Postpartum histoplasmosis in an HIV-negative woman: a case report and phylogenetic characterization by internal transcribed spacer region analysis

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.

Identificación molecular de Aspergillus fumigatus aislados de pacientes con aspergilosis invasiva / Molecular identification of Aspergillus fumigatus isolated from patients with invasive aspergillosis

RESUMEN El objetivo del estudio fue identificar molecularmente cepas de aspergillus aislados de pacientes con aspergilosis invasiva (AI), que fueron tipificadas primariamente como Aspergillus fumigatus sensu lato por métodos fenotípicos convencionales. Se trabajó con 20 cepas de la micoteca de la sección de micología del Instituto de Medicina Tropical "Daniel A. Carrión". Para obtener el ADN fúngico se emplearon las técnicas de choque térmico, tratamiento enzimático y columnas de silica-gel; y se almacenó a -20 0C para conservarlo. En el procedimiento de la reacción en cadena de la polimerasa en tiempo real (qPCR) se incluyeron primers marcados con fluorocromo, los cuales amplificaron las secuencias específicas de A. fumigatus. La fluorescencia se midió con el termociclador al final de la fase de hibridación de cada ciclo. Se identificó molecularmente que sólo el 50% de las cepas estudiadas pertenecen a la especie Aspergillus fumigatus sensu stricto.

ABSTRACT The objective of the study was to identify molecularly-isolated strains of Aspergillus from patients with invasive aspergillosis (IA); these strains were primarily typed as Aspergillus fumigatus sensu lato by conventional phenotypic methods. We worked with 20 strains from the mycology section of the Institute of Tropical Medicine "Daniel A. Carrión." To obtain the fungal DNA, thermal shock, enzymatic treatment, and silica gel column techniques were used; and it was stored at -20°C to preserve it. The real-time polymerase chain reaction (qPCR) procedure included fluorochrome-labeled primers, which amplified the specific sequences of A. fumigatus. Fluorescence was measured with the thermocycler at the end of the hybridization phase of each cycle. It was molecularly-identified that only 50% of the strains studied belong to the species Aspergillus fumigatus sensu stricto.

Cryptococcal meningitis epidemiology: 17 years of experience in a State of the Brazilian Pantanal

Abstract INTRODUCTION: This study aimed to describe cryptococcal meningitis (CM) cases and the associated demographic, clinical, and microbiological data obtained from cities in the State of Mato Grosso do Sul in the Midwestern region of Brazil. METHODS: The data from 129 patients with laboratory-confirmed CM admitted from 1997 to 2014 were retrospectively reviewed. The molecular types of Cryptococcus neoformans and Cryptococcus gattii isolated from cerebrospinal fluid were analyzed to determine their geographic distribution. RESULTS: The patients had a mean age of 37 years and consisted mostly of men (76.7%). Most of the Cryptococcus isolates were obtained from patients infected with human immunodeficiency virus (HIV) and included 105 (87.5%) and 5 (55.6%) isolates of C. neoformans and C. gattii complexes, respectively. A restriction fragment length polymorphism (RFLP) analysis of URA5 revealed that most of the isolates were C. neoformans molecular type VNI (89.1%), whereas the molecular types VGII (7%) and VNII (3.9%) were observed less frequently. Notably, 65% of the cases with a time from symptom onset to laboratory diagnosis of more than 60 days resulted in fatalities, and sequelae were observed among the patients who survived. CONCLUSIONS: The present study documents the occurrence of neurocryptococcosis, which is mainly caused by C. neoformans VNI, in Mato Grosso do Sul, Brazil, with probable autochthonous cases in the Brazilian Pantanal, the world's largest tropical wetland, and a biome where cryptococcosis has not yet been explored.

Evaluación del Aspergillus lateral flow device para el diagnóstico de aspergilosis invasora, experiencia en un hospital universitario / Evaluation of the lateral flow Aspergillus assay for the diagnosis of invasive aspergillosis, experience in a university hospital

Resumen Introducción: El diagnóstico de aspergilosis invasora (AI) se realiza mediante criterios clínicos y microbiológicos los que incluyen marcadores séricos. Recientemente, el test inmunocromatográfico Aspergillus lateral flow device (LFD), ha sido evaluado como método para diagnóstico de AI. Objetivo: Evaluar el desempeño de este test para el diagnóstico de AI. Material y Método: Estudio transversal en que se evaluaron muestras de suero y lavado bronco-alveolar (LBA) procesadas para galactomanano provenientes de pacientes adultos con sospecha de AI, atendidos en el Hospital Clínico de Red de Salud UCCHRISTUS. Resultados: Se procesó un total de 142 muestras de 98 pacientes, correspondientes a AI probada 5,6%, AI probable 41,5%, AI posible 12,7% y ausencia de AI 40,1%. Al confrontar los resultados con las categorías diagnósticas según criterios EORTC/MSG se obtuvo una sensibilidad y especificidad de LFD para diagnóstico de AI de 70,9 y 53,5% para muestras de suero y 83,3 y 38,5% para muestras de LBA. La concordancia entre galactomanano y LFD fue de 62,4% (54,1-69,9) con un índice Kappa de 0,202 (0,03682-0,3669). Conclusiones: Aspergillus LFD presentó una adecuada sensibilidad; sin embargo, la especificidad fue baja por lo que un resultado positivo requiere ser confirmado.

Background: The incidence of invasive aspergillosis is increasing. Its diagnosis is based on clinical and microbiological criteria which include the determination of serological markers such as galactomannan. Recently, the Aspergillus lateral flow device, an inmunocromatograph assay has been described for its diagnosis. Aim: To evaluate the performance of the lateral flow device for the diagnosis of invasive aspergillosis (IA) in adult patients. Material and Method: In this cross-sectional study, frozen samples that had been previously evaluated for galactomannan from patients classified with proven/probable/possible or no AI according to the EORTC/MSG criteria were selected. Results: A total of 142 samples from 98 patients were processed, corresponding to proven AI 5.6%, probable IA 41.5%, possible IA 12.7% and no-IA 40.1%. The sensitivity and specificity of the Aspergillus lateral flow was 70.9% and 53.5% for serum samples and 83.3% and 38.5% for BAL samples. The concordance between the galactomannan and Aspergillus lateral flow was 62.4% (54.1 - 69.9) with a Kappa index of 0.202 (0.03682 - 0.3669). Conclusions: We observed a good sensitivity but low specificity, a positive result need a confirmatory test.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.

The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital

ABSTRACT Cryptococcosis, a systemic disease caused by the fungus Cryptococcus neoformans/ Cryptococcus gattii is more severe in immunocompromised individuals. This study aimed to analyze the epidemiology of the disease, the molecular characteristics and the antifungal susceptibility of C. neoformans isolated from patients treated in a Brazilian university hospital. This retrospective study was conducted in the Clinical Hospital, Federal University of Uberlândia, and evaluated cases of cryptococcosis and strains of C. neoformans isolated from 2004 to 2013. We evaluated 41 patients, 85% of whom were diagnosed with AIDS. The fungus was isolated from the cerebrospinal fluid (CSF) of 21 patients (51%); 19.5% had fungemia and in 24% the agent was isolated from the CSF and blood, concurrently. Meningoencephalitis was the most frequent (75%) manifestation of infection. Despite adequate treatment, the mortality of the disease was 58.5%. Most isolates (97.5%) presented the VNI genotype (serotype A, var. grubii) and one isolate was genotyped as C. gattii (VGI); all the isolates were determined as mating type MATa and showed susceptibility to the tested antifungals (fluconazole, voriconazole, amphotericin B and 5-flucytosine). Although AIDS detection rates remain stable, opportunistic infections such as cryptococcosis remain as major causes of morbidity and mortality in these patients.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens

ABSTRACT The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Prevalencia de Encephalitozoon intestinalis y Enterocytozoon bieneusi en pacientes VIH positivos de Maracaibo, Venezuela / Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in HIV positive patients to Maracaibo, Venezuela

Los microsporidios pueden provocar infecciones emergentes y oportunistas en individuos inmunocomprometidos de todo el mundo. Se realizó éste estudio para identificar las especies de microsporidios intestinales presentes en pacientes con VIH-SIDA del Servicio Autónomo Hospital Universitario de Maracaibo (SAHUM). Se recolectaron 50 muestras fecales de individuos con diagnóstico confirmado de VIH durante los años 2007-2008; se obtuvieron las cifras de CD4 de solo 42 pacientes. Las muestras se analizaron mediante PCR separadas para la identificación de Encephalitozoon intestinalis y Enterocytozoon bieneusi. Las especies de microsporidios presentaron un 36% de prevalencia, 10 pacientes presentaron Encephalitozoon intestinalis, 4 Enterocytozoon bieneusi y 4 ambas especies. Se determinó una relación inversamente proporcional y estadísticamente significativa entre el contaje de CD4 y la presencia de microsporidios en la muestra fecal. Es destacable la elevada prevalencia de especies de microsporidios observada en los pacientes VIH estudiados, donde predominó E. intestinalis.

Microsporidioses are considered emerging and opportunistic infections in immunocompromised individuals worldwide. The purpose of this study was to identify the species of intestinal microsporidia in patients with HIV-AIDS from the Servicio Autónomo Hospital Universitario de Maracaibo, Venezuela (SAHUM). Fecal samples were collected from 50 patients with confirmed diagnosis of HIV, during the years 2007 and 2008; the CD4 values were obtained from 42 patients. The samples were analyzed by separate PCRs to identify Encephalitozoon intestinalis and Enterocytozoon bieneusi. Microsporidia species showed a 36% prevalence: ten patients had Encephalitozoon intestinalis, four Enterocytozoon bieneusi and four both species. An inverse and statistically significant relationship between the CD4 count and the presence of microsporidia in the fecal sample was also found. It is remarkable the high prevalence of microsporidia species observed in the HIV patients studied, with a predominance of E. intestinalis.

Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large-Subunit D1/D2, and beta-Tubulin

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.

Molecular analysis and dimorphism of azole-susceptible and resistant Candida albicans isolates / Análise molecular e dimorfismo de isolados de Candida albicans suscetíveis e resistentes aos azólicos

INTRODUCTION: Candida albicans is responsible for superficial or systemic infections known as candidiasis, which may be found in infected tissue as unicellular budding yeasts, hyphae, or pseudohyphae. In this study, the effects of both fluconazole and itraconazole antifungal agents on the hyphal formation and genotypic characterization of C. albicans isolates classified as either susceptible or resistant were investigated. METHODS: The hyphal production of five C. albicans isolates under the action of antifungal agents was investigated by culturing yeast on growth medium and on hyphal induction medium. The genotypic characterization was carried out for 13 isolates of C. albicans using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. RESULTS: The dimorphism analysis showed that the hyphal formation was higher in resistant than in the susceptible isolates to both azoles. The RAPD-PCR method identified the formation of two different groups. In group A, four resistant and two susceptible isolates were clustered, and in group B, one resistant and six susceptible isolates were clustered. CONCLUSIONS: Considering that hyphal formation was higher in resistant isolates in the presence of azole drugs, we confirmed that the hyphal production is closely related to susceptibility to azoles. These drugs may affect the morphogenesis of C. albicans depending on their susceptibility to these drugs. In relation to RAPD-PCR, most resistant isolates classified in group A and susceptible isolates in group B demonstrated that this method presented a similar standard between the two groups, suggesting that by this technique, a strong correlation between genotypes and fluconazole-resistant samples may be found.

INTRODUÇÃO: Candida albicans é responsável por infecções superficiais ou sistêmicas conhecidas como candidíase, encontrada em tecidos infectados na forma de leveduras brotantes unicelulares, hifas ou pseudohifas. Neste estudo, os efeitos de agentes antifúngicos como o fluconazol e o itraconazol sobre a formação de hifas e caracterização genotípica de isolados de C. albicans suscetíveis ou resistentes foram investigados. MÉTODOS: A produção de hifas de cinco isolados de C. albicans, sob a ação de antifúngicos foi investigada pelo cultivo da levedura em meios de crescimento e de indução de hifas. A caracterização genotípica foi realizada para 13 isolados de C. albicans pelo método de RAPD-PCR. RESULTADOS: A análise do dimorfismo mostrou que a formação de hifas foi maior nos isolados resistentes do que nos suscetíveis aos antifúngicos. O método de RAPD-PCR identificou a formação de dois diferentes grupos. No grupo A, foram agrupados quatro isolados resistentes e dois suscetíveis e no grupo B um resistente e seis suscetíveis. CONCLUSÕES: Considerando que a formação hifal foi maior em isolados resistentes na presença de azólicos, concluimos que a produção hifal está muito relacionada a suscetibilidade a estes fámacos. Estes antifúngicos podem alterar a morfologia de C. albicans em dependência da sua suscetibilidade. No método de RAPD-PCR, o encontro da maioria dos isolados resistentes classificados como pertencentes ao grupo A e suscetíveis ao grupo B demonstrou que este método apresentou um padrão semelhante entre os dois grupos, sugerindo que por este método pode ser detectado uma estreita correlação entre genótipos e amostras resistentes ao fluconazol.

Infección diseminada por Penicillium marneffei en un paciente HIV-positivo: Primera observación en la República Argentina / Disseminated infection due to Penicillium marneffei related to HIV infection: First observation in Argentina

Se presenta el primer caso humano de peniciliosis por Penicillium marneffei observado en la República Argentina. El paciente era un joven de 16 años, HIV-positivo, procedente de un área rural del sur de China. El paciente fue internado en el Hospital "F. J. Muñiz" por padecer una neumonía grave con insufciencia respiratoria aguda. El agente causal fue aislado de un lavado broncoalveolar y se lo observó en un citodiagnóstico de piel. La identifcación de P. marneffei fue confrmada por las características fenotípicas del aislamiento y la amplifcación del ADNr. El enfermo padecía una infección muy avanzada por HIV que condujo a la aparición simultánea de infecciones por citomegalovirus, Pneumocystis jirovecii y procesos bacterianos nosocomiales. Este complejo cuadro derivó en una evolución fatal.

The frst case observed in Argentina of AIDS-related human penicillosis is herein presented. The patient was a six- teen year-old young man coming from a rural area of southern China. He was admitted at the F. J. Muñiz Hospital of Buenos Aires city with severe pneumonia and adult respiratory distress. Penicillium marneffei was isolated from bronchoalveolar lavage fuid and was microscopically observed in a skin cytodiagnosis. P. marneffei identifcation was confrmed by rRNA amplifcation and its phenotypic characteristics. The patient suffered an advanced HIV infection and also presented several AIDS-related diseases due to CMV, nosocomial bacterial infections and Pneumocystis jirovecii which led to a fatal outcome.

Dipodascus capitatus (Geotrichum capitatum): infecção sistêmica fatal em paciente com leucemia mielocítica aguda / Dipodascus capitatus (Geotrichum capitatum): fatal systemic infection on patient with acute myeloid leukemia

As infecções causadas por Dipodascus capitatus são raras e de difícil tratamento. Aqui se relata um caso em paciente com leucemia mielocítica aguda. O isolamento fúngico ocorreu a partir de hemocultura e a identificação fenotípica baseou-se em métodos micológicos clássicos; a identificação genotípica foi realizada através do sequenciamento da região D1/D2 do 26 rDNA. Os testes de suscetibilidade foram realizados através do Etest® e microdiluição em caldo. A antifungicoterapia foi ineficaz, registrando-se óbito da paciente no 17° dia após o diagnóstico. Os autores comparam o caso com relatos similares e discutem a emergência destas infecções bem como suas dificuldades diagnósticas e terapêuticas.

The infections caused by Dipodascus capitatus are rare, and the treatment is difficult. We reported a case of a patient with acute myeloid leukemia. The fungus was first isolated from hemocultures, and the phenotypic identification was based on mycological methods. The genotyping was carried out by sequencing the region D1/D2 from 26 rDNA. The susceptibility tests were assayed by Etest® and by the microdilution technique. None of the antifungal treatments employed were effective. The patient died on day 17 after the mycological diagnosis. The authors discussed the emergence of such infections as well as the difficulty regarding the diagnosis and treatment.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) on stromata of Cyttaria hariotii in northwestern Patagonian Nothofagus forests

The occurrence and distribution of Xanthophyllomyces dendrorhous associated with Cyttaria hariotii parasitizing three Nothofagus species (N. dombeyi, N. antarctica and N. pumilio) in northwestern Patagonia (Argentina), as well as the factors that may affect this distribution were herein studied. Between 2000 and 2007, samples were obtained from 18 different locations. Based on physiological tests and morphological characteristics of sexual structures, 72 isolates were identified as X. dendrorhous. Representative strains were studied by MSP-PCR fingerprinting and sequence analysis of the ITS region. MSP-PCR fingerprints were similar for the newly isolated strains, and were also identical to the profiles of the strains previously found in this region. Patagonian strains appear to be a genetically uniform and distinct population, supporting the hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. X. dendrorhous was recovered from N. dombeyi and N. antarctica. Approximately half the sampling sites and samples were positive for X. dendrorhous, but the isolation recovery rate was low. X. dendrorhous was absent in the early stages of ascostromata maturation, becoming more abundant in later stages. The present work represents a step forward in the understanding of the natural distribution and ecology of this biotechnologically relevant yeast.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) asociado a estromas de Cyttaria hariotii en bosques de Nothofagus en el noroeste de la Patagonia. Se estudió la ocurrencia y la distribución de Xanthophyllomyces dendrorhous asociado a Cyttaria hariotii en tres especies de Nothofagus (N. dombeyi, N. antarctica y N. pumilio) del noroeste de la Patagonia (Argentina), y los factores que podrían afectar esta distribución. El muestreo se realizó entre 2000 y 2007 en 18 sitios diferentes. Según las pruebas fisiológicas y las características morfológicas de las estructuras sexuales, 72 de los aislamientos obtenidos se identificaron como X. dendrorhous. Se estudiaron cepas representativas mediante la técnica de MSP-PCR fingerprinting y secuenciación de la región ITS. Los perfiles de MSP-PCR fueron similares, tanto entre los nuevos aislamientos como entre estos y los de cepas previamente obtenidas en la región. Aparentemente, las cepas patagónicas forman una población genéticamente uniforme y distinta de otras poblaciones. Esto apoya la hipótesis de que la asociación con diferentes especies hospedadoras ha determinado la diferenciación genética de X. dendrorhous en todo el mundo. X. dendrorhous se recuperó de N. dombeyi y de N. antarctica. Aproximadamente la mitad de los sitios de muestreo y de muestras fueron positivos para X. dendrorhous, pero la tasa de aislamiento fue muy baja. X. dendrorhous está ausente en estadios tempranos de maduración de ascostromas y se hace más abundante en estadios más tardíos. El presente trabajo contribuye al mejor entendimiento de la distribución natural y la ecología de esta levadura, de relevancia biotecnológica.

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.

Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis / 대한진단검사의학회지

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.

A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples / Semi-nested PCR para a detecção molecular de Paracoccidioides brasiliensis em amostras de tecido

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.

INTRODUÇÃO: A paracoccidioidomicose é uma infecção sistêmica causada pelo Paracoccidioides brasiliensis. MÉTODOS: Neste estudo, uma semi-nested PCR foi desenvolvida para o diagnóstico da paracoccidioidomicose. Os oligonucleotídeos iniciadores ITS1 e ITS4 foram usados na primeira reação, enquanto os oligonucleotídeos iniciadores MJ03 e ITS1 foram usados na segunda reação. A semi-nested PCR foi usada para investigar biopsias de cinco pacientes com lesões orais que se assemelhavam a paracoccidioidomicose. RESULTADOS: A semi-nested PCR foi positiva para quatro amostras e negativa para a amostra de um paciente, posteriormente diagnosticado com leishmaniose. CONCLUSÕES: A semi-nested PCR descrita aqui é útil para o diagnóstico da paracoccidioidomicose.

Brote de histoplasmosis en la Escuela de Cadetes de la Base Aérea de Morón, Provincia de Buenos Aires, República Argentina / Histoplasmosis outbreak in Morón, Buenos Aires Province, Argentina

Se describe un brote de histoplasmosis que afectó a 6 cadetes de la Fuerza Aérea Argentina, sin antecedentes patológicos previos. Todos consultaron por problemas respiratorios después de haber limpiado un hangar. En ese recinto se encontraron abundantes deyecciones de animales, presuntamente de palomas y murciélagos. Los pacientes sufrieron fiebre, mialgias, taquipnea y tos no productiva. Las radiografías y tomografías de tórax mostraron imágenes pulmonares micronodulares, engrosamiento de los tabiques interalveolares y adenopatías hiliares. Todos tuvieron una evolución favorable y no requirieron tratamiento antifúngico. Las pruebas de inmunodifusión y contrainmunoelectroforesis con antígenos de Histoplasma capsulatum fueron positivas, al igual que las intradermorreacciones con histoplasmina. Se recogieron 5 muestras de tierra del lugar, las que fueron inoculadas por vía intraperitoneal a 20 hámsteres. De los cultivos de hígado y bazo de dichos animales se consiguió aislar la fase micelial de H. capsulatum. La cepa aislada se comparó con las obtenidas de 12 pacientes argentinos utilizando perfiles genéticos y se observó un clado único con más de 96% de similitud, lo que confirma la homogeneidad de las cepas argentinas. Si bien la histoplasmosis es endémica en la Pampa húmeda, este es el primer brote totalmente documentado al sur del paralelo 34°.

An histoplasmosis outbreak affecting 6 previously healthy Air Force cadets is herein presented. The patients suffered from fever and respiratory symptoms after having cleaned an abandoned hangar soiled with pigeons and bat droppings. They all presented fever, myalgia, tachypnea, and nonproductive cough. Chest X-ray and CT scan studies showed disseminated reticulonodular images affecting both lungs. Hiliar adenomegalies were also observed. All patients achieved a favourable outcome without antifungal treatment. Both serologic tests searching for specificic antibodies (immunodiffusion and counterimmunoelectrophoresis) and histoplasmin skin tests were positive in all cases. Five soil samples mixed with pigeons and bat droppings were collected from the hangar. Suspensions of these samples were inoculated into 20 hamsters by intraperitoneal injection; mycelial phase of H. capsulatum was isolated from liver and spleen cultures. The genetic profile of this strain was compared with 12 isolates obtained from Argentinean patients, and a great degree of homogeneity was observed (> 96% similarity). Although histoplasmosis is endemic in the wet Pampas, this is the first epidemic outbreak reported south of the 34th parallel.

Identificação por PCR e sensibilidade a antifúngicos de isolados clínicos vaginais de Candida sp / Identification by PCR and antifungal susceptibility of vaginal clinical Candida sp isolates

INTRODUÇÃO: A candidíase vaginal é uma condição que afeta um grande número de mulheres em idade fértil. Candida albicans é a espécie mais frequentemente isolada de secreção vaginal, entretanto, outras espécies mais resistentes às drogas antifúngicas podem ser isoladas de amostras clínicas vaginais. MÉTODOS: Foram identificadas as espécies de 30 isolados vaginais de Candida sp por PCR utilizando o primer universal ITS4 e primers espécie-específicos para C. albicans, C. glabrata, C. tropicalis e C. krusei. A sensibilidade destes isolados frente à anfotericina B, fluconazol e voriconazol foi determinada pelo método de macrodiluição M27-A2 do CLSI. RESULTADOS: Através dos ensaios de PCR, 28 isolados foram caracterizados como C. albicans e 2 isolados apresentaram amplificação para os primers específicos de C. albicans e C. glabrata. A concentração inibitória mínima para anfotericina B variou de 0,03µg/mL a 0,25µg/mL, para o fluconazol de 0,125µg/mL a 16µg/mL e para o voriconazol de 0,03µg/mL a 0,25µg/mL. CONCLUSÕES: A identificação de Candida ao nível de espécie através de ensaios de PCR deve ser relevante para o gerenciamento clínico destas infecções.

INTRODUCTION: Vaginal candidiasis is a condition that affects innumerous fertile women. Candida albicans is the most frequently isolated species from vaginal discharge; however, other different species that are more resistant to antifungal drugs can be identified in vaginal clinical samples. METHODS: The species of 30 vaginal Candida isolates was identified by PCR using the universal ITS4 primer and species-specific primers for C. albicans, C. glabrata, C. tropicalis and C. krusei. The sensitivity pattern to amphotericin B, fluconazole and voriconazole was assessed using the CLSI M27-A2 macrodilution method. RESULTS: The PCR assay revealed 28 C. albicans and 2 samples showed amplification for C. albicans and C. glabrata primers. The minimum inhibitory concentration for amphotericin B ranged from 0.03µg/mL to 0.25µg/mL, for fluconazole from 0.125µg/ml to 16µg/mL and for voriconazole from 0.03µg/mL to 0.25µg/m. CONCLUSIONS: Identification at Candida species level by PCR assay could be relevant for clinical management of these infections.