GLI meeting

Stakeholders Roundtable on DST diagnostic technologies

Diagnosis
for
choosing the appropriate remedy
--- Genoscholar ---

April 29th, 2015

NIPRO CORPORATION
 2

Drug-resistant M. tuberculosis in the world

（1994-2007） world.
World Health Organization. Anti-tuberculosis drug resistance in the
Report no. 4. WHO/HTM/TB/2008.394. Geneva, World Health
(%) Organization, 2008.
50

45

40
35
35

30 27.7

25 New
20.1 17 Previous
20 17.5
15.3
15 10.9
10.3
10.3
10
3.7
5 2.5 2.9

0
Any INH Any RFP Any SM Any EB Any res. MDR
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 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.
gyrBA operon for FQsR

embCAB operon for EMBR

rpoB for RFPR

rpsL for aminoglycosidesR

M. tuberculosis
H37Rv
pncA for PZAR
4.4 Mbp Drug gene Sensitivity
rrs for aminoglycosidesR
(estimated)
INH katG, inhA, mabA 60 – 80 %
etc.
katG for INHR
fabG1-inhA operon for INHR
RFP rpoB 95 %
PZA pncA 72 - 97%
FQs gyrA 90 %
KM rrs, eis 80 %
SM rrs, rpsL 60 – 80%
EB embB 70 %
Genoscholar kit

TB
R? or S?

Resistant- Sensitive-
TB TB

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The lineup of our product
PZA strip NTM+MDRTB strip INH strip

Control Control
Control inhA 1, 2
M. avium
mabA 1, 2
M. Intracellulare
M. kansasii

M. tuberculosis

rpoB S1 - S5

pncA 1 - 48 R2, R4a, R4b, R5 katG 1 - 40

inhA S6, S7
katG S8 - S11

R9a, R9b

identification of NTM
resistance to resistance to
resistance to Rifampicin
Pyrazinamide Isoniazid
Isoniazid
 Genoscholar・PZA TB
• 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.

Pyrazinamide
Resistance

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 Principle pyrazinamide resistance and pncA
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.

PZA（prodrug）
pncA (pirazinamidase) 100【bp】
3 AlaGlu
96 Lys Thr
10 Gln Pro 136 AspTyr
cytoplasm 12 Asp Ala 51 His Gln 148 ArgSer
14CysTrp 54ProLeu 175 Met Ile
175 MetVal
72 CysTrp
PZase Initiation Termination

（PncA） codon ATG codon TGA

(60 GlyGly) 132 GlySer 493 Insertion C

- 133 IleThr 420 Insertion G
Primer 10
pyrazinoic acid 218 Insertion 10 nt

（POA-） .
Sekiguchi et al., J Clin Microbiol, 2007

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 PZA strip detects whole region of pncA.
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.
pncA (pirazinamidase) 100【bp】
3 AlaGlu
96 Lys Thr
10 Gln Pro 136 AspTyr
12 Asp Ala 51 His Gln 148 ArgSer
14CysTrp 54ProLeu 175 Met Ile
175 MetVal
72 CysTrp
Initiation Termination
codon ATG codon TGA

(60 GlyGly) 132 GlySer 493 Insertion C

133 IleThr 420 Insertion G
Primer 10
218 Insertion 10 nt
.
Sekiguchi et al., J Clin Microbiol, 2007

PZA strip

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 Clinical trial ～cultured isolates～

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

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)

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

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Genoscholar TB・NTM+MDR
• Detect Rifampicin and Isoniazid resistance in 1 day with only 1
single test.
• Identify not only M. tuberculosis but also M. avium, M. avium
M. intracellulare
M. intracellulare and M. kansasii. M. kansasii
M. tuberculosis
• It is possible to get result from direct sample or cultured material.
• Easy visual interpretation. Rifampicin

• Highly sensitive and specific detection.

Isoniazid

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Flow of TB diagnostics
Conventional method NIPRO

Sample Sample
Preparation Preparation

Identification Identification Identification Identification

of TB of NTM of TB of NTM

DST

DST

Identification of TB or NTM
and DST in one test!
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Background of NTM situation in Japan
Incident rate of NTM

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

• NTM patients in Japan gradually increase.
NTM treatment

M. avium
CAM／AZM／EB／RFP
M. intracellulare
M. kansasii
RFP／INH／EB
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).
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.

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Results of identification of Mycobacterium
In clinical trial
No. of isolates No. of isolates identified using by LiPA with NTM/MDR-TB strip
Mycobacterium identified using by
spp. conventional
methodsa M. tuberculosis M. avium M. intracellulare M. kansasii negative

M. tuberculosis 316 316

M. avium 71 71
M. intracellulare 51 51
*1
M. kansasii 54 53 1
Other spp. of NTM 62 62
*1 : M. kansasii subtype III
Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)
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 Clinical trial ～cultured isolates～
LiPA
Sensitivity
Mutant Wild type
98.9%(87/88)
M. tuberculosis
Specificity
RIF – Resistant 87 1
97.3% (220/226)
RIF – Susceptible 6 220

LiPA
Sensitivity
Mutant Wild type
61.6%(85/138)
M. tuberculosis
Specificity
INH – Resistant 85 53
100% (176/176)
INH – Susceptible 0 176
Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)
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 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
inhA
material. mabA

• Easy visual interpretation.

• Highly sensitive and specific detection of isoniazid resistance.

katG

Isoniazid
Resistance

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 INH strip detects many region of katG.
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.

inhA mabA katG

S315

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 Clinical trial ～cultured isolates～

LiPA
Sensitivity
Mutant Wild type
90.6%(125/138)
M. tuberculosis
Specificity
INH – Resistant 125 13
100% (176/176)
INH – Susceptible 0 176

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

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 Clinical trial ～cultured isolates～
LiPA
INH strip
Sensitivity Mutant Wild type

M. tuberculosis
Isoniazid 90.6%(125/138)
INH – Resistant 125 13
Resistance
INH – Susceptible 0 176

NTM+MDRTB strip LiPA

Sensitivity Mutant Wild type

61.6%(85/138) M. tuberculosis

INH – Resistant 85 53

INH – Susceptible 0 176

Isoniazid
Resistance
Mitarai S et al. J Clin Microbiol., 50, 884-90. (2012)
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 Outline of test procedure
Specimen
( Sputum or Culture )

DNA extraction

Thermal cycler

DNA Amplification
Multi-Blot NS-4800

Hybridization is
automatically done
DNA Detection up to 48 tests per
run.

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Summary
Quick 1-Day detection

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

High sensitivity of INH resistance

High sensitivity of PZA resistance

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Thank you
for your attention.

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24
以下，参考スライド

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Treatment of tuberculosis is
done by using 3 or 4 drugs for long time.
For example …

Rifampicin Isoniazid Pyrazinamide Other are used

for 4 months.

M. tuberculosis

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

some drugs.
・Therefore it is important to choose some appropriate drugs.
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 Results of Drug susceptibly test by LiPA
in Japan clinical trial
LiPA result
Drug No. of
Sensitivity Specificity
susceptibility M. tuberculosis No. of No. of (%) (%)
testing resultb isolates resistant susceptible

RIF NTM/MDR-TB strip

c
Resistant 88 87 1 98.9 97.3
c
Susceptible 226 6 220

INH INH strip

d
Resistant 138 125 13 90.6 100
Susceptible 176 0 176

INH NTM/MDR-TB strip

e 61.6 100
Resistant 138 85 53
Susceptible 176 0 176

PZA PZA strip

f 89.7 96.0
Resistant 58 52 6
f
Susceptible 250 10 240

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

Outline of test procedure
Specimen
( Sputum or Culture )

DNA extraction step

Thermal cycler

DNA Amplification
step
Multi-Blot NS-4800

DNA Detection

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Results of Drug susceptibly test by LiPA
in Japan clinical trial
LiPA result
Drug No. of
Sensitivity Specificity
susceptibility M. tuberculosis No. of No. of (%) (%)
testing resultb isolates resistant susceptible

RIF NTM/MDR-TB strip

c
Resistant 88 87 1 98.9 97.3
c
Susceptible 226 6 220

INH INH strip

d
Resistant 138 125 13 90.6 100
Susceptible 176 0 176

INH NTM/MDR-TB strip

e 61.6 100
Resistant 138 85 53
Susceptible 176 0 176

PZA PZA strip

f 89.7 96.0
Resistant 58 52 6
f
Susceptible 250 10 240
Mitarai S, Kato S, Ogata H et al. J Clin Microbiol., 50, 884-90. (2012)
Raito to NTM for mycobacterium species
Drug resistance
10～20% M.tuberculosis NTM
50～70 % 30～50 %

MAC
M. kansasii
（ M. avium＋ M. intracellulare ）
About 20 %
70～80 % Other species

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

・Non tuberculosis mycobacterium infection is increased recently.

・Drug efficacy differ according to mycobacterium species.

・It is important to identify mycobacterium species. 30