Search

Background: The Trichosporonaceae family comprises a large number of basidiomycetes widely distributed in nature. Some of its members, especially Trichosporon asahii, have the ability to cause human infections. This ability is related to a series of virulence factors, which include lytic enzymes production, biofilm formation, resistance to oxidising agents, melanin and glucuronoxylomannan in the cell wall, metabolic plasticity and phenotypic switching. The last two are poorly addressed within human pathogenic Trichosporonaceae., Objective: These factors were herein studied to contribute with the knowledge of these emerging pathogens and to uncover mechanisms that would explain the higher frequency of T. asahii in human infections., Methods: We included 79 clinical isolates phenotypically identified as Trichosporon spp. and performed their molecular identification. Lactate and N-acetyl glucosamine were the carbon sources of metabolic plasticity studies. Morphologically altered colonies after subcultures and incubation at 37°C indicated phenotypic switching., Results and Conclusion: The predominant species was T. asahii (n = 65), followed by Trichosporon inkin (n = 4), Apiotrichum montevideense (n = 3), Trichosporon japonicum (n = 2), Trichosporon faecale (n = 2), Cutaneotrichosporon debeurmannianum (n = 1), Trichosporon ovoides (n = 1) and Cutaneotrichosporon arboriforme (n = 1). T. asahii isolates had statistically higher growth on lactate and N-acetylglucosamine and on glucose during the first 72 h of culture. T. asahii, T. inkin and T. japonicum isolates were able to perform phenotypic switching. These results expand the virulence knowledge of Trichosporonaceae members and point for a role for metabolic plasticity and phenotypic switching on the trichosporonosis pathogenesis., (© 2022 Wiley-VCH GmbH.)

Background: Black fungi of the Herpotrichiellaceae family are agents of chromoblastomycosis and phaeohyphomycosis. There are few therapeutic options for these infections and it is common to associate antifungal drugs in their treatment., Objectives: To investigate the Medicines for Malaria Venture (MMV) Pathogen Box® for possible compounds presenting synergism with antifungal drugs used to treat black fungal infections., Methods: An initial screening of the Pathogen Box® compounds was performed in combination with itraconazole or terbinafine at sub-inhibitory concentrations against Fonsecaea pedrosoi. Hits were further tested against eight Herpotrichiellaceae using the checkerboard method., Findings: No synergism was observed with terbinafine. MMV687273 (SQ109) and MMV688415 showed synergism with itraconazole against F. pedrosoi. Synergism of these compounds was confirmed with some black fungi by the checkerboard method. SQ109 and itraconazole presented synergism for Exophiala dermatitidis, F. pedrosoi, F. monophora and F. nubica, with fungicidal activity for F. pedrosoi and F. monophora. MMV688415 presented synergism with itraconazole only for F. pedrosoi, with fungicidal activity. The synergic compounds had high selectivity index values when combined with itraconazole., Main Conclusions: These compounds in combination, particularly SQ109, are promising candidates to treat Fonsecaea spp. and E. dermatitidis infections, which account for most cases of chromoblastomycosis and phaeohyphomycosis.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii , which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii , Trichosporon japonicum , Apiotrichum montevideense , Trichosporon inkin , Trichosporon faecale , Cutaneotrichosporon debeurmannianum , and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Andrade, Araújo, Brito-Santos, Figueiredo-Carvalho, Zancopé-Oliveira, Frases and Almeida-Paes.)

Cryptococcosis is a systemic fungal disease acquired from contaminated environments with propagules of the basidiomycetous yeasts of the Cryptococcus neoformans and C. gattii species complexes. The C. neoformans species complex classically comprises four major molecular types (VNI, VNII, VNIII, and VNIV), and the C. gattii species complex comprises another four (VGI, VGII, VGIII, and VGIV) and the newly identified molecular type VGV. These major molecular types differ in their epidemiological and ecological features, clinical presentations, and therapeutic outcomes. Generally, the most common isolated types are VNI, VGI, and VGII. The epidemiological profile of cryptococcosis in domestic cats is poorly studied and cats can be the sentinels for human infections. Therefore, the present study aimed to determine the molecular characterization of Cryptococcus spp. isolated from domestic cats and their dwellings in the metropolitan area of Rio de Janeiro, Brazil. A total of 36 Cryptococcus spp. strains, both clinical and environmental, from 19 cats were subtyped using multilocus sequence typing (MLST). The ploidy was identified using flow cytometry and the mating type was determined through amplification with specific pheromone primers. All strains were mating type alpha and 6/36 were diploid (all VNII). Most isolates (63.88%) were identified as VNII, a rare molecular type, leading to the consideration that this genotype is more likely related to skin lesions, since there was a high percentage (68.75%) of cats with skin lesions, which is also considered rare. Further studies regarding the molecular epidemiology of cryptococcosis in felines are still needed to clarify the reason for the large proportion of the rare molecular type VNII causing infections in cats.

Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease. Trichosporon asahii and Trichosporon mucoides share with Cryptococcus species the ability to produce GXM. To check whether other opportunistic species in the Trichosporonaceae family produce GXM-like polysaccharides, extracts from 28 strains were produced from solid cultures and their carbohydrate content evaluated by the sulfuric acid / phenol method. Moreover, extracts were assessed for cryptococcal GXM cross-reactivity through latex agglutination and lateral flow assay methods. Cryptococcus neoformans and Saccharomyces cerevisiae were used as positive and negative controls, respectively. In addition to T. asahii, the species Trichosporon inkin, Apiotrichum montevideense, Trichosporon japonicum, Trichosporon faecale, Trichosporon ovoides, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis are also producers of a polysaccharide immunologically similar to the GXM produced by human pathogenic Cryptococcus species. The carbohydrate concentration of the extracts presented a positive correlation with the GXM contents determined by titration of both methodologies. These results add several species to the list of fungal pathogens that produce glycans of the GXM type and bring information about the origin of potential false-positive results on immunological tests for diagnosis of cryptococcosis based on GXM detection.

Bloodstream infections (BSI) caused by Candida species are the fourth cause of healthcare associated infections worldwide. Non-albicans Candida species emerged in the last decades as agents of serious diseases. In this study, clinical and microbiological aspects of six patients with BSI due to the Meyerozyma (Candida) guilliermondii species complex from an oncology reference center in Brazil, were evaluated. To describe demographic and clinical characteristics, medical records of the patients were reviewed. Molecular identification of the isolates was performed by ITS1-5.8S-ITS2 region sequencing. Antifungal susceptibility was evaluated by the EUCAST method and the minimal inhibitory concentrations (MIC) assessed according to the epidemiological cutoff values. Virulence associated phenotypes of the isolates were also studied. Ten isolates from the six patients were evaluated. Five of them were identified as Meyerozyma guilliermondii and the others as Meyerozyma caribbica. One patient was infected with two M. caribbica isolates with different genetic backgrounds. High MICs were observed for fluconazole and echinocandins. Non-wild type isolates to voriconazole appeared in one patient previously treated with this azole. Additionally, two patients survived, despite infected with non-wild type strains for fluconazole and treated with this drug. All isolates produced hemolysin, which was not associated with a poor prognosis, and none produced phospholipases. Aspartic proteases, phytase, and esterase were detected in a few isolates. This study shows the reduced antifungal susceptibility and a variable production of virulence-related enzymes by Meyerozyma spp. In addition, it highlights the poor prognosis of neutropenic patients with BSI caused by this emerging species complex., Lay Abstract: Our manuscript describes demographic, clinical and microbiological characteristics of patients with bloodstream infection by the Meyerozyma guilliermondii species complex at a reference center in oncology in Brazil., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Sporotrichosis in immunocompromised patients has a high morbidity and may cause deaths. Particularly, patients with acquired immunodeficiency syndrome (AIDS) with low T CD4 counts develop a chronic disease, with severe and widespread forms. Recently, the ability of Sporothrix brasiliensis, the main agent of zoonotic sporotrichosis, to increase its virulence in a diabetic patient without HIV infection was described. Since it was a unique finding, it is not known how often this occurs in patients with chronic and refractory sporotrichosis. The aim of this study is to compare sequential Sporothrix isolates obtained from patients with sporotrichosis and AIDS in order to detect changes in virulence-related phenotypes and acquisition of antifungal resistance during the evolution of the disease. Fungal growth in different substrates, antifungal susceptibility, thermotolerance, resistance to oxidative stress, and production of hydrolytic enzymes were evaluated. Correlations were assessed between clinical and phenotypic variables. Sixteen isolates, all identified as S. brasiliensis, obtained from five patients were studied. They grew well on glucose and N-acetyl-D-glucosamine, but poorly on lactate. Except from isolates collected from two patients, which were non-wild type for terbinafine, they were considered wild type for the antifungal drugs tested. Thermotolerance of the isolates was moderate to high. Except for phytase and phospholipase, isolates were able to produce virulence-related enzymes on different levels. Changes in all studied phenotypes were observed during the course of the disease in some patients. The results show that the HIV-driven immunosuppression is more relevant than fungal phenotypes on the unfavorable outcomes of disseminated sporotrichosis.

Microsporum canis is a zoophilic dermatophyte and the most common fungus isolated from dogs and cats worldwide. To invade skin, this pathogen uses different enzymes, which may be associated with virulence, that contribute to the fungal pathogenicity. The aim of this study is to compare the expression of enzymes that may be associated with virulence, and thermotolerance of M. canis strains isolated from dogs, cats, and humans. The in vitro expression of the enzymes keratinase, catalase, urease, hemolysin, and aspartic protease was evaluated in 52 M. canis strains recently isolated from 14 human patients, 12 dogs, 15 symptomatic, and 11 asymptomatic cats. In addition, thermotolerance was assessed by comparative analysis of fungal growth at 25 °C and 35 °C. Keratinase activity was low in 34 and moderate in 18 strains. Aspartic-protease activity was low in 7, moderate in 33, and high in 12 strains. Hemolysin activity was low in 44 and moderate in 8 strains. All strains were classified as low producers of catalase. All but three strains produced urease in vitro, with a broad range of activity. The strains presented in vitro growth at the two studied temperatures were classified as presenting low (36.5%), medium (44.3%), or high (19.2%) thermotolerance. There was no statistically significant difference in the new putative virulence-associated factors studied among the different hosts, which suggests that they may have a similar role on human, cat, and dog infection. Also, no difference was observed between strains isolated from symptomatic and asymptomatic cats. This suggests that these factors have a limited impact on the fate of feline dermatophytosis caused by M. canis.

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., Competing Interests: The authors have declared that no competing interests exist.

Cryptococcosis, a potentially fatal mycosis in humans, is acquired via exposure to exogenous environmental sources. This study aimed to investigate the frequency, genetic diversity, and virulence of cryptococcal strains isolated from indoor dust in the Rio Negro micro-region of the Brazilian Amazon. A total of 8.9% of the studied houses were positive, recovering nine Cryptococcus neoformans VNI and 16 C. gattii VGII isolates, revealing an endemic pattern in domestic microenvironments. The International Society for Human and Animal Mycology (ISHAM) consensus multilocus sequence typing (MLST) scheme for the C. neoformans/C. gattii species complexes identified two sequence types (STs), ST93 and ST5, amongst C. neoformans isolates and six STs amongst C. gattii isolates, including the Vancouver Island Outbreak ST7 (VGIIa) and ST20 (VGIIb), the Australian ST5, and ST264, ST268 and ST445, being unique to the studied region. Virulence studies in the Galleria mellonella model showed that five C. gattii strains and one C. neoformans strain showed a similar pathogenic potential to the highly virulent Vancouver Island outbreak strain CDR265 (VGIIa). The findings of this study indicate that humans can be exposed to the agents of cryptococcosis via house dust, forming the basis for future studies to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical infections., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Infections caused by Rhodotorula spp. are increasing worldwide. This study identified, through the light of the new taxonomic advances on the subphylum Pucciniomycotina, 16 isolates from blood cultures and compared their antifungal susceptibility on microdilution and gradient diffusion methods. Internal transcriber spacer sequencing identified Rhodotorula mucilaginosa (n = 12), Rhodotorula toruloides (n = 2), Rhodotorula dairenensis (n = 1), and Cystobasidium minutum (n = 1). Amphotericin B was the most effective drug. A good essential agreement was observed on MIC values of amphotericin B and voriconazole determined by the two methods. Therefore, the gradient method is useful for susceptibility tests of R. mucilaginosa against these drugs.

Cryptococcal meningitis is a several disease common in late stage of HIV infection. Detection of cryptococcal antigen (CrAg) is an important for early diagnosis of this invasive mycosis. The pre-emptive treatment for isolated antigenemia prevents the onset of meningoencephalitis. Screening CrAg in patients with low CD4 count is cost-effective in countries with prevalence of antigenemia above 3%. However, in Brazil, the number of prevalence studies on cryptococcosis and HIV is insufficient. The objective of this study is to estimate the prevalence of CrAg and describe clinical characteristics from a cohort of patients followed at a reference center in Brazil. CrAg screening was performed in 89 inpatients with CD4 count ≤200 cells/mm 3 or WHO stage III/IV from the National Institute of Infecttious Disease, Rio de Janeiro. Patients with isolated antigenemia received pre-emptive therapy with fluconazole and patients with meningoencephalitis were treated with Amphotericin B. Individuals were followed up for 12 months. Prevalence of serum CrAg was 11.23%, cryptococcal meningoencephalitis 6.74% and isolated antigenemia 4.81%. None of the patients with isolated antigenemia developed meningoencephalitis during the follow up. Signs and symptoms of meningoencephalitis were unspecific or absent. Our study suggests the need of CrAg screening in Brazil and highlights that lumbar puncture is mandatory in all individuals CrAg positive to exclude asymptomatic meningoencephalitis., (© 2019 Blackwell Verlag GmbH.)

Early diagnosis, efficient clinical support, and proper antifungal therapy are essential to reduce death and sequels caused by cryptococcosis. The emergence of resistance to the antifungal drugs commonly used for cryptococcosis treatment is an important issue of concern. Thus, the in vitro antifungal susceptibility of clinical strains from northern Brazil, including C. neoformans VNI (n = 62) and C. gattii VGII (n = 37), to amphotericin B (AMB), 5-flucytosine, fluconazole, voriconazole, and itraconazole was evaluated using the Etest and Vitek 2 systems and the standardized broth microdilution (CLSI-BMD) methodology. According to the CLSI-BMD, the most active in vitro azole was voriconazole (C. neoformans VNI modal MIC of 0.06 μg/ml and C. gattii VGII modal MIC of 0.25 μg/ml), and fluconazole was the least active (modal MIC of 4 μg/ml for both fungi). Modal MICs for amphotericin B were 1 μg/ml for both fungi. In general, good essential agreement (EA) values were observed between the methods. However, AMB presented the lowest EA between CLSI-BMD and Etest for C. neoformans VNI and C. gattii VGII (1.6% and 2.56%, respectively, P < .05 for both). Considering the proposed Cryptococcus spp. epidemiological cutoff values, more than 97% of the studied isolates were categorized as wild-type for the azoles. However, the high frequency of C. neoformans VNI isolates in the population described here that displayed non-wild-type susceptibility to AMB is noteworthy. Epidemiological surveillance of the antifungal resistance of cryptococcal strains is relevant due to the potential burden and the high lethality of cryptococcal meningitis in the Amazon region., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 °C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.

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., Competing Interests: The authors have declared that no competing interests exist.

Tinea capitis caused by Microsporum audouinii is reported herein from two Brazilian schoolchildren, which are brothers. Arthroconidia were evidenced on direct examination of scalp hair, and a fungus of the genus Microsporum was isolated from cultures of each patient. The isolated fungi were classified as M. audouinii by visualization of species-specific structures, including: pectinate hyphae, chlamydospores, and fusiform macroconidia, sterile growth with characteristic brown pigment in rice grains, and through DNA sequencing of the internal transcriber spacer region. Patients were refractory to ketoconazole, but the two cases had a satisfactory response to oral terbinafine. All M. audouinii infections described in this century were reviewed, and to our knowledge, this is the first literature description of this species from South America. Misidentification of M. audouinii with Microsporum canis can occur in this area, leading to erroneous data about the occurrence of this species.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole-trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.

Background: Sporothrix brasiliensis is the most virulent sporotrichosis agent. This species usually responds to antifungal drugs, but therapeutic failure can occur in some patients. Antifungal susceptibility tests have been performed on this species, but no clinical breakpoints (CBPs) are available. In this situation, minimal inhibitory concentration (MIC) distributions and epidemiological cutoff values (ECVs) support the detection of identification of resistant strains., Objectives: To study the MIC distributions of five antifungal drugs against S. brasiliensis and to propose tentative ECVs., Methods: MICs of amphotericin B (AMB), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), and terbinafine (TRB) against 335 S. brasiliensis strains were determined by the Clinical and Laboratory Standards Institute broth microdilution method., Findings: The proposed ECV, in µg/mL, for AMB, ITR, KET, POS, and TRB were 4.0, 2.0, 1.0, 2.0, and 0.25, respectively. Percentages of wild-type strains in our population for the above antifungal drugs were 98.48, 95.22, 95.33, 100, and 97.67%, respectively., Main Conclusions: These ECVs will be useful to detect strains with resistance, to define CBPs, and to elaborate specific therapeutic guidelines for S. brasiliensis. Rational use of antifungals is strongly recommended to avoid the emergence of resistant strains and ensure the therapeutic effectiveness of sporotrichosis.

Eighty-nine patients with clinical suspicion of leishmaniasis were referred for differential diagnosis. Sporothrix schenckii sensu lato was isolated in Novy-MacNeal-Nicolle + Schneider media in 98% of 64 patients with final diagnosis of sporotrichosis. This medium may be suitable for diagnosis of sporotrichosis in areas where cutaneous leishmaniasis is also endemic., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence.

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types-VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.

Two commercial methods, the Etest and Vitek 2, were compared with the Clinical and Laboratory Standards Institute broth microdilution method to determine the susceptibility of Candida parapsilosis complex to amphotericin B, caspofungin, fluconazole, voriconazole, and itraconazole. One-hundred bloodstream isolates of C. parapsilosis complex from three hospitals in Rio de Janeiro city, Brazil, between 1998 and 2006 were analyzed. C. parapsilosis sensu stricto (61 %) was the predominant species, followed by C. orthopsilosis (37 %) and C. metapsilosis (2 %). Most isolates were susceptible to the tested drugs. However, one C. parapsilosis sensu stricto isolate was considered resistant for amphotericin B. The essential agreement was 100 % between the methods, except for itraconazole (96.3 %). The categorical agreement varied for fluconazole and itraconazole by Etest and for amphotericin B and fluconazole by Vitek 2. This study reinforces the suitability of the commercial methods in routine clinical microbiology laboratories for antifungal susceptibility testing.