Validation of MALDI-TOF MS database expansion with spectra of in house bacterial strains isolated in the pharmaceutical industry

Introduction: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique used to identify microorganisms quickly and accurately, but instruments databases do not include all the bacterial diversity of the pharmaceutical industry environment, making their constant expansion essential. Objectives:In order to comply with regulatory guidelines and standards, the process of validating a technique involving alternative identification methods to expand the MALDI-TOF MS database is necessary.Methodology: According to RDC 166/2017 and other compendia, the parameters required for the validation of alternative microbiological methods for microorganisms' identification are accuracy, specificity, robustness and precision.The aim of this work was to validate the expansion of the MALDI Biotyper (Bruker) database with strains isolated locally at Bio-Manguinhos.To achieve this goal, four bacterial strains previously unidentified by MALDI-TOF MS were analyzed using the 16S rRNA full gene sequencing.The data obtained were compared with the EzBioCloud platform, considering 98.7% as the cut-off point for species-level identification.The strains were plated on sheep's blood agar and incubated at 32.5C for 24 h.The super extraction technique was used to obtain the proteins, and each strain was analyzed in eight replicates on the MALDI Biotyper . Results:The obtained spectra were inserted into the equipment's database, according to the manufacturer's instructions.To comply with the specificity and accuracy criteria, the strains were analyzed in 5 replicates by 2 analysts on 2 different days.For the robustness parameter, the strains were plated on tryptone soy agar and analyzed in 5 replicates by 2 analysts.Accuracy was assessed using the true positive rate of the data obtained for the evaluated parameters.All strains were correctly identified with an A level of consistency, scores between 2 and 3, and a true positive rate of 100%. Conclusion:The creation of a customized and validated database will optimize microorganism identification in the microbiological control laboratory, meeting regulatory agency requirements and ensuring data integrity, making it possible to obtain results quickly, safely, and at low cost.