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Laboratory diagnosis of mycobacteria, with a focus on the bacteriological proof
VELKOVÁ, Martina
Laboratory methods play an important role in disease diagnostic. Although there are currently available fast molecular genetic methods and methods based on cell response, basic diagnostic method of direct evidence is still microscopy and cultivation. Microscopy (staining by Ziehl-Neelsen and fluorescence microscopy) is the primary method for most of the specimens, especially for sputum. The sensitivity of this method is not very high because for the detection of a positive finding in 1 mm3 is needed at least 105 microbes. However, the method is important because it can fastly proof extensive clinical and epidemiological serious diseases. The ?Golden standard? is today the cultivation on solid egg media. Nowadays it is filled in automatic detection system, which accelerates the time of detection. The thesis was carried out in the Laboratory of Medical Microbiology, Bacteriology department, Hospital Ceske Budejovice in the period from 1.1.2013 till 6.6.2013, and included 400 sputa, which were processed and examined. The purpose was to compare the effectiveness of decontamination methods with HCl and with NALC, and to monitor the recovery and detection for the automatic detection system and conventional cultivation. In the automatic system using the BACTECTM MGITTM 960 Non-Radiometric fluorescent technology is cultivation in liquid Middlebrook 7H9 medium supplemented with antimicrobials (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocilin). The classical culture used solid egg culture medium: Lowenstein-Jensen and culture medium by Ogawa. The individual results then show that the method of HCl had the overall contamination of 1 % of the samples. For each methods in 5 % BACTECTM MGITTM 960 and 4 % in the conventional cultivation. In comparison the method NALC, had the overall contamination of 21 % of the samples. For the individual methods in 26 % of BACTECTM MGITTM 960 and 50 % in the conventional cultivation. From these results it is obvious that the method with the NALC has lower efficiency, the proportion of contamination compared to the method with HCL increased by 20 %. Decontamination with the NALC method recommended by the manufacturer for BACTECTM MGITTM 960 was found to be unsatisfactory and was canceled. Comparing the recovery and the detection of the strains isolated in individual methods showed that the BACTECTM MGITTM 960 exhibits greater sensitivity than conventional cultivation, since the total of 17 strains isolated BACTECTM MGITTM 960 captured 15 against 10 strains isolated in conventional cultivation. There is also a significantly shorter time to detect positive samples. The average detection time for BACTECTM MGITTM 960 was 16.2 days, while a conventional culturing was 31 days. It was confirmed that the BACTECTM MGITTM 960 system achieves better results, but optimization is achieved by combining the two methods used. There is an apparent decrease in detection of mycobacteria from the processed statistical data of the strains isolated during the period 2010 - 2012, but a substantial reduction does not occur. Noticeable is only the decline of the isolated stains of Mycobacterium bovis BCG, which can be explained by the fact that the vaccination against tuberculosis is since 2011 no longer carried out across the board, but according to the new legislation in force only in high-risk groups. An interesting fact is that every year the highest laboratory detection is found in the age group of over 60 years.
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