Cheap and rapid in-house method for direct identification of positive blood cultures by MALDI-TOF MS technology

摘要

BackgroundPresence of microorganisms in the bloodstream is a life-threatening situation that requires rapid identification and treatment. Pathogen identification is of great significance, enabling adjustment of antibiotic care.In most clinical microbiology laboratories, the traditional method for microbial identification includes sampling of a blood specimen from the positive blood culture, subculturing it onto solid agars for 18–24?h, and bacterial identification according to biochemical features and antimicrobial susceptibility testing. The primary disadvantage of this method is that causative pathogen identification is performed only after colony growth and isolation, which leads to a prolonged turnaround time, especially when handling slow growing microorganisms such as anaerobic bacteria and yeasts. As time from diagnosis to appropriate antimicrobial care strongly influences mortality, shortening the turnaround time of pathogen recognition is extremely important [1, 2].In recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based technology has become useful for highly specific and sensitive identification of bacteria and yeasts from clinical samples. In this technique, a laser beam irradiates microorganism colonies and ionizes their proteins. As each microorganism has a different ionized protein profile, a different mass spectrum is created by the MALDI-TOF MS device. Whenever a new sample is subject to MS analysis, the software compares its profile to a database in order to define the microorganism. The first devices enabled pathogen recognition from one colony, grown on solid agar [3]. Later, new protocols were developed for identification of microorganisms directly from sterile body fluids such as cerebrospinal fluid, in order to save the culturing time [4]. Additional in-house methods enabled direct identification from blood cultures following various preparation protocols. Most of these protocols require hemolysis of red blood cells [2] and several blood centrifugations for the separation of blood cells and microorganisms [1, 2]. Along with laboratory-developed procedures, several commercial kits were developed, saving the handling time of solution preparation and additional equipment purchase. One such kit is the MALDI SepsiTyper Kit (Bruker Daltonics, Bremen, Germany), which was described in various studies as an efficient and successful tool for identification of bacteria and yeasts directly from blood cultures [5]. Successful microbial identification in this assay, as well as in others, depends on the bacterial amount that is collected from the culture pellet. The primary disadvantage of ready-to-use kits is their high cost.In the current study we compared bacteria and yeast identification by MALDI SepsiTyper with a simple and cost-saving method using a VACUETTE? Z Serum Sep Clot Activator tube, which is frequently used in clinical laboratories and enables separation of microorganisms from blood cells.