What is a line probe assay?

What is a Line Probe Assay?

A line probe assay (LPA) is a rapid molecular diagnostic test that uses reverse hybridization technology with membrane-bound oligonucleotide probes to detect specific DNA sequences, primarily used to identify genetic mutations conferring drug resistance in pathogens like Mycobacterium tuberculosis and Hepatitis B virus. 1

Technical Methodology

The line probe assay operates through a hybridization-based detection system:

• PCR amplification of target DNA sequences is performed first, followed by hybridization of the amplified products to specific oligonucleotide probes immobilized on membrane strips 1
• The assay can detect single nucleotide mismatches and contains a series of short membrane-bound oligonucleotide probes that bind to specific mutation sites 1
• Results are visualized as colored lines on the membrane strip, with each line corresponding to a specific genetic sequence or mutation 2, 3

Clinical Applications

Tuberculosis Drug Resistance Detection

Line probe assays are widely used for rapid detection of drug-resistant tuberculosis:

• Detects resistance mutations to rifampicin, isoniazid, fluoroquinolones, ethambutol, and second-line injectables (streptomycin, amikacin, capreomycin) 2, 3
• Can be performed directly on smear-positive sputum specimens or on cultured isolates, with results available within 1-2 days compared to weeks for conventional culture-based methods 2, 4
• The GenID and GenoType MTBDRplus are commercially available LPA systems with sensitivities ranging from 77.8% to 96.4% for detecting MDR-TB in direct sputum samples 5, 6

Hepatitis B Virus Resistance Testing

In HBV management, line probe assays identify resistance mutations to antiviral medications:

• Detects mutations in the HBV polymerase gene that confer resistance to nucleoside/nucleotide analogs like lamivudine, adefovir, and entecavir 1
• Used as an alternative to direct sequencing for genotypic resistance testing when virologic breakthrough occurs during treatment 1
• Can detect viral mutants that constitute as little as 5% of the total viral population, making it more sensitive than direct PCR sequencing which requires approximately 20% 1

Diagnostic Performance Characteristics

Sensitivity Considerations

• Smear-positive specimens: LPA demonstrates excellent sensitivity (85.6% to 93.3%) for detecting Mycobacterium tuberculosis DNA 2, 5
• Smear-negative specimens: Sensitivity drops significantly to 22.7% to 77.8%, with high rates of invalid results (up to 30.8%) 5, 6
• For drug resistance detection in TB, sensitivity ranges from 84.2% to 100% for rifampicin, 75% to 90.6% for isoniazid, and 85.7% to 100% for fluoroquinolones 3, 5

Specificity and Accuracy

• Specificity for drug resistance detection is generally excellent (91.7% to 100%) across most antibiotics 3, 4
• For HBV resistance testing, LPA provides accurate identification of known resistance mutations but requires separate probe sets for each mutation of interest 1

Critical Limitations and Pitfalls

Technical Constraints

• Cannot detect unknown or novel mutations - only identifies mutations for which specific probes have been designed 1
• Requires separate sets of reactions for each mutant of interest, making comprehensive resistance screening complex 1
• Invalid results are common in smear-negative specimens, particularly for second-line injectable resistance testing (up to 23.6% invalid rate) 5

Clinical Context Requirements

• Molecular results must always be confirmed by phenotypic culture-based drug susceptibility testing, as LPA is an adjunct rather than replacement for conventional methods 1, 3
• Performance depends heavily on specimen quality and bacterial load - acid-fast bacilli detection in sputum strongly predicts LPA success 5
• For HBV, direct sequencing remains the gold standard because it can detect all mutations, whereas LPA only identifies pre-specified resistance mutations 1

Comparison with Alternative Methods

• More sensitive than direct PCR sequencing (5% vs 20% detection threshold for minority variants) but less sensitive than MALDI-TOF mass spectrometry (<1% detection) 1
• Faster than culture-based methods (1-2 days vs 4-8 weeks) but slower than GeneXpert (hours) 2
• Less expensive and complex than whole genome sequencing but provides limited information on resistance mechanisms 1