Evaluation of rapid identification and susceptibility testing methods in the diagnosis of bacteremia

Bloodstream infections are a critical issue that leads to serious morbidity and mortality, especially in hospital settings. While treatment should ideally begin within the first hour to increase the chances of survival in patients with sepsis, traditional culture methods delay targeted treatment by 48-72 hours. The aim of this study is to evaluate the laboratory performance of multiplex PCR (rapid pathogen diagnosis) and Rapid Antimicrobial Susceptibility Testing (RAST) methods, developed to overcome this critical diagnostic delay, by comparing them with current routine practices. This study was conducted with 50 isolates obtained from 50 different patients with positive blood cultures at the Van Yüzüncü Yıl University Medical Microbiology Laboratory between 01.09.2024 and 31.05.2025. Positive blood culture bottles were examined by gram staining for preliminary reporting and simultaneously subcultured onto solid media. Species identification was performed using an automated system (Phoenix M50) and antimicrobial susceptibility testing was conducted using the disk diffusion method. A multiplex PCR (Bio-Speedy Sepsis qPCR MX-30L Panel) method was also used to detect pathogens and resistance genes. The RAST method involved inoculating 125±25 µl of undiluted blood directly from positive blood culture bottles onto Mueller-Hinton agar, with results read at 4, 6, 8, and 16-20 hours according to EUCAST RAST breakpoints. The standard disk diffusion method served as the reference method. Among the 50 isolates examined, the most frequently encountered species were Staphylococcus aureus n=15 (30%), Klebsiella pneumoniae n=11 (22%), and Pseudomonas aeruginosa n=11 (22%). When PCR results were compared using the automated system (for genus and species identification) and the disk diffusion method (for resistance profile) as reference methods, consistency was detected in 49 (98%) of the 50 isolates at the genus level, 46 (92%) at the species level, and 43 (86%) at the resistance gene level. While 40 isolates (80%) showed full concordance across all parameters, partial concordance was observed in 10 isolates (20%). Regarding the RAST results, disk readability rates increased with incubation time, reaching 86.90% at the 4th hour, 96.89% at the 6th and 8th hours, and 100% at the 16th-20th hours. The categorical agreement rates of the method were found to be high at all time points: 96.86% at the 4th hour, 96.24% at the 6th hour, 96.97% at the 8th hour, and 98.43% at the 16th-20th hours. In the study, very major error rates at the 4th, 6th, 8th, and 16th-20th hours were found to be 0.63%, 0.39%, 0.72%, and 0.71%, respectively; major error rates were 5.79%, 7.48%, 3.33%, and 1.42%, respectively; and minor error rates were 0.00%, 0.23%, 0.46%, and 0.22%, respectively. The combined use of multiplex PCR (rapid diagnosis) and HADT shortens the time to antibiotic susceptibility testing by 32-48 hours, accelerating the transition to targeted treatment. The high concordance between EUCAST HADT breakpoints and final results demonstrates the reliability of this early assessment. This approach has critical potential for reducing patient morbidity/mortality and controlling antimicrobial resistance. Keywords: Rapid Antimicrobial Susceptibility Test (RAST), bacteremia, Polymerase Chain Reaction (PCR)