Evaluation of a standardized antibiotic microplate (Sensititre® MYCOTB MIC)

for susceptibility testing of MDR-TB isolates to 1st and 2nd line drugs:

comparison with the agar proportion method

Moses Joloba, MD, PhD

for the

TB-Clinical Diagnostics Research Consortium

Disclosure

• Thermo Fisher Scientific donated Sensititre® MYCOTB MIC plates and provided training in the inoculation and reading of plates

Background

• Reference phenotypic methods for Mtb DST are qualitative and based on drug critical concentrations

• Limitations include lack of standardization: • Variations in drug sources

• Variations in-lab preparation of drug stock solutions

• Variations in solid agar media types and preparations

• DST for some 2nd line drugs is particularly challenging

• A standardized, manufacturing quality-assured method based on MIC determination may have advantages

Background

• TREK Sensititre® MYCOTB MIC plate (Thermo Fisher Scientific, USA) – a dry micro-dilution plate containing lyophilized

antibiotics, with concentrations prepared and quality controlled by manufacturer

– Configured for determination of MICs to 1st and 2nd line TB drugs

– Currently little published information about test performance, especially with resistant Mtb isolates

Sensititre® MYCOTB Panels

5

TREK Sensititre® MYCOTB MIC plate o

flo

xaci

n

mo

xifl

oxa

cin

rifa

mp

icin

amik

acin

stre

pto

myc

in

rifa

bu

tin

PAS

eth

ion

amid

e

cycl

ose

rin

e

iso

nia

zid

kan

amyc

in

eth

amb

uto

l

Design & Setting

• Multi-site study using archived Mtb isolates with previously characterized DST – MDR, XDR, other DR patterns, pan-susceptible

• Reference labs in Kampala, Uganda and Masan, Republic of Korea

• Site staff received training in TREK MYCO TB and APM methods

• Regular quality monitoring

Methods • Strains thawed, sub-cultured twice onto 7H10

• APM

– Colonies inoculated into 7H9 broth, adjusted to McFarland 1.0, serial dilutions made in 7H9

– Agar plates containing critical drug concentrations inoculated

– Incubated 37°C, read at 21 days.

• TREK MYCO TB

– Colonies inoculated into saline/tween solution with glass beads, mixed, adjusted to McFarland 0.5, settled x 15 min, dilution made in 7H9

– 100 µl inoculated into each well

– Results read at each of 7, 10, 14, 21 days by two independent readers

– Results considered interpretable if adequate growth in the (+) control wells

Analysis

• Two approaches used for comparison of MYCO TB with APM (reference comparator):

– ‘Categorical’

• MYCO TB considered ‘susceptible’ if MIC was ≤ APM CC

• MYCO TB considered ‘resistant’ if MIC > APM CC

• Sensitivity, specificity, agreement calculated

– ‘Conditional’ • As above, with allowance for +/- one dilution around

APM CC

Example: ISONIAZID (APM CC 1.0 µg/ml)

0.03 ********************

***************

0.06 ********************

***********

0.12 ****

0.25 ******

0.5 *** *********

1 **** **

2 **************** *

4 ******************

******************

***** *

>4 ******************

***********

Resistant Susceptible

0.03 ********************

***************

0.06 ********************

***********

0.12 ****

0.25 ******

0.5 *** *********

1 **** **

2 **************** *

4 ******************

******************

***** *

>4 ******************

***********

Resistant Susceptible

Blue indicates discordance

• MYCO TB considered ‘susceptible’ if MIC was ≤ APM CC

• MYCO TB considered ‘resistant’ if MIC > APM CC

CATEGORICAL CONDITIONAL

TREK

MYC

O T

B M

IC

APM STATUS

Flow diagram of isolates tested

185 isolates

inoculated onto APM and MYCO TB

182 isolates with

results for both APM and MYCO TB

3 isolates

excluded from the analysis

insufficient growth APM (2) insufficient growth MYCO TB (1)

% of test isolates resistant by APM (reference comparator method)

Drug, µg/ml % resistant

Drug, µg/ml % resistant

Isoniazid 0.2 61 Ofloxacin 2.0 28

Isoniazid 1.0 51 Moxifloxacin 0.5 24

Rifampin 1.0 52 Moxifloxacin 2.0 10

Rifabutin 1.0 39 Streptomycin 2.0 18

Ethambutol 5.0 48 Streptomycin 10.0 14

Ethambutol 10.0 24 Amikacin 4.0 23

Kanamycin 5.0 27

Cycloserine 25 8

Ethionamide 5.0 38

PAS 2.0 28

Time to interpretation of results, by method

• APM: 21 days

• MYCO TB: median 10 days

0

10

20

30

40

50

60

70

80

7 10 14 21

Korea

Uganda

MYCO-TB: first scheduled reading day with control well growth sufficient for interpretation

% o

f is

ola

tes

MYCO TB: % agreement of MIC between 2 independent readers

70

75

80

85

90

95

100IN

H

RIF

RB

T

EMB

OFL

MX

F

STR

AM

I

KA

N

CY

C

ETH

PA

S

Korea Uganda

% a

gre

em

en

t

Sensitivity of MYCO TB for detection of drug resistance, using APM as comparator method

Sen

siti

vity

Specificity of MYCO TB for detection of drug resistance, using APM as comparator method

Spec

ific

ity

Categorical concordance of MYCO TB and APM results

Co

nco

rdan

ce

Conditional concordance of MYCO TB (+/- one dilution) and APM results

Co

nco

rdan

ce

Categorical (dark blue) and conditional (light blue) concordance

of MYCO TB and APM results

Co

nco

rdan

ce

• Towards understanding the nature of MYCO TB vs APM discordance for: – Moxifloxacin

– Ethambutol

• Towards understanding the nature of the poor sensitivity of MYCO TB for detection of resistance for cycloserine (using APM as reference standard)

% of isolates with discordant results (+/- 1 dilution) by MYCO TB and APM, by drug

0

2

4

6

8

10

12

INH

0.2

INH

1.0

RIF

1.0

RB

T 1

.0

EMB

5.0

EMB

10

.0

OFL

2.0

MX

F 0

.5

MX

F 2

.0

STR

2.0

STR

10

.0

AM

I 4.0

KA

N 5

.0

CY

C 2

5

ETH

5.0

PA

S 2

.0

MYCO TB-S / APM-R MYCO TB-R / APM-S

% o

f is

ola

tes

Moxifloxacin discordance driven by relatively large number of isolates characterized as Susceptible by APM yet Resistant by MYCO TB.

This is not much affected by APM critical concentration or by definition of concordance

MOXIFLOXACIN, APM 0.5 ug/ml

0.06 *****

0.12 ******************

*************

0.25 ******************

******************

********

0.5 ******************

**************

1 * **************

2 ****** ****

4 ******** *

8 ************** ***

>8*************** ****

Resistant Susceptible

MOXIFLOXACIN, APM 2 ug/ml

0.06 *****

0.12 ******************

*************

0.25 ******************

******************

********

0.5 ******************

**************

1 ***************

2 * *********

4 * ********

8 ***** ************

>8 ************ *******

Resistant Susceptible

TREK

MYC

O T

B M

IC

APM STATUS

In-depth analysis of moxifloxacin discordance (MYCO TB-R / APM-S): gyrA or gyrB mutations detected in all but one isolate sequenced to date

ID QRDR Sequence MYCO TB MIC APM 0.5 ug/ml APM 2.0 ug/ml

K1 90 GCG-GTG 2 ug/ml Sens Sens

K2 90 GCG-GTG 2 Sens Sens

K4 90 GCG-GTG >8 Res Sens

K9 91 TCG-CCG >8 Res Sens

K13 No mutation (gyr A, B) 4 Sens Sens

K14 gyrB 540 GAA-GAC 8 Res Sens

K15 91 TCG-CCG 8 Res Sens

K16 94 GAC-GGC 8 Res Sens

K18 90 GCG-GTG 8 Sens Sens

K22 94 GAC-GCC 8 Sens Sens

K25 94 GAC-TAC >8 Sens Sens

K27 94 GAC-AAC >8 Sens Sens

K29 90 GCG-GTG 4 Sens Sens

K32 94 GAC-GGC 8 Res Sens

Ethambutol discordance driven by relatively large number of isolates characterized as Resistant by APM yet Susceptible by MYCO TB.

This is not somewhat affected by definition of concordance, since 1/3 to 1/2 of discordant isolates have MYCO TB MICs around the APM CC.

TREK

MYC

O T

B M

IC

APM STATUS

ETHAMBUTOL, APM 5 ug/ml

0.5 ***** *********

1

*******

******************

******************

********

2******

******************

***************

4***************** ****

8 *****************

*****************

****** *****

16 *********

32 **

>32 *

Resistant Susceptible

ETHAMBUTOL, APM 10 ug/ml

0.5 * *************

1

*

******************

******************

**************

2****

******************

*****************

4 ******* **************

8 *****************

****

******************

******

16 ****** ***

32 **

>32 *

Resistant Susceptible

Cycloserine: sensitivity of MYCO TB for detection of resistance was 40.0%; some but not all of this explained by MYCO TB MICs around the APM CC

CYCLOSERINE, APM 25 ug/ml

2 ********

4* ******************

8***

******************

******************

******************

16

*****

******************

******************

******************

*****************

32 ***** ****************

64 *

128

256

>256

Resistant Susceptible

TREK

MYC

O T

B M

IC

APM STATUS

Summary

• The MYCO TB test was feasible – Shorter time to results than APM – Excellent agreement between 2 readers (except for highly

resistant PAS isolates) – No test ‘failures’ (e.g. contamination)

• Concordance was high between MYCO TB and APM results, except for MXF and EMB – For MXF, sequencing to date confirms MYCO TB results

• We are working to identify the most suitable MYCO TB critical concentrations

• This work was funded by contract #HHSN2722000900050C from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, USA.

Acknowledgements

• Makerere University, Kampala, Uganda:

– W. Ssengooba, F. Mumbowa, C. Namaganda

• International Tuberculosis Research Center, Republic of Korea:

– J. Lee, J. Park, Y. Yeuni, SN Cho, CE Barry 3rd, L. Via

• Johns Hopkins University, Baltimore, MD, USA:

– D. Armstrong, SE Dorman

• University of Medicine & Dentistry, New Jersey, USA:

– D. Alland

• Boston University Medical Center, Boston, MA, USA:

– G. Chien, S. Armakovitch, J. Ellner, D. Hom

• TREK/Thermo Fischer Scientific: Nadine Sullivan

0.00

0.20

0.40

0.60

0.80

1.00

0.00 0.20 0.40 0.60 0.80 1.00

Sensi

vity

1-Specificity

ROCCurve-Cycloserine(25ug/mL)

Reference

CYCCri calConcentra ons

64->256ug/mL

32ug/mL

16ug/mL

8ug/mL

4ug/mL

2ug/mL

0.80

0.85

0.90

0.95

1.00

0.00 0.05 0.10 0.15 0.20

Sensi

vity

1-Specificity

ROCCurve-Rifampin(1ug/mL)

RIFCri calConcentra ons

4ug/mL

8ug/mL

2ug/mL

1ug/mL0.25/0.5ug/mL

0.12ug/mL

Using ROC analysis to determine critical concentrations for a revised (non-MIC) MYCO TB plate for

testing multiple Mtb isolates