Cryptococcus neoformans species complex são fungos oportunistas que desencadeiam meningite, tanto em pacientes imunocompetentes como em indivíduos hígidos. Este trabalho teve como objetivo estudar as espécies circulantes de Cryptococcus neoformans species complex isolados no Estado de Sergipe tanto a partir do LCR (líquido céfalo raqueanao) de pacientes com meningite infecciosa como de diversos substratos ambientais. Foram utilizadas amostras do LCR que deram entrada no IPH/HEMOLACEN/SE no período de 2001 a 2007. Foram realizadas 104 coletas ambientais dentro do Estado de Sergipe, das quais 55 foram obtidas a partir de excretas de aves e quatro de amostras de fezes de morcegos coletadas em cavernas. As demais foram obtidas de materiais de origem vegetal, perfazendo 45 amostras: 21 de Eucalyptus spp., oito de material de troncos vegetais em decomposição, 10 de restos vegetais coletados em duas praças e seis de Anacardium occidentale L. (cajueiro). O líquor foi examinado por microscopia direta e cultivado a 25ºC e a 37ºC. No período de 2005 a 2007 foram obtidos 141 isolados ambientais e 35 isolados clínicos de Cryptococcus neoformans species complex. Foram determinadas inferências ecológicas e epidemiológicas dos isolados obtidos além de realização de provas para identificação presuntiva fenotípica em meioNíger e, das espécies em meio CGB, teste de sensibilidade frente aosantifúngicos, caracterização enzimática de interesse biotipar e/ou marcador de linhagens, determinação de mating type sexual utilizando PCR e PCR Fingerprintig com iniciador M13. Nas linhagens de Cryptococcus spp. isoladas de líquor de pacientes com meningite infecciosa no Estado de Sergipe prevaleceu a espécie C. neoformans. Em relação às linhagens ambientais, somente uma amostra, dentre 140, foi identifcada como C. gattii. Todas as amostras oriundas de excretas de aves foram positivas no isolamento da levedura. Os isolados clínicos de C. neoformans e C. gattii oriundos de pacientes com meningite criptocócica no Estado de Sergipe apresentaram valores altos de concentração inibitória mínima (CIM) para os agentes antifúngicos testados, o que simboliza alta taxa de resistência, principalmente à Anfotericina B e ao Fluconazol. Em relação ao mating type sexual foi evidenciando que a maioria das linhagens clínicas foi identificada como MAT, confirmando o caráter de virulência que esta levedura desenvolve. Entretanto, no isolados ambientais o MATa foi mais prevalente, evidenciando o caráter menos virulento destas linhagens frente às de origem clínica. Cryptococcus neoformans foi a única espécie amplificada por PCR fingerprinting. Houve presença, dentre as amostras locais, de três perfis genéticos distintos epertencentes a uma mesma espécie. _________________________________________________________________________________________ ABSTRACT: Cryptococcus neoformans species complex are opportunistic fungi that cause meningitis in both immunocompetent patients and in healthy individuals. This work aimed to study the species of circulating Cryptococcus neoformans species complex isolated from northeastern Brazil from both the CSF (cerebrospinal fluid) of patients with infectious meningitis as various environmental substrates. Samples of CSF were received at IPH/HEMOLACEN/SE in the period 2001 to 2007. Were carried out 104 environmental samples within the state of Sergipe, of which 55 were obtained from the feces of birds and four stool samples collected from bat caves. The other samples were obtained from plant materials, totaling 45 samples: 21 from Eucalyptus spp., eight from material of decomposing trunks of plants, 10 from plant collected in two squares, six from Anacardium occidentale L. - (Cashew). CSF was examined by direct microscopy by staining with Indian ink and cultured in Sabouraud agar with chloramphenicol at 25 °C and 37 ºC. In the period from 2005 to 2007 were obtained from 141 environmental isolates and35 clinical isolates of Cryptococcus neoformans species complex. Ecological and epidemiological inferences were determined. In addition characteristics of isolates were carried out by tests for presumptive phenotypic identification, through Niger medium and species into the CGB test, sensitivity to the antifungal, enzymatic characterization of biotypes of interest and/or marker lines, determining sexual mating type using PCR and PCR Fingerprintig with primer M13. Among the strains of Cryptococcus spp. isolated from CSF of patients with infectious meningitis in the state of Sergipe, in the period from 2001 to 2009, prevaled the species C. neoformans. Among the environmental strains, only one sample of 140, was identified as C. gattii. All samples from thefeces of birds were positive in the isolation of yeast. Clinical isolates ofCryptococcus neoformans and C. gattii originating from patients withcryptococcal meningitis in the state of Sergipe exhibited higher values ofminimum inhibitory concentration (MIC) to the antifungal agents tested, which symbolizes high rate of resistance, especially to amphotericin B and fluconazole. In relation to sexual mating type was evident that most of the clinics were MAT strains, confirming the character of virulence of this yeast. However, among the environmental isolates the MATa was more prevalent, showing the character of these less virulent strains. Cryptococcus neoformans was the only species amplified by PCR fingerprinting. There was a presence among the local samples of three different genetic profiles belonging to the same species.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-12-26T16:15:27Z No. of bitstreams: 2 Dissertação - Lucas Daniel Quinteiro de Oliveira 2017.pdf: 3361217 bytes, checksum: f1ea8dda888ba50d64581258f8ca97cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Olha a unidade acadêmica é IPTSP e não FM on 2017-12-27T10:44:54Z (GMT) / Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-12-27T12:11:57Z No. of bitstreams: 2 Dissertação - Lucas Daniel Quinteiro de Oliveira 2017.pdf: 3361217 bytes, checksum: f1ea8dda888ba50d64581258f8ca97cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-28T09:29:42Z (GMT) No. of bitstreams: 2 Dissertação - Lucas Daniel Quinteiro de Oliveira 2017.pdf: 3361217 bytes, checksum: f1ea8dda888ba50d64581258f8ca97cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-28T09:29:42Z (GMT). No. of bitstreams: 2 Dissertação - Lucas Daniel Quinteiro de Oliveira 2017.pdf: 3361217 bytes, checksum: f1ea8dda888ba50d64581258f8ca97cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-26 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The Cryptococcus neoformans complex comprises two species responsible for causing cryptococcosis, the main manifestation being meningoencephalitis in immunocompromised hosts. Its pathogenicity is related to virulence factors. The treatment of this disease is difficult because the drugs present high toxicity, resistance of the fungus and little therapeutic efficiency. Research with biologically active natural products has shown that essential oils (OEs) have important antifungal activity. Thus, the present study had as objective to evaluate the antifungal efficacy and action on the virulence factors in C. neoformans complexes of OEs of Cinamomum cassia (Cinnamon of China) and Cybopogom flexuosus (Lemon Grass). By means of the microdilution technique in broth, OEs had MIC ranging from 8 to 256 μg / ml and CFM from 16 to 512 μg / ml. C. cassia promoted a 47.8% reduction in capsule size and inhibited melanin synthesis in 30% of the isolates. The evaluation of this OE on the activity of exoenzymes phospholipase, urease and DNAse demonstrated inhibition in 15%, 30% and 25% of the evaluated isolates respectively. C. flexuosus reduced the size of the capsule by 48.7%, inhibited the production of melanin, urease, phospholipase and DNAse by 30%, 30%, 25% and 15% respectively. The data demonstrated that the isolates did not produce proteinase and the hemolytic activity was reduced by 15% for C. cassia and 45% C. flexuous. The results highlight the potential of these OEs in the treatment of cryptococcosis as well as the inhibition of virulence factors. These data are promising, and further research is needed to develop of a new drug. / O complexo Cryptococcus neoformans compreende duas espécies, responsáveis por causar criptococose, sendo a principal manifestação a meningoencefalite em imunocomprometidos. Sua patogenicidade está relacionada aos fatores de virulência. O tratamento desta enfermidade é dificultado devido aos fármacos apresentarem alta toxicidade, resistência do fungo e pouca eficiência terapêutica. Pesquisa com produtos naturais biologicamente ativos tem demonstrado que os óleos essenciais (OEs) apresentam importante atividade antifúngica. Assim, o presente estudo teve como objetivo avaliar a eficácia como antifúngico e ação sobre os fatores de virulência em espécies do complexo C. neoformans dos OEs de Cinamomum cassia (Canela da China) e Cybopogom flexuosus (Capim limão). Por meio da técnica de microdiluição em caldo, os OEs apresentaram CIM variando entre 8 a 256 μg/ml e CFM de 16 a 512 μg/ml. C. cassia promoveu uma redução de 47,8% no tamanho da cápsula e inibiu a síntese de melanina em 30% dos isolados. A avaliação deste OE sobre da atividade das exoenzimas fosfolipase, urease e DNAse demonstrou inibição em 15%, 30% e 25% dos isolados avaliados respectivamente. C. flexuosus reduziu o tamanho da cápsula em 48,7%, inibiu a produção de melanina, urease, fosfolipase e DNAse em 30%, 30%, 25% e 15% respectivamente. Os dados demonstraram que os isolados não produziram proteinase e a atividade hemolítica foi reduzida em 15% para C. cassia e 45% C. flexuosos. Os resultados enaltecem o potencial desses OEs no tratamento da criptococose assim como na inibição dos fatores de virulência. Esses dados são promissores, sendo necessário pesquisas mais aprofundadas para o desenvolvimento de um novo fármaco.

Antifungal drug resistance is a growing problem. Several mechanisms contribute to the development of resistance to antifungal agents. In South Africa, little is known about the antifungal susceptibility of local yeast isolates. The aim of this study was therefore to determine the antifungal susceptibility profile of local Candida species and Cryptococcus neoformans isolates and the molecular mechanisms of resistance to different antifungal agents in Pretoria. A total of 250 yeast isolates were collected from the diagnostic laboratory in the Department of Medical Microbiology at the University of Pretoria-National Health Laboratory Services. The isolates were subcultured on Sabouraud dextrose agar media for purity of yeast colonies. Identification to species level was performed using biochemical techniques. The antifungal susceptibility of 87 isolates was determined by the Etest for three azole antifungals (fluconazole, posaconazole and voriconazole), amphotericin B and caspofungin. Clinical breakpoint susceptibility was determined according to the CLSI clinical breakpoint reference methods. Polymerase chain reaction was performed on C. albicans isolates to amplify the ERG11 gene and the FKS1 gene. Sequencing was done for the amplification products and the sequence data were analysed by the CLC genome workbench software. Among the 250 isolates collected, Candida species accounted for 82.8% and C. neoformans accounted for 17.2% of the isolates. C. albicans was the most commonly isolated (76.8% of Candida species), of which 30% were resistant to caspofungin. Fluconazole resistance was detected in 56.7% of C. parapsilosis isolates, the highest fluconazole resistance among Candida species. Cross-resistance was found between fluconazole and voriconazole. Resistance to posaconazole was detected in 66.7% of C. glabrata isolates, whilst all other Candida species and C. neoformans isolates were fully susceptible. Furthermore, C. neoformans var. gattii isolates were less susceptible to azole antifungal agents than C. neoformans var. neoformans isolates. Molecular alterations are one of the mechanisms of resistance to azole and caspofungin antifungal agents. The amino acid substitutions D116E, K128T and V437I were detected in the ERG11 gene of two azole susceptible isolates. The new amino acid substitution E517Q was detected in the ERG11 gene of a resistant isolate. The S642L substitution was detected in the FKS1 gene of all the isolates that were caspofungin resistant and caspofungin susceptible. C. albicans was the most commonly isolated yeast species in Pretoria. Cross-resistance was detected between fluconazole and voriconazole. Therefore, these two agents are not a good alternative to use in the treatment of resistant isolates. With Candida species and C. neoformans isolates, there was less resistance to posaconazole than to fluconazole and voriconazole. The identification of the two varieties of C. neoformans is important in order to establish the differences in their antifungal susceptibility. Resistance to azole and caspofungin antifungal agents existed without the previously described molecular alterations in the ERG11 and FKS1 genes of resistant isolates. Further studies are required to explain the role of new amino acid substitutions, as well as the involvement of other mechanisms in resistance to antifungal drugs. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Medical Microbiology / unrestricted

No description available.

Um die Rolle von Interleukin-12p75 (IL-12p75) und Interleukin-12p40 (IL-12p40) in der Abwehr einer Kryptokokken-Infektion im Mausmodell zu untersuchen, wurden Mäuse auf 129Sv/Ev Stammhintergrund intraperitoneal und intranasal mit Cryptococcus neoformans (C. neoformans) infiziert. Dabei wurden die Unterschiede im Infektionsverlauf und in der Immunreaktion von Wildtyp-, IL-12p35-/- und IL-12p35/p40-/--Mäusen analysiert. Unterschiede zwischen den Wildtyp- und den IL-12p35-/--Mäusen lassen auf die Bedeutung von IL-12p75 schließen, wogegen Unterschiede zwischen IL-12p35-/-- und IL-12p35/p40-/--Mäusen auf die Rolle von IL-12p40 schließen lassen. Untersucht wurden sowohl die Erregerkonzentration in den Organen, Antigenspiegel im Blut, histologische Veränderungen und Serumantikörperkonzentrationen. Nach intraperitonealer Infektion war die Keimbelastung der Organe bei den Wildtyp-Mäusen geringer als bei beiden IL-12-/--Mausstämmen. Bei Wildtyp-Mäusen waren nicht nur weniger lebende Kryptokokken in den Organen zu finden, sondern auch weniger Kryptokokken Antigen im Serum als bei beiden IL-12-/--Mäusen nachweisbar. Das zeigt, dass IL-12p75 für die Kontrolle der intraperitonealen Infektion mit C. neoformans notwendig ist. IL-12p40 hatte ähnlich wie IL-12p75, wenn auch in etwas geringerem Masse, eine protektive Rolle bei der Erregerabwehr. Ohne IL-12p40 war eine Kontrolle der Infektion auf einem geringen Niveau der Keimbelastung nicht möglich. Besonders deutlich wurde dieses Phänomen beim Antigentiter bei den IL-12p35/p40-/--Mäusen. Durch das Fehlen von IL 12p40 wurde bei den IL-12p35/p40-/--Mäusen viel mehr Antigen über das Blut im Serum verteilt als bei den IL-12p35-/-- oder den Wildtyp-Mäusen. Die Wirtsreaktion bei einer Infektion mit C. neoformans geht mit der Bildung von Granulomen einher. Ohne IL-12p75 kam es zwar noch zur Bildung von Granulomen, diese zeigten aber eine veränderte zelluläre Zusammensetzung. Die IL-12p35/p40-/--Mäuse waren nicht zur Ausbildung von typischen Granulomen fähig. Bei ihnen kam es zu einer vermehrten Ansammlung von Kryptokokken fast ohne Entzündungszellen. IL-12p40 ist also für die Ausbildung einer zellulären Entzündungsreaktion notwendig. IL-12p40 ist auch für die Antikörperbildung gegen C. neoformans erforderlich. Die IL 12p35/p40-/--Mäuse waren kaum in der Lage, spezifische Antikörper gegen C. neoformans zu bilden. IL-12p75 ist für die Ausbildung einer Th1-Antwort notwendig. Infizierte Wildtyp-Mäuse produzierten doppelt soviel IgG2a, welches für ein Th1-Antwort typisch ist, wie die IL 12p35-/--Mäuse. Der intranasale Infektionsweg kommt der natürlichen aerogenen Infektion recht nahe. Deshalb wurde – zusätzlich zur intraperitonealen Infektion - dieser Infektionsweg zur Untersuchung der Immunantwort gegen C. neoformans berücksichtigt. Auch bei intranasaler Infektion ist IL-12p75 für die Kontrolle der Keimbelastung der Organe notwendig. Interessanterweise war die Keimbelastung der Lunge bei den IL-12p35-/--Mäusen etwas höher als bei den IL-12p35/p40-/--Mäusen. Bei den Wildtypmäusen war die Dissemination der Kryptokokken aus der Lunge in die Milz und ins Gehirn gering. Ein Fehlen von IL-12p75 bewirkte allerdings eine Besiedlung besonders des Gehirns. Nach intranasaler Infektion kam es in der Lunge von Wildtyp-Mäusen zu atypischen Granulomen mit zentraler Einschmelzung von Gewebe und Kryptokokken. Diese Reaktion war bei den IL-12p35-/--Mäusen noch stärker ausgeprägt als bei den Wildtyp-Mäusen. Bei den IL-12p35/p40-/--Mäusen blieb eine Gewebsreaktion größerer Areale aus. Es waren nur eine Aktivierung des BALT zu sehen. IL-12p40 ist demnach auch nach intranasaler Infektion für eine zelluläre Entzündungsreaktion notwendig. Möglicherweise kann sich diese Eigenschaft von IL-12p40 bei intranasaler Infektion in einer immunpathologischen Reaktion äußern, die bei IL-12p35-/--Mäusen für eine massive Infiltration der Lunge mit Entzündungszellen verantwortlich ist. Der Gehalt an Kryptokokken-spezifischen Antikörpern war nach intranasaler Infektion fünf- bis zehnmal höher als nach intraperitonealer Infektion. Der intranasale Infektionsweg zeigte also eine wesentlich ausgeprägtere humorale Antwort. Der Typ der Immunantwort schien sich im Gegensatz zur intraperitonealen Infektion in Richtung Th2 (d. h. verstärkte Antikörperbildung) verschoben zu haben. Sowohl nach intraperitonealer wie auch nach intranasaler Infektion mit C. neoformans lassen sich die immunstimulatorischen Aktivitäten von IL-12p75 und von IL-12p40 nachweisen, auch wenn diese sich in Abhängigkeit vom Infektionsweg etwas unterschiedlich manifestieren. / To analyse the role of interleukin-12p75 (IL-12p75) and interleukin-12p40 (IL-12p40) in the defence against Cryptococcus neoformans (C. neoformans) a murine infection model was established and studied. Mice of wild-tpye 129Sv/Ev background as well as IL-12p35-/- and IL-12p35/p40-/- 129Sv/Ev mice were infected intraperitoneally or intranasally with C. neoformans. The differences between the immune response of these genotypes were analysed. Comparing wild-type and IL-12p35-/--mice allows for conclusions related to the importance of IL-12p75, comparing IL-12p40-producing IL-12p35-/- mice with IL-12p35/p40-/- mice shows the importance of IL-12p40. Fungal organ burden, serum antigen levels, inflammatory cell responses, and antibody production were examined. The fungal organ load in wild-type mice was smaller than in both mutant IL-12-/--mice. In wild-type mice fewer cryptococci were found in organs and less cryptococcal antigen in serum than in IL-12p35-/- and IL-12p35/p40-/- mice. This underlines the importance of IL 12p75 for the control of the infection with C. neoformans. In addition, IL-12p40 was found to have a similar but weaker role as IL-12p75 in protection against C. neoformans. In the absence of IL-12p40 IL-12p35/p40-/- mice developed higher antigen titers than IL-12p35-/- and wild-type mice. The host response against infection with C. neoformans is associated with granuloma formation. Recruitment of inflammatory cells to granulomas was altered in the absence of IL 12p75. In addition, IL-12p40 contributed significantly to granuloma formation since IL 12p35/p40-/- mice developed no or only very poor granulomatous responses. Therefore, IL 12p40 is required for inflammatory cell responses. IL-12p40 was also found to be required for antibody production against C. neoformans. Infected IL-12p35/p40-/--mice had only very low levels of specific antibodies against C. neoformans. IL-12p75 is known to be essential for protective Th1 response against intracellular microorganisms. Th1 responses are commonly associated with the production of IgG2a. Infected wild-type mice produced 2-fold higher IgG2a levels than IL-12p35-/--mice. To adapt the infection model more to the natural infection mode the intraperitoneal infection route was changed to an intranasal route. Following intranasal infection IL-12p75 also proved to be necessary for control of the fungal organ load. Interestingly the organ load was higher in IL-12p35-/--mice than in IL-12p35/p40-/-mice which suggest a role of IL-12p40 in cell recruitment. Following intranasal application of cryptococci fungal dissemination to spleen and brain was reduced as compared to the intraperitoneal infection route. Without IL-12p75 dissemination of C. neoformans to the brain occured. This shows that IL-12p75 is involved in control of dissemination from lung to brain. The inflammatory response of IL-12p35-/--mice was stronger than the tissue response of wild-type mice. The massive tissue reactions of IL-12p35-/--mice caused big areas of diffuse cellular infiltration in their lungs. In IL-12p35/p40-/--mice inflammatory responses could be observed only in the peribronchial tissue. This shows that IL-12p40 is not only needed for a cellular inflammatory response following intraperitoneal but also following intranasal infection. Following intranasal infection IL-12p40 can induce immunopathological effects. Intranasal infection of mice with C. neoformans resulted in five to ten times higher antibody responses than intraperitoneal infection. This suggests that intranasal infection of mice results in a more Th2-biased humoral response. In summary, these experiments show that besides IL-12p75 also IL-12p40 contributes to cellular immunity against C. neoformans. The immunostimulatory properties of both, IL 12p75 and IL-12p40, can be observed after intraperitoneal and intranasal infection routes with similar but also distinct manifestations.

Orientador: João Claudio Thomeo / Banca: Daniela Alonso Bocchini Martins / Banca: Tatiane Iembo / Resumo: A criptococose é uma micose sistêmica que afeta principalmente o sistema nervoso central, causando meningite. Causada pela inalação de propágulos de fungos do gênero Cryptococcus sp, C neoformans acomete hospedeiros com imunodepressão celular, cujo maior contingente é representado por indivíduos com AIDS. A fonte principal de aquisição desta levedura são fezes de aves, principalmente pombos (Columba livia). C. gattii infecta indivíduos aparentemente imunocompetentes, sendo isolado principalmente em espécies de eucaliptos. C. albidus e C. laurentii aparece em casos de meningites, fungemias, pneumonias, abscessos pulmonares e infecções cutâneas. C.uniguttulatus é encontrado no trato gastrointestinal, fezes de pássaros e contaminante de leitos de animais e habitats de roedores. Termo-tolerância, parede celular, cápsula, adesão, e produção de enzimas são fatores importantes de virulência do gênero. São José do Rio Preto é a 5ª cidade entre os 100 municípios do estado de São Paulo em de casos de AIDS. Analisou-se morfologicamente e bioquimicamente 48 amostras clínicas e 155 ambientais. Amostras de líquor resultaram em 30 isolados da espécie C. neoformans, 17 de C. gattii e um de C. luteolus. Foram identificados 26 isolados de C.albidus, 22 de C. laurentii, 9 de C. neoformans, 5 de C. gattii e um de C.uniguttulatus nas amostras ambientais. A concentração inibitória mínima para antifúngicos foi determinada para todos os isolados por meio do teste de microdiluição em caldo padronizado pelo NCCLS. Os resultados obtidos mostraram resistência à anfotericina B e itraconazol nas cepas clínicas de C. neoformans e C. gattii, e as cepas de C.albidus e C. laurentii apresentaram resistência a 5-fluorcitosina, itraconazol e anfotericina B / Abstract: Cryptococcosis is a systemic mycosis that mainly affects the central nervous system, causing meningitis. Caused by inhalation of seedlings of fungi of the genus Cryptococcus sp, C. neoformans affects hosts with immunosuppression cellular, whose largest contingent is represented by individuals with AIDS. The main source of acquiring this yeast is feces of birds, especially pigeons (Columba livia). C. gattii infects individuals apparently immunocompetent, being isolated mainly in species of eucalyptus. C. albidus and C. laurentii appear in cases of meningitis, nosocomial fungemia, pneumonia, lung abscesses and skin infections. C. uniguttulatus is found in the gastrointestinal tract and feces of birds and contaminant of beds of animals and habitats of rodents. Thermo-tolerance, cell wall, capsule, accession, and production of enzymes are important factors in the virulence of the genus. São José do Rio Preto is the 5th city among the 100 cities in the state of São Paulo in cases of AIDS. We analyzed morphologically and biochemically 48 clinical samples and 155 environmental. Liquor samples resulted in 30 isolates of the species C. neoformans, 17 of C. gattii and one of C. luteolus. We identified 26 isolates of C. albidus, 22 of C. laurentii, 9 of C. neoformans, 5 of C. gattii and one of C. uniguttulatus in environmental samples. The minimal inhibitory concentration for antifungal agents was determined for all isolates by means of the test of broth microdilution standardized by the NCCLS. The results obtained showed resistance to amphotericin B and itraconazole in clinical strains of C. neoformans and C. gattii, C. albidus and C. laurentii exhibited resistance to 5-fluorocytosine, itraconazole and amphotericin B / Mestre

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.

<p>Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, <italic>C. neoformans</italic> can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to <italic>C. neoformans in vitro</italic> and <italic>in vivo</italic>. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo</italic> by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to <italic>C. neoformans</italic> and, consequently, impart insight into potential future treatment strategies for cryptococcosis.</p> / Dissertation

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.