National tuberculosis Seminar
Santat Sudrungrot
Laboratory Officer, MHD
IOM Damak NEPAL
Laboratory management: 14 July 2014
Pokhara
Content
• Liquid Culture by MGIT960 System
• Tuberculosis Identification tool
 MPT64 Antigen detection
 Line Probe Assay (MTBDRplus)
• GeneXpert Technology
2
Why Liquid Culture?
• Current practice, largely relies on
microscopy and solid culture with
the following drawback:
 Microscopy: although very
specific, but low sensitivity,
cannot identify MDR, cannot
differentiate between
NTM&MTB
 Solid Culture: more sensitive, but
growth of MTB requires 4-8
weeks, thus delay treatment.
• Expanding culture capacity as a
result of epidemic of HIV-TB coinfection and MDR TB
• Liquid culture reduce result
timeline
 Weeks to days for MTB
identification
 From month (28-42 d) to week
for DST (as little as 10 d)
• Increased 10% case yield
compare to solid culture.
HOWEVER, Liquid Culture
• Prone to contamination: ~5-10%
cannot yield result
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Liquid Culture
MGIT960 system
Product name: Mycobacterium Growth
Indicator Tube (MGIT) and drug
susceptibility testing (DST)
Manufacturer: Becton, Dickinson and
Company (BD). Endorsed by WHO (2007).
Implementation in endemic countries is
ongoing. Both of these products are
already in use in high income countries
and in the private sector.
Intend use where culture and biosafety
facilities exist
Capacity: The BACTEC MGIT 960 system , 960 tubes /up to 8,000 specimens per year
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MGIT
Mycobacterium Growth Indicator Tube
 Acceptable specimen types are digested and
decontaminated clinical specimens (except
urine) and sterile body fluids (except blood).
 Made from unbreakable plastic tubes
containing enriched culture media.
The BBL™ MGIT™ Mycobacteria
Growth Indicator Tube
supplemented with BBL™ MGIT™
OADC enrichment and BBL™
MGIT™ PANTA™ antibiotic
mixture, when appropriate, is
intended for the detection and
recovery of mycobacteria.
 At the bottom of the tube is a silicone plug
containing chemicals that become
fluorescent when bacteria consume oxygen
during the process of growth, making
detection possible using either manual or
automated system
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Liquid Culture
MGIT960 system
MGIT can also be used to perform DST, which is done by comparing the growth of
mycobacteria with and without the addition of drugs used to treat TB.
Despite having been developed over a
decade ago, the advantages of MGIT for
TB detection were not reaching most
endemic settings for several reasons.
This was primarily due to
 Cost of the test
 Lack of a simple means to confirm the
growth of M. tuberculosis species in
positive tubes.
 Lack of data demonstrating that the
use of liquid culture was feasible in
resource-constrained settings.
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Cost Related
Particular
Conc. Smear
Direct
Cost
Indirect
Cost
Total ($)
0.4
0.2
0.6$
(57 NPR)
2$
Culture Solid
1
1
Culture Liquid
3
3
Identification
(MPT64)
6
2
8$
(760 NPR)
1st Line DST
(S,H,R,E,Z)
13
3
16$
(1,520 NPR)
(190 NPR)
6$
(570 NPR)
Direct Cost:
Cost of tube, kit per test
Indirect Cost:
Cost of other
consumables, biosafety
per test
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Liquid Culture Procedure
Sputum
Collection
Decontamination
Concentration
Culture
Inoculation
Smear
2x LJ Solid media
MGIT
Incubator
8 weeks
MGIT 960
Incubation 6
weeks
buff to yellow,
rough wrinkled
colonies
Reading by
Fluorescent
Microscopy
Identification
• ZN Staining
• MPT64, LPA, Biochemistry tests
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Identification
MGIT Positive
tube
Sub-culture to LJ to later
observe morphology
ZN Staining
Cord Formation
LPA
MPT64
OPTIONAL
Biochemistry Test (NO3+,
Niacin, Catalase, PNB)
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MTB rapid speciation
MPT64 Antigen Detection
 Secretory Protein specific to MTBc in
culture
 Immuno-chromatography test platform
 Applicable for both Liquid& Solid
 Result available within 15 mins
 Less technical required
 Testing environment requires as to
identification&DST.
Commercially available from
manufacturer:




Tauns Co. Ltd (Endorsed by WHO 2007)
SD Bioline MPT64
IDTBc
Cost: 3-6 $
 May give negative result in case of
MPT64 gene mutant strain
 Weakly positive in case of other
protein interferences (Mixed growth
with other bacteria/NTM)
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Line Probe Assay : LPA
Identification of the M. tuberculosis complex and its resistance to Rifampicin and/or Isoniazid
from pulmonary clinical specimens or cultivated samples
BIOSAFETY
LABORATORY DESIGN
 Procedures require digestion,
decontamination and concentration of
clinical specimens prior to DNA extraction.
 Producing aerosols (Vortex, centrifuge,
pipetting sol. In and out).
To reduce the risk of DNA amplicon cross
contamination; the following separated
room are required;
 Processing of smear-positive specimens:
BSL2
 Processing of positive cultures would
require BSL3 facilities
2. DNA extraction room
1. Reagents Preparation room
3. PCR amplification, hybridization and post
amplification room
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Equipment
In addition to the equipment required for initial digestion-decontamination
of sputum specimens (such as BSCs and safety centrifuges),
LPA specific equipment
1. Thermal cycler,
2. Shaking platform incubator
and water bath,
3. Water bath
4. Micro centrifuge and tubes,
5. Hybridization instrument,
fridge, freezer,
6. Micropipettes and pipette
tips, and PCR tubes.
PRINCIPLE and PROCEDURE
1. DNA Extraction
 Direct sputum specimen
 Culture specimen
3. Amplicon identification
by reverse hybridization
Chromogen (MBT/BCIP)
Alkaline Phosphatase
2. Multiplex amplification
with biotinylated primers
 rpoB primer
 katG primer
 inhA primer
Colour reaction
Streptavidin
Biotin
Biotin-labelled
single stranded
DNA-probeamplified target
Nitrocellulose strip
27 Reaction zones



(1) conjugate control (CC)
(2) amplification control (AC)
(3) M. tuberculosis complex-specific control (TUB)



(4)rpoB amplification control
(5-12) rpoBwild-type probes WT1 to WT8 (505 to 533)
(13-16) 4 x rpoB mutant probes (probes MUT1, MUT2A, MUT2B,
and MUT3) in codons D516V, D526Y, H526D, and S531L,
respectively



(17) katG amplification control
(18) katG codon 315 wild-type probe
(19-20) two katG codon 315 mutant probes (probes MUT1 and
MUT2) with AGC-ACC (S315T1) and AGC-ACA (S315T2)
mutations, respectively


(21) inhA amplification control
(22-23) inhA wild-type probes WT1 and WT2 covering positions
−15 and −16 of the gene regulatory region
(24-27))four inhA mutant probes (probes MUT1, MUT2, MUT3A,
and MUT3B) with mutations C→T at position −15, A→G at
position −16, T→C at position −8, and T→A at position −8,
respectively. M, colored marker.

The targeted genes and Specific probes
RESULT INTERPRETATION
• In order to give a valid result, all six expected control
bands should appear correctly. Otherwise, the result is
considered invalid
 The absence of at least one of the wild-type bands or the
presence of mutation bands in each drug resistance-related
gene implies that the sample tested is resistant to the
respective antibiotic.
 Presence of all the wild-type probes and there is no detectable
mutation within the region examined, the sample tested is
susceptible to the respective antibiotic.
GeneXpert Orientation
CONTENT
What is GeneXpert & Xpert MTB/RIF?
What are the benefits and limitations?
Test performance and accuracy?
How to operate GeneXpert system?
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What is GeneXpert?
• Based on the principle of Polymerase Chain Reaction (PCR)
Automates and integrates (1) Sample preparation, (2) Nucleic acid
Amplification, and (3) Real time detection of the target sequence
• Suitable for in vitro diagnostic and research based applications that
require hands-off processing of patient samples (specimens) and provides
both summarized and detailed test results data in tabular and graphic
formats.
• The GeneXpert system can only be used with the GeneXpert cartridge
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GeneXpert Dx system
First launched in 2004
by Cepheid Inc. (CA)
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®
Xpert
MTB/RIF
Detects M. tuberculosis as well as rifampicin resistance-conferring mutations
directly from sputum, in an assay providing results within two hours.
» Using Xpert lysis reagent – Sample is prepared in
leak proof container15 minutes
» Simply apply prepared sample to cartridge and load
into instrument
» Sample will be purified& concentrated. DNA is
extracted, amplified and detected right inside the
single-used cartridge
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®
Xpert
MTB/RIF
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Other Xpert Cartridge
Xpert® CT/NG
90 minute detection and differentiation of Chlamydia trachomatis (CT)
and Neisseria gonorrhoeae (NG)
Xpert® Flu
Accurate determination of Flu A & Flu B and identification of 2009 H1N1
in just over one hour
Xpert® MRSA/SA SSTI
On-demand testing for Methicillin-resistant Staphylococcus aureus (MRSA)
and Staphylococcus aureus (SA)
Xpert® MRSA
On-demand MRSA testing. Available in 10 and 120 test kits.
And many more
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CONTENT
What is GeneXpert & Xpert MTB/RIF?
What are the benefits and limitations?
Test performance and accuracy?
How to operate GeneXpert system?
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Benefits
Rapid MTB& MDR diag. could prompt
treatment decision
Time to result
• 2 hours
Biosafety
• Equivalent to Smear microscopy
• Safe processing – reagent for Xpert test
kills viable bacteria in sputum
• Convenient waste disposal - cartridge
Lab infrastructure
• Not required sophisticated BSL-3 as for
culture
• Not required 3 separated rooms as for
other NAATs
Technical skill
• Less required – only 3 simple steps of
specimen preparation
Diagnostic method
(Drugre, IAS 2009)
Limit of detection
( CFU/ml)
Liquid Culture
MGIT 960
10-100
NAAT
(LAMP/Xpert)
50-150
Antigen
150-10,000
Line Probe Assay
10,000
Fluorescent
microscopy
10, 000
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Limitations
• Reliable result critically is dependent upon proper specimen
collection, handling and storage (Detection of MTB depends on
number of bacteria presented in sample)
• Positive test result does not necessarily indicate the presence of
viable organism
• Test result might be affected by concurrent antibiotics therapy.
Therefore, therapeutic success or failure cannot be assessed by
using this test because DNA might persist following anti-microbial
therapy.
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Operational Challenges
• Require stable power supply, to minimize unnecessary test
error
• GeneXpert dx system is controlled solely through computer
(desktop or laptop)- infected by computer virus could stop
the whole machine operation.
• The most commonly-deployed GeneXpert device (GX4) has a
limited throughput, and larger systems (or linked devices),
with throughputs of up to 1000 tests/day, will carry higher
capital costs.
• Still costly- for High TB burden countries
4 modules machine cost $17,000
Cartridge costs $10
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CONTENT
What is GeneXpert & Xpert MTB/RIF?
What are the benefits and limitations?
Test performance and accuracy?
How to operate GeneXpert system?
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Test Performance
(Ref.: Oct-2013 WHO Policy Update Xpert MTB/RIF)
Algorithm
Sensitivity
Initial test replace smear microscopy
MTB Detection
88%
99%
From 22 studies, 9008 participants
Ad-on test after smear negative
MTB Detection
68%
99%
From 23 Studies, 7151 participants
People Living with HIV
MTB Detection
79%
n/a
From 7 studies, 1,789 participants
Rifampicin Resistant
17 studies
95%
Extra-pulmonary TB
Specificity
Specimen
Sensitivity
Specificity
Lymph
node
84.9%
92.5%
CSF
79.5%
98.6%
Pleural
fluid
43.7%
98.1%
Gastric
Lavage
83.8%
98.1%
From 12 studies ; other sample types e.g.
ascetic fluid, pericardial fluid, urine, blood
and stool were limit and not considered for
analysis
99%
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Rif resistant Vs MDR marker?
How often is a rifampicin resistant case also resistant to
isoniazid?
• Data from FIND global project show that frequently patients
with rifampicin resistance are MDR. But not always;
 82% of new rifampicin resistant cases were MDR
 87% of previously treated rifampicin resistant cases are
also MDR-TB.
 Among high prevalence populations, the median
proportion is closer to 92%
The answer is “PRETTY GOOD but NOT PERFECT”
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GeneXpert Result
30
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BSL-2/BSL-3
•Access to the laboratory is restricted when
work is being conducted.
Safety equipment
•Appropriate personal protective equipment
(PPE)is worn, including lab coats and gloves.
Eye protection and face shields can also be
worn, as needed.
•All procedures that can cause infection from
aerosols or splashes are performed within a
biological safety cabinet
(BSC)
.
•An autoclave or an alternative method of
decontamination is available for proper
disposals.
Facility construction
•The laboratory has self-closing doors.
•A sink and eyewash are readily available.
•Laboratorians are under medical surveillance
and might receive immunizations for microbes
they work with.
•Access to the laboratory is restricted and
controlled at all times.
Safety equipment
•Appropriate PPE must be worn, and respirators
might be required
.
•All work with microbes must be performed
within an appropriate BSC
.
Facility construction
•A hands-free sink and eyewash are available
near the exit.
•Exhaust air cannot be recirculated, and the
laboratory must have sustained directional
airflow by drawing air into the laboratory from
clean areas towards potentially contaminated
areas.
•Entrance to the lab is through two sets of selfclosing and locking doors
.
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