Line Probe Assay (LPA) — Comprehensive Answer

Definition

Principle

Reverse Hybridization (Core Concept)

Step-by-Step Process

Components on the Strip (MTBDRplus as example)

Commercially Available LPAs for TB

Genetic Targets and Resistance Mutations Detected

Interpretation Logic:

• WT band present + MUT band absent → Susceptible
• WT band absent + MUT band present → Resistant (specific known mutation)
• WT band absent + MUT band absent → Resistant (unknown/novel mutation — requires sequencing)
• WT band present + MUT band present → Mixed infection or heteroresistance

Specimen Requirements

• Direct specimen: AFB smear-positive sputum (without prior culture)
• Indirect specimen: positive liquid or solid culture
• Processing requires Biosafety Level 2 (or Class II laminar flow cabinet minimum)
• DNA must be extracted with adequate decontamination

Performance Characteristics

WHO Recommendations

1. Screening of patients at risk of MDR-TB (prior treatment, contact of MDR case, high-burden settings)
2. Use directly on AFB smear-positive sputum — WHO Policy 2008
3. MTBDRsl for second-line drug resistance (pre-XDR and XDR-TB screening)

Advantages

1. Rapid — results in 24–48 hours from smear-positive specimen, far faster than culture-based DST (weeks)
2. Simultaneous — detects organism identity AND multiple drug resistance mutations in one test
3. Direct specimen use — can be applied to smear-positive sputum without prior culture
4. High specificity — stringent hybridization conditions prevent cross-reactivity
5. Multiplexing — many probes on a single strip
6. Standardized — visual band pattern is semi-quantitative and interpretable with a reading card
7. Cost-effective for high-burden settings compared to sequencing

Limitations

1. Does not detect all resistance mutations — only covers known mutations; novel mutations (WT absent, MUT absent) require DNA sequencing
2. Lower sensitivity in smear-negative or paucibacillary specimens — sensitivity drops to 40–60% in AFB smear-negative sputum
3. Cannot assess phenotypic resistance level (e.g., low-level vs. high-level INH resistance)
4. Does not replace culture — culture remains gold standard; LPA cannot determine viability or assess all drugs
5. Requires PCR facility — biosafety considerations; cannot be used as a point-of-care test
6. Resistance to pyrazinamide not detected by MTBDRplus (requires a separate assay)
7. Mixed infection/contamination can give ambiguous results

LPA vs. Other Rapid Diagnostics

Applications Beyond TB

• Hepatitis C virus (HCV) genotyping — INNO-LiPA HCV assay (genotypes 1–6)
• HIV drug resistance — detection of reverse transcriptase/protease mutations
• Hepatitis B — lamivudine resistance mutations
• HPV genotyping — high-risk vs. low-risk types
• Hepatitis C genotyping for treatment selection (e.g., direct-acting antivirals are genotype-dependent)

Summary for Exam

LPA = Reverse hybridization + PCR amplification + membrane-bound probes + colorimetric detection

Key assay: GenoType MTBDRplus — detects rpoB (RIF resistance) + katG/inhA (INH resistance) = MDR-TB diagnosis within 1–2 days from smear-positive sputum

Wild-type band loss = resistance; Mutation band appearance = specific known mutation; WHO-endorsed for MDR-TB screening

• Tietz Textbook of Laboratory Medicine, 7th Edition (Hybridization Arrays, p. 2665–66)
• Henry's Clinical Diagnosis and Management by Laboratory Methods (Susceptibility Testing, p. 1366)
• Fishman's Pulmonary Diseases and Disorders, 2-Volume Set (Nucleic Acid Amplification, p. 2296)
• Harrison's Principles of Internal Medicine, 22nd Ed. (Drug-Resistant TB, p. 1441)