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1.
Med Mycol ; 53(1): 22-7, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25526778

RESUMEN

Sporotrichosis and cutaneous leishmaniasis are skin infections with similar clinical manifestations but different treatment methods. The present study aimed to evaluate qPCR and conventional PCR for differential detection of the etiological agents of both infections in multiplex format. Assays were designed using two sets of reported primers: SS1/SS2, designed on the 18S ribosomal RNA gene from Sporothrix spp., and JW11/JW12, designed on the kinetoplast DNA (kDNA) minicircles of Leishmania spp. qPCR detected 200 fg of DNA per reaction for both Sporothrix and Leishmania. Melting curve analysis revealed two distinctive Tm peaks for Sporothrix spp. (85.5°C), and Leishmania spp. (82.6°C). A detection limit of 20 pg was determined for the diagnosis of both with conventional PCR. No other clinically important organisms were detected by either PCR or qPCR. However, a Blast analysis on GenBank databases, using as query the sequence of the PCR fragment obtained with primers SS1/SS2, showed 100% identity to environmental fungi of the Ophiostomales order. Lower percentages of identity (≤80%), with mismatches at primers' sequence regions were obtained for other environmental or clinically important fungi. Proper handling of clinical samples is required to avoid false negatives due to contamination with environmental fungi of the Ophiostomales order.


Asunto(s)
Leishmania/aislamiento & purificación , Leishmaniasis Cutánea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sporothrix/aislamiento & purificación , Esporotricosis/diagnóstico , ADN de Hongos/genética , ADN de Cinetoplasto/genética , ADN Protozoario/química , Diagnóstico Diferencial , Humanos , Leishmania/clasificación , Leishmania/genética , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Sporothrix/clasificación , Sporothrix/genética , Esporotricosis/microbiología , Esporotricosis/patología
2.
BMC Infect Dis ; 15: 94, 2015 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-25880588

RESUMEN

BACKGROUND: Sporotrichosis is a cutaneous and subcutaneous fungal disease of humans and other mammals, known to be caused by the Sporothrix schenckii species complex, which comprises four species of clinical importance: S. brasiliensis, S. globosa, S. luriei, and S. schenckii sensu stricto. Of them, S. globosa and S. schenckii s. str. show global distribution and differences in global frequency as causal agents of the disease. In the Americas, only three species are present: S. schenckii s. str., S. brasiliensis (so far, only reported in Brazil), and S. globosa. In Venezuela, since the first case of sporotrichosis reported in 1935, S. schenckii have been considered its unique etiological agent. In the present work, the presence of more than one species in the country was evaluated. METHODS: By phenotypic key features and molecular phylogeny analyses, we re-examined 30 isolates from diverse Venezuelan regions belonging to the fungi collection of Instituto de Biomedicina, Caracas, Venezuela, and national reference center for skin diseases. All isolates were collected between 1973 and 2013, and maintained in distilled water. RESULTS: Sporotrichosis in Venezuela is mainly caused by S. schenckii s. str. (70%). However, a significant proportion (30%) of sporotrichosis cases in the country can be attributable to S. globosa. A correlation between intraspecific genotypes and clinical presentation is proposed. CONCLUSIONS: Our data suggest that sporotrichosis various clinical forms might be related to genetic diversity of isolates, and possibly, to diverse virulence profiles previously reported in the S. schenckii species complex. Sporothrix globosa was found to be the causative agent of 30% of sporotrichosis for the Venezuelan cases re-examined, the highest frequency of this species so far reported in the Americas. The high genetic variability presented by S. schenckii s. str. indicates that species distinction based on phenotypic key features could be a challenging and uncertain task; molecular identification should be always employed.


Asunto(s)
ADN de Hongos/análisis , Sporothrix/genética , Esporotricosis/epidemiología , Variación Genética , Genotipo , Humanos , Epidemiología Molecular , Filogenia , Sporothrix/aislamiento & purificación , Esporotricosis/microbiología , Venezuela/epidemiología
3.
PLoS Genet ; 7(10): e1002345, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22046142

RESUMEN

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.


Asunto(s)
Onygenales/genética , Paracoccidioides/genética , Paracoccidioidomicosis/microbiología , Proteínas Quinasas/genética , Metabolismo de los Hidratos de Carbono/genética , Sistemas de Liberación de Medicamentos , Evolución Molecular , Genoma Fúngico , Genoma Mitocondrial/genética , Humanos , Familia de Multigenes/genética , Onygenales/enzimología , Paracoccidioides/enzimología , Filogenia , Proteolisis , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN
4.
Antimicrob Agents Chemother ; 54(12): 5391-4, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20937789

RESUMEN

Five Paracoccidioides brasiliensis isolates were grown in the presence of caspofungin (0 to 1 µg/ml). Inhibition of the yeast phase ranged from 20 to 65%, while in the mycelial form it ranged from 75% to 82%. Such variability was loosely related to the amount of cell wall ß-1,3-glucan. No association with point mutations in the ß-1,3-glucan synthase was detected. Caspofungin induced physical changes and cytoplasmic deterioration in both fungal phases.


Asunto(s)
Antifúngicos/farmacología , Equinocandinas/farmacología , Paracoccidioides/efectos de los fármacos , Proteínas Bacterianas/genética , Caspofungina , Glucosiltransferasas/genética , Lipopéptidos , Micelio/citología , Micelio/efectos de los fármacos , Micelio/genética , Paracoccidioides/citología , Paracoccidioides/genética , Mutación Puntual/genética , Levaduras/citología , Levaduras/efectos de los fármacos , Levaduras/genética
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