Synergy Testing

7/28/2019 Synergy Testing

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Extremely Drug-resistant

organisms:Synergy Testing

LIM TZE PENG

Principal Pharmacist

Singapore General Hospital


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Acinetobacter baumannii& Pseudomonas aeruginosa

Emerging Gram-negative bacilli Part of the ESKAPE group of organisms1

Enterococcus faecium

Staphylcoccus aureus

Klebsiella pneumoniae

Acinetobacter baumannii

Pseudomonas aerugionosa

Enterobacter spp.

Background

1. Helen W. Boucher, Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clin Infect Dis2009; 48:1-12


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What is the difference between

MDR

XDR

PDR

Definitions

1. Magiorakos, A. P., A. Srinivasan, et al. (2011). "Multidrug-resistant, extensive ly drug-resistant and pandrug-resistant bacteria: aninternational expert proposal for inte rim standard definitions for acquired resistance." Clin Microbiol Infec t.


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What is the difference between

Synergistic

Bactericidal

Inhibitory

Antagonistic

Definitions


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Typical MIC profileStrain

Antibiotic18351 18352 27640 9447 11171

Ampicillin/Sulbactam 32/16 32/16 64/32 32/16 32/16

Ciprofloxacin 16 16 16 16 16

Gentamicin 64 64 64 64 64

Imipenem 64 32 32 64 64

Meropenem 128 64 32 64 64Aztreonam 128 64 32 128 128

Piperacillin/Tazobactam 256 256 256 256 256

Polymyxin B 64 32 128 16 16

Tigecycline 4 4 16 4 4Ceftazidime 128 128 128 128 128

Amikacin 128 128 128 128 128

Cefepime 128 128 128 128 64

Rifampicin 2 4 256 256 256


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Strain

Antibiotic18351 18352 27640 9447 11171

Ampicillin/Sulbactam 32/16 32/16 64/32 32/16 32/16

Ciprofloxacin 16 16 16 16 16

Gentamicin 64 64 64 64 64

Imipenem 64 32 32 64 64

Meropenem 128 64 32 64 64Aztreonam 128 64 32 128 128

Piperacillin/Tazobactam 256 256 256 256 256

Polymyxin B 64 32 128 16 16

Tigecycline 4 4 16 4 4Ceftazidime 128 128 128 128 128

Amikacin 128 128 128 128 128

Cefepime 128 128 128 128 64

Rifampicin 2 4 256 256 256

Typical MIC profile


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Decreasing new antibacterials approved for use

Only 2 new antibacterials in the last 6 years

Doripenem (2007), Ceftaroline (2013)

Multi-pronged approach Judicious use of existing agents

Efficient infection control

Antimicrobial Stewardship Program Combination therapy

Potential solutions


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Often used in clinical practice TB & HIV

Increasingly used in MDRA. baumannii& P.aeruginosa

Enhanced pharmacodynamic effect(synergism)

Enhanced bactericidal effect Suppress emergence of resistance

Combination therapy


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1. Maragakis LL, Perl TM. Acinetobacte r baumannii: epidemiology , antimicrobial resistance, and treatment options. Clin Infect Dis.2008 Apr 15;46(8):1254-63.


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Various methodologies Checkerboard method

Time-kill studies In-vitro pharmacodynamic models

Types of combination studies


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1. Hsieh MH, Yu CM, Yu VL, Chow JW. Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infec t Dis. 1993 May-Jun;16(4):343-9.

Fractional Inhibitory Concentration

Index

Synergy: FIC index < 0.5

Additive: FIC index = 1

Antagonism: FIC index > 4


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Limitations of FIC index

Based on Loewe additivity

Assume similar & linear concentration-time

relationship

Subjective endpoints

Cloudy vs Clear wells


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0

1

2

3

4

5

6

7

8

9

0 2 4 8 12 24

Time (Hours)

Log10[cfu/ml]

Growth control

Polymyxin B

Rifampicin

Polymyxin B & Rifampicin

Time-kill studies (TKS)

Antibiotic synergy:

2 log decrease incfu/ml

1. National Committe e for Clinical Laboratory Standards. 1999. Methods for Determining Bactericidal Activity of AntimicrobialAgents. Approved Guideline M26-A., vol. 19. NCCLS, Wayne, PA, USA


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Limitations of TKS Clinical relevance

24 hour endpoint

At least one of the drugs must be present in a concentration

which does not affect the growth curve of the test organism when

used alone.1

Pharmacokinetic factors ignored

Resource management

Labour & Time intensive

1. Antimicrobial Agents & Chemotherapy: Instructions to Authors


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Hollow-fiber infection model allowing simulation of human PKin vitro. (Tam, JID 2007)

bacteria

Drug

EliminationDistribution

Hollow-Fiber System


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Aims

To elucidate efficacious antibioticcombinations against PDRA. baumannii


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Methods

Time-kill studies (TKS)

Maximal clinically achievable concentrations

Hollow Fiber Infection Model (HFIM) In-vivo environment simulation


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Time Kill Study (TKS)


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Susceptibility resultsAntimicrobial

TTSH 112(mg/l)

TTSH 105(mg/l)

SGH 8879(mg/l)

Meropenem 32 (R) 64 (R) >32 (R)

Polymyxin B 1 (S) 1 (S) 2 (S)

Rifampicin1 4 4 2

Tigecycline 4 (I) 0.5 (S) 2 (S)

1There are currently no international standards for rifampicin and susceptibility

testing against Acinetobacter baumannii


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Pharmacokinetic Data

AntimicrobialSimulated

FREE drug

conc (mg/l)

Maximum clinicalachievable FREE

drug conc (mg/L)

Corresponding maximumclinical dose

Meropenem 64 64 (plasma) 2g q8h over 3h infusion

Polymyxin B 2 2 (plasma) At least 1 MU q12h

Rifampicin 2 2 (plasma) PO 600mg q12h

Tigecycline 2 2 (tissues) 100mg q12h

1. Jaruratanasir ikul S, et al. Compariso n of the pharmacodynamics of mero penem in patients with ventilator- asso ciated pneumonia following

administration by 3-hour inf usion or bolus in jection . Antimicrob Agents Chemother. 2005 Apr;49(4):1337-9.2. Kwa AL, Lim TP, Low JG, Hou J, Kurup A, Pr ince RA, Tam VH. Pharmacokinetics of polymyxin B1 in patients with multidr ug-resistant Gram-

negative bacterial in fections. Diagn Microbiol In fect Dis. 2008 Feb ;60(2):163-7. Epub 2007 Oct 4.3. Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. Concentration -dependent Mycobacterium tuberculosis killing and

prevention of resistance by rifamp in. Antimicro b Agents Chemother. 2007 Nov;51(11):3781-8. Epub 2007 Aug 27.

4. Rod vold KA, Gotfried MH, Cwik M, Ko rth- Brad ley JM, Dukart G, Ellis-Gr osse EJ. Serum, tissue an d b ody fluid concentration s of tigecycline after asingle 100 mg dose. J Antimicrob Chemother. 2006 Dec;58(6):1221-9.


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0

2

4

6

8

10

0 4 8 12 1 6 20 24

Time (h)

Log10CFU/ml

Pl acebo

Rif + PB

Rif + Mero

PB + Mero

Tige + PB

Tige + Ri f

Tige + Me r

0

2

4

6

8

10

0 4 8 12 16 2 0 24

Time (h)

Log10CFU/ml

Placebo

PB

Mero

Rif

Tige

TKS SGH AB 8879

Microbiological responses of AB againstvarious antibiotic combinations

Microbiological responses of ABagainst various antibiotics


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Objective

To evaluate the efficacy of :

Polymyxin B and Rifampicin or

PolymyxinB and Tigecycline or

Tigecycline and Rifampicin combined against PDR AB

from our local hospitals.


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Methods Bacteria

361 AB strains collected from National AntimicrobialResistance, Singapore

MIC testing (microtitre)

31 PDR AB with OXA-23, OXA-51 b-lactamases &ISAba1OXA complex

Time-kill studies performed with the 31 PDR AB

strains

Baseline inoculums of 5 log10 CFU/ml


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Pharmacokinetic Data

Antimicrobial

Simulated FREE

drug conc

(mg/l)

Corresponding maximum

clinical dose

Polymyxin B 2 (serum trough) At least 1 MU q12h

Rifampicin 2 (serum peak) PO 600mg q12h

Tigecycline 2 (tissue peak) 100mg q12h

Kwa AL, Lim TP, Low JG, Hou J, Kurup A, Prince RA, Tam VH. Pharmacokinetics of polymyxin B1 in patients withmultidrug-resistant Gram-negative bacterial infections. Diagn Microbiol Infect Dis. 2008 Feb;60(2):163-7. Epub 2007 Oct 4.

Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. Concentration-dependent Mycobacterium

tuberculosis killing and prevention of resistance by rifampin. Antimicrob Agents Chemother. 2007 Nov;51(11):3781-8. Epub

2007 Aug 27.

Rodvold KA, Gotfried MH, Cwik M, Korth-Bradley JM, Dukart G, Ellis-Grosse EJ. Serum, tissue and body fluidconce ntrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006 Dec;58(6):1221-9.


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0

1

2

3

4

5

6

7

8

9

0 2 4 8 12 24

Time (Hours)

Log10[cfu/ml]

Growth control

Polymyxin B

RifampicinPolymyxin B & Rifampicin

Combination timekill

Antibiotic synergy:

2 log decrease in

cfu/ml

2 log decrease incfu/ml from original

inoculum


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MIC results (31 PDR AB strains)

Susceptibility (%)

Antibiotics MIC50 (mg/L) MIC90 (mg/L) Range (mg/L) R I S

Polymyxin B 1 2 0.5 2 100

Rifampicin 6 64 1 64

Tigecycline 4 32 0.5 32

Resistant to all antibiotics


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Single TKS results24 hour mean bacteria burden after exposure to various antibiotics alone(1-2 log reductions strains denoted in red)Tigecycline Polymyxin B Rifampicin

AB

strain

Starting

inocula

Mean Mean Mean

8 5.22

12 5.27

16 5.23

17 5.03

23 5.35

25 5.44

28 5.30

32 5.30

41 5.38

59 5.25

60 5.36

69 5.25

70 5.26

88 5.28

91 5.20

3.50

3.74 3.29

3.40

4.02

3.12

3.00

97 5.33 6.94 4.85 5.62

7.13 8.07

8.60 5.72 7.77

7.72 8.69

9.14

7.94 4.94 5.99

6.86 5.72 7.83

8.08 8.61 8.73

5.11 7.91

7.99 8.04 5.56

8.08 5.13 9.17

7.97 8.46

8.16 5.48 8.97

8.33 8.60

7.17 4.84 6.11

7.14 8.58

8.53


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Single TKS results (continued)24 hour mean bacteria burden after exposure to various antibiotics alone(1-2 log reduction strains denoted in red)Tigecycline Polymyxin B Rifampicin

AB

strain

Starting

inocula

Mean Mean Mean

98 5.54

102 5.21

104 5.16

126 5.20

128 5.42

129 5.19 4.07

138 5.38

170 5.37

174 5.26 3.57

112 5.18

8879 5.01

14101 5.40

3160 5.17

13631 5.43

48038 5.32

7.72 4.31 5.59

6.63 5.09 8.05

6.84 5.05 7.70

8.77 5.59 5.43

8.71 7.86 5.82

8.84 8.28

6.77 4.40 8.36

7.62 5.42 8.49

6.35 8.24

7.45 4.73 7.52

5.25 4.23 7.56

5.38 5.71 8.33

8.93 4.75 8.60

7.93 5.59 8.86

8.90 4.78 8.18


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24 hour mean bacteria burden after exposure to various antibiotic combinations(modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)

Tigecycline + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline

AB

strain

Starting

inocula

Mean Mean Mean

8 5.22 0.00

12 5.27 0.00 0.00

16 5.23 0.80

17 5.03 0.00 0.00 0.65

23 5.35

25 5.44 0.00

28 5.30 3.57

32 5.30 0.00 2.31

41 5.38

59 5.25 4.10 3.24

60 5.36 0.80

69 5.25 4.10

70 5.26 4.09

88 5.28 2.42 0.00

91 5.20 0.00 0.00

97 5.33 0.00 0.00

6.81 5.11

7.19

4.70 3.56

4.95 4.85 4.82

4.73 5.01

5.53 4.80

5.41

4.77 4.54 5.10

8.67

6.65 5.37

7.64 5.08

6.84 4.66

4.90

4.65

4.25

Combination TKS results


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Combination TKS results (continued)24 hour mean bacteria burden after exposure to various antibiotic combinations(modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)Tigecycline + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline

AB

strain

Starting

inocula

Mean Mean Mean

98 5.54 0.00 0.00

102 5.21 0.00

104 5.16

126 5.20 2.60 2.48

128 5.42 2.69 0.00

129 5.19 2.28

138 5.38 3.86 4.15

170 5.37 3.79 3.66

174 5.26

112 5.18 0.00 0.00 0.00

8879 5.01 0.00 1.69 0.00

14101 5.40

3160 5.17

13631 5.43

48038 5.32

4.84

5.35 4.86

4.48 4.85 4.57

1.60

5.49

5.93 4.73

5.33

8.29

5.59 4.65 4.63

4.27 5.27 7.16

6.70 4.62 4.59

5.91 5.44 5.94

6.03 5.44 5.41


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Time-Kill Results Polymyxin B alone

6 out of 31 strains showed a reduction of 1-2 log10 CFU/ml

in bacterial density compared to baseline at 24 hrs 25 out of 31 strains show insignificant reduction (< 1 log10

CFU/ml) or higher inoculums (approx 8 log10 CFU/ml) at 24hrs

Tigecycline or rifampicin alone

Either < 2 log10 CFU/ml drop at 24 hrs from baseline

inoculums for 2 & 1 strain(s) in tigecycline & rifampicinrespectively

Or increase of > 2 log10 CFU/ml at 24 hrs from baselineinoculums


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Combination Time-Kill Results Polymyxin B+ rifampicin

14 out of 31 strains achieve > 2 log10 CFU/mldecrease from baseline inoculum, at 24 hrs

Polymyxin B + tigecycline

10 out of 31 strains

Tigecycline + rifampicin

8 out of 31 strains


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Time-Kill Results None of the antibiotics combinations demonstrated

modified bactericidal activity against 14 out of 31strains Polymyxin + rifampicin, polymyxin +tigecycline

demonstrated 1-2 log10 CFU/ml reduction in 5 & 4 strains

respectively from baseline at 24hr. Total 6 (28,59, 69, 70,138, 170)

Tigecycline + rifampicin is at least additive in 7 strains (23,104, 174, 14101, 3160, 13631, 48038)

Polymyxin + rifampicin is additive to 1 strain (41) Polymyxin alone demonstrated the lowest bacteria burden for

5 strains (23, 174, 3160, 13631, 48038) at 24 hr ~ 3.5-5.6 log10CFU/ml


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TKS results24 hour mean bacteria burden after exposure to various antibiotic combinations(modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)Tigecycline + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline

AB

strain

Mean Mean Mean

98 0.00 0.00 4.84

102 5.35 0.00 4.86

104 4.48 4.85 4.57

126 2.60 2.48 1.60

128 5.49 2.69 0.00

129 5.93 4.73 2.28

138 5.33 3.86 4.15

170 8.29 3.79 3.66

174 5.59 4.65 4.63

112 0.00 0.00 0.00

8879 0.00 1.69 0.00

14101 4.27 5.27 7.16

3160 6.70 4.62 4.59

13631 5.91 5.44 5.94

48038 6.03 5.44 5.41


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Pharmacokinetic/Pharmacodynamic Modelling

of Polymyxin B, Rifampicin and Tigecyclineagainst Pandrug-resistantAcinetobacter

baumanniiin an In-vitro Model

T.P. Lim1, T.Y. Tan2, W. Lee1, Sasikala. S.2, T.T. Tan1, L.Y. Hsu3, A.L. Kwa1

1Singapore General Hospital, 2Changi General Hospital. 3National University Hospital

ECCMID 2010, Vienna, Austria


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HFIM 2 representative strains used to validate the

results in hollow-fiber infection model (HFIM)

TTSH AB 112

SGH AB 8879


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HFIM l


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HFIM results

0

2

4

6

8

10

0 1 2 3 4 5

LogCFU/ml

Days

Placebo

Polymyxin B 1MU q12h

Rifampicin 600mg q12h

Tigecycline 100mg q12h

Polymyxin B 1MU q12h +

Rifampicin 600mg q12h

Polymyxin B 1MU q12h +Tigecycline 100mg q12h

Tigecycline 100mg q12h +Rifampicin 600mg q12h

HFIM l


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HFIM results

0

2

4

6

8

10

0 1 2 3 4 5

Days

L

ogCFU/m

l

Tota l

Res ista nt

Polymyxin B regimen simulated

Polymyxin B resistant isolates plated on drug-supplemented

media at 3X MIC.


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Antibiotic Combinations against


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Antibiotic Combinations against

MDR Bacteria

Trial and Error

Countless permutations

Different combinationseffectiv e for differentstrains1

Certain combinations maylead to antagonism2,3

Guided by in-vitroTesting Avoid use of antagonistic

combinations Identify effectiv e

combinations

1. Lim TP et al. (2009) I Antibiot (Tokyo).2. Aaron SD et al, (2000) . Am J Respir Cr itCare Med 161: 1206-1212.3. Lang BJ et. al (2000). Am J Respir CritCare Med 162: 2241-2245.

M th d l Ad t Li it ti


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Methodology Advantages Limitations

Time-kill (TK) method Gold-standardMeasures bactericidal

activity

Describes extent of kill over24 hours

Time-consumingLimits no. of combinations

tested

Need for repetitive samplingResults likely retrospective in

nature

Multiple CombinationBactericidal Testing(MCBT) method

Fast turn-around timeLarge no. of antibioticcombinations tested

Nov el methodLimitations not f ullyelucidated

MCBT method TK method


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Inoculation of bacteria Bacteria at standard concentration

added

More than 80 combinations tested

Day 0Isolates

received

TIME-LINE

Day -1

Day 1

Day 2

MCBT method

Preparation of microtiter plates Addition of one or two antibiotic(s) to

well

Prepared in bulk and stored till required

Sampling and plating of bacteria Preliminary results (based on turbid

wells)

Contents of wells sampled and plated

Bacteria Counts

Counts enumerated based on growthon plates

Preparation ofmicrotiter

plates

Inoculation ofbacteria

Sampling and

plating of bacteria

Bacteria Counts

Preparation of drugs /Inoculating bacteria

Addition of one/two antibiotics toflasks

Standard concentration of bacteriaadded

Up to 20 flasks tested

Sampling and plating of bacteria Contents of flasks sampled and

plated

Bacteria Counts

Counts enumerated based ongrowth on plates

Sampling and plating

of bacteria

Bacteria Counts

Preparation of drugs/Inoculating bacteria


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ACI BAUMAN

- - - - - - - - - - - -POLYMYXIN + RIFAMPICIN

POLYMYXIN + TIGECYCLINE

POLYMYXIN + AZTREONAM

RIFAMPICIN + TIGECYCLINE

MEROPENEM +AZTREONAM

AZTREONAM + LEVOFLOXA CIN

POLYMYXIN + MEROPENEM MEROPENEM + AZTREONAM

RIFAMPICIN + MEROPENEM

LEVOFLOXACIN + TIGECYCLINE

Legend

At least inhibitory No utility


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Collaborators Changi General Hospital

Dr Tan Thean Yen

National University Hospital

A/P Hsu Li Yang

Tan Tock Seng Hospital

Dr David Lye A*STAR (IBN)

A/P Yang Yi-Yan

Parkw ay Health

Dr AsokKurup

University of Houston

A/P Vincent Tam

Singapore General Hospital Dr Tan Thuan Tong

Dr Tan Ban Hock

Dr Jenny Low

NUS (Pharmacy)

A/P Eric Chan

NUS (Chemical and

Biomolecular Engineering ) A/P Xie Jianping

A/P Leong Tai

University of Monash

A/P Li Jian.


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Thank You!

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Synergy Testing