Mycobacterial Interspersed Repetitive-Unit Variable Number of Tandem Repeats (MIRU-VNTR) genotyping

Primary typing of Mycobacterium tuberculosis complex (MTBC) isolates using Variable Number of Tandem Repeat analysis based molecular typing to determine relatedness of strains.

Solid or liquid media growth of pure culture.  

• For solid media growth, isolated colonies on plated media. MTBC species must show visible growth and be no more than 4 weeks old.
•  For liquid culture, a minimum volume of 4 mL of actively growing culture. 
• If the culture has inadequate growth, contamination, or mixed Mycobacterium species, the submitted culture will be rejected and a resubmission will be requested. 

N/A (submit culture only).

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

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.

The first positive culture isolated from a TB patient is preferred for TB genotyping.  If required, subsequent cultures can be tested.  It should be noted on the requisition if a previous isolate from the patient has been tested or if the culture is a part of a contamination investigation.

Notification of sample submission must be emailed or faxed prior to shipping isolates. A requisition for the NRCM must be completed and signed off by the supervisor/designate of the submitting Provincial/Territorial Healthcare 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, identification, and susceptibility results, if available. Requested testing for urgent submissions should be accompanied with justification. The NRCM will provide results to the submitting Provincial/Territorial Healthcare laboratory, who is then responsible for disseminating results to the appropriate local lab or physician.

Cultures will be rejected if appropriate documentation is incomplete or missing.

MIRU-VNTR patterns will be determined as per the “Multilocus Variable Number Tandem Repeat Genotyping of Mycobacterium tuberculosis Technical Guide”.  The 24 locus MIRU-VNTR patterns will be provided with a dendrogram including other closely related strains from the requesting province.  The order of the loci is 0154-MIRU 02, 0580-MIRU 04, 0960-MIRU 10, 1644-MIRU 16, 2059-MIRU 20, 2531-MIRU 23, 2687-MIRU 24, 2996-MIRU 26, 3007-MIRU 27, 3192-MIRU 31, 4348-MIRU 39, 0802-MIRU 40, 0424-Mtub 04, 0577-ETR C, 1955-Mtub 21, 2163b-QUB 11b, 2165-ETR A, 2347-Mtub 29, 2401-Mtub 30, 2461-ETR B, 3171-Mtub 34, 3690-Mtub 39, 4052-QUB 26, 4156-QUB 4156.

Routine testing from culture: 30 calendar days from the date of specimen receipt. 

1. Technical guidelines can be accessed in the Multilocus Variable Number Tandem Repeat Genotyping of Mycobacterium tuberculosis Technical Guide available at the following website: http://www.miru-vntrplus.org/MIRU/files/MIRU-VNTRtypingmanualv6.pdf
2. Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, and Locht C. (2001). Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.
3. Blackwood KS, Wolfe JN, Kabani AM. (2004) Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten? J Clin Microbiol. 42:5001-6.
4. de Beer J, Kremer K, Kodmon C, Supply P, van Soolingen. (2011). First Worldwide Proficiency study on Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis complex strains. J Clin Microbiol. 50: 662-669.
5. Christianson S, Wolfe J, Orr P, Karlowsky J, Levett PN, Horsman GB, Thibert L, Tang P, Sharma MK. Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada. 2010. Tuberculosis 90:31-38