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Mycobacteria DNA Sequencing

Cultivation of mycobacteria from specimens submitted for AFB culture is challenging due to their slow growth rate, finicky nutritional requirements, and numerous biochemical tests needed for definitive identification. Mycobacteria can take as long as 6 weeks to grow from a specimen. Antimicrobial susceptibility testing requires an additonal 7-10 days to perform after mycobacterial species identification has been made. The majority of isolates are Mycobacterium avium-intracelluare, followed by the M. fortuitum-chelonae group. Fewer than 10 Mycobacterium tuberculosis are isolated per year.

Innovations in technology have enhanced detection & identification of mycobacteria in recent years. One example is an automated liquid media system for incubation of AFB cultures, in addition to traditional solid culture media. This system automatically analyzes samples for growth every 60 minutes and has improved recovery time for M. tuberculosis by several days. Cultures positive for mycobacterial growth are tested by nucleic acid probes to differentiate M. tuberculosis, M. avium-intracellulare, and M. kansasii. These probes are performed as soon as growth is detected from liquid media, often before growth is visible on solid media. Direct amplified (PCR) testing for M. tuberculosis from respiratory samples is also available as a send-out test, with results available within 24 to 48 hours.

The most recent addition to the mycobacteriologists’ toolbox is nucleic acid sequencing. This technique is particularly useful to identify mycobacteria species for which nucleic acid probes are unavailable, including rapid-growing mycobacteria species of the M. fortuitum-chelonae group. The technique requires isolation of bacterial DNA, amplification, sequencing, and data analysis. There are some limitations to sequencing in that some species within a group cannot be differentiated, for example M. chelonae from M. abscessus. However, the majority of identifications are available within 48 hours, as opposed to 2-3 weeks required for phenotypic identification through biochemical testing. 

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