Diagnostics of blood-borne infections is still an urgent problem of modern medicine. The main causative agents of septic conditions are gram-positive microorganisms particularly Staphylococcus aureus, enterococci, etc., whereas clinical significance of isolated coagulase-negative staphylococci is ambiguous. Escherichia coli, Klebsiella pneumonia and other enterobacteria, as well as Acinetobacter baumannii prevail among the gram-negative flora. Modern possibilities of accelerated identification of microorganisms derived from positive blood cultures based on mass-spectrometry consist of two approaches. Firstly, the manufacturers' developed consumables for mass spectrometry are proposed, and secondly, there are domestic developments of accelerated sample preparation protocols developed by microbiological laboratories. The approaches used have a number of advantages and disadvantages, but to summarize, the use of the proposed methods in routine practice is quite limited. At the same time, the need to accelerate the issuing a microbiological conclusion related to nosology is great being associated with improved outcomes. In this regard, the aim of the study was to evaluate convergence and accuracy of results for accelerated identification of microorganisms derived from positive blood cultures in blood-borne infections. The study included 87 positive blood cultures, the identification of pathogens from them occurred in four ways: the classical microbiological analysis of blood for sterility, pathogen identification directly from the vial without isolating a pure culture, as well as two sample preparation methods based on ethylenediaminetetraacetic acid (EDTA) and potassium ethylenediaminetetraacetate disubstituted (EDTA-K2) as wash additives. It was found that gram-positive and gram-negative flora were isolated from the blood almost evenly often. When evaluating an influence of biomaterial used for mass spectrometry, it turned out that use of wash additives increases chances of successful identification of bacteria from a blood sample. The influence of tinctorial properties on the results of determining the species assignment of isolates was also evaluated. Identification of gram-positive flora is more accurate, since some pathogens were not identified without washing additives, and when using EDTA-K2 and the corresponding acid, assignment to the genus was obtained in the same samples. A similar pattern was also characteristic of gram-negative flora. At the same time, modern manufacturers of laboratory equipment and reagents allow to standardize sample preparation procedures in the protocols used. The effects of EDTA-K2, which allowing to use it as a washing component, are associated with the binding of calcium and magnesium ions in solution, which reduces the adhesion of bacterial cells to blood cells, thereby contributing to better mass spectrometry of microbial sediments with accelerated identification of microorganisms from positive blood cultures. Thus, use of the described additives can provide high quality, timely and adequate diagnostics of serious and life-threatening conditions such as blood-borne infections.
The culture method continues to be the “gold” standard for microbiological diagnosis of bloodstream infections. This is primarily due to the fact that the definition of the etiology of a generalized infectious process determines the etiotropic antibiotic therapy. To do this, it is necessary to conduct periodic microbiological monitoring of the prevailing microflora. To do this, in the present study, a retrospective analysis of the results of a microbiological blood test for sterility was performed in case of suspected bloodstream infections in a multidisciplinary hospital to assess the influence of analytical stage factors on the laboratory data obtained. Automatic hematological cultivators were used, identification was carried out based on the biochemical characteristics of microorganisms, as well as using time-of-flight mass spectrometry with matrix-activated laser desorption / ionization (MALDI-TOF MS). More than 10,000 research results were analyzed, the average microflora seeding rate was 15.1%. The analysis of the isolated microflora was carried out in 2 groups of positive results: at the beginning, the data obtained in the presence of growth in two vials at once were evaluated, then the positive results of blood cultures obtained in any one vial from a pair were studied. The predominance of gram-positive flora in the structure of microorganisms isolated from whole blood was revealed, the influence of cultivation conditions and the composition of thenutrient medium on the isolated flora was not found, however, a number of microorganisms, due to the specific characteristics of metabolism, were characterized by growth under strictly defined cultivation conditions. The presented study actualizes the need for constant microbiological monitoring in order to determine the prevailing hospital microflora, which can contribute to a timely response in order to limit the spread of highly virulent, aggressive, resistant strains of microorganisms leading to the development of generalized bloodstream infections.
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