Line Probe Assay

Line probe assays (LPA) are molecular strip tests that detect Mycobacterium tuberculosis complex and its resistance mutations by PCR amplification followed by reverse hybridisation of the amplicon to specific probes fixed on a nitrocellulose strip.

First line LPA (GenoType MTBDRplus, Nipro NTM+MDRTB) detects rpoB mutations (rifampicin resistance), katG (high level isoniazid resistance) and inhA promoter mutations (low level isoniazid resistance and cross-resistance to ethionamide).

Second line LPA (GenoType MTBDRsl) detects gyrA and gyrB mutations (fluoroquinolone resistance) and rrs and eis promoter mutations (injectable aminoglycoside and capreomycin resistance). Results are available within 1 to 2 days (compared with weeks for phenotypic DST) and can be done on smear positive sputum directly or on culture isolates.

The assays are used to confirm drug resistant tuberculosis and to design regimens in multidrug resistant (MDR) and rifampicin resistant disease. For first line LPA on smear positive sputum, sensitivity is about 96% for rifampicin resistance and 90% for isoniazid resistance, with specificity of 98 to 99%.

For second line LPA, sensitivity is about 86% for fluoroquinolones and about 87% for injectable drugs on smear positive specimens (lower in smear negative samples), with specificity over 98%. The assays do not replace culture based DST for all drugs.

Indications. Rifampicin resistance detected by Xpert MTB/RIF (to test isoniazid resistance and fluoroquinolone resistance). Smear positive sputum or culture isolates for initial DST in presumptive MDR-TB. Previously treated patients (relapse, failure, loss to follow up). Contacts of MDR-TB. Treatment failure or lack of conversion at 2 to 3 months.

Selection of the regimen for rifampicin resistant disease (confirm fluoroquinolone susceptibility before the shorter all-oral regimen). Detection of mixed or heteroresistant populations and resistance surveillance.

Specimen. Smear positive sputum (direct, 2 to 5 mL after decontamination) or a culture isolate (liquid or solid). Smear negative sputum can be tested after culture growth. DNA is extracted by heat lysis or commercial kit in a dedicated laboratory area.

Reference values. The strip has control bands (conjugate, amplification, M. tuberculosis complex, locus controls) and probe bands. For each gene, wild type probes cover the region and mutation probes identify common mutations. Results are reported as susceptible (all wild type bands present and no mutation band) or resistant (loss of a wild type band, or presence of a mutation band).

Interpretation of first line LPA.

• The commonest rpoB mutation is S531L (about 60 to 70% of rifampicin resistance), then H526Y and H526D. Some rpoB mutations (L511P, D516V, H526N, L533P) can give low level resistance that phenotypic testing may call susceptible, and these still respond poorly to rifampicin.
• katG S315T gives high level isoniazid resistance (minimum inhibitory concentration over 1 micrograms/mL). inhA promoter mutations give low level resistance, and high dose isoniazid may still add effect.
• Second line LPA: gyrA or gyrB mutation indicates fluoroquinolone resistance (levofloxacin and moxifloxacin), and loss of wild type bands in rrs (A1401G) or eis promoter indicates aminoglycoside resistance (kanamycin, amikacin, capreomycin). A fluoroquinolone resistant, rifampicin resistant case is pre-extensively drug resistant disease and requires a specialist regimen.
• Indeterminate or invalid results (no control band, weak amplification, or missing bands of the target gene) are repeated or sent for culture based DST or sequencing.
• A negative (susceptible) LPA does not exclude resistance caused by mutations outside the targets, and it is not used to confirm susceptibility to other drugs (pyrazinamide, ethambutol, bedaquiline, linezolid).

Critical values and findings needing immediate action.

• Rifampicin resistance with fluoroquinolone resistance on second line LPA: pre-extensively drug resistant tuberculosis, refer to a specialist service, design a regimen without fluoroquinolones (bedaquiline, pretomanid, linezolid, plus other agents).
• Isoniazid resistance with rifampicin susceptibility: modify first line therapy (rifampicin, ethambutol, pyrazinamide and a fluoroquinolone for 6 months).
• Rifampicin resistance confirmed: infection control, public health notification and contact tracing.
• Discordance between Xpert and LPA or phenotypic results: urgent resolution by sequencing, because treatment depends on it.
• Failure of treatment with newly acquired resistance: repeat testing and reconsider regimen.

Interferences and pitfalls.

• Only the common resistance mutations in selected genes are detected. Mutations outside the probe regions, in other genes or in efflux pumps are missed, and heteroresistance at low proportions (under 10 to 20%) may not be seen.
• Sensitivity is lower on smear negative direct specimens, and many are invalid or indeterminate.
• Contamination of amplicons (carry over) leads to false results, so strict separation of pre-PCR and post-PCR rooms, unidirectional workflow and negative controls are needed.
• Silent mutations and phylogenetic polymorphisms can cause loss of a wild type band with no phenotypic resistance, and results need interpretation in context with other tests.
• Non-tuberculous mycobacteria give no M. tuberculosis band and are not reported as resistant.
• The assay needs trained staff, a thermocycler, water bath or automated hybridisation instrument, and quality control, and a typical run handles up to 12 to 48 samples.
• Newer drugs (bedaquiline, delamanid, pretomanid, linezolid) are not covered by the classic strips, and targeted next generation sequencing or whole genome sequencing fills this gap.
• A susceptible LPA result should not by itself justify stopping other tests when clinical response is poor.

Special groups.

• Children: use isolates from culture or Xpert positive specimens, since direct sputum is difficult to obtain.
• Pregnancy: DST results are used to design a safe regimen, with specialist input.
• HIV: more extrapulmonary and smear negative disease, so culture isolates are tested more often.
• Previously treated patients: higher rates of resistance and mixed infection.
• Regions: LPA brought rapid DST to national programmes, but the need for a molecular laboratory limits use, and it is increasingly replaced by Xpert MTB/XDR and targeted sequencing.

Follow up tests. Phenotypic DST (liquid culture), targeted next generation sequencing or whole genome sequencing, Xpert MTB/RIF or Xpert MTB/XDR, culture conversion monitoring monthly in drug resistant disease, HIV test, ECG for QT, audiometry, renal and liver function, visual acuity (ethambutol and linezolid), full blood count (linezolid), contact tracing, and treatment adherence support.