The Impact of Paracoccidioides spp Infection on Central Nervous System Cell Junctional Complexes.

Paracoccidioidomycosis (PCM), a systemic mycosis caused by the fungus Paracoccidioides spp. is the most prevalent fungal infection among immunocompetent patients in Latin America. The estimated frequency of central nervous system (CNS) involvement among the human immunodeficiency virus (HIV)/PCM-positive population is 2.5%. We aimed to address the impact of neuroparacoccidioidomycosis (NPCM) and HIV/NPCM co-infection on the tight junctions (TJ) and adherens junction (AJ) proteins of the CNS. Four CNS formalin-fixed paraffin-embedded (FFPE) tissue specimens were studied: NPCM, NPCM/HIV co-infection, HIV-positive without opportunistic CNS infection, and normal brain autopsy (negative control). Immunohistochemistry was used to analyze the endothelial cells and astrocytes expressions of TJ markers: claudins (CLDN)-1, -3, -5 and occludin; AJ markers: ß-catenin and E-cadherin; and pericyte marker: alpha-smooth muscle actin. FFPE CNS tissue specimens were analyzed using the immunoperoxidase assay. CLDN-5 expression in the capillaries of the HIV/NPCM coinfected tissues (mixed clinical form of PCM) was lower than that in the capillaries of the HIV or NPCM monoinfected (chronic clinical form of PCM) tissues. A marked decrease in CLDN-5 expression and a compensatory increase in CLDN-1 expression in the NPCM/HIV co-infection tissue samples was observed. The authors suggest that Paracoccidioides spp. crosses the blood-brain barrier through paracellular pathway, owing to the alteration in the CLDN expression, or inside the macrophages (Trojan horse).

Geographic distribution of patients affected by Cryptococcus neoformans/Cryptococcus gattii species complexes meningitis, pigeon and tree populations in Southern Brazil.

Cryptococcal meningitis is mainly caused by members of the C. neoformans/C. gattii species complexes. The ecological niches of Cryptococcus species have extensively been studied, but its epidemiological relationship with meningitis cases is still unknown. In this study, we estimate the relationship between cryptococcal meningitis cases and tree and pigeon populations, the classical niches of members of C. neoformans/C. gattii sensu lato. We analysed the records of every patient whose cerebrospinal fluid culture yielded Cryptococcus spp. during the last 30 years at Clinical Hospital of Curitiba. Data about Curitiba's pigeon and tree distribution were obtained from Curitiba's Secretaries of Zoonosis and Environment archives. We used ArcGis9 software to plot the distribution of the pigeon and tree populations in this city as well as cryptococcal meningitis cases, distinguishing them according to the causal agent in C. neoformans or C. gattii s.l. In total, 489 cryptococcal cultures were documented, with 140 corresponding to patients eligible for this study (134 affected by C. neoformans s.l. and 6 by C. gattii s.l.). The map showed a relationship between C. neoformans s.l. patients and pigeon population. C. gattii s.l. patients were associated with neither tree nor pigeon populations, but lived close to large unbuilt, unforested areas.

Echinocandin resistance in two Candida haemulonii isolates from pediatric patients.

We report 3 cases of patients with Candida haemulonii isolates that were obtained from hemocultures. In 2 of the 3 cases, isolates exhibited resistance to echinocandins and fluconazole. This is the first report of an echinocandin-resistant species of this fungus in pediatric patients.

Paracoccidioidomycosis in a dog: case report of generalized lymphadenomegaly.

Paracoccidioidomycosis (PCM) is a severe systemic mycosis, endemic in Latin America and highly prevalent in Brazil, where it ranks eighth as a mortality cause among infectious and parasitic diseases in humans. The disease in animals has been little explored. It is observed that armadillos can harbor the fungus at high frequencies, although the active disease has not been well documented in this wild mammal. Dogs are susceptible to experimental infection, and the naturally acquired PCM-disease was reported only recently in a dog from Brazil. The present work reports the second case of naturally acquired PCM in a 6-year-old female dog that presented emaciation, lymphadenomegaly, and hepatosplenomegaly. Biochemical and pulmonary radiographic evaluation did not reveal any abnormalities. PCM was diagnosed by clinical findings, culturing, immunohistochemistry, and histopathology of popliteal lymph node. The fungus was recovered from popliteal lymph node, and the molecular analysis showed respective sequencing similarities of 99 and 100% for 803 nucleotides of the Gp43 gene and 592 nucleotides from the ITS-5.8S region of Paracoccidioides brasiliensis. Immunohistochemistry revealed severe lymphadenitis and presented numerous yeasts, which reacted against the gp43 antibody. Histopathology revealed a severe granulomatous lymphadenitis associated with numerous single or multiple budding yeasts. After diagnosis, the dog was successfully treated with itraconazol for 2 years. Veterinarians should be aware of the importance of considering PCM for differential diagnosis, especially in dogs from PCM-endemic areas, whose monophagocytic system involvement is evident.

A Model for Trans-Kingdom Pathogenicity in Fonsecaea Agents of Human Chromoblastomycosis.

The fungal genus Fonsecaea comprises etiological agents of human chromoblastomycosis, a chronic implantation skin disease. The current hypothesis is that patients acquire the infection through an injury from plant material. The present study aimed to evaluate a model of infection in plant and animal hosts to understand the parameters of trans-kingdom pathogenicity. Clinical strains of causative agents of chromoblastomycosis (Fonsecaea pedrosoi and Fonsecaea monophora) were compared with a strain of Fonsecaea erecta isolated from a living plant. The clinical strains of F. monophora and F. pedrosoi remained concentrated near the epidermis, whereas F. erecta colonized deeper plant tissues, resembling an endophytic behavior. In an invertebrate infection model with larvae of a beetle, Tenebrio molitor, F. erecta exhibited the lowest survival rates. However, F. pedrosoi produced dark, spherical to ovoidal cells that resembled muriform cells, the invasive form of human chromoblastomycosis confirming the role of muriform cells as a pathogenic adaptation in animal tissues. An immunologic assay in BALB/c mice demonstrated the high virulence of saprobic species in animal models was subsequently controlled via host higher immune response.

Risk factors for candidemia mortality in hospitalized children.

OBJECTIVE: To evaluate risk factors associated with death due to bloodstream infection caused by Candida spp. in pediatric patients and evaluate the resistance to the main anti-fungal used in clinical practice. METHODS: This is a cross-sectional, observational, analytical study with retrospective collection that included 65 hospitalized pediatric patients with bloodstream infection by Candida spp. A univariate analysis was performed to estimate the association between the characteristics of the candidemia patients and death. RESULTS: The incidence of candidemia was 0.23 cases per 1000patients/day, with a mortality rate of 32% (n=21). Clinical outcomes such as sepsis and septic shock (p=0.001), comorbidities such as acute renal insufficiency (p=0.01), and risks such as mechanical ventilation (p=0.02) and dialysis (p=0.03) are associated with increased mortality in pediatric patients. The resistance and dose-dependent susceptibility rates against fluconazole were 4.2% and 2.1%, respectively. No resistance to amphotericin B and echinocandin was identified. CONCLUSION: Data from this study suggest that sepsis and septic shock, acute renal insufficiency, and risks like mechanical ventilation and dialysis are associated with increased mortality in pediatric patients. The mortality among patients with candidemia is high, and there is no species difference in mortality rates. Regarding the resistance rates, it is important to emphasize the presence of low resistance in this series.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis.

Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15-25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile.

In vitro susceptibility and molecular characterization of Candida spp. from candidemic patients.

BACKGROUND: Candida species are the main cause of hospital acquired fungal bloodstream infections. The main risk factors for candidemia include parenteral nutrition, long-term intensive care, neutropenia, diabetes, abdominal surgery and the use of central venous catheters. The antifungal drugs used to treat candidemia are mainly the echinocandins, however some isolates may be resistant to these drugs. AIMS: This work aims to evaluate the in vitro susceptibility patterns of various Candida species isolated from blood samples and provide their identification by molecular characterization. METHODS: Antifungal susceptibility testing was performed using the broth microdilution method. The sequencing of the ITS and D1/D2 regions of rDNA was used for molecular characterization. RESULTS: Seventy-four of the 80 isolates were susceptible to anidulafungin, 5 were intermediate, and 1 was resistant. For micafungin 67 were susceptible, 8 were intermediate and 5 were resistant. All isolates were susceptible to amphotericin B. Lastly, 65 isolates were susceptible to fluconazole, 8 were dose-dependent and 4 were resistant. The molecular identification corroborated the phenotypic data in 91.3% of the isolates. CONCLUSIONS: Antifungal susceptibility data has an important role in the treatment of candidemia episodes. It was also concluded that the molecular analysis of isolates provides an accurate identification and identifies genetic variability within Candida species isolated from patients with candidemia.

Risk factors for candidemia mortality in hospitalized children / Fatores de risco de mortalidade por candidemia em crianças internadas

Abstract Objective: To evaluate risk factors associated with death due to bloodstream infection caused by Candida spp. in pediatric patients and evaluate the resistance to the main anti-fungal used in clinical practice. Methods: This is a cross-sectional, observational, analytical study with retrospective collection that included 65 hospitalized pediatric patients with bloodstream infection by Candida spp. A univariate analysis was performed to estimate the association between the characteristics of the candidemia patients and death. Results: The incidence of candidemia was 0.23 cases per 1000 patients/day, with a mortality rate of 32% (n = 21). Clinical outcomes such as sepsis and septic shock (p = 0.001), comorbidities such as acute renal insufficiency (p = 0.01), and risks such as mechanical ventilation (p = 0.02) and dialysis (p = 0.03) are associated with increased mortality in pediatric patients. The resistance and dose-dependent susceptibility rates against fluconazole were 4.2% and 2.1%, respectively. No resistance to amphotericin B and echinocandin was identified. Conclusion: Data from this study suggest that sepsis and septic shock, acute renal insufficiency, and risks like mechanical ventilation and dialysis are associated with increased mortality in pediatric patients. The mortality among patients with candidemia is high, and there is no species difference in mortality rates. Regarding the resistance rates, it is important to emphasize the presence of low resistance in this series.

Resumo Objetivo: Avaliar os fatores de risco associados ao óbito por infecção da corrente sanguínea causada pela Candida spp em pacientes pediátricos e avaliar a resistência ao principal antifúngico usado na prática clínica. Métodos: Este é um estudo transversal, observacional e analítico com coleta retrospectiva que incluiu 65 pacientes pediátricos internados com infecção da corrente sanguínea por Candida spp. Foi feita uma análise univariada para estimar a associação entre as características dos pacientes com candidemia e o óbito. Resultados: A incidência de candidemia foi de 0,23 casos em cada 1.000 pacientes/dia, com taxa de mortalidade de 32% (n = 21). O resultado clínico como sepse e choque séptico (p = 0,001), comorbidades como insuficiência renal aguda (p = 0,01) e riscos como ventilação mecânica (p = 0,02) e diálise (p = 0,03) estão associados ao aumento da mortalidade em pacientes pediátricos. As taxas de resistência e susceptibilidade dose-dependente contra o fluconazol foram de 4,2% e 2,1%, respectivamente. Não foi identificada resistência à anfotericina B e equinocandina. Conclusão: Os dados de nosso estudo sugerem que a sepse e o choque séptico, a insuficiência renal aguda e riscos como ventilação mecânica e diálise estão associados ao aumento da mortalidade em pacientes pediátricos. A mortalidade entre pacientes com candidemia é alta e não há diferença nas taxas de mortalidade entre as espécies. Sobre a resistência, é importante enfatizar a presença de baixa resistência nesta série.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis

Abstract Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15–25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile.

Cryptococcus neoformans Isolated from Passerine and Psittacine bird excreta in the state of Paraná, Brazil.

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.

Pesquisa de Cryptococcus neoformans e Candida spp. em excretas de psitacídeos e passeriformes cativos / Research on Cryptococcus neoformans and Candida spp. in the excretas of captive psittacisittacines and passeriformes / Investigación de Cryptococcus neoformans y Candida spp. en excretas de psittacidae y psittaciformes cautivos

As leveduras podem causar diversas doenças no homem e animais. Nas aves, as leveduras estão envolvidas principalmente em lesões no trato respiratório e digestório. Entre as leveduras patogênicas, Cryptococcus neoformans vem se destacando pela alta prevalência de criptococose humana em pacientes imunodeprimidos. Assim, os objetivos deste estudo foram identificar C. neoformans e outras leveduras patogênicas na cloaca e coana de passeriformes e psitaciformes e em excretas coletadas do fundo de gaiolas de aviários. Foram obtidas 29 amostras de 15 aves manifestando algum sinal respiratório, provenientes do Ambulatório de Animais Selvagens da UFPR (n=6) e da Clínica Veterinária Vida Livre (n= 23). As amostras foram semeadas em Ágar Sabouraud e Ágar Níger e mantidas a 300ºC por até 30 dias. Todas as colônias foram analisadas quanto à macro e micromorfologia. Para aquelas identificadas como leveduras, foram realizadas as provas bioquímicas: assimilação de carbono e nitrogênio e formação de tubo germinativo para identificação de Candida albicans. As amostras de excreta dos aviários (n=8) foram misturadas com solução fisiológica contendo antibiótico e o sobrenadante foi semeado em Ágar Níger. Nenhuma amostra das aves apresentou resultado positivo para C. neoformans, porém identificaram-se amostras positivas para C. albicans (duas amostras de coana), C. famata (uma amostra de coana) e C. tropicalis (uma amostra de coana). As excretas foram negativas para C. neoformans. Portanto, apesar de não ter sido isolado C. neoformans, outras leveduras patogênicas foram isoladas, demonstrando a importância dessas aves como possíveis veiculadoras de doenças para humanos.

The yeasts can cause many diseases in man and animals. On birds, the yeasts are involved mainly in respiratory and digestive tract lesions. Among pathogenic yeast, Cryptococcus neoformans is an important cause of human cryptococcisis associated with immunocompromised states. The purpose of this study is to identify the occurrence of C. neoformans and other pathogenic yeasts in cloacae and choana from passeriformes and psittacines as well as in excretas from poultry cages. Twenty nine samples from fifteen birds showing some respiratory symptom, from Veterinary Hospital of UFPR (n = 6) and Vida Livre Veterinary Clinic (n = 23), were collected. The samples were spread in Sabouraud dextrose Agar and Staib medium and kept at 30°C and observed for 30 days. All colonies were analyzed with respect to its micro and macromorphology. Biochemical assays were conducted for samples presenting yeasts: carbon and nitrogen assimilation profile and germ tube for Candida albicans identification. Samples from birds’ extracts (n = 8) were diluted in sterile saline solution with antibiotic and the supernatant was inoculated in spread on Niger seed agar. All samples were negative for Cryptococcus neoformans, however, C. albicans (two samples from choana), C. famata (one sample from choana) and C. tropicalis (choana) were found. Excretas from bird cages were negative to C. neoformans. Results suggested that birds harbor various pathogenic species of yeast, but not C. neoformans, and the result showed potential danger to carry diseases to humans.

Las levaduras pueden causar diversas enfermedades en el hombre y animales. En las aves, las levaduras están involucradas principalmente en lesiones en el tracto respiratorio y digestivo. Entre las levaduras patogénicas, Cryptococcus neoformans viene destacándose por la alta incidencia de cryptococcus humana en pacientes inmune deprimidos. Así, el objetivo de este estudio fueron identificar C. neoformans y otras levaduras patogénicas en la cloaca y coana de psittacidae y psittaciformes y en excretas colectadas de las jaulas de pajareras. Fueron obtenidas 29 muestras de quince (15) aves manifestando algún señal respiratorio, provenientes del Ambulatorio de Animales Salvajes de la UFPR (n=6) y de la Clínica Veterinaria Vida Livre (n= 23). Las muestras fueron sembradas en Ágar Sabouraud y Ágar Níger y mantenidas a 30ºC hasta 30 días. Todas las colonias fueron analizadas cuanto a la macro y micromorfología. Para aquellas identificadas como levaduras, fueron realizadas las pruebas bioquímicas: asimilación de carbono y nitrógeno, y formación de tubo germinativo para identificación de Candida albicans. Las muestras de excreta de los pajareros (n=8) fueron mezcladas con solución fisiológica conteniendo antibiótico y el sobrenadante fue sembrado en Ágar Níger. Ninguna muestra de las aves presentó resultado positivo para C. neoformans, pero se identificaron muestras positivas para C. albicans (dos muestras de coana), C. famata (una muestra de coana) y C. tropicalis (una nuestra de coana). Las excretas fueron negativas para C. neoformans. Por lo tanto, a pesar de no haber sido aislado C. neoformans, otras levaduras patogénicas fueron aisladas, demostrando que esas aves son posibles transmisoras de enfermedades para los seres humanos.