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 co-
infection 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
3
 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
4
 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.

 At the bottom of the tube is a silicone plug

containing chemicals that become
fluorescent when bacteria consume oxygen
The BBL™ MGIT™ Mycobacteria during the process of growth, making
Growth Indicator Tube detection possible using either manual or
supplemented with BBL™ MGIT™ automated system
OADC enrichment and BBL™
MGIT™ PANTA™ antibiotic
mixture, when appropriate, is
intended for the detection and
recovery of mycobacteria.
5
 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.

 Cost of the test

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
 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.
6
 Liquid Culture Procedure

Sputum
Collection Decontamination Concentration

Culture Smear
Inoculation

Reading by
2x LJ Solid media MGIT Fluorescent
Microscopy
Incubator MGIT 960
8 weeks Incubation 6
weeks

buff to yellow,
rough wrinkled
colonies Identification
• ZN Staining
• MPT64, LPA, Biochemistry tests

8
 Identification
MGIT Positive Sub-culture to LJ to later
tube observe morphology

ZN Staining

OPTIONAL
Cord Formation

LPA Biochemistry Test (NO3+,

MPT64
Niacin, Catalase, PNB)

9
 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

 May give negative result in case of
manufacturer: MPT64 gene mutant strain
 Tauns Co. Ltd (Endorsed by WHO 2007)
 SD Bioline MPT64  Weakly positive in case of other
 IDTBc protein interferences (Mixed growth
 Cost: 3-6 $ with other bacteria/NTM)

10
 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, To reduce the risk of DNA amplicon cross

decontamination and concentration of contamination; the following separated
clinical specimens prior to DNA extraction. room are required;
 Producing aerosols (Vortex, centrifuge,
pipetting sol. In and out). 1. Reagents Preparation room

 Processing of smear-positive specimens: 2. DNA extraction room

BSL2
 Processing of positive cultures would 3. PCR amplification, hybridization and post
require BSL3 facilities amplification room

11
 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 3. Amplicon identification

 Direct sputum specimen by reverse hybridization
 Culture specimen

Chromogen (MBT/BCIP)

Colour reaction
Alkaline Phosphatase

Streptavidin
Biotin
Biotin-labelled
single stranded
2. Multiplex amplification DNA-probeamplified target
with biotinylated primers
 rpoB primer
 katG primer Nitrocellulose strip
 inhA primer
 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?

17
 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

18
 GeneXpert Dx system

First launched in 2004

by Cepheid Inc. (CA)

19
 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

20
Xpert ® MTB/RIF

21
 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

22
 CONTENT

What is GeneXpert & Xpert MTB/RIF?

What are the benefits and limitations?

Test performance and accuracy?

How to operate GeneXpert system?

23
 Benefits
Rapid MTB& MDR diag. could prompt
treatment decision
Diagnostic method Limit of detection
Time to result
(Drugre, IAS 2009) ( CFU/ml)
• 2 hours
Biosafety
Liquid Culture 10-100
• Equivalent to Smear microscopy
MGIT 960
• Safe processing – reagent for Xpert test
kills viable bacteria in sputum
NAAT 50-150
• Convenient waste disposal - cartridge
(LAMP/Xpert)
Lab infrastructure Antigen 150-10,000
• Not required sophisticated BSL-3 as for
culture Line Probe Assay 10,000
• Not required 3 separated rooms as for
other NAATs Fluorescent 10, 000
Technical skill microscopy
• Less required – only 3 simple steps of
specimen preparation 24
 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.

25
 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
26
 CONTENT

What is GeneXpert & Xpert MTB/RIF?

What are the benefits and limitations?

Test performance and accuracy?

How to operate GeneXpert system?

27
 Test Performance
(Ref.: Oct-2013 WHO Policy Update Xpert MTB/RIF)

Algorithm Sensitivity Specificity Extra-pulmonary TB

Initial test replace smear microscopy Specimen Sensitivity Specificity

MTB Detection 88% 99% Lymph 84.9% 92.5%

node
From 22 studies, 9008 participants

Ad-on test after smear negative CSF 79.5% 98.6%

MTB Detection 68% 99% Pleural 43.7% 98.1%

fluid
From 23 Studies, 7151 participants
Gastric 83.8% 98.1%
People Living with HIV
Lavage
MTB Detection 79% n/a
From 12 studies ; other sample types e.g.
From 7 studies, 1,789 participants ascetic fluid, pericardial fluid, urine, blood
and stool were limit and not considered for
Rifampicin Resistant analysis
17 studies 95% 99%
28
 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”

29
GeneXpert Result

30
31

•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) . near the exit.
•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.
BSL-2/BSL-3
•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
•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 self-
closing and locking doors .
32