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Hain GenoType Line Probe Assay Test

The document provides an overview of the HAIN GenoType Line Probe Assay (LPA) for rapid detection of mutations associated with drug resistance in Mycobacterium tuberculosis complex (MTBC). The LPA uses PCR and reverse hybridization to identify MTBC and detect mutations for drugs like rifampin and isoniazid. It should be performed in laboratories with the proper infrastructure, equipment, and biosafety precautions. The LPA involves extracting DNA from clinical samples, amplification, reverse hybridization, and analysis of test strips to identify the presence or absence of control and drug bands which determines drug resistance profiles.

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272 views

Hain GenoType Line Probe Assay Test

The document provides an overview of the HAIN GenoType Line Probe Assay (LPA) for rapid detection of mutations associated with drug resistance in Mycobacterium tuberculosis complex (MTBC). The LPA uses PCR and reverse hybridization to identify MTBC and detect mutations for drugs like rifampin and isoniazid. It should be performed in laboratories with the proper infrastructure, equipment, and biosafety precautions. The LPA involves extracting DNA from clinical samples, amplification, reverse hybridization, and analysis of test strips to identify the presence or absence of control and drug bands which determines drug resistance profiles.

Uploaded by

pacome abdul
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Hain GenoType Line Probe

Assay: Overview and


Training

Presented by Pacôme Achimi, PhD


Cermel-TB Lab/Gabon

TB Diagnostics Workshop, 8-12


July 2019,
HAIN Genotype Line Probe Assay
 (LPA)
The LPA uses PCR and reverse hybridization methods for
rapid detection of mutations associated with drug resistance.

 The LPA is designed to identify MTBC and simultaneously


detect mutations associated with drug resistance.
 The LPA is should be performed in laboratories with a proven
capacity to conduct molecular testing including appropriate
laboratory infrastructure and equipment.

 This must also include the necessary biosafety precautions


and the prevention of contamination.
LPA Procedures: Key Steps

 1. DNA extraction a. Clinical


pulmonary specimens
(decontaminated) b. Cultured
isolates (solid or liquid cultures)

 2. Amplification

 3. Reverse Hybridization

 4. Analysis
LPA Test Controls
 Conjugate Control (CC) -Included on Strip
• Demonstrates efficient conjugate binding and substrate
reaction
• Line must be present for a valid result

 Amplification Control (AC)-Included on Strip


• Demonstrates successful amplification

 Negative Control (NC)-Recommended Test/Batch


• Contains water instead of DNA to control for contamination
• Only the CC and AC bands should be present on this strip
MTBDRplus Assay: Tests for Mutations in INH and Rifampin
 rpoB: If mutation present, this may correlate with resistance to Rifampin
 inhA: If mutation present, this may correlate with resistance to INH (low
level resistance)
 katG: If mutation present, this may correlate with resistance to INH (high
level resistance)
Interpreting Line Probe
Assay Strips
 Step 1: Look at the "CC” band,
“AC”band and “TUB” band.

 Conjugate Control (CC) Band: This


should be present for each test (if not,
the test is invalid)
 Amplification Control (AC) Band:
This band should be present for each
test (if not, the test is invalid)

 M. tuberculosis complex (TUB)


Band: This band should hybridize with
all members of the MTB complex. A
positive (+) test result shows the band
present, while a negative (-) test result
shows the band as absent.
Interpreting Line Probe Assay Strips(2)

 Step 2: Look at the drug control


bands (called Locus controls, these
are the non-WT or MUT bands)

 For a valid test, there should be a band present for


each of the drug control bands.
 If the TUB band is present (indicating an MTBC
positive result) and the drug control band is absent,
the results for that particular drug are indeterminate.
 If the TUB band is absent (indicating an MTBC
negative result), there should be NO drug control
bands present for that particular sample*.
 (*NOTE: If TUB band is negative but there is still an
evaluable susceptibility pattern, MTB complex is
suspected, but test should be repeated)
GenoType MTBDRplus
version 2.0
Attributes and advantages
Highly sensitive and specific (98.9%, 100%)
Detects both Rifampicin and Isoniazid
Meant for low and high throughput labs
Short Turn around time of 5hrs
Affordable – ~$10 per test

Disadvantages
Cannot be used as point of care test
Requires biosafety facilities
12/11/19
Benefits of the assay (1)

 Highly Sensitive and specific: Test can


be performed from +ve and –ve sputum
smear samples

 Efficient diagnosis: Patients previously


tested and are MDR-TB positive don’t
need to give out another sputum
sample

 Rapid XDR-TB results generated within 4


hours
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Benefits of the assay (2)

 Improved version contains extra genes


(gyrB gene) in fluoroquinolones. This
reduces negative/positives cases

 Enables early patient isolation and


therapy initiation preventing spread of
the strain

 Laboratory procedure remains the same


12/11/19
Thank you for your attention

12/11/19

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