Evaluating extraction-free PCR for rapid detection of beta-lactam antibiotic resistance in urinary tract infection

The rapid detection of beta-lactam antibiotic resistance is crucial for guiding effective antimicrobial therapy and controlling the spread of resistant bacterial strains. CTX-M Group 1 extended-spectrum beta-lactamases (ESBLs) are among the most prevalent resistance determinants in Gram-negative bacteria, particularly Escherichia coli and Klebsiella pneumoniae, which are major causes of urinary tract infections (UTIs). Conventional molecular diagnostic methods for detecting CTX-M genes rely on nucleic acid extraction before polymerase chain reaction (PCR) amplification. However, these processes are time-consuming, labor-intensive, and resource-intensive, limiting their accessibility in low-resource and high-throughput laboratory settings. This study evaluates Direct-to-PCR (D2P) extraction-free technology as an alternative to traditional extraction-based methods for detecting CTX-M Group 1 genes. A comparative analysis was conducted using reference microbial isolates and clinical urine samples, testing D2P alongside silica column- and magnetic bead-based extraction methods. Quantitative PCR results demonstrated that D2P achieved comparable sensitivity and specificity to traditional extraction methods while significantly reducing sample processing time and cost. Statistical analysis revealed no significant differences (p > 0.05) in cycle threshold (Ct) values between D2P and conventional extraction-based methods, supporting its feasibility as a rapid, cost-effective alternative. The findings suggest that D2P technology may enhance antibiotic resistance surveillance, clinical diagnostics, and infection control programs by enabling faster, extraction-free molecular detection of ESBL-producing pathogens. Further studies should assess its performance in diverse sample matrices and clinical settings.