Outbreak of equine pythiosis in a southeastern region of Brazil: Environmental isolation and phylogeny.

Pythiosis is a disease caused by the oomycete Pythium insidiosum, mainly reported in equines, dogs and humans and directly transmitted through contaminant zoospores in aquatic environments. We report the first outbreak of equine pythiosis in five equines. Wound samples were submitted for diagnostic testing including mycological culture and nested PCR. Treatment approaches consisted of conventional and alternative therapies. Microbiological analyses were performed using water samples from the riverbanks close to where the animals had grazed. All animals were positive for P. insidiosum cultures, and two animals responded successfully to alternative therapy (ozone therapy). After culture and molecular analysis of environmental samples, the presence of P. insidiosum in one section of the Tietê River was confirmed through a 99% sequence identity. Phylogenetic analyses using the cytochrome oxidase II gene showed that the animal isolates clustered in clade I and the environmental isolates clustered in clade III. Although the environmental and wound isolates belonged to different genetic clades, we concluded that the Tietê River is an important source of infection by P. insidiosum and that research concerning environmental isolation of P. insidiosum from rivers and lakes should be strongly facilitated in Brazil.

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen's hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.

Rabies virus and Histoplasma suramericanum coinfection in a bat from southeastern Brazil.

Bats are essential to the global ecosystem, but their ability to harbour a range of pathogens has been widely discussed, as well as their role in the emergence and re-emergence of infectious diseases. This paper describes the first report of coinfection by two zoonotic agents, rabies virus (RABV) and the fungus Histoplasma suramericanum in a bat. The bat was from the Molossus molossus species, and it was found during the daytime in the hallway of a public psychiatric hospital in a municipality in São Paulo State, southeastern Brazil. RABV infection was diagnosed by the direct fluorescent antibody test and mouse inoculation test. The fungus was isolated by in vitro culture. Both diagnoses were confirmed by molecular techniques. Phylogenetic analysis showed that the fungus isolate had proximity to H. suramericanum in the Lam B clade, while the RABV isolate was characterized in the Lasiurus cinereus lineage. Since the M. molossus bat was found in a peri-urban transition area (urban/peri-urban), the possibility of cross-species transmission of this RABV lineage becomes more plausible, considering that this scenario may provide shelter for both M. molossus and L. cinereus. These are relevant findings since there has been an increase in bat populations in urban and peri-urban areas, particularly due to environmental modifications and anthropogenic impacts on their habitat. Thus, the detection of two zoonotic agents in a bat found in a public hospital should raise awareness regarding the importance of systematic surveillance actions directed towards bats in urban areas.

Inferring putative virulence factors for Pythium insidiosum by proteomic approach.

Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening disease that affects human and animals, has difficult diagnosis, and therapy. Studies on protein characterization of P. insidiosum are scarce, so we aimed to determine the protein profile of P. insidiosum by mass spectrometry and bioinformatics strategies targeting in proteins that may act as putative virulence factors. Therefore, an extraction protocol was standardized to obtain the total proteins of P. insidiosum. By the analysis of Image Master 2D Platinum software, it was found that 186 spots ranging between 12 and 89 KDa and isoelectric point from 4 to 7. By the analysis of 2D-SDS-PAGE it was possible to visualize and excise 103 spots, which were hydrolyzed with trypsin and submitted to mass spectrometry, resulting in the identification of 36 different proteins. Three of them were classified as proteins supposedly related to virulence factors due to its functions, such as glucan 1,3-beta glucosidase, Heat shock protein (Hsp) 70 and enolase. These results may contribute to a better understanding of the virulence factors of this medically important oomycete, as well as to subsidize new studies on diagnosis and therapeutic approaches.

Paracoccidioidomycosis manifested by sarcoidosis-like cutaneous lesions and caused by Paracoccidioides brasiliensis sensu stricto (S1a)

Abstract: Molecular studies have shown more than one species of the genus Paracoccidioides to be the causal agent of paracoccidioidomycosis. Efforts have been made to correlate the identified species with epidemiological and clinical data of patients, aiming to determine the real meaning and impact of new species. Bearing this objective in mind, the authors report a clinical case of paracoccidioidomycosis, from São Paulo state, Brazil, that manifested as uncommon sarcoid-like cutaneous lesions and was caused by Paracoccidioides brasiliensis sensu stricto (S1a). The patient was treated with itraconazole 200mg/day for 12 months, with complete clinical remission.

Paracoccidioidomycosis manifested by sarcoidosis-like cutaneous lesions and caused by Paracoccidioides brasiliensis sensu stricto (S1a).

Molecular studies have shown more than one species of the genus Paracoccidioides to be the causal agent of paracoccidioidomycosis. Efforts have been made to correlate the identified species with epidemiological and clinical data of patients, aiming to determine the real meaning and impact of new species. Bearing this objective in mind, the authors report a clinical case of paracoccidioidomycosis, from São Paulo state, Brazil, that manifested as uncommon sarcoid-like cutaneous lesions and was caused by Paracoccidioides brasiliensis sensu stricto (S1a). The patient was treated with itraconazole 200mg/day for 12 months, with complete clinical remission.

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects.

Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Molecular detection of Histoplasma capsulatum in insectivorous and frugivorous bats in Southeastern Brazil.

Bats are considered to play a significant role in the epidemiology of histoplasmosis, worldwide. We investigated the occurrence of H. capsulatum in lung samples from 89 bats, from urban areas in Southeastern Brazil, using nested PCR based on ribosomal DNA. Fungal DNA was detected in 31/89 samples (34.8%), of which 13/31 were Molossids (41.9%), 4/31 Eumops spp. (12.9%), 2/31 Artibeus lituratus (6.5%), and 12/31 others (38.7%). This is the first report of natural infection by H. capsulatum in A. lituratus in Southeastern Brazil, which reinforces the importance of these synanthropic animals in the epidemiology of histoplasmosis in urban areas.

Short communication: The first report of Cyberlindnera rhodanensis associated with clinical bovine mastitis.

An acute case of clinical mastitis in a Holstein cow from second lactation is reported here. A milk sample from the affected quarter was cultured on 5% bovine blood agar and incubated at 37°C for 72 h. After 24 h of incubation, numerous colonies of yeast were observed: the Candida characteristic was not detected by CHROMagar Candida (Difco, Franklin Lakes, NJ). The DNA extraction of the isolate was performed, and DNA was subjected to amplification and sequencing of the D1/D2 region of the large subunit rRNA gene. The sequences were aligned using Mega 7.0 and used for searching GenBank by BLASTn (Basic Local Alignment Search Tool for nucleotides), revealing 98% of identity with Cyberlindnera rhodanensis. To date, this is the first report of this yeast associated with clinical bovine mastitis.

Paracoccidioidomycosis: Current Perspectives from Brazil.

BACKGROUND: This review article summarizes and updates the knowledge on paracoccidioidomycosis. P lutzii and the cryptic species of P. brasiliensis and their geographical distribution in Latin America, explaining the difficulties observed in the serological diagnosis. OBJECTIVES: Emphasis has been placed on some genetic factors as predisposing condition for paracoccidioidomycosis. Veterinary aspects were focused, showing the wide distribution of infection among animals. The cell-mediated immunity was better characterized, incorporating the recent findings. METHODS: Serological methods for diagnosis were also compared for their parameters of accuracy, including the analysis of relapse. RESULTS: Clinical forms have been better classified in order to include the pictures less frequently observesiod. CONCLUSION: Itraconazole and the trimethoprim-sulfamethoxazole combination was compared regarding efficacy, effectiveness and safety, demonstrating that azole should be the first choice in the treatment of paracoccidioidomycosis.

Stryphnodendron adstringens and purified tannin on Pythium insidiosum: in vitro and in vivo studies.

BACKGROUND: Pythium insidiosum is the etiological agent of pythiosis, an emerging life-threatening infectious disease in tropical and subtropical regions. The pathogen is a fungus-like organism resistant to antifungal therapy, for this reason, most cases need extensive surgical debridments as treatment, but depending on the size and anatomical region of the lesion, such approach is unfeasible. We investigate the fungicidal effect and toxicity of crude bark extract of Stryphnodendron adstringens and commercially available tannin on Pythium insidiosum both in vitro and in vivo. METHODS: Standardized fragments of mycelia of fifteen isolates of P. insidiosum were tested with different concentrations of bark extract (10 to 30% v/v) and tannin (0.5, 1.0 and 1.5 mg/mL). For in vivo study, fifteen rabbits were experimentally infected with zoospores of P. insidiosum and treated by oral and intralesional applications of bark extract and tannin. Acute toxicity tests with both substances were also performed in rats. RESULTS: In vitro studies showed fungicidal effect for both substances at different concentrations and the SEM showed alteration on the cell wall surface of the pathogen. All infected rabbits developed a firm nodular mass that reached around 90 mm2 ninety days after inoculation, but neither the intralesional inoculation of tannin, nor the oral administration of crude extract and tannin were able to promote remission of the lesions. CONCLUSIONS: Lesions developed by rabbits presented an encapsulated abscess being quite different of naturally acquired pythiosis, which is characterized by ulcerated lesions. Since no toxicity was observed in rats or rabbits inoculated with these products, while in vitro experiments showed direct antifungal effect, therapeutic activity of S. adstringens and tannin should be clinically tested as an alternative for healing wounds in naturally acquired pythiosis.

Paracoccidioidomycosis: current perspectives from Brazil

BACKGROUND: This review article summarizes and updates the knowledge on paracoccidioidomycosis. P lutzii and the cryptic species of P. brasiliensis and their geographical distribution in Latin America, explaining the difficulties observed in the serological diagnosis. OBJECTIVES: Emphasis has been placed on some genetic factors as predisposing condition for paracoccidioidomycosis. Veterinary aspects were focused, showing the wide distribution of infection among animals. The cell-mediated immunity was better characterized, incorporating the recent findings. METHODS: serological methods for diagnosis were also compared for their parameters of accuracy, including the analysis of relapse. RESULTS: Clinical forms have been better classified in order to include the pictures less frequently observesiod. CONCLUSION: Itraconazole and the trimethoprim-sulfamethoxazole combination was compared regarding efficacy, effectiveness and safety, demonstrating that azole should be the first choice in the treatment of paracoccidioidomycosis.

Pythium insidiosum: inhibitory effects of propolis and geopropolis on hyphal growth

Abstract Propolis and geopropolis are resinous products of bees showing antimicrobial effects. There is no data concerning their action against Pythium insidiosum - the causative agent of pythiosis, a pyogranulomatous disease of the subcutaneous tissue that affects mostly horses, dogs and humans. Fragments of 15 isolates of P. insidiodum were incubated with propolis and geopropolis extracts and evaluated for up to seven days to detect the minimal fungicidal concentration (MFC). Propolis inhibited three isolates at 1.0 mg mL-1 after 24 h and all other isolates at 3.4 mg mL-1. Geopropolis led to more variable results, exerting predominantly a fungistatic action than a fungicidal one. Propolis was more efficient than geopropolis in inhibiting P. insidiosum since lower concentrations led to no growth after 24 h. This effect may be due to propolis chemical composition, which has more active compounds than geopropolis. Propolis seemed to be a good candidate for in vivo studies, since treatment with conventional antifungal compounds is difficult in most of the cases, requiring extensive surgical debridement.

Pythium insidiosum: inhibitory effects of propolis and geopropolis on hyphal growth.

Propolis and geopropolis are resinous products of bees showing antimicrobial effects. There is no data concerning their action against Pythium insidiosum - the causative agent of pythiosis, a pyogranulomatous disease of the subcutaneous tissue that affects mostly horses, dogs and humans. Fragments of 15 isolates of P. insidiodum were incubated with propolis and geopropolis extracts and evaluated for up to seven days to detect the minimal fungicidal concentration (MFC). Propolis inhibited three isolates at 1.0mgmL-1 after 24h and all other isolates at 3.4mgmL-1. Geopropolis led to more variable results, exerting predominantly a fungistatic action than a fungicidal one. Propolis was more efficient than geopropolis in inhibiting P. insidiosum since lower concentrations led to no growth after 24h. This effect may be due to propolis chemical composition, which has more active compounds than geopropolis. Propolis seemed to be a good candidate for in vivo studies, since treatment with conventional antifungal compounds is difficult in most of the cases, requiring extensive surgical debridement.

Environmental Mapping of Paracoccidioides spp. in Brazil Reveals New Clues into Genetic Diversity, Biogeography and Wild Host Association.

BACKGROUND: Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques. METHODS/PRINCIPAL FINDINGS: Aerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts. CONCLUSIONS/SIGNIFICANCE: Data may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.

Sporothrix schenckii sensu stricto isolated from soil in an armadillo's burrow.

Sporotrichosis is a polymorphic disease of man and animals caused by traumatic implantation of propagules into the skin and subcutaneous tissue. Pathogenic species includes S. brasiliensis, S. schenckii, S. globosa and S. luriei. The disease is remarkable for its occurrence as sapronoses and/or zoonosis outbreaks in tropical and subtropical areas; although, the ecology of the clinical clade is still puzzling. Here, we describe an anamorphic Sporothrix strain isolated from soil in an armadillo's burrow, which was located in a hyper endemic area of Paracoccidioidomycosis in Brazil. This isolate was identified as S. schenckii sensu stricto (Clade IIa) based on morphological and physiological characteristics and phylogenetic analyses of calmodulin sequences. We then discuss the role of the nine-banded armadillo Dasypus novemcinctus as a natural carrier of Sporothrix propagules to better understand Sporothrix sources in nature and reveal essential aspects about the pathogen's eco-epidemiology.

Photodynamic therapy in Pythium insidiosum - an in vitro study of the correlation of sensitizer localization and cell death.

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen's cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish.

INTRODUCTION: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. METHODS: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. RESULTS: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. CONCLUSIONS: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish / Avaliação da ocorrência de fungos patogênicos em estruturas traumatogênicas de peixes fluviais

INTRODUCTION: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. METHODS: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. RESULTS: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. CONCLUSIONS: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

INTRODUÇÃO: Infecções fúngicas na pele humana (como a esporotricose) podem se manifestar após traumatismos por peixes. O objetivo deste trabalho é procurar fungos patogênicos para o homem em peixes fluviais. MÉTODOS: Extração de arcadas dentárias Serrassalmus maculatus (piranha) e Hoplias malabaricus (traíra), ferrões de Pimelodus maculatus (mandis), raios da nadadeira dorsal de Plagioscion spp. (corvina) e Tilapia spp. para a realização do cultivo em agar Mycosel. Algumas culturas foram submetidas à extração de DNA para a identificação molecular pelo seqüenciamento da região ITS-5.8S do rDNA. RESULTADOS: As culturas mostraram que a maioria das leveduras era Candida spp. e o sequenciamento também permitiu a identificação de Phoma spp. e Yarrowia lipolytica. CONCLUSÕES: Embora a pesquisa para S. schenckii tenha sido negativa, a presença de fungos do gênero Phoma e Candida revela o potencial patogênico desta via de infecção. O gênero Phoma está envolvido em alguns casos de feohifomicoses, micoses subcutâneas causadas por fungos dematiáceos com relatos de infecções em órgãos e sistemas humanos. As estruturas traumatizantes de alguns peixes fluviais apresentam fungos patogênicos e esta pode ser uma importante via de infecção que deve ser considerada em algumas regiões do Brasil, uma vez que há um grande número de pescadores e peixes traumatogênicos.