Comparative diagnostic evaluation of real-time PCR and culture for detecting pathogens in podiatric wound infections

Culture-based wound infection diagnostics have well-recognized limitations in detecting fastidious, anaerobic, or polymicrobial pathogens. This study compared the performance of a PCR wound panel with traditional culture. Dual-swab specimens from 93 clinical wound cases underwent both culture and PCR testing. Analyses included organism-level concordance, Gram stain correlation, and resistance gene detection. Logistic regression, receiver operating characteristic (ROC), and latent class analysis (LCA) were used to evaluate diagnostic performance. Under the conventional (culture-referenced) framework, PCR sensitivity was 98.3% and specificity was 73.5%. LCA (model M3) estimated PCR sensitivity at 95.6% and specificity at 91%. PCR detected 110 clinically significant pathogens missed or ambiguously reported (~30%) by culture. A logistic model incorporating <i>16S rRNA</i> Ct values and Gram morphology found that 73.8% of PCR-only detections had infection probabilities >75%. Applying these probabilities and resistance gene codetection data in a symmetric framework improved PCR specificity from 45% to 86% with 95% sensitivity, while culture specificity remained high but suffered notable underdetection of known clinically significant pathogens. Many PCR-only detections, especially in polymicrobial wounds, were supported by high bacterial burden, Gram concordance, and resistance markers. For three key pathogens (<i>Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus agalactiae</i>), analysis showed PCR specificity ≥95% when compared to culture, suggesting that culture underdetection may partly reflect colony selection and prioritization during workup. These results underscore the known limitations of culture in resolving polymicrobial podiatric wound infections and highlight PCR's role in providing significantly faster and more comprehensive organism-level detection to guide clinical decisions.IMPORTANCEChronic wounds, including diabetic foot infections, are a major cause of hospitalization and amputation worldwide. Conventional culture often requires several days and may miss fastidious or coexisting pathogens, delaying targeted therapy. In this study, we demonstrate that a real-time PCR wound panel provides same-day results with broader organism detection while maintaining strong concordance for key pathogens, such as <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>. By revealing additional clinically significant bacteria supported by high organism burden, Gram-stain concordance, and resistance-gene detection, molecular testing provides a more comprehensive view of the wound microbial profile. These results support PCR as a rapid, broad-range, and potentially first-line diagnostic method that complements culture and enables earlier, more informed treatment decisions.