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Molecular Differentiation of M. tuberculosis complex (MTBC)

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Differentiation (speciation) of Mycobacterium tuberculosis complex (MTBC) members through the analysis of whole genome sequencing data. The MTBC is a closely related group of species that encompasses Mycobacterium tuberculosis (including the non-validly published 'M. tuberculosis var. canettii’ (1,2) , 'M. tuberculosis var. orygis' (2,3) and some rare animal lineages), M. bovis, M. bovis BCG, M. africanum (lineages 5 and 6) (4), M. caprae, M. microti, M. pinnipedii (5).

Test Category:
Molecular Differentiation
Mycobacterium tuberculosis
Illnesses and Diseases:
  • Tuberculosis (TB)

This test will be performed on cultures previously identified as MTBC and it is not appropriate for clinical samples. This method can be performed on solid or liquid media growth, though solid media is optimal. For cultures on solid media, visible growth that is less than 6 weeks old is required, and isolated colonies on plated media are preferred. Liquid cultures must be flagged as positive on the Bactec MGIT 960 and have a minimum volume of 4 ml. Cultures with inadequate growth or volume, and cultures contaminated with other bacterial or mycobacterial species will be rejected.

Collection Method:


Specimen Processing, Storage and Shipping:

Ship all cultures at room temperature (DO NOT freeze) for overnight delivery, and prior to Wednesday each week to ensure receipt by Friday.

Transportation of Dangerous Goods:

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.


Patient Criteria:

This test will be performed on all submitted cultures identified as MTBC, as well as cultures identified as MTBC at the NRCM.

Accompanying Documentation:

Notification of sample submission must be emailed or faxed prior to shipping isolates. Samples should be shipped attention: Melissa Rabb, Head, Reference and Diagnostic Services, NRCM/NML at 204-789-6081 / 204-789-6038.  A requisition for the NRCM must be completed and signed off by the supervisor/designate of the submitting laboratory and include the source of specimen, patient gender, date of birth, clinical history, submitting laboratory identifier and submitter information.  Please also include isolate characteristics:  microscopy, pigmentation, culture characteristics, growth rate/temperature, and identification.  Requested testing for urgent submissions should be accompanied with justification.


Submitted cultures will be rejected and a resubmission requested if there is inadequate growth, or there is contamination. Cultures will be rejected if appropriate documentation and justification is incomplete or missing.

Methods and Interpretation of Results:

Species identification will be accomplished through whole genome sequence analysis of phylogenetically informative single nucleotide polymorphisms (6,7) using the bioinformatics tool Biohansel (8). This method can identify all validly published members of the M. tuberculosis complex, as well as some non-validly published species such as 'M. tuberculosis var. canettii’ (1,2) and 'M. tuberculosis var. orygis' (2,3) and some rare animal lineages.

Turnaround Time:

Routine testing from culture: 20 calendar days from the date of specimen receipt.

Phone #: (204) 789-2136 or (204) 789-6038
Fax: (204) 789-2036
  1. Pfyffer GE, Auckenthaler R, van Embden JD, van Soolingen D. Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg Infect Dis. 1998;4: 631–634. doi:10.3201/eid0404.980414.
  2. Riojas MA, McGough KJ, Rider-Riojas CJ, Rastogi N, Hazbón MH. Phylogenomic analysis of the species of the Mycobacterium tuberculosis complex demonstrates that Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii are later heterotypic synonyms of Mycobacterium tuberculosis. Int J Syst Evol Microbiol. 2017; doi:10.1099/ijsem.0.002507.
  3. van Ingen J, Rahim Z, Mulder A, Boeree MJ, Simeone R, Brosch R, et al. Characterization of Mycobacterium orygis as M. tuberculosis complex subspecies. Emerg Infect Dis. 2012;18: 653–655. doi:10.3201/eid1804.11088.
  4. Coll F, McNerney R, Guerra-Assunção JA, Glynn JR, Perdigão J, Viveiros M, et al. A robust SNP barcode for typing Mycobacterium tuberculosis complex strains. Nat Commun. 2014;5: 4812. doi:10.1038/ncomms5812.
  5. Cousins DV, Bastida R, Cataldi A, Quse V, Redrobe S, Dow S, et al. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov. Int J Syst Evol Microbiol. 2003;53: 1305–1314. doi:10.1099/ijs.0.02401-0.
  6. Niemann S, Harmsen D, Rüsch-Gerdes S, Richter E. Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis. J Clin Microbiol. 2000;38: 3231–3234. Available:
  7. Samuel Lipworth, Rana Jajou, Albert de Neeling, Phelim Bradley, Wim van der Hoek, Gugu Maphalala, et al. SNP-IT Tool for Identifying Subspecies and Associated Lineages of Mycobacterium tuberculosis Complex. Emerging Infectious Disease journal. 2019;25: 482. doi:10.3201/eid2503.180894.
  8. A robust genotyping scheme for Salmonella enterica serovar Heidelberg clones circulating in North America. Geneviève Labbé, James Robertson, Peter Kruczkiewicz, Marisa Rankin, Matthew Gopez, Chad R. Laing, Philip Mabon, Kim Ziebell, Aleisha R. Reimer, Lorelee Tschetter, Gary Van Domselaar, Sadjia Bekal, Kimberley A. MacDonald, Linda Hoang, Linda Chui, Danielle Daignault, Durda Slavic, Frank Pollari, E. Jane Parmley, Elissa Giang, Lok Kan Lee, Jonathan Moffat, Joanne MacKinnon, Roger Johnson, John H.E. Nash. [Manuscript in preparation].