1,114 research outputs found
Prospects for the development of a subunit vaccine against Mycobacterium ulcerans disease (Buruli ulcer)
No full textBuruli ulcer (BU) is a slow progressing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. It presents in different clinical forms ranging from small non-ulcerative nodules to large ulcers and sometimes multiple ulcerations. The highest prevalence of the focally occurring disease is found in rural areas of West African countries. Both the mode of transmission as well as the potential environmental reservoir of M. ulcerans remain unidentified to date. In Cameroon, the remote Mapé dam region has recently been identified as a new BU endemic area. To assess the age-adjusted prevalence and local geographic distribution of BU, a house-by-house survey in the Bankim health district was conducted. Results showed that children between the age of five to 15 and elderly people were over proportionally affected by BU. To confirm the clinical diagnosis of BU during and after the health survey in Bankim, fine needle aspirates and swabs from undermined ulcer edges were transported to the Swiss Tropical and Public Health Institute for laboratory confirmation by quantitative real time PCR. In parallel we developed a protocol for primary culture initiation of M. ulcerans from patient lesions after a long time span between sampling and processing in a BSL3 culture laboratory. The established two sets of Cameroonian patient isolates from the Mapé and the Nyong river valleys were used for a comparative genome sequencing study revealing the presence of two distinct phylogenetic clonal complexes in Cameroon. Despite the fact that BU can be treated with antibiotics, the socioeconomic impact of the disease on affected populations remains devastating. As long as it is not clearly known how the disease is contracted, interruption of transmission is not an option. A vaccine against M. ulcerans on the other hand could be used both as preventive measure and therapeutically. While sero-epidemiological studies imply the presence of protective immunity in some individuals, no vaccine is available to date. Within the framework of the EU funded collaborative project BuruliVac we investigated the potential for developing a protein subunit vaccine against M. ulcerans by delivering vaccine candidate antigens with three different systems: i. as recombinant proteins with an adjuvant, ii. as vesicular stomatitis virus replicon and iii. incorporated into a genetically modified mouse malaria parasite (Plasmodium berghei) in an infection - treatment - approach. All three formulations were assessed for their immunogenicity and their protective potential in a mouse model of BU. Although all three vaccination approaches elicited strong humoral immune responses, no full protection was observed for any of the formulations. However a slight partial protection was seen for a replicon - prime / recombinant protein boost regimen with a vesicular stomatitis virus replicon incorporating an expression cassette for the M. ulcerans protein MUL2232. Additionally, a transient delay of foot pad swelling was observed in mice receiving infection - treatment - vaccination with P. berghei expressing MUL4987. Despite the mainly extracellular nature of M. ulcerans in infected tissue, antibody production against the protein vaccine candidates thus does not seem to be sufficient for protection. Considering marked differences between the mouse footpad model of BU and the disease in humans, we aimed at developing an animal model that better reflects local pathogenesis and host-pathogen interactions in the human BU lesions. Hence, we developed the pig as novel animal model for BU and showed that the observed histopathological changes in the infected pig skin closely represent those of human BU. Therefore the pig model has great potential for the validation of new therapeutic and prophylactic interventions
Development of a malaria vaccine candidate based on virosome technology
No full textMalaria is an infectious disease caused by protozoan pathogens of the genus
Plasmodium. The most important species affecting humans is P. falciparum
transmitted by the bite of female Anopheles mosquitoes during a blood meal. About
40% of the world’s population is exposed to the parasite and 350 to 500 million cases
of disease and more than one millions deaths are reported every year. Almost 80%
of these cases occur in sub-Saharan Africa, where mainly children younger than five
years and pregnant women are affected. Due to problems with arising resistance of
mosquitoes against insecticides and parasites against drugs, the development of a
malaria vaccine is an urgent need. It has been shown more than 30 years ago that
sterile protection against malaria infection is feasible by vaccination with irradiated
sporozoites. Since then many malaria vaccine candidates have been developed but
there is no vaccine on the market to date.
We have established a strategy to develop synthetic peptides of P. falciparum
antigens for inclusion in a multi-stage multivalent malaria subunit vaccine based on
the immunopotentiating reconstituted influenza virosome (IRIV) technology. IRIVs are
an already registered delivery/adjuvant system based on liposomes incorporating
influenza surface proteins hemagglutinin and neuraminidase. IRIVs enhance and
facilitate the delivery of antigens to antigen presenting cells. This technology allows a
stepwise lead peptide optimization based on parasite-binding properties of antibodies
elicited after immunization of experimental animals with virosomally-formulated
peptide-phospholipid conjugates. In this thesis the development steps of three
peptide antigens are described: UK-39 a peptide derived from the sporozoite antigen
circumsporozoite protein (CSP), AMA49-CPE derived from the blood-stage protein
apical membrane antigen 1 (AMA-1) and FB-23 derived from the blood-stage protein
serine repeat antigen 5 (SERA5). All optimized peptides induced parasite crossreactive
antibodies in experimental animals. Virosomal formulations of the antigens
UK-39 and AMA49-CPE were carried into a phase 1 clinical trial. Both vaccine
components were safe and immunogenic in malaria-naïve volunteers. Two
immunizations with appropriate doses of UK-39 or AMA49-CPE were enough to
induce high titers of parasite-binding antibodies. Combined delivery of the two
peptides did not interfere with the development of an antibody response to either of
the two antigens. The antibody responses were affinity maturated and long-lived,
indicating the formation of B cell memory. Purified total IgG from UK-39 immunized
volunteers inhibited sporozoite migration, invasion and development in a dose
dependent manner. Further clinical trials with this two-component vaccine candidate
are ongoing and new peptide candidates like FB-23 have been developed and are
now ready for preclinical and clinical profiling
Overview: Mycobacterium ulcerans disease (buruli ulcer)
No full textEnhanced international research efforts since the establishment of the Global BU Initiative in 1998 by the WHO have helped to advance our understanding of the epidemiology, and pathogenesis of Mycobacterium ulcerans infections. Improved methods to cultivate the extremely slow-growing pathogen from BU lesions have laid the groundwork for a variety of studies using M. ulcerans isolates, including the analysis of the genome and proteome of the pathogen, as well as drug susceptibility testing and analyses of host-pathogen interactions in vitro and in animal models. The identification of specific, high-copy number target sequences in the genome of M. ulcerans has enabled the development of diagnostic tests and assays to detect the pathogen in the environment. Important research questions remain about the reservoir(s) of M. ulcerans in aquatic environments, factors leading to or promoting transmission to hosts, and host-pathogen interactions resulting in chronic infection versus spontaneous healing
Monoclonal antibodies as tools in antigen detection assay and vaccine development : design of a sensitive detection test for Brucella bacteria and profiling of the malaria vaccine candidate antigen reticulocyte-binding homolog 2 (PfRH2)
No full textWe aimed at identifying immunodominant Brucella antigens for implementation in new detection tools or for subunit vaccine development. In particular, our strategy was to produce Brucella cell surface antigen-specific monoclonal antibodies (mAbs) for the development of an antigen capture assay for the detection of Brucella cells as potential bio threat agents in complex samples. We generated a panel of Brucella lipopolysaccharide (LPS)-specific mAbs by immunising mice with inactivated B. melitensis and B. abortus cells. The mAbs recognised Brucella species with ‘smooth’ LPS independently of the way how the bacterial cells were inactivated. Two mAbs were implemented into a bead-based Luminex assay detecting ‘smooth’ Brucella spp. with species-dependent detection limits of 2 x 102 to 8 x 104 cells per mL. Integration of the Luminex assay into a multiplex format enabled simultaneous detection of Brucella spp. and three other bio threat agents within a single sample. The developed Luminex assay may be applied for the detection of whole Brucella cells both in natural Brucella outbreak and in bioterrorism attack scenarios.
We also tried to generate mAbs against Brucella cell surface proteins from mice immunised with inactivated whole Brucella cells. While serum antibody responses against both LPS and protein antigens were seen in Western blotting analyses, attempts to generate protein-specific mAbs failed, most likely due to the immunodominant nature of the LPS. Western blot analyses with Brucella lysate also identified antibodies against some immunodominant Brucella proteins in the serum of cattle naturally infected with Brucella spp., however, identification of the recognised proteins with a Brucella-specific peptide microarray failed.
In a second part of the thesis we aimed at evaluating the potential of the Plasmodium falciparum reticulocyte-binding homolog 2 (PfRH2) present in the rhoptries as a malaria blood stage vaccine antigen. We produced PfRH2-specific mAbs by immunising mice with the 40kDa receptor-binding domain of PfRH2. The PfRH2-specific mAbs cross-reacted with the natural PfRH2 protein present in schizont stage parasites and showed a rhoptry-characteristic staining pattern in immunofluorescence microscopy. However when evaluated in functional in vitro and in vivo assays PfRH2 specific mAbs showed no inhibitory effect on erythrocyte invasion. Furthermore, the invasion-inhibitory effect of mAbs specific for the cysteine-rich protective antigen (PfCyRPA) was not enhanced by PfRH2-specific mAbs
Development of new molecular genetic tools to study "Mycobacterium ulcerans" infection (Buruli ulcer)
No full textBuruli ulcer is an infectious disease caused by an environmental pathogen, Mycobacterium
ulcerans, which is the third major mycobacterial pathogen of man, after M. tuberculosis and
M. leprae. Since 1980, dramatic increases in the incidence of Buruli ulcer have been reported
from West African countries, sometimes associated with man-made environmental changes.
After the first international conference on Buruli ulcer in 1998 (Yamoussoukro meeting),
attention has been drawn to the severity of this neglected disease and to its many poorly
understood features. Since then new initiatives have been undertaken to promote control and
research efforts. Within the framework of WHO identified research priorities, the present PhD
project focused on the development of new molecular genetic tools to investigate M. ulcerans
epidemiology and pathology.
Apart from the association of Buruli ulcer with swampy environments, little is known about
risk factors, environmental reservoirs and pathways of transmission. One factor that impairs
research on these issues is the lack of suitable fine typing methods to track different M.
ulcerans subclones and their spreading within a community. The comprehension of the
population structure itself and of the mechanisms leading to genetic variability also suffers
from this lack of tools. For this reason, we developed a new plasmid-based microarray
approach, which was used to perform a comparative genomic analysis of 30 M. ulcerans
strains, from different geographical origins. Fifteen large sequence polymorphisms were
identified affecting genes of all major functional categories. Results obtained with this
prototype microarray demonstrated that insertional/deletional events, often associated with
insertion sequences are the most important mechanisms of genetic diversification in M.
ulcerans. Analysis of strain diversity with a larger microarray should represent a suitable tool
for micro-epidemiological studies.
Within the framework of a Buruli ulcer survey in Cameroon, an optimized diagnostic PCR
was developed. The method, operating on genetic material extracted directly from swab
samples, demonstrated the usefulness of such highly sensitive technique for epidemiological
studies. Neglected Buruli ulcer foci have been rediscovered and an association between Buruli
ulcer cases and slow flowing water basins have been reconfirmed.
A quantitative PCR specific for M. ulcerans DNA, the IS2404 real-time PCR, was developed
with the aim to gain insights into the pathology of the disease. The very high sensitivity and
specificity of the method allowed the quantitative assessment of the dissemination of the
mycobacteria in Buruli ulcer lesions, and its comparison with histopathological changes.
Although the heaviest mycobacterial burden was detected in the central foci of the lesions, we
could measure significant amounts of mycobacterial DNA and microcolonies in samples from
peripheral regions and occasionally in healthy appearing excised tissue margins. Additional
peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing.
Even when granulomas provided evidence for the development of cell-mediated immunity,
development of satellite lesions by contiguous spreading was not completely prevented. The
technique offers also the potential to predict recurrences: in one case we could demonstrate
that a relatively small number of mycobacteria that have spread into healthy appearing tissue
can lead to the development of a recrudescence. These data altogether support the concept that
wider surgical excision improves the chance of healing of Buruli ulcer. The application of our
approach for assessing the mycobacterial burden in excision margins, combined with long
term follow-up of patients, should help to improve current guidelines for surgical treatment of
Buruli ulcer.
It is becoming more and more evident that mycobacterial spreading can occur even at distant
sites from the original primary ulcer, producing so called “metastatic lesions”. The
contribution of re-activation versus re-infection is not clear, neither is the mode of spreading
into the body known. In the case of a HIV+ patient, we could report the insurgence of
multifocal aggressive lesions leading to osteomyelitis. The time span interposing between the
primary Buruli manifestations and the recurrence at the new sites, together with the physical
distance of the patient from the endemic area, is such to argue about eventual persistence of
M. ulcerans in an immunocompromised individual
Genetic diversity and immune evasion of bacterial pathogens
No full textWithin the framework of this thesis the genetic diversity of three bacterial pathogens, Mycobacterium ulcerans, Neisseria meningitidis and Streptococcus agalactiae was investigated. The aim of these analyses was to contribute to the understanding of how genetic properties of the pathogens contribute to immune evasion. Implications of the findings for vaccine design are discussed.
Mycobacterium ulcerans
Buruli ulcer is a disease of skin and soft tissue caused by the bacterial pathogen Mycobacterium ulcerans. M. ulcerans has recently diverged from an M. marinum progenitor through the acquisition of a virulence plasmid, lateral gene transfer and reductive evolution. Isolates of M. ulcerans deriving from different regions of the globe can be associated with two distinct lineages, either the ancestral or the classical lineage. Here, we show that the two copies of the esxB-esxA gene cluster present in the genome of M. marinum are both deleted from the genome of M. ulcerans strains belonging to the highly virulent classical lineage. Members of the ancestral M. ulcerans lineage instead retained copies of the esxB-esxA gene cluster. Additionally, the hspX gene was present in the strains of the ancestral lineage and absent in the classical lineage. Our results indicate that M. ulcerans is adapting to an environment that is screened by immune recognition mechanisms by loss of highly immunogenic proteins.
Neisseria meningitidis
Certain hypervirulent lineages of Neisseria meningitidis, a commensal of the human nasopharynx, are a major cause of meningitis and septicaemia. Here we have investigated subcapsular antigens of serogroup A Neisseria meningitidis strains isolated in the course of longitudinal colonization and disease surveys in the African meningitis belt. In the course of clonal waves of colonization and disease we observed no sequence diversification of the outer membrane proteins PorA, PorB
and FetA. In contrast, high variability in the expression of Opa proteins was observed due to changing numbers of pentamer repeats within the open reading frames of the four opa genes opaA, opaB, opaD and opaJ. Furthermore, we found some exchange of alleles of the opa genes OpaA and OpaJ by horizontal gene transfer. Herd immunity may thus be a stronger driving force for diversification of Opa proteins than for other outer membrane proteins.
Streptococcus agalactiae
While Streptococcus agalactiae, the group B streptococcus (GBS), is traditionally considered a neonatal pathogen, it is also emerging as a significant cause of morbidity in adults. Here we have analysed the population structure of GBS isolates, collected from carriers and clinical cases in Kenya. Multi-locus sequence typing differentiated the 173 strains analyzed into 22 sequence types (STs), including 5 novel STs. A close correlation between STs and distinct capsular serotypes was found with the disease isolates being more diversified with respect to both STs and capsular serotypes than carrier isolates. The STs and capsular serotypes most prevalent in Kenya were also commonly found in many other regions of the world.
In this investigation, the highest genetic variablility was found in our GBS collection. In the N. meningitidis isolates collected during clonal waves of meningococcal colonization and disease, there was a striking lack of diversification, with the exception of the opa genes. The most conserved bacterial pathogen in this study was M. ulcerans, where no genetic variability could be found within a geographic region.
No vaccines exist to date against M. ulcerans or S. agalactiae, and an affordable universal vaccine against N. meningitidis is urgently needed. The technological advances in whole genome sequencing are likely to facilitate efforts towards finding suitable candidate antigens for subunit vaccines
Identification and characterization of "Plasmodium falciparum" and "Mycobacterium ulcerans" antigens as potential vaccine components and targets for serological test and molecular typing methods
No full textThe increase of Plasmodium falciparum’s drug resistance and the resurgence of
Mycobacterium ulcerans disease through environmental changes aggravate health
problems caused by these pathogens.
Buruli ulcer, caused by M. ulcerans, is after tuberculosis and leprosy the third most
common mycobacterial human infection and the most poorly understood of these three
diseases. It is characterized by chronic, necrotizing ulceration of subcutaneous tissues and
the overlying skin. M. ulcerans is a slow-growing mycobacterium which multiplies extracellularly
in Buruli ulcer lesions. There is a broad antigenic overlap between mycobacterial
species, which complicates the analysis of adaptive immune responses and hampers the
development of specific sero-diagnostic tests for M. ulcerans in areas where BCG
vaccination has been implemented and tuberculosis is endemic. In an effort to identify
immunodominant antigens of M. ulcerans, we have generated panels of monoclonal
antibodies from mice immunized with this pathogen. Cross-reactivity studies with other
mycobacterial species performed by Western blot and immunofluorescence assays have
identified immunodominant epitopes with a limited cross-species distribution (18kDa and
the 34-37kDa proteins). In contrast, the majority of antigens were spread widely amongst
different mycobacterial species. One set of non-crossreactive monoclonal antibodies
recognized an 18kDa protein of M. ulcerans that is associated with the cell-wall fraction,
and expressed in Buruli ulcer lesions. The target protein was identified by massspectroscopy
as the M. ulcerans orthologue of the M. leprae 18kDa small heat shock
protein, which has no orthologues in the genomes of M. bovis and M. tuberculosis. Human
anti-18kDa small heat shock protein antibodies were found in the serum of all Buruli ulcer
patients tested, but not in sera from Europeans volunteers and only rarely in sera from
Africans living in Buruli ulcer non-endemic regions. Reactivity of sera from a large
proportion of people living in a Buruli ulcer endemic area and in contact with Buruli ulcer
patients indicated that an 18kDa small heat shock protein-based serological test is suitable
to detect exposure to M. ulcerans.
Since M. ulcerans shows only very limited genetic diversity, standard multi-locus
sequence typing of housekeeping genes is not a suitable tool for molecular epidemiological
analysis of Buruli ulcer. Among the monoclonal antibodies exhibiting broad inter-species
cross-reactivity, one group recognized the M. ulcerans orthologue of mycobacterial
laminin-binding protein. DNA sequence analysis demonstrated that the corresponding
hupB gene from M. ulcerans isolates of diverse geographical origin exhibited considerable
diversity based both on insertional/deletional polymorphism and on single base exchanges.
Dominance of non-conservative exchanges was indicative of a diversifying selection
pressure. Sequences analysis of a set of such variable genes may develop into a new tool
for genetic fingerprinting of isolates.
There is great need to identify new malaria vaccine and drug targets. Monoclonal
antibodies were used to characterize a novel conserved protein of P. falciparum designated
D13. Western blot analysis demonstrated that D13 is stage-specifically expressed during
schizogony in asexual blood stages of the parasite. It has a functionally essential role in
parasite biology, since anti-D13 monoclonal antibodies have parasite growth inhibitory
activity. The D13 protein may represent a suitable target for a malaria vaccine design.
Immunofluorescence analysis with monoclonal antibodies specific for glyceraldehydes-3-
phosphate dehydrogenase (pfGAPDH) and pfAldolase showed that pfGAPDH and
pfAldolase colocalise in early stages of both liver and asexual blood stage parasite
development. However, during schizogony, unlike pfAldolase, pfGAPDH was enriched in
the apical region of the parasites. In addition, Western blot analyses demonstrate that
pfGAPDH is in both the membrane-containing pellet and supernatant fractions. These
results have provided evidence that pfGAPDH exerts non-glycolytic function(s) in P.
falciparum; including possibly a role in vesicular transport and biogenesis of apical
organelles. This data together with the limited amino acid sequence identity with human
GAPDH suggest that the pfGAPDH could be a promising safe target for drug treatment
Epidemiology of Buruli ulcer in the Mapé Basin of Cameroon
No full textBuruli ulcer (BU) is a neglected tropical disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. The disease has been reported from over 30 countries with most cases coming from West Africa. While it is commonly accepted that BU is not acquired by human-to-human transmission both the environmental reservoir of the pathogen and the mode of transmission to humans remain to be identified. Clinically BU presents with a wide range of forms which includes non-ulcerative lesions and ulcers with undermined edges. Much of the pathology of a M. ulcerans infection is believed to be caused by its unique ability to produce the macrolide toxin called mycolactone which causes tissue necrosis and local immunosuppression. BU can be diagnosed by microscopy, polymerase chain reaction (PCR), culturing and histology, however due to lack of access to laboratory facilities, cases are often diagnosed based on clinical symptoms only. Historically, BU was treated using wide scale excision, but since 2004 the WHO recommends the use of streptomycin and rifampicin daily for 8 weeks to treat the infection.
In the framework of this PhD thesis we have established a new BU field research site in the Mapé Basin of Cameroon and studied various aspects of BU epidemiology, differential diagnosis and transmission at this location. As a basis for our research, we conducted an exhaustive house-by-house survey for BU, leprosy and yaws in the Bankim Health District. Following the survey we closely monitored and studied all BU cases detected in the region. By supporting local laboratory diagnosis with real time-PCR (RT-PCR) and culturing, we were able to identify and describe a case of cutaneous tuberculosis which was initially diagnosed as BU. This patient highlighted the importance of further support to improve clinical differential diagnosis of BU in remote endemic areas. Eighty-eight of all the RT-PCR confirmed BU cases identified in the Mapé Basin in the course of our research were studied in detail. By mapping the patients’ homes we were able to describe the distribution of BU in the area and to identify highly endemic communities. Based on the population age data collected in the survey we were also able to compute the age adjusted cumulative incidence rate of BU, revealing that children below the age of five were underrepresented among cases of BU. By analysing serological responses of patients and community members from BU endemic areas in the Mapé Basin and Ghana against an immunodominant antigen of M. ulcerans, we have observed a similar late onset of seroconversion, indicating that BU transmission intensifies when preschool children start moving further away from home. To identify potential sites of M. ulcerans transmission, we screened sites of environmental contact at the homes and farms of laboratory confirmed BU patients for the presence of M. ulcerans DNA. In this analysis we identified three RT-PCR positive permanent village water sites and by studying one of these sites longitudinally in great detail we obtained evidence that M. ulcerans can persist in underwater detritus. This niche of M. ulcerans may represent an environmental reservoir and a source of infection of the pathogen. To further elucidate BU transmission pathways we have generated a set of clinical isolates of M. ulcerans from the Mapé Basin for a phylogeographic analysis of the distribution of the currently circulating haplotypes of M. ulcerans based on whole genome sequencing. By the routine culturing from clinical specimens and the evaluation of different transport media and decontamination methods we were further able to develop an optimized protocol for the primary isolation of M. ulcerans after long term storage of samples.
Our interdisciplinary research approach including biomedical and social science research elements, including future behavioural studies in young children, may eventually help to elucidate transmission and to improve control of BU
"Mycobacterium ulcerans" disease and treatment : a histopathological perspective
No full textSummary: Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing skin disease with the highest prevalence in West-African countries. The current WHO treatment recommendation is a combination of the two antibiotics rifampicin, given orally and streptomycin, requiring daily injections. Although antibiotic treatment reduced recurrence rates below 2% and circumvents surgical excision in part of the patients, toxic side effects, immunopathological adverse events and other wound healing disorders may occur. In this thesis we conducted detailed histopathological studies to better characterize responses to antibiotic treatment and the nature of paradoxical reactions.
In the case of non-ulcerative plaque lesions, we observed in half of all patients either an enlargement or ulceration of lesions during antibiotic therapy. Histopathological analysis after completion of antibiotic treatment revealed the persistence of extensive necrotic areas besides hallmarks of successfully treated BU lesions, like infiltration, granuloma formation and loss of solid staining of the mycobacteria. Where removal of the necrotic tissue by the immune system is not efficient enough, lesions are ulcerating, leading to the discharge of necrotic tissue. Based on the clinical and histopathological data it is suggested to support healing of such plaque lesions by surgical débridement. While our data demonstrate that the antibiotic therapy efficiently destroys M. ulcerans infection foci, they also indicate that proper wound management during and after chemotherapy is for advanced BU lesions as important as the antibiotic treatment itself.
Secondary lesions may occur at distant body sites during and after chemotherapy. Our analysis of such secondary lesions revealed hallmarks of inactive M. ulcerans infection. Emergence of secondary lesions during antibiotic treatment may thus be the result of immune-mediated paradoxical reactions driven by mycobacterial antigens and immunostimulators at sites of clinically inconspicuous infection foci. These lesions may, however, also have arisen from new infections or mycobacteria that have survived chemotherapy. In this case our histopathological findings would indicate that after priming during the successful treatment of the initial lesion, the immune system is capable of eliminating new infection foci. Our results demonstrate that no additional antibiotic treatment is necessary, when such secondary lesions appear.
To reduce the danger of toxic side effects and to obviate the need for daily injections, an entirely oral treatment without streptomycin would be preferable. In the framework of this thesis we analyzed tissue samples from a first clinical treatment trial using clarithromycin instead of streptomycin. Histopathological findings and efficacy were comparable to the current treatment, leading to the planning of a WHO-sponsored large multi-center trial comparing the efficacy of eight weeks treatment with clarithromycin and rifampicin versus streptomycin and rifampicin.
To gain deeper insights into the histopathological changes in the very early phase of M. ulcerans infection, we conducted in parallel to the analysis of clinical samples, longitudinal histopathological studies in experimentally infected mice. Many features found in the mouse model, such as an early wave of infiltrating neutrophils, as well as the formation of B-cell clusters and the loss of solid Ziehl-Neelsen staining of mycobacteria during chemotherapy, have correlates in human BU lesions. The mouse infection model thus appears to be suitable for the preclinical evaluation of new drug treatments and of candidate vaccines.
Taken together the results described in this thesis demonstrate that histopathology is an important tool to strengthen diagnosis of BU, to evaluate new treatment regimens and to come to a better understanding of paradoxical reactions emerging during antibiotic treatment of BU.
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Zusammenfassung: Das Buruli-Ulkus (BU) ist eine nekrotisierende Hauterkrankung, die von Mycobacterium ulcerans hervorgerufen wird und vor allem in den tropischen Regionen West-Afrikas auftritt. Gegenwärtig empfiehlt die Weltgesundheits-organisation (WHO) eine systemische Chemotherapie mit dem oral verabreichten Antibiotikum Rifampicin kombiniert mit einer täglichen Injektion von Streptomycin. Nach dieser Therapie treten bei weniger als 2% der Patienten Rückfälle auf und eine chirurgische Zusatzbehandlung der Läsion ist bei vielen Patienten nicht erforderlich. Allerdings können toxische Nebenwirkungen sowie immunpathologische Reaktionen und andere Wundheilungsstörungen auftreten. In der vorliegenden Arbeit wurden detaillierte histopathologische Studien durchgeführt, um die therapeutische Wirksamkeit der Chemotherapie des BU besser zu verstehen und die Natur von paradoxen Reaktionen zu charakterisieren.
Bei vielen Patienten mit zunächst geschlossenen BU Plaques kommt es während der Chemotherapie zur Ulzeration. Unsere histopathologischen Analysen zeigten, dass auch nach Beendigung der vollständigen Antibiotika-Behandlung noch grosse nekrotische Gewebebereiche zurückbleiben können. Daneben sind andererseits auch typische Merkmale einer behandelten BU-Läsion wie Infiltration, Granulom-Bildung und Verlust der vollständigen Anfärbbarkeit der Mycobakterien zu beobachten. Unvollständige Resorption des nekrotischen Gewebes durch das Immunsystem führt jedoch häufig zur Ulzeration und Abstossung des nekrotischen Gewebes. Basierend auf unseren histopathologischen und klinischen Daten erscheint es angezeigt, die vollständige Ausheilung von Plaques durch chirurgisches Debridement zu beschleunigen. Die Befunde zeigen, dass M. ulcerans Bakterien durch die Chemotherapie mit Rifampicin und Streptomycin zwar effektiv abgetötet werden, dass aber vor allem im fortgeschrittenen Stadium eine gute Wundversorgung für den Heilungserfolg beim BU ebenso wichtig ist.
Bei einem Teil der BU Patienten entwickeln sich während oder nach Abschluss der Chemotherapie sekundäre Läsionen. Bei der Analyse solcher Läsionen haben wir charakteristische Merkmale einer inaktiven M. ulcerans Infektion gefunden. Der Auslöser für die Entwicklung von Sekundärläsionen könnte daher eine paradoxe Immunreaktion gegen noch im Gewebe vorhandene mykobakterielle Antigene bei klinisch zuvor nicht auffälligen Infektionsherden sein. Möglich wäre allerdings auch, dass sich nach der Chemotherapie durch überlebende Mycobakterien oder durch Neuinfektion neue Läsionen gebildet haben. In diesem Fall würden unsere Ergebnisse darauf hindeuten, dass die neuen Infektionsherde durch eine während der Erstinfektion gebildete protektive Immunantwort zerstört worden sind. Insgesamt zeigen unsere Untersuchungen, dass eine erneute Antibiotikagabe beim Auftreten von sekundären Läsionen nicht erforderlich ist.
Zur Reduzierung der Gefahr toxischer Nebenwirkungen und zur Vermeidung der täglich erforderlichen Injektionen, wäre eine vollständig orale Chemotherapie ohne Streptomycin anstrebenswert. Bei einer ersten klinischen Studie, bei der Streptomycin durch Clarithromycin ersetzt wurde, waren Wirksamkeit und histopathologische Beobachtungen vergleichbar zu den Ergebnissen mit der gegenwärtigen Standardtherapie. Basierend auf diesen Ergebnissen plant die Weltgesundheitsorganisation eine multizentrische klinische Studie, bei der 8 Wochen Rifampicin/Clarithromycin mit 8 Wochen Rifampicin/Streptomycin verglichen werden soll.
Um einen tieferen Einblick in die frühen Phasen der M. ulcerans Infektion zu bekommen, wurden longitudinale histopathologische Untersuchungen mit experimentell infizierten Mäusen durchgeführt. Viele histopathologische Befunde, wie das Auftreten einer ersten Infiltration mit Neutrophilen in der Frühphase der Infektion und die Entwicklung von B-Zell-Anhäufungen und der Verlust der gleichmässigen Anfärbbarkeit der Mycobakterien während der Chemotherapie haben Entsprechungen bei der BU Erkrankung beim Menschen. Das Mausmodell scheint daher geeignet zu sein, neue Therapien und Kandidaten-Impfstoffe präklinisch zu testen.
Insgesamt zeigen unsere Ergebnisse, dass die Histopathologie nicht nur ein sehr aussagekräftiges Diagnosewerkzeug ist, sondern dass sie auch einen wichtigen Beitrag zur Beurteilung neuer Behandlungsmethoden und zur Erforschung paradoxer Reaktion während der Antibiotika Behandlung leisten kann
Development and application of new approaches to study the epidemiology of "mycobacterium ulcerans" disease (Buruli ulcer) in Ghana
No full textMycobacterium ulcerans, causing the devastating skin disease Buruli ulcer (BU), has evolved from the fish pathogen M. marinum. By the acquisition of a virulence plasmid (pMUM) a progenitor of M. ulcerans has gained the unique property of producing a polyketide-derived macrolide toxin termed mycolactone. The toxin is responsible for the characteristic chronic ulcerative pathology of BU. Adoption of pMUM was probably the key event in the evolution of M. ulcerans, constituting a population bottleneck that led to the development of a new species with a highly clonal population structure. Subsequent reductive evolution is indicative for the adaptation to more stable environmental niches and led to the emergence of three M. ulcerans lineages considered as different ecotypes. M. ulcerans lineage 3, responsible for BU infections in Africa and Australia, is characterized by extremely low levels of genetic diversity. This genetic monomorphism has long hindered molecular epidemiological studies aiming at the identification of transmission pathways and environmental sources of M. ulcerans.
Our whole genome sequence comparison of Ghanaian M. ulcerans strains representing three previously identified variable number of tandem repeat (VNTR) types in Ghana, enabled the identification of single nucleotide polymorphisms (SNPs). Real-time PCR-based SNP typing assays at the detected SNP loci facilitated medium-throughput analyses of a comprehensive collection of Ghanaian M. ulcerans isolates. Typing results of M. ulcerans strains isolated between 1999 and 2007 from patients residing in the BU endemic Densu River Basin and another BU endemic area in the Amansie West district of Ghana demonstrated the presence of two different clonal complexes in the two regions. Local clustering of certain haplotypes within the Densu River Basin revealed that M. ulcerans is very focally transmitted, which excludes some of the currently discussed modes of transmission. Our recently developed temperature-switch PCR-based typing strategy is used by a reference laboratory in Ghana to monitor the spatio-temporal distribution and further spread of the detected M. ulcerans variants in that region.
M. ulcerans infection foci are commonly connected with close proximity to human-disturbed aquatic environments. While BU in Ghana is highly associated with residence along the lower part of the Densu River, where the construction of an impoundment has led to the formation of a lake and wetlands surrounding this area, no infections have so far been reported from villages located further upstream. However, results of our sero-epidemiology studies indicated that healthy inhabitants of communities both downstream (BU endemic) and upstream (BU non-endemic) of the Densu River are equally exposed to M. ulcerans. About one third of healthy individuals from both areas showed specific humoral responses against the M. ulcerans 18KDa small heat shock protein. Moreover, environmental samples collected in the BU endemic and non-endemic communities showed no significant difference in PCR positivity targeting M. ulcerans sequences.
Elusive transmission pathways and reservoirs of M. ulcerans combined with the lack of a vaccine against BU complicate the prevention of this disease. Since the current strategy to control BU relies on early case detection and rapid treatment, the identification of alternative drugs is of high importance. Therefore a compound screening platform for M. ulcerans was developed within the framework of this thesis and is currently used to screen compound libraries for new scaffolds which may eventually have the potential to replace rifampicin.
Taken together, the spatio-temporal patterns of M. ulcerans haplotypes in the Densu River Valley as well as the observed clustering of BU cases in certain communities along the Densu River indicate that the emergence of M. ulcerans infection foci cannot be solely explained by an interaction between M. ulcerans and the environment. We hypothesize that BU patients with ulcerative lesions containing vast loads of mycobacteria may play an active role in the dissemination of M. ulcerans in the environment. This would have major implications for strategies to better control the disease and remains to be investigated further. ---------- Zusammenfassung: Mycobacterium ulcerans, der Erreger der gravierenden Hauterkrankung Buruli-Ulkus (BU), hat sich aus dem Fischpathogen M. marinum entwickelt. Durch den Erwerb eines Virulenzplasmids (pMUM) hat ein M. ulcerans-Vorfahre die einzigartige Fähigkeit erlangt, ein Makrolid-Toxin namens Mycolakton zu produzieren, das für die BU-typische chronisch-ulzerative Pathologie verantwortlich ist. Die Aufnahme von pMUM war vermutlich das Schlüsselereignis für die Entstehung des Erregers und führte durch diesen genetischen „Flaschenhalseffekt“ zu der Entwicklung einer neuen Spezies mit hochklonaler Populationsstruktur. Die darauf folgende reduktive Evolution deutet auf die Anpassung an eine stabilere ökologische Nische hin und führte zu der Entwicklung von drei M. ulcerans Abstammungslinien, die verschiedene Ökotypen darstellen. Da die M. ulcerans Abstammungslinie 3, die BU Infektionen in Afrika und Australien hervorruft, nur minimale genetische Diversität aufweist, war die Erforschung der Herkunft und Übertragungswege von M. ulcerans durch Feintypisierung von Isolaten lange Zeit unmöglich.
Vergleiche der Genomsequenzen von Vertretern der drei in Ghana vorhandenen M. ulcerans VNTR (Variable Number of Tandem Repeat) Varianten ermöglichte die Identifizierung von Einzelnukleotid-Polymorphismen (single nucleotide polymorphisms, SNPs). Mit Hilfe der identifizierten SNP-Positionen wurden Real-Time PCR-basierende Assays entwickelt, mit denen M. ulcerans Isolate aus Ghana typisiert werden konnten. Die Typisierung von M. ulcerans Stämmen, die zwischen 1999 und 2007 von Patienten aus dem BU endemischen Densu Flussgebiet und einer anderen endemischen Region im Amansie West Bezirk isoliert wurden, hat gezeigt, dass in den beiden Regionen zwei unterschiedliche klonale M. ulcerans Komplexe vorherrschen. Da auch innerhalb des Densu Flussgebietes die Verbreitung der meisten M. ulcerans Varianten nur auf ein bestimmtes Teilgebiet begrenzt war, können einige der derzeit diskutierten Übertragungswege des offensichtlich sehr fokal übertragenen Erregers ausgeschlossen werden.
M. ulcerans Infektionsbrennpunkte sind im Allgemeinen mit aquatischen Umgebungen assoziiert, die durch den Menschen verändert worden sind. So tritt BU in Ghana vorwiegend entlang des unteren Densu Flussbereichs auf, in dem durch den Bau eines Staudamms ein See mit umliegenden Feuchtgebieten entstanden ist, während aus Dörfern im oberen Flussbereich noch keine Krankheitsfälle gemeldet wurden. Unsere sero-epidemiologischen Studien haben gezeigt, dass dennoch sowohl Bewohner aus Gemeinden flussabwärts (BU endemisch) als auch weiter flussaufwärts (nicht BU endemisch) gleichermassen mit M. ulcerans in Kontakt kommen. Bei etwa einem Drittel der gesunden Studienteilnehmer aus beiden Flussgebieten konnten spezifische Antikörper gegen das so genannte „18KDa small heat shock protein“ von M. ulcerans nachgewiesen werden.
Da Herkunft und Übertragungswege von M. ulcerans unklar sind und bis heute kein Impfstoff verfügbar ist, wird BU weniger durch Prävention, als vielmehr mit der Behandlung von Patienten in möglichst frühen Krankheitsstadien bekämpft. Daher ist die Suche nach alternativen Chemotherapeutika essentiell. Wir haben im Rahmen dieser Arbeit eine Screening-Plattform für M. ulcerans entwickelt, die nunmehr dazu verwendet wird, ausgewählte Wirkstoffbibliotheken auf neue Substanzen zu durchsuchen, die im besten Fall das Potential haben Rifampicin zu ersetzen.
Zusammengefasst, weisen sowohl die räumlich-zeitliche Verbreitung von M. ulcerans Varianten im Densu Flussgebiet als auch das fokale Auftreten von BU in bestimmten Gemeinden entlang des Flusses darauf hin, dass die Entstehung von M. ulcerans Infektionsbrennpunkten nicht nur auf eine Wechselwirkung zwischen M. ulcerans und der Umwelt zurückzuführen ist. Basierend auf unseren Beobachtungen vermuten wir, dass BU Patienten mit bakteriell hoch belasteten ulzerierenden Läsionen eine wichtige Rolle bei der Verbreitung des Erregers in der Umwelt spielen. Dies könnte bedeutende Konsequenzen für die Vorgehensweise bei der Bekämpfung von BU haben und liefert einen wichtigen Ausgangspunkt für zukünftige Forschung
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