Bedaquiline, Levofloxacin and Clofazimine Susceptibility Testing

Susceptibility of Mycobacterium tuberculosis or non-tuberculous mycobacterium (NTM) isolates to bedaquiline, levofloxacin, and clofazimine by broth microdilution method as as described by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Note that clofazimine MIC testing for NTM species is included in our standard AST panels.

Solid or liquid media growth of pure culture.

• For solid media growth, isolated colonies on plated media.
• • Mycobacterium tuberculosis complex (MTBC) species must show visible growth and be no more than 4 weeks old. 
  • Rapidly growing mycobacterium species must be no more than 10 days old
  • Slowly growing mycobacterium species mustbe 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

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.

Tuberculosis is a reportable disease and susceptibility testing for all cases in Canada is mandatory.

For NTMs: Respiratory samples must meet the ATS recommendations for clinical significance. For non-respiratory samples, the CLSI guidelines state, “susceptibility testing of NTM should be considered only for those isolates believed to be clinically significant. Isolates from blood, other sterile body fluids or tissues are generally considered clinically significant”. Justification and clinical history must be provided with the requisition form. Samples submitted for bedaquiline testing must be clinically significant AND isolates must be from patients who develop bacteremia while on macrolide therapy or from patients who relapse while on macrolide therapy or other NTM cases with clinical justification. Testing will be performed when physician intends to use bedaquiline for treatment. 

Notification of sample submission must be emailed or faxedprior to shipping isolates.A requisition for the NRCM and/or the ‘Susceptibility Testing of Bedaquiline, Clofazimine and Levofloxacin for Mycobacterium tuberculosis isolates’ requisition 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, and identification. 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.

The testrequest must be accompanied by MTBC first and/or second line susceptibility testing results OR justification as to why testing is required. Cultures will be rejected if appropriate documentation and justification is incomplete or missing.

Testing is performed on MTBDST panels (Thermofisher). There are currently no CLSI guidelines for the interpretation of bedaquiline, levofloxacin, or clofazimine MICs for MTBC or NTMs. MIC values for these antibiotics will be provided without interpretations (1). Bedaquiline susceptibility testing for NTMs is currently being validated by the NRCM and results should be considered experimental.

The specimen turnaround time is 45 calendar days from the date of specimen receipt. This TAT includes sample processing and culture growth times. 

1.CLSI. Susceptibility Testing of Mycobacteria, Nocardia and Other Aerobic Actinomycetes. 3^rd ed. CLSI standard M24. Wayne, PA: Clinical and Laboratory Standards Institute; 2023.

2.Inderlied CB and Pfyffer GE.  (2003)  Susceptibility Test Methods: Mycobacteria, p. 1149 – 77.  In Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, and Yolken RH (ed).  Manual of Clinical Microbiology, 8th ed.  American Society for Microbiology, Washington, D.C.

3.Sabine Rüsch-Gerdes, Gaby E. Pfyffer, Manuel Casal, Maureen Chadwick, and Salman Siddiqi. 2006. Multicenter Laboratory Validation of the BACTEC MGIT 960 Technique for Testing Susceptibilities of Mycobacterium tuberculosis to Classical Second-Line Drugs and Newer Antimicrobials. J. Clin. Microbiol. 44: 688-692.

4.Gaby E. Pfyffer, Donald A. Bonato, Adeleh Ebrahimzadeh, Wendy Gross, Jacqueline Hotaling, John Kornblum, Adalbert Laszlo, Glenn Roberts, Max Salfinger, Franziska Wittwer, and Salman Siddiqi. 1999. Multicenter Laboratory Validation of Susceptibility Testing of Mycobacterium tuberculosis against Classical Second-Line and Newer Antimicrobial Drugs by Using the Radiometric BACTEC 460 Technique and the Proportion Method with Solid Media. J. Clin. Microbiol. 37: 3179-3186.

5.Annika Krüüner, Malcolm D. Yates, and Francis A. Drobniewski. 2006 Evaluation of MGIT 960-Based Antimicrobial Testing and Determination of Critical Concentrations of First- and Second-Line Antimicrobial Drugs with Drug-Resistant Clinical Strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 44: 811-818.

6.M. K. Sharma, Thibert L., Chedore P., Shandro C., Jamieson F., Tyrrell G., Christianson S., Soualhine H., Wolfe J. (2011).  A Canadian Multicentre Laboratory Study for Standardized Second-line Antimicrobial Susceptibility testing of Mycobacterium tuberculosis.  J. Clin. Microbiol. 49(12): 4112-6.