Mycobacterium – TB Susceptibility Testing

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
This page provides phenotypic drug susceptibility testing (pDST) and whole genome sequencing (WGS) information for the Mycobacterium tuberculosis complex (MTBC) at Public Health Ontario (PHO). The causative agent(s) of tuberculosis (TB) disease include the members of the MTBC, which differ from other members of the genus in that they are not known to replicate in the environment; the major ecological niche of MTBC is tissue of humans and other warm-blooded animals1. This page is for information specific to susceptibility testing and antimicrobial resistance (AMR) predictions for the MTBC. For information regarding other testing options for mycobacteria (MTBC and non-tuberculosis mycobacteria), refer to the following PHO webpages:

Real-time PCR:

Culture:

Non-tuberculosis mycobacteria:

Updates

  • Updated test information to include whole genome sequencing for complex member (variant) identification, lineage assignment, and genotypic antimicrobial resistance (AMR) predictions.

Testing Indications

In accordance with the Canadian Tuberculosis Standards, 8th edition2, pDST should be routinely performed for all first-positive-culture isolates obtained from each new TB case. WGS can be used to predict antimicrobial resistance by interrogating ≥90% of the MTBC genome and detecting resistance-associated mutations at specific regions of the genome. AMR predictions by WGS are presumptive as they do not provide direct evidence for resistance. The use of WGS does not eliminate the need for conventional culture-based pDST; pDST remains the gold standard method for determining susceptibility of the organism to a specific drug and will continue to be performed on MTBC isolates.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

Mycobacterium – TB Susceptibility Testing

M. tuberculosis complex culture

N/A

Appropriate solid or liquid media to support growth of M. tuberculosis complex

Submission and Collection Notes

1

Complete all fields of the requisition form, including:

  1. Test(s) requests and testing indications/criteria for testing
  2. Patient Setting & Signs/Symptoms
  3. Specimen Information
  4. Patient Information
  5. Submitter/Health Care Provider (HCP) Information
2

Submit viable growth of M. tuberculosis complex culture on solid or in liquid Mycobacterium culture medium.

3

Contaminated or non-viable cultures will not be tested for pDST and/or WGS.

Limitations

MTBC WGS was developed with its performance characteristics determined by Public Health Ontario for clinical testing. It has not been cleared or approved by Health Canada. The absence of mutations by genotypic AMR prediction does not exclude the possibility of other contributory mechanisms of resistance. Results must be interpreted with caution and confirmed by pDST. Refer to the current edition of the Canadian Tuberculosis Standards for recommended treatment regimens of tuberculosis disease.

In settings of high levels of additional non-tuberculosis organism present in the culture or non-viable MTBC, WGS and/or pDST may not be performed.

Storage and Transport

Label the culture container with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.
Before packaging, place culture container(s) in a biohazard bag and insert the completed requisition in the pocket on the outside of the sealed biohazard bag. Continue to incubate if transport is delayed more than one hour. All clinical specimens should be shipped to PHO’s laboratory sites as soon as possible and in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

MTBC pDST is performed daily from Monday to Friday at PHO’s laboratory-Toronto. Turnaround time (TAT) for first-line MTBC pDST is up to 14 calendar days for rifampin, isoniazid, and ethambutol and up to 21 calendar days for pyrazinamide from the date of the culture report. TAT for second-line pDST is up to 14 calendar days after the first-line pDST results are reported.

WGS of MTBC is performed weekly at PHO’s laboratory-Toronto. TAT for WGS is up to 21 calendar days from the date of the culture report.

Test Methods

pDST for the MTBC is performed using the BACTEC™ MGIT 960 method, Becton Dickinson, MD. For information on the BACTEC™ MGIT 960 see BACTEC™ MGIT 960 User’s Manual. The intended use of MTBC WGS is to provide MTBC complex member (variant) identification, lineage assignment, and genotypic predictions of antimicrobial resistance. Sequencing involves MTBC library preparation of genomic DNA, followed by a short-read WGS method. Generated sequences are processed using bioinformatics tools and analyzed using a laboratory-developed pipeline.

The first culture of MTBC isolated from a patient is routinely tested for phenotypic susceptibility and genotypic AMR predictions by WGS to each of the four first-line anti-TB drugs isoniazid, rifampin, ethambutol and pyrazinamide. Genotypic AMR predictions for moxifloxacin are also provided for the first culture of MTBC isolated from a patient. pDST and WGS are repeated at three months if cultures are still positive.

Testing details for pDST:

  • MTBC isolates resistant to INH at 0.1mg/L are also tested at a concentration of 0.4 mg/L and to moxifloxacin. As per the Clinical and Laboratory Standards Institute M24S3, moxifloxacin pDST by the critical concentration method using the BACTEC™ MGIT 960 method predicts levofloxacin susceptibility.
  • Any test that shows resistance to rifampin or any two of the first-line drugs is further tested for susceptibility to second-line drugs.
  • Second-line drugs tested at PHO’s laboratory include amikacin, capreomycin, ethionamide, kanamycin, linezolid, moxifloxacin, para-aminosalicylic acid, rifabutin, and streptomycin.
  • Intrinsic resistance to pyrazinamide is commonly observed for M tuberculosis variant bovis and bovis BCG. Refer to the current edition of the Canadian Tuberculosis Standards for recommended treatment regimens of tuberculosis disease.

Testing details for WGS:

  • WGS is not performed on MTBC isolates as a standalone orderable test.
  • MTB complex (variant) identification and lineage assignment is provided for the main phylogenetic groups, designated as lineages 1 through 9, and animal adapted strains.
  • In the setting of multiple genes analyzed for genotypic AMR predictions for a single drug, any instance of resistance-associated mutation(s) detected suggests that resistance is predicted for the drug even if other genes may have not have mutations detected.
  • In the setting of no resistance-associated mutations detected for the four first-line anti-TB drugs (isoniazid, rifampin, ethambutol and pyrazinamide) and moxifloxacin, release of genotypic AMR prediction results to second-line drugs require approval from the PHO Microbiologist.
  • Genotypic AMR predictions for second-line drugs include amikacin, capreomycin, ethionamide, kanamycin, linezolid, levofloxacin, streptomycin, clofazimine, bedaquiline, and delamanid.
  • Lower sensitivity for genotypic AMR prediction for pyrazinamide relative to other first-line anti-TB drugs is observed with WGS of MTBC. In settings where no high confidence resistance mutations are detected for the drug, resistance cannot be ruled out and should be confirmed by pDST.
  • Genotypic AMR predictions of borderline resistance rpoB mutations for rifampin and mutations conferring low increases in the minimum inhibitory concentration for ethionamide and streptomycin may not be detected by the BACTEC™ MGIT 960 method for pDST.
  • Phenotypic resistance to pyrazinamide is commonly observed for M. tuberculosis variant canettii that is not detected by genotypic AMR prediction. Confirmation by phenotypic drug susceptibility testing is recommended.

Interpretation

For Phenotypic Drug Susceptibility Testing:
pDST of first- and second-line drugs is performed using the BACTEC™ MGIT 960 and critical concentration method, i.e., the lowest concentration of drug that inhibits 95% of wildtype strains of MTBC that have never been exposed to the drug. An isolate is resistant if ≥1% of the test population grows in the presence of the critical concentration of the drug. Logarithmic growth of the test population in the presence or absence of drug over the incubation period is measured by the BACTEC™ MGIT 960 system in Growth Units (GU). Each pDST test for an isolate is deemed complete when the GU value of the control tube reaches 400. In settings where high levels of additional non-tuberculosis organism are present in the culture pDST may be cancelled.

Result Interpretation
Susceptible GU <100 in drug tube
Resistant GU ≥100 in drug tube
Indeterminate Logarithmic growth of test population not observed


For Whole Genome Sequencing:

Human-adapted MTBC is classified into nine main phylogenetic groups, designated lineage 1 through lineage 9. Lineages 1-4, and 7-9 are M. tuberculosis sensu stricto (M. tuberculosis var. tuberculosis) whereas lineages 5 and 6 are largely limited to West Africa and are traditionally referred to as Mycobacterium africanum (M. tuberculosis var. africanum). Animal adapted MTBC variants include bovis, caprae, microti, mungi, orygis, pinnipedii, suricattae, dassie, and chimpanzee; lineage designations have not been established for all animal adapted variants. M. tuberculosis var. canettii is the phylogenetically closest relative of the MTBC and does not have a lineage corresponding to the nine main phylogenetic groups (lineages 1-9)4-6. MTB complex member identification (WGS) is reported according to the most current MTBC nomenclature published in the International Journal of Systematic and Evolutionary Microbiology7. Identification of M. tuberculosis var. canettii will have a corresponding lineage result of “Not applicable”. In settings of mixed infection or multiple populations of MTBC within a sample, MTB complex member and/or lineage may not be able to be determined by WGS and will be reported as “Unable to determine variant/lineage”.

The following antimicrobial agents and gene targets are analyzed by WGS for genotypic AMR predictions and reported by PHO’s laboratory. Predictions of resistance are determined based on the World Health Organization (WHO) Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance (2nd ed)8.

Antimicrobial Agent Genes Analyzed
Isoniazid (INH) katG, inhA, ahpC
Rifampin (RIF) rpoB
Ethambutol (EMB) embB
Pyrazinamide (PZA) pncA
Moxifloxacin (MOX) gyrB, gyrA
Amikacin (AK) rrs, eis
Capreomycin (CAP) rrs, tlyA
Ethionamide (ETA) inhA, ethA
Kanamycin (KAN) rrs, eis
Linezolid (LIN) rplC, rrl
Levofloxacin (LEV) gyrB, gyrA
Streptomycin (SM) rrs, rpsL, gid
Clofazimine (CFZ) Rv0678, pepQ
Bedaquiline (BDQ) atpE, Rv0678, pepQ
Delamanid (DLM) Ddn

In settings where sufficient gene coverage and read depth is obtained by WGS, genotypic AMR prediction results will be either “Resistance predicted in gene(s) analyzed ([gene(s) specific to drug])” or “No high confidence resistance mutations detected. Cannot rule out resistance.” in the laboratory test report. Gene(s) with low coverage and/or read depth will have a result of “Unable to determine resistance prediction at gene target(s). Cannot rule out resistance” in the laboratory test report. In settings where novel and/or borderline resistance mutations are detected, the laboratory test report will indicate “Consultation with PHO Microbiologist recommended”.

Test Possible Results Comment
MTB complex member identification (WGS)

M. tuberculosis var. tuberculosis
M. tuberculosis var. africanum
M. tuberculosis var. bovis
M. tuberculosis var. bovis BCG
M. tuberculosis var. caprae
M. tuberculosis var. microti
M. tuberculosis var. pinnipedii
M. tuberculosis var. canettii
M. tuberculosis var. mungi
M. tuberculosis var. orygis
M. tuberculosis var. suricattae
M. tuberculosis var. dassie
M. tuberculosis var. chimpanzee
Unable to determine variant

Phenotypic resistance to pyrazinamide commonly observed for Mycobacterium tuberculosis variant canettii that is not detected by genomic antimicrobial resistance prediction. Confirmation by phenotypic drug susceptibility testing is recommended.
MTB complex lineage (WGS) 1 – Indo-Oceanic
2 – East Asian / Beijing
3 – East-African-Indian
4 – Euro-American
5 – West Africa
6 – West Africa
7 – Horn of Africa / Ethiopia
8 – African Great Lakes region
9 – East Africa
La1 – bovis
La2 – orygis
La3 – caprae
Not applicable
Unable to determine lineage
 
[Drug] (molecular prediction)

Resistance predicted in gene(s) analyzed ([gene(s) specific to drug]).

No high confidence resistance mutations detected. Cannot rule out resistance.

Unable to determine resistance prediction at gene target(s). Cannot rule out resistance.

Consultation with PHO Microbiologist recommended.

MTB complex genomic antimicrobial resistance prediction was developed with its performance characteristics determined by Public Health Ontario for clinical testing. It has not been cleared or approved by Health Canada. The absence of mutations does not exclude the possibility of other contributory mechanisms of resistance. Results must be interpreted with caution and confirmed by phenotypic drug susceptibility testing.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition. Specimens that are positive for Mycobacterium tuberculosis complex are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

  1. Martin, I., Pfyffer, G. E., & Parrish, N. (2023). Mycobacterium: General Characteristics, Laboratory Processing, Staining, Isolation, and Detection Procedures. Manual of Clinical Microbiology (12th Edition). Washington D.C.: American Society of Microbiology.
  2. Marcel A. Behr, Simon Grandjean Lapierre, Dennis Y. Kunimoto, Robyn S. Lee, Richard Long, Inna Sekirov, Hafid Soualhine & Christine Y. Turenne (2022) Chapter 3: Diagnosis of tuberculosis disease and drug-resistant tuberculosis, Canadian Journal of Respiratory, Critical Care, and Sleep Medicine, 6:sup1, 33-48.
  3. CLSI 2023. M24S Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes (2nd ed). Clinical and Laboratory Standards Institute. Wayne, PA, USA.
  4. Ngabonziza J, Loiseau C, Marceau M, et al. A sister lineage of the mycobacterium tuberculosis complex discovered in the african great lakes region. Nature Communications. 2020;11(2917).
  5. Napier G, Campino S, Merid Y, et al. Robust barcoding and identification of mycobacterium tuberculosis lineages for epidemiological and clinical studies. Genome Med. 2020;12(1):114-020-00817-3.
  6. Coscolla M, Gagneux S, Menardo F, et al. Phylogenomics of mycobacterium africanum reveals a new lineage and a complex evolutionary history. Microb Genom. 2021;7(2):10.1099/mgen.0.000477.
  7. 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. 2018 Jan;68(1):324-332.
  8. World Health Organization. (‎2023)‎. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, 2nd ed. World Health Organization. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, 2nd ed. License: CC BY-NC-SA 3.0 IGO.
Updated 3 Feb 2025