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Objective. To present the results of using in-house method for rapid identification of fungi from funguspositive bottles with routine conventional culture-based identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in patients with bloodstream infection. Materials and Methods. Prospective study was performed from 2016 to 2019 at the National Research Center for Hematology, Moscow. During the study period, all blood cultures (BC) bottles obtained from hematological patients were incubated in the BACTEC FX system (Becton Dickinson, USA). Positive BC bottles were examined by Gram stain. In house method was used after Gram stain was positive for yeast cells or hyphae. For that, BC media was transfer from fungus-positive bottles into tube. In house method included series section steps consisted from centrifugation and extraction of fungal proteins by adding of sodium dodecyl sulfate. Routine conventional culture-based identification on Sabouraud with chloramphenicol agar (bioMerieux, France) for yeasts and on Sabouraud dextrose agar (Oxoid, UK) for molds was used simultaneously with the in house method. Results. During the study period, 16 fungus-positive bottles were obtained from which were isolated in monoculture 14 (87.5%) Candida spp.: C. parapsilosis (n = 5), C. tropicalis (n = 4), C. albicans (n = 3), C. krusei (n = 1), C. guilliermondii (n = 1), one (6.3%) Rhodotorula mucilaginosa and one (6.3%) Fusarium dimerum. The in house method resulted in 75% (12/16) and 68.8% (11/16) identification rate at the genus and species level of fungi, respectively. The identification of fungi to species level was confirmed by conventional culture-based method in all cases. The median time from the start of vial incubation in BACTEC FX system to identification of fungi by in house method was less than conventional culture-based identification: 36 hrs 20 min vs 55 hrs 31 min (p = 0.028). Conclusions. A high rate of correct direct species identification and significant reduction in time to verification of fungi from fungus-positive bottles by in house method were obtained. The proposed in house method should be recommended for use in real microbiology practice to reduce the time for submitting results of identification to clinical units.

Objective. To assess pathogen identification from positive blood cultures by MALDI-TOF mass spectrometry. Materials and Methods. At the first stage, an in vitro study using MALDI-TOF MS and including 11 pathogen species (K. pneumoniae, E. coli, P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, S. haemolyticus, E. faecalis, E. faecium, C. albicans, C. parapsilosis) was performed. This study involved hourly attempts to identify the above listed pathogens by MALDI-TOF MS (Vitek MS, bioMerieux) during incubation until their correct identification. At the second (clinical) stage, a total of 360 positive blood cultures obtained from 187 patients undergoing cardiac surgery were tested. Two accelerated techniques of pathogen identification from the positive blood cultures were studied concurrently with the routine method (MALDI-TOF MS assay following 24-hour incubation). A total of 300 blood samples (284 single cultures) were tested by the short-term incubation technique. A total of 211 blood samples (201 single cultures) were tested by the direct identification technique (with no culture on a solid medium). A total of 151 blood samples were tested by these two identification techniques concurrently. Results. At the first stage, the incubation time (on blood agar) required to complete identification was no more than 5 hours and 6 hours for vials with polymer granules and vials with active charcoal, respectively. The incubation times for Candida spp. were 10 to 22 hours. The identification by MALDI-TOF MS following short-term incubation on blood agar was successful for 259/284 (91.2%) of pathogens, including 97%, 94.5%, and 43.5% of Gram-negative rods, Gram-positive cocci, and Candida spp., respectively; the mean incubation times were 2.7, 3.9 and 5 hours, respectively. The results of successful pathogen identification coincided with those obtained by the routine method in all cases. The identification to species level by the direct MALDI-TOF MS assay was correct for 163/201 (81.1%) of pathogens, including 94%, 75.2%, and 40% of Gram-negative rods, Gram-positive cocci, and Candida spp., respectively. A turnaround time for this technique, including sample preparation, was no more than 1 hour. The concurrent use of the both accelerated techniques provided the overall successful identification in 95% of cases, including 98.4%, 96%, and 50% of Gram-negative rods, Gram-positive cocci, and Candida spp., respectively (Cohen’s kappa for comparison with the routine method was 0.92). Conclusions. The accelerated techniques of pathogen identification from blood cultures by MALDI-TOF MS provide rapid and reliable identification of microorganisms causing bacteremia, which in combination with local microbiological monitoring data may contribute to early appropriate antimicrobial therapy.