Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/3422 |
Resumo: | Sporotrichosis is a subcutaneous mycosis that affects humans and animals, and it is caused by dimorphic melanized fungi belonging to the genus Sporothrix. Sporotrichosis is mainly acquired by traumatic implantation of the fungus into the dermis through scratches inflicted by domestic animals or by handling plants. This study approaches: a) the in vitro susceptibility of Sporothrix schenckii complex species to photodynamic inactivation using methylene blue as a photosensitizer and laser as the light source; b) the combination of amphotericin B plus posaconazole in a murine model of systemic infection by S. brasiliensis and S. schenckii sensu stricto, seeking to enhance the treatment of disseminated sporotrichosis; c) the interference of fungal melanin in the in vitro susceptibility of S. brasiliensis and S. schenckii sensu stricto to amphotericin B and itraconazole. In the photodynamic therapy study we have applied methylene blue as a photosensitizing agent and Light emitting diode (InGaAlP) against Sporothrix schenckii complex species in an in vitro assay. The viability of the conidia was determined through the counting of colony forming units. The activity of amphotericin B and posaconazole alone or in combination was evaluated by using a microdilution checkerboard method. For the in vivo study, OF-1 mice were intravenously (i.v.) infected with a lethal inoculum of 2 x 107 CFU/animal. Two strains of S. brasiliensis and two of S. schenckii were tested. Animals received amphotericin i.v. at suboptimal dose i.e., 0.3 mg/kg, in combination with oral posaconazole at 2.5 mg/kg, 5 mg/kg or 10 mg/kg BID. Results were compared with their respective monotherapies and efficacy was evaluated by prolongation of survival and reduction of tissue burden in spleen and liver. In the study of the interference of melanin in the susceptibility of S. brasiliensis and S. schenckii sensu stricto, yeast cells were cultivated in minimal medium with or without L-DOPA in order to induce the production of melanin. Microdilution and time-kill methods were used to determine the antifungal activity against yeast cells with different amounts of melanin. The laser light (InGaAlP) together with a methylene blue photosensitizer were able to significantly reduce the growth of all the tested Sporothrix strains. In vitro data demonstrated a synergistic effect of the combination and although posaconazole alone was effective against sporotrichosis, efficacy was increased when combined with amphotericin B. The maximum efficacy of the combinations was obtained with posaconazole 5 mg/kg. The killing assay showed that melanization protected isolates within the Sporothrix schenckii complex from amphotericin B, particularly in the lower concentrations tested. The conclusions of the present study are: a) the in vitro inhibition of Sporothrix spp. isolates by laser light deserves in vivo experimental and clinical studies since it may be a promising treatment for cutaneous and subcutaneous sporotrichosis; b) posaconazole in combination with suboptimal doses of amphotericin B could be an option in the treatment of disseminated sporotrichosis especially when itraconazole or amphotericin B at optimal doses are contraindicated; c) combination studies of amphotericin B with inhibitors of melanin are required in order to avoid this effect. |
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2016-10-182016-10-182015-02-12MARIO, Débora Alves Nunes. Sporothrix schenckii complex: Photodynamic Inactivation, Influency of Melanin in Antifungal Activity and Drug Combination. 2015. 91 f. Tese (Doutorado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2015.http://repositorio.ufsm.br/handle/1/3422Sporotrichosis is a subcutaneous mycosis that affects humans and animals, and it is caused by dimorphic melanized fungi belonging to the genus Sporothrix. Sporotrichosis is mainly acquired by traumatic implantation of the fungus into the dermis through scratches inflicted by domestic animals or by handling plants. This study approaches: a) the in vitro susceptibility of Sporothrix schenckii complex species to photodynamic inactivation using methylene blue as a photosensitizer and laser as the light source; b) the combination of amphotericin B plus posaconazole in a murine model of systemic infection by S. brasiliensis and S. schenckii sensu stricto, seeking to enhance the treatment of disseminated sporotrichosis; c) the interference of fungal melanin in the in vitro susceptibility of S. brasiliensis and S. schenckii sensu stricto to amphotericin B and itraconazole. In the photodynamic therapy study we have applied methylene blue as a photosensitizing agent and Light emitting diode (InGaAlP) against Sporothrix schenckii complex species in an in vitro assay. The viability of the conidia was determined through the counting of colony forming units. The activity of amphotericin B and posaconazole alone or in combination was evaluated by using a microdilution checkerboard method. For the in vivo study, OF-1 mice were intravenously (i.v.) infected with a lethal inoculum of 2 x 107 CFU/animal. Two strains of S. brasiliensis and two of S. schenckii were tested. Animals received amphotericin i.v. at suboptimal dose i.e., 0.3 mg/kg, in combination with oral posaconazole at 2.5 mg/kg, 5 mg/kg or 10 mg/kg BID. Results were compared with their respective monotherapies and efficacy was evaluated by prolongation of survival and reduction of tissue burden in spleen and liver. In the study of the interference of melanin in the susceptibility of S. brasiliensis and S. schenckii sensu stricto, yeast cells were cultivated in minimal medium with or without L-DOPA in order to induce the production of melanin. Microdilution and time-kill methods were used to determine the antifungal activity against yeast cells with different amounts of melanin. The laser light (InGaAlP) together with a methylene blue photosensitizer were able to significantly reduce the growth of all the tested Sporothrix strains. In vitro data demonstrated a synergistic effect of the combination and although posaconazole alone was effective against sporotrichosis, efficacy was increased when combined with amphotericin B. The maximum efficacy of the combinations was obtained with posaconazole 5 mg/kg. The killing assay showed that melanization protected isolates within the Sporothrix schenckii complex from amphotericin B, particularly in the lower concentrations tested. The conclusions of the present study are: a) the in vitro inhibition of Sporothrix spp. isolates by laser light deserves in vivo experimental and clinical studies since it may be a promising treatment for cutaneous and subcutaneous sporotrichosis; b) posaconazole in combination with suboptimal doses of amphotericin B could be an option in the treatment of disseminated sporotrichosis especially when itraconazole or amphotericin B at optimal doses are contraindicated; c) combination studies of amphotericin B with inhibitors of melanin are required in order to avoid this effect.A esporotricose é uma micose subcutânea que afeta homens e animais, e é causada por fungos dimórficos melanizados pertencentes ao gênero Sporothrix. Pode ser principalmente adquirida por implantação traumática do fungo na derme através da arranhadura de animais domésticos ou manipulação de plantas contaminadas. O presente estudo investigou: a) a suscetibilidade in vitro de espécies do Complexo Sporothrix schenckii à inativação fotodinâmica; b) a combinação de anfotericina B e posaconazol em modelo murino de infecção sistêmica por S. brasiliensis e S. schenckii stricto sensu, buscando melhorar o tratamento da esporotricosse disseminada; c) a interferência da melanina fúngica na suscetibilidade in vitro de S. brasiliensis e S. schenckii stricto sensu à anfotericina B e itraconazol. No estudo da terapia fotodinâmica foi aplicado azul de metileno como fotossensibilizador e emissão de luz diiodo (InGaAlP) frente espécies do complexo Sporothrix schenckii em ensaio in vitro. A viabilidade dos conídios foi determinada através de contagem de unidades formadoras de colônia (UFC). A atividade da anfotericina B e posaconazol sozinhos ou combinados foi avaliada segundo a técnica de microdiluição em caldo e técnica de checkerboard. No estudo in vivo, camundongos OF-1 foram infectados com um inóculo de 2 x 107 UFC/animal. Foram utilizadas duas cepas de S. brasiliensis e duas cepas de S. schenckii. Os animais receberam doses de 0.3 mg/kg de anfotericina B em combinação com 2.5 mg/kg e 5 mg/kg de posaconazol. Os resultados foram comparados com as respectivas monoterapias e a eficácia foi avaliada através do prolongamento da sobrevivência e redução da carga fúngica no fígado e baço. No estudo da interferência da melanina na suscetibilidade de S. brasiliensis e S. schenckii stricto sensu à anfotericina B e itraconazol, células leveduriformes foram cultivadas em meio mínimo com e sem L-DOPA. Métodos de microdiluição e tempo de morte foram utilizados para determinar a atividade dos antifúngicos frente às células com diferentes quantidades de melanina. A luz laser (InGaAlP) em conjunto com o azul de metileno foram capazes de reduzir significativamente o crescimento de todas as cepas de Sporothrix testadas. Os dados in vitro demonstraram um efeito sinérgico da combinação de posaconazol e anfotericina B, e, apesar de o posaconazol 5 mg/kg sozinho apresentar eficácia contra a esporotricose, teve sua eficiência aumentada quando combinado com anfotericina B 0,3 mg/kg. A técnica de tempo de morte mostrou que a melanização protegeu os isolados do complexo Sporothrix schenckii da anfotericina B, particularmente na concentração mais baixa testada. Desta forma, as conclusões do presente estudo são: a) a inibição fotodinâmica in vitro de isolados de Sporothrix merece estudos experimentais e clínicos, uma vez que pode ser um tratamento promissor para a esporotricose cutânea e subcutânea; b) o posaconazol em combinação com dose baixa de anfotericina B pode ser uma opção para o tratamento de esporotricose disseminada, especialmente quando itraconazol e anfotericina B são contraindicados; c) estudos de combinação de anfotericina B com inibidores de melanina são necessários a fim de evitar o efeito protetor da melanina contra as defesas do organismo e contra antifúngicos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de Santa MariaPrograma de Pós-Graduação em Ciências FarmacêuticasUFSMBRAnálises Clínicas e ToxicológicasComplexo Sporothrix schenckiiTerapia fotodinâmicaCombinação de antifúngicosMelaninaSporothrix schenckii complexPhotodynamic therapyAntifungal combinationMelaninCNPQ::CIENCIAS DA SAUDE::FARMACIAComplexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacosSporothrix schenckii complex: photodynamic inactivation, influency of melanin in antifungal activity and drug combinationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisAlves, Sydney Hartzhttp://lattes.cnpq.br/0330782478769631Pereira, Daniela Isabel Brayerhttp://lattes.cnpq.br/3382450720179401Santurio, Janio Moraishttp://lattes.cnpq.br/6316012260769979Zanette, Régis Adrielhttp://lattes.cnpq.br/3166371422900794Schwarzbold, Alexandre Vargashttp://lattes.cnpq.br/5057171339596380http://lattes.cnpq.br/0731595522786356Mario, Débora Alves Nunes201000000000400300300300300300300bfe6016c-9c1e-42ca-ab1d-db5453ed090cbaf55de6-47bc-4c60-b985-412ab10daa0d8cf0f648-751e-463b-8607-0f38cce0874cfcf059a8-da92-4056-88e2-8facd44a897a9f4b7354-7051-4bdf-a038-0f46d7f9740cc0617b86-51f9-4f01-a452-308ec6c69eb5info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações do UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALMARIO, DEBORA ALVES NUNES.pdfapplication/pdf1082111http://repositorio.ufsm.br/bitstream/1/3422/1/MARIO%2c%20DEBORA%20ALVES%20NUNES.pdf92ed56c3a37ea9bd7d91624cad0a62d9MD51TEXTMARIO, DEBORA ALVES NUNES.pdf.txtMARIO, DEBORA ALVES NUNES.pdf.txtExtracted texttext/plain170522http://repositorio.ufsm.br/bitstream/1/3422/2/MARIO%2c%20DEBORA%20ALVES%20NUNES.pdf.txt2724b5efca2e9fab924cf8d2b55e3508MD52THUMBNAILMARIO, DEBORA ALVES NUNES.pdf.jpgMARIO, DEBORA ALVES NUNES.pdf.jpgIM Thumbnailimage/jpeg4910http://repositorio.ufsm.br/bitstream/1/3422/3/MARIO%2c%20DEBORA%20ALVES%20NUNES.pdf.jpgc04b96c9d3ee9bdbe0faa4512c7cee51MD531/34222022-05-11 12:03:05.511oai:repositorio.ufsm.br:1/3422Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2022-05-11T15:03:05Biblioteca Digital de Teses e Dissertações do UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.por.fl_str_mv |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
dc.title.alternative.eng.fl_str_mv |
Sporothrix schenckii complex: photodynamic inactivation, influency of melanin in antifungal activity and drug combination |
title |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
spellingShingle |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos Mario, Débora Alves Nunes Complexo Sporothrix schenckii Terapia fotodinâmica Combinação de antifúngicos Melanina Sporothrix schenckii complex Photodynamic therapy Antifungal combination Melanin CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
title_full |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
title_fullStr |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
title_full_unstemmed |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
title_sort |
Complexo Sporothrix schenckii: inativação fotodinâmica, influência da melanina na atividade dos antifúngicos e combinação de fármacos |
author |
Mario, Débora Alves Nunes |
author_facet |
Mario, Débora Alves Nunes |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Alves, Sydney Hartz |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/0330782478769631 |
dc.contributor.referee1.fl_str_mv |
Pereira, Daniela Isabel Brayer |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/3382450720179401 |
dc.contributor.referee2.fl_str_mv |
Santurio, Janio Morais |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/6316012260769979 |
dc.contributor.referee3.fl_str_mv |
Zanette, Régis Adriel |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/3166371422900794 |
dc.contributor.referee4.fl_str_mv |
Schwarzbold, Alexandre Vargas |
dc.contributor.referee4Lattes.fl_str_mv |
http://lattes.cnpq.br/5057171339596380 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/0731595522786356 |
dc.contributor.author.fl_str_mv |
Mario, Débora Alves Nunes |
contributor_str_mv |
Alves, Sydney Hartz Pereira, Daniela Isabel Brayer Santurio, Janio Morais Zanette, Régis Adriel Schwarzbold, Alexandre Vargas |
dc.subject.por.fl_str_mv |
Complexo Sporothrix schenckii Terapia fotodinâmica Combinação de antifúngicos Melanina |
topic |
Complexo Sporothrix schenckii Terapia fotodinâmica Combinação de antifúngicos Melanina Sporothrix schenckii complex Photodynamic therapy Antifungal combination Melanin CNPQ::CIENCIAS DA SAUDE::FARMACIA |
dc.subject.eng.fl_str_mv |
Sporothrix schenckii complex Photodynamic therapy Antifungal combination Melanin |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
Sporotrichosis is a subcutaneous mycosis that affects humans and animals, and it is caused by dimorphic melanized fungi belonging to the genus Sporothrix. Sporotrichosis is mainly acquired by traumatic implantation of the fungus into the dermis through scratches inflicted by domestic animals or by handling plants. This study approaches: a) the in vitro susceptibility of Sporothrix schenckii complex species to photodynamic inactivation using methylene blue as a photosensitizer and laser as the light source; b) the combination of amphotericin B plus posaconazole in a murine model of systemic infection by S. brasiliensis and S. schenckii sensu stricto, seeking to enhance the treatment of disseminated sporotrichosis; c) the interference of fungal melanin in the in vitro susceptibility of S. brasiliensis and S. schenckii sensu stricto to amphotericin B and itraconazole. In the photodynamic therapy study we have applied methylene blue as a photosensitizing agent and Light emitting diode (InGaAlP) against Sporothrix schenckii complex species in an in vitro assay. The viability of the conidia was determined through the counting of colony forming units. The activity of amphotericin B and posaconazole alone or in combination was evaluated by using a microdilution checkerboard method. For the in vivo study, OF-1 mice were intravenously (i.v.) infected with a lethal inoculum of 2 x 107 CFU/animal. Two strains of S. brasiliensis and two of S. schenckii were tested. Animals received amphotericin i.v. at suboptimal dose i.e., 0.3 mg/kg, in combination with oral posaconazole at 2.5 mg/kg, 5 mg/kg or 10 mg/kg BID. Results were compared with their respective monotherapies and efficacy was evaluated by prolongation of survival and reduction of tissue burden in spleen and liver. In the study of the interference of melanin in the susceptibility of S. brasiliensis and S. schenckii sensu stricto, yeast cells were cultivated in minimal medium with or without L-DOPA in order to induce the production of melanin. Microdilution and time-kill methods were used to determine the antifungal activity against yeast cells with different amounts of melanin. The laser light (InGaAlP) together with a methylene blue photosensitizer were able to significantly reduce the growth of all the tested Sporothrix strains. In vitro data demonstrated a synergistic effect of the combination and although posaconazole alone was effective against sporotrichosis, efficacy was increased when combined with amphotericin B. The maximum efficacy of the combinations was obtained with posaconazole 5 mg/kg. The killing assay showed that melanization protected isolates within the Sporothrix schenckii complex from amphotericin B, particularly in the lower concentrations tested. The conclusions of the present study are: a) the in vitro inhibition of Sporothrix spp. isolates by laser light deserves in vivo experimental and clinical studies since it may be a promising treatment for cutaneous and subcutaneous sporotrichosis; b) posaconazole in combination with suboptimal doses of amphotericin B could be an option in the treatment of disseminated sporotrichosis especially when itraconazole or amphotericin B at optimal doses are contraindicated; c) combination studies of amphotericin B with inhibitors of melanin are required in order to avoid this effect. |
publishDate |
2015 |
dc.date.issued.fl_str_mv |
2015-02-12 |
dc.date.accessioned.fl_str_mv |
2016-10-18 |
dc.date.available.fl_str_mv |
2016-10-18 |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
MARIO, Débora Alves Nunes. Sporothrix schenckii complex: Photodynamic Inactivation, Influency of Melanin in Antifungal Activity and Drug Combination. 2015. 91 f. Tese (Doutorado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/3422 |
identifier_str_mv |
MARIO, Débora Alves Nunes. Sporothrix schenckii complex: Photodynamic Inactivation, Influency of Melanin in Antifungal Activity and Drug Combination. 2015. 91 f. Tese (Doutorado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
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http://repositorio.ufsm.br/handle/1/3422 |
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Análises Clínicas e Toxicológicas |
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Universidade Federal de Santa Maria |
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