Test ID: MTBPZ
Mycobacterium tuberculosis Complex, Pyrazinamide Resistance by pncA DNA Sequencing

Detection of genotypic resistance to pyrazinamide by Mycobacterium tuberculosis complex isolates

• Infectious Specimen Shipping Guidelines

DNA Sequencing

Specimen Type: Organism

Container/Tube: Middlebrook 7H10 agar slant

Specimen Volume: Isolate

Collection Instructions:

1. Organism must be in pure culture, actively growing.

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

Additional Information:

1. Specimen source and suspected organism identification are required.

2. See Infectious Specimen Shipping Guidelines in Special Instructions for shipping information.

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.

Pyrazinamide resistance not detected

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."

Wild-type 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.

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.

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 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

(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 FDA-approved, rapid broth method(VersaTREK, TREK Diagnostic Systems, Cleveland, OH) 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, Sparks, MD), 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. The 2 x 2 table for pncA sequencing versus arbitrated broth susceptibility testing is shown below in Table 2.

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

(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

nt=nucleotide

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 wild-type (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 single nucleotide polymorphism (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, Foster City, CA) and no interoperator or interinstrument differences in performance were noted.

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;45:595-599

2. Dormandy J, Somoskovi A, Kreiswirth BN, et al: 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;131:497-501

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

Organisms identified as Mycobacterium tuberculosis complex using the Mycobacterium tuberculosis AccuProbe(GenProbe, San Diego, CA) are lysed using the PrepMan Ultra lysis buffer. Using the pncA primers described by Shenai and colleagues, an approximately 700 bp-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 wild-type pncA sequence using MicroSeq Microbial ID software (v2.0). A custom library of non-wild-type 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, et al: Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing. Indian J Tuberc 2009;56:82-90)

1 day per week, Varies

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