Molecular tuberculosis (TB) diagnostic methods

Nucleic acid amplification tests (NAATs)Line probe assays (LPAs)Whole genome sequencing (WGS)Targeted next-generation sequencing (tNGS)
MechanismAmplify the DNA using PCR, and detect a particular nucleic acid sequencePCR-based tests that use LPA as detection (detect the binding pattern of DNA amplification products to probes that target specific parts of the Mycobacterium tuberculosis genome, resistance-associated mutations to anti-TB drugs, or the wild-type DNA sequence)Analyse the entire genomic DNA sequence at a single timeFocus on amplicons (DNA amplification products) or targets known to have strong associations with mutations
AdvantagesDetect specific mutations associated with resistance to selected anti-TB drugsDetect resistance to a wide range of first-line and second-line agents and provide mutation-specific data for common variantsCan identify low frequency variantsCan identify low frequency variants in targeted regions with high confidence
Can be used directly on clinical specimensCan be used directly on clinical specimensImprove detection of various types of mutationsCan be used directly on clinical specimens
Short turn-around timeShort turn-around time (5 h)Assess a broader range of drugs compared with phenotypic DSTShorten turn-around time if performed from clinical specimens
Provide unbiased detection of mutations mediating low
or moderate MIC
Can also be used for surveillance and source investigation
LimitationsCannot identify low frequency variantsLow availability of supply chain
Sometimes follow-up actions (e.g. sequencing) are needed to guide appropriate TB treatmentNeed high technical and analytical skills Need high storage size and security
High cost (capital investment costs, running costs, data storage costs)
Need for culture
High cost, but less than WGS; tNGS requires adding the PCR step; the decrease in cost could be linked to the running of many samples in the same flow cell
Longer turn-around time than other mWRDs methods
Difficulty interpreting whole-genome variation data in the context of the high number of rare variants
Example of platformsXpert MTB/RIF and Xpert MTB/RIF Ultra (Cepheid); Truenat (Molbio); Abbott RealTime MTB and Abbott RealTime MTB RIF/INH (Abbott); BD MAX MDR-TB (Becton Dickinson); cobas MTB and cobas
MTB-RIF/INH (Roche); FluoroType MTBDR and FluoroType MTB (Hain Lifescience/Bruker)
GenoType MTBDRplus v1 and v2, and GenoType MTBDRsl (Hain Lifescience/Bruker); Genoscholar NTM+MDRTB II, and Genoscholar PZA-TB II (Nipro)Miseq, MiniSeq, NextSeq, HiSeq (Illumina); Personal Genome Machine (Ion Torrent); PacBio RS II (Pacific Biosciences); MinION (Oxford Nanopore Technologies)

DST: drug-susceptibility testing; MIC: minimum inhibitory concentration; mWRD: molecular WHO-recommended rapid diagnostic tests.