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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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The Interaction of Borrelia Outer Surface Proteins with Tick Salivary Proteins
PAKTAN, Aylin
The aim of this thesis was to investigate the interaction of the salivary gland protein, Salp15, in relation to the outer surface protein C, OspC, using in silico analysis, as well as trying to amplify Salp15 from different tick species found in same geographical areas and observing the growth of Borrelia in the midgut and salivary glands of these different tick species in vitro.
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Úloha osy PD-1/PD-L1 při infekci \kur{Borrelia burgdorferi} u myší
PALOUNKOVÁ, Anna
Borrelia burgdorferi, the causative agent of Lyme disease, induces upregulation of inhibitory immune checkpoint PD-L1 in mice. We studied if the blockade of PD-1/PD-L1 axis by neutralizing antibodies influences the proliferation of T lymphocytes and cytokine milieu in imunological synapsis between murine dendritic cells and T cells in vitro.
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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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Adapting Genetic Methods For Transposon Mutagenesis And Fluorescent Markers In Borrelia
WEBER, Peter
The aim of this Bachelor thesis was the examination and generation of different mutants in various Borrelia species. While several assays were used to examine aspects of B. burgdorferi transposon mutants, genetic tools were applied in order to generate fluorescent markers in B. afzelii and a vector construct in B. burgdorferi. This work should provide an overview of various genetic methods and their methodology that have been developed in Borrelia bacteria.
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Detection of persistent forms of Borrelia burgdorferi sensu stricto in infected mice after antibiotic treatment
RAMZY, Ida
Antibiotic treatment of Lyme disease is a challenging procedure with unexpected outcomes that may arise sometime. According to standard protocols recommended by the Centers for Disease Control and Prevention, USA (CDC) for early Lyme disease, a short course of oral antibiotics cures the majority of cases. This thesis deals with the analysis of the efficiency of 2 majorly used antibiotics, doxycycline and amoxicillin, in elimination of Borrelia infection on laboratory mice model after 2 weeks of treatment. Our results confirmed the presence of persistent forms of spirochetes in mice tissues after antibiotic treatment using the spirochete cultivation method in liquid MKP medium.
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Exploration of the tick-Borrelia molecular interactions by employing the transcriptomic approaches
MAHMOOD, Sazzad
Along with climate change and increased sharing of habitat, ticks are coming into more frequent contact with humans. The hard tick Ixodes scapularis and Ixodes ricinus are known disease vectors in Northern America and Europe, respectively. Along with many other pathogenic microorganisms, these ticks spread Borrelia sp. by ectoparasitic blood feeding. Borrelia afzelii is the major European Lyme disease pathogen spread by I. ricinus. Our study focuses on differential gene expression in I. ricinus salivary gland and midgut, induced in the nymphal stage by B. afzelii infection. Tick genes upregulated by infection are considered to play essential roles for the acquisition, persistence, and transmission of Borrelia. We have determined 32,897 full length sequences of tick mRNA from B. afzelii infected/noninfected tick salivary glands and the whole body. In addition, we have obtained MACEseq (Massive Analysis of cDNA Ends) from both midgut and salivary glands while the nymphs were non-infected or infected with B. afzelii during three different phases of blood-feeding. From the MACE database, we obtained 250-500 bp 3'-end sequences with raw quantitative expression values. Total reads, unique sequences and protein coding tick genes from midgut samples were 38,199,641, 88,825 and 24,276, and from salivary gland were 74,651,134, 93,096 and 26,179, respectively. After filtering, using several criteria, expression was validated by qPCR. Hence, the validated genes may most likely interact with Borrelia in its acquisition, persistence, or transmission to the vertebrate host. In our study, RNA interference approaches and vaccination were implemented in order to investigate the impact of upregulated tick midgut and salivary gland genes on Borrelia transmission to C3H mice.
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