Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A coccidioidomicose e a histoplasmose sÃo micoses sistÃmicas que acometem o homem e animais, causadas por espÃcies de fungos dimÃrficos, com Ãnfase para Coccidioides posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. SÃo consideradas doenÃas profundas importantes, podendo culminar em diversas complicaÃÃes secundÃrias. Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e o aumento da ocorrÃncia de doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas no mundo. Apesar da existÃncia de terapias eficazes com antifÃngicos contra a coccidioidomicose e a histoplasmose, a busca por novas drogas para o tratamento destas doenÃas se faz necessÃria. Ciprofloxacina à uma droga antibacteriana clÃssica do grupo das fluoroquinolonas, que inibe a atividade catalÃtica da DNA girase e topoisomerase IV, essenciais na replicaÃÃo e transcriÃÃo do DNA bacteriano. Estudos verificaram que ciprofloxacina pode atuar na DNA girase dos fungos. Assim, o presente estudo visou avaliar o efeito inibitÃrio in vitro de ciprofloxacina (CIP) isolada e em combinaÃÃo com os antifÃngicos anfotericina B (AMB), itraconazol (ITC), voriconazol (VRC) e caspofungina (CAS) frente à C. posadasii e Histoplasma capsulatum var. capsulatum. Foram utilizados 16 cepas de C. posadasii na fase filamentosa, 16 cepas de H. capsulatum var. capsulatum na fase filamentosa e 9 cepas de H. capsulatum var. capsulatum na fase leveduriforme. O estudo foi conduzido em ensaio de macrodiluiÃÃo e microdiluiÃÃo em caldo, descritos nos documentos M-38A e M-27A2, padronizados pelo Clinical Laboratory Standards Institute (CLSI), sendo utilizados para C. posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. A interaÃÃo das drogas foi analisada atravÃs do cÃlculo do Ãndice da ConcentraÃÃo InibitÃria FracionÃria (FICI), definido como a soma das relaÃÃes entre a concentraÃÃo inibitÃria mÃnima (CIM) de cada droga em combinaÃÃo e a CIM da mesma droga isolada, considerando os valores menores ou iguais a 0,5 indicativos de sinergismo. Com relaÃÃo Ãs cepas de C. posadasii, foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,125≤CIM≤12,5 ug mL-1) com ITC (0,0078≤CIM≤0,125 ug mL-1) (n=13/16), CIP (3,125≤CIM≤12,5 ug mL-1) com VRC (0,0078≤CIM≤0,0312 ug mL-1) (n=13/16) e CIP (3,125≤CIM≤12,5 ug mL-1) com CAS (2≤CIM≤8 ug mL-1) (n=14/16). Para as cepas de H. capsulatum na fase filamentosa, tambÃm foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,906≤CIM≤62,5 ug mL-1) com ITC (0,00006≤CIM≤0,0078 ug mL-1) (n=14/16) e CIP (31,25≤CIM≤125 ug mL-1) com VRC (0,0156≤CIM≤0,125 ug mL-1) (n=16/16). No tocante Ãs cepas de H. capsulatum na fase leveduriforme, foram observadas poucas interaÃÃes sinÃrgicas nas combinaÃÃes de drogas testadas. Nenhuma das associaÃÃes de drogas testadas apresentou antagonismo. Os dados obtidos apontam uma nova alternativa para o tratamento da coccidioidomicose e da histoplasmose, sendo necessÃrios novos estudos que visem investigar os mecanismos de aÃÃo dessas combinaÃÃes de drogas no metabolismo celular fÃngico, bem como o delineamento de experimentos in vivo para confirmar a significÃncia desses achados. / Coccidioidomycosis and histoplasmosis are systemic mycoses that occur in humans and other animals and are caused by the dimorphic fungi Coccidioides posadasii and Histoplasma capsulatum var. capsulatum, respectively. They are considered important deep mycoses that can lead to several secondary complications. In the past years, the improvement of the techniques applied in mycological diagnosis and the increase in the occurrence of immunocompromising diseases have caused a great impact in the incidence of deep and opportunistic mycoses in the world. In spite of the existence of effective antifungal therapy against coccidioidomycosis and histoplasmosis, the pursue of new drugs to treat theses diseases is necessary. Ciprofloxacin is a classic antibacterial drug that belongs to the group of fluoroquinolones, which inhibit the catalytic activity of DNA gyrase and topoisomerase IV, which are essential in bacterial DNA replication and transcription. Some studies have shown that ciprofloxacin can act on fungal DNA gyrase. Thus, the present study aimed at evaluating the in vitro inhibitory effect of ciprofloxacin (CIP), when associated with amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC) or caspofungina (CAS), on C. posadasii and H. capsulatum var. capsulatum. Sixteen strains of C. posadasii in the filamentous phase and 16 and 9 strains of H. capsulatum in the filamentous and yeast-like phase, respectively, were used. Broth macrodilution and microdilution assays were performed, as described in the documents M38-A and M27-A2, respectively, of the Clinical Laboratory Standards Institute (CLSI). Drug interaction was analyzed by calculating the fractional inhibitory concentration index (FICI), which is defined as the sum of the ratios between the minimal inhibitory concentration (MIC) of each combined drug and the MIC of the same drug isolatedly. Values of FICI smaller or equal to 0.5 indicate the occurrence of synergy. Concerning the isolates of C. posadasii, synergistic interactions were observed for all combinations, especially for the associations of CIP (3.125≤CIM≤12.5 ug mL-1) with ITC (0.0078≤CIM≤0.125 ug mL-1) (n=13/16), CIP (3.125≤CIM≤12.5 ug mL-1) with VRC (0.0078≤CIM≤0.0312 ug mL-1) (n=13/16) and CIP (3.125≤CIM≤12.5 ug mL-1) with CAS (2≤CIM≤8 ug mL-1) (n=14/16). For the isolates of H. capsulatum in the filamentous phase synergistic interactions were also observed for all combinations, with emphasis to the associations of CIP (3.906≤CIM≤62.5 ug mL-1) with ITC (0.00006≤CIM≤0.0078 ug mL-1) (n=14/16) and CIP (31.25≤CIM≤125 ug mL-1) with VRC (0.0156≤CIM≤0.125 ug mL-1) (n=16/16). For H. capsulatum in yeast-like phase, few synergistic interactions were observed for the tested drug combinations. None of the tested combinations presented antagonism. The obtained data may point at a new alternative for the treatment of coccidioidomycosis and histoplasmosis. Thus, it is necessary to investigate the mechanisms of action of these drug combinations on the fungal cellular metabolism and to perform in vivo experiments to confirm the relevance of these findings.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / As infecÃÃes fÃngicas sistÃmicas causadas por leveduras do gÃnero Candida sÃo consideradas micoses oportunistas de alto risco em ambientes hospitalares. Essas infecÃÃes representam importante desafio terapÃutico, em razÃo do surgimento de espÃcies resistentes a antifÃngicos, associados a altos Ãndices de mortalidade. Por esta razÃo, este trabalho objetivou: Verificar a freqÃÃncia de fungemias no Hospital Geral Dr. CÃsar Cals (HGCC) e Hospital Infantil Albert Sabin (HIAS); identificar os agentes etiolÃgicos implicados; determinar o perfil de sensibilidade aos antifÃngicos e avaliar o perfil genotÃpico das espÃcies isoladas de pacientes com quadros de recorrÃncia. Para isso foram recuperadas, de 4 627 hemoculturas, 55 hemoculturas positivas para leveduras do HGCC, e, de 5 316 hemoculturas, 87 positivas para leveduras do HIAS. Destas, 23 do HGCC e 43 pacientes do HIAS que possuÃam histÃrias clÃnicas com dados completos entraram neste estudo. O diagnÃstico micolÃgico foi realizado por meio das caracterÃsticas bioquÃmicas e morfolÃgicas dos agentes etiolÃgicos isolados. O teste de sensibilidade aos antifÃngicos - anfotericina B, fluconazol, itraconazol e cetoconazol - utilizou o mÃtodo de microdiluiÃÃo em caldo, descrito no documento M27-A2 do NCCLS. As tÃcnicas de genotipagem foram realizadas por eletroforese em campo pulsÃtil (PFGE) e amplificaÃÃo aleatÃria do DNA (RAPD). Entre o perÃodo do junho do 2000 a junho do 2002, no HGCC, foram analisadas 4 627 hemoculturas, sendo positivas 1051, das quais 55 (5,2%) foram positivas para leveduras do gÃnero Candida. Em contrapartida, no perÃodo de junho do 2001 a junho do 2002, no HIAS, foram analisadas 5316 hemoculturas, sendo positivas 1520 amostras, das quais 87 (5,72%) foram positivas para leveduras dos gÃneros Candida e Rhodotorula. A principal espÃcie envolvida, dentro do gÃnero Candida, foi a C. parapsilosis com 36% e 42% no HGCC e HIAS, respectivamente. Os principais fatores de risco foram: a antibioticoterapia prÃvia (n=19; 90%), cateter venoso central, nutriÃÃo parenteral, sondagem gÃstrica , ventilaÃÃo mecÃnica, cirurgia e prematuridade. Quanto à resistÃncia a drogas antifÃngicas, foi observado que anfotericina B apresentou um Ãndice de resistÃncia de 4%, fluconazol- 52%, itraconazol- 50% e cetoconazol- 86%. A tÃcnica do PFGE caracterizou um perfil cromossÃmico de 6 bandas para C. parapsilosis e 4 para C. tropicalis. Esta tÃcnica permitiu a diferenciaÃÃo de uma cepa com padrÃo de bandas alterado para o Ãltimo episÃdio do paciente 4. A tÃcnica de RAPD caracterizou um padrÃo de distribuiÃÃo de bandas predominante para C. parapsilosis e foi capaz de caracterizar 4 perfis genÃmicos, indicando re-infecÃÃo em 3 pacientes e 2 perfis genÃmicos foram encontrados para C. tropicalis . Este ensaio demonstra a emergÃncia das Candida nÃo-albicans, especialmente a C. parapsilosis, como principal responsÃvel pelos casos de candidemias em dois hospitais de indicaÃÃo terciÃria de Fortaleza, associado a uma alta resistÃncia in vitro aos antifÃngicos. A variabilidade genÃtica encontrada nas C. parapsilosis e C. tropicalis, mediante as tÃcnicas do PFGE e RAPD, revelaram quadros de re-infecÃÃo, os quais erguem a possibilidade de que estas sejam decorrentes de uma contaminaÃÃo entre pacientes ou indiretamente, por intermÃdio de trabalhadores da saÃde. / The systemic infections caused by yeasts of the genus Candida are considered opportunist mycoses of high risk in hospital environments. These infections represent important therapeutical challenge, by reason of arising of species resistent to some antifungals, associated with high rate of mortality. Therefore, this work sought: To verify the frequency of fungemias at Dr. CÃsar Cals Hospital and Albert Sabin Children Hospital; identify the etiological agents implicated; determine the sensitivety profile to antifulgal and evaluate the genotypical profile of the species isolated from patients with recurrency. For this, were recuperated from 4627 hemcultures, 55 yeast-positive hemocultures at HGCC and from 5316 hemocultures 87 yeast-positive hemocultures at HIAS. From them, 23 patients at HGCC and from 43 at HIAS that had clinical histories with complet data entered this study. The mycological diagnosis was performed through biochemical and morphological characteristics of the etiological isolated. The test of sensitivety to antifungal- amphotericin B, fluconazole, itraconazole and cetoconazole- utilized the microdilution method in broth, reported in the document M27-A2 of the NCCLS. The genotyping techniques were performed through PFGE (Pulsed Field Gel Electrophresis), RAPD (Randomly amplified polymorphic DNA analysis). Within the period from june of 2000, to june of 2002 at HGCC, were analysed 4627 hemocultures, being positives 1051, of which 55 (5,2%) were positive for yeast of the genus Candida. On the other hand, in the period from june of 2001 to july of 2002 at HIAS, were analysed 5316 hemocultures, of which 87 (5,72%) were yeast-positive of the genus Candida and Rhodotorula.The main specie involved in the genus Candida, was C. parapsilosis with 36% and 42% of the cases at HGCC and HIAS respectively. The main risk factors associated with candidemia, were: the previous antibiotic therapy, central venous catheter, parenteral nutrition, gastric probe, mechanical ventilation, surgery and prematurity. About the resistance to antifungals drugs was observed that amphotericin B showed a resistance level of 4%, fluconazole-56%, itraconazole-52% and cetoconazole- 86%. The PFGE technique caracterized a cromossomic profile of 6 bands for C. parapsilosis and 4 for C. tropicalis. This technique permited the diferentiation of one cepa with changed pattern of bands for the last episode of the patient 4. The technique of RAPD chacacterized a distribution pattern of predominant bands for C. parapsilosis and was capable of characterize 4 genetical profiles, denoting reinfection in 3 patients and 2 genomic profiles were found for C. tropicalis. This assay shows the emergency of the non- albicans Candida, specially the C. parapsilosis, as main responsible for the cases of candidemia in the two hospitals of tertiary indication of Fortaleza, associated with a high resistance in vitro to antifungals. The genetical variability found in C. parapsilosis and C. tropicalis through the PFGE and RAPD techniques, revealed reinfection pictures, which rises the possibility that these be due to a contamination among patients or indirectly through the healthy workers.