Test Id : TBPZA

If resistance to pyrazinamide is detected, the reflex sequencing test or confirmation of resistance will be performed at an additional charge.

If the broth microdilution test is unable to be performed (eg, due to reagent shortages, failure of the isolate to grow in the test medium), the reflex sequencing test will be performed at an additional charge.

To test for first-line agents, isoniazid, rifampin and ethambutol, order TB1LN / Antimicrobial Susceptibility, Mycobacterium tuberculosis Complex, First Line, Varies.

Isolates identified as Mycobacterium bovis and Myocobacterium bovis bacille Calmette-Guerin (BCG) will not be accepted as they are intrinsically resistant to pyrazinamide.

If organism identification is not provided, CTB / Mycobacteria and Nocardia Culture, Varies or CTBID / Culture Referred for Identification, Mycobacterium and Nocardia, Varies must also be ordered and will be charged separately.

1. See Infectious Specimen Shipping Guidelines

2. Place specimen in a large infectious container (T146) and label as an etiologic agent/infectious substance.

Specimen source and suspected organism identification are required.

Specimen Type: Organism

Supplies: Infectious Container, Large (T146)

Container/Tube: Middlebrook 7H10 agar slant

Specimen Volume: Isolate

Collection Instructions: Organism must be in pure culture, actively growing.

• Infectious Specimen Shipping Guidelines

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.

Susceptibility testing of Mycobacterium tuberculosis complex isolates growing in pure culture against pyrazinamide

This test is not useful for Mycobacterium bovis and Myocobacterium bovis bacille Calmette-Guerin (BCG) isolates as they are intrinsically resistant to pyrazinamide.

If resistance to pyrazinamide is detected, the reflex sequencing test or confirmation of resistance will be performed at an additional charge.

If the broth microdilution test is unable to be performed (eg, due to reagent shortages, failure of the isolate to grow in the test medium), the reflex sequencing test will be performed at an additional charge.

Initial treatment regimens for Mycobacterium tuberculosis complex often include isoniazid, rifampin, ethambutol, and pyrazinamide (PZA). Susceptibility testing of M tuberculosis complex isolates against these antimycobacterial agents is a key component of patient management.

The Clinical and Laboratory Standards Institute provides consensus protocols for the methods, antimycobacterial agents, and critical concentrations of each agent to be tested to permit standardized interpretation of M tuberculosis complex susceptibility test results.

This test uses a US Food and Drug Administration cleared commercial system for rapid broth susceptibility testing of M tuberculosis complex against PZA. The literature indicates that broth testing of PZA can, at times, produce falsely resistant results, so resistance to PZA by the broth method is automatically confirmed by pncA DNA sequencing. Variants in the pncA gene and upstream promoter region have been reported to account for the majority (70%-97%) of PZA-resistant isolates. However, 3% to 30% of PZA-resistant isolates do not have a corresponding pncA variant and other genes (eg, rpsA) may also play a role.

A separate test is available for testing of the other primary agents (isoniazid, rifampin and ethambutol). If desired, this must be ordered separately; TB1LN / Antimicrobial Susceptibility, Mycobacterium tuberculosis Complex, First Line, Varies.

Results are reported as susceptible or resistant.

Mycobacterium tuberculosis complex isolates are reported as susceptible or resistant to pyrazinamide at the critical concentration.

For isolates determined to be resistant by the broth method, confirmatory testing using pncA DNA sequencing is automatically performed and the presence or absence of pncA variations associated with pyrazinamide resistance is reported. Some variants associated with pyrazinamide resistance may occur outside of the pncA promoter and gene region and may therefore not be confirmed by DNA sequencing of this target. Presence of a pncA mutation associated with resistance confirms the broth susceptibility testing results. Absence of a pncA mutation does not rule out possible resistance.

Susceptibility testing should be performed on pure culture isolates of Mycobacterium tuberculosis complex.

1. Nahid P, Mase SR, Migliori GB, et al. Treatment of Drug-Resistant Tuberculosis. An Official ATS/CDC/ERS/IDSA Clinical Practice Guideline [published correction appears in Am J Respir Crit Care Med. 2020 Feb 15;201(4):500-501

2. Dormandy J, Somoskovi A, Kreiswirth BN, Driscoll JR, Ashkin D, Salfinger M. Discrepant results between pyrazinamide susceptibility testing by the reference BACTEC 460TB method and pncA DNA sequencing in patients infected with multidrug-resistant W-Beijing Mycobacterium tuberculosis strains. Chest. 2007;131(2):497-501. doi:10.1378/chest.06-1899

3. Chedore P, Bertucci L, Wolfe J. Potential for erroneous results indicating resistance when using the BACTEC MGIT 960 system for testing susceptibility of mycobacterium tuberculosis to Pyrazinamide. J Clin Microbiol 2010;48(1):300-301

4. Campbell PJ, Morlock GP, Sikes RD, et al. Molecular resistance of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2011;55(5):2032-2041

5. Shi W, Zhang X, Jiang X, et al. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 2011;333(6049):1630-1632

6. Clinical and Laboratory Standards Institute (CLSI). Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes. 3rd ed. CLSI standard M24. CLSI; 2018

7. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes. 2nd ed. CLSI supplement M24S. CLSI; 2023

8. Espasa M, Salvado M, Vicente E, et al. Evaluation of the versaTREK system compared to the Bactec MGIT 960 system for first-line drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol. 2012;50:488-491

9. Somoskovi A, Dormandy J, Parson LM, et al. Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA mutation in "Mycobacterium canettii" and the reliable and rapid predictor of pyrazinamide resistance. Confirmation of pyrazinamide resistance is done using Sanger dideoxy sequencing of approximately 700bp of the pncA gene and promoter region. J Clin Microbiol. 2007;45(2):595-599

10. Jureen P, Werngren J, Toro JC, Hoffner S: Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2008;52(5):1852-1854

This test is based on presence or absence of growth of an Mycobacterium tuberculosis complex isolate in broth cultures in the presence of critical concentrations of the antimycobacterial drug pyrazinamide. One of two FDA-cleared platforms (BD MGIT 960 or the VersaTREK) will be used. (Brown-Elliott, BA, Cirillo DM, Musser KA, Rowlinson M-C. Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes. In: Carroll KC, Pfaller MA, eds. Manual of Clinical Microbiology, 13th Edition. ASM Press, 2023)

The BACTEC MGIT 960 platform measures fluorescence in a Mycobacterial Growth Indicator Tube (MGIT) containing an M tuberculosis complex isolate in the presence of a critical concentrations of pyrazinamide (Package insert: BACTEC MGIT 960 SIRE Kit, BD Diagnostics, 9/2019)

The VersaTrek platform inside bottles containing M tuberculosis complex isolates in the presence of a critical concentration of the antimycobacterial PZA. (Package insert: VersaTREK Mycobacteria Detection and Susceptibility Testing system, TREK Diagnostics, 04/2014)

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This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

87188-Susceptibility, Mycobacterium tuberculosis Complex, Pyrazinamide

87153-Mtb PZA Confirmation, pncA Sequencing (if appropriate)