Quanta Matrix Inc. Rapid Antimicrobial Susceptibility Test Using Single Cell Morphological Analysis (SCMA) in Plastic Microfludic Chips Sunghoon Kwon

Quanta Matrix Inc.

Rapid Antimicrobial Susceptibility Test Using Single Cell Morphological Analysis (SCMA) in Plastic Microfludic Chips

Sunghoon Kwon

Professor, Department of Electrical Engineering, Seoul National University, Korea CEO, Quanta Matrix Inc. , Korea

Helping life scientists with technologies

MAC 2.0

antibiotic s Bacteri a



Seoul National University

Antibiotics is one of the most important invention saving lives

Alexander Fleming (1881-1955)

“It is not difficult to make microbes resistant to

penicillin and people may die as a result of bacteria becoming resistant to antibiotics in the future.”

-In Nobel Lecture, 1945-

Discovery of penicillin in 1928

Nobel Prize, 1945

Fleming’s prediction has come true : Superbugs!

Drug-resistant bacteria (MRSA) have found refuge

Seoul National University

Faster Antibiotic Susceptibility Test (AST) is needed

Rapid and accurate Antibiotic treatment

Gentamicin

2μg/mL (MIC value)

P. aeruginosa

Identification of bacteria Among ~60 Clinical pathogens

Choosing the proper antibiotic (amount) out of

~60 clinical antibiotics

Resistance or Sensitive?

Blood Lab  Department of Infectious Diesease

* Takes 3-4 days

to get this report

Seoul National University

Antibiotic Susceptibility Test (AST) Using Turbidity

Bacteria

Growth Inhibition At Over MIC

Growth At Under MIC Antibiotics

Minimum Inhibitory Concentration (MIC) :

the lowest concentration of an antimicrobial that will inhibit the visible growth

S R

AST for Determination of MIC

Different Antibiotics

Concen tr ations

ATM CAZ VAN

Growth Non -Gr owth

Points MIC Inoculating Bacteria from Patients

Control 0.5 μg/mL 1 μg/mL 2 μg/mL 4 μg/mL 8 μg/mL 16 μg/mL

Gentamicin

Control

0.5 μg/mL

1 μg/mL

2 μg/mL

4 μg/mL

8 μg/mL

16 μg/mL

Incubation for 16-20 hours

MIC

Vancomycin

The most

Susceptible

Seoul National University

Conventional AST : SLOW!!!

Blood-sampling (8-10ml)

Blood culture bottles

Growth detecting machine

Microdilution Test

VITEK AST kit

Culture on Blood agar plate

(Pure culture)

Bacterial Colony

Dilution tube

- A few hours or 1 day -

Overnight (16~24 hours)

Blood Culture (0.5~1 day) Pure Culture (1 day)

AST (0.5 - 1 day)

antibiotics

concentration

10

⁵

CFU/mL

10

⁸

CFU/

5ⅹ10

⁶

CFU/mL

≤ 10 CFU/mL

10

⁸

cells/colony

~10

⁸

CFU/mL

1

^st

culture 2

^nd

culture

3

^rd

culture

Needs of ‘Same Day Result’

Rapid AST

• VITEK2 and Phoenix : the most AST’s time-to-result is 9~14h

• Antibiotics can be prescribed on following working day.

• QM RAST : Results come out within 4 hours

• Antibiotics can be prescribed on the same day.

Saving 20hrs

Seoul National University

v

Antibiotics Bacteria in the

agarose

Agarose Bacteria

Bacterial Capture

and Release

Bacteria In the blood

sample

Analyzer

Our RAST System : 3 hour AST

Single Cell

Imaging

The Idea : Single Cell Tracking AST

Seoul National University

Difficulty of Single Bacterium Imaging: It’s moving!

v

A)

Inlet Antibiotic

Well Microfluidic

Channel B)

outlet

Integrating Microfluidic Chip with 96 Well Plate (MAC 2.0)

96 well form

For covering whole Range of candidate

Imaging with Temperature

Control

Seoul National University

- Cell and matrix (agarose) Loading

- Liquid medium (chemical solution) Loading

Process of MAC 2.0 RAST

0 hr

1 hr

2 hr

3 hr

High Thoughput AST Chip

Seoul National University

A)

B)

0 h 1.5 h 3 h

Morphological Determination of Antibiotic Susceptibility – Normal case

0 h 1.5 h 3 h

P. aeruginosa

Amikacin Susceptible

Amikacin

Resistance

MIC Determination from Time Lapse Images

Amika cin Conce ntratio n [μ g/mL]

[0 h] [1 h] [2 h] [3 h]

P. aeruginosa

MIC

0 0.5

1 2

4 8

Growth

Non- Growth

Minimum Inhibitory

20 μm

Seoul National University

v

v MIC determination

through

imaging processing

P. aeruginosa ATCC 27853 with Amikacin

CLSI MIC 1~4 ug/ml MAC MIC

4 ug/ml

0 μg/ml

0.5 μg/ml

1 μg/ml

2 μg/ml

4 μg/ml

8 μg/ml

MIC

4 μg/ml 2 μg/ml 1 μg/ml 0.5 μg/ml

0 μg/ml

8 μg/ml

Morphological Determination of Antibiotic Susceptibility – Filament growth case

0 h 1.5 h 3 h

Aztreonam ( β -Lactam) Susceptible

Aztreonam ( β -Lactam) Resistance

No dividing, No growth

Seoul National University

RAST based on Single Cell Morphological Analysis

(SCMA)

Gram Negative Strains

E. coli

ATCC 25922

Antimicrobial MAC QC range

Amikacin 0.5~1 0.5~4

Amoxicillin/Clavulanic Acid 4/2 2/1~8/4

Ampicillin 2~4 2~8

Aztreonam 0.12 0.06~0.25

Cefazolin 2 1~4

Cefepime 0.03~0.06 0.015~0.12

Cefotaxime 0.06 0.03~0.12

Cefoxitin 2 2~8

Ceftazidime 0.5 0.06~0.5

Ciprofloxacin 0.004~0.008 0.004~0.015

Gentamicin 0.25 0.25~1

Imipenem 0.12 0.06~0.25

Norfloxacin 0.03~0.06 0.03~0.12

meropenem - -

Piperacillin 2 1~4

Piperacillin/Tazobactam 1/4~2/4 1/4~4/4

Tetracycline 1 0.5~2

Trimethoprim/Sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5

Ticarcilin - -

Ticarcillin/Clavulanic acid - -

Tobramycin - -

Time to Result 3 hr 16~20 hr

Gram Positive Strains

S. aureus

ATCC 29213

Antimicrobial MAC QC range

Ampicillin 1 0.5~2

Amoxicillin/Clavulanic Acid 0.25/0.12~0.5/0.25 0.12/0.06~0.5/0.25

Ciprofloxacin 0.5 0.12~0.5

Clindamycin 0.12 0.06~0.25

erythromycin 0.25 0.25~1

Gentamicin 0.25~0.5 0.12~1

Imipenem 0.03 0.015~0.06

Levofloxacin 0.12~0.25 0.06~0.5

Linezolid 1~4 1~4

Oxacillin 0.5 0.12~0.5

Norfloxacin - -

Penicillin 0.5 0.25~2

Rifampin 0.004~0.008 (6 hr) 0.004~0.015

Streptomycin High Level - -

Teicoplanin - -

Tetracycline 0.12~0.5 0.12~1

Trimethoprim/sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5

Vancomycin 1 0.5~2

Time to Result 4 hr 16~20 hr

Validation of Morphological Analysis AST

using Standard Strains

22

J. Choi, S. Kwon et al., Science Translational Medicine, 2014

FDA Requirement

mE ME VME CA

<1 0%

<

3%

<1.

5%

>9 0%

Clinical Samples Summary of Clinical Sample Test

Methicillin-Resistant Staphylococcus aureus (MRSA) Vancomycin-Resistant Enterococci (VRE)

Extended-Spectrum Beta-Lactamases (ESBL)

minor Error (mE) Major Error (ME)

Very Major Error (VME)

Categorical Agreement (CA)

Seoul National University

v Seoul National University

Biophotonics & Nano Engineering Lab.

Sung-Weon Ryu Sang-Woo Ha

Hye-Jin Kim Sunghoon Kwon

Yong-Gyun JungJungil Choi

Eun-Geun Kim

• BiNEL & QuantaMatrix : Devices

• Prof. Eui Jong Kim & Prof. Sanghoon Song @ Seoul National University Hospital, Korea : Clinical study

• Prof. Seungwuk Lee, Catholic Hospital, Korea : Clinical Study

• Prof. Hee Chan Kim & Jungchan Lee @ SNU : Image processing

• Dr. Sungwon Ryu @ Korean Tuberculosis Institutes: Clinical Study

Acknowledgements

QM & KTI

Thank you