A Dual-Phase Strategy to Integrate the VITEK MS RUO Database into Routine Clinical Practice: From Validation of Analytical Performance to Implementation of an Automated Workflow

ABSTRACT Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is pivotal in clinical microbiology. The VITEK MS Research Use Only (RUO) database offers broader species coverage, yet its clinical adoption is hindered by insufficient performance validation against the approved in vitro diagnostic (IVD) database and inefficient manual operational workflows. Objective This two-phase study first aimed to develop and evaluate an automated integrated workflow to enhance laboratory efficiency and diagnostic capability. Methods The RUO database’s two-tier identification architecture was utilized and in-house automated relay software was developed to parse IVD results and fully automate RUO reanalysis. Phase 1 (Mar 2021–Jun 2022) involved parallel manual testing of 2,432 isolates with both databases to analyze concordance and supplementary performance. Phase 2 (Jul–Nov 2022) prospectively incorporated 3,954 isolates to implement and assess the “IVD screening – automated RUO reanalysis” workflow. Results Phase 1 demonstrated high RUO-IVD concordance (95.7% species/genus agreement). The RUO database correctly identified 98.9% of isolates and provided valid supplementary identification for 84.4% (108/128) of IVD-failed cases, with Tier 2 contributing 28.9%. Phase 2 revealed that the integrated workflow increased the overall identification rate from 95.5% to 98.7%, with Tier 2 contributing an additional 14.5%. The automated software reduced reanalysis turnaround time by > 75%, saving consumables and labor. Conclusion The VITEK MS RUO database is a reliable and complementary tool to the IVD database. Integration with automated software creates an efficient, compliant clinical workflow, providing a practical model to enhance pathogen identification for infectious disease management. IMPORTANCE The present study bridges the validation-to-application gap for the VITEK MS RUO database. We confirm its high concordance (95.7%) and complementary value to the IVD database, quantify the impact of manual workflow inefficiency and introduce an automated software solution. The strategy highlights the key role of Reference Spectra (Tier 2) in expanding coverage and simultaneously improves diagnostic efficacy (∼99% ID rate), operational efficiency (> 75% time saved) and cost-effectiveness, offering a practical model to accelerate pathogen reporting and to guide therapy.