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Optimization of a loop-mediated isothermal amplification method for a detection of Haemophilus influenzae infection.
Zadáková, Kateřina ; Jeřábek, Petr (advisor) ; Svášková, Dagmar (referee)
Haemophilus influenzae is a gramnegative, rod-shaped and predominantly human pathogen. Infection caused by this microorganism is especially dangerous for children under five years of age, who are mainly at risk of pneumonia and meningitis.Althought vaccination is available against its most dangerous type b, it still causes problems, especially in developing countries. Due to the insufficient vaccination, strains that are more resistant to the antibiotics are more spreading in these countries. Equipment requirement of current detection options for H. influenzae infection(e.g. polymerase chain reaction) causes, that reliable detectionis a problem in poor countries. Loop-mediated isothermal amplification could be a cheap and reliable method. The aim of this bachelor thesis was the design of sets of primers and their applicationinthe LAMP reaction. The designed primers targetedthe ompP6 gene, which was alreadythe target of LAMP reaction in some studies and is also a frequent target in PCR detection.As a part of the optimization, different volumes of the reaction mixtures and multiple detection options were tested -colorimetric detectionusinganacid-base indicator and fluorescence detection using different dyes monitored in real time. Key words: Haemophilus influenzae, loop-mediated isothermal...
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Comparison of different molecular-biological approaches for detecting the presence of DNA of the pathogenic bacterium Haemophilus influenzae
Smrčka, Tomáš ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Haemophilus influenzae is one of the main initiators of meningitis and pneumonia in children. Implementation of fast, cheap and instrumentally accessible method for detection of this pathogen would enable an early and targeted treatment of patients. The development of amplification methods in the last decades enables, apart from commonly used PCR method, application of alternative approaches, such as the LAMP. The focus of this Bachelor thesis was the study (research) of the alternative method LAMP as a tool for detection of Haemophilus influenzae. The LAMP method was successfully implemented for Haemophilus influenzae, however, it has contended with the false positive results of negative control in case of longer incubation times. Therefore, the optimized LAMP method was designed in presence of deoxyuridine triphosphate and uracil-DNA glycosylase. Its aim was to change the structure of LAMP products via the incorporation of uracils to amplified regions of DNA and subsequent removal of uracils with influence of uracil-DNA glycosylase, and therefore prevent their replication during potential contamination of reaction mixtures and consequently reduce the risk of false positive results of negative controls to minimum. The concentration of deoxyuridine triphosphate in reaction mixtures was optimized...
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The Spanish Flu Pandemic 1918/19 with particular reference to the Bohemian Lands and Central European relations
Salfellner, Harald ; Hlaváčková, Ludmila (advisor) ; Fialová, Ludmila (referee) ; Pock, Lumír (referee)
Charles University First Medical Faculty Study programme: History of Medicine Summary of dissertation The Spanish Flu Pandemic 1918/19 with particular reference to the Bohemian Lands and Central European relations Dr. med. univ. Harald Salfellner Prague, 2017 Summary Towards the end of the First World War, in 1918 and 1919, humanity faced a previously unparalleled flu pandemic; within a few months, more people had been killed than in all the battles of the 1914-18 war put together. The precise number of victims is unknown but is today generally reckoned at between 20 and 50 million. The whole world was affected by the Spanish flu, with the exception of a few remote islands, and Europe, already bled to death by industrialised warfare, was particularly hard hit. In summer 1918, the pandemic reached Bohemia in an early, relatively benign wave. A few weeks later, thousands were struck down in Prague in a second and far more deadly phase of the illness. In October 1918, as the First Czechoslovakian Republic arose from the ashes of the multiethnic Austrian state, and the masses celebrated in the cities, thousands of feverish patients were coughing behind drawn curtains, and facing an uncertain fate. In the USA, the flu pandemic - the greatest health disaster of the 20th century - has been the subject of many...
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Beta-lactamase positive Haemophilus influenzae in children
BROMOVÁ, Pavla
This Bachelor thesis, called {\crqq}Beta-lactamase Positive Haemophilus influenzae in Children`` deals with the basic properties and current occurrence of the bacteria Haemophilus influenzae, and also with the occurrence or other hemofils (Haemophilus species) in children, which occurred in the biological material sent to the microbiological laboratory- Laboma s.r.o. in České Budějovice, where some of them were producers of {$\beta$}-lactamase. The occurrence of these enzymes might be a serious problem for the right choice of antibiotics for the treatment of diseases caused by these hemofils. The essential objective was to collect and assess all data about {$\beta$}-lactamase positive hemofils in the material from airways of children from birth to 15 years of age and with a certain numbers of diagnoses in the aforementioned microbiological laboratory. The data for my thesis was collected in the second half of 2008 and first half of 2009 and was later statistically processed into graphic figures and charts in the form of results. I have also learned to use an easy and used diagnostic method for the verification of {$\beta$}-lactamase ({$\beta$}-LACTAstrip test), during which the color was changed in the zone of the indicator paper if {$\beta$}-lactamase was present. I have also visited pediatricians, who co-operate with this laboratory. They were willing to reply to four questions in my questionnaire. The main objective of these questions was to focus on the treatment of ill children whose biological material from upper airways shows present {$\beta$}-lactamase positive hemofils. From the collected data I managed to reject hypothesis No. 1 by calculating the average relative quantity from the total occurrence of hemofils producing {$\beta$}-lactamase, and I have found that the production of these enzymes is lower by 50 %. Then, I confirmed hypothesis No. 2 that more common producers of {$\beta$}-lactamase are other hemofils (H. species) than H. influenzae. From replies provided by pediatricians, I found out that all of these pediatricians take the presence of {$\beta$}-lactamase into consideration when they chose antibiotics and this confirmed hypothesis No. 3. This Bachelor thesis can be used as an argument for pediatricians to take into consideration the presence of {$\beta$}-lactamase in the biological material of producers, when they choose antibiotics.
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