Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing

Area of Science:

• Microbiology
• Genetics
• Medical Diagnostics

Background:

• Pyrazinamide (PZA) is a crucial first-line drug for tuberculosis (TB) treatment.
• Accurate PZA susceptibility testing is essential for effective TB management and preventing drug resistance.
• The pncA gene is frequently associated with PZA resistance in Mycobacterium tuberculosis complex (MTBC).

Purpose of the Study:

• To compare the performance of the phenotypic MGIT 960 TB system with enzymatic (Wayne assay) and genotypic (pncA sequencing) methods for PZA susceptibility testing.
• To determine the prevalence of Mycobacterium bovis in PZA-resistant MTBC isolates.
• To identify novel mutations in the pncA gene associated with PZA resistance.

Main Methods:

• Phenotypic PZA susceptibility testing was performed using the MGIT 960 TB system.
• Enzymatic activity of pyrazinamidase was assessed using the Wayne assay.
• The pncA gene was amplified by PCR and sequenced to detect mutations.
• Spoligotyping was used to identify M. bovis in PZA-resistant isolates.

Main Results:

• The MGIT 960 system demonstrated higher concordance with pncA gene sequencing results compared to the Wayne assay.
• Of 33 PZA-resistant isolates by MGIT 960, 31 were Wayne assay negative.
• PncA gene sequencing identified diverse mutations in 32 of 33 resistant isolates, with 15 novel mutations reported; no M. bovis was detected.

Conclusions:

• The MGIT 960 system is a reliable method for PZA susceptibility testing, showing better agreement with genotypic analysis.
• A significant proportion (85%) of multidrug-resistant TB (MDR-TB) isolates tested were resistant to PZA.
• The study highlights the importance of molecular methods for accurate PZA resistance detection and the growing challenge of PZA resistance in MDR-TB.