Line Probe Assay in Tuberculosis Diagnostics

Technical Methodology

Line probe assays operate through a hybridization-based detection system that uses PCR amplification of target DNA sequences, followed by hybridization of amplified products to specific oligonucleotide probes immobilized on membrane strips 1
The assay can detect single nucleotide mismatches using a series of short membrane-bound oligonucleotide probes that bind to specific mutation sites 3, 1
LPAs can detect viral or bacterial mutants that constitute as little as 5% of the total population, making them more sensitive than direct PCR sequencing which requires approximately 20% of the population to harbor the mutation 3, 1

The European Respiratory Society notes that LPA 1 is the initial molecular test for detecting multidrug-resistant tuberculosis (MDR-TB) after rifampicin resistance is identified 4, 2
LPA 2 is used after MDR-TB is confirmed to detect extensively drug-resistant tuberculosis (XDR-TB) and guide treatment regimen selection 4
For patients with confirmed rifampicin resistance on GeneXpert, immediately perform LPA 1 to confirm MDR-TB and detect isoniazid resistance patterns, as recommended by the European Respiratory Society 4, 2

If LPA 1 confirms MDR-TB, immediately perform LPA 2 to detect fluoroquinolone resistance, as this determines eligibility for the shorter MDR-TB regimen versus longer regimens, according to the European Respiratory Society 4
If LPA 2 shows fluoroquinolone susceptibility, patient is eligible for the standardized all-oral bedaquiline-containing shorter regimen (9-11 months), as recommended by the European Respiratory Society 4
If LPA 2 shows fluoroquinolone resistance, patient requires an individualized longer regimen (18-20 months) or enrollment in operational research for the BPaL regimen (bedaquiline, pretomanid, linezolid), according to the European Respiratory Society 4

The American Association for the Study of Liver Diseases notes that molecular results from line probe assays must always be confirmed by phenotypic culture-based drug susceptibility testing 1
Individual probes are required for each mutation, and single-nucleotide polymorphisms that have no effect on phenotype can impair probe binding and produce false-negative results 5, 6