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Most of the members of the Gram-negative genus Bordetella cause severe infections of the respiratory tract in their hosts. B. pertussis and specific lineages of B. parapertussis infect humans and cause the disease known as whooping cough, a highly contagious respiratory disease that is currently on the rise even in highly vaccinated populations. Therefore, a more detailed understanding of the B. pertussis interactions with the host is crucial. B. pertussis produces a great variety of virulence factors, majority of which is regulated by the two- component BvgAS system. These factors assist the pathogen in the colonization of the host and evasion of the host immune system. The studies on host-pathogen interactions use both in vitro and in vivo infection models, which complement each other appropriately. Recently, it was demonstrated that B. pertussis escape killing and persists in macrophages, suggesting that B. pertussis can be considered as a facultative intracellular pathogen. This ability may allow the pathogen cells to persist within the host and potentially spread to the new host. The aim of this bachelor thesis was to summarize the knowledge on the host-pathogen interactions between B. pertussis and its host with focus on in vitro and in vivo infection models. The attention is paid especially...

The aim of this study was to challenge the conclusion presented in the current recommendations of the EU Perstrain Group (European group of reference laboratories) [17] that immunoblotting methods are not appropriate serological methods for the diagnosis of pertussis because their results cannot be quantified. To consider benefits of these methods for the diagnosis of Bordetella infections was another aim of this work.The residual sera from routine testing intended for disposal and results of routine tests for Bordetella infections performed in Spadia Lab in 2015 and 2016 were used in this study. The test samples were anonymized. Standard commercial ELISA and immunoblot kits were used for analyses.Using the TestLine Clinical Diagnostics Company kits, we have shown that, contrary to the conclusion of the EU Perstrain Group, quantification of the immunoblot results is possible and that these methods can improve the diagnosis of Bordetella infections, ultimately making it more effective.

Diphtheria, tetanus and pertussis are very serious diseases threatening the lives of children and adults. However, these diseases are preventable by vaccination. After the World Health Organization was founded in 1946, its experts rapidly developed a vaccination schedule for the pediatric population. The former Czechoslovakia, a founding member of this organization, actively participated in the implementation of vaccination against many infectious diseases, including vaccinations against tetanus, diphtheria and pertussis. Mass vaccination of children could not be implemented in all countries of the European Region. The main problem was differences in the economic situation since the mass vaccination of the pediatric population represents a significant financial burden. It was only in the early 1970´s that the pediatric immunization program was initiated in developing countries. The article compares the impact of vaccination in different continents (Europe, Asia, Africa) since 1980. High immunization coverge is essential for a reduction in the incidence of the above infectious diseases. Cases of the diseases reported between 1980 and 2016 are cited in the article. Trends in these infectious diseases are related to the economic and political changes that occurred at the turn of the millennium.

To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered.DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967-1980, 1990-2007, and 2008-2015 (June).Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967-1980 while MT27 was the most prevalent in 1990-2007 and 2008-2015 (June). The DI was the lowest (0.49) in 2008-2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967-1980 and 0.654 for 1990-2007).MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.

Pertussis is a respiratory disease caused by the Gram-negative encapsulated bacterium Bordetella pertussis. Despite the high vaccination coverage rate and addition of new booster doses to the immunisation scheme (in response to the epidemiological situation), pertussis is on the rise not only in the Czech Republic but also in many other countries. The age groups at highest risk are infants and, to a lower extent, newborns who can get infected before receiving the first dose of vaccine and develop a severe course of the disease, often requiring admission to hospital. The most common source of infection are adults or adolescents from the childs close environment who experience a mild course of the disease because of the previous vaccination. The immune response induced by the currently available acellular vaccines does not last. It can be reasonably assumed that pertussis has been underreported. Multiple studies have shown mutations in the causative bacterium that confer higher pathogenicity to it, either as a result of enhanced production of pertussis toxin or loss of some antigens. Possible strategies to control these negative trends are to develop novel more effective vaccines using new adjuvants or to use whole-cell vaccines. Maternal vaccination in pregnancy trimester 3 also turned out to be effective.pertussis - vaccination - epidemiology - diagnosis - newborns.

Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...

Comparison of antigenic structures of Bordetella pertussis (B. pertussis) strains isolated from 1967 to 2010 in the Czech Republic.Seventy strains of B. pertussis were referred to the National Reference Laboratory (NRL) for Pertussis and Diphtheria within the surveillance of pertussis from all over the Czech Republic (CR) between 1967 and 2010. To study the strains, the analysis was performed of the genome sequences encoding the surface immunogenic structures--the pertussis toxin S1 subunit gene (ptxA), pertactin gene region 1 (prnA), type 3 fimbriae gene (fim3)--and pertussis toxin promoter (ptxP) responsible for the regulation of the production of pertussis toxin.For the study set of B. pertussis strains, the sequencing analysis revealed changes in all genomic regions studied. The isolates from three periods differ in the allelic profile. In period I (19671978) with the use of whole cell pertussis vaccine (wP), the following two profiles were the most common: ptxP(1), ptxA(2), prnA(1), fim3(1) and ptxP(1), ptxA(1), prnA(3), fim3(1). In period 2 (19902007) with the switch to acellular pertussis vaccine (aP), the most common profile was: ptxP(3), ptxA(1), prnA(2), fim3(2). Period 3 (20082010) with the use of aP was characterized by the predominance of the following two profiles which had never been found in period 1: ptxP(3), ptxA(1), prnA(2), fim3(2) and ptxP(3) ptxA(1), prnA(2), fim3(1).Sequencing of the genomic regions ptxP, ptxA, prnA, and fim3 of B. pertussis strains isolated in the CR between 1967 and 2010 confirmed changes in the allelic variants of these regions. The incidence of strains carrying the new allelic variants was increasing after 1995 at the expense of those carrying the original variants. The study results can be interpreted as a partial genetic escape of pathogenic strains of B. pertussis beyond the reach of the pertussis vaccines.

To characterize the epidemiological situation of pertussis in children under one year of age in the Czech Republic in 1997-2013.The study cohort consisted of children under one year of age with laboratory confirmed pertussis reported to the communicable disease system from 1997 to 2013. A total of 265 pertussis cases were reported in children under one year of age over the study period. Selected demographic data, need for hospitalization, and vaccination history were evaluated in the study cohort.Children under one year of age have shown a steady upward trend in reported cases of pertussis since the 1990s. The reported incidence of pertussis in this age group was the lowest in 1998 (1.1/100,000 population) and the highest in 2013 (31.3/100,000). In 1997-2013, 265 pertussis cases were reported in children under one year of age, 128 females and 137 males, to the communicable disease system in the Czech Republic. Most of these children, nearly 77%, developed pertussis within the first four months of life. Of the 265 children, 79% were not vaccinated before the onset of the disease and 21% were immunized with at least one dose of pertussis vaccine before developing the disease. As many as 75% of the children with pertussis needed hospitalization. Most of them, nearly 81%, were hospitalized with pertussis in the first four months of life and 90% of them in the first six months of life.In 1997-2013, an upward trend was observed in pertussis cases in children under one year of age. Most children developed the disease within the first four months of life while not vaccinated against pertussis. This fact unambiguously supports the "cocoon" strategy, i.e. vaccination of the closest contacts of the child, and a booster dose at 25 years of age. At the same time, a question arises whether to provide vaccination to pregnant women.

Bordetella pertussis, a gram-negative bacterium, is a human pathogen which affects the upper respiratory tract. It is the causative agent of whooping cough or pertussis. B. pertussis produces several virulence factors consisting of toxins and adhesins. Whole cell vaccine and subsequent acellular vaccine were developed against pertussis in the past. However, a gradual increase of pertussis incidences has been reported in the last twenty years. This thesis provides basic information about B. pertussis and whooping cough. The main aim of the herein presented work is to summarize the contemporary epidemiologic situation along with determining reasons for increased pertussis cases. In addition, possible solutions for the present situation are proposed.

The goal of this study is to summarize the results of the detection of Bordetella pertussis (BP) and Bordetella parapertussis (BPP) by a real-time polymerase chain reaction (RT-PCR) assay and serological methods. In 2008-2010, 73 patients of the Department of Clinical Immunology and Allergology of the Centre for Immunology and Microbiology, Public Health Institute in Ústí nad Labem were screened for pertussis. They were selected according to the WHO and ECDC criteria, i. e. they presented with a persistent cough lasting more than two weeks. Direct detection of BP and BPP DNA from nasopharyngeal wash specimens was performed using a RT PCR assay. The serological responses were evaluated by a direct agglutination test for the detection of total antibodies and by enzyme-linked immunosobent assay (ELISA) for the detection of IgG, IgA, and IgM antibodies against pertussis toxin. Forty-two patients were positive for BP and/or BPP, 19 of them by RT-PCR (group A) and 23 by serology (group B). Ten group A patients (52.6%) were also positive by serology. Our results show that pertussis needs to be a consideration in persistent cough. We believe that increased awareness of the medical community, along with improved laboratory tests will result in increased detection of pertussis that is still considered by many physicians as a childhood infection.

We present a case report of a small boy with cystic fibrosis complicated by pertussis and pneumothorax. The child had not been vaccinated against Bordetella pertussis due to the failure to thrive and was infected with the bacterium at the age of 4 months. The course of the disease was severe, with respiratory distress and spontaneous pneumothorax. The diagnosis of pertussis was serologically confirmed. A suspicion for cystic fibrosis increased after an unusually prolonged course of the disease and a history of the failure to thrive. A comprehensive diagnostic procedure revealed Pseudomonas aeruginosa respiratory infection, anemia, pancreatic insufficiency, a positive sweat test and the presence of two CFTR gene mutations. The patient, treated with comprehensive cystic fibrosis therapy, recovered from acute respiratory illness and started to thrive soon.

The thesis deals with the problem of the occurrence of whooping cough in the South Bohemian Region in the period of 2003-2012. Despite the high level of vaccination the incidence of this illness after a significant fall in the 1980s has been growing constantly since 1993. This trend evidently also applies in other developed countries with a high rate of vaccination. The main aim is to draw up an overview of the occurrence of whooping cough in the South Bohemian Region in the monitored period of the last 10 years, compare this occurrence with other regions in the Czech Republic and map out the changes which have occurred in whooping cough vaccination. Quantitative and qualitative research was chosen to process the empirical part. Data for quantitative research was acquired from the database of infectious diseases (EPIDAT), and consisted of reported cases of whooping cough. Another essential group of data were demographic figures about the number and age structure of the population. The second qualitative part was drawn up on the basis of a literary search. I acquired additional information by communicating with individual territorial workplaces of the Regional Hygiene Station of the South Bohemian Region based in České Budějovice. Using the processed data I discovered that the incidence of whooping cough in the South Bohemian Region does not have an ascending trend in 2003-2012 unlike the trend in the whole of the Czech Republic. The highest occurrence of whooping cough in the Czech Republic in the monitored period was reported in the 10-14 age group. The research also showed that in the period of 2003-2012 individual regions differed greatly in the occurrence and can be divided into two groups ? regions with the highest reached incidence below 10 cases/100,000 people and regions with incidence with the highest reached incidence above 10 cases/100,000 people which includes the South Bohemian Region. There have been many changes in the vaccination of whooping cough and the most important was the transition from whole cell vaccine to acellular in 2007 and adding a 6th booster dose between the 10th and 11th year in 2009. The greatest attention should be paid above all to increase information or test the knowledge of the lay public, especially women preparing for pregnancy and people taking care of newborn babies and infants about the occurrence, potential risks and possibilities of vaccination against this infection even at an adult age. In my opinion a further necessity is the consolidation of diagnostics, definition of a case and procedures of surveillance throughout the Czech Republic, education of paediatricians and general practitioners about the illness and topicality of the problem, an appeal to report the occurrence of this infection to epidemiologists, observing the isolation of the patient, monitoring possible epidemiological connections between cases and making use of the offer of the NRL (National Reference Laboratory) services against whooping cough and diphtheria. This thesis can be used in practice as a summary of information material for the lay and professional public about the occurrence of whooping cough in the South Bohemian Region and throughout the Czech Republic and overview of vaccination against whooping cough. It can be an incentive for further anti-epidemic measures and the need for further research into this problem.

Bordetella pertusis is a Gram-negative, aerobic, non-spore-forming coccobacillus. Although it's strictly human pathogen, it's possible to infect other mammals at laboratory conditions. Transmission among hosts is mediated via respiratory tract droplets. Infection could be direct, host to host, alternatively by contaminated environment. Bordetella colonizes upper respiratory tract, wherefrom descends into lungs and causes disease known as whooping cough or pertussis leading to 195 000 deaths of 16 mil. incidences per year (according to WHO report from 2010). More than twenty years before, respectively to found pertussis toxin, that time intensively under examination, pertussis was marked as toxin-mediated disease. In the course of time, more other virulence factors were revealed, that could be divided into groups of adhesins, toxins and others. Adhesins are filamentous haemagglutinin, pertactin and fimbriae. Toxins include pertussis toxin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin and lipopolysaccharide. Most of virulence factors are regulated by two component system Bvg. However, it is needed lots of other factors for successful infection as for example autotransporters or so called siderophores serving as iron acquisition from environment. Secretion of virulence factors is mediated by its...

Current Issues Linked to Bordetella pertussis and Bordetella parapertussis Abstract Bordetella pertussis and Bordetella parapertussis are causative agents of infections affecting airways. These agents are Gram-negative, aerobic coccobacillus of the genus Bordetella and the family Brucellaceae. Subjects infected with bacteria Bordetella pertussis develops into a highly contagious infection called whooping cough. Those infected with Bordetella parapertussis show milder clinical symptoms than those infected with Bordetella pertussis. The highly contagious infection affects the airways and is considered to be one of the most serious infections found in children. Before proper vaccine was discovered, this infection caused high mortality especially among one-year-old babies and younger. The most vulnerable group of subjects comprises of infants. Infection is even observed in subjects of 10+ years of age and may infect even the adults that are identified as the source of infection who subsequently infect their children. In case a subject is suspected of being infected with the genus Bordetella pertussis or Bordetella parapertussis, it is important to test a proper biological sample, i.e. epipharynx swab. Objective of the thesis: The objective of the thesis is to acquire practical skills in laboratory work. This concerns bacterial culture tests of epipharynx swabs taken from subjects suspected of being infected with bacteria Bordetella parapertussis and Bordetella pertussis on one hand, and assessment of sensitivity to antibiotics in case of isolated genera Bordetella parapertussis and Bordetella pertussis on the other. Also, it is meant to identify to what extent the two bacteria differ from each other. Another objective was to find out and assess the data collected in the region of Jindřichův Hradec and gathered from samples tested by Laboma s.r.o. microbiological laboratory that concerned the occurrence of the bacteria Bordetella parapertussis and Bordetella pertussis in each of the past 3 years. The data gathered in relation to the occurrence of the bacterium were processed statistically. Subsequently, medical procedures carried out in case of suspicion of airways infection caused by the bacteria Bordetella pertussis were scrutinized. The information was collected by questionnaires. Hypotheses: Hypothesis 1: Culture diagnosis of Bordetella parapertussis will be less time demanding than diagnosis of Bordetella pertussis. Hypothesis 2: The occurrence of Bordetella pertussis and Bordetella parapertussis is higher in autumn and spring periods. Hypothesis 3: The occurrence of Bordetella pertussis and Bordetella parapertussis is most frequent among the children of 10-14 years of age. Conclusion: The outcomes of this bachelor?s thesis show that cultures of Bordetella pertussis and Bordetella parapertussis are demanding bacteria in terms of growth. Bordetella parapertussis differs from Bordetella pertussis not only in terms of the course of bacterias cultures, but also in terms of appearance, biochemical properties, and sensitivity to antibiotics. Hypotheses 1, 2, and 3 were confirmed following the outcomes of the performed research.

To analyze the incidence of pertussis in the Czech Republic, influencing factors and, in particular, the effect of vaccination on pertussis morbidity, clinical seriousness of the disease and circulation of Bordetella pertussis in the population. To study the causes of defective diagnosis and reporting and to propose remedial measures.Data on pertussis morbidity were obtained from the archives of the National Institute of Public Health and the public health information systems ISPO and EPIDAT. Mortality data were taken from the above sources and literature. The case definition was used as specified in the Methodical Guidance for Pertussis Surveillance and the EC Directive. Laboratory diagnosis was based on culture and serology. An at least fourfold increase in the serum antibody titer found within an experiment was considered as a positive result.Pertussis together with diphteria and measles used to be among the most dangerous infections in childhood. The oldest mortality data date back to 1890 when 62 deaths per 100,000 population were reported. In 1951, the death rate still reached 3.6/100,000. The pertussis morbidity peaked in 1955 with 540 cases per 100,000 population. Vaccination against pertussis since the early 1950's led to a rapid reduction of morbidity in children. Nevertheless, higher pertussis morbidity rates were observed at 2-4-year intervals (in the so- called epidemic years). The lowest morbidity rates were reported during the 1980's. However, a stable upward trend has been observed over the following years. In 2006, a neonate died from pertussis. The age specific morbidity rates in 1980 through 2000 were highest in children under one year of age. This fact together with the regularly increased morbidity rates observed at 2-3-year intervals indicate that Bordetella pertussis still circulates in the population. While in the 1980's, the cases of pertussis were reported almost exclusively in children under one year of age, in the 1990's, they became more common also among children 1-4 years of age and started to be prevalent in children 10-14 years of age since 2001.A high immunisation coverage (97%) with five doses of high quality whole-cell vaccine of Czech origin introduced into practice in 1958 played a crucial role in the reduction of pertussis morbidity from more than 500/100,000 in the mid-1950's to less than 0.5/100,000 in the 1980's. Nevertheless, this strategy did not lead to elimination of the causative agent in the population. It is evident that the current immunization scheme with the use of the available vaccines cannot solve the epidemiological situation. Since the efficacy of the currently available acellular pertussis vaccines is, at the best, the same as that of the whole-cell vaccines, any improvement of the current status cannot be expected. Only effective active surveillance, the use of new more immunogenic pertussis vaccines and revaccination of older age groups can result in desirable outcomes.

In the differential diagnosis of protracted irritating cough we should always consider the possibility of pertussis. Serology performed primarily in a late stage of the disorder does not always provide a clear answer. We wanted to verify whether a quantitative determination of IgG antibodies to the pertussis toxin (IgG-PT) could help establish a clear diagnosis.Between 1 January and 30 June 2005 we performed serological investigations in 139 children presenting with an irritable cough or after application of an acellular pertussis vaccine. In 95 children we compared the serological response of agglutination antibodies and IgG-PT.After vaccination the children presented with different levels of antibodies and these were not always identical in two types of serological response. Children with clinical manifestations of pertussis presented a good formation of antibodies, but the two kinds of antibodies often persisted for long periods at unchanged levels.The determination of IgG-PT can assist the diagnosis of the disease, but this investigation alone cannot yield a clear-cut confirmation of pertussis.

In the course of six months the authors investigated 32 children aged 1-32 months with pertussoid cough. This number included one child where pertussis was confirmed in a 25-month-old properly vaccinated child and parapertussis in an 8-month-old incompletely vaccinated infant. In the remaining children by cultivation or serological examination a different aetiology was found (RS virus, adenovirus, parainfluenza 2, M. pneumoniae, H. influenzae, Branhamella catarrhalis).