005 to 16 mg/L)

MGIT-REMA ON OTSUKA PANEL Code #strain MIC (OTSK) REMA 7H11 MGIT 1 MGIT 2

OTSK N0268 R > 32 > 16 > 16 > 16

OTSK N1002 R 1 1 1 1

OTSK N0185 R > 32 > 16 > 16 > 16

OTSK N0652 R 0,5 1 0,25 0,25

OTSK N0339 S 0,03 0,004 0,016 0,008

OTSK N0085 S 0,008 0,016 0,008 0,004

OTSK N0001 S 0,016 0,016 0,016 0,008

OTSK N0082 S 0,016 0,016 0,008 0,008

OTSK N0299 S 0,008 0,016 0,016 0,01

OTSK N0156 S 0,004 0,004 0,004 0,008

OTSK N0400 S 0,008 0,004 0,004 0,016

OTSK N0117 S 0,008 0,008 0,016 0,008

OTSK N0110 S 0,004 0,016 0,008 0,008

OTSK N0678 S 0,008 0,002 0,004 0,008

OTSK N0097 S 0,008 0,004 0,016 0,016

OTSK N0667 S 0,004 0,004 0,004 0,004

OTSK N0946 S 0,008 0,004 0,004 0,004

OTSK N0193 S 0,03 0,125 0,03 0,03

H37RV 27294 S 0,004 0,016 0,004 0,008

WGS ON OTSUKA PANEL

Code #strain MIC ddn (Rv3547) fgd (Rv0407) fbiA (Rv3261) fbiB (Rv3262) fbiC (Rv1173) Lineage

OTSK N0268 R Insertion pos: 3986911 Phe 320 (silent) wt wt wt Beijing

OTSK N1002 R pos 3987149 CA->C DEL Phe 320 (silent) wt wt wt Beijing

OTSK N0185 R INSERTION + GTCA (pos: 3987023) wt wt wt Trp678Gly Leu 55 (silent) LAM

OTSK N0184 R Pos 3987023 G->GTCA INS wt wt wt Trp678Gly Leu 55 (silent) LAM

OTSK N0652 R Leu107Pro wt wt wt wt LAM

OTSK N0339 S wt wt wt wt wt LAM


OTSK N0001 S wt Phe 320 (silent) wt wt wt Beijing


OTSK N0299 S wt wt wt wt Asp375Asn LAM


OTSK N0400 S wt Phe 320 (silent) Val 5 (silent) wt wt EAI "Manila"

OTSK N0117 S



OTSK N0097 S wt wt wt wt Try678Gly Leu 55 (silent) LAM

OTSK N0667 S wt wt wt wt Lys 8 (silent) Euro-Am Sup

OTSK N0946 S wt wt wt wt wt Euro-Am Sup


SELECTION OF CLINICAL STRAINS: n 288

America 1%

Africa 23%

Asia 33%

Europe 43%

Susceptible 29%

NO-MDR 9%

MDR 32%

pre-XDR 16%

XDR 14%

Beijing 38%

Delhi-CAS 5%

EAI 8%

Haarlem 10%

LAM 10%

AFRI II 1%

TUR 1%

Ural 6%

Euro-America

n Superlineage …

Ghana 1%

MIC IN REMA AND MGIT

DLM MIC [µg/ml] ≤0,03 0,06 0,125 0,25 0,5 ≥1 Total

Susceptible 78 0 0 0 0 0 78

MDR 105 0 0 0 0 3 108

pre-XDR 55 0 0 0 0 0 55

XDR 29 0 0 0 0 1 30

NO_MDR 17 0 0 0 0 0 17

Total number of strains 284 0 0 0 0 4 288

DLM MIC [µg/ml] ≤0,03 0,06 0,125 0,25 0,5 ≥1 Total

Susceptible 72 0 0 0 0 0 72

MDR 83 2 0 0 0 3 88

pre-XDR 42 0 0 0 0 0 42

XDR 15 0 0 0 0 1 16

NO_MDR 3 0 0 0 0 0 3

Total number of strains 215 2 0 0 0 4 221

EXTENDED MIC in REMA

and MGIT

CORRELATION MGIT-REMA

Table 1. Correlation of drug susceptibility results obtained for 221 clinical strains tested both in MGIT and REMA. Conventional breakpoint of 0.2 mg/L determined by agar proportion method was considered when assigning the definition of Susceptible (S) or Resistant (R)

Beijing subfamily

W148

CHARACTERIZATION OF THE 4 DLM RESISTANT STRAINS

Strain Lineage Phenotipic DST SNP ddn (Rv3547) fbiA (Rv3261)

DLM_R1 Beijing MDR tGg->tAg W88STOP wt



DLM_R4 Beijing XDR Aag->Tag wt K250STOP

3 Strains resistant harboured a stop codon mutation in ddn (W88STOP) and 1 FbiA (K250STOP) .

SNPs IN GENES INVOLVED IN DLM

ACTIVATION

Genome analysis of the 167 DLM susceptible strains revealed eleven polymorphisms in the five genes associated to drug activation ddn, fgd1, fbiA, fbiB, fbiC leading to amino acid exchanges in 39 (22.8%) out of 171 strains (Table 2).

gene Nucleotide change Amino-acid change number of strains with

the SNP

ddn Cgg->Tgg R72W 2

gaG->gaC E83D 1

fbiA cAg->cGg Q120R 4

aCg->aTg T302M 2

fbiB

cTg->cGg K447R 1

aAg->aGg L448R 1

Ttc->Ctc F220L 8

fbiC Acg->Gcg T273A 5

aCc->aTc T681I 1

fgd1 aAg->aTg* K270M* 13

Aag->Gag* K296E* 1

Two of the identified SNPs were previously described as lineage-specific mutation of Haarlem (K270M) and M. africanum WA2 (K296E) genotypes.

MULTICENTER STUDY FOR THE VALIDATION OF BREAKPOINT

Validation of breakpoint in MGIT using 75 clinical isolates per study site plus 25 isolates from the original panel in four to six SR laboratories (400 – 500 tests in total)

CONCLUSION

DST for DLM can be performed in both MGIT and REMA.

We propose 0.125 mg/L as a breakpoint to screen for DLM sensitivity to this new drug

Pre-exposure high level resistance was observed on clinical strains

Low level resistance was not observed

WGS analysis in genes involved in the activation pathways show presence of several SNPs non related to an increased MIC

STOPcodons in the same genes are clearly associated to high level of resistance

ACKNOWLEDGEMENTS

Otsuka Becton Dickinson

GMBH, Gouting H. Hoffman S. Hoffman L. Nedialkova

San Raffaele Scientific Institute,Milano: S. Battaglia E. Borroni A. Cabibbe A. Trovato E. Schena

Research Center, Borstel M. Merkel C. Upatel S.Niemann

SRL network WHO - LDR

SNPs in genes involved in DLM activation

Genome analysis of the 167 DLM susceptible strains revealed eleven polymorphisms in the five genes associated to drug activation ddn, fgd1, fbiA, fbiB, fbiC leading to amino acid exchanges in 39 (22.8%) out of 171 strains (Table 2).

Two of the identified SNPs were previously described as lineage-specific mutation of Haarlem (K270M) and M. africanum WA2 (K296E) genotypes.

Phenotype Lineage fgd1 fbiA fbiB fbiC ddn Rv3547 nt change MIC

MDR Beijing wt wt wt wt Trp88STOP TGG->TGA ≥ 32



DR EAI wt wt wt wt Arg72Trp AGG->TGG 0,002

MDR Ural wt wt wt wt Glu83Asp GAG->GAT 0,001

MDR-AG EAI wt wt wt wt Arg72Trp AGG->TGG 0,004

MDR M. africanum WA2 Lys296Glu* wt wt wt wt AAG->GAG 0,001

MDR Harlem Lys270Ser* wt wt wt wt AAG->ATG 0,004

XDR Beijing Lys250STOP wt wt wt wt AAG->TAG ≥ 32

MDR-FQ Eur-Am Superlineage wt Thr302Met wt wt wt ACG->ATG 0,001

MDR Eur-Am Superlineage wt Thr302Met wt wt wt ACG->ATG 0,002

DR Eur-Am Superlineage wt Gln120Arg Phe220Leu wt wt CAA->CGA;TTC->TTA 0,0016

DR Eur-Am Superlineage wt Gln120Arg wt wt wt CAA->CGA 0,008

S Eur-Am Superlineage wt Gln120Arg wt wt wt CAA->CGA 0,004

S Eur-Am Superlineage wt Gln120Arg wt wt wt CAA->CGA 0,008

MDR Beijing wt wt Phe220Leu wt wt TTC->TTA 0,004

MDR Delhi/CAS wt wt Lys448Arg wt wt AAG->AGA 0,008

S Eur-Am Superlineage wt wt Leu447Arg wt wt CTA->CGA 0,004

S Eur-Am Superlineage wt wt wt Thr273Ala wt ACT->GCT 0,002


S Eur-Am Superlineage wt wt Phe220Leu Thr273Ala wt TTC->TTA 0,008


S Beijing wt wt wt wt Thr681Ile ACC->ATC 0,004

DELAMANID SUSCEPTIBILITY TESTING IN AN AUTOMATED …

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