Mycobacterium – MTBC and Rifampin/Isoniazid Resistance PCR

Background
This page provides real-time PCR-based diagnostic testing information for Mycobacterium tuberculosis complex (MTBC) and mutations associated with resistance to rifampin (RIF) and isoniazid (INH) at Public Health Ontario (PHO). The MTBC is the causative agent(s) of tuberculosis (TB) disease and these organisms 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 animals.¹ For information regarding other testing options for mycobacteria (MTBC and non-tuberculosis mycobacteria) culture and phenotypic susceptibility testing, refer to the following PHO webpages:

Culture:

Susceptibility Testing:

Updates
Real-time PCR testing for Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) detection is now discontinued and replaced by the BD MAX™ MDR-TB Assay to simultaneously detect MTBC DNA and mutations associated with resistance to two first-line anti-TB drugs, rifampin (RIF) and isoniazid (INH). See “Testing Methods” for more detailed information.

*Uncontrolled print copy. Valid only on day of print: 17 July 2024.

Testing Indications

In accordance with the Canadian Tuberculosis Standards, 8^th edition, it is recommended that in all new smear positive patients, at least one acid-fast bacilli positive respiratory sample be tested with a Health Canada-approved or –validated laboratory-developed nucleic acid amplification test (NAAT).² In people with signs and symptoms of pulmonary TB, moderate complexity automated NAATs may be used on respiratory specimens for detection of pulmonary TB, RIF resistance and INH resistance, rather than culture and phenotypic drug-susceptibility testing (DST).³

Acceptance/Rejection Criteria

Frozen specimens and blood will not be tested.
Fresh specimens or concentrate mixed with fixative will not be tested.
FFPE samples submitted without the required components and information as indicated under Specimen Collection and Handling will not be tested, and will be returned to the submitting laboratory.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Fresh respiratory, body fluids, gastric lavage, abscess contents/aspirate	5.0 ml	Tuberculosis Kit Order#: 390042
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Fresh tissue or bone biopsy, skin lesions, faeces	1.0 gram	Tuberculosis Kit Order#: 390042
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Fresh CSF	2.0 ml	Tuberculosis Kit Order#: 390042
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Fresh urine	40.0 ml	Tuberculosis Kit Order#: 390042
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Concentrated Specimens	0.75 ml	Sterile screw-capped tube
MTBC and Rifampin /Isoniazid Resistance PCR	General Test Requisition	Formalin Fixed Paraffin Embedded (FFPE) block (see note 4 & 5) and H&E stained slide (see note 5)	N/A	N/A

Submission and Collection Notes

Complete all fields of the requisition form, including:

Test(s) requests and testing indications/criteria for testing
Patient Setting & Signs/Symptoms
Specimen Information
Patient Information
Submitter/Health Care Provider (HCP) Information

Collect fresh specimens using aseptic technique in sterile containers without fixative. Frozen specimens will not be tested. Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Acid-fast bacilli (AFB) smear negative specimens will be tested only upon request and must be requested within 72 hours of the date and time the specimen was received to ensure specimen integrity. Requests should be considered for patients in whom tuberculosis is highly likely and have significant risk factors for tuberculosis. A negative result cannot be used to “rule out” tuberculosis. The physician must contact the Microbiologist, the laboratory’s Operational Lead, or PHO Customer Service Centre for approval of the assay. Requests written on the requisition will not be accepted unless prior approval is obtained.

FFPE samples should only be submitted when there is a strong suspicion of TB disease clinically and epidemiologically, and there is evidence observed on histopathological examination of the tissue (e.g. caseating/necrotizing granulomata visualized on histopathology or acid-fast organisms seen on ZN staining). This information should be part of the histopathology report. A copy of the full histopathology report including microscopic findings must be submitted together with the requisition and FFPE sample. Fresh tissue is the specimen of choice, FFPE samples should only be submitted if fresh tissue is not available.

For corresponding H&E stained slide(s) accompanying an FFPE sample, mark on the slide the suspected area (e.g. granulomatous inflammation, caseating/necrotizing granulomata, organisms visualized by ZN etc.) to improve the potential for molecular detection of MTBC DNA. The FFPE sample and slide will be returned to the submitter after testing has been completed. A core biopsy may be used in its entirety for testing. Please identify TB as priority if necessary.

Timing of Specimen Collection

Freshly collected specimens or processed concentrates must be sent to PHO’s laboratory within 72 hours from the time of collection to maintain sample integrity.

Limitations

A negative PCR result from smear negative specimens and FFPE samples does not exclude the possibility of TB disease. FFPE samples are not recommended for testing due to DNA fragmentation and should only be submitted when fresh tissue is not available.

Storage and Transport

Fresh specimens should be stored at 2-8°C following collection or concentration if transport is anticipated to be delayed for more than one hour. Before packaging, place specimen container(s) in a biohazard bag and insert the completed requisition in the pocket on the outside of the sealed biohazard bag. Arrange shipment of specimens to PHO’s laboratory as soon as possible. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Real-time PCR for detection of MTBC and RIF/INH resistance is performed daily, Monday to Friday (excluding holidays) at PHO’s Toronto laboratory.

Turnaround time (TAT) for fresh specimens is up to 4 days from receipt at PHO’s laboratory. TAT for FFPE samples may take up to 10 days from receipt at PHO’s laboratory.

Test Methods

The BD MAX™ Multi Drug Resistant Tuberculosis (MDR-TB) multiplex real-time PCR assay is performed on AFB smear positive specimens from new, untreated patients. Repeat testing is conducted on AFB smear positive specimens from patients who have had no AFB positive cultures for the past three years. A patient with a treated and resolved past history of TB may give a positive test result for several years post treatment and/or may give a negative result after a short period of patient treatment.

This test detects MTBC DNA in specimen concentrates (exception of CSF). This test contains a multicopy and single copy genomic target for MTBC DNA detection. In specimens where MTBC DNA is detected, the test detects mutations in the rifampin resistance determining region (RRDR) of the rpoB gene associated with rifampin resistance and mutations in the katG gene and inhA promoter region, both of which are associated with isoniazid resistance. This test has been validated by PHO for respiratory and non-respiratory specimens including tissue, body fluids, CSF, urine, stool, and FFPE specimens. The absence of RIF and INH resistance detection does not exclude the possibility of other contributory mechanisms of resistance. Results must be interpreted with caution and confirmed by phenotypic drug susceptibility testing.

This test targets DNA from viable and non-viable bacteria, and cannot be used to monitor the progression or success of treatment of patients with anti-TB therapy, or whether a patient may be infectious. Due to differences in sensitivity among the targets used for the detection of MTBC DNA and rifampin and isoniazid resistance mutations, recollection of specimens for the purpose of molecular detection of resistance is not recommended and should be determined by culture and phenotypic drug susceptibility testing.

Results are qualitative (i.e., “Detected” or “Not Detected”) and cannot be used to determine bacillary load.

Interpretation

The following table provides possible test results with associated interpretations:

MTB complex by Real-Time PCR Result	Rifampin and Isoniazid resistance by Real-Time PCR Result(s)	Comments
MTBC DNA Detected	Not Detected	Results should be interpreted in the context of patient clinical history and other findings.
MTBC DNA Detected	Detected	Results should be interpreted in the context of patient clinical history and other findings. Molecular detection of determinants of drug resistance is presumptive; results must be confirmed by phenotypic drug susceptibility testing.
MTBC DNA Detected	Unable to detect resistance to [rifampin] and/or [isoniazid] due to poor amplification or insufficient DNA in specimen	Results should be interpreted in the context of patient clinical history and other findings.
MTBC DNA Not Detected	Not tested	Results should be interpreted in the context of patient clinical history and other findings.
Indeterminate result due to inhibition	Not tested	Repeat collection and testing is recommended if clinically indicated.
Indeterminate result which may be due to interfering substances in specimen or test run failure		Repeat collection and testing is recommended if clinically indicated.

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 with molecular detection of Mycobacterium tuberculosis complex are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

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.
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.
WHO consolidated guidelines on tuberculosis, Module 2: Screening, Systematic Screening for tuberculosis results. Accessed from: https://tbksp.org/en/node/4 on June 2, 2024