Molecular tuberculosis (TB) diagnostic methods
| Nucleic acid amplification tests (NAATs) | Line probe assays (LPAs) | Whole genome sequencing (WGS) | Targeted next-generation sequencing (tNGS) | |
| Mechanism | Amplify the DNA using PCR, and detect a particular nucleic acid sequence | PCR-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 time | Focus on amplicons (DNA amplification products) or targets known to have strong associations with mutations |
| Advantages | Detect specific mutations associated with resistance to selected anti-TB drugs | Detect resistance to a wide range of first-line and second-line agents and provide mutation-specific data for common variants | Can identify low frequency variants | Can identify low frequency variants in targeted regions with high confidence |
| Can be used directly on clinical specimens | Can be used directly on clinical specimens | Improve detection of various types of mutations | Can be used directly on clinical specimens | |
| Short turn-around time | Short turn-around time (5 h) | Assess a broader range of drugs compared with phenotypic DST | Shorten 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 | ||||
| Limitations | Cannot identify low frequency variants | Low availability of supply chain | ||
| Sometimes follow-up actions (e.g. sequencing) are needed to guide appropriate TB treatment | Need 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 platforms | Xpert 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.