MEDRANO, Delia Jessica Astete. Perfil de sensibilidade de cepas de Coccidioides posadasii a associação de drogas antimicrobianas. Fortaleza,2010. 146 f. Tese (Doutorado) - Universidade Federal do Ceará. Faculdade de Medicina. Programa de Pós-Graduação em Ciências Médicas, Fortaleza, 2010. / Submitted by denise santos (denise.santos@ufc.br) on 2011-10-05T15:37:12Z No. of bitstreams: 1 2010_tese_djamedrano.pdf: 3128064 bytes, checksum: 0f3a21eec28eb85ca0c5d157d3bdc8aa (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2011-10-07T16:22:43Z (GMT) No. of bitstreams: 1 2010_tese_djamedrano.pdf: 3128064 bytes, checksum: 0f3a21eec28eb85ca0c5d157d3bdc8aa (MD5) / Made available in DSpace on 2011-10-07T16:22:43Z (GMT). No. of bitstreams: 1 2010_tese_djamedrano.pdf: 3128064 bytes, checksum: 0f3a21eec28eb85ca0c5d157d3bdc8aa (MD5) Previous issue date: 2010 / The coccidiodomicose is a systemic infection, predominantly lung caused by dimorphic fungi Coccidioides immitis and C. posadasii. Brazil is among the countries with endemic areas of coccidiodomicose, confined to the semiarid areas of Northeast Brazil. This scenario leads to a larger investigation into new strategies for an injunction against C.posadasii. Based on this context, the purpose of this study was to investigate new strategies, to evaluate the in vitro interaction of the major antifungal agents used in the treatment of coccidioidomycosis, analyze the inhibitory effect in vitro antimicrobial drugs, such as sulfamethoxazole and trimethoprim (SMX / TMP ) associated or not with amphotericin B (AMB) and evaluate the in vitro interaction of antituberculosis drugs with antifungal drugs against C. posadasii strains isolated in northeastern Brazil. The study was conducted in test macrodilution according to the protocol described in document M-38A, standardized by the Clinical Laboratory Standars Institute (CLSI, 2002). The synergistic interaction of the drugs was evaluated by the Checkerboard method. This study showed that the eight combinations of two antifungal drugs such as amphotericin B with azole derivatives and voriconazole with derivative azole and caspofungin, respectively showed inhibitory effect in vitro against the isolates of C. posadasii, but only a combination of voriconazole with amphotericin B showed synergistic interaction, whose minimum inhibitory concentration (MIC) was reduced by 15 times when compared with the MIC of the drug tested alone. SMX/TMP had an inhibitory effect in vitro against strains of C. posadasii, however, decreased the MIC of AMB at 5,85 times when associated with SMX / TMP. Regarding the 24 combinations of antifungal drugs with antituberculosis drugs tested showed any inhibitory effect in vitro against the isolates of C. posadasii. Of the 24 combinations, synergistic interactions were observed in 16 associations, of which, amphotericin B with rifampicin, itraconazole or rifampicin with ethambutol or pyrazinamide, ethambutol with fluconazole and voriconazole with ethambutol MICs were reduced ten-fold when compared with the MICs of drugs tested in isolation. Future studies in animal models are needed to confirm the usefulness of these combinations. This work is a source of support for new investigations of combinations that result in synergistic interactions, as well as to determine the correlation of results in vivo. / A coccidioidomicose é uma infecção sistêmica, predominantemente pulmonar, causada pelos fungos dimórficos Coccidioides immitis e C. posadasii. O Brasil está na relação de países com áreas endêmicas da coccidioidomicose (RESTREPO, 2006), circunscritas às zonas do semiárido da região do Nordeste (COX & MAGEE, 2004). Esse cenário leva a uma investigação maior para novas estratégias inibitórias contra Coccidioides posadasii. Baseando-se neste contexto, buscou-se estudar novas estratégias, como avaliar a interação in vitro dos principais antifúngicos utilizados no tratamento da coccidioidomicose, analisar o efeito inibitório in vitro de drogas antimicrobianas, como o sulfametoxazol com trimetoprim (SMX/TMP) associado ou não com anfotericina B (AMB), bem como avaliar a interação in vitro das drogas antituberculose com drogas antifúngicas frente a cepas de C. posadasii isoladas no Nordeste brasileiro. O estudo foi conduzido em ensaio de macrodiluição, de acordo com o protocolo descrito no documento M-38A, padronizado pelo Clinical Laboratory Standars Institute (CLSI, 2002). A interação sinérgica das drogas foi avaliada pelo método do Checkerboard. Este estudo mostrou que as oito combinações entre duas drogas antifúngicas testadas como anfotericina B com derivados azólicos e voriconazol com derivados azólicos e caspofungina respectivamente apresentaram efeito inibitório in vitro frente aos isolados de C. posadasii; porém apenas uma combinação voriconazol com anfotericina B exibiu interação sinérgica, cuja concentração inibitória mínima (CIM) foi reduzida em 15 vezes, quando se comparou com a CIM da droga testada isoladamente. O SMX/TMP mostrou efeito inibitório in vitro com frente a cepas de C. posadasii, entretanto, houve diminuição da CIM da AMB em cerca de 5,85 vezes quando associado à SMX/TMP. Com relação às 24 combinações entre drogas antifúngicas com drogas antituberculose testadas todas apresentaram efeito inibitório in vitro frente aos isolados de C. posadasii. Das 24 combinações, foram observadas interações sinérgicas em 16 associações, das quais anfotericina B com rifampicina; itraconazol com rifampicina ou etambutol ou pirazinamida; fluconazol com etambutol e voriconazol com etambutol foram reduzidas as CIMs em dez vezes, ao se comparar com a CIMs da droga testada isoladamente. Futuros estudos em modelos animais são necessários para confirmar a utilidade destas combinações. Este trabalho é fonte de apoio para novas investigações de combinações que resultem em interações sinérgicas, bem como para determinar a correlação de resultados in vivo.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A coccidioidomicose à uma infecÃÃo sistÃmica, predominantemente pulmonar, causada pelos fungos dimÃrficos Coccidioides immitis e C. posadasii. O Brasil està na relaÃÃo de paÃses com Ãreas endÃmicas da coccidioidomicose (RESTREPO, 2006), circunscritas Ãs zonas do semiÃrido da regiÃo do Nordeste (COX & MAGEE, 2004). Esse cenÃrio leva a uma investigaÃÃo maior para novas estratÃgias inibitÃrias contra Coccidioides posadasii. Baseando-se neste contexto, buscou-se estudar novas estratÃgias, como avaliar a interaÃÃo in vitro dos principais antifÃngicos utilizados no tratamento da coccidioidomicose, analisar o efeito inibitÃrio in vitro de drogas antimicrobianas, como o sulfametoxazol com trimetoprim (SMX/TMP) associado ou nÃo com anfotericina B (AMB), bem como avaliar a interaÃÃo in vitro das drogas antituberculose com drogas antifÃngicas frente a cepas de C. posadasii isoladas no Nordeste brasileiro. O estudo foi conduzido em ensaio de macrodiluiÃÃo, de acordo com o protocolo descrito no documento M-38A, padronizado pelo Clinical Laboratory Standars Institute (CLSI, 2002). A interaÃÃo sinÃrgica das drogas foi avaliada pelo mÃtodo do Checkerboard. Este estudo mostrou que as oito combinaÃÃes entre duas drogas antifÃngicas testadas como anfotericina B com derivados azÃlicos e voriconazol com derivados azÃlicos e caspofungina respectivamente apresentaram efeito inibitÃrio in vitro frente aos isolados de C. posadasii; porÃm apenas uma combinaÃÃo voriconazol com anfotericina B exibiu interaÃÃo sinÃrgica, cuja concentraÃÃo inibitÃria mÃnima (CIM) foi reduzida em 15 vezes, quando se comparou com a CIM da droga testada isoladamente. O SMX/TMP mostrou efeito inibitÃrio in vitro com frente a cepas de C. posadasii, entretanto, houve diminuiÃÃo da CIM da AMB em cerca de 5,85 vezes quando associado à SMX/TMP. Com relaÃÃo Ãs 24 combinaÃÃes entre drogas antifÃngicas com drogas antituberculose testadas todas apresentaram efeito inibitÃrio in vitro frente aos isolados de C. posadasii. Das 24 combinaÃÃes, foram observadas interaÃÃes sinÃrgicas em 16 associaÃÃes, das quais anfotericina B com rifampicina; itraconazol com rifampicina ou etambutol ou pirazinamida; fluconazol com etambutol e voriconazol com etambutol foram reduzidas as CIMs em dez vezes, ao se comparar com a CIMs da droga testada isoladamente. Futuros estudos em modelos animais sÃo necessÃrios para confirmar a utilidade destas combinaÃÃes. Este trabalho à fonte de apoio para novas investigaÃÃes de combinaÃÃes que resultem em interaÃÃes sinÃrgicas, bem como para determinar a correlaÃÃo de resultados in vivo. / The coccidiodomicose is a systemic infection, predominantly lung caused by dimorphic fungi Coccidioides immitis and C. posadasii. Brazil is among the countries with endemic areas of coccidiodomicose, confined to the semiarid areas of Northeast Brazil. This scenario leads to a larger investigation into new strategies for an injunction against C.posadasii. Based on this context, the purpose of this study was to investigate new strategies, to evaluate the in vitro interaction of the major antifungal agents used in the treatment of coccidioidomycosis, analyze the inhibitory effect in vitro antimicrobial drugs, such as sulfamethoxazole and trimethoprim (SMX / TMP ) associated or not with amphotericin B (AMB) and evaluate the in vitro interaction of antituberculosis drugs with antifungal drugs against C. posadasii strains isolated in northeastern Brazil. The study was conducted in test macrodilution according to the protocol described in document M-38A, standardized by the Clinical Laboratory Standars Institute (CLSI, 2002). The synergistic interaction of the drugs was evaluated by the Checkerboard method. This study showed that the eight combinations of two antifungal drugs such as amphotericin B with azole derivatives and voriconazole with derivative azole and caspofungin, respectively showed inhibitory effect in vitro against the isolates of C. posadasii, but only a combination of voriconazole with amphotericin B showed synergistic interaction, whose minimum inhibitory concentration (MIC) was reduced by 15 times when compared with the MIC of the drug tested alone. SMX/TMP had an inhibitory effect in vitro against strains of C. posadasii, however, decreased the MIC of AMB at 5,85 times when associated with SMX / TMP. Regarding the 24 combinations of antifungal drugs with antituberculosis drugs tested showed any inhibitory effect in vitro against the isolates of C. posadasii. Of the 24 combinations, synergistic interactions were observed in 16 associations, of which, amphotericin B with rifampicin, itraconazole or rifampicin with ethambutol or pyrazinamide, ethambutol with fluconazole and voriconazole with ethambutol MICs were reduced ten-fold when compared with the MICs of drugs tested in isolation. Future studies in animal models are needed to confirm the usefulness of these combinations. This work is a source of support for new investigations of combinations that result in synergistic interactions, as well as to determine the correlation of results in vivo.

Nikkomycin Z (NikZ) is a chitin synthase inhibitor with antifungal efficacy against Coccidioides spp. and other endemic fungi. Dogs suffer a rate and range of natural coccidioidomycosis similar to humans and were considered an excellent model for initially testing NikZ against naturally acquired disease. Twelve dogs with coccidioidal pneumonia that had been present for an average of three months were treated with 250 mg (5 - 15 kg) or 500 mg (> 15 - 30 kg) twice daily for 60 days. Nine dogs completed the course of treatment and seven dogs had improvement in disease based on radiographs, clinicopathological parameters, physical examination findings, and subjective assessment by the owners; three dogs had resolution or near resolution of disease. Based on this small study, NikZ shows efficacy to treat naturally acquired coccidioidomycosis and merits further development for trials in humans.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Coccidiodomycosis is a systemic infection, predominantly pulmonary, caused by the geophilic and dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. In Brazil, coccidioidomycosis is associated with semi-arid areas in the Northeastern region of this country, which is considered one of the endemic areas of this disease in South America. These pathogens are morphologically indistinguishable species, but they exhibit molecular differences. Different molecular techniques have been described for the characterization of these species. The nuclear ribosomal DNA (rDNA) from Coccidioides spp. has been described as an important molecular marker for the identification, taxonomy and phylogeny. Currently, there are still shortages of maps for epidemiological approaches in order to direct the correlation between populations of Coccidioides spp. and the outbreaks of coccidiomycosis. Given the above, this study aimed at performing the molecular identification of 18 clinical and environmental isolates of C. posadasii, from Northeastern Brazil, maintained in the fungal collection of the Specialized Medical Mycology Center (CEMM), through PCR, as well as, to analyze the genetic diversity of these isolates by sequencing of 18S-28S regions of nuclear rDNA. The identification of the isolates was performed through PCR, using specific primers Coi9-1F and Coi9-R. The sequencing of the 18S-28S rDNA regions was performed through the method of chain termination by dideoxynucleotides, using the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The results confirmed the identification of all strains included in this study as belonging to the species C. posadasii. The phylogenetic tree based on 18S-28S rDNA region of C. posadasii from CEMM and Coccidioides spp. from Genbank. reveals the formation of a unique cluster encompassing the following strains CEMM 05-2-063, CEMM 05-2-064, CEMM 05-2-066 and CEMM 05-2-065, in a properly sustained branch, which apparently seems to group these isolates according to their geographical origin. The strains of C. posadasii showed lower genetic divergence in the ITS1 and ITS2 regions, when compared to strains of C. immitis. Analyses did not detect differences between strains of clinical origin and those of environmental origin. Further studies involving the analysis of fast evolving markers, such as microsatellites, can provide evidences to determine whether the groups found in this study are derived from a lineage of clonal reproduction. / A coccidioidomicose à uma infecÃÃo sistÃmica, predominantemente pulmonar, causada pelos fungos dimÃrficos e geofÃlicos, Coccidioides immitis e Coccidioides posadasii. No Brasil, a coccidioidomicose està associada a locais situados na zona semi-Ãrida da regiÃo Nordeste, considerada uma das Ãreas endÃmicas da doenÃa na AmÃrica do Sul. Estes patÃgenos consistem em espÃcies morfologicamente indistinguÃveis, mas que exibem diferenÃas moleculares peculiares. O DNA ribossÃmico nuclear (rDNA) de Coccidioides spp. tem sido apontado como importante marcador molecular utilizado na identificaÃÃo, taxonomia e filogenia. Atualmente, ainda hà escassez de mapas de abordagens epidemiolÃgicas para direcionar a correlaÃÃo entre as populaÃÃes de Coccidioides spp. com os surtos de coccidioidomicose. Diante do exposto, este estudo teve por objetivo, realizar a identificaÃÃo molecular de 18 isolados clÃnicos e ambientais de C. posadasii, oriundos do Nordeste brasileiro, mantidos na Micoteca do Centro Especializado em Micologia MÃdica (CEMM), atravÃs da tÃcnica de PCR, bem como, analisar a diversidade genÃtica destes isolados por meio do seqÃenciamento das regiÃes 18S-28S do rDNA nuclear. A identificaÃÃo dos isolados foi realizada por PCR utilizando os primers especÃficos Coi9-1F e Coi9-R. O sequenciamento das regiÃes 18S-28S rDNA foi realizado pelo mÃtodo da terminaÃÃo da cadeia pelo didesoxinucleotÃdeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). Os resultados confirmaram a identificaÃÃo de todas as cepas incluÃdas neste estudo, como pertencentes à espÃcie C. posadasii. A Ãrvore filogenÃtica, baseada na regiÃo 18S-28S rDNA de C. posadasii do CEMM, juntamente com sequÃncias de Coccidioides spp. depositadas no Genbank. revela a formaÃÃo de um cluster exclusivo englobando as cepas CEMM 05-2-063, CEMM 05-2-064, CEMM 05-2-065 e CEMM 05-2-066, em um ramo adequadamente sustentado, que aparentemente parece agrupar estes isolados segundo sua origem geogrÃfica. As cepas de C. posadasii apresentaram menor Ãndice de divergÃncia genÃtica nas regiÃes ITS1 e ITS2, quando comparadas Ãs cepas de C. immitis A anÃlise nÃo detectou diferenÃas entre as cepas de origem clÃnica e as de origem ambiental. Estudos posteriores envolvendo a anÃlise de marcadores de evoluÃÃo mais rÃpida, os microssatÃlites, podem fornecer evidÃncias para determinar se os agrupamentos encontrados neste estudo sÃo resultantes de uma linhagem de reproduÃÃo clonal.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A ordem Chiroptera mostra-se como reservatÃrio para vÃrios agentes infecciosos, incluindo protozoÃrios, bactÃrias, vÃrus e fungos. A fim de investigar a a eco-epidemiologia de Histoplasma capsulatum no Nordeste do Brasil, foram capturados 83 morcegos em Ãreas urbanas e rurais no estado do CearÃ, em coletas diurnas e noturnas, durante o perÃodo de agosto de 2010 a marÃo de 2011. ApÃs eutanÃsia, fragmentos de baÃo, fÃgado e pulmÃo foram removidos, macerados e alÃquotas de 100μL foram semeadas em placas contendo Ãgar BHI modificado, as quais foram incubadas por atà seis semanas nas temperaturas de 28ÂC e 35ÂC. AlÃquotas remanescentes de cada material macerado foram estocadas a -20ÂC. Embora o fungo nÃo tenha sido isolado em nenhuma amostra, apÃs 21 dias de incubaÃÃo à 35ÂC foi observado o crescimento de uma colÃnia fÃngica sugestiva de Coccidioides spp. em amostra de pulmÃo da espÃcie Carollia perspicillata. Fragmentos de pulmÃo do morcego retirados do estoque a -20ÂC e analisados ao microscÃpio Ãptico revelaram estruturas esfÃricas semelhantes a forma parasitÃria do fungo. A colÃnia foi investigada por meio do teste de reversÃo in vivo em modelo murino, com observaÃÃo dos animais inoculados por atà quatro semanas. A identificaÃÃo molecular das culturas foi realizada por reaÃÃo de PCR especÃfica para C. posadasii, empregando os oligonucleotÃdeos Coi9-R e Coi9-F. Por fim, amostras de macerados dos pulmÃes, baÃo e fÃgado foram investigadas quanto à presenÃa de anticorpos ou antÃgenos especÃficos para C. posadasii, bem como quanto à presenÃa de anticorpos anti-Histoplasma, por meio da tÃcnica de imunodifusÃo. EsfÃrulas caracterÃsticas de Coccidioides spp. foram visualizadas em amostras de pulmÃo dos camundongos infectados experimentalmente. AnÃlise molecular das culturas isoladas de pulmÃo de C. perspicillata, bem como dos ÃrgÃos dos camundongos, mostraram banda de aproximadamente 630 pb, caracterÃstica de C. posadasii. Por meio de reaÃÃo de imunodifusÃo, foram detectados dois animais reagentes para antÃgenos de Coccidioides. Os animais pertenciam Ãs espÃcies Glossophaga soricina e Desmodus rotundus e um animal da espÃcie G. soricina anticorpo-positivo para Coccidioides. NÃo foi observada presenÃa de anticorpos anti-Histoplasma em nenhuma das amostras. O presente estudo mostra o primeiro isolamento de C. posadasii em quirÃpteros. Adicionalmente, de forma inÃdita, detectou-se de antÃgenos e anticorpos especÃficos para C. posadasii nestes animais. Faz-se necessÃrio o estudo dos quirÃpteros na ecoepidemiologia do patÃgeno. / The order Chiroptera is shown as a reservoir for various infectious agents, including protozoan, bacteria, virus and fungi. To investigate the eco-epidemiological aspects of Histoplasma capsulatum in Northeast Brazil, 83 bats were captured in urban and rural areas in CearÃ, collected in day and night, during the period August 2010 to March 2011. After euthanasia, fragments of sleen, liver and lung were removed, macerated and 100 μL aliquots were plated on agar containing modified BHI, which were incubated for up to six weeks at temperatures of 28 ÂC and 35 ÂC. Aliquots of each remaing macerated material stored at -20 ÂC. Although the fungus was not isolated in any samples, after 21 days at 35 ÂC was observed the growth of a fungal colony suggestive of Coccidioides spp. in lung samples of the species Carollia perspicillata. Fragments of the bet lung removed from storage at -20 ÂC and analyzed by optical microscopy revealed spherical structures similar to the shape of the parasitic fungus. The colonies were investigated by means of the reversion in vivo in a murine model, with observation of the animals incoculated up to four weeks. The molecular identification of cultures was performed by PCR specific for C. posadasii, using the oligonucleotides Coi9-R and Coi9-F. Finally, samples of macerated lung, spleen and liver were investigated for the presence of antibodies or antigens specific for C. posadasii, as well as the presence of anti-Histoplasma, by immunodiffusion. Spherules of Coccidioides spp. features were visualized in lung samples of mice experimentally infected. Molecular analysis of cultures isolated from lungs of C. perpicillata, as well as organs of mice, showed approximately 630 pb band, characteristic of C. posadasii. By immunodiffusion reaction, two animals were detected reagents for Coccidioides antigens. The animals belonged to the species Glossophaga soricina and Desmodus rotundus and an animal of the species G. soricina antibody-positive for Coccidioides. There was no presence of anti-Histoplasma is any sample. The present study shows the first isolation of C. posadasii in bats. Additionally, in an unprecedented manner, we detected antigens and antibodies specific to C. posadasii these animals. Its is necessary to the study of bats in ecoepidemiology the pathogen.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.