Diagnosis

for

choosing the appropriate remedy

--- Genoscholar ---

April 29th, 2015

NIPRO CORPORATION

GLI meeting

Stakeholders Roundtable on DST diagnostic technologies

Drug-resistant M. tuberculosis in the world

（1994-2007）

10.3

3.7

10.9

2.5

17

2.9

27.7

17.5

20.1

10.3

35

15.3

0

5

10

15

20

25

30

35

40

45

50

Any INH Any RFP Any SM Any EB Any res. MDR

New

Previous

(%)

2

World Health Organization. Anti-tuberculosis drug resistance in the

world. Report no. 4. WHO/HTM/TB/2008.394. Geneva, World Health

Organization, 2008.

2

Molecular mechanisms of drug resistant TB Resistance of M. tuberculosis to antimycobacterial drugs is the consequence of naturally

occurring, spontaneous mutations in genes that encode either the target of the drug, or enzymes

that are involved in drug activation.

M. tuberculosis

H37Rv

4.4 Mbp

gyrBA operon for FQsR

rpoB for RFPR

rpsL for aminoglycosidesR

rrs for aminoglycosidesR

fabG1-inhA operon for INHR katG for INHR

pncA for PZAR

embCAB operon for EMBR

Drug gene Sensitivity

(estimated)

INH katG, inhA, mabA

etc.

60 – 80 %

RFP rpoB 95 %

PZA pncA 72 - 97%

FQs gyrA 90 %

KM rrs, eis 80 %

SM rrs, rpsL 60 – 80%

EB embB 70 %

4

Genoscholar kit

Resistant-

TB

Sensitive-

TB

TB

R? or S?

The lineup of our product

Control

pncA 1 - 48

PZA strip

resistance to

Pyrazinamide

Control

rpoB S1 - S5

R2, R4a, R4b, R5

inhA S6, S7

katG S8 - S11

R9a, R9b

NTM+MDRTB strip

M. avium

M. Intracellulare M. kansasii

M. tuberculosis

inhA 1, 2

katG 1 - 40

mabA 1, 2

INH strip

Control

identification of NTM

resistance to Rifampicin

Isoniazid

resistance to

Isoniazid

Pyrazinamide

Resistance

6

• This kit is used genetic technology.

• Detect Pyrazinamide resistance in 1 day with only 1 single test.

• It is possible to get result from direct sample or cultured material.

• Easy visual interpretation.

• Highly sensitive and specific detection of Pyrazinamide resistance.

Genoscholar・PZA TB

7

Pyrazinamide (PZA) is a prodrug and is converted to the its acid form, pyrazinoic acid, by

the pyrazinamidase (PZase). PZA resistant TB is defective for PZase activity and

mutations of PZase gene (pncA) are major mechanism of PZA resistant. About 72 - 97% of

PZA-resistant TB have a mutation in pncA, although the distribution of pncA mutations is

dispersed along the gene.

Sekiguchi et al., J Clin Microbiol, 2007

.

Primer 10

100【bp】 pncA (pirazinamidase)

Initiation

codon ATG

Termination

codon TGA

10 GlnPro 12 AspAla

14CysTrp 51 HisGln

54ProLeu

96 LysThr 136 AspTyr

148 ArgSer 175 MetIle

493 Insertion C

3 AlaGlu

72 CysTrp

175 MetVal

132 GlySer

133 IleThr

(60 GlyGly)

420 Insertion G

218 Insertion 10 nt

PZA（prodrug）

pyrazinoic acid

（POA-）

PZase

（PncA）

-

cytoplasm

Principle pyrazinamide resistance and pncA

8

pncA is about 560 bp gene and the distribution of pncA mutations is dispersed

along the gene.

PZA strip has 48 probes that cover whole region of pncA. This provides high

sensitivity to detect PZA resistant TB.

Sekiguchi et al., J Clin Microbiol, 2007

.

Primer 10

100【bp】 pncA (pirazinamidase)

Initiation

codon ATG

Termination

codon TGA

10 GlnPro 12 AspAla

14CysTrp 51 HisGln

54ProLeu

96 LysThr 136 AspTyr

148 ArgSer 175 MetIle

493 Insertion C

3 AlaGlu

72 CysTrp

175 MetVal

132 GlySer

133 IleThr

(60 GlyGly)

420 Insertion G

218 Insertion 10 nt

PZA strip detects whole region of pncA.

PZA strip

9

LiPA PZA

Mutant Wild type

M. tuberculosis

PZA - Resistant 52 6*1

PZA– Susceptible 10*2 240

*1 : wild type (n=6)

*2 : Thr168Ile (n=2), Val147Ile (n=1), Gly162Ser (n=4), Gly17Asp (n=2), Gly132Asp (n=1)

Sensitivity Specificity

89.7% (52/58) 96.0% (240/250)

Clinical trial ～cultured isolates～

Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)

10

• Detect Rifampicin and Isoniazid resistance in 1 day with only 1

single test.

• Identify not only M. tuberculosis but also M. avium,

M. intracellulare and M. kansasii.

• It is possible to get result from direct sample or cultured material.

• Easy visual interpretation.

• Highly sensitive and specific detection.

Isoniazid

Rifampicin

M. avium M. intracellulare

M. kansasii M. tuberculosis

Genoscholar TB・NTM+MDR

11

Flow of TB diagnostics

Identification

of NTM

Sample

Preparation

Identification

of TB

DST

Conventional method

Identification

of TB

Identification

of NTM

DST

Sample

Preparation

NIPRO

Identification of TB or NTM

and DST in one test!

Background of NTM situation in Japan

• Incident rate (2007) : 5.7 per 100,000 pop per year

• NTM patients in Japan gradually increase.

Incident rate of NTM

RFP／INH／EB

CAM／AZM／EB／RFP

13

M. avium

M. intracellulare

M. kansasii M. tuberculosis

MAC infection is treated with 2 or 3 antimicrobials for

at least 12 months. Commonly used first-line drugs

include macrolides (clarithromycin or azithromycin),

ethambutol, and rifamycins (rifampin, rifabutin).

NTM treatment

First-line regimen for M. kansasii consists of rifampin

plus ethambutol plus isoniazid plus pyridoxine, with

the treatment duration continuing until sputum culture

results are negative for 12 months.

M. tuberculosis M. avium M. intracellulare M. kansasii negative

M. tuberculosis 316 316

M. avium 71 71

M. intracellulare 51 51

M. kansasii 54 53 1

Other spp. of NTM 62 62

Mycobacterium

spp.

No. of isolates

identified using by

conventional

methodsa

No. of isolates identified using by LiPA with NTM/MDR-TB strip

Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)

Results of identification of Mycobacterium

In clinical trial

14

*1 : M. kansasii subtype III

*1

15

Sensitivity

98.9%(87/88)

Specificity

97.3% (220/226)

Clinical trial ～cultured isolates～

Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)

LiPA

Mutant Wild type

M. tuberculosis

RIF – Resistant 87 1

RIF – Susceptible 6 220

Sensitivity

61.6%(85/138)

Specificity

100% (176/176)

LiPA

Mutant Wild type

M. tuberculosis

INH – Resistant 85 53

INH – Susceptible 0 176

16

Genoscholar・INH TB

• Detect isoniazid resistance in 1 day with only 1 single test.

• It is possible to get result from direct sample or cultured

material.

• Easy visual interpretation.

• Highly sensitive and specific detection of isoniazid resistance.

Isoniazid

Resistance

inhA

mabA

katG

17

Most frequent mutations are located on S315 in katG and promoter region of

inhA, but many mutations are reported on other region. INH strip has 43 katG

probes and detect other mutations on katG.

INH strip detects many region of katG.

S315

inhA mabA katG

18

Clinical trial ～cultured isolates～

Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)

Sensitivity

90.6%(125/138)

Specificity

100% (176/176)

LiPA

Mutant Wild type

M. tuberculosis

INH – Resistant 125 13

INH – Susceptible 0 176

19

Clinical trial ～cultured isolates～

Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)

Sensitivity

61.6%(85/138)

Sensitivity

90.6%(125/138)

LiPA

Mutant Wild type

M. tuberculosis

INH – Resistant 125 13

INH – Susceptible 0 176

Isoniazid

Resistance

Isoniazid

Resistance

LiPA

Mutant Wild type

M. tuberculosis

INH – Resistant 85 53

INH – Susceptible 0 176

NTM+MDRTB strip

INH strip

Outline of test procedure

DNA extraction

DNA Detection

Specimen

( Sputum or Culture )

Multi-Blot NS-4800

Thermal cycler

20

DNA Amplification

Hybridization is

automatically done

up to 48 tests per

run.

22

Summary

Quick 1-Day detection

High sensitivity of INH resistance

High sensitivity of PZA resistance

Detect NTM/TB and RFP/INH resistance in same time

Thank you for your attention.

23

24

以下，参考スライド

25

26

M. tuberculosis

Treatment of tuberculosis is

done by using 3 or 4 drugs for long time.

are used

for 4 months.

For example …

But… ・There are M. tuberculosis that have resistance for one or

some drugs.

・Therefore it is important to choose some appropriate drugs.

Isoniazid Pyrazinamide Rifampicin Other

27

No. of

resistant

No. of

susceptible

RIF

Resistant 88 87 1c

Susceptible 226 6c

220

INH

Resistant 138 125 13d

Susceptible 176 0 176

INH

Resistant 138 85 53e

Susceptible 176 0 176

PZA

Resistant 58 52 6f

Susceptible 250 10f

240

Drug

susceptibility

testing resultb

No. of

M. tuberculosis

isolates

LiPA result

NTM/MDR-TB strip

61.6 100

Sensitivity

(%)

Specificity

(%)

98.9 97.3

NTM/MDR-TB strip

INH strip

PZA strip

89.7 96.0

90.6 100

Mitarai S, Kato S, Ogata H et al. J Clin Microbiol., 50, 884-90. (2012)

Results of Drug susceptibly test by LiPA

in Japan clinical trial

Outline of test procedure

DNA Amplification

step

DNA extraction step

DNA Detection

Specimen

( Sputum or Culture )

Multi-Blot NS-4800

Thermal cycler

28

No. of

resistant

No. of

susceptible

RIF

Resistant 88 87 1c

Susceptible 226 6c

220

INH

Resistant 138 125 13d

Susceptible 176 0 176

INH

Resistant 138 85 53e

Susceptible 176 0 176

PZA

Resistant 58 52 6f

Susceptible 250 10f

240

Drug

susceptibility

testing resultb

No. of

M. tuberculosis

isolates

LiPA result

NTM/MDR-TB strip

61.6 100

Sensitivity

(%)

Specificity

(%)

98.9 97.3

NTM/MDR-TB strip

INH strip

PZA strip

89.7 96.0

90.6 100

Mitarai S, Kato S, Ogata H et al. J Clin Microbiol., 50, 884-90. (2012)

Results of Drug susceptibly test by LiPA

in Japan clinical trial

30

Raito to NTM for mycobacterium species

MAC

（ M. avium＋ M. intracellulare ）

70～80 %

M. kansasii

About 20 % Other species

Drug resistance 10～20% NTM

30～50 % M.tuberculosis

50～70 %

・Non tuberculosis mycobacterium infection is increased recently.

・Drug efficacy differ according to mycobacterium species.

・It is important to identify mycobacterium species.

Resistance to Anti-TB drug Sensitive to Anti-TB drug