National tuberculosis Seminar

Santat SudrungrotLaboratory Officer, MHDIOM Damak NEPALLaboratory management: 14 July 2014Pokhara

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Content

• Liquid Culture by MGIT960 System

• Tuberculosis Identification tool MPT64 Antigen detection Line Probe Assay (MTBDRplus)

• GeneXpert Technology

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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

Liquid Culture MGIT960 system

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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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MGITMycobacterium Growth Indicator Tube

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.

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 during the process of growth, making detection possible using either manual or automated system

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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.

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

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Cost Related

Particular Direct Cost

Indirect Cost Total ($)

Conc. Smear 0.4 0.2 0.6$ (57 NPR)

Culture Solid 1 1 2$(190 NPR)

Culture Liquid 3 3 6$(570 NPR)

Identification (MPT64) 6 2 8$

(760 NPR)1st Line DST (S,H,R,E,Z) 13 3 16$

(1,520 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

SmearCulture Inoculation

2x LJ Solid media MGIT

Identification• ZN Staining• MPT64, LPA, Biochemistry tests

Incubator8 weeks

Reading by Fluorescent Microscopy

MGIT 960 Incubation 6

weeks

buff to yellow, rough wrinkled colonies

ConcentrationDecontamination

Identification

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ZN Staining

MGIT Positive tube

Sub-culture to LJ to later observe morphology

LPA MPT64

Cord FormationOPTIONAL

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

Procedures require digestion, decontamination and concentration of clinical specimens prior to DNA extraction.

Producing aerosols (Vortex, centrifuge, pipetting sol. In and out).

Processing of smear-positive specimens: BSL2

Processing of positive cultures would require BSL3 facilities

BIOSAFETY

To reduce the risk of DNA amplicon cross contamination; the following separated room are required;

1. Reagents Preparation room

2. DNA extraction room

3. PCR amplification, hybridization and post amplification room

LABORATORY DESIGN

Equipment

LPA specific equipment1. Thermal cycler, 2. Shaking platform incubator and

water bath, 3. Water bath4. Micro centrifuge and tubes, 5. Hybridization instrument,

fridge, freezer, 6. Micropipettes and pipette tips,

and PCR tubes.

In addition to the equipment required for initial digestion-decontamination of sputum specimens (such as BSCs and safety centrifuges),

Chromogen (MBT/BCIP)

Alkaline Phosphatase

StreptavidinBiotin

Nitrocellulose strip

DNA-probe

Biotin-labelled single stranded amplified target

Colour reaction

PRINCIPLE and PROCEDURE

2. Multiplex amplification with biotinylated primers rpoB primer katG primer inhA primer

3. Amplicon identification by reverse hybridization1. DNA Extraction

Direct sputum specimen Culture specimen

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(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

(1) conjugate control (CC) (2) amplification control (AC) (3) M. tuberculosis complex-specific control (TUB)

(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

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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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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 systemGeneXpert Dx system

First launched in 2004 by Cepheid Inc. (CA)

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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

Xpert® MTB/RIF

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Xpert® MTB/RIF

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Other Xpert Cartridge

Xpert® CT/NG90 minute detection and differentiation of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)

Xpert® FluAccurate determination of Flu A & Flu B and identification of 2009 H1N1 in just over one hour

Xpert® MRSA/SA SSTIOn-demand testing for Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA)

Xpert® MRSAOn-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

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

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 NAATsTechnical skill• Less required – only 3 simple steps of

specimen preparation

Rapid MTB& MDR diag. could prompt treatment decision

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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 $17,000Cartridge 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 Specificity

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 Resistant17 studies 95% 99%

Extra-pulmonary TB

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

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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

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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 self-closing and locking doors .