Ann Clin Microbiol 2025;28(3):18. Challenges and advances in mycobacterial molecular typing
| Technique | Principle | Advantages | Limitations | MTB applications | NTM applications | Ref |
|---|---|---|---|---|---|---|
| Spoligotyping | PCR detection of presence/absence of unique spacer sequences in the direct repeat locus | Rapid, low DNA input (10 fg), standardized, suitable for degraded samples | Low discriminatory power, not suitable for outbreak confirmation | Lineage typing, strain tracking in low-resource settings | Limited, low resolution between NTM species | [22] |
| RFLP | RFLP analysis based on the insertion sequence IS6110 (for MTB); for NTM, IS1245, IS901, IS1311, or groES-based RFLP variants | High discriminatory power, stable profiles (IS6110 for MTB); for NTM polymorphic patterns with BsaAI, PvuII, NruI (IS1245), PvuII (IS901/IS1311), or BamHI/BstNI/HpaI (groES PCR-RFLP) | Labor intensive, requires large DNA amounts, not suitable for low-copy IS6110 strains (MTB); for NTM, band clustering with certain enzymes (Examples: PvuII in IS1245/IS1311), unexpected low-molecular-size fragments, limited standardization | Historical gold standard for outbreak studies | IS1245-based RFLP yields polymorphic patterns with BsaAI and NruI easier to detect, suggesting unidentified insertion elements; IS901 RFLP identifies 6 profiles (E, F, G, M, Q, V) linked to flocks and transmission (Examples: pheasants/goshawks); IS1311 RFLP with PvuII differentiates M. avium subspecies from human/animal origins, often combined with MIRU-VNTR; groES-based PCR-RFLP differentiates M. avium (25 isolates) and M. intracellulare (20 isolates) using HgaI polymorphism, complementing high-performance liquid chromatography | [20,21,34,53,54] |
| MIRU-VNTR | PCR-based detection of variable number of tandem repeats at multiple loci | High resolution, reproducible, database compatible, digital outputs | Less discriminatory than WGS, low-copy-number strains reduce resolution | Current global standard for MTB genotyping | Applied to select NTM species (Example: M. intracellulare) | [23] |
| 16S rRNA sequencing | Amplification and sequencing of the conserved 16S ribosomal RNA gene | Broad-range detection, widely available, applicable to direct specimens | Cannot distinguish closely related species, low resolution (Example: M. avium vs. M. intracellulare) | Rarely used owing to poor discriminatory power | Common for genus-level identification, limited for subspecies resolution | [26] |
| hsp65, rpoB, gyrB sequencing | Gene-targeted sequencing of heat-shock protein, RNA polymerase, or gyrase genes | High species-level resolution, suitable for clinical ID, complements 16S rRNA | Still limited for some MAC/MABC species, not all targets validated | Limited, mainly used for species confirmation | Widely used for NTM species and subspecies discrimination, routine tool in clinical mycobacteriology labs | [25] |
| MLST | Sequencing of internal fragments of multiple housekeeping genes | Portable between laboratories, good for phylogenetics, population structure analysis | Low resolution for outbreak tracing, requires multiple loci sequencing | Rarely used | Increasingly used for taxonomy and evolutionary analysis | [30] |
| WGS | Sequencing of the entire genome | Highest resolution; detects SNPs, drug resistance, and phylogeny | Costly, bioinformatics heavy, requires high-quality DNA and infrastructure | Drug resistance prediction, outbreak tracing, evolution | Drug resistance prediction, outbreak tracing, evolution, resolves relapse vs. re-infection, novel species ID, resistance markers | [14,15,29,31] |
Abbreviations: MTB, Mycobacterium tuberculosis; NTM, nontuberculous mycobacteria; Ref, reference; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; MIRU-VNTR, mycobacterial interspersed repetitive unit–variable number tandem repeat; ID, identification; MAC, Mycobacterium avium complex; MABC, Mycobacterium abscessus complex; WGS, whole-genome sequencing; SNP, single nucleotide polymorphism; MLST, multilocus sequence typing.