"Mycobacterium ulcerans" disease and treatment : a histopathological perspective

Summary: 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. ---------- 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

Experimental infection of the pig with Mycobacterium ulcerans : a novel model for studying the pathogenesis of Buruli ulcer disease

Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available.; For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2×107 and 2×106 colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans.; Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions

Geographic distribution, age pattern and sites of lesions in a cohort of buruli ulcer patients from the mapé basin of cameroon

Buruli ulcer (BU), a neglected tropical disease of the skin, caused by Mycobacterium ulcerans, occurs most frequently in children in West Africa. Risk factors for BU include proximity to slow flowing water, poor wound care and not wearing protective clothing. Man-made alterations of the environment have been suggested to lead to increased BU incidence. M. ulcerans DNA has been detected in the environment, water bugs and recently also in mosquitoes. Despite these findings, the mode of transmission of BU remains poorly understood and both transmission by insects or direct inoculation from contaminated environment have been suggested. Here, we investigated the BU epidemiology in the Mapé basin of Cameroon where the damming of the Mapé River since 1988 is believed to have increased the incidence of BU. Through a house-by-house survey in spring 2010, which also examined the local population for leprosy and yaws, and continued surveillance thereafter, we identified, till June 2012, altogether 88 RT-PCR positive cases of BU. We found that the age adjusted cumulative incidence of BU was highest in young teenagers and in individuals above the age of 50 and that very young children (>5) were underrepresented among cases. BU lesions clustered around the ankles and at the back of the elbows. This pattern neither matches any of the published mosquito biting site patterns, nor the published distribution of small skin injuries in children, where lesions on the knees are much more frequent. The option of multiple modes of transmission should thus be considered. Analyzing the geographic distribution of cases in the Mapé Dam area revealed a closer association with the Mbam River than with the artificial lake

Gambiense human african trypanosomiasis sequelae after treatment: a follow-up study 12 years after treatment

The clinical presentation of Human African Trypanosomiasis (HAT) due to; Trypanosoma brucei gambiense; is well known, but knowledge on long-term sequelae is limited. In the frame of studies conducted between 2004 and 2005 in the Democratic Republic of the Congo (DRC), the prevalence of HAT related signs and symptoms were evaluated before the start of treatment and at the end of treatment. To explore possible long-term sequelae, the same clinical parameters were assessed in 2017 in 51 first stage and 18 second stage HAT patients. Signs and symptoms 12-13 years after treatment were compared to before and immediately after treatment and to controls matched for sex and age (±5 years). In first stage HAT patients, the prevalence of all signs and symptoms decreased compared to before treatment but were still higher after 12-13 years than immediately at the end of treatment and in the control group. In second stage HAT patients, all HAT-specific findings had continuously decreased to the point where they were in the range of the healthy control group. In a selection of oligosymptomatic first stage HAT patients, no trypanosomes were detected in the blood by microscopic examination or PCR. An oligosymptomatic presentation of HAT due to the persistence of parasites in compartments, where first stage HAT medications do not penetrate, could not be ruled out

Experimental infection of the pig with Mycobacterium ulcerans: a novel model for studying the pathogenesis of Buruli ulcer disease.

BACKGROUND Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available. METHODOLOGY/PRINCIPAL FINDINGS For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2 × 10(7) and 2 × 10(6) colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans. CONCLUSION/SIGNIFICANCE Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Secondary bacterial infections of buruli ulcer lesions before and after chemotherapy with streptomycin and rifampicin

Buruli ulcer (BU), caused by Mycobacterium ulcerans is a chronic necrotizing skin disease. It usually starts with a subcutaneous nodule or plaque containing large clusters of extracellular acid-fast bacilli. Surrounding tissue is destroyed by the cytotoxic macrolide toxin mycolactone produced by microcolonies of M. ulcerans. Skin covering the destroyed subcutaneous fat and soft tissue may eventually break down leading to the formation of large ulcers that progress, if untreated, over months and years. Here we have analyzed the bacterial flora of BU lesions of three different groups of patients before, during and after daily treatment with streptomycin and rifampicin for eight weeks (SR8) and determined drug resistance of the bacteria isolated from the lesions. Before SR8 treatment, more than 60% of the examined BU lesions were infected with other bacteria, with Staphylococcus aureus and Pseudomonas aeruginosa being the most prominent ones. During treatment, 65% of all lesions were still infected, mainly with P. aeruginosa. After completion of SR8 treatment, still more than 75% of lesions clinically suspected to be infected were microbiologically confirmed as infected, mainly with P. aeruginosa or Proteus miriabilis. Drug susceptibility tests revealed especially for S. aureus a high frequency of resistance to the first line drugs used in Ghana. Our results show that secondary infection of BU lesions is common. This could lead to delayed healing and should therefore be further investigated

Histopathological Changes and Clinical Responses of Buruli Ulcer Plaque Lesions during Chemotherapy: A Role for Surgical Removal of Necrotic Tissue?

The tropical necrotizing skin disease Buruli ulcer (BU) caused by Mycobacterium ulcerans is associated with extensive tissue destruction and local immunosuppression caused by the macrolide exotoxin mycolactone. Chemotherapy with a combination of rifampicin and streptomycin for 8 weeks is the currently recommended treatment for all types of BU lesions, including both ulcerative and non-ulcerative stages (plaques, nodules and edema). Our histopathological analysis of twelve BU plaque lesions revealed extensive destruction of sub-cutaneous tissue. This frequently led to ulceration during antibiotic treatment. This should not be mistaken as a failure of the antimycobacterial chemotherapy, since we found no evidence for the persistence of active infection foci. Large necrotic areas were found to persist even after completion of antibiotic treatment. These may disturb wound healing and the role of wound debridement should therefore be formally tested in a clinical trial setting

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc; 1; :aa; 3; . While the cytochrome bc; 1; :aa; 3; is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc; 1; :aa; 3; to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment

Single Nucleotide Polymorphism Typing of Mycobacterium ulcerans Reveals Focal Transmission of Buruli Ulcer in a Highly Endemic Region of Ghana

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted