RESUMEN
Background: The introduction of a method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF mass spectrometry) into the practice of laboratories significantly increased the identification of acid-resistant bacteria (ARB). Methods: Seventy-four nontuberculous mycobacteria (NTM) cultures identified by deoxyribonucleic acid (DNA) hybridization, polymerase chain reaction, Sanger sequencing, and MALDI-ToF mass spectrometry. Results: Analysis of the identification results obtained by the methods of DNA hybridization and Sanger sequencing showed a complete match only for 67.6% of samples of the total number of cultures included in the study. The partial match of the identification results was 68.9%. When comparing the results of the identification of 74 samples obtained by MALDI-ToF mass spectrometry to the results obtained by sequencing, full match of identification of Mycobacterium chimaera/Mycobacterium intracelullare, Mycobacterium porcinum/Mycobacterium peregrinum and Mycobacterium tuberculosis complex was found for 90.5% of the samples; the partial match of the results - for 4.1%.. DNA hybridization as a method for identifying NTM showed acceptable sensitivity and specificity; however, for mass spectrometry, a significantly higher sensitivity with comparable specificity was determined. Conclusions: Mass spectrometry is an important element in the modern system of species identification of microorganisms. The optimization of sample preparation protocols and assessment of the impact on the identification of new techniques of cultivation of microorganisms can significantly improve the quality of identification of microorganisms from the ARB group. In this case, accurate species identification and the development of algorithms for its application will improve the diagnosis of diseases caused by ARB.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Micobacterias no Tuberculosas/genética , ADNRESUMEN
Background: Problem with mass spectrometers is the difficulty in identifying some genera of acid-fast bacteria (AFB). Due to the peculiarities of the colony architectonics (the formation of dry colonies with a complex structure) and the structure of the cell wall, the probability of obtaining a sufficient amount of ribosomal proteins is significantly reduced. This problem is partially solved using an extended method of direct application and extraction with formic acid, which can significantly improve the quality of the identification. Methods: The study analyzed strains of microorganisms obtained during the examination of patients with suspected tuberculosis. In total, 287 strains of nontuberculous mycobacteria (NTM) were obtained. In addition, 63 strains of the most common bacteria from the AFB group were analyzed. Matrix-assisted laser desorption/ionization (MALDI) was used. Three main methods of sample preparation of microorganisms according to the manufacturer's recommendations for use with the MALDI-time-of-flight (ToF) mass spectrometry method were used in the work: Direct coating method, extended direct coating method, and formic acid extraction method. Results: When evaluating the influence of the cultivation medium on the result of NTM identification by MALDI-ToF mass spectrometry, statistically significant results of the influence of the medium on the result of NTM identification were revealed for all compared parameters. Conclusions: Optimization of sample preparation protocols and assessment of the impact on the identification of new methods of cultivating microorganisms can significantly improve the quality of the identification of both clinically significant microorganisms from the AFB group and saprophytic microflora, the clinical significance of which has not been proven at the moment.
Asunto(s)
Actinomycetales , Micobacterias no Tuberculosas , Humanos , Bacterias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Rayos LáserRESUMEN
Background: The variety of morphological and cultural characteristics of acid-resistant bacteria (ARB) makes it possible to use microscopy and estimate the growth rate and pigment formation when cultivating on solid egg media for preliminary identification only as additional indicative methods. It is necessary to develop new approaches for the cultivation and primary identification of ARB isolated from the biological material. It will allow to obtain data on the prevalence, structure, epidemiological, and clinical features of infectious processes caused by opportunistic ARB. Methods: Three hundred and sixty strains of ARB were isolated from the various biological materials obtained from the patients during the examination for tuberculosis. All biological material samples were negative on Mycobacteria tuberculosis complex. Species identification of all bacteria was performed by matrix-assisted lazer desorption/ion-ization time-of-flight mass spectrometry. The cultural characteristics of ARB were evaluated on a universal chromogenic media. As a selective additive, a mixture of bacitracin and polymyxin sulfate which had no effect on ARB was tested to suppress concomitant Gram-positive and Gram-negative microflora. Results: Cultural characteristics were identified and described for all tested representatives of fast-growing nontuberculous mycobacteria (NTM), as well as for all types of nocardia, gordonia, and streptomycetes. Representatives of other genera of ARB on a universal chromogenic media gave meager growth or did not show it at all. When inoculated on a universal chromogenic media with a selective addition, 100% of the strains from the ARB group showed abundant or moderate growth. Incubation time for fast-growing species was up to 7 days; for slow-growing species, it was up to 28 days. Concomitant control strains of Gram-positive and Gram-negative bacteria on universal chromogenic media with selective growth additive did not show the growth. Conclusions: The use of a universal chromogenic media allows to preliminarily identify NTM and other ARB by cultural characteristics. The addition of bacitracin and polymyxin sulfate does not reduce the growth properties of ARB, which can be used when working with both biological materials and for the isolation of pure ARB cultures from mixtures with other bacteria.