Susceptibilidade de Fonsecaea spp. aos antifúngicos: relações com a produção de melanina fúngica, associação de fármacos e aspectos clínicos dos pacientes

Made available in DSpace on 2018-05-21T13:29:56Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) rowena_coelho_ini_mest_2017.pdf: 1835410 bytes, checksum: 49248c79932cd1eee8ad1eefa28b4429 (MD5)Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.A cromoblastomicose (CBM) é uma micose crônica que afeta a pele, tecidos subcutâneos e apresenta distribuição global, com predominância em áreas rurais de países tropicais e subtropicais. No Brasil, o gênero Fonsecaea é o principal responsável pela CBM. Fonsecaea pedrosoi, F. nubica, F. monophora e F. pugnacius só se diferenciam entre si por análises genotípicas, já que fenotipicamente são muito semelhantes. Os agentes de CBM produzem e secretam melanina, importante fator de virulência em vários fungos patogênicos. O acúmulo de melanina proporciona resistência e integridade à parede celular frente a diversos compostos químicos, dentre os quais podem estar incluídos os fármacos antifúngicos. O tratamento da CBM, em geral, é longo e sujeito a recaídas, com muitos casos crônicos altamente resistentes à terapia. Várias formas de intervenção são relatadas na literatura e não há um padrão-ouro de tratamento. Assim, a avaliação in vitro da susceptibilidade aos agentes antifúngicos pode ser uma forma de orientar o tratamento desta micose. Os objetivos desse estudo foram avaliar a susceptibilidade in vitro aos antifúngicos, isoladamente ou em combinação, de 20 isolados obtidos de 17 pacientes com CBM, investigar a influência da melanina fúngica na susceptibilidade desses isolados e verificar possíveis relações entre susceptibilidade e resposta terapêutica dos pacientes. A caracterização molecular dos isolados foi realizada com base na amplificação e sequenciamento da região ITS do rDNA, na qual foram identificadas três espécies: F. monophora (n=10), F. pedrosoi (n=5) e F. nubica (n=5) A determinação da concentração inibitória mínima (CIM) in vitro foi realizada pelo método da microdiluição em caldo, de acordo com o protocolo M38-A2 recomendado pelo Clinical and Laboratory Standards Institute. Os fármacos testados foram: anfotericina B (AMB), flucitosina (5-FC), terbinafina (TRB), fluconazol (FLZ), itraconazol (ITZ), cetoconazol (KTZ), posaconazol (PSZ), voriconazol (VRZ), ravuconazol (RVZ), caspofungina (CAS) e micafungina (MFG). TRB e VRZ apresentaram boa atividade in vitro, enquanto os fármacos FLZ, 5-FC, AMB e MFG apresentaram CIMs mais elevadas. Foi estudada a associação de ITZ/TRB, AMB/5-FC e ITZ/CAS pelo método tabuleiro de xadrez, e observou-se interação sinérgica de três isolados de F. monophora frente à combinação AMB/5-FC. Os pacientes apresentaram CBM moderada ou grave e a terapia com ITZ não foi suficiente para cura completa na maioria dos casos, exigindo abordagens cirúrgicas adjuvantes. Foi avaliado o efeito da inibição da melanina pelo triciclazol na susceptibilidade aos fármacos que apresentaram elevados CIMs no teste in vitro. O fármaco 5-FC apresentou uma melhor atividade frente aos isolados não melanizados. Em contrapartida, MFG e FLZ não mostraram atividade na maioria dos isolados, havendo uma diminuição da ação do fármaco após inibição da melanina. Os resultados deste estudo apontam para uma predominância de F. monophora, que é a segunda espécie de Fonsecaea da América do Sul, em pacientes atendidos no INI/Fiocruz, especialmente naqueles nascidos e residentes no estado do Rio de Janeiro, Brasil (100%). TRB e VRZ, bem como a associação AMB/5-FC, podem ser melhor estudados no contexto clínico da CBM devido às baixas CIMs e sinergismo, respectivamente.Chromoblastomycosis (CBM) is a chronic mycosis that affects the skin, subcutaneous tissues and presents a global distribution, predominantly in rural areas of tropical and subtropical countries. In Brazil, the Fonsecaea genus comprises the major agents of CBM. Fonsecaea pedrosoi, F. nubica, F. monophora, and F. pugnacius differ only by means of genotypic analysis, since they are phenotypically very similar. The CBM agents produce and secrete melanin, an important virulence factor for several pathogenic fungi. The accumulation of melanin provides to the cell wall resistance and integrity against several chemical compounds, possibly also including antifungal drugs. The treatment of CBM in general is long and refractory, with many chronic cases highly resistant to therapy. Several forms of intervention are reported in the literature and there is no gold standard of treatment. Thus, the evaluation of in vitro susceptibility to antifungal agents may be a way to guide the treatment of this mycosis. The objectives of this study were to evaluate the in vitro susceptibility to antifungal drugs, isolated or in combination, of 20 isolates obtained from 17 patients with CBM, to investigate the influence of fungal melanin on the susceptibility of these isolates and to verify possible relationships between susceptibility and therapeutic response of the patients. The molecular characterization of the isolates was performed based on the amplification and sequencing of the ITS region rDNA, in which we identified three species: F. monophora (n=10), F. pedrosoi (n=5), and F. nubica (n=5) The in vitro minimal inhibitory concentration (MIC) determination was performed by the broth microdilution method, according to the M38-A2 protocol recommended by the Clinical and Laboratory Standards Institute. The drugs tested were amphotericin B (AMB), flucytosine (5-FC), terbinafine (TRB), fluconazole (FLZ), itraconazole (ITZ), ketoconazole (KTZ), posaconazole (PSZ), voriconazole (VRZ), ravuconazole (RVZ), caspofungin (CAS) and micafungin (MFG). TRB and VRZ showed good activity in vitro, while FLZ, 5-FC, AMB and MFG presented higher MICs. The association of ITZ/TRB, AMB/5-FC and ITZ/CAS was studied by the chequerboard method, and a synergistic interaction of three F. monophora isolates was observed against the AMB/5- FC combination. Patients had moderate or severe CBM and ITZ therapy was not sufficient for complete cure in most of the CBM cases, requiring adjuvant surgical approaches. The effect of melanin inhibition by tricyclazole on susceptibility to drugs that showed high MICs in the in vitro test was evaluated. The drug 5-FC presented a better activity against non-melanized isolates. On the other hand, MFG and FLZ showed no activity in most isolates, with a decrease in the action of the drug after inhibition of melanin. The results of this study point to a predominance of F. monophora, which is the second Fonsecaea species in South America, in patients attending INI/Fiocruz, especially those born and residing in the state of Rio de Janeiro, Brazil (100%). TRB and VRZ, as well as the AMB/5-FC association, may been better studied in the clinical context of CBM due to low MICs and synergism, respectively

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 μM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 μM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 μM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 μM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection

Does DHN-Melanin Always Protect Fungi against Antifungal Drugs? The <i>Fonsecaea</i>/Micafungin Paradigm

Several human pathogenic fungi produce melanin. One of its properties during parasitism is the protection against antifungal drugs. This occurs with the agents of chromoblastomycosis, in which DHN-melanin reduces antifungal susceptibility to terbinafine and itraconazole. Since these agents are resistant to some antifungal drugs, we investigated the role of DHN-melanin on the Fonsecaea susceptibility to amphotericin B, micafungin, fluconazole, and flucytosine, drugs that usually present high minimal inhibitory concentrations (MIC) to this genus. Seven strains from three Fonsecaea human pathogenic species were treated with tricyclazole, a DHN-melanin inhibitor, and the MIC of the treated and untreated cells were compared. A survival assay was performed to confirm the alterations in the susceptibility of strains with reduced melanization, and the chitin levels of the strains were estimated by fluorescence. Tricyclazole did not affect fluconazole and flucytosine MIC, while melanin inhibition increased susceptibility to amphotericin B. Surprisingly, DHN-melanin inhibition decreased the susceptibility to micafungin. Survival assays confirmed this result on five strains. Cell wall chitin levels of the strains were not associated with the decrease in micafungin susceptibility. The results show that DHN-melanin does not have a role in the intrinsic resistance of Fonseacaea spp. to amphotericin B, fluconazole, and flucytosine, and its inhibition may promote micafungin resistance

Does DHN-Melanin Always Protect Fungi against Antifungal Drugs? The Fonsecaea/Micafungin Paradigm

Several human pathogenic fungi produce melanin. One of its properties during parasitism is the protection against antifungal drugs. This occurs with the agents of chromoblastomycosis, in which DHN-melanin reduces antifungal susceptibility to terbinafine and itraconazole. Since these agents are resistant to some antifungal drugs, we investigated the role of DHN-melanin on the Fonsecaea susceptibility to amphotericin B, micafungin, fluconazole, and flucytosine, drugs that usually present high minimal inhibitory concentrations (MIC) to this genus. Seven strains from three Fonsecaea human pathogenic species were treated with tricyclazole, a DHN-melanin inhibitor, and the MIC of the treated and untreated cells were compared. A survival assay was performed to confirm the alterations in the susceptibility of strains with reduced melanization, and the chitin levels of the strains were estimated by fluorescence. Tricyclazole did not affect fluconazole and flucytosine MIC, while melanin inhibition increased susceptibility to amphotericin B. Surprisingly, DHN-melanin inhibition decreased the susceptibility to micafungin. Survival assays confirmed this result on five strains. Cell wall chitin levels of the strains were not associated with the decrease in micafungin susceptibility. The results show that DHN-melanin does not have a role in the intrinsic resistance of Fonseacaea spp. to amphotericin B, fluconazole, and flucytosine, and its inhibition may promote micafungin resistance

Cryptococcosis due to Cryptococcus gattii VGII in southeast Brazil: The One Health approach revealing a possible role for domestic cats

Two cats infected by C. gattii, presented lesions on the nasal region and respiratory signs. Strains were typed as molecular type VGII, mating type alpha, MLST subtypes ST442 and ST185. Since Rio de Janeiro is known as an endemic area for C. neoformans VNI, these cases might be a warning for a possible emergence of C. gattii VGII in southeast Brazil. Keywords: Cryptococcus gattii, VGII, MLST, Cryptococcosis, Ca

Preservation Methods in Isolates of <i>Sporothrix</i> Characterized by Polyphasic Approach

Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by eight pathogenic species of the Sporothrix genus. Different ex situ preservation methods are used around the world to maintain the survival, morphophysiological and genetic traits of fungal strains isolated from patients with sporotrichosis for long terms. The main aim of the present study was to evaluate the survival, phenotypic and genotypic stability of Sporothrix strains after preservation on PDA slant stored at 4 °C, sterile water and cryopreservation at −80 °C, for a period of time of 6, 12, 18 and 24 months of storage. Eight clinical Sporothrix isolates were identified based on a polyphasic approach consisting of classical macro- and micro-morphological traits, biochemical assays, proteomic profiles by MALDI-TOF MS and molecular biology. According to the final identification, one strain was identified as S. schenckii (CMRVS 40428) and seven strains were re-identified as S. brasiliensis (CMRVS 40421, CMRVS 40423, CMRVS 40424, CMRVS 40425, CMRVS 40426, CMRVS 40427 and CMRVS 40433). In addition, it was observed that the isolates survived after the different time points of storage in distilled water, PDA slant and cryopreservation at −80 °C. For fungi preserved in water, low polymorphisms were detected by the partial sequencing of β-tubulin. Cryopreservation at −80 °C induced morphological changes in one single isolate. The proteomic profiles obtained by MALDI-TOF MS after preservation showed differences among the methods. In conclusion, preservation on agar slant stored at 4 °C was the most effective method to preserve the eight clinical Sporothrix strains. This method produced less change in the phenotypic traits and kept the genetic integrity of all strains. Agar slant stored at 4 °C is a simple and inexpensive method and can be especially used in culture collections of limited funding and resources

Paracoccidioidomycosis due to Paracoccidioides brasiliensis S1 plus HIV co-infection

<div><p> BACKGROUND Paracoccidioidomycosis (PCM) is one of the most important systemic mycoses in Latin America and the leading fungal cause of mortality in non-immunosuppressed individuals in Brazil. However, HIV/PCM co-infection can increase the clinical severity in these co-infected patients. This co-infection is rarely reported in the literature mainly because of the different epidemiological profiles of these infections. Furthermore, PCM is a neglected and non-notifiable disease, which may underestimate the real importance of this disease. The advent of molecular studies on the species of the genus Paracoccidioides has expanded the knowledge regarding the severity and the clinical spectrum in PCM. In this context, the development of studies to describe the association of the Paracoccidioides phylogenetic cryptic species in vulnerable populations, such as HIV-infected patients, appears relevant. OBJECTIVE To describe the clinical, epidemiological, therapeutic and prognostic aspects in HIV/PCM co-infected patients, along with the molecular identification of the Paracoccidioides species involved in these cases. METHODS The investigators performed a molecular and clinical retrospective study involving HIV/PCM co-infected patients, from a reference centre for PCM care in the endemic area of Rio de Janeiro, Brazil, from 1998 to 2015. Molecular identification of the fungal strains was done by amplification of partial sequences of arf and gp43 genes. FINDINGS Of 89 patients diagnosed with PCM by fungal isolation in the culture, a viable isolate was recovered for molecular analysis from 44 patients. Of these 44 patients, 28 (63.6%) had their serum samples submitted for enzyme immunoassay tests for screening of HIV antibodies, and 5 (17.9%) had a positive result. All cases were considered severe, with a variable clinical presentation, including mixed, acute/subacute clinical forms and a high rate of complications, requiring combination therapy. Paracoccidioides brasiliensis S1 was the species identified in all cases. CONCLUSIONS HIV/PCM co-infection can change the natural history of this fungal disease. The authors reinforce the need to include HIV screening diagnostic tests routinely for patients with PCM.</p></div

Molecular identification and antifungal susceptibility profiles of clinical strains of Fonsecaea spp. isolated from patients with chromoblastomycosis in Rio de Janeiro, Brazil.

BACKGROUND:Chromoblastomycosis (CBM) is a difficult-to-treat chronic subcutaneous mycosis. In Brazil, the main agent of this disease is Fonsecaea pedrosoi, which is phenotypically very similar to other Fonsecaea species, differing only genetically. The correct species identification is relevant since different species may differ in their epidemiologic aspects, clinical presentation, and treatment response. METHODOLOGY/PRINCIPAL FINDINGS:Partial sequencing of the internal transcribed spacer (ITS) was used to identify twenty clinical isolates of Fonsecaea spp. Their in vitro antifungal susceptibility was determined using the broth microdilution method, according to the M38-A2 protocol. Amphotericin B (AMB), flucytosine (5FC), terbinafine (TRB), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), posaconazole (POS), voriconazole (VRC), ravuconazole (RVC), caspofungin (CAS), and micafungin (MFG) were tested. The association between ITC/TRB, AMB/5FC, and ITC/CAS was studied by the checkerboard method to check synergism. The available patients' data were correlated with the obtained laboratory results. Fonsecaea monophora (n = 10), F. pedrosoi (n = 5), and F. nubica (n = 5) were identified as CBM' agents in the study. TRB and VRC were the drugs with the best in vitro activity with minimal inhibitory concentrations (MIC) lower than 0.25 mg/L. On the other hand, FLC, 5FC, AMB, and MFG showed high MICs. The AMB/5FC combination was synergistic for three F. monophora strains while the others were indifferent. Patients had moderate or severe CBM, and ITC therapy was not sufficient for complete cure in most of the cases, requiring adjuvant surgical approaches. CONCLUSIONS/SIGNIFICANCE:F. monophora, the second most frequent Fonsecaea species in South America, predominated in patients raised and born in Rio de Janeiro, Brazil, without cerebral involvement in these cases. TRB, VRC, and the AMB/5FC combination should be further investigated as a treatment option for CBM

Fatal septic shock caused by Paracoccidioides brasiliensis phylogenetic species S1 in a young immunocompetent patient: a case report

Submitted by Sandra Infurna ([email protected]) on 2019-01-30T12:47:36Z No. of bitstreams: 1 jessicaR_lima_etal_IOC_2018.pdf: 1592300 bytes, checksum: e37e95450872afd75af0ec6711008788 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-01-31T15:38:43Z (GMT) No. of bitstreams: 1 jessicaR_lima_etal_IOC_2018.pdf: 1592300 bytes, checksum: e37e95450872afd75af0ec6711008788 (MD5)Made available in DSpace on 2019-01-31T15:38:43Z (GMT). No. of bitstreams: 1 jessicaR_lima_etal_IOC_2018.pdf: 1592300 bytes, checksum: e37e95450872afd75af0ec6711008788 (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Dermatologia Infecciosa. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Departamento de Medicina Intensiva. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Departamento de Medicina Intensiva. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Micobacterioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Dermatologia Infecciosa. Rio de Janeiro, RJ. Brasil.The authors report the first case of fatal septic shock, a rare clinical presentation of paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis S1. We also provide an immunological evaluation of the patient. Severe clinical signs such as organ dysfunction and digital gangrene occurred in this case. The patient presented a remarkable cell activation profile and diminished percentage of peripheral blood T regulatory cells. A decrease in anti-inflammatory IL-1RA plasma level showed the potential for endothelium damage, probably contributing to a vasculitis process. Together with P. lutzii, P. brasiliensis appears to be involved in severe cases of PCM