Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis

Area of Science:

• Microbiology
• Genetics
• Infectious Diseases

Background:

• Pyrazinamide (PZA) is crucial for treating tuberculosis (TB).
• PZA resistance is primarily linked to mutations in the pncA gene.
• Current drug susceptibility testing for PZA requires precise pH control, limiting its reliability.

Purpose of the Study:

• To develop and evaluate a rapid melting curve assay for detecting pncA gene mutations associated with PZA resistance.
• To assess the assay's performance on clinical Mycobacterium tuberculosis isolates.

Main Methods:

• A melting curve assay was designed to target specific regions of the pncA gene.
• The assay was validated using 271 Mycobacterium tuberculosis complex isolates from Swaziland.
• Genotyping for pncA mutations was performed and compared to phenotypic susceptibility data.

Main Results:

• The assay can detect non-wild type pncA genotypes in as little as 2.5 hours with a detection limit of 3750 gene copies/mL.
• Sensitivity was 83% (95% CI 75-89%) and specificity was 100% (95% CI 98-100%) in identifying pncA mutations.
• The assay correctly identified 118 isolates with known pncA mutations.

Conclusions:

• The melting curve assay shows potential as a rapid rule-in test for PZA susceptibility by identifying wild-type pncA.
• The assay's high specificity suggests it can reliably rule out PZA resistance in most cases.
• False resistant results (mutant pncA but susceptible to PZA) are possible and require further investigation for complete PZA susceptibility assessment.