What are LPA (Line Probe Assay) 1 and 2?

Line Probe Assay (LPA) 1 and 2 in Tuberculosis Diagnostics

LPA 1 (first-line LPA) detects resistance to rifampicin and isoniazid, while LPA 2 (second-line LPA) detects resistance to fluoroquinolones and second-line injectable agents (amikacin, kanamycin, capreomycin). 1, 2

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

Clinical Applications by LPA Type

LPA 1 (First-Line LPA - GenoType MTBDRplus)

This assay is the initial molecular test for detecting multidrug-resistant tuberculosis (MDR-TB) after rifampicin resistance is identified. 3, 2

• Detects mutations in the rpoB gene (rifampicin resistance) at codons 516,526, and 531. 4, 5
• Detects mutations in the katG gene (primarily katG315) and inhA promoter region (position -15) for isoniazid resistance. 4, 5
• The sensitivity for detecting isoniazid resistance is 77.4% (95% CI: 65.5-86.2) among Hr-TB isolates and 94.3% (95% CI: 80.4-99.4) among MDR-TB isolates, with 100% specificity. 5
• The sensitivity for detecting rifampicin resistance is 96.4% with 100% specificity in smear-positive sputum samples. 4

LPA 2 (Second-Line LPA - GenoType MTBDRsl)

This assay is used after MDR-TB is confirmed to detect extensively drug-resistant tuberculosis (XDR-TB) and guide treatment regimen selection. 3, 6

• Detects mutations conferring resistance to fluoroquinolones (ofloxacin, moxifloxacin, levofloxacin) in the gyrA and gyrB genes. 6
• Detects mutations conferring resistance to second-line injectable agents (amikacin, kanamycin, capreomycin) in the rrs and eis genes. 6
• The sensitivity for detecting fluoroquinolone resistance is 90.7% (95% CI: 80.1-96.0%) with 98.1% specificity. 6
• The sensitivity for detecting amikacin resistance is 100% (95% CI: 91.8-100%) with 99.4% specificity. 6
• The sensitivity for detecting extensive drug resistance is 92.3% (95% CI: 75.9-97.9%) with 99.6% specificity. 6

Critical Clinical Algorithm

For patients with confirmed rifampicin resistance on GeneXpert, immediately perform LPA 1 to confirm MDR-TB and detect isoniazid resistance patterns. 3, 2

1. 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. 3

2. If LPA 2 shows fluoroquinolone susceptibility: Patient is eligible for the standardized all-oral bedaquiline-containing shorter regimen (9-11 months). 3

3. 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). 3

Critical Limitations and Pitfalls

The line probe assay cannot detect unknown or novel mutations—it only identifies mutations for which specific probes have been designed. 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. 3
• 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
• In smear-negative sputum samples, LPA 1 shows only 77.8% sensitivity for detecting M. tuberculosis, with high rates of invalid results (30.8% in one study), making it unreliable for direct testing in smear-negative specimens. 4
• LPA 2 showed 23.6% invalid results for second-line injectable resistance testing despite repeat testing. 7

Turnaround Time Advantage

Implementation of LPA 2 significantly reduces turnaround time by 93.3% compared to conventional culture-based drug susceptibility testing, from weeks to days. 6

• This rapid dissemination of second-line DST results guides initiation of appropriate treatment, thereby reducing transmission and improving treatment outcomes. 6
• LPA testing provides results within 1-2 days compared to 4-8 weeks for phenotypic culture-based methods. 6

Specimen Requirements

LPA 1 and 2 perform optimally on smear-positive sputum samples or cultured isolates. 4, 6

• For smear-positive direct testing, collect at least 2-3 mL of sputum with visible purulence. 4
• For smear-negative samples, culture the specimen first and perform LPA on the cultured isolate rather than attempting direct testing. 4
• The diagnostic sensitivity of LPA depends strongly on the detection of acid-fast bacilli in the sputum specimen. 7