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Serological diagnosis of borreliosis deseases
Sližová, Ivana ; Chmelař,, Dittmar (referee) ; Lochman,, Ivo (advisor)
The aim of present master’s thesis was to compare the results of serological methods for diagnosing borreliosis that are commonly used in Spadia laboratories (ELISA, immunoblots) in terms of recommendation on how and when to indicate and interpret them. The theoretical part is focusing on the characteristics and history of borreliosis, microbiological description of Borrelia, immune system and pathogenesis of the disease as well as the therapy and prevention. The experimental part is focusing on the analysis of results obtained from common examinations of antibodies to Borrelia made in Spadia Lab laboratories from January 1st 2014 to December 31st 2015. Screening of antibodies to Borrelia made by ELISA in IgM and IgG was done for all samples according to recommendation of CDC. In 2014 the ELISA screening was done using ELISA kits from Euroimmun and Evolis sample processors whereas in 2015 it was done using DiaSorin’s CLIA kits on Liaison analyser. Positive results were then confirmed by Westernblot or lineblot alternatively if the physician did not ask otherwise. It must be remembered that ELISA and Westernblot belong among serological methods that are using antibodies, i.e. substances produced by the immune system. The immune system plays the key role in protecting the body against infection and the antibodies are its important tool. Serological methods belong among immunoassay methods, which is still not standardized. Diagnosis of infections cann‘t be based only on antibody testing. It is necessary to assess the results in the context of the entire clinical picture, history and in the case of antibodies it is recommended retesting with an interval.
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Problems of laboratory examination in Lyme disease
HAVLEOVÁ, Lucie
Lyme borreliosis is a multisystem infectious disease most often transmitted by ticks of the genus Ixodes. In the Czech Republic, specifically by ticks Ixodes ricinus. The initiator of this disease is a complex of spirochetal bacteria Borrelia burgdorferi sensu lato. The incidence of the disease is relatively often as several thousand cases are reported in the Czech Republic every year. However the knowledge about Lyme borreliosis is relatively extensive and simultaneously an intensive research is constantly ongoing, the opinions of experts on this issue are still unfolding accordingly. So far, it is still rather difficult to diagnose Lyme disease, especially because of the various and sometimes non-specific clinical symptoms that might often lead to failure in diagnosis. As no effective vaccine has yet been developed, the only way to reduce the risk of infection is prevention and protection against the ticks. This work investigates and summarizes current knowledge about the history, etiology, epidemiology, pathogenesis, clinical course, prevention, therapy, and diagnosis of Lyme disease. Patients suspected of having contracted Lyme disease were tested using a two-level screening, firstly by applying ELISA method in order to select either positive patients or patients with uncertain results so that a confirmation Western blot method could follow subsequently. The outcome of my work confirmed the well-known fact that Lyme disease is seasonal. The higher number of positive samples in the summer months is related to the increased activity of ticks during this period. However, the results of positive patients who are divided into groups by age or gender represent very interesting findings. Particularly the higher incidence of the positive samples can be found among women.
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Detekce perzistentních borelií \kur{Borrelia burgdorferi} sensu lato ve tkáních savců přeléčených antibiotiky
OPELKA, Jakub
Lyme disease (LD) is a tick-borne illness caused by certain species of the spirochetes of Borrelia burgdorferi sensu lato complex. LD in humans affects multiple organs and although it is reported rarely as a cause of the death, it reduces the life quality of millions people around the world. All stages of LD are mainly treated with antibiotics doxycycline and amoxiciline. In 10 - 20 % cases, the symptoms of the disease persist even after treatment, but majority of practical physicians attribute these symptoms to psychosomatic problems. In this thesis the results of immunohistochemical research have shown that persistent forms of borrelia with a frequency of 0,16 - 0,3 individuals per 1 mm3 still exist in mammalian tissues after treatment with commonly prescribed antibiotics according to medical protocols. The ability to recultivate B. burgdorferi sensu lato from the tissues of antibiotic-treated mice confirmed their viability and suggest that relapse of infection can be caused by original infection.
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Preparation of clonal populations of \kur{Borrelia} from North American isolates in purpose to detect the presence of \kur{Borrelia} species commonly distributed in Europe.
SMOLENOVÁ, Štěpánka
Up to now, 22 spirochete species from the Borrelia burgdorferi sensu lato complex have been described. Three of them are generally known to cause Lyme borreliosis disease in humans in Europe: B. garinii, B. afzelii and B. burgdorferi sensu stricto. In the USA, only B. burgdorferi sensu stricto has been officially recognized as the LB agent. B. garinii and B. afzelii are currently regarded as species occurring exclusively in Europe. The newly tested method of Borrelia cultivation on hard soil and its separation into individual clones may lead to isolation of spirochete clonal cultures and confirmation of the presence of the European Borrelia species on the territory of the USA, which would confirm the theory of spirochete transfer over the ocean with migrating birds.
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Potenciální antimikrobiální účinky vybraných přírodních látek
MAŠKOVÁ, Hana
This study was focused on potential antiviral and antibacterial effects of viniferin and some derivatives of adamantane. The most suitable concentration was determined using flow cytometer. The antiviral effect on tick-borne encephalitis virus infection in glioblastoma cells was determined. Virus concentration was determined using plaque assay. The antibacterial effect was detected by the incubation of bacterial strains with suitable concentration of selected natural substances. The rate of bacterial growth was determined using absorbance measurement at 600 nm and plating on agar plates.
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Serological diagnosis of borreliosis deseases
Sližová, Ivana ; Chmelař,, Dittmar (referee) ; Lochman,, Ivo (advisor)
The aim of present master’s thesis was to compare the results of serological methods for diagnosing borreliosis that are commonly used in Spadia laboratories (ELISA, immunoblots) in terms of recommendation on how and when to indicate and interpret them. The theoretical part is focusing on the characteristics and history of borreliosis, microbiological description of Borrelia, immune system and pathogenesis of the disease as well as the therapy and prevention. The experimental part is focusing on the analysis of results obtained from common examinations of antibodies to Borrelia made in Spadia Lab laboratories from January 1st 2014 to December 31st 2015. Screening of antibodies to Borrelia made by ELISA in IgM and IgG was done for all samples according to recommendation of CDC. In 2014 the ELISA screening was done using ELISA kits from Euroimmun and Evolis sample processors whereas in 2015 it was done using DiaSorin’s CLIA kits on Liaison analyser. Positive results were then confirmed by Westernblot or lineblot alternatively if the physician did not ask otherwise. It must be remembered that ELISA and Westernblot belong among serological methods that are using antibodies, i.e. substances produced by the immune system. The immune system plays the key role in protecting the body against infection and the antibodies are its important tool. Serological methods belong among immunoassay methods, which is still not standardized. Diagnosis of infections cann‘t be based only on antibody testing. It is necessary to assess the results in the context of the entire clinical picture, history and in the case of antibodies it is recommended retesting with an interval.
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Citlivost spirochet Lymské boreliózy ke komplementu různých druhů zvířat chovaných v zoologických zahradách: potenciál rezervoárových hostitelů vybraných exotických obratlovců
TICHÁ, Lucie
Reaction of vertebrate serum complement with different Borrelia burgdorferi sensu lato species was used as a basis in determining reservoir hosts among domesticated and wild animals. Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii were tested for their sensitivity to serum of exotic vertebrate species housed in 5 zoos located in the Czech Republic. We confirmed that different Borrelia species have different sensitivity to host serum. We found that different tolerance to Borrelia infection possessed by hosts might alter among the individuals of the same genera or species and is not affected by host's age or sex. From all zoo animals included in our study, carnivores demonstrated the highest apparent reservoir competency for Lyme borreliosis spirochetes.
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Sensitivity of spirochetes from \kur{Borrelia burgdorferi} sensu lato complex to human complement: infection potential of selected species
TICHÁ, Lucie
Sensitivity of spirochetes from Borrelia burgdorferi sensu lato complex to serum complement of humans of different age and sex was analyzed. Complement-mediated Borrelia killing was observed in different combination of serum and selected Borrelia genospecies. The obtained results confirmed that age itself does not influence the sensitivity of human to Borrelia infection. However, the females seem to be more vulnerable to it. Each of ten tested Borrelia species was proved to be potentially infective for human in different ratio. The clear separation of all ten checked Borrelia species into two groups was revealed after the reaction with human sera: species with low sensitivity to human serum complement (mortality below 1 percent) and species with higher sensitivity (mortality over 3-4 percent).
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Preparation of monoclonal antibodies against Borrelia
ŠVEJDOVÁ, Veronika
In my bachelor thesis I deal with preparation of monoclonal antibodies against antigens of Borrelia. In theoretical section I describe genus Borrelia and antibodies. The introduction deals with the history of discovery of the spirochete that causes Lyme disease, vectors of this disease and classification of Borreliae according to geographical occurrence. Taxonomic categorization and description of the bacterium followers. It is focused on flagellum, arrangement of genetic material and on external surface antigens important for this bacterium. In the next section I describe Borrelia vector - host interactions and the development of immune response during the disease. Dermal symptoms of the Lyme disease are described in detail. A few words about relaxing fever are added. This chapter ends with description of laboratory diagnostics of Borrelia burgdorferi. Next, I focused on the characterization of antibodies and on preparation of monoclonal antibodies by hybridoma technology. I briefly describe ways of their utilization. Practical part of Bachelor thesis was carried out in the laboratories of Faculty of Science. Here, with the help of hybridoma technology, I fused myeloma cells with splenocytes of immunized mice producing hybridomas. These hybridomas I cultivated in medium, which I subsequently tested for occurrence of antibodies by ELISA. Hybridomas having high productivity were cloned, frozen and stored in liquid nitrogen. Finally I purified monoclonal antibodies, concentrated them and tested their titre.
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