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Laboratory diagnostics of Lyme disease
DOSTÁLOVÁ, Simona
Lyme borreliosis is the most common infectious disease that is transmitted by ticks infected with Borrelia burgdorferi bacteria (anthropozoonosis). The diagnosis of this disease is based on the detection of specific IgG and IgM class immunoglobulins by specific laboratory tests. This bachelor thesis focuses on the evaluation of the clinical condition of patients by the attending physician (see case reports) in accordance with the results of laboratory tests. The correct diagnosis is based on the clinical symptoms, anamnesis and laboratory findings of each patient, which is used to guide adequate antibiotic treatment. In this work a comparison of two methods used for detection of antibodies against Borrelia burgdorferi, the chemiluminescence immunoassay (CLIA) method and the confirmatory Western blotting method used in the Clinical Laboratory of DIA-GON MP, s.r.o. in Cheb, is performed. The CLIA method provides rapid and sensitive screening for IgG and IgM antibodies, while Western blotting serves as a confirmatory test with higher specificity. The evaluation of the results and their interpretation are based on the detection of specific antibodies against Borrelia by Western blotting. The last part of the bachelor thesis is a search of an article from the Czech Hydrometeorological Institute, which cooperates with the State Institute of Health with the support of the Ministry of Health of the Czech Republic. It discusses tick activity in relation to their seasonal occurrence. On the basis of this article, I make an assessment of the seasonal occurrence of Lyme borreliosis in 2015-2017 with respect to the average temperatures in that period.
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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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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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Knowledge of Pupils Secondary School in the Area of Selected Zoonoses
Svobodová, Barbora ; Pavlasová, Lenka (advisor) ; Říhová, Dagmar (referee)
The subject of the dissertation deals with the secondary school pupil's awareness of zoonoses. Zoonoses are the diseases transmited from animals to human beings. Those are one of the topics of biology study currently. The main aim of the dissertation is theoretical definition of the matter in the first place. For knowledge testing have been chosen these four illness: Toxoplasmosis, Lyme boreliossis, Rabies and Creuzfeldt-Jakobo disease. With these chosen illness the main research goal is to provide a comprehensive collection of information about the details such as disease transfer, spread of the disease, disease development, cure and avoidance. The research part of the dissertation tries to find out the extent of knowledge about the chosen kinds of zoonoses among the pupils at specialized secondary schools. As a tool of quantitative research has been used a pupil's didactic test. As based on the evaluated results we may say that the pupil's knowledge at explored schools is relatively satisfactory.
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Antimicrobial and antiborrelial characterization of Dermacentor marginatus defensin.
CHRUDIMSKÁ, Tereza
Ticks, as blood sucking arthropods, are able to transmit various pathogens. Their immune system involves many antimicrobial molecules to fight against them. Among these antimicrobials, defensins, a 5.3 kDa peptides, play an important role in rapid immune answer. In this study we examined the antimicrobial spectrum of Dermacentor marginatus defensin (def DM) with respect to the fact that Dermacentor variabilis ticks are not able to successfully maintain and transmit Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. Expression of the def DM gene was detected in hemolymph, midgut and salivary glands. Defensin was isolated from hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic mature peptide def DM revealed an anti-Gram-positive bacterial role as well as borreliacidal activity, with concentration dependent influence. These results suggest a possible role in the clearing of borrelia spirochetes ingested by D. marginatus ticks.
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Lyme borreliosis diagnostics using in vitro cellular immune response testing
Prokopová, Tereza ; Drbal, Karel (advisor) ; Melter, Oto (referee)
Lyme borreliosis is a multisystemic disease affecting skin, joints, heart and central nervous system. The disease is caused by spirochetes of Borrelia burgdorferi sensu lato complex. These bacteria are spread by ticks of Ixodes genus. In 2016 there were almost 4,000 newly infected individuals reported in the Czech Republic. Contemporary serological diagnostics of Lyme borreliosis is not sensitive nor specific enough and does not even correlate with the pathology of the disease in the early or late phases. For the correct diagnosis of the disease it is necessary to detect the pathogen and its genotype. For this reason we had aimed at two goals. Through the digital droplet PCR (ddPCR) method we detected Borrelia-specific DNA and its genotype. The detection limit of borrelial DNA was set on gDNA samples isolated from the tick. Detection threshold for the initial amount of 1 ng of tick gDNA is at the range of 10-17 g of specific borrelial DNA. Borrelia spp. coinfection was detected in 5 out of 12 tested samples. The most frequent type was B. garinii which was detected in 5 samples. On the basis of published sequences for virulent factors we have designed specific primers in conserved regions of the genes flanking their variable segments to be PCR amplified. Gene variability will be monitored through...
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Knowledge of Pupils Secondary School in the Area of Selected Zoonoses
Svobodová, Barbora ; Pavlasová, Lenka (advisor) ; Říhová, Dagmar (referee)
The subject of the dissertation deals with the secondary school pupil's awareness of zoonoses. Zoonoses are the diseases transmited from animals to human beings. Those are one of the topics of biology study currently. The main aim of the dissertation is theoretical definition of the matter in the first place. For knowledge testing have been chosen these four illness: Toxoplasmosis, Lyme boreliossis, Rabies and Creuzfeldt-Jakobo disease. With these chosen illness the main research goal is to provide a comprehensive collection of information about the details such as disease transfer, spread of the disease, disease development, cure and avoidance. The research part of the dissertation tries to find out the extent of knowledge about the chosen kinds of zoonoses among the pupils at specialized secondary schools. As a tool of quantitative research has been used a pupil's didactic test. As based on the evaluated results we may say that the pupil's knowledge at explored schools is relatively satisfactory.
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Epidemiological aspects of Lyme Borreliosis in Czech Republic
Fišerová, Tereza ; Janovská, Daniela (advisor) ; Čelko, Alexander (referee)
The aim of this bachelor work was to collect and to evaluate the data on the incidence of Lyme borreliosis in the Czech Republic in the last ten years (2003-2012). First, a comparison of the incidence of this disease in the administrative regions of the Czech Republic was made, which indicated noticeable differences between the regions. Then, the data for the South Bohemia Region and Vysočina Region were analyzed in a more detail to identify the main factors, which could contribute to the increased incidence of this disease. A brief history of the incidence of Lyme borreliosis worldwide and its etiology is also provided, together with an overview of its clinical manifestations and spread, as well as the corresponding laboratory diagnostic methods and methods of prevention.
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Serological evidence of Lyme borreliosis
NOVÁKOVÁ, Iveta
This bachelor thesis deals with Lyme borreliosis (Lyme disease). Lyme disease has a worldwide distribution. It is caused by several species of the Borrelia bacteria that vary according to geographic occurrence. In our country, this disease is transmitted, above all, by hard ticks of the genus Ixodes ricinus (Other genera are prevalent in other parts of the world.). These ticks are present in a wet environment, e.g. in the woods or in high grass. This poses a higher risk for people who stay or work in similar sites; i.e. lumberjacks, but also for children who spend their free time in the great outdoors. According to symptoms Lyme disease can be divided into three stages: stage 1 is called early localized Lyme borreliosis, stage 2 is called early disseminated Lyme disease, and stage 3 is called late disseminated, or persistent Lyme disease. The most frequently reported typical early sign is a rash called erythema migrans (EM). EM is an oval reddish skin eruption created at the site of the tick bite; this skin condition may gradually disappear. Other symptoms are: borrelial lymphocytoma, conjunctivitis, neuroborreliosis, Lyme carditis, etc. Therefore, generally recommended preventive measures should be used before each trip to an area infested with ticks in order to avoid contact with the ticks. Lyme disease is treated with antibiotics that are administered to a patient upon confirmation of the disease. As a rule the following antibiotics are prescribed: cephalosporin, macrolide, tetracycline or penicillin, depending on at what stage of the disease medication is prescribed and whether the patient shows typical Lyme disease symptoms. Laboratory diagnostics of Lyme disease relies on direct and indirect methods. The direct methods include: cultivation of Borrelia, hybridization, immunoelectron microscopy, and the PCR method (polymerase chain reaction) applied to prove the presence of borrelial DNA. Of all the methods, PCR is most often the method of choice. The indirect diagnostic methods are: indirect immunofluorescence, Western blot (WB) and ELISA tests (enzyme linked Immunosorbent Assay). These methods are used mainly to establish specific antibodies of the class IgM and IgG. The aim of this thesis was to analyze patients' samples for the presence of Lyme disease by means of the ELISA test and the Western blot, to test these methods, and to evaluate their results. The individual samples of serums, liquors and punctuates to be analyzed were provided by the Teaching Hospital in Pilsen, specifically by the Department of Serology and Parasitology. The samples must be diluted before examination. The method ELISA involved the use of a device manufactured by Dynex, DSX (an automated ELISA system). This device determines IgM and IgG antibodies in samples and facilitates analysis of up to 96 patient samples during a single measurement. The author of this thesis used this opportunity to try the manual determination of the ELISA test whose use is rather infrequent, especially in situations where a large number of samples need analyzing. The further examination of the IgM and IgG antibodies was accomplished with a Western blot, specifically by means of a commercial set for manual determination. The author applied the ELISA test to a total of 90 samples in the IgM class. 4 samples were borderline and 4 were positive. The same method was used to analyze the same 90 samples in the IgG class where 4 samples were positive. The Western blot method was used to examine 98 samples. The IgM class was borderline in 5 samples and positive in 18 samples. The IgG class was borderline in 5 samples and positive in 30 samples. Following laboratory examination the results of serological tests and clinical symptoms must be considered by a physician who suggests a suitable method to prove Lyme disease and exclude others.
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