Dissertations / Theses on the topic 'GBS infection'

Group B Streptococcus (GBS) is a species of gram positive bacteria representing a significant human pathogen; namely, as the most prolific cause of neonatal disease and mortality globally, but also increasingly reported in adult disease (especially among the elderly and those with compromised immune systems). The first chapter of this thesis reviews the extensive literature covering GBS; with a focus on classification and disease. Selected virulence factors are also discussed. In chapter 2, eight strains of GBS were selected for whole genome sequencing by Third Generation, Pacific Biosystems (PacBio) sequencing technology. A protocol was optimised to provide sufficient, high quality genomic preparations from multiple strains of GBS – suitable for the PacBio technology. Using a sequenced strain of GBS from chapter 2, an infection model was optimised in chapter 3 for the purpose of providing quality RNA for co-transcript analysis. U937 human monocytes were infected with GBS (strain 874391) and the host/pathogen RNA prepared from the same reaction (monocytes with internalised GBS) – with an emphasis on yielding sufficient pathogen RNA; which can sometime be an impediment for co-transcript studies. Pathogen RNA derived from the optimised infection protocol was demonstrated to amplify with RT-qPCR for 12 tested GBS genes (cylE, 1010, rib, czcD, pil2B, cpsE, scpB, htp, cfb, copA, hvgA and maeA). In chapter 4, RT-qPCR was used to analyse differential gene expression from the mixed, host/pathogen RNA. Twelve human genes and 12 GBS genes were assessed for differential gene expression. Seven of the tested human genes (IL8, IL1A, IL1B, IL10, TNF, LMO2 and MCP-1) and 6 of the tested GBS genes (scpB, 1010, rib, czcD, htp, hvgA) were significantly upregulated in RNA derived from the infection samples. Of the GBS genes tested, htp was the most upregulated. An htp knockout mutant of GBS strain 874391 (Δhtp) was constructed for chapter 5 of this thesis to assess the impact of htp transcription on GBS survival in an infection context. The infection assays optimised in chapter 3 were performed with the Δhtp GBS construct. Contrary to expectation, the Δhtp GBS construct survived the internalised environment of the monocytes in significantly higher numbers than the wild-type over 48 hours of infection.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Carbohydrates are among the most abundant molecules found on the cell surfaces of bacteria, parasites, and viruses. Apart from the conventional roles of carbohydrates as energy sources and structural polymers, carbohydrates are also associated with cancer metastasis, protein stabilization, pathogen infection and the immune response. Cells of our body have sensors made out of carbohydrates on outer surface of plasma membrane and acts as sensors and can detect many kinds of stimuli, and can signal the immune system to respond. Carbohydrate-protein molecular recognition processes have pivotal roles in infections and in immune response to pathogens. To date, several vaccines based on isolated capsular polysaccharides (CPSs) are marketed against infectious diseases. However, the use of isolated capsular polysaccharide poses several limitations, as natural sources are generally limited and the isolation is very challenging. Additionally, the isolated polysaccharides are heterogeneous and often contains impurities. Furthermore, limited protection of certain CPS antigens impairs the efficiency of vaccines. To overcome limitations associated with isolated polysaccharides, synthetic oligosaccharides present an effective alternative with great potential to understand glycan immunology and rationally design effective antigens. Consequently, characterization and reconstruction of carbohydrate epitopes with authentic composition has become one of the major target in glycoscience. To this end, strategies are needed to facilitate the streamlined design and generation of these antigens. This thesis concerns the development of an effective synthetic strategy to obtain Group B Streptococcus (GBS) type II oligosaccharide for vaccine development. GBS, a Gram-positive bacterium, inhabits the intestinal and genitourinary tract of 10‐30% of humans. GBS is one of the primary causes of bacterial infections among neonates and pregnant women, resulting in many severe diseases such as sepsis, meningitis, abortion, and so on. Type II GBS is one of the predominant GBS serotypes and is associated with about 15% of the invasive infections in adults and infants; therefore, represents an important human pathogen. The development of effective preventive vaccine against GBS is much needed to help pregnant women protect their newborns. This thesis describes the effective synthetic strategy to synthesize GBS type II oligosaccharide to be applied for vaccine development. Herein, we present a new and convenient synthesis of the repeating unit of GBS type II capsular polysaccharide. The structure of GBS type II was elucidated in 1983 and the repeating unit of GBS type II is a heptasaccharide composed of α-Neu5Ac (2-3)-ß-D-Gal-(1-4)- ß-D-GlcNAc-(1-3)-[-ß-D-Gal-(1-6)]-ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc. The presented synthetic strategy is based on the five subcomponents derived from the retro synthetic analysis. Suitably protected lactosamine and lactose derivatives are pivotal building blocks in our synthesis and both disaccharide fragments have been achieved from the cheap and readily available lactose. Having started from two disaccharides saves the efforts of glycosylation and reduces the number of synthetic steps. The building blocks have been obtained in good overall yield following the optimized synthetic approach. The synthesis of backbone linear chain trisaccharide [ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc] and pentasaccharide [ß-D-Gal-(1-4)-ß-D-GlcNAc-(1-3)-ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc] has been achieved in excellent yield (~80% yield). The final steps of the synthesis comprise- the incorporation of ß-D-Gal unit into the linear chain pentasaccharide (currently ongoing) followed by the enzymatic introduction of sialic acid (NeuNAc unit) and subsequent deprotection to yield the repeating unit of GBS type II capsular polysaccharide. To conclude, in this thesis we present an efficient and easy handling synthetic approach to the heptasaccharide repeating unit of GBS type II. Readily available and cheap dairy side-product lactose has been used as a key structure in the presented scheme, allowing the efficient synthesis of the pentasaccharide backbone of the target compound. The synthetic GBS II fragments will be used for glycan array and structural studies and immunochemical characterization with specific monoclonal antibodies. This thesis comprises of four main chapters and the experimental section containing the methods and synthetic procedures for the discussed schemes. Chapter one is a general introduction and deals with the necessity and the social importance of the described project. Chapter two of the thesis outlines the scientific background and pathogenesis of GBS, carbohydrates and their biological importance, and general introduction of vaccines and how the carbohydrates can be used as a suitable vaccine candidate. Chapter two establishes the importance of synthetic carbohydrates and how the synthetic carbohydrates can be used to develop suitable effective vaccines against GBS diseases. Chapter three of the thesis contains the general introduction and structural features of GBS II CPS and the retrosynthetic analysis of GBS II CPS to identify the building blocks for the synthesis of GBS CPS II. Chapter four of the thesis summarizes the synthetic strategies and results to achieve the building blocks described in chapter three and the recombination of fragments to achieve the final molecule GBS II CPS repeating unit. The last part of the thesis will consists of the experimental methods and synthetic procedures to achieve the proposed molecule along with the characterization data.

Urinary tract infection (UTI) is one of the most common infections and costs millions of dollar each year to the health care industry. Escherichia coli is the causative agent responsible for over 80% of UTIs. However, other pathogens such as Group B streptococcus (GBS), a Grampositive bacterium, also cause UTI. In addition, GBS is an important commensal microbe in the female genital tract. Researchers have used various murine models to study mechanisms of disease pathogenesis for UTI and microbe colonisation of the genital tract. However, there is a lack of understanding of the pathogenic mechanisms and microbe virulence factors involved in UTI and genital tract colonisation due to both E. coli and GBS. This thesis reports on a series of experiments using murine models to better understand the pathogenesis of E. coli and GBS infection in the urogenital tract. It explores the use of several murine models to better understand host responses to these infections. In one series of experiments on both E. coli and GBS, murine models were utilised to define the global host immune response to these bacteria in UTI on a genome-wide scale with the aid of microarrays. Another series of experiments used a murine model of E. coli UTI to study the virulence factor α-hemolysin and how this factor influences host responses. The thesis also describes the development of a novel GBS long-term genital tract colonisation model in mice that was used to define the nature of GBS colonisation and survival in this infection. Thus, a series of novel murine models are described in the thesis that were applied in various host response studies and virulence assays to better understand how E. coli and GBS survive and cause infection in the urogenital tract in vivo. The application of murine models to study urogenital tract infection as described in this thesis provides researchers with a solid platform to examine the effects of virulence factors, and host responses in more detail in future studies. One particular area for future research relates to the specific series of experiments in which a murine UTI model was used to test the efficacy of prophylactic use of a novel E. coli 83972 ‘probi otic strain’ expressing a P fimbriae oligosaccharide receptor mimic to prevent acute infection. In this way, the data described in this thesis also provides vital new insight into how murine models can be used to test possible novel treatment strategies for preventing UTI.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Group A Streptococcus (GAS) is a globally disseminated pathogen that causes >500,000 deaths yearly and is ranked as ninth leading infectious cause of human mortality by the World Health Organisation. The spectrum of disease ranges from superficial infections of the skin and epithelium to invasive and systemic infections. Although the interaction of GAS with neutrophils has been extensively studied much remains to be discovered about the role of macrophages, which are the first line of defence encountered by invading pathogens. In this study, the aim was to establish a means of deriving macrophages from primary monocytes and to study both the efficiency of macrophage killing of GAS and the macrophage cell death response to GAS infection. Here, we report that monocyte-derived macrophages are able to take up and kill GAS during in vitro infection. Production of reactive oxygen species by macrophages was elicited during infection, but not nearly in as high amounts as produced by neutrophils. Investigating the type of cell death induced by GAS, markers for both apoptosis and necroptosis can be found after 8 hours of infection. These results highlight that macrophages indeed are participating in the clearance of GAS and more studies are needed to understand the roles of macrophages in early control of GAS infection.

Twenty four percent of all HIV infections in the United States occur among adults aged 50 and older (mature adults), yet little is understood of the dynamics of HIV infection among this group in Texas. Data from 1999 to 2009 examined the relationship between HIV spatial and temporal patterns affecting socio-economic and demographic variables including poverty, gender, race/ethnicity and mode of exposure. Results revealed highest HIV infection rates among White homosexual men, Black males engaged in IV-drug use, Black female heterosexuals and minorities in poverty. Concentrations of HIV infection among mature adults were located primarily in urban centers of Houston and Dallas and indicated increasing HIV infection rates from 1999 to 2009. These results will assist future allocation of resources by zip code in urban areas for this understudied population.

WN virus has spread for over 60 years creating endemic and epidemic areas throughout Africa, Asia, and Europe, affecting human, bird, and equine populations. Its 1999 appearance in New York shows the ability of the virus to cross barriers and travel great distances, emerging into new territories previously free of infection. Spreading much faster than expected, WN virus has infected thousands of birds, equine, and humans throughout the conterminous United States (US). Case and serological studies performed in the Eastern hemisphere prior to 1999 offer detailed descriptions of endemic and epidemic locations in regards to geography, land cover, land use, population, climate, and weather patterns. Based on the severity of WN activity within each study area, the patterns associated with these environmental factors allow for the identification of values associated with different levels of risk. We can then model the landscape of the disease within the US and identify areas of high risk for infection. State and county public health officials can use this model as a decision-making tool to allocate funding for disease prevention and control. Dynamic factors associated with increased transmission, such as above average temperature and precipitation, can be closely monitored and measures of prevention can be implemented when necessary. In turn, detailed information from higher resolution analyses can be documented to an online GIS (Geographic Information System) that would contribute to a global collaboration on outbreaks and prevention of disease.
Master of Science

Das 1995 entdeckte GB-Virus C (GBV-C) gehört als Pegivirus zur Familie der Flaviviridae und ist nichtpathogen. In Industrieländern sind 2 bis 12,5 % der gesunden Bevölkerung und bis zu 45 % der Personen aus Risikokollektiven, z.B. Patienten mit Infektionen mit dem humanen Immundefizienzvirus Typ 1 (HIV-1) oder dem Hepatitis-C-Virus (HCV), virämisch. Die Mehrzahl der klinischen Studien und Metaanalysen zu GBV-C/HIV-1-Koinfektionen zeigten, dass GBV-C mit einem verlangsamten Krankheitsverlauf und einer erhöhten Überlebenswahrscheinlichkeit von GBV-C/HIV-1-koinfizierten Patienten korreliert. In der Hemophilia Growth and Development Study konnte dieser Effekt bei GBV-C/HCV-/HIV-1-infizierten Kindern und Jugendlichen jedoch nur bedingt nachgewiesen werden. Dafür wurde ein Zusammenhang zwischen einer GBV-C/HCV-Koinfektion und dem Ausheilen der HCV-Infektion beobachtet und in einer weiteren Patientenkohorte aus der Anti-D-Studie bestätigt. GBV-C/HCV-koinfizierte Patienten haben schlechtere Chancen, die HCV-Infektion auszuheilen. Der Einfluss von GBV-C auf die HIV-1-Replikation wurde in Zellkulturexperimenten untersucht. Es zeigte sich, dass sich die verschiedenen GBV-C-Isolate hinsichtlich ihrer inhibitorischen Kompetenz unterschieden. Folgende mögliche Ursachen wurden untersucht: 1.) die IRES-Aktivität als Indikator für die Translationseffizienz, 2.) die NS5A-Sequenz des in der Literatur beschriebenen HIV-1-inhibitorisch aktiven 16mer-Peptids sowie 3.) die E2-Sequenz und die HIV-1-inhibitorische Wirkung von 18mer-E2-Peptiden. Es konnten weder Unterschiede in der IRES-Aktivität noch in der NS5A-Sequenz zwischen den unterschiedlich inhibitorisch-kompetenten GBV-C-Isolaten nachgewiesen werden. Im E2-Protein hingegen wurden zwei für alle HIV-1-nichtinhibitorischen GBV-C-Isolate einheitliche Mutationen, E143K/H und T204A, identifiziert. Diese könnten eine Ursache für die Varianz in der Fähigkeit, HIV-1 zu inhibieren, darstellen. Die Mutation an Position E143 ist an der Oberfläche des nativen E2-Proteins exponiert und spielt möglicherweise im Hemmmechanismus eine wichtige Rolle. Hinweise darauf gaben die Untersuchungen mit synthetischen 18mer-Peptiden, von denen das Peptid mit dem größten inhibitorischen Potenzial die Aminosäure an Position 143 beinhaltete. Eine mögliche Theorie des Wirkmechanismus des E2-Proteins wäre wie folgt denkbar: Das E2-Protein interagiert über eine Domäne um die Aminosäure E143 mit dem gp41 des HIV-1, verhindert somit die Fusion von Virus- und Zellmembran und in der Folge den Eintritt des HIV-1 in die Zielzelle.

With a case fatality rate higher than the 1918 Spanish Flu pandemic, H5N1 highly pathogenic avian influenza represents a threat to global public health. Efforts to identify locations with the greatest potential for pandemic emergence, as well as how the virus is spreading, may help minimize this threat. First detected in Egypt in 2006, H5N1 viruses have resulted in the deaths of millions of birds in both commercial and backyard poultry flocks, and more than 350 human infections, the most of any country, have been confirmed. Human outbreaks have been so far constrained by poor viral adaptation to non-avian hosts. There are two evolutionary mechanisms by which the H5N1 avian influenza virus could acquire pandemic potential: 1) via reassortment as a result of coinfection with another subtype (such as low pathogenic avian influenza H9N2); and/or 2) via antigenic drift and the accumulation of randomly occurring genetic changes found to improve viral fitness, herein called key substitutions (KS). Both mechanisms were investigated using geospatial methods including ecological niche modeling and hot spot analyses to predict locations with elevated potential for pandemic emergence. Using ecological niche modeling environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt were identified, with niches differing markedly by subtype. Niche estimates were combined using raster overlay to estimate co-infection potential, with known occurrences used for validation. Co-infection was successfully predicted with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991). 41 distinct KS in H5N1 were detected in Egyptian isolates, including 17 not previously reported in Egypt. Phenotypic consequences of detected KS were varied, but the majority have been implicated in improving mammalian host adaptation and increasing virulence. Statistically significant spatial clustering of high KS rates was detected in the northwestern portion of the Nile River delta in the governorates of Alexandria and Beheira. To investigate how the virus spreads between poultry farms, landscape genetics techniques were employed. Viral genetic sequences were evaluated using phylogenetics to determine viral relatedness between samples, then distance models representing competing diffusion mechanisms were created using road networks and a least-cost path model designed to approximate wild waterbird travel using niche modeling and circuit theory. Spatial correlations were evaluated using Mantel tests, Mantel correlograms, and multiple regression of distance matrices within causal modeling and relative support frameworks. Samples from backyard farms were most strongly correlated with least cost path distances, implicating wild bird diffusion, while samples from commercial farms were most strongly correlated with road network distances, implicating human-mediated diffusion. Results were largely consistent across gene segments. Identifying areas at risk of co-infection can help target spaces for increased surveillance. Similarly, detecting spatial hot spots of KS highlight areas of concern for pandemic emergence from antigenic drift. Demonstration of different diffusion mechanisms by farm type should inform both surveillance and biosecurity practices. Knowledge of where to focus intervention efforts, both spatially and strategically, allows limited public health resources to be targeted most effectively. By detecting where in the country pandemic influenza is likely to emerge and identifying how the virus is spreading between farms, this work contributes to efforts to predict and prevent the next influenza pandemic.

Urinary tract infections (UTIs) are among the most common infections caused by both Gram-negative and Gram-positive bacteria, acquired in the community and hospitals. There are three main groups of UTIs: (i) lower UTIs that affect the urethra or the bladder, (ii) upper UTIs that affect the kidneys, or (iii) asymptomatic bacteriuria (ABU). Group B Streptococcus (GBS) is a Gram-positive bacteria known to cause a variety of infections in neonates, pregnant women, the elderly or immunocompromised individuals. GBS has been estimated to cause 1-2% of all single organism UTIs. GBS has been shown to form biofilms, on abiotic and biotic surfaces, protecting it from killing by antibiotics or host immune cells and promotes host colonisation. Various factors have been shown to affect the biofilm forming ability of GBS. Here we determined that LB supplemented with glucose was the optimal media for biofilm formation by a strong biofilm forming strain. We then investigated the biofilm forming phenotype of 292 clinical GBS isolates that presented with asymptomatic, acute, or recurrent infection. We found that there was no significant difference in the biofilm forming ability across the clinical presentations. We also showed a significant reduction in the biofilm forming ability of a strong biofilm forming strain and its isogenic maeK and maeE mutants in LB supplemented with 1% glucose. A multiplex PCR screen for genes encoding bsaB, pil1, pil2a, and pil2b found that there was no significant difference in the number of strains that had the right sized fragments for all four genes across the three clinical presentations. We also found that there was a significant difference in the proportion of strains that had the right sized fragments for the pil genes across the three different levels of biofilm activity under shaking conditions. High biofilm forming strains had the lowest proportion of strains that possessed all four genes, compared to low and medium biofilm formers. Lastly, we assessed the haemolytic activity of the strains by growing them on tryptic soy agar plates supplemented with 5% horse blood and found that asymptomatic strains had a significantly higher number of strains with high haemolytic activity compared to acute and recurrent strains.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
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Clostridium perfringens is the causative agent of gas gangrene. A lethal infection in mice requires a large inoculum suggesting that the immune system is involved in inhibiting disease. Human monocytic cells and neutrophils killed C. perfringens in vitro when complement was present. Macrophages and neutrophils co-localized with C. perfringens in vivo when bacterial numbers were low. Depletion of neutrophils and monocytes in mice revealed that monocytic cells play a role in inhibiting C. perfringens gas gangrene in mice infected with an intermediate dose. C. perfringens can persist in the tissues and this could be mediated by persistence within macrophages. To examine if the toxin perfringolysin O (PFO) could mediate this, less active variants of PFO were used to examine what occurs between phagosomal escape and cell lysis. The mutant forms of PFO did mediate phagosomal escape in macrophages and were found within macrophages at higher numbers than wild-type C. perfringens. Our data were preliminary but may indicate that less active PFO mediates intracellular persistence. To investigate the role of sialidase in C. perfringens gas gangrene we made nanI-, nanJ-, and nanI-/nanJ- mutants. We observed that NanI is responsible for the majority of sialidase activity of C. perfringens strain 13, that NanJ is an extracellular sialidase, and that these genes are transcriptionally regulated by sialic acid. Murine infection trials revealed that these sialidases may be protective for mice during infection. In conclusion, murine monocytes inhibit disease onset and C. perfringens sialidase enhances mouse survival. However, the toxin PFO if less active promotes the survival of C. perfringens with macrophages.
Master of Science

This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia. Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers. In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis. In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution. In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens. Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection. In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.

Chlamydia is a sexually transmitted infection. The infection rates have increased in the last decade. During 2008 the trend rates declined in all counties and regions in Sweden. Chlamydia infections have a geographical spatial pattern differences. This is seen from the national surveillance data report from the Swedish Institute for infectious disease control (SMI). The overall aim with the study is to conduct a geographical analysis of Chlamydia infections and explain the differences in the spatial pattern. The study is limited to the counties and a region that have been using Becton Dickinson laboratorial analysis methods. The study is based on statistics on Chlamydia infections incidence rates, from the SMI. The statistics did raise questions regarding the spatial patterns differences. Therefore some of the chosen counties and region were contacted through both telephone and email. Further information search was conducted on internet sites for some of the government authorities: The National Board of Health and Welfare, Swedish National Institute of Public Health etc. The mentioned authorities along with the SMI, play important roles in the area of sexually transmitted infections (STI).

Geographical spatial pattern differences of Chlamydia in the chosen counties and region can be explained on the basis of several factors. Statistics have been influenced by the fact that individuals have tested themselves in other areas/locations and not the locations where they have their permanent addresses (home areas). The quantities of taken samples and the effectiveness regarding the processes taken to reduce Chlamydia infections have led to detection of several cases and control of further infections. The public has more place access and opportunities of carrying out the tests (both in the region, the counties and through internet). The possibility to conduct detailed geographical studies has been limited because of the missing data at the municipality level. However, the study has raised questions that need further analysis. The previous research of Chlamydia infections has shown that other countries for example USA have succeeded in conducting detailed research studies of Chlamydia infections by using  geographic information systems (GIS) methods. Are there possibilities to conduct such research in Sweden with the current Swedish statistics on Chlamydia infections?

 

Klamydia är en sexuell överförbar infektion. Klamydiainfektioner har under det senaste decenniet ökat på nytt i Sverige. År 2008 skedde en trendminskning i alla landsting och regioner. Utgångspunkten är att det finns mycket kunskap i samhället om klamydia och skyddsåtgärder för sjukdomen. Geografiska skillnader vad gäller spridning av klamydiainfektioner förekommer, vilket framgår av statistik från alla 21 landsting/regioner iSverige. Det övergripande syftet med studien är att göra en geografisk analys av klamydiainfektioner och ge en förklaring till förekommande skillnader av det rumsliga mönstret. Studien begränsar sig till landsting och regioner som använt sig av Becton Dickinsons testmetod och har utgått ifrån statistik från Smittskyddsinstitutet (SMI). Därefter har de berörda landstingen och regionerna kontaktats via både mail och telefon. Vidare informationssökning har skett hos de olika aktörerna inom klamydiaområdet såsom socialstyrelsen, statens folkhälsoinstitut osv.

Klamydiainfektioner har utvecklats i olika takt i de valda landstingen/regionen genom åren. Det rumsliga mönstret kan förklaras utifrån flera faktorer. Statistik påverkas av att individer testar sig på andra orter än där de är mantalsskrivna (hemkommuner). Antal tagna provmängder och effektivitet vad gäller handläggning av klamydiainfektioner leder tillupptäckt av flera fall och bromsar utvecklingen. Även tillgänglighet till platser för testninghar ökat i vissa län.

Möjlighet att genomföra en grundlig geografisk analys begränsas på grund av avsaknad avdata på kommunnivån. Däremot har studien lett till frågor som man bör söka svar på. Tidigare forskning har visat att andra länder exempelvis USA har lyckats genomföradetaljerade studier av klamydiainfektioner med hjälp av geografiskt informationssystem(GIS). Finns möjligheter att genomföra sådana klamydiastudier i Sverige med nuvarandesvensk data?

At the intersection of geography and public health, the field of spatial epidemiology seeks to use the tools of geospatial analysis to answer questions about disease. In this work we explore two areas: the use of geostatistical modeling as an extension of niche modeling, and the use of mobility metrics to augment modeling for epidemic responses. Niche modeling refers to the practice of using statistical methods to relate the underlying spatially distributed environmental variables to an outcome, typically presence or absence of a species. Such work is common in disease ecology, and often focuses on exploring the range of a disease vector or pathogen. The technique also allows one to explore the importance of each underlying regressor, and the effect it has on the outcome. We demonstrate that this concept can be extended, through geostatistical modeling, to explore non-logistic phenomena such as incidence. When combined with weather forecasts, such efforts can even predict incidence of an upcoming season, allowing us to estimate the total number of expected cases, and where we would expect to find them. We demonstrate this in Chapter 2, by forecasting the incidence of melioidosis in Australia given weather forecasts a year prior. We also evaluate the efficacy of this technique and explore the impact of environmental variables such as elevation on melioidosis. But these techniques are not limited to free-living and vector-borne pathogens. We theorize that they can also be applied to diseases that spread exclusively by person-to-person contact. Exploring this allows us to find areas of underreporting, as well as areas with unusual local forcing which might merit further investigation by the health department. We also explore this in Chapter 4, by relating the incidence of hepatitis C in rural Virginia to demographic data. The West African Ebola Outbreak of 2014 demonstrated the need to include mobility in predictive disease modeling. One can no longer assume that neglected tropical diseases will remain contained and immobile, and the assumption of random mixing across large areas is unwise. Our efforts with modeling mobility are twofold. In Chapter 3, we demonstrate the creation of mobility metrics from open source road and river network data. We then demonstrate the usefulness of such data in a meta-population patch model meant to forecast the spread of Ebola in the Democratic Republic of Congo. In Chapter 4, we also demonstrate that mobility data can be used to strengthen outbreak detection via hotspot analysis, and to augment incidence models by factoring in the incidence rates of neighboring areas. These efforts will allow health departments to more accurately forecast incidence, and more readily identify disease hotspots of atypical size and shape.
Doctor of Philosophy
The focus of this work is called “spatial epidemiology”, which combines geography with public health, to answer the where, and why, of disease. This is a growing field, and you’ve likely seen it in the news and media. Have you ever seen a map of the United States turning red in some virus disaster movie? The real thing looks a lot like that. After the Ebola outbreak of 2014, public health agencies wanted to know where the next one might hit. Now that there is another outbreak, we need to ask where and how will it spread? What areas are hardest hit, and how bad is it going to get? We can answer all these questions with spatial epidemiology. Our work adds to two aspects of spatial epidemiology: niche modeling, and mobility. We use niche modeling to determine where we could find certain diseases, usually those that are spread by insects or animals. Consider Lyme disease, you get it from the bite of a tick, and the tick gets it from a white-footed mouse. But both the mice and ticks only live in certain parts of the country. With niche modeling we can determine where those are, and we can also guess at what makes those areas attractive to the mice and ticks. Is it winter harshness, summer temperatures, rainfall, and/or elevation? Is it something else? In Chapter 2, we show that you can extend this idea. Instead of just looking at where the disease is, what if we could guess how many people will get infected? What if we could do so, a year in advance? We show that this can be done, but we need a good idea of what the weather will be like next year. In Chapter 4, we show that you can do the same thing with hepatitis C. Instead of Lyme’s ticks and mice, hepatitis C depends on drug-use, unregulated tattooing, and unsafe sex. And like with Lyme, these things are only found in certain places. Instead of temperature or rainfall, we now need to find areas with drug-problems and poverty. But we can get an idea of this from the Census Bureau, and we can make a map of hepatitis C as easily as we did for Lyme. But hepatitis C spreads person-to-person. So, we need some idea of how people move around the area. This is where mobility comes in. Mobility is important for most public health work, from detecting outbreaks to estimating where the disease will spread next. In Chapter 3, we show how one could create a mobility model for a rural area where few maps exist. We also show how to use that model to guess where the next cases of Ebola will show up. In Chapter 4, we show how you could use mobility to improve outbreak and hotspot detection. We also show how it’s used to help estimate the number of cases in an area. Because that number depends on how many cases are imported from the surrounding areas. And the only way to estimate that is with mobility.

Introduction: Oil/gas communities across Northeastern British Columbia are experiencing rapid in-migration of young, primarily male workers in response to an economic ‘boom’ in the oil/gas sectors. Accompanying the ‘boom’ has been a rise in rates of sexually transmitted infections (STIs) among young people, with Chlamydia rates among youth in the Northeast exceeding the provincial average by 22%. Previous research indicates that socio-cultural and structural determinants of youth sexual behaviour and access to STI testing are important for understanding youth sexual health disparities – and represent key targets for STI prevention efforts. No other research has explored STIs in this rapidly developing, under-resourced context. Therefore, objectives of this thesis were to: (1) Examine how socio-cultural and structural features related to the oil/gas ‘boom’ affect the sexual behaviour of young people in Fort St. John (FSJ), BC; (2) Gather the perspectives of youth and their service providers on the socio-cultural and structural barriers to STI testing in FSJ; (3) Develop recommendations to improve the accessibility of STI testing. Results: Participants identified 4 main ways in which the socio-cultural and structural conditions created by the ‘boom’ affect sexual behaviours, fuelling the spread of STIs in FSJ: mobility of oil/gas workers; binge partying; high levels of disposable income; and gendered power dynamics. As well, 5 key barriers to STI testing among youth were identified: limited opportunities for access; geographic inaccessibility; local social norms; limited information; and negative interactions with providers. Discussion: These data indicate that the conditions fostered by the ‘boom’ in FSJ exacerbate sexual health inequalities among young people. They can be more widely contextualized as an example of the unintended – but not unexpected – health and social implications of a resource-extraction ‘boom’, illustrating the fallacy of ‘development’ as representing uniformly positive ‘progress’. Recommended actions include STI prevention and testing service delivery models that incorporate a locally tailored public awareness campaign, outreach to oil/gas workers, condom distribution, expanded clinic hours and drop-in appointments, specialized training for health care providers, and intersectoral partnerships between public health, non-profit organizations, and industry. An ongoing knowledge translation internship has been undertaken to implement some of these recommendations.

Ticks are of global interest as the pathogens they spread can cause diseases that are of importance to both human health and economies. In Scotland, the most populous tick species is the sheep tick Ixodes ricinus, which is the vector of pathogens causing diseases such as Lyme borreliosis and Louping-ill. Recently, both the density and spread of I. ricinus ticks have grown across much of Europe, including Scotland, increasing disease risk. Due to the nature of the tick lifecycle they are particularly dependent on environmental factors, including temperature and habitat type. Because of this, the recent increase in tick-borne disease risk is believed to be linked to climate change. Many mathematical models have been used to explore the interactions between ticks and factors within their environments; this thesis begins by presenting a thorough review of previous modelling of tick and tick-borne pathogen dynamics, identifying current knowledge gaps. The main body of this thesis introduces an original mathematical modelling framework with the aim to further our understanding of the impact of climate change on tick-borne disease risk. This modelling framework takes into account how key environmental factors influence the I. ricinus lifecycle, and is used to create predictions of how I. ricinus density and disease risk will change across Scotland under future climate warming scenarios. These predictions are mapped using Geographical Information System software to give a clear spatial representation of the model predictions. It was found that as temperatures increase, so to do I. ricinus densities, as well as Louping-ill and Lyme borreliosis risk. These results give a strong indication of the disease risk implications of any changes to the Scottish environment, and so have the potential to inform policy-making. Additionally, the models identify areas of possible future research.

L'interaction des glycosphingolipides de la membrane plasmique avec les protéines de liaison aux glucides (lectines) est d'une importance vitale pour l'infection de la cellule hôte par divers virus et bactéries. Dans ce travail, nous avons exploré l’interaction des lectines LecA de la bactérie P. aeruginosa et de la sous-unité B de la toxine Shiga (StxB) de S. dysenteriae avec son récepteur à membrane plasmatique, le globotriaosylcéramide (Gb3). De plus, nous avons étudié l'interaction de la bactérie complète P. aeruginosa avec Gb3. Afin de déchiffrer l'interaction lectine-Gb3 en l'absence d'autres composants cellulaires, nous avons utilisé les systèmes de membrane artificielle - vésicules unilamellaires géantes (GUV) et bicouches lipidiques supportées (SLB). Nous avons observé la liaison de la lectine en utilisant différents modes de microscopie à fluorescence (confocal, TIRF, etc.). Nous examinons la liaison des deux lectines aux domaines membranaires de différents ordres et compositions. Nous avons constaté que StxB préfère des domaines membranaires plus ordonnés alors que LecA est moins préférentiel. De plus, les deux lectines induisent la réorganisation des domaines membranaires: StxB stabilise les domaines ordonnés, les réduit et induit la formation des nouveaux domaines ordonnés. D'autre part, LecA ainsi que la bactérie P. aeruginosa induisent la dissolution des domaines ordonnés. Nous pensons que ces processus de réorganisation membranaire sont cruciaux pour l’infection bactérienne
The interaction of plasma membrane glycosphingolipids with the carbohydrate binding proteins (lectins) is of vital importance for the infection of the host cell by various viruses and bacteria. In this work, we explored the interaction of the lectins LecA from the bacterium P. aeruginosa and B subunit of Shiga toxin (StxB) from S. dysenteriae with its plasma membrane receptor globotriaosylceramide (Gb3). Moreover, we studied the interaction of the complete bacterium P. aeruginosa with Gb3. In order to decipher the lectin-Gb3 interaction in absence of other cellular components we employed the artificial membrane systems – Giant unilamellar vesicles (GUVs) and Supported lipid bilayers (SLBs). We observed the lectin binding using different modes of fluorescence microscopy (confocal, TIRF, etc…). We examine the binding of both lectins to the membrane domains of different ordere and composition. We found that StxB prefers more ordered membrane domains whereas LecA is less preferential. Moreover, both lectins induce the reorganization of the membrane domains: StxB stabilizes ordered domains, shrinks them and induces the formation of the novel ordered domains. On the other hand LecA, as well as the bacterium P. aeruginosa induce the dissolution of the ordered domains. We believe, these membrane reorganization processes are crucial for the bacterial infection

Contents of the present thesis were published in the following articles. 1. Suzuki S, Mori K, Higashino A, Iwasaki Y, Yasutomi Y, Maki N, Akari H. 2016. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey. Microbiol Immunol 60:26-34. Copyright © 1999-2016 John Wiley & Sons, Inc.2. Yoshida T, Suzuki S, Iwasaki Y, Kaneko A, Saito A, Enomoto Y, Higashino A, Watanabe A, Suzuki J, Inoue K, Kuroda T, Takada M, Ito R, Ito M, Akari H. 2013. Efficient in vivo depletion of CD8+ T lymphocytes in common marmosets by novel CD8 monoclonal antibody administration. Immunol Lett 154:12-17.Copyright © 2013 Elsevier B. V.
Kyoto University (京都大学)
0048
新制・課程博士
博士(理学)
甲第19546号
理博第4206号
新制||理||1604(附属図書館)
32582
京都大学大学院理学研究科生物科学専攻
(主査)教授 明里 宏文, 教授 岡本 宗裕, 教授 中村 克樹
学位規則第4条第1項該当

INTRODUÇÃO: O vírus da hepatite G (GBV-C) foi descoberto simultaneamente por dois grupos de pesquisa que buscavam identificar um agente causador de hepatite pós-transfusional não-A, não-B e não-C. Trata-se de um vírus linfotrópico que replica primariamente em células mononucleares do sangue periférico (PBMC), baço e medula óssea, sendo a replicação hepática menos importante. Sabe-se que, após um período de viremia assintomática, a maioria dos carreadores virais clareia o vírus ao longo do tempo havendo surgimento do anticorpo contra a glicoproteína do envelope viral, anti-E2. Uma vez que, provavelmente, não exista nenhuma associação entre o GBV-C e qualquer doença identificada até o momento, o mesmo tem sido considerado um vírus humano não patogênico. Entretanto, alguns estudos demonstraram haver uma interação benéfica entre o GBV-C e HIV a ponto de retardar a progressão da infecção pelo HIV para aids. OBJETIVOS: Avaliar a prevalência da viremia e de anticorpos contra a glicoproteína anti-E2 e estudar o efeito da interação GBV-C e HIV ao longo do acompanhamento de pacientes femininas baseado em valores da média e mediana de linfócitos T CD4+ , da carga viral do HIV e da carga viral quantitativa do GBV-C RNA. MÉTODOS: Foram estudas 248 pacientes femininas portadoras da infecção pelo HIV acompanhadas por um período de cerca de 10 anos, tendo como término de estudo a data limite de 01/01/2008, ou a data da última consulta no ambulatório ou a data do óbito. RESULTADOS: Das 115 pacientes expostas, 57 eram GBV-C RNA positivas (23%) e 58 eram anti-E2 positivas (23%). Não houve achado da presença concomitante do GBV-C RNA e do anticorpo anti-E2 nas pacientes estudas. Com relação à contagem de linfócitos T CD4+ e de carga viral basal do HIV no início do estudo, não observamos diferença estatística entre os valores em relação aos grupos (GBV-C RNA-/anti-E2- , GBV-C RNA+/anti-E2 - e GBV-C RNA -/anti-E2+), sendo o p=0,36 e 0,713, respectivamente. O risco relativo de óbito para o grupo GBV-C+/anti-E2- foi 63% menor do que para o grupo GBV-C-/anti-E2-. CONCLUSÃO: A prevalência da viremia por GBV-C RNA e a do anticorpo anti-E2 foi de 23%, perfazendo uma frequência de exposição ao GBV-C de 46%. Em análise multivariada, apenas a carga viral do HIV, maior que 100.000 cópias/mL, e manifestação de doença oportunística ao longo do acompanhamento das pacientes estiveram associadas à melhora da sobrevida. Provavelmente, o uso da terapia antirretroviral para o HIV foi fator limitante na análise de efeito protetor do GBV-C RNA em nossa casuística
INTRODUCTION: GB virus C (GBV-C) was discovered by two research groups that aimed to identify a possible agent responsible for a non-A, non-B and non-C pos-transfusion hepatitis. There is remarkable evidence that GBV-C is a lymphotropic virus that primarily replicates in PBMCs, spleen and bone marrow. This virus does not appear to be hepatotropic and does not replicate effectively in hepatocytes. After an asymptomatic viremia period, most subjetcs clear the virus over time with the development of antibody against a viral envelope glycoptrotein (anti-E2). Since there is probably no association between GBV-C and any identify disease, this virus has been considered not pathogenic. However, according to some studies, GBV-C co-infection in HIV seropositive patients is associated with a slower disease progression and longer survival after AIDS development in HIV infected patients. Objective: To evaluate the prevalence of GBV-C viremia, anti- E2 antibody and assess the effect of the interaction of GBV-C and HIV in an exclusive female cohort, in a follow up period of up to 10 years. Methodos: Two hundred forty-eight HIV infected women were enrolled in the study. Follow-up sample was obtained from 248 patients. Laboratorial variables as mean and median of values of CD4, HIV and GBV-C quantitative viral load, and clinical parameters as HAART use, OIs influence on survival were investigated. Results: One hundred and fifteen subjects were exposed to GBV-C: 57 were GBV-C RNA positive (23%) and 58 were anti-E2 antibody positive (23%). Viral RNA with concomitant anti-E2 antibodies was not found in any patient. There was no statistical difference among three studied groups (GBV-C RNA-/anti-E2 - , GBV-C RNA+/anti-E2- and GBV-C RNA -/anti-E2+), regarding CD4+ and viral load baselines (p=0,360 and 0,713, respectively). Relative risk of death for GBV-C RNA+/anti-E2- group was 63% lower than the GBV-C-/anti-E2 group. Conclusion: Forty-six percent of the enrolled individuals were exposed to GBV-C virus. Multivariate analysis demonstrated that only HIV load higher than 100.000 copies/mL and opportunistic disease during the follow-up period were associated to longer survival after AIDS development. Probably, antiretroviral therapy for HIV in our study blurred the observation of a putative protective effect related to GBVC RNA

La fonction principale du poumon est d'alimenter le sang en oxygène et d'enlever le dioxyde de carbone du sang. Le poumon s'empare de l'oxygène présent dans l'air ambiant dans lequel il rejette le dioxyde de carbone prélevé dans le sang. Cet échange est rendu possible par le processus de ventilation pulmonaire qui fait entrer et sortir périodiquement un volume d'air ambiant. D'un point de vue idéalisé, la ventilation peut être caractérisée par deux paramètres : la vitesse maximale de l'air dans la trachée (l'amplitude) et la fréquence respiratoire (la période). Le but de cette thèse est d'étudier et de modéliser le processus de transport et d'échanges d'oxygène et de dioxyde de carbone dans le poumon. Le transport de gaz est modélisé par des équations de convection-diffusion-réaction dans un poumon idéalisé. Une analyse mathématique du modèle a été réalisée afin de prouver l'existence d'une solution unique ainsi que la périodicité asymptotique en temps. Des simulations numériques ont été réalisées pour étudier un large éventail de configurations physiologiques. Dans le cas d'un poumon humain en bonne santé, les quantités de gaz échangées prédites par notre modèle sont proches de la physiologie. Les énergies visqueuse et élastique dépensées lors de l'inspiration ont ensuite été minimisées en supposant que nos besoins en oxygène peuvent être représentés dans notre modèle par une contrainte du flux d'oxygène échangé avec le sang. Des simulations ont été réalisées pour l'homme, mais aussi pour tous les mammifères en utilisant les lois allométriques. Les prédictions de notre modèle montrent que les paramètres de ventilation chez les mammifères pourraient être optimisés pour dépenser le moins d'énergie possible. Ensuite, nous nous sommes concentrés sur la ventilation pulmonaire d'un humain souffrant d'une infection pulmonaire. La propagation d'une infection bronchique a été modélisée de manière idéalisée et nous avons étudié comment la ventilation est affectée par la réponse du système immunitaire à travers l'inflammation de la paroi bronchique. Nos résultats montrent que la localisation de la zone de transition entre convection et diffusion influence principalement la quantité d'oxygène échangée avec le sang. L'emplacement de cette transition peut être affecté par l'infection et donc altérer l'efficacité de la ventilation et modifier la configuration optimale. Enfin, pour mieux comprendre l'efficacité d'un traitement médicamenteux délivré sous forme d'aérosol, nous avons modélisé le dépôt de particules d'aérosol dans la première bifurcation des bronches du poumon humain. Notre modèle prend en compte l'évolution du rayon des particules due à l'échange de vapeur d'eau et l'évolution de la température des particules due au changement du milieu environnant. Nos résultats montrent que la modélisation de ces paramètres est importante pour représenter plus précisément le dépôt des particules sur les parois des bronches. Ces travaux permettent de mieux comprendre comment le processus de ventilation pulmonaire est ajusté et comment il est affecté par les pathologies pulmonaires. De plus, il souligne comment la ventilation peut être utilisée efficacement pour administrer des médicaments dans le corps humain
The main function of the lung is to supply the blood with oxygen and to drain the carbon dioxide from it. The lung captures the oxygen present in the ambient air where it rejects the carbon dioxide taken from the blood. This exchange results from the process of the lung's ventilation that repeatedly makes a volume of ambient air enter and leave the lung. In an idealized view, the ventilation can be characterized by two parameters: the maximum air velocity in the trachea (the amplitude) and the breathing frequency (the period). The goal of this thesis is to study and model the process of oxygen and carbon dioxide transport and exchanges in the lung. Gas transport is modeled by convection-diffusion-reaction equations in an idealized lung. A mathematical analysis of the model has been performed to prove the existence of a unique solution along with an asymptotic periodicity in time. Numerical simulations were performed to study a wide range of physiological configurations. In the healthy human case, the amounts of gas exchanged predicted by our model are close to physiology. The viscous and elastic energies spent during inspiration were then minimized assuming that our body needs in oxygen can be represented in our model by a constraint on the oxygen flow exchanged with the blood. Simulations were carried out for humans but also for any mammals using allometric scaling laws. The predictions of our model show that the ventilation parameters in mammals might be optimized to cost as little energy as possible. Then, we focused on the lung's ventilation of a human subject suffering from a pulmonary infection. The spread of a bronchial infection has been modeled in an idealized way and we studied how the ventilation is affected by the response of the immune system through bronchi wall inflammation. Our results show that the location of the transition zone between convection and diffusion mainly influence the quantity of oxygen exchanged with the blood. The location of this transition can be affected by the infection and hence alter the efficiency of the ventilation and modify its optimal configuration. Finally, to better understand the efficiency of a drug treatment delivered by aerosol, we modeled the deposit of aerosol particles in the first bifurcation of the bronchi of the human lung. Our model takes into account the evolution of the radius of the particles due to the exchange of water vapor and the evolution of the temperature of the particles due to the change of the surrounding environment. Our results show that the modeling of these parameters is important to represent more accurately the deposit of the particles on the walls of the bronchi. This work allows to better understand how the process of lung’s ventilation is adjusted and how it is affected by lung’s pathologies. Moreover, it highlights how ventilation can be used efficiently as a way to deliver drugs in the body

Understanding worldwide declines in reptiles due to factors such as habitat loss and emerging infectious disease has become an increasingly important focus in conservation biology. Here, I use novel approaches from the field of landscape genetics to combine spatial genetic data with landscape data at both regional and local spatial scales to explore natural and anthropogenic landscape features that shape population structure and gene flow in a federally threatened reptile, Gopherus polyphemus. I also utilize approaches from the field of spatial epidemiology to examine the extent to which environmental variables can be used to predict the seroprevalence of an associated pathogen Mycoplasma agassizzi in gopher tortoise populations. Using mitochondrial data, I find evidence of a historical barrier to gene flow that appears to coincide with the Apalachicola River. I also discover low genetic diversity and evidence of population bottlenecks in the western portion of the range. My evaluation at the regional scale shows that dispersal is limited by geographic distance, areas of low elevation and major roads ways. A finescale study reveals no evidence of spatial genetic structure within a 14 x 35 km area. However, soil type is significantly correlated with pairwise genetic distances between individuals, suggesting that this variable influences fine-scale population structure in the gopher tortoise. In addition to soil, high density canopy cover is an important factor impeding gene flow at the local level for females, while land cover type explains some of the genetic variance between males. Finally, temperature and precipitation appear to be important predictors of the seroprevalence of the pathogen Mycoplasma agassizii in gopher tortoises. The probability of an individual testing seropositive for exposure to this disease increased with high temperature and low precipitation values. The methods presented in this dissertation evaluate novel approaches for assessing the influence of environmental variables on population structure, dispersal and disease occurrence and could be applied in future studies of other threatened and endangered taxa.

Disease maps are powerful tools for depicting spatial variations in disease risk and its underlying drivers.  However, producing effective disease maps requires careful consideration of the statistical and spatial properties of the disease data. In fact, the choice of mapping method influences the resulting spatial pattern of the disease, as well as the understanding of its underlying population characteristics. New developments in mapping methods and software in addition to continuing improvements in data quality and quantity are requiring map-makers to make a multitude of decisions before a map of disease burdens can be created. The impact of such decisions on a map, including the choice of appropriate mapping method, not been addressed adequately in the literature. This research demonstrates how choice of mapping method and associated parameters influence the spatial pattern of disease. We use four different disease-mapping methods – unsmoothed choropleth maps, smoothed choropleth maps produced using the headbanging method, smoothed kernel density maps, and smoothed choropleth maps produced using spatial empirical Bayes methods and 5-years of zip code level HIV incidence (2007- 2011) data from Dallas and Tarrant Counties, Texas. For each map, the leading population characteristics and their relative importance with regards to HIV incidence is identified using a regression analysis of a CDC recommended list of socioeconomic determinants of HIV. Our results show that the choice of mapping method leads to different conclusions regarding the associations between HIV disease burden and the underlying demographic and socioeconomic characteristics. Thus, the choice of mapping method influences the patterns of disease we see or fail to see. Accurate depiction of areas of high disease burden is important for developing and targeting appropriate public health interventions.

Mandatory notification of disease forms the backbone of disease surveillance in New Zealand and overseas. Notification data is used by public health professionals and academics to identify cases requiring public health control, monitor disease incidence and distribution, and in epidemiological research. However, there is emerging evidence that notification rates do not accurately reflect the true extent of notifiable diseases within the community, resulting in the underascertainment of many notifiable cases. While adequate surveillance does not necessarily require that all cases of notifiable disease be captured, the systematic underascertainment of disease can have significant implications for perceived spatial and demographic trends in disease prevalence; potentially threatening the credibility of spatial epidemiological research by under or overestimating the burden of disease in different populations. There is evidence that systematic underascertainment occurs as a result of the differential actions of laboratories and general practitioners. It has also been recognised that that underascertainment can be influenced by a patient's willingness to seek medical attention and participate in laboratory tests. However, few studies have investigated whether these factors systematically influence notification either in New Zealand or overseas. Furthermore, the discipline of health geography has been slow to engage with this topic of public health importance, despite the inherently spatial nature of the processes involved, and the close ties to the geographic literature on health service utilization and healthcare provision. This thesis explores the spatial and temporal variation in notification rates in New Zealand for the period 1997-2005 and the potential relationships between notification rates and different variables. Unlike many underascertainment studies, which have used individual data and capture-recapture methods, data constraints inspired a unique ecological approach to investigating the factors which may be associated with notification in New Zealand. Variables were divided into categories based on Anderson's behavioural model for healthcare utilization and the influence of these variables on notification was determined through multiple regression analyses. The main findings of this research indicate that in New Zealand notification rates have increased during the period 1997-2005 and that there is a north-south gradient in notifications, with substantially lower rates in the North Island than in the South Island. Furthermore, it is also evident that the variables associated with notification vary according to disease, spatial aggregation and spatial scale. Notification rates are significantly associated with a range of predisposing and enabling factors which might influence patient choice to consult for many frequently underascertained diseases. More variation in enteric diseases is explained by the independent variables analysed than the variation in non-enteric diseases. However, further research into these relationships, and underascertainment in general, is required before firm conclusions can be drawn.

Objective: Criminal liability for the sexual-transmission of HIV raises complex questions for both clinicians and service-users regarding their responsibilities and legal obligations to disclose information to others. This is the first research study to address the impact of these issues upon everyday clinical and professional management in the UK. The prevalence and incidence of clinical and HIV-legal issues reported by the 107 psychologists sampled are reported. Design: A cross-sectional approach comprising two components was utilised: Firstly, questionnaire survey (Response rate 22%) scoping the experiences of practice issues among psychologists from sexual-health and generic settings. Attitudes towards HIV-prosecutions and various measures of professional self-efficacy were also collected. Secondly, three focus groups (N=15) exploring the impact of practice issues upon clinicians’ likely confidentiality breaking behaviours. Methods: Clinical and legal issues are presented. Further statistical analyses explored the interaction of various demographic, clinical and attitudinal variables upon clinician’s perceived self-efficacy. Focus Group transcripts analysed using Thematic Analysis (Data-driven approach) with eight emergent themes. Results: Although no direct involvements in police investigations reported, two instances of psychology notes being subpoenaed plus multiple ‘near miss’ clinical experiences described. High proportions of sexual-health psychologists experienced HIV-clients disclosing problematic behaviours, including intentional transmission (9%; N=5) and/or ‘reckless’ behaviour (72%). Focus groups expressed high levels of anxiety regarding these scenarios associated to multiple influences (interpersonal, clinician, professional and service factors). Quantitative and qualitative results were triangulated to provide a detailed analysis of how psychologists manage the clinical impact of the issues. Conclusions: Psychologists broadly supported HIV-prosecutions for intentional transmission (81%) but only limited support around ‘reckless’ cases (44%), particularly among those sexual-health experienced. Those ‘critical’ attempted to mitigate the impact of legal issues by proactively raising awareness among HIV-clients and resisting overly-defensive service changes; whereas those ‘less-critical’ were more accepting. Clinical, training and therapeutic implications are briefly considered.

A bactéria estreptococos do grupo B (EGB), comensal dos tratos intestinal e geniturinário de adultos saudáveis, é a principal causa de infeções bacterianas em recém-nascidos, incluindo a meningite. A morbidade associada é elevada e até 50 % das crianças que sobrevivem à infeção apresentam sequelas neurológicas permanentes. Para o desenvolvimento de novas estratégias terapêuticas e neuro protetoras é fundamental uma maior compreensão sobre a fisiopatologia da doença. No entanto, os métodos clássicos de quantificação da infeção não permitem estudos longitudinais, pois implicam a morte do animal. Assim, o objetivo deste estudo foi o desenvolvimento de um novo sistema bioluminescente com base na luxABCDE que permita monitorizar a disseminação da EGB, através do uso de imagens in vivo em murganhos. Para isso, a estirpe hipervirulenta da EGB (BM110) foi transformada com o plasmídeo pSL101P32, que contém o operão luxABCDE (EGB BM110+pSL101P32). Estudos in vitro mostraram que o sinal de bioluminescência e densidade ótica foram proporcionais ao número de bactérias. Além disso, a presença do plasmídeo não afetou o metabolismo da EGB, uma vez que a sua taxa de crescimento não foi alterada e o plasmídeo permaneceu estável na ausência de pressão seletiva durante 8 dias. A virulência da EGB BM110+pSL101P32 no contexto da colonização do hospedeiro foi avaliada através da capacidade de adesão e invasão às linhas celulares raw 264.7 e Caco-2. Nenhuma diferença significativa foi detetada em relação à estirpe selvagem. De forma a avaliar a virulência in vivo, murganhos recém-nascidos foram inoculados intraperitonealmente com uma dose letal da estirpe selvagem ou bioluminescente. Não foram observadas diferenças na taxa de sobrevivência nem na carga bacteriana presente no cérebro, fígado, pulmões e intestino entre os grupos. No entanto, a bioluminescência só foi detetada em crias infetadas com um inóculo inferior e apenas na região abdominal (local da infeção), o que sugere que este sistema tem uma limitada capacidade de deteção. Assim, foi gerada outra estirpe luminescente, usando um novo plasmídeo que contém a luciferase do pirilampo “red-shifted” [FFluc(PTA)], uma vez que este apresenta uma maior capacidade de penetração dos tecidos. Resultados preliminares revelaram que a emissão de luz pode ser expressa em função do crescimento celular e a bioluminescência obtida foi maior do que com a luxABCDE. Estes resultados sugerem que o FFluc(PTA) é um sistema promissor para monitorizar a disseminação da EGB em recém-nascidos através de técnicas de imagem in vivo, permitindo o estudo de meningite neonatal comprovada.
Group B Streptococcus (GBS), a commensal of the intestinal and genitourinary tracts of healthy adults, is the main bacterial cause of severe neonatal invasive disease, including meningitis. Morbidity is high and up to 50 % of surviving infants experience long-term neurologic sequalae. To define new therapeutic and neuroprotective strategies, we must gain deeper insights into the pathophysiology of disease. The classical methods for quantifying infection do not allow longitudinal studies as it entails the animal death. Thus, the aim of this work was the development of a luxABCDE-based bioluminescent reporter system to monitor GBS dissemination, using whole-mouse in vivo imaging. For that purpose, a hypervirulent GBS strain (BM110) was transformed with the plasmid pSL101P32, containing the luxABCDE operon (GBS BM110+pSL101P32). After confirming that GBS was efficiently transformed, the new strain was validated in vitro. The obtained results revealed that the bioluminescence signal and optical density were proportional to the number of bacteria. Moreover, the presence of the plasmid did not affect GBS fitness [measured by GBS growth rate and compared with wild-type (WT) strain] and the plasmid remained stable in the absence of selective pressure, for at least 8 days. The virulence of GBS BM110+pSL101P32 in the context of host colonization was evaluated in vitro by analyzing the adhesion and invasion ability to the raw 264.7 and Caco-2 cell lines, relative to the WT strain. No significant differences were detected between both strains. The infectivity was also evaluated in vivo by inoculating pups intra-peritoneally with a lethal dose of the WT strain or its bioluminescent derivate. No differences were observed between groups either in the survival rate or in bacterial load present in brain, liver, lungs and gut. However, the bioluminescence signal was only detected in living pups infected with a lower inoculum and only in the abdominal area (infection zone), suggesting that this lux-based reporter system has a limited ability of being detected. Thus, we decided to use another plasmid, FFluc(PTA), containing the red-shifted firefly luciferase that has a higher ability to penetrate deeper tissues. Our preliminary results revealed that the light emission can be expressed as a function of cell growth. Importantly, the radiance signal was higher than the one obtained with the luxABCDE operon. These results suggest that FFluc(PTA) is a promising reporter system to monitor GBS dissemination in neonates using in vivo imaging technics, allowing the study of pups with proved meningitis.

Verotoxin produced by Enterohemorrhagic E. coli is comprised of a catalytic A subunit and a receptor Gb3 binding B subunit pentamer. VT causes protein synthesis inhibition by ribosomal inactivation in Gb3 positive cells via receptor mediated endocytosis and retrograde transport to the ER. We propose that verotoxin is a novel inhibitor for HIV-1 infection. Experiments conducted using VT treated Jurkat-C T cells and PHA/IL-2 activated human PBMCs reveal the anti-HIV-1 property of VT is receptor Gb3 independent since the catalytic A subunit alone is sufficient for inhibition. Possible mechanism of action involves mild inhibition of protein synthesis and cell proliferation. Recent findings in our lab suggest Gb3 is a natural resistance factor for HIV-1 infection, which was further investigated by selecting a Gb3 low subpopulation in THP-1 cells using VT treatment. Selected THP-1 cells were completely resistant to HIV-1 infection, however decreased surface CXCR4 expression may be a cause.

GB virus C (GBV-C) is a common, apathogenic virus that can inhibit human immunodeficiency virus (HIV) replication in vitro. Persistent coinfection with GBV-C has been associated with improved survival among HIV-infected adults while loss of GBV-C viremia has been associated with poor survival. If GBV-C does inhibit HIV replication, it is possible that GBV-C infection may reduce mother-to-child-transmission (MTCT) of HIV. This study investigated whether maternal or infant GBV-C infection was associated with reduced MTCT of HIV infection. The study population consisted of 1,783 pregnant women from three Bangkok perinatal HIV transmission studies (1992-94, 1996-7, 1999-2004). We tested plasma collected at delivery for GBV-C RNA, GBV-C antibody, and GBV-C viral genotype. If maternal GBV-C RNA was detected, the four- or six-month infant specimen was tested for GBV-C RNA. Rates of MTCT of HIV in GBV-C-infected and GBV-C-uninfected women and infants were compared using multiple logistic regression as were associations with MTCT of GBV-C and prevalence of GBV-C infection. The prevalence of GBV-C infection (i.e. presence of RNA or antibody) was 33% among HIV-infected women and 15% among HIV-uninfected women. Forty-one percent of GBV-C-RNA-positive women transmitted GBV-C to their infants. Only two of 101 (2.0%) GBV-C-RNA-positive infants acquired HIV infection compared to 162 (13.2%) of 1,232 of GBV-C-RNA-negative infants (RR 0.15, p<0.0001). This association remained after adjustment for maternal HIV viral load, antiretroviral prophylaxis, CD4+ count and other covariates. MTCT of HIV was not associated with presence of maternal GBV-C RNA or maternal GBV-C antibody. Maternal receipt of antiretroviral therapy was associated with increased MTCT of GBV-C, as was high GBV-C viral load, vaginal delivery and absence of infant HIV infection. GBV-C infection among women was independently associated with more than one lifetime sexual partner, intravenous drug use and HIV-infection. We observed a higher prevalence of GBV-C infection among HIV-infected compared to HIV-uninfected pregnant women in Thailand, likely due to common risk factors. Antiretroviral therapy appears to increase MTCT of GBV-C. Infant GBV-C acquisition, but not maternal GBV-C infection, was significantly associated with reduced MTCT of HIV. Mechanisms for these later two associations are unknown.
Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero J, Kaul R, Kulkarni P, McConnell MS, Mock PA, Culnane M, McNicholl J, Roongpisuthipong A, Chotpitayasunondh T, Shaffer N, Butera S. 2008. Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. J Infect Dis 197(10):1369-1377. Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero JW, Kaul R, Kulkarni P, McConnell MS, Mock PA, McNicholl JM, Vanprarar N, Asavapiriayanont S, Shaffer N, Butera ST. 2009. Mother-to-child transmission of GB virus C in a cohort of women coinfected with GB virus C and HIV in Bangkok, Thailand. J Infect Dis 200:227-235.

No
Infection is the major reason of GTR/GBR membrane failure in clinical application. In this work, we developed GTR/GBR nanofiber membranes with localized drug delivery function to prevent infection. Metronidazole (MNA), an antibiotic, was successfully incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (0, 1, 5, 10, 20, 30, and 40 wt% polymer). To obtain the optimum anti-infection membrane, we systematically investigated the physical-chemical and mechanical properties of the nanofiber membranes with different drug contents. The interaction between PCL and MNA was identified by molecular dynamics simulation. MNA released in a controlled, sustained manner over 2 weeks and the antibacterial activity of the released MNA remained. The incorporation of MNA improved the hydrophilicity and in vitro biodegradation rate of PCL nanofibers. The nanofiber membranes allowed cells to adhere to and proliferate on them and showed excellent barrier function. The membrane loaded with 30% MNA had the best comprehensive properties. Analysis of subcutaneous implants demonstrated that MNA-loaded nanofibers evoked a less severe inflammatory response than pure PCL nanofibers. These results demonstrate the potential of MNA-loaded nanofiber membranes as GTR/GBR membrane with antibacterial and anti-inflammatory function for extensive biomedical applications.

Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients.
Thesis (Ph.D.)-University of Natal, 2003.

Das 1995 entdeckte GB-Virus C (GBV-C) gehört als Pegivirus zur Familie der Flaviviridae und ist nichtpathogen. In Industrieländern sind 2 bis 12,5 % der gesunden Bevölkerung und bis zu 45 % der Personen aus Risikokollektiven, z.B. Patienten mit Infektionen mit dem humanen Immundefizienzvirus Typ 1 (HIV-1) oder dem Hepatitis-C-Virus (HCV), virämisch. Die Mehrzahl der klinischen Studien und Metaanalysen zu GBV-C/HIV-1-Koinfektionen zeigten, dass GBV-C mit einem verlangsamten Krankheitsverlauf und einer erhöhten Überlebenswahrscheinlichkeit von GBV-C/HIV-1-koinfizierten Patienten korreliert. In der Hemophilia Growth and Development Study konnte dieser Effekt bei GBV-C/HCV-/HIV-1-infizierten Kindern und Jugendlichen jedoch nur bedingt nachgewiesen werden. Dafür wurde ein Zusammenhang zwischen einer GBV-C/HCV-Koinfektion und dem Ausheilen der HCV-Infektion beobachtet und in einer weiteren Patientenkohorte aus der Anti-D-Studie bestätigt. GBV-C/HCV-koinfizierte Patienten haben schlechtere Chancen, die HCV-Infektion auszuheilen. Der Einfluss von GBV-C auf die HIV-1-Replikation wurde in Zellkulturexperimenten untersucht. Es zeigte sich, dass sich die verschiedenen GBV-C-Isolate hinsichtlich ihrer inhibitorischen Kompetenz unterschieden. Folgende mögliche Ursachen wurden untersucht: 1.) die IRES-Aktivität als Indikator für die Translationseffizienz, 2.) die NS5A-Sequenz des in der Literatur beschriebenen HIV-1-inhibitorisch aktiven 16mer-Peptids sowie 3.) die E2-Sequenz und die HIV-1-inhibitorische Wirkung von 18mer-E2-Peptiden. Es konnten weder Unterschiede in der IRES-Aktivität noch in der NS5A-Sequenz zwischen den unterschiedlich inhibitorisch-kompetenten GBV-C-Isolaten nachgewiesen werden. Im E2-Protein hingegen wurden zwei für alle HIV-1-nichtinhibitorischen GBV-C-Isolate einheitliche Mutationen, E143K/H und T204A, identifiziert. Diese könnten eine Ursache für die Varianz in der Fähigkeit, HIV-1 zu inhibieren, darstellen. Die Mutation an Position E143 ist an der Oberfläche des nativen E2-Proteins exponiert und spielt möglicherweise im Hemmmechanismus eine wichtige Rolle. Hinweise darauf gaben die Untersuchungen mit synthetischen 18mer-Peptiden, von denen das Peptid mit dem größten inhibitorischen Potenzial die Aminosäure an Position 143 beinhaltete. Eine mögliche Theorie des Wirkmechanismus des E2-Proteins wäre wie folgt denkbar: Das E2-Protein interagiert über eine Domäne um die Aminosäure E143 mit dem gp41 des HIV-1, verhindert somit die Fusion von Virus- und Zellmembran und in der Folge den Eintritt des HIV-1 in die Zielzelle.

Obwohl seit der Einführung des Penicillins ein wirksames Medikament gegen Streptokokken der Lancefield Gruppe A (GAS) existiert, bei welchem bislang keine Resistenzen beschrieben wurden, bleiben GAS-Infektionen auch heute noch ein großes gesundheitspolitisches Problem, das sowohl die Morbidität als auch die Mortalität der Menschen weltweit beeinflusst. GAS können ein breites Spektrum an Erkrankungen beim Menschen verursachen. Dazu zählen nicht nur unkomplizierte Racheninfektionen mit und ohne Scharlach oder Hautinfektionen wie Erysipel oder Impetigo, sondern auch invasive sowie Folgeerkrankungen. 1928 wurde als Typ-spezifische, Antikörperbildung-induzierende Substanz das M-Protein beschrieben, welches durch das emm-Gen kodiert und seither zur Beschreibung der Epidemiologie von GAS verwendet wird. Eine der Hauptfunktionen des auf der Oberfläche von GAS verankerten M-Proteins besteht darin, die Phagozytose durch polymorphkernige Leukozyten zu verhindern, was zu den wichtigsten Abwehrmechanismen von Infektionen mit GAS gezählt wird. Obwohl seit einigen Jahrzehnten große Anstrengungen unternommen wurden, bleibt ein sicherer und effektiver Impfstoff bisher ein unerreichtes Ziel. In der hier vorliegenden Studie wurde, anhand der über den Zeitraum vom 11.03.2006 bis 19.05.2012 am Universitätsklinikum Freiburg gesammelten Daten und Isolaten, die regionale Epidemiologie von Infektionen pädiatrischer Patienten durch GAS retrospektiv untersucht. Mit insgesamt 566 Isolaten und zugehörigen klinischen Daten stellt diese Studie die bisher größte unizentrische epidemiologische Untersuchung von pädiatrischen Erkrankungen mit emm-Typisierung von GAS in Deutschland dar. Dabei wurde besonders auf Zusammenhänge zwischen den molekularepidemiologischen Daten, basierend auf der emm-Typisierung, und den anonymisierten klinischen Informationen eingegangen. In die Kohorte konnten insgesamt 566 Fälle eingeschlossen werden. Bei 405 Fällen wurde eine Racheninfektion festgestellt, wovon bei wiederum 75 Fällen zusätzlich die Diagnose Scharlach gestellt wurde, 34 Kinder stellten sich mit einer Hautinfektion vor, 21 mit einer akuten Otitis media, 19 mit einer anogenitalen Infektion, acht mit einer invasiven Infektion und zwei mit einer Harnwegsinfektion. Als Kolonisation durch GAS ohne Krankheitswert wurden 77 Fälle gewertet, davon 48 mit pharyngealer Kolonisation. In der molekularepidemiologischen Untersuchung konnten drei neue emm-subtypen entdeckt werden, welche als emm29.13, emm36.7 sowie emm75.5 erstbeschrieben und deren Sequenzen in der Datenbank des CDC hinterlegt wurden. Über die gesamte Kohorte hinweg wurde Typ emm12 bei 19% aller Fälle gefunden und lag somit am häufigsten vor, gefolgt von emm1 und emm4 mit je 14% sowie emm28 und emm89 mit je 11%. Bei Betrachtung der emm-Cluster zeigte sich E4 mit 31% am häufigsten, danach folgten Cluster A-C4 mit 19%, A-C3 und E1 mit jeweils 14%. Unter den 405 Fällen mit GAS-Tonsillopharyngitis lag emm12 mit knapp 20% am häufigsten vor, gefolgt von emm4 mit 15%, emm1 mit 14%, emm89 mit 13% und emm28 sowie emm3 mit je 9%. Hinsichtlich der Cluster wurde E4 dort mit knapp 30% am häufigsten festgestellt, gefolgt von A-C4 mit 20%, E1 mit 15%und A-C3 mit 14%. In der vorliegenden Studie konnte gezeigt werden, dass sich die emm-Typ- sowie die emm-Cluster-Epidemiologie in Abhängigkeit von der klinischen Manifestation unterscheidet. Auch wenn sich die Verteilungen grundsätzlich ähnelten, traten emm4 bzw. die Cluster A-C5 und E1 bei Patienten mit Tonsillopharyngitis mit Scharlach auch nach Bonferroni-Korrektur signifikant häufiger auf als bei solchen mit Tonsillopharyngitis ohne Scharlach. Erste Hinweise hierfür wurden im Rahmen dieser Arbeit in einer Vorabauswertung zu dieser Kohorte 2013 erstmals beschrieben und durch Ergebnisse folgender, internationaler Studien gestützt. Bei anogenitalen Infektionen wurde in knapp 80% der Fälle Cluster E4 und in 58% emm28 festgestellt, so-dass hier ein deutlich eingeengtes Erregerspektrum vorlag. Verglichen zu allen Fällen mit Tonsillopharyngitis wurden bei anogenitaler Infektion Typ emm28 und Cluster E4 signifikant häufiger isoliert. Für Hautinfektionen konnte kein signifikanter Unterschied der emm-Typ-Verteilung im Vergleich zu Racheninfektionen insgesamt gefunden werden. Es zeigte sich jedoch Cluster E4 signifikant häufiger bei Patienten mit einer Hautinfektion als bei solchen mit Scharlach. Insgesamt zeigte sich im konkreten Vergleich zu einer französischen Studie eine weitgehende Übereinstimmung hinsichtlich der Epidemiologie der emm-Typen und -Cluster, jedoch auch einzelne Differenzen. Diese Unterschiede waren signifikant für emm6 und emm22, ebenso wie für Cluster M6. Weiterhin bestätigte unter anderen die Studie von d´Humieres et al. die Häufung von emm4 bei Scharlach-Patienten, was die Aussage der vorgelegten Ergebnisse unter-streicht. Weiterhin wurde zur Untersuchung der longitudinalen Entwicklung der emm-Typen und emm-Cluster die Verteilung des Zeitraumes vom 01.04.2006 und 31.03.2007 mit dem vom 01.05.2011 bis 30.04.2012 verglichen. Zumindest für den vergleichsweise kurzen zeitlichen Abstand konnten nach Adjustierung keine signifikanten Veränderungen der Epidemiologie hinsichtlich einzelner emm-Typen bzw. -Cluster beobachtet werden. In bisherigen Studien wurde die Pathogenität eines Stammes meist anhand des klinischen Erscheinungsbildes bestimmt. In dieser Studie wurde weiterführend untersucht, inwiefern sich einzelne emm-Typen bzw. emm-Cluster auch in quantitativ messbaren Parametern wie u.a. dem C-reaktiven Protein (CRP) sowie der Leukozytenzahl im Blut unterscheiden. Bei Patienten mit Tonsillopharyngitis zeigte sich lediglich Cluster E4 signifikant häufiger mit einem CRP-Wert über 35 mg/l assoziiert. Für die Leukozytenzahl war ein solcher Zusammenhang dagegen nicht nachweisbar. Da die verwendeten Analysen jedoch Störfaktoren unterlagen und ein Kausalzusammenhang zwischen der Pathogenität des Erregers und der Auslenkung der ge-nannten Parameter im Rahmen des Studiendesigns nicht bewiesen werden konnte, lassen diese Ergebnisse keine abschließende Beurteilung zu. Der bereits entwickelte 30-valente Impfstoff zeigte anhand der enthaltenen M-Antigene eine gute Übereinstimmung mit den in dieser Studie gefundenen emm-Typen. Dabei waren die Antigene von 19 der 25 in dieser Studie registrierten emm-Typen in dem Impfstoff enthalten, was jedoch unter Berücksichtigung der Kreuzreaktivitäts-Hypothese für emm-Cluster zu einem Deckungsgrad von 99,8% aller untersuchten Fälle (565 von 566) führt. Insgesamt erwies sich der unizentrische Charakter der hier vorgelegten Studie in gewisser Hinsicht als Vorteil gegenüber multizentrischen Studien, da hierdurch zu bestimmten Fragen Informationen ohne den Einfluss regionaler Besonderheiten ausgewertet werden konnten. Inwiefern regionale Prävalenzen einzelner emm-Typen oder deren Pathogenitätspotential ent-scheidend für die Epidemiologie insbesondere invasiver Infektionen sind, kann anhand dieser Studie nicht abschließend beurteilt werden. Zur näheren Untersuchung dieses Sachverhaltes wären weiterführende Studien in größerem Maßstab notwendig. Zusammenfassend wurde mit dieser Arbeit eine umfassende epidemiologische Untersuchung anhand der molekularepidemiologischen Erregeranalyse unter Einschluss klinischer Aspekte an einer großen pädiatrischen Kohorte durchgeführt. Die gewonnenen Erkenntnisse leisten einen Beitrag zur Aufklärung der regionalen wie auch internationalen Epidemiologie von GAS und bieten wichtige Grundlagen sowie Ansätze für nachfolgende Untersuchungen, insbesondere für die Impfstoffentwicklung gegen GAS.:1 Einleitung 7 2 Theoretische Grundlagen 8 2.1 Epidemiologie 8 2.2 Taxonomie 10 2.3 Infektionspathologie 11 2.4 Aufbau des M-Proteins 14 2.5 Die Bedeutung des M-Proteins 16 2.6 emm-Genetik 18 2.7 Therapie und Prophylaxe 20 3 Ziele der Studie 22 4 Patienten, Material und Methoden 23 4.1 Mikrobiologische Isolate und klinische Daten 23 4.1.1 „Klinische Krankheitsbilder“ 25 4.1.2 Modifizierter Centor-Score 26 4.1.3 Grunderkrankungen 27 4.1.4 Paraklinik 27 4.2 Geräte und Materialien 29 4.2.1 Geräte und Hilfsmittel 29 4.2.2 Verbrauchsmaterialien 30 4.2.3 Molekulare Diagnostiksysteme 31 4.2.4 Primer für PCR und Sequenzierung 31 4.2.5 Medien und Lösungen 31 4.3 Mikrobiologische Methoden 32 4.3.1 Mikrobiologische Proben 32 4.3.2 Kultur von GAS 32 4.3.3 Latex-Agglutinationstest auf Gruppe A Antigen 33 4.3.4 Kryokonservierung der Isolate 34 4.4 Molekulargenetische Methoden 35 4.4.1 DNA-Isolierung 35 4.4.2 Photometrische Bestimmung der DNS-Konzentration und Einstellung 36 4.4.3 Polymerase-Kettenreaktion (PCR) 37 4.4.4 Agarose-Gelelektrophorese 37 4.4.5 DNA Sequenzierung 39 4.4.6 Sequenz-basierte Typisierung 41 4.5 Statistische Auswertung 42 5 Ergebnisse 43 5.1 Ausgewertete mikrobiologische Proben und retrospektive Daten 43 5.2 Analyse der Kohorte 45 5.2.1 Analyse der Altersverteilung 45 5.2.2 Analyse der Geschlechtsverteilung 48 5.2.3 Analyse der saisonalen Verteilung 49 5.2.4 Analyse der klinischen Symptome 51 5.2.5 Analyse von Vorerkrankungen und Versorgungsform 55 5.3 Laborbefunde 58 5.3.1 Analyse der semiquantitativen Wachstumsdichte der Abstrich-Kulturen 58 5.3.2 Blutparameter: C-Reaktives Protein (CRP) 59 5.3.3 Blutparameter: Leukozytenzahl 59 5.4 Molekulare Epidemiologie des emm-Typs 60 5.5 Erstbeschreibung neuer emm-Subtypen 60 5.6 emm-Typ- bzw. Cluster-Verteilung und klinische Manifestation 61 5.6.1 emm-Verteilung bei Tonsillopharyngitis 61 5.6.2 emm-Verteilung bei akuter Otitis media (AOM) 63 5.6.3 emm-Verteilung bei Hautinfektionen 64 5.6.4 emm-Verteilung bei anogenitalen Infektionen 65 5.6.5 emm-Verteilung bei invasiven Infektionen 68 5.6.6 emm-Verteilung bei asymptomatischer Rachen-Kolonisierung 68 5.7 Analyse der Altersverteilung für emm-Typen und -Cluster 70 5.8 Infektionsparameter und Korrelation zur molekularen Epidemiologie 71 5.8.1 Temperatur: 71 5.8.2 CRP: 72 5.8.3 Leukozytenzahl: 73 5.9 Patienten mit wiederholten Vorstellungen 73 5.9.1 Antibiotische Therapie 75 5.10 Resistenzlage 76 5.11 Analyse der molekularen Epidemiologie über der Zeit 78 6 Diskussion 80 6.1 Vorbemerkung 80 6.2 Kohorte 81 6.2.1 Alter 81 6.2.2 Geschlecht 82 6.2.3 Jahreszeit 82 6.2.4 Vorerkrankungen 83 6.2.5 Klinische Symptome 84 6.2.6 Diagnostischer Wert der semiquantitativen Wachstumsdichte 85 6.2.7 Rezidive 85 6.3 emm-Typ und –Cluster-Epidemiologie 86 6.3.1 emm-Typen und -Cluster bei Tonsillopharyngitis 86 6.3.2 emm-Typen und –Cluster bei Anogenitalinfektionen 86 6.3.3 emm-Typen und -Cluster bei invasiven Infektionen 87 6.3.4 emm-Typ und -Cluster-Epidemiologie im Vergleich zu anderen Studien 87 6.3.5 Longitudinale Betrachtung der emm-Typ-Epidemiologie 91 6.3.6 Vergleich der emm-Typen und Cluster-hinsichtlich des Alters der Patienten 92 6.3.7 emm-Typen und -Cluster hinsichtlich Infektionsparametern 92 6.3.8 Deckungsgrad mit 30-valentem M-Protein-basiertem GAS-Impfstoff 95 6.3.9 emm-Typ / -Cluster – Antibiotika-Resistenz 96 6.4 Ausblick 97 7 Zusammenfassung 98 8 Summary 101 9 Anhang 104 10 Abbildungsverzeichnis 112 11 Tabellenverzeichnis 114 12 Abkürzungsverzeichnis 116 13 Literaturverzeichnis 118 14 Anlage 1 127 15 Anlage 2 129 16 Danksagung 131
Although - since the introduction of penicillin - there has been an effective drug against strep-tococci of Lancefield Group A (GAS), in which no resistance has been described so far, GAS infections remain a major healthcare policy problem, which affects both morbidity and mortality of people worldwide. GAS can cause a wide range of disorders in humans. These include un-complicated pharyngeal infections with and without scarlet fever as well as skin infections such as erysipelas or impetigo but also invasive as well as secondary complications. In 1928, the M-protein encoded by the emm gene was described as a type-specific, antibody -inducing substance and has since been used to characterize the epidemiology of GAS. One of the main functions of the M protein, anchored on the surface of GAS, is to evade phagocytosis by polymorphonuclear leukocytes, which is one of the most important defense mechanisms against infections by GAS. Although major efforts have been made for several decades, a safe and effective vaccine remains an unreached goal. In this study, the regional epidemiology of infections of pediatric patients by GAS was retro-spectively investigated on the basis of data and isolates collected from 11.03.2006 to 19.05.2012 at the University Medical Center in Freiburg. With a total of 566 isolates and asso-ciated clinical data, the present study provides the largest uni-centric epidemiological study of pediatric diseases with emm-typing of GAS so far in Germany. Particular attention was paid to associations between molecular epidemiology, based on emm-typing, and anonymized clinical data. The total cohort included 566 cases, thereof 405 cases of pharyngeal infection, 75 of which were additionally diagnosed with scarlet fever, 34 children presented with a skin infection, 21 with an acute otitis media, 19 with an anogenital infection, eight with an invasive infection and two with an urinary tract infection. In 77 cases colonization by GAS was estimated as having no clinical relevance of those 48 isolates were isolated from pharyngeal swabs. In the molecular investigation, three new emm subtypes were discovered which were first de-scribed as emm29.13, emm36.7 as well as emm75.5. These sequences were entered into the database of the CDC. Over the entire cohort, emm12 was found in 19% of all cases and was thus the most frequent, followed by emm1 and emm4, with 14% each, emm28 and emm89, with 11% each. When considering emm-clusters, E4 was the most frequent with 31%, followed by cluster A-C4 with 19%, A-C3 and E1 with 14%. Among the 405 cases with GAS-related pharyngeal infection, emm12 was the most common with almost 20%, followed by emm4 with 15%, emm1 with 14%, emm89 with 13% and emm28 as well as emm3 with 9% each. With respect to the clus-ters, E4 was found to be the most common with around 30%, followed by A-C4 with 20%, E1 with 15% and A-C3 with 14%. In the present study it was shown that emm-types as well as emm-clusters differed depending on the clinical manifestation. Although the distributions were basically similar, emm4 as well as clusters A-C5 and E1 were significantly more common in patients with tonsillopharyngitis and scarlet fever, even after Bonferroni correction, than those with tonsillopharyngitis but without the diagnosis of scarlet fever. These findings were first described in a preliminary evaluation of this cohort in 2013 and supported by the results of consecutively published international stud-ies. In anogenital infections, cluster E4 was found in almost 80% and emm28 in 58%, indicat-ing a clearly narrowed spectrum. Compared to cases with tonsillopharygitis, emm28 and clus-ter E4 were significantly more frequently isolated in anogenital infections. For skin infections no significant difference could be found in the emm-distribution compared to tonsillopharyngi-tis. However, cluster E4 was found to be significantly more common in patients with a skin infection than in those with scarlet fever. Overall, in a direct comparison to a French study, there was a wide agreement regarding the epidemiology of emm-types and -clusters, but also some differences. These differences were significant for emm6, emm22, as well as for cluster M6. Furthermore, among others, the study by d´Humieres et al. confirmed the accumulation of emm4 in scarlet fever patients, which un-derlines the statement of the presented results. Furthermore, in order to investigate the longitudinal development of emm-types and emm-clusters, the distribution in the period from 01.04.2006 to 31.03.2007 was compared to that from 01.05.2011 to 30.04.2012. At least for the comparatively short period, no significant changes in the epidemiology of individual emm-types or -clusters could have been observed after adjusting the p-values. In previous studies, the pathogenicity of a strain was determined by its association to the clini-cal picture. In addition, this study investigated the extent to which individual emm-types or emm-clusters also differed in quantitatively measurable parameters such as the C-reactive protein (CRP) and the leukocyte count in the blood. In cases of tonsillopharyngitis, cluster E4 was found significantly associated with a CRP value above 35 mg/l. For the leukocyte count such a difference was not detectable. However, since the values were subject to confounding factors, a causal link between pathogenicity of certain emm-types and the deflection of the mentioned parameters could not be proved within the framework of this study. Therefore, these results do not allow to draw final conclusions. The existing 30-valent M-protein based vaccine would show a good agreement with the corre-sponding emm-types of the cohort used here. The antigens of 19 of the 25 different emm-types registered in this study were included in the vaccination model, which corresponds to a vaccine coverage of 99.8% (565 of 566) of all strains examined here, if cross-reactivity of GAS strains within an emm-cluster was taken into consideration. Overall, the uni-centric character of the study presented here provided in certain aspects an advantage over multi-centric studies, as differences and similarities between different clinical pictures, excluding regional differences as well as comprehensive clinical information on the cases, could be emphasized. The extent to which regional prevalence of individual emm-types or their pathogenicity potential are decisive for the epidemiology of invasive infections could not be conclusively assessed by this study. For a closer look at this issue, further studies on a larger scale would be necessary. In summary, this work presents a comprehensive epidemiological investigation on molecular epidemiologic pathogen analysis including clinical aspects in a large pediatric cohort. The find-ings contribute to the elucidation of the regional an international epidemiology of GAS and pro-vide important basics as well as approaches for subsequent investigations, especially for vac-cine development against GAS.:1 Einleitung 7 2 Theoretische Grundlagen 8 2.1 Epidemiologie 8 2.2 Taxonomie 10 2.3 Infektionspathologie 11 2.4 Aufbau des M-Proteins 14 2.5 Die Bedeutung des M-Proteins 16 2.6 emm-Genetik 18 2.7 Therapie und Prophylaxe 20 3 Ziele der Studie 22 4 Patienten, Material und Methoden 23 4.1 Mikrobiologische Isolate und klinische Daten 23 4.1.1 „Klinische Krankheitsbilder“ 25 4.1.2 Modifizierter Centor-Score 26 4.1.3 Grunderkrankungen 27 4.1.4 Paraklinik 27 4.2 Geräte und Materialien 29 4.2.1 Geräte und Hilfsmittel 29 4.2.2 Verbrauchsmaterialien 30 4.2.3 Molekulare Diagnostiksysteme 31 4.2.4 Primer für PCR und Sequenzierung 31 4.2.5 Medien und Lösungen 31 4.3 Mikrobiologische Methoden 32 4.3.1 Mikrobiologische Proben 32 4.3.2 Kultur von GAS 32 4.3.3 Latex-Agglutinationstest auf Gruppe A Antigen 33 4.3.4 Kryokonservierung der Isolate 34 4.4 Molekulargenetische Methoden 35 4.4.1 DNA-Isolierung 35 4.4.2 Photometrische Bestimmung der DNS-Konzentration und Einstellung 36 4.4.3 Polymerase-Kettenreaktion (PCR) 37 4.4.4 Agarose-Gelelektrophorese 37 4.4.5 DNA Sequenzierung 39 4.4.6 Sequenz-basierte Typisierung 41 4.5 Statistische Auswertung 42 5 Ergebnisse 43 5.1 Ausgewertete mikrobiologische Proben und retrospektive Daten 43 5.2 Analyse der Kohorte 45 5.2.1 Analyse der Altersverteilung 45 5.2.2 Analyse der Geschlechtsverteilung 48 5.2.3 Analyse der saisonalen Verteilung 49 5.2.4 Analyse der klinischen Symptome 51 5.2.5 Analyse von Vorerkrankungen und Versorgungsform 55 5.3 Laborbefunde 58 5.3.1 Analyse der semiquantitativen Wachstumsdichte der Abstrich-Kulturen 58 5.3.2 Blutparameter: C-Reaktives Protein (CRP) 59 5.3.3 Blutparameter: Leukozytenzahl 59 5.4 Molekulare Epidemiologie des emm-Typs 60 5.5 Erstbeschreibung neuer emm-Subtypen 60 5.6 emm-Typ- bzw. Cluster-Verteilung und klinische Manifestation 61 5.6.1 emm-Verteilung bei Tonsillopharyngitis 61 5.6.2 emm-Verteilung bei akuter Otitis media (AOM) 63 5.6.3 emm-Verteilung bei Hautinfektionen 64 5.6.4 emm-Verteilung bei anogenitalen Infektionen 65 5.6.5 emm-Verteilung bei invasiven Infektionen 68 5.6.6 emm-Verteilung bei asymptomatischer Rachen-Kolonisierung 68 5.7 Analyse der Altersverteilung für emm-Typen und -Cluster 70 5.8 Infektionsparameter und Korrelation zur molekularen Epidemiologie 71 5.8.1 Temperatur: 71 5.8.2 CRP: 72 5.8.3 Leukozytenzahl: 73 5.9 Patienten mit wiederholten Vorstellungen 73 5.9.1 Antibiotische Therapie 75 5.10 Resistenzlage 76 5.11 Analyse der molekularen Epidemiologie über der Zeit 78 6 Diskussion 80 6.1 Vorbemerkung 80 6.2 Kohorte 81 6.2.1 Alter 81 6.2.2 Geschlecht 82 6.2.3 Jahreszeit 82 6.2.4 Vorerkrankungen 83 6.2.5 Klinische Symptome 84 6.2.6 Diagnostischer Wert der semiquantitativen Wachstumsdichte 85 6.2.7 Rezidive 85 6.3 emm-Typ und –Cluster-Epidemiologie 86 6.3.1 emm-Typen und -Cluster bei Tonsillopharyngitis 86 6.3.2 emm-Typen und –Cluster bei Anogenitalinfektionen 86 6.3.3 emm-Typen und -Cluster bei invasiven Infektionen 87 6.3.4 emm-Typ und -Cluster-Epidemiologie im Vergleich zu anderen Studien 87 6.3.5 Longitudinale Betrachtung der emm-Typ-Epidemiologie 91 6.3.6 Vergleich der emm-Typen und Cluster-hinsichtlich des Alters der Patienten 92 6.3.7 emm-Typen und -Cluster hinsichtlich Infektionsparametern 92 6.3.8 Deckungsgrad mit 30-valentem M-Protein-basiertem GAS-Impfstoff 95 6.3.9 emm-Typ / -Cluster – Antibiotika-Resistenz 96 6.4 Ausblick 97 7 Zusammenfassung 98 8 Summary 101 9 Anhang 104 10 Abbildungsverzeichnis 112 11 Tabellenverzeichnis 114 12 Abkürzungsverzeichnis 116 13 Literaturverzeichnis 118 14 Anlage 1 127 15 Anlage 2 129 16 Danksagung 131

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched.
Health Studies
M.Sc. (Life Sciences (Microbiology))

Anti-microbial proteins (AMP) are the key effector arm of the innate immune system. The prevalence of AMP in single-celled eukaryotes to humans shows its importance during the course of evolution. The first report for the role of the anti-microbial peptide in clearing infection was given by Alexander Fleming in 1990’s through the discovery of Penicillin and Lysozyme. The search for antimicrobial agents in human granulocytes was begun by Ehrlich in 1870’s but the first successful isolation of an antimicrobial agent from rabbit neutrophils was done by Zeya and Spitznagel in 1969. Later work by Peter Elshbach and his group on AMPs in rabbit neutrophils brought to light an AMP that can increase the permeability of the bacterial membrane. This AMP named as Bactericidal/permeability-increasing protein (BPI) was further isolated from human neutrophils. Since then many studies have been carried out to understand the mode of action of BPI, which culminated in understanding the new functional activity of this protein viz opsonisation, LPS neutralization and anti-angiogenic function. Knowing to the role of BPI as an anti-inflammatory agent, multiple studies have tried to use BPI for treating endotoxic shock. Dysregulation of BPI expression is associated with various inflammatory diseases like Crohn’s Disease (CD), Ulcerative colitis (UC) and Infectious enteritis’s. Mutations in BPI are also linked to susceptibility to various infections. Even though there are several studies focusing on the functional aspects of BPI, the regulation of BPI expression is poorly understood. Knowing the clinical importance of dysregulation of BPI, it is vital to understand the regulation of BPI expression during the course of bacterial infection. The Thesis is divided into four chapters. As the main aim of this study is to understand the regulation of BPI expression, in Chapter 1 we introduce the known facts about the protein. A brief overview of the mode of action and regulation of BPI is discussed in this chapter. The subsequent sections describe the diseases associated with Dysregulation of BPI and the use of BPI as a therapeutic agent in various diseases. Towards the end, the objective of the present study is discussed. BPI is primarily known to be expressed in human neutrophils and epithelial cells. Previous studies have shown that among innate immune cells, murine BPI is expressed only in dendritic cells and neutrophils, but not in macrophages. Based on these results, it was presumed that BPI is not expressed in human macrophages. In Chapter 2, we report the presence of BPI in human macrophages. Our studies revealed increased expression of BPI in human macrophages stimulated with various PAMPs (Pathogen-associated molecular patterns) viz., LPS, flagellin as well as during bacterial infection. Further, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. BPI was induced in both M1 and M2 differentiated macrophages suggesting its role in limiting Gram-negative bacteria and parasitic infection. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages. This chapter concludes with a discussion on the importance of BPI expression in human but not murine macrophages. The importance of maintaining an active replicating niche by STM to evade interaction with BPI is also discussed. As the first line of defense against invading pathogens, intestinal epithelium produces various antimicrobial proteins (AMP) that help with clearance of pathogen. The precise mechanism of AMP regulation in intestinal epithelium is not clear. Intestinal epithelium being a primary entry point for various pathogens, we tried to understand the regulation of BPI expression in the intestine during the course of bacterial infection. In Chapter 3, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S.aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium levels as a Danger-associated molecular pattern (DAMP) associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that BPI expression in the murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium & Shigella flexneri) whereas mutants which don’t cause intestinal damage (STM fliC & STM invC), didn’t induce BPI expression. These findings have a huge impact on our current understanding of AMP response during inflammatory bowel diseases (IBD). Our results suggest that dysregulation of BPI expression might be an effect rather than a cause of IBD. This chapter concludes with a discussion on the importance of potassium efflux associated with membrane damage as an important signal that helps in discriminating the invading pathogen from the pool of gut microflora. Bactericidal/permeability-increasing protein had been shown to possess anti-inflammatory and endotoxin neutralizing activity by interacting with LPS of Gram-negative bacteria. Even though rBPI (recombinant BPI) has cleared phase III clinical trials for treating endotoxemia, the high cost of purified BPI provided by pharmaceutical companies makes it inaccessible or unavailable for the common man. In Chapter 4, we examined the feasibility of using murine BPI (mBPI) expressed on halophilic Archaeal gas vesicle nanoparticles (GVNPs) for the treatment of endotoxemia in high-risk patients, using a murine model of D-galactosamine-induced endotoxic shock. Halobacterium sp. NRC-1 was used to express the N-terminal 199 amino acid residues of mBPI fused to the GVNP GvpC protein, and bound to the surface of the haloarchaeal GVNPs. Our results indicate that delivery of mBPIN-GVNPs increase the survival rate of mice challenged with lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine. Additionally, the mBPIN-GVNP-treated mice displayed reduced symptoms of inflammation including inflammatory anemia, recruitment of neutrophils, liver apoptosis and pro-inflammatory serum cytokine levels. This chapter concludes with a discussion of the advantages of using mBPIN-GVNPs over purified protein in treating endotoxic shock.