Line Probe Assay (LPA) for Tuberculosis -Introduction, Principle

Introduction

Table of Contents

The Line Probe Assay (LPA) is a rapid molecular diagnostic test used to detect Mycobacterium tuberculosis complex (MTBC) and its resistance to anti-tuberculosis drugs. It identifies specific mutations in the genes associated with drug resistance by hybridizing amplified DNA onto membrane strips.

The LPA is a high-throughput technology recommended by the World Health Organization (WHO) for the rapid detection of drug-resistant TB. It is categorized into:

First-line LPA (FL-LPA): Detects resistance to Isoniazid (INH) and Rifampicin (RIF).
Second-line LPA (SL-LPA): Detects resistance to Fluoroquinolones (FQ) and Second-line Injectable Drugs (SLID) like Amikacin and Kanamycin.

Principle

The test is based on DNA Strip Technology, involving three main steps:

DNA Extraction: Isolating DNA from clinical samples (smear-positive sputum) or culture isolates.
Multiplex PCR: Amplifying specific regions of the TB genome using biotin-labeled primers.
Reverse Hybridization: The amplified DNA is denatured and hybridized to membrane-bound probes. If the DNA matches the probe (wild-type or mutant), a color reaction occurs via a streptavidin-conjugated enzyme, appearing as a visible band.

Test Requirements

1. Specimen Requirements

Acceptable Samples:
- Sputum: Freshly collected (spot or morning), purulent, or muco-purulent samples.
- Other Respiratory Material: Bronchial washings or alveolar lavage.
- Culture Isolates: Growth from solid (LJ) or liquid (MGIT) media.
Smear Status:
- Direct testing is primarily recommended for smear-positive specimens.
- For smear-negative specimens, the test is typically performed on the culture isolate once growth is detected.
Storage & Transport:
- Temperature: Store specimens at 2-8°C.
- Transport: Must be transported to the lab as soon as possible, ideally within 1–2 days.
- Stability: Specimens should not be older than 4 days for optimal decontamination.

2. Laboratory Infrastructure

To prevent cross-contamination of DNA, the laboratory must be divided into at least three physically separate rooms with a unidirectional workflow:

Room 1 (DNA Extraction): Where clinical samples are processed, and DNA is isolated.
Room 2 (Pre-amplification): Dedicated to preparing the PCR Master Mix (must be kept free of any amplified DNA).
Room 3 (Amplification & Post-amplification): Where PCR occurs and the final hybridization/detection on strips is performed.

3. Equipment & Reagents

Core Equipment:
- Biosafety Cabinet (BSC): A Type II BSC is mandatory for handling infectious samples.
- Thermal Cycler: For PCR amplification of target TB genes.
- Twin Incubator/Shaking Water Bath: For the hybridization and washing steps.
- Centrifuge: For specimen concentration and DNA extraction.
Key Reagents: Specific commercial kits (e.g., GenoType® MTBDRplus) containing membrane strips, primers, nucleotide mix, polymerase, and various buffers (hybridization, stringent wash, and rinse solutions).
Power Supply: An Uninterrupted Power Supply (UPS) is critical, as temperature fluctuations or power cuts during PCR or hybridization can ruin results.

4. Biosafety & Personnel

Biosafety Levels:
- BSL-2: Required for processing smear-positive clinical specimens.
- BSL-3: Mandatory if the assay is being performed on positive cultures or if manipulating live TB strains.
Staff Training: Personnel must be specifically trained in molecular techniques and strict aseptic practices to avoid “false positive” results caused by DNA contamination.

Procedure

The procedure is typically performed in a specialized lab with separate areas to prevent contamination:

Step 1: DNA Extraction: DNA is extracted from the sample using chemical or physical lysis.
Step 2: Master Mix Preparation: PCR reagents and specific primers are prepared.
Step 3: Amplification: A thermocycler amplifies the target genes (e.g., rpoB for Rifampicin, katG and inhA for Isoniazid).
Step 4: Hybridization: The PCR products are added to the LPA strips in a shaking water bath or automated processor. Probes on the strip include:
- Conjugate Control (CC): Confirms the development process.
- Amplification Control (AC): Confirms successful PCR.
- MTB Control (TUB): Confirms the presence of MTBC DNA.
Step 5: Detection: The strips are washed, and a substrate is added to visualize the bands.

Result Interpretation

Interpretation is based on the presence or absence of “Wild Type” (WT) and “Mutant” (MUT) bands on the strip:

Susceptible: All WT bands are present, and NO MUT bands are present.
Resistant:
- Absence of at least one WT band.
- OR the presence of a MUT band.
- Invalid: If the TUB, AC, or CC bands do not appear, the test must be repeated.

Keynotes

Specimen Requirements: Primarily used for smear-positive sputum samples or cultured isolates. It is not recommended for smear-negative samples due to low sensitivity.
Turnaround Time: Results are typically available within 24 to 48 hours, significantly faster than traditional culture-based drug susceptibility testing (DST).
Laboratory Requirements: Requires a high-level biosafety setup (BSL-2 or higher) and strict spatial separation to avoid PCR crossover contamination.
Limitation: It only detects known mutations included on the strip; resistance caused by rare or unknown mutations will not be identified.
Line Probe Assay (LPA) is a molecular tool that uses PCR and reverse hybridization to rapidly detect M. tuberculosis and resistance-conferring mutations for first-line and second-line drugs, providing results in under 48 hours.

Line Probe Assay (LPA) for Tuberculosis -Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, and Keynotes

Introduction

Principle

Test Requirements

Procedure

Result Interpretation

Keynotes

Further Readings

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