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Laboratory diagnostics of Hepatitis B
JANŮ, Denisa
Hepatitis B is an inflammatory viral disease of the liver. The etiological agent is a DNA virus from the Hepadnaviridae family. It is transmitted through the blood and secretions of an infected person. The greatest danger of the disease lies in the transition to chronicity. Chronic infection carries a high risk of developing liver cirrhosis and hepatocellular carcinoma. Hepatitis B is widespread throughout the world. The most reliable prevention is vaccination and avoidance of risky behavior and contact with persons with risky behavior, especially drug users and promiscuous persons. The aim of this bachelor thesis is to get acquainted with the disease of viral hepatitis B and to describe the current possibilities of diagnostics of hepatitis B markers using the COBAS e601 device from ROCHE by electrochemiluminescent immunoassay and quantification of viral DNA on the COBAS 4800 from ROCHE by real time PCR and evaluation of results using both methods. The set of examined samples comes from the laboratory of infectious serology and virology of the company VIDIA-DIAGNOSTIKA. The results show that HBsAg is the most investigated marker of hepatitis B. Positive findings make up only 0.67%. The ratio of HBsAg positive men and women in 2019 was almost balanced. Most HBsAg positive patients were aged 30-40 years. I also examined the prevalence of anti-HBc Ig total antibodies. The positivity of anti-HBc Ig total was confirmed in 4.27% of examinations. The ratio of anti-HBc Ig total positive men and women in 2019 was also almost balanced and the most positive were at the age of 30-40 years.
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Gene expression of enzymes involved in the regulation of apoptosis in rat moycardium - effect of chronic and acute hypoxia
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee)
Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...
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Detection of five garlic viruses using real- time PCR analysis: methodology for practice
Mitrová, Katarína ; Svobodová, Leona ; Ovesná, Jaroslava
The objective of the assay is detection of five viruses in garlic using real- time PCR analysis. . This assay allows to identify five of the viruses in the varieties of garlic - Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Shallot latent virus (SLV) and Garlic mite-borne filamentous virus (GMbFV) based on the detection of gene sequences for the coat protein by SYBR Green real-time PCR. This method was shown in our work to be a suitable tool for the detection of highly variable pathogens, such as garlic viruses. The method brings a new procedure for detection of these viruses.
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Laboratory methods using in diagnostic morbus Bechterew
FAROVÁ, Veronika
The aim of my bachelor thesis was to get familiar with laboratory methods, which are used in the diagnostics of morbus Bechterew. Very important for the diagnostics of morbus Bechterew is the marker HLA-B27, which is identified by screening, with the help of flow cytometer and then is the presence confirmed or refuted with the help of molecular-biological methods. Other laboratory examination, which is essential when it comes to interception and observation of the activities of morbus Bechterew, is the determination of the C-reactive protein level, which is an indicator of inflammatory reaction in the body of the patient. The last examination I got familiar with was the determination of the sedimentation speed of erythrocytes, which is used as an indicator of ongoing inflammation in the body. In the theoretical part of the thesis I focused primarily on the definition of morbus Bechterew. I dealt with the question what are the symptoms of the disease, then also according to which criteria is the disease diagnosed and what treatment is applied. In the second part of the theoretical part I focused primarily on the major histocompatibility complex, which is highly connected with morbus Bechterew. I dealt mostly with the structure and presentation of antigens by the means of MHC molecules of class I. I also mentioned the structure and the function of C-reactive protein. In the practical part I described laboratory methods used in diagnostics of morbus Bechterew. The first method I tried was screening examination of HLA-B27 marker by the means of flow cytometry. I introduced how the following processes should look like: collection of biological material, preparation of the sample and the analysis made on the flow cytometer. At the end of this part I put an example of the analysis results, how do the results look like when they are positive, negative, or in the grey zone. As a second method I introduced genetic typing of HLA-B27 molecule, which is made by the means of real time PCR. I mentioned the preanalytical part and the process of preparation. Moreover I dealt with the measuring of C-reactive protein by the means of nephelometry and then I also mentioned examination with the help of a test strip. The last method I tried was measuring the sedimentation speed of erythrocytes, I mentioned the principle of method, the process of measuring and the measured results as well. In the last part of my bachelor thesis I worked with the data I got from Motol University Hospital, more precisely from the Department of Immunology, 2nd Faculty of Medicine of Medicine, Charles University in Prague and Motol University Hospital. During the year 2017, overall 452 patients were tested on the presence of HLA-B27 marker and 75 of them tested positive. Then I worked only with HLA-B27 positive patients. I researched how much is the disease represented in both sexes, what age do the HLA-B27 positive patients have and what is their diagnosis.
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Gene expression of enzymes involved in the regulation of apoptosis in rat moycardium - effect of chronic and acute hypoxia
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee)
Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...
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Determination of causal agent of ring rot of potato, Clavibacter michiganensis subsp. sepedonicus bacteria in breeding and multiplicated materials
Pánková, Iveta ; Krejzar, Václav
The methodology includes a method of artificial infection of plants and potato tubers as a prerequisite for the study of migration and localization of the pathogen in tubers and within the plant vascular tissues, time schedule and recommended size of samples of individual parts of plants and potato tubers during the growing season, depending on external conditions and potato genotype, how to deal with samples of the plants and potato tubers prior to treatment, method of processing samples of individual plant parts and tuber depending on the detection method and the optimization of methods for detection of the pathogen Clavibacter michiganensis subsp. sepedonicus. In compliance with the proposed methodological procedure should be interrupted vertical transmission of the pathogen in the process of breeding, the possibility of detection of the pathogen in breeding and propagation material and reduction of suspicious samples should be increased and the late positive determination of ring rot in the official phytosanitary and company checks abroad should be reduced.
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Diagnostics of Clostridium difficile as nosocomial infections in Nemocnice ČB a.s. using methods of molecular biology.
ŠTĚRBOVÁ, Denisa
Nosocomial infections caused by Clostridium difficile represent a substantial part of hospital-acquired infections in Czech hospitals. Intoxication by toxins produced by Clostridium difficile leads to serious damage to gastrointestinal tract and life of the patient may be in danger. The progress of intoxication could be quick, this is why reliable and time-efficient diagnostic methods are of great importance for efficient treatment of the patients. Bacterial toxins are not produced during the whole life cycle of Clostridium difficile. This is why it is better to detect bacterial DNA which is always present in the bacterial cells, not the toxins. In Nemocnice ČB a.s. (České Budějovice municipal hospital) I compared methods based on toxins detection (?hyplex? ClosTox? and ?hyplex? ClosTox 027? by BAG Health Care) with a method based on DNA detection (real-time PCR ?Xpert C. Difficile? by Cepheid). I found out the real-time PCR method is much quicker. It takes one hour to prepare the samples and to obtain analytical result for this method. Both tests based on toxin detection are much more time consuming. It takes up to 5 hours to complete them. I conclude the real-time PCR is much quicker analytical method and it allows Clostridium difficile detection during all life phases of the bacteria.
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