As per the Integrated Drug-resistant Tuberculosis (DR-TB) Diagnostic Algorithm:

• Nucleic Acid Amplification Tests (NAAT) are preferred for the initial detection of Rifampicin (R) resistance
• Line Probe Assays (LPA) are preferred for the detection of Isoniazid (H), Fluoroquinolones (FQ) and second-line injectable (SLI) drugs resistance.

When rifampicin resistance is detected by NAAT (see figure below):

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:

• MgCl₂ :1.5 - 6 mM
• Buffer (pH 8.3 - 8.8)
• DNA polymerase (Taq polymerase):  0.5 - 2.5 U
• Target DNA: <1µg  
• Primers: Short DNA sequences to select the region to be amplified

Molecular Basis: Introduction to Polymerase Chain Reaction(PCR) Technology

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:

Disposal of infectious samples and used cartridges in Truenat laboratories is an essential activity which needs to be performed to keep everyone safe and prevent TB infection in healthcare workers.

General biosafety requirements for Truenat laboratory include:

External Quality Assurance (EQA) ensures quality of Truenat laboratory is maintained by comparing their results through retesting and panel testing by a higher level laboratory (Intermediate Reference Laboratory (IRL)/ National Reference Laboratory (NRL)).

EQA for Truenat laboratory is done by: