Accelerated Direct Identification of Gram-Positive and Gram-Negative Bacteria Using Matrix-Assisted Laser Desorption...

Bloodstream infections (BSIs) are associated with high morbidity and mortality, particularly in critically ill patients. Delayed bacterial identification may result in inappropriate empirical therapy and poor outcomes. Direct identification from positive blood culture bottles can significantly reduce turnaround time (TAT). While direct matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been evaluated for gram-negative bacilli (GNB), data on gram-positive cocci (GPC) remain limited. This study assessed direct MALDI-TOF MS performance for both GPC and GNB to support timely sepsis management.

The objective of the study was to compare direct bacterial identification by MALDI-TOF MS with the standard method of overnight incubation and processing using MALDI-TOF MS and to evaluate the reduction in TAT.

This prospective study was conducted from September 2024 to September 2025 at the Department of Microbiology of a tertiary care centre in North India. Adult ICU patients with two or more blood culture bottles flagged positive by BACT/ALERT® 3D (bioMérieux SA, Marcy-l'Étoile, France) and showing monomicrobial morphology on Gram stain (yeast excluded) were included, yielding 251 isolates. Direct MALDI-TOF MS was performed from positive broth; the standard method involved subculture on blood agar and MacConkey agar with 18-24 hours aerobic incubation at 37°C, followed by MALDI-TOF MS. Concordance was assessed using categorical agreement and Cohen's kappa.

Among 251 monomicrobial isolates, 103 (41.0%) were GPC and 148 (59.0%) were GNB by the standard method. Direct MALDI-TOF MS achieved species-level identification in 82.5% of GPC and 85.1% of GNB isolates. Genus-level agreement was 96.1% for GPC and 85.8% for GNB; species-level agreement was 82.5% and 83.8%, respectively. Overall concordance was 84.1%, with almost perfect agreement (Cohen's κ = 0.82). Mean TAT decreased by 23.82 hours, with the Wilcoxon signed-rank test confirming a highly significant reduction (p < 0.001).

Identification accuracy was slightly higher for GNB compared with GPC. The higher misidentification rate observed among GPC isolates may reflect the spectral similarity among closely related species. Significant reduction in TAT demonstrates the potential for earlier targeted antimicrobial therapy and improved clinical decision-making in critically ill patients.

Direct MALDI-TOF MS from positive blood cultures demonstrated high concordance with the standard method while significantly reducing TAT. The approach performed robustly for both GPC and GNB. This accelerated workflow can facilitate the timely optimization of antimicrobial therapy, strengthen antimicrobial stewardship, and may improve clinical outcomes in critically ill patients.