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The Line Probe Assay (LPA) is a strip based rapid molecular technique based on polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis and resistance to anti-TB drugs.
LPA detects resistance to Rifampicin (R), Isoniazid (H), Fluroquinolones (FQ); (Ofloxacin, Ofx; Levofloxacin, Lfx; Moxifloxacin) and second-line injectable (SLI); (Kanamycin, Km; Amikacin, Am) drugs.
Principle of LPA
Anti-TB drugs and molecular mechanism of multi-drug resistance with regards to First Line- Line Probe Assay (FL-LPA) and Second Line- LPA (SL-LPA) is described here.
Rifampicin (RIF)
Polymerase Chain Reaction (PCR) is a laboratory technique to amplify Deoxyribose Nucleic Acid (DNA).
The PCR mix consists of:
TrueNAT assays are Polymerase Chain Reaction (PCR) tests performed using freeze dried PCR reagents and primers contained in microtubes mixed with eluted DNA (Trueprep device) and amplified on Truelab device.
The primers for PCR are designed to amplify ribonucleoside-diphosphate reductase gene - nrdZ gene in Mycobacterium tuberculosis (MTB) genome.
Analytical Exclusivity (Primer Specificity) test were done by the manufacturer to determine the specificity of the TrueNAT Assays by:
The TrueNAT laboratory should maintain a document/ records of Truelab and Trueprep usage and maintenance using the logs and/ or checklist. This helps to:
Representative documents to maintain record for use and maintenance are shown in table 1(A) - (B) and figure below.
The limitations of TrueNAT Assays are:
“Indeterminate” result on completion of the test run with TrueNAT MTB-Rif Dx means Polymerase Chain Reaction (PCR) melting temperature (Tm) values did not meet the requirements for Rif resistance determination.
Implications
Troubleshooting TrueNAT MTB Rif Dx “Indeterminate”
Samples collected for TrueNAT tests are tested immediately (if collected at TrueNAT facility) or transported on the same day to nearest linked TrueNAT facility.
TrueNAT MTB and TrueNAT MTB Rif Dx Assay are intended to be used for detection of Mycobacterium tuberculosis bacteria (for TB diagnosis) and to detect resistance to rifampicin (Rif), respectively.
Positioning of TrueNAT MTB and TrueNAT MTB Rif Dx Assay
TrueNAT MTB and TrueNAT MTB Rif Dx Assay are suitable as point-of-care tests for use in peripheral laboratories and microscopy centres due to the following operational features:
Ensuring accurate sputum microscopy results is paramount to maintain optimal sensitivity and specificity of sputum microscopy.
In this regard, External Quality Assurance (EQA) is an important aspect of the National TB Elimination Programme (NTEP) Laboratory Network. Different levels of EQA activities are undertaken to ensure that the system follows a standard protocol and remains efficient. The timely completion of EQA activities is important.