Test Catalog

Test Id : MTBPZ

Mycobacterium tuberculosis Complex, Pyrazinamide Resistance by pncA DNA Sequencing, Varies

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detection of genotypic resistance to pyrazinamide by Mycobacterium tuberculosis complex isolates

Reflex Tests
Lists tests that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial tests.

Test Id Reporting Name Available Separately Always Performed
MTBVP Mtb PZA Confirmation, pnc A Sequence No, (Bill Only) No

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

When this test is ordered, the reflex test may be performed and charged.

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Name
A short description of the method used to perform the test

DNA Sequencing

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.


Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Mtb PZA Resistance, pncA Sequencing

Lists additional common names for a test, as an aid in searching

(AFB) Acid-Fast Bacilli

Acid-Fast Bacilli (AFB)

Antibiotic Susceptibility

Antimicrobial Susceptibility, Mycobacterium tuberculosis

Bacillus, Acid-Fast

MTB (Mycobacterium tuberculosis)

Mycobacterium tuberculosis (MTB)

Mycobacterium tuberculosis Complex Susceptibility-Broth

Susceptibility Testing

Susceptibility, Mycobacterium tuberculosis

TB (Tuberculosis)

Tubercle Bacilli: Mycobacterium tuberculosis

Tuberculosis (TB)

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

When this test is ordered, the reflex test may be performed and charged.

Specimen Type
Describes the specimen type validated for testing


Shipping Instructions

1. See Infectious Specimen Shipping Guidelines.

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

Necessary Information

Specimen source and suspected organism identification are required.


Question ID Description Answers
Q00M0041 Specimen Source (Required) and Organism Identification (Required unless concurrent identification test is ordered)

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

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.

Special Instructions
Library of PDFs including pertinent information and forms related to the test


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

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Agar plate Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Varies Ambient (preferred)

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detection of genotypic resistance to pyrazinamide by Mycobacterium tuberculosis complex isolates

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

When this test is ordered, the reflex test may be performed and charged.

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

The protein product of the Mycobacterium tuberculosis complex pncA gene is an enzyme that is responsible for activation of the prodrug pyrazinamide (PZA). DNA sequencing of the Mycobacterium tuberculosis complex pncA gene can be used to detect mutations that correlate with in vitro PZA resistance.(1,2) The sequencing result can be available in as little as 1 day after the Mycobacterium tuberculosis complex isolate grows in culture, thereby providing a more rapid susceptibility result than the average 10 to 14 days required by phenotypic broth methods.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

Pyrazinamide resistance not detected

Provides information to assist in interpretation of the test results

Polymorphisms in the pncA gene that have been previously correlated in our laboratory with pyrazinamide (PZA) resistance will be reported as "Mutation was detected in pncA suggesting resistance to pyrazinamide."


Wildtype pncA or a silent pncA gene polymorphism (ie, no change in the amino acid translation) will be reported as "No mutation was detected in pncA."


New polymorphisms in the pncA gene that have not previously been seen in our laboratory will require additional testing using a reference broth method to determine their correlation with PZA resistance.

Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

According to the literature,(3) 72% to 97% of pyrazinamide (PZA)-resistant clinical isolates carry mutations in the pncA gene or promoter region. However, other resistance mechanisms (eg, changes in PZA uptake or increased PZA efflux) will not be detected by this method.  


Correlation of the in vitro sequencing result with clinical presentation is strongly recommended.

Supportive Data

The correlation between pncA sequencing results and in vitro broth susceptibility test results was evaluated using 21 reference strains of Mycobacterium tuberculosis complex with known broth susceptibility profiles. Nine of 21 isolates were from the American Type Culture Collection (ATCC) and 12 of 21 isolates were from completed and closed Proficiency Testing (PT) testing events from the Center for Disease Control and Prevention (CDC), the College of American Pathologists (CAP), or the New York State Department of Health. Isolates demonstrating a polymorphism by sequencing were resequenced and all isolates had identical results between the first and second sequencing evaluation. Results are presented in Table 1.

Table 1. Accuracy of pncA Sequencing for Reference/PT Isolates


ATCC or PT Isolate broth susceptibility result

% Categorical agreement



pncA wild-type or silent SNP(a)




pncA polymorphisms



(a)SNP=single nucleotide polymorphism; see Table 3 for a description of the silent SNPs detected; a silent SNP does not result in an amino acid change.


pncA sequencing was also compared to a US Food and Drug Administration (FDA)-approved, rapid broth method(VersaTREK, TREK Diagnostic Systems) for 141 Mycobacterium tuberculosis complex isolates consisting of 96 clinical isolates and 45 reference strains (ATCC and closed PT). Any discordant results were resolved by additional testing using either the BACTEC 460 or BACTEC MGIT 960 broth methods (Becton Dickinson), which are also FDA-approved. Any isolate that had a polymorphism or that had a sequencing result that did not correlate with the broth susceptibility testing result was resequenced and identical results were found for all isolates between the first and second sequencing run. See Table 2 for pncA sequencing versus arbitrated broth susceptibility testing().


Table 2. Accuracy of pncA Sequencing vs Arbitrated Broth Susceptibility Testing

Sequencing result

Arbitrated(a) broth susceptibility testing result

% Categorical agreement



pncA wild-type or a silent SNP




pncA polymorphisms



(a) for 30 isolates with discrepant VersaTREK broth and pncA sequencing results, a second broth method (either BACTEC MGIT 960 or BACTEC 460TB) was performed to determine whether the VersaTREK or sequencing result was correct.

-Sensitivity versus arbitrated broth methods=102/102 x 100=100%

-Specificity vs arbitrated broth methods=39/39 x 100=100%

-Very major error rate=0%

-Major error rate=0%


Table 3 provides a list of the pncA polymorphisms found in the validation of this method.


Table 3. pncA Nucleotide Polymorphisms Detected In House During Validation

Nucleotide position(S) in pncA coding region

Codon change

Amino acid change

Pyrazinamide broth susceptibility result





106 and 107

GC insertion
































1 nt deletion








1 nt deletion
































7 nt deletion




1 nt deletion




Silent SNPs were seen at nt positions 195, 222, 306, 408


Interday precision was evaluated by sequencing Mycobacterium tuberculosis (ATCC 27294, also known as H37Rv, PZA susceptible), Mycobacterium bovis (ATCC 19210, PZA resistant), and water (negative control) 12 times over 10 days. Mycobacterium tuberculosis ATCC 27294 gave a 100% match to the wildtype (wt) pncA sequence 12 of 12 times with good specimen quality scores (> or =37) and an average consensus length of 682 + /-15 bases. Similarly, Mycobacterium bovis ATCC 19210 had a SNP present at pncA amino acid position 169, which is consistent with published literature reports for this organism. The 169 SNP was seen 12 of 12 times with good specimen quality scores (> or =40) and an average consensus length of 701 +/-9 bases. Interday precision was done by 2 operators using 2 ABI sequencers (Applied Biosystems) and no interoperator or interinstrument differences in performance were noted.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

1. 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. J Clin Microbiol. 2007;45(2):595-599

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 multi-drug resistant W-Beijing Mycobacterium tuberculosis strains. Chest. 2007;131(2):497-501

3. Somoskovi A, Parson LM, Salfinger M: The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res. 2001;2(3):164-168

4. Bouzouita I, Cabibbe AM, Trovato A, Draoui H, Ghariani A, Midouni B, Essalah L, Mehiri E, Cirillo DM, Slim-Saidi L. Is sequencing better than phenotypic tests for the detection of pyrazinamide resistance? Int J Tuberc Lung Dis. 2018 Jun 1;22(6):661-666. doi:10.5588/ijtld.17.0715

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Description
Describes how the test is performed and provides a method-specific reference

Organisms identified as Mycobacterium tuberculosis complex using the Mycobacterium tuberculosis AccuProbe (GenProbe) are lysed using the PrepMan Ultra lysis buffer. Using the pncA primers described by Shenai and colleagues, an approximately 700 base pair-polymerase chain reaction (PCR) product is generated that flanks the entire pncA gene and the upstream promoter region. The PCR product is cleaned and sequenced using the Big Dye terminator v 1.1 Cycle Sequencing reagents (Applied Biosystems). Results are analyzed versus the wildtype pncA sequence using MicroSeq Microbial ID software. A custom library of non-wildtype sequences was constructed in MicroSeq. An exact match to the custom nucleotide library is required to report the result.(Shenai S, Rodrigues C, Sadani M, Sukhadia N, Mehta A: Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing. Indian J Tuberc. 2009;56(2):82-90)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information


Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.


Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

7 to 21 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

1 year

Performing Laboratory Location
Indicates the location of the laboratory that performs the test


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Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

87153-Mtb PZA Confirmation, pncA Sequence

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
MTBPZ Mtb PZA Resistance, pncA Sequencing 46245-7
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
MTBPZ Mtb PZA Resistance, pncA Sequencing 46245-7

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

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Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports