Evaluation of a simplified agar-based population analysis profiling as a practical alternative for detecting heteroresistance in clinical bacterial isolates

Rapid and accurate detection of antibiotic heteroresistance (HR) causing clinical bacterial infections is essential for prompt and effective treatment. However, current detection methods vary across laboratories. This study aimed to evaluate a Simplified Agar-based Population Analysis Profiling (SA-PAP) method as a practical alternative to the reference Population Analysis Profiling (PAP) for HR detection, and to describe the screening performance of a modified E-test. We collected 56 carbapenem-resistant Escherichia coli (CREC) and 305 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. HR to tigecycline (in CREC), vancomycin, and teicoplanin (in MRSA) was assessed using the modified E-test, SA-PAP, and PAP (reference method). The screening performance of the modified E-test was evaluated descriptively, while the diagnostic agreement of SA-PAP with PAP was assessed using Cohen’s kappa. By PAP, HR prevalence was 37.5% (21/56) to tigecycline in CREC, and 34.1% (104/305) and 53.8% (163/305) to vancomycin and teicoplanin in MRSA, respectively. Compared to PAP, the modified E-test showed variable and lower sensitivities (66.7% for CREC/tigecycline, 70.2% for MRSA/vancomycin, and 48.8% for MRSA/teicoplanin), indicating a substantial risk of false-negative results. In contrast, SA-PAP demonstrated excellent agreement (Cohen’s kappa > 0.90 for all combinations) with sensitivities of 100%, 100%, and 99.4%, and specificities of 97.1%, 95.0%, and 97.9%, respectively. Heteroresistance is highly prevalent among clinical CREC and MRSA isolates, underscoring the need for its detection. SA-PAP offers a reliable, simpler, and more economical alternative to the reference PAP method for HR confirmation. The modified E-test may serve as a rapid initial screen but has significant limitations due to its suboptimal sensitivity. Laboratories should consider adopting SA-PAP for accurate HR identification.