## 6 Detection, Identification, and Differentiation Tools for Mycobacteria
Amongst those tests is INNNO-LiPA MYCOBACTERIA v2 which is a reverse hybridization DNA probe assay capable of identifying 16 different species of mycobacteria [348]. Three of the probes included in the assay were found to cross-hybridize with some isolates rarely found in clinical samples [348]. More recently, it was also reported the misidentification of _M. singematis_ for _M. fortuitum_ complex in a case study event [349]. Another similar test is GenoType NTM-DR which is capable of identification of the more relevant clinical NTM species, as well as detection of resistance to both macrolides and aminoglycos [350]. In recent years, this assay has been tested for identification of MAC and _M. abscessus_ with 92% accuracy for reference strains and 100% for clinical isolates, though three MAC reference strains were misidentified as _M. intracellular_[350]. In another study with MAC isolates, 91% of isolates were identified correctly to the species level [351]. Regarding the differentiation of NTM, most techniques have been adapted from MTC differentiation protocols. For most NTM, the gold standard for differentiation still is Pulsed-Field Gel Electrophoresis (PFGE). This technique has high costs and requires technical expertise, with a long-lasting protocol that put it aside for clinical settings, but still one the most discriminatory for NTM [352]. PFGE was applied to several NTM species (e.g., _M. kansasii_[353], MAC [354], _M. gordonae_[355], _M. haemophilium_[356]), with relative success. Another technique that appears to achieve good results in NTM differentiation is Random Amplified Polymorphic DNA (RAPD) analysis. One of its low points is the fact that differences in technical applications may influence reproducibility [352]. However, it remains useful for NTM differentiation and it was used in species like _M. abscessus_[357], _M. mucogenicum_[186], _M. phocaicum_[186], _M. gordonae_[358]. In the particular case of _M. avium_, the differentiation has been based on the restriction fragment length polymorphism (RFLP) typing of _IS1245_, which appears as reference for this species [359]. A promising tool in the range of molecular techniques is whole-genome sequencing (WGS). In addition to the ability for extensive species characterization and categorization that is particularly relevant for the _Mycobacterium_ genus, WGS enables the discovery of novel biomarkers helpful in the context of pathogen identification [17]. Recently, it was proposed the use of the _ku_ gene as a biomarker for _Mycobacterium_ genus [360]. In that study, 79.7% of all known mycobacterial species and all submitted genomes were considered for comparative genomics, indicating that this gene is widely distributed and conserved across _Mycobacterium_ species [360]. [PERSON] and coworkers (2018) indicate that ten conserved signature indels and nine conserved signature proteins are shared by most members of the _Mycobacterium_ genus [13]. Those biomarkers have the potential to be used as targets in PCR-based methods, Enzyme-Linked Immunosorbent Assay (ELISA), etc. Although WGS requires DNA purity and quantity, which is sometimes harder to achieve while working with environmental samples, it allows answering to both identification and differentiation questions. Small quantities of DNA can be overcome using whole-genome amplification techniques. In addition to those fundamental elements for isolate identification and differentiation, WGS can also provide insights into antibiotic resistance patterns in environmental isolates, epidemiological issues, etc., that may be relevant in the clinical context. A long-lasting discussion on the pros and cons of culture-dependent and culture-independent approaches remains. The main advantage of the culture-dependent approach consists of the detection of viable mycobacteria (colony formation) within the clinical sample, which increases the probability of these being the causative disease agent. However, the time needed for mycobacteria cultivation can be high with some species (e.g., MAP) being extremely fastidious.