Epidemiology of Mycobacterium ulcerans disease in the Bankim Health Distrit of Cameroon and monitoring of the healing process of Buruli Ulcer lesions

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans which, if untreated, can lead to extensive tissue destruction and ulceration. The disease has been reported from over 30 countries with the highest prevalence in West Africa. Generally it is assumed that M. ulcerans is acquired from environmental sources, but BU is considered a “mysterious disease” because the natural reservoir and the mode of transmission are still not identified. Clinically BU presents with a spectrum of forms ranging from non-ulcerative lesions to large ulcers. The gold standard for diagnosing BU is IS2404 qPCR, which is a sophisticated technology not applicable in the field, where BU is often diagnosed on the basis of clinical signs and symptoms only. Direct microscopic smear examination after Ziehl-Neelsen staining, which has a low sensitivity, is the only point-of-care laboratory diagnostic method currently available. Since 2004, the WHO recommends to treat BU with a combination of streptomycin and rifampicin daily for 8 weeks. While this specific treatment is highly effective in killing the bacteria, healing of large ulcers may require long periods of time. The Bankim Health District (HD) in the Mapé dam basin of Cameroon has been recently identified as BU endemic area and a new BU field research site was established by us in 2010. Within the framework of this thesis, we have contributed to strengthening of the local BU treatment and research site by the implementation of a surveillance and documentation system to promote a continuous case detection and follow up of patients, to investigate the pathway of transmission and to perform a comprehensive spatio-temporal distribution analysis of BU in the area. Local clinical and microscopic diagnosis was re-confirmed by qPCR, bacterial culture and histopathology performed in Basel. The in-depth analysis on 148 qPCR confirmed cases underlined that BU is a pediatric disease in Africa and that the lesions occur mainly at the limbs with no differences amongst males and females. We obtained information on the exact geographical origin of 136 qPCR positive BU patients through mapping of their houses and farms. Results revealed for the majority of patients residence or agricultural activities close to the Mbam river. Sites of environmental contact of BU patients were screened to search for potential reservoirs of M. ulcerans. At one village water site, DNA of M. ulcerans, was persistently found over more than 2 years, indicating that the pathogen may persist in detritus. Because some of the BU lesions healed very fast, while others showed an impaired healing process, we analyzed tissue samples in detail for the presence of wound healing and scarring biomarkers. Using the histopathological approach, we evaluated the use of markers of cell activation, myofibroblast formation and matrix deposition for the monitoring of the healing of BU lesions. While α-smooth muscle actin-positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as procollagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype. Healing wounds showed dermal tissue remodelling by apoptosis, and increased cytokeratin 16 expression in the epidermis. Taken together, the results described in this thesis were obtained by a multidisciplinary approach. They contribute to our understanding of BU epidemiology and transmission, as well as of pathogenesis, wound healing and may eventually help to improve diagnosis, treatment and prevention of BU

Immunohistochemical monitoring of wound healing in antibiotic treated buruli ulcer patients

While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression.; Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While α-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis.; We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients

Exudate collection using wound sponges - An easy, non-invasive and reliable method to explore protease activities in ulcers

Proteases are important for wound healing, but in excessive amounts or left uncontrolled, they may cause healing impairment or other severe wound complications. Point-of-care testing for protease activities in wounds may be useful for monitoring the effectiveness of treatment, and for early identification of wounds that potentially fail to heal. Here we describe an easy, noninvasive method to collect wound fluid for evaluating the protease milieu of wounds. Wound fluids were collected using sterile sponges applied between wound surface and normal wound dressing. Wound fluid could be easily squeezed or centrifuged out of the sponges and was tested for gelatinase (MMP-2 and MMP-9) activities by gel zymography. In addition, we measured polymorphonuclear granulocyte elastase levels by ELISA. Both gelatinases were remarkably stable in sponge derived fluids, as no significant loss was observed even when samples were stored for 3 days at room temperature. Protease levels were highly diverse amongst patients and, in some cases, showed substantial variations in the course of the treatment. The here described wound sponge approach represents a patient-friendly and reliable method to collect wound fluid for evaluating wound healing relevant biomarkers, such as matrix metalloproteinases

Evaluation of a PfHRP-2 based rapid diagnostic test versus microscopy method among HIV-positive and unknown serology patients in Ouagadougou, Burkina Faso

We evaluated the performance of a malaria rapid diagnostic test (RDT; Malaria Quick Test(®); Cypress Diagnostic) compared with the standard thick-smear microscopy method using blood samples from human immunodeficiency virus (HIV)-infected individuals and individuals of unknown HIV status collected in Ouagadougou, Burkina Faso. Our results show that 42.1% of 114 HIV-infected patients were concordantly RDT- and thick smear-positive, and 55.3% were concordantly negative. Sensitivity and specificity of the RDT test were 100.0% and 95.4%, respectively, with 5.9% false-positive results and a total agreement of 97.4%; 127 patients with unknown HIV serology were analyzed; of them, 40.9% were RDT- and thick smear-positive, and 46.4% concordantly negative. Sensitivity and specificity were 100.0% and 78.6%, respectively, with 23.5% false-positive results and a total agreement of 87.4%. Malaria Quick Test(®) is rapid and effective for the diagnosis of malaria and has a high sensitivity, confirming its use in general and HIV patients in particular

Psychotherapy trainees' epistemological assumptions influencing research-practice integration

Over the last few decades a growing number of psychotherapy scholars as well as psychotherapy researchers have joined a paradigm shift, moving from a reductionist to a complexity-oriented epistemology. Many authors recognize that when human subjectivity is the object of intervention and study, it is appropriate to resist simplification and to assume a more complex approach. While this paradigm shift is taking place not only in psychology but also in other disciplines, many psychotherapists still share the assumption that psychotherapy practice and psychotherapy research have opposite values; hence, they are worlds that cannot be reconciled. Considering this as one of the main reasons preventing a useful integration of evidence-based practice and clinical training in psychotherapy, we conducted an online survey of 126 Italian trainees from three differently-oriented psychotherapy institutes (cognitive-behavioral, relational-psychoanalytic and relational-systemic) to explore the epistemology underling the clinical and research practices. After presenting a clinical vignette, we asked questions about diagnostic considerations, case formulations, and treatment plans; we also asked questions about participants' involvement in research projects or in research methodology courses and about willingness to be involved in future research studies in their clinical practice. We found some significant differences among trainees with different orientations, but in general most of the responses reflected a positivistic epistemology underlying both clinical and research activities. These findings suggest that a deeper awareness of one's own epistemological assumptions could help trainees foster a more theory-coherent and research-informed clinical practice

Increase of Cytokeratin 16 expression by keratinocytes during antibiotic therapy.

<p>Histological sections were stained by immunohistochemistry with an anti-Cytokeratin 16 antibodies and were counterstained with Haematoxylin. While healthy skin was completely devoid of Cytokeratin 16 staining (A1), some staining was observed (A2) in the epidermal layer of untreated BU lesions (T1). Staining intensity and epidermal thickness increased in samples collected during (T2) and after completion (T3) of antibiotic therapy (A3, A4). After completion of therapy (T3) heterogeneous staining (B, Overview), with some areas of the epidermal layer showing much weaker Cytokeratin 16 staining (Region 1) than others (Region 2) was observed.</p

Increased expression of the ECM proteins tenascin, fibronectin and pro-collagen 1 in healing BU lesions.

<p>Serial histological sections were stained with antibodies against αSMA and the ECM proteins tenascin, fibronectin and pro-collagen 1 and counterstained with Haematoxylin. Panel A represents a typical lesion before commencement of antibiotic therapy (T1) and Panel B and C typical tissue specimens from two patients after completion of therapy (T3). Whereas no or only weak staining for αSMA, tenascin, fibronectin and pro-collagen 1 was observed before therapy (A1–A4), tissues turned strongly positive for all four markers after completion of treatment (B1–B4 and C1–C4). Staining of ECM proteins was most prominent in areas containing many αSMA positive myofibroblasts.</p

Emergence of apoptotic fat cells after completion of antibiotic therapy.

<p>Histological sections were stained by immunohistochemistry with anti-CC3 antibodies and were counterstained with Haematoxylin. Infiltrated necrotic areas (A, C, E) and fat cell layers (B, D, F) of the subcutaneous tissues are displayed. Before treatment some of the infiltrating cells showed CC3 staining (A). No staining was observed in the subcutaneous layer (B). During treatment (C, D) only very few cells showed CC3 staining. After treatment substantial numbers of infiltrating cells were CC3-positive (E) and in addition larger numbers of CC3-positive fat cells were found (F).</p