MMed (Dermatology), Faculty of Health Sciences, University of the Witwatersrand / Introduction: Cutaneous tuberculosis (TB) accounts for about 2 - 3% of all cases of tuberculosis. It is as a result of direct infection of the skin or immune responses to antigenic components of Mycobacterium tuberculosis, known as tuberculids. In sub-Saharan Africa around 70% of patients with tuberculosis are co - infected with the human immunodeficiency virus (HIV). The prevalence of HIV in South Africa is approximately 11.4%. There are no studies in South Africa on the manifestations of cutaneous tuberculosis in the setting of HIV infection. Aims: The objectives of this study were to determine the clinical and histopathological spectrum of cutaneous tuberculosis in the Johannesburg area and to assess the correlation of HIV infection and CD4 count, on the clinical and pathological presentation. Patients and Methods: This was a prospective, hospital-based study conducted over a period of 3 ½ years from Oct 2004 - March 2008. A total of 74 patients diagnosed with cutaneous tuberculosis who were seen during the above mentioned period were enrolled for the study. Patients were enrolled from the three academic hospitals, Johannesburg, Chris Hani Baragwanath and Helen Joseph. Inclusion criteria included patients aged 10 years and older diagnosed with cutaneous tuberculosis. Patients from whom consent could not be obtained and those with lesions caused by non-tuberculous mycobacteria were excluded from the study. v The diagnosis was based on clinical and histopathological features as well as supportive diagnostic tests. Data was captured onto Epi-Info spreadsheet and then analyzed using STATA data software. Results: The entire clinical spectrum of lesions of cutaneous TB was seen with the exception of the nodular and phlebetic tuberculids. Erythema induratum, a tuberculid was the most common form of cutaneous TB accounting for more than a third (36.5%) of all cases. Scrofuloderma was the most common true infection accounting for about 29.7% of all cases. HIV-TB co-infection rate was 61.4%. The histology ranged from a granulomatous inflammation with absence of bacilli to a diffuse inflammation with abundance of bacilli. Conclusions: Despite the high prevalence of TB and high TB-HIV co-infection rate, cutaneous tuberculosis infection is still relatively uncommon. There is a however a relative increase in the frequency of true infections particularly scrofuloderma in comparison with the studies done previously here in South Africa. The association between HIV positive status and true infections was statistically significant with p = 0.024 and was not found to be statistically significant between HIV positive status and tuberculids with p = 0.71.

Habilitationsschrift--Marburg. / "Literatur-Verzeichnis": p. 58.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-28T09:47:26Z No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-28T09:49:33Z (GMT) No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-28T09:49:33Z (GMT). No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Millions of people die every year due to tuberculosis (TB), an infectious disease that have effective treatment, can be prevented and is curable. One of the greatest problems faced by this disease is the latent infection (LTBI), where individuals do not manifest clinical symptoms, is a reservoir of the causing agent, Mycobacterium tuberculosis (Mtb), and can reactive the disease at any time during their life span. Moreover, TB can affect any organ in the body, such as the skin, causing extrapulmonary TB, a rare form of TB, the TB skin. Some of these forms may be more severe than pulmonary TB, causing serious consequences to the patient, contributing to the high mortality rate of this disease. In this context, understanding the immunological events related to the interaction between pathogen and host the development of active disease or latent infection is a crucial point that can contribute to the control of TB. Thus, the objectives of this study were to evaluate the specific immune response of CD8+ T cells and cytokines in cutaneous tuberculosis, latent and active pulmonary TB. Thirty six patients with pulmonary TB, one patient with cutaneous TB and 36 healthy controls, classified as LTBI (N = 13) or negative (TST-, N = 23) by the tuberculin skin test were recruited and the peripheral blood mononuclear cells, plasma and sera from those individuals were collected to perform flow cytometry, ELISA and multiplex bead array analysis. It was observed that patients with active pulmonary TB presented TCD8+ cells with a regulatory profile, expressing IL-10 and TGF-β in a direct relation to the bacillary load. The same profile was observed in the individual with cutaneous TB, an extra-pulmonary form of TB. The findings observed in this studyco nclude that Mtb can modulate CD8+ T cell response in lung and skin tuberculosis, demonstrating the importance of studies assessing the immune interaction between the pathogen and the host. / A tuberculose (TB) é uma doença causada por Mycobacterium tuberculosis, principal agente etiológico da TB humana. Milhões de pessoas morrem todo ano em decorrência da TB, doença infecciosa que tem prevenção, tratamento e cura. Um dos maiores problemas enfrentados com essa doença é a infecção latente (TBIL), onde o indivíduo não manifesta os sintomas clínicos e constitui um reservatório da bactéria, podendo desenvolver a doença ativa em qualquer momento. Além disso, a TB pode afetar qualquer órgão do corpo, como por exemplo, a pele, causando uma forma rara de TB extrapulmonar, a TB cutânea. Algumas dessas formas podem ser mais severas que a TB pulmonar, trazendo consequências graves ao paciente, contribuindo para o alto índice de mortalidade dessa enfermidade. Nesse contexto, entender os eventos imunológicos relacionados à interação entre patógeno e o hospedeiro no desenvolvimento da doença ativa ou da infecção latente é um ponto crucial que pode contribuir para o controle da TB. Diante disso, os objetivos desse trabalho foram avaliar a resposta imune específica de células TCD8+ e citocinas na tuberculose cutânea, latente e pulmonar ativa. Para isso, foram recrutados 36 pacientes com TB pulmonar ativa, 01 paciente com TB cutânea e 36 controles sadios classificados quanto à prova tuberculínica em indivíduos com infecção latente (TBIL = 13) ou não (PT-=23). Foram obtidas, de todos os pacientes, as células mononucleares do sangue periférico, o plasma e o soro para realização dos ensaios de citometria de fluxo e ELISA. Foi observado que os pacientes com TB pulmonar ativa apresentam um perfil regulador de células TCD8+ específicas, com expressão de IL-10 e TGF-β relacionados com a carga bacilar quando comparado aos indivíduos com TBIL e controles sadios PT negativa. Esse mesmo perfil também foi observado e descrito no caso clínico do paciente com TB cutânea. Diante de todos os achados observados nesse trabalho podemos concluir que Mtb pode modular a resposta de células TCD8+ na tuberculose pulmonar e cutânea, demonstrando a importância de estudos que avaliem a interação imunológica entre o patógeno e o hospedeiro tais como este.