Diagnostics for MAC

The following section focuses on diagnostic tools and classification schemes that are typically em­ployed for members of the MAC. How these tests do, and do not, aid in the identification of MAP is reviewed.

5.3.1 Culture and traditional methods for classification of MAC

Definitive diagnosis of a mycobacterial infection requires culture of the organism from a clinical specimen, followed by identification using estab­lished techniques. Both liquid and solid (agar- or egg-based) media can be used for mycobacterial culture. However, no single medium or growth condition will permit the successful isolation of all mycobacteria and therefore protocols may vary between laboratories. Mycobacterium intra- cellulare and the classical M. avium strains can grow on any standard mycobacterial media, with or without 10% CO₂. While the MAC grows well at 3 7°C incubation, M. avium strains may grow best at an increased temperature of 40­4 2 °C (Kent and Kubica, 1985). MAC requires >7 days for growth and 3-4 weeks to reach ma­turity. Mycobacterial cultures are typically kept up to 6-8 weeks before being considered nega­tive. However, these standard conditions are insufficient for routine isolation of MAP. The organism’s extremely slow growth rate and re­quirement for mycobactin supplementation pre­clude detection. Even when present in immense quantities, it can take several months to detect MAP in the clinical setting (Richter et al., 2002).

Traditionally, speciation of non- tuberculous mycobacteria was based on phe­notypic characteristics such as pigmentation, growth rate, growth temperature and bio­chemical activities. The MAC is considered non­pigmented, although some strains may present with bright yellow pigmentation (e.g.

For decades, serotyping was used for clas­sification of MAC strains. Together, M. avium and M. intracellulare comprise 28 different se- rovars (Saito et al., 1990; Wayne et al., 1993). Serotyping relies on the presence of serovar- specific glycopeptidolipids (GPLs) (Brennan et al., 19 78; Brennan and Goren, 19 79). MAP isolates do not produce GPLs and therefore can­not be serotyped. However, this is not a diagnos­tically useful characteristic since GPL mutants of M. avium (Belisle et al., 1993) and other non- serotypeable MAC clones (De Smet et al., 1996) are also encountered. Skilled technicians can also use high-performance liquid chromatog­raphy (HPLC) of mycolic acids to successfully separate some MAC species (Butler et al., 1992), but differentiation of MAP from other M. avium subspecies is not possible (Dei et al., 1999).

Matrix-associated laser desorption/ioniza- tion-time-of-flight mass spectrometry (MALDI- TOF MS) has become a routine method for bacterial identification. This method can distin­guish M. avium from M. intracellulare, but com­mercial MALDI-TOF databases do not contain profiles for all validated species, nor are all spe­cies and subspecies of MAC typically evaluated in published studies.

5.3.2 DNA sequencing for the identification of MAC

DNA-based analysis, including WGS, confirms that the MAC includes more than M. avium and M. intracellulare. Most validated MAC species can be identified by targeted sequencing of the full-length 16S rRNA gene. The exceptions are M. intracellulare and M. paraintracellulare, and M. marseillense and M. yongonense (Tortoli et al., 2017). Additional targets, including ITSs, hsp65 and rpoB allow further differentiation of MAC species and subspecies. Within M. avium, eight ITS sequevars have been reported. Because MAP strains typically present with the Mav-A seque- var, ITSs cannot be used to distinguish them from other M. avium subspecies (Turenne et al., 2006). Similarly, the 441 base pair (BP) region at the 5' end of the hsp65 gene that is widely used for speciation of mycobacteria (Telenti et al., 1993) does not effectively differentiate M. avium subspecies. In contrast, the 3' ‘tail end’ of the hsp65 gene can simultaneously identify spe­cies as well as host-associated subtypes (Turenne et al., 2006). MAP is represented by two seque- vars, one for each of the MAP- C and Map- S lin­eages. Another hsp65 sequevar encompasses both of the bird-associated subspecies (i.e. subsp. avium and silvaticum together). rpoB sequencing is also capable of distinguishing among subspe­cies of M. avium, including MAP (Higgins et al., 2011).

5.3.3 Other molecular assays for detection or identification of MAC

A scheme based on three insertion sequences, IS1245, IS901 and IS900, has been used to de­fine the subspecies of M.

A non-sequencing-based method for reli­able differentiation of M. avium subsp. silvaticum and M. avium subsp. avium has only recently been described. This high-resolution melt meth­od can detect differences as small as a single nu­cleotide polymorphism (Ronai et al., 2015).

5.3.4 Commercial assays for the identification of MAC

Genetic variation is the basis for a number of commercial assays currently available for the de­tection and/or speciation of mycobacteria. These tests offer rapid turnaround time and greater accuracy than conventional methods and thus contribute to improved patient care. Some of the first, introduced in the early 1990s and still used today, are the AccuProbe^® Culture Identification Tests (Hologic) (Saito et al., 1989). Currently six tests are available for mycobacteria. Each targets ribosomal RNA and permits identification from a positive culture. In addition to individual tests for the M. tuberculosis complex, M. kansasii, M. gordonae, M. intracellulare and M. avium, there is a MAC test that can identify any MAC organ­ism to the complex level (Lebrun et al., 1992; Viljanen et al., 1993). However, on rare occa­sions the MAC test may cross-react with other mycobacteria (Tortoli et al., 2010). Because the target of AccuProbe is ribosomal RNA, this assay cannot distinguish MAP from other M.

Reverse hybridization line probe assays (LPA) are also commercially available. In ad­dition to other non-tuberculous mycobacteria (NTM), the Inno-LiPA® MYCOBACTERIA v2 (Fujirebio) can identify M. avium, two subsets of M. intracellulare and ‘MAIS complex' (Tortoli et al., 2003; Lebrun et al., 2005). However, the target is the ITS region, which does not permit resolution of MAP. Hain Lifescience markets sev­eral LPAs for mycobacteria. These detect species­specific 23S rDNA sequences. The GenoType^® Mycobacteria Direct can be used for identifica­tion directly from clinical specimens, whereas the GenoType^® Mycobacterium CM (Common Mycobacteria), AS (Additional Species) and NTM-DR (which can specifically identify M. chi­maera) tests require positive cultures. The Speed- oligo Mycobacteria assay (Vircell) is the latest LPA that includes M. avium and M. intracellulare. It targets both the 16S rRNA gene and adjacent ITSs but performance evaluations are limited to date. Again, since these LPAs target the riboso­mal operon, none has the capacity to differenti­ate MAP from other M. avium.

5.4