Differential identification of Sporothrix spp. and Leishmania spp. by conventional PCR and qPCR in multiplex format.

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.

Molecular epidemiology of human sporotrichosis in Venezuela reveals high frequency of Sporothrix globosa.

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.

[In vitro susceptibility of isolates of Paracoccidioides spp complex to systemic antifungals using the microdilution method]. / Sensibilidad in vitro de aislados del complejo Paracoccidioides spp a antifúngicos sistémicos utilizando el método de microdilución.

Broth microdilution, the reference method recommended by the Clinical Laboratory Standards Institute (CLSI), is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this work, in vitro susceptibility of the Paracoccidioides complex (n=19) to systemic antifungals: amphotericin B, 5-flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole and caspofungin, was evaluated using the microdilution method (Document M27-A3, M27-S3), with some modifications such as: culture time in Sabouraud dextrose agar (7-10 days), RPMI 1640 medium supplemented with 2% glucose and the incubation time (7, 8 and 18 days). The sensitivity in vitro was variable; the majority of Paracoccidioides isolates was susceptible to ketoconazol (73.7%), followed by voriconazole (68.4%), itraconazole (63.1%), amphotericin B (52.6%), fluconazole (47.4%), 5-flucytosine (42.1%) and caspofungin (5%). The overall resistance was mainly to caspofungin (94.7%), followed by 5-flucytosine (52.6%) and amphotericin B (47.4%). Fifty-three percent of the isolates were susceptible-dose dependent to fluconazole followed by itraconazole (15.7%) and 5-fluorocytosine (5.3%). Amphotericin B, itraconazole and voriconazole were the most potent antifungal drugs against Paracoccidioides spp (CMI: 0.03-1 microg/mL). Based on these results, we tentatively propose a microdilution assay protocol for susceptibility testing of Paracoccidioides spp to antifungal drugs. This method may be clinically useful to predict resistance, even though further studies are needed.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium]. / Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Geographical distribution and ecological niche modeling of the etiological agents of human sporotrichosis in Venezuela.

The geographical distribution and ecological niche of the two circulating species of the Sporothrix genus in Venezuela was established. For this, 68 isolates of Sporothrix spp. from patients of different regions of the country were analyzed. A molecular taxonomy analysis was conducted using a fragment of the calmodulin gene (CAL), and ITS regions, confirming the presence of S. schenckii (62%) and S. globosa (38%). Computational models of ecological niche for each species were obtained by the maximum entropy method using the MaxEnt software, which predicted the best environmental conditions for the presence of the two species. These models predict that the main variables influencing the presence of S. schenckii were altitude and annual mean temperature, while for S. globosa, the more influent variable was the land use, with 82% of S. globosa located at urban areas vs 56% for S. schenckii. The results here presented could contribute to understand the specific environmental factors that might modulate the occurrence of Sporothrix spp. as well as its transmission. To our knowledge, our analyses show for the first time Sporothrix spp.-specific ecological niche data, a valuable tool to promote evidence-based public health policymaking within endemic areas of sporotrichosis.

[Isolation of Sporothrix schenckii from environmental samples in Venezuela]. / Aislamiento de Sporothrix schenckii del medio ambiente en Venezuela.

The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis frequently found in Latin America. The isolation of this fungus from the environment and other sources has been widely reported. Nevertheless, to our knowledge this fungus has not been isolated from the endemic areas of Venezuela. In studies related to a clinical case of sporotrichosis in "Colonia Tovar", produced by traumatism after manipulating soil samples, the fungus was isolated from the soil of that particular area. This is the first report of the isolation of S. schenckii from environmental sources in an endemic area of Venezuela.

Detection of Candida dubliniensis in patients with candidiasis in Caracas, Venezuela.

Candida species are responsible for 80% of all nosocomial fungal infections. In 1995 a new yeast species was described, Candida dubliniensis which shares with Candida albicans characteristics. We have studied 109 yeast isolates identified as C. albicans to investigate the presence of C. dubliniensis by microbiological studies and PCR using DUBR/DUBF primers. Positive results using microbiological tools were between 90 and 98%. Two morphological and physiological of the 80 DNA examined samples (2.5%) showed a PCR product of 288 bp which allow the identification of C. dubliniensis. This is the first report in Venezuela of identification of this species using a PCR approach.

[Physiological comportment and in vivo sensitivity of Sporothrix schenckii isolates maintained for 18 years by two preservation methods]. / Comportamiento fisiológico y de sensibilidad in vitro de aislamientos de Sporothrix schenckii mantenidos 18 años por dos métodos de preservación.

We compared two methods for the preservation of fungi, Castellani's method and repeated passage in Sabouraud medium-agar, on five isolates of Sporothrix schenckii that were preserved for 18 years at room temperature by both procedures. They were evaluated for viability of the strains, growth rate, morphological and physiological characteristics, and in vitro sensitivity to iodide, itraconazole, terbinafine and posaconazole. 100% viability was observed in all of the isolates, with slower growth rate on strains preserved in water compared to strains periodically re-cultured. The typical morphological feature of these fungi was preserved by both methodologies. With regard to enzymatic activity, both groups gave urease reactions and were beta glucosidase-positive. Nevertheless, complete inhibition of the capacity to hydrolyse starch was observed only on the isolates preserved in water. This group also was more sensitive to potassium iodide at a concentration of 10 microM in the in vitro sensitivity tests.

[Superficial mycoses: comparative study between type 2 diabetic patients and a non-diabetic control group]. / Frecuencia de micosis superficiales: estudio comparativo en pacientes diabéticos tipo 2 y en individuos no diabéticos.

Superficial mycoses are considered to affect more frequently patients with type 2 diabetes mellitus (DM-2), specially onychomycosis and Tinea pedis. The purpose of this study was to compare the dermatophytoses, candidiasis and Pitiriasis versicolor frequency between 40 patients with DM-2 and 40 healthy persons of either sex, 40 years old or more. Clinical, metabolic, mycologic and inmunologic studies against Candida albicans, were carried out. Both diabetics 75% (30/40) and controls 65% (26/40) presented a high frequency of superficial mycoses (no significant difference p = 0.329). Pitiriasis versicolor was not detected in diabetic patients. They presented Tinea unguium, concomitant with Tinea pedis, with a higher frequency. The predominant dermatophyte was Trichophyton rubrum 18/23 (78%) in diabetics and 8/16 (50%) in non diabetics. Candida was isolated as commensal from oral mucous: 23/40 (58%) in diabetics and 21/40 (52%) in non diabetics (serotipo A was the more frequent), and from onychomycosis: 11/40 (28%) in diabetics and 12/40 (30%) in non diabetics. The immunological response was the same in both groups: celular 100%, humoral 20%. No statistical correlation among superficial mycoses, blood glucose level, glycosylated hemoglobin values or the time suffering the disease was observed. The high susceptibility to dermatophytes and Candida sp. infection showed to be associated with age and no with the diabetic type 2 condition in those patients.

[Mycoses in Venezuela: Working Groups in Mycology reported cases (1984-2010)]. / Las micosis en Venezuela: casuística de los Grupos de Trabajo en Micología (1984-2010).

BACKGROUND: In 1984 the Venezuelan Work Groups in Mycology (VWGM) were created introducing an innovative approach to the study of the mycoses in Venezuela. AIM: To study the occurrence of the mycoses in Venezuela. METHODS: Review the reported cases of mycoses by the newsletter Boletín Informativo Las Micosis en Venezuela (VWGM) from 1984 to 2010. RESULTS: The data collected showed 36,968 reported cases of superficial mycoses, 1,989 of deep systemic cases, and 822 of localized mycoses. Pityriasis dermatophytosis was the most common superficial infection, and paracoccidioidomycosis and histoplasmosis the most frequent deep systemic infection. Chromoblastomycosis was the most frequently diagnosed subcutaneous infection. The data provided showed the distribution by geographical area for each of the fungal infections studied, which may help to establish the endemic areas. DISCUSSION: Superficial mycosis is a public health problem due to its high morbidity and is probably responsible for some of the outbreaks in high-risk groups. Paracoccidioidomycosis and histoplasmosis were reported more often, which agrees with earlier reports prior to the formation of the VWGM. Cases of sporotrichosis and chromoblastomycosis in Venezuela can be considered unique due to the high number of cases. This study highlights the contribution of the VWGM to the behavior of the mycoses in Venezuela, its incidence, prevalence, and the recognition of these infections as a problem of public health importance. The VWGM should keep working in this endeavor, not only reporting new cases, but also unifying the clinical and epidemiological criteria, in order to properly monitor the evolving epidemiological changes reported in these types of infections.

[Technical evaluation of nested PCR for the diagnosis of experimental sporotrichosis]. / Evaluación de la técnica PCR anidada para el diagnóstico de la esporotricosis experimental.

BACKGROUND: Sporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis. AIM: To determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures. METHODS: BALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure. RESULTS: The pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice. CONCLUSIONS: In the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.

Brote de histoplasmosis aguda en escolares de el Mayar, Estado Sucre, Venezuela / Outbreak of acute histoplasmosis in schoolchildren from El Mayar, Sucre State, Venezuela

Introducción: La histoplasmosis es una infección sistémica causada por un hongo dimorfo, Histoplasma capsulatum. Crece en suelos que contienen grandes cantidades de excretas tanto de aves como de murciélagos, en las proximidades del suelo de los gallineros y alrededor de árboles que albergan aves y murciélagos, en el interior o alrededor de las cuevas. La aerosolización de las microconidias residentes en estos suelos es la causante de las manifestaciones de la enfermedad Objetivo: Dar a conocer un brote de esta enfermedad relacionado con una actividad de remoción de suelos en espacio abierto. Método: Estudio observacional descriptivo de reporte de serie de casos que se inició con la identificación del caso índice. Se realizaron tomas de muestras tanto a los niños del colegio como al personal docente, así como de las fuentes de contagio común. Resultados: Se identificaron 7 niños, edad promedio de 11 años, predominantemente del sexo femenino y sin enfermedad predisponente condicionante. El diagnóstico de la enfermedad se realizó por estudio de material clínico (esputo, médula ósea, tejido pulmonar) con coloraciones especiales. Se aisló Histoplasma capsulatum del suelo donde estos niños residen y donde se desarrolló la actividad de remoción y rastrillo. Se estimó una tasa de ataque del 58 % % con una letalidad del 14 %. Conclusión: Se ha realizado el primer reporte para el país de un brote de histoplasmosis en espacio abierto relacionado con remoción y rastrillo de suelo, reconociendo un área de endemicidad previamente no determinada.

Introduction: Histoplasmosis is a systemic infection caused by a dimorphic fungus, Histoplasma capsulatum. It grows in soils with the consequent contamination of large amounts of birds and bats excreta, near barn soils and around trees that harbor birds and bats, and also inside or around caves. Aerosolization of microconidia residents in these soils is the cause of the transmision of the disease Objective: To report an outbreak of this disease related to an activity of soil removal in an open space. Methods: This is a descriptive observational study of a case series that began with the identification of the index case. Samples were obtained from school children and teachers, as well as from common sources of infection. Results: 7 children, predominantly females with an average age of 11 years, with no predisposing disease were included. Diagnosis was performed by special colorations of sputum, bone marrow and lung tissue. Histoplasma capsulatum was isolated from the soil where these children reside and where the activity of removal and rake was performed. An attack rate of 58% was estimated with a fatality rate of 14%. Conclusion: This was the first report in Venezuela of an outbreak of histoplasmosis in open space associated with soil removal and rake, recognizing an area of endemicity previously undetermined.

Evaluación morfológica y bioquímica de aislados clínicos de Trichophyton spp / Morphological and biochemical evaluation of clinical isolates of Trichophyton spp

Debido al pleomorfismo y variabilidad macroscópica que presenta el género Trichophyton, 1os métodos de identificación basados exclusivamente en caracteres morfológicos no son suficientes para su clasificación. El objetivo fue evaluar las características morfológicas y bioquímicas de aislados clínicos identificados como Trichophyton spp. Se estudiaron 98 cultivos, identificados previamente por macro y micromorfología y se reevaluaron por: microcultivo en lámina, perforación del pelo in vitro, hidrólisis de urea y uso de agares Trichophyton y BCP-MS-G. El análisis morfológico y las pruebas de hidrólisis de urea y perforación del pelo arrojaron 51% T. rubrum, 24% T. mentagrophytes, 16,6% T. interdigitale, 7% T. tonsurans y 2% Trichophyton spp. La prueba de urea evidenció un error de 9,4%. La evaluación mediante el BCP-MS-G permitió diferenciar T. rubrum tipo A algodonoso de T. interdigitale, e identificar Trichophyton spp. como T. rubrum tipo B granular. La concordancia entre la evaluación previa y la reevaluación fue de un 70% y entre las pruebas morfofisiológicas y BCP-MS-G fue de 96%. Se detectó un 30% de sobrediagnóstico de T. rubrum. Se sugiere el empleo del medio BCP-MS-G para diferenciar T. interdigitale de T. rubrum tipo A y otros biotipos de T. rubrum tipo B.

Due to the pleomorphism and macroscopic variability of the Trichophyton genus, identification methods based solely on the morphological characteristics are insufficient for its classification. The objective was to evaluate the morphological and biochemical characteristics of clinical isolates of Trichophyton spp. The study included 98 isolates previously identified by macro and micromorphology that were reassessed by means of microculture sheet, in vitro hair perforation, urea hydrolysis and culture on Trichophyton and BCP-MS-G agars. For morphological analysis, urea hydrolysis tests and in vitro hair perforation results were: T. rubrum 51%, T. mentagrophytes 24%, T. interdigitale 16.6%, T. tonsurans 7% and Trichophyton spp. 2%. The urea hydrolysis test showed 9.4% error. Culture on the BCP-MS-G medium allowed to differentiate T. rubrum cottony A type from T. interdigitale and the identification of Trichophyton spp. as T. rubrum granular B type. The agreement between the previous assessment and reassessment was 70% and when morphophysiological and BCP-MS-G culture were considered, agreement reached 96%. There was 30% overdiagnosis for T. rubrum. Cultures on BCP-MS-G agar are suggested to differentiate T. interdigitale from T. rubrum A type and other biotypes of T. rubrum B type.

Sensibilidad in vitro de aislados del complejo Paracoccidioides spp a antifúngicos sistémicos utilizando el método de microdilución / In vitro susceptibility of isolates of Paracoccidioides spp complex to systemic antifungals using the microdilution method

El método de referencia, microdilución en caldo, recomendado por el Instituto de Estándares Clínicos y de Laboratorios (CLSI), no está disponible para hongos dimórficos, como los del género Paracoccidioides. En este trabajo se evaluó la sensibilidad in vitro del Complejo Paracoccidoides (n=19) frente a los antifúngicos sistémicos: anfotericina B, 5-fluorocitosina, ketoconazol, itraconazol, fluconazol, voriconazol y caspofungina empleando el método de microdilución (Documento M27-A3 y M27-S3), con algunas modificaciones: tiempo de cultivo en medio de Sabouraud dextrosa agar (7-10 días), medio RPMI 1640 suplementado con glucosa al 2%, tiempo de incubación (7, 8 y 18 días). La sensibilidad in vitro fue variable; la mayoría de los aislados de Paracoccidioides fueron sensibles a ketoconazol (73,7%), seguido de voriconazol (68,4%), itraconazol (63,1%), anfotericina B (52,6%), fluconazol (47,4%), 5-fluorocitosina (42,1%) y caspofungina (5%). La resistencia global fue mayor ante caspofungina (94,7%), seguido de 5-fluorocitocina (52,6%) and anfotericina B (47,4%). El 53% de los aislados fue sensible dosis dependiente a fluconazol, 15,7%, itraconazol y 5,3% a 5-fluorocitosina. Anfotericina B, itraconazol y voriconazol fueron los antifúngicos más potentes contra Paracoccidioides spp (CMI: 0,03-1µg/mL). Basándose en estos resultados, se propone tentativamente un protocolo de ensayo de microdilución para las pruebas de sensibilidad de Paracoccidioides spp frente a fármacos antimicóticos. Esta metodología podría ser clínicamente útil para predecir el desarrollo de resistencias, aunque son necesarios más estudios.

Broth microdilution, the reference method recommended by the Clinical Laboratory Standards Institute (CLSI), is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this work, in vitro susceptibility of the Paracoccidioides complex (n=19) to systemic antifungals: amphotericin B, 5-flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole and caspofungin, was evaluated using the microdilution method (Document M27-A3, M27-S3), with some modifications such as: culture time in Sabouraud dextrose agar (7-10 days), RPMI 1640 medium supplemented with 2% glucose and the incubation time (7, 8 and 18 days). The sensitivity in vitro was variable; the majority of Paracoccidioides isolates was susceptible to ketoconazol (73.7%), followed by voriconazole (68.4%), itraconazole (63.1%), amphotericin B (52.6%), fluconazole (47.4%), 5-flucytosine (42.1%) and caspofungin (5%). The overall resistance was mainly to caspofungin (94.7%), followed by 5-flucytosine (52.6%) and amphotericin B (47.4%). Fifty-three percent of the isolates were susceptible-dose dependent to fluconazole followed by itraconazole (15.7%) and 5-fluorocytosine (5.3%). Amphotericin B, itraconazole and voriconazole were the most potent antifungal drugs against Paracoccidioides spp (CMI: 0.03-1µg/mL). Based on these results, we tentatively propose a microdilution assay protocol for susceptibility testing of Paracoccidioides spp to antifungal drugs. This method may be clinically useful to predict resistance, even though further studies are needed.

Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto / Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium

En este estudio se desarrolló y se evaluó el ensayo por inmunoabsorción ligado a enzimas (ELISA), para la detección de anticuerpos en sueros de pacientes con esporotricosis, para lo cual se empleó un antígeno crudo de Sporothrix schenckii sensu stricto obtenido a partir de la forma micelial. Los sueros positivos para esporotricosis fueron ensayados por otras técnicas serológicas: inmunodifusión doble (IDD) y contrainmunoelectroforesis (CIE). El ensayo fue validado utilizando sueros de otras patologías como histoplasmosis, paracoccidioidomicosis, tuberculosis, leishmaniasis, lupus y sueros de individuos sanos como controles negativos. Se encontró una especificidad de 100 % con las técnicas utilizadas y una sensibilidad del antígeno de S.schenckii sensu stricto, por encima del 98% para IDD, CIE y ELISA. Estos resultados demuestran la alta sensibilidad y especificidad del antígeno de S. schenckii sensu stricto, para el diagnóstico de la esporotricosis, empleando las técnicas de IDD, CIE y ELISA. Los resultados sugieren, que este antígeno podría ser usado en conjunto con otras pruebas convencionales para el diagnóstico diferencial y puede ser útil para monitorizar la evolución de la enfermedad y respuesta al tratamiento.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Trichosporon spp como agente causal de onicomicosis / Trichosporon spp as a causative agent of onychomycosis

Se evaluó la incidencia de aislados de la levadura de Trichosporon spp. a partir de las muestras clínicas procesadas en el Laboratorio de Micología del Instituto de Biomedicina en un lapso de 10 años (2000-2010). Encontrándose un total de 14 casos, lo que represento una incidencia del 0,13%, los casos mostraron 71% de positividad en el examen directo. El 57% de los casos fueron uñas de mano y pie, al contrario de lo reportado en Candida, las uñas de pie fueron las mas afectadas (50%) por el Trichosporon spp. en este estudio

The incidence of Trichosporon spp. yeast isolates from clinical samples tested in the Mycology Laboratory at the Institute of Biomedicine over a period of 10 years (2000-2010) was evaluated. There were a total of 14 cases, representing an incidence of 0.13%; the cases showed 71% positivity on direct examination. 57% of the cases were in nails of the hand and foot. In contrast to cases reported for Candida, toenails were the most affected (50%) by Trichosporon spp. in this study

Evaluación del inmunoanálisis enzimático en el diagnóstico y seguimiento de pacientes con coccidioidomicosis / Evaluation of the enzymatic immune analysis in the diagnosis and follow-up of coccidioidomycosis patients

Este estudio evaluó el inmunoanálisis enzimático (IAE) para el diagnóstico y seguimiento de pacientes con coccidioidomicosis (CDM). Se utilizaron 360 muestras de suero: 38 provenientes de pacientes con diagnóstico clínico, micológico e inmunológico de CDM, 100 de individuos sanos, 50 de pacientes sensibilizados a la coccidioidina y 172 con otras patologías, empleando dos exoantígenos de Coccidioides spp. La sensibilidad del IAE fue de 71,1% para ambos antígenos, con especificidad de 98% (Ag 1) y 96% (Ag 2). Los valores predictivos positivos fueron de 93,1% (Ag 1) y 87,1% (Ag 2), y negativos de 89,9% (Ag 1) y 89,7% (Ag 2), con razones de verosimilitud positiva de 35,6 (Ag 1) y 17,8 (Ag 2) y negativa de 0,3 para ambos antígenos. La potencia global del IAE se estimó en 90,6% (Ag 1) y en 89,1% (Ag 2). El índice Kappa reflejó una buena concordancia con la IDD. No se observó correspondencia entre las absorbancias detectadas por el IAE y el título de anticuerpos específicos obtenido mediante IDD en el seguimiento de pacientes con CDM. Se observaron reacciones cruzadas con las muestras de suero de pacientes con paracoccidioidomicosis e histoplasmosis. Se concluyó que el IAE puede ser una técnica útil en el diagnóstico, más no en el seguimiento de pacientes con CDM.

This study evaluated the enzymatic immune analysis (EIA) procedure for the diagnosis and follow-up of coccidioidomycosis (CDM) patients. Three hundred and sixty (360) serum samples were studied using two exoantigens of Coccidioides spp.: 38 obtained from patients with clinical, mycological and immunological CDM diagnosis, 100 from healthy individuals, 50 from individuals sensitized to coccidiodine, and 172 from individuals with other pathologies. It was estimated a 71.1% of sensitivity for both antigens, with a 98% specificity (Ag 1), and 96% (Ag 2), positive predictive values of 93.1% (Ag 1) and 87.1% (Ag 2), and negative predictive values of 89.9% (Ag 1) and 89.7% (Ag 2). The positive verisimilitude rates were 35.6 (Ag 1) and 17.8 (Ag 2), and 0.3 negative verisimilitude rates for both antigens. The global potency of the EIA was estimated as 90.6% (Ag 1) and 89.1% (Ag 2). The Kappa index reflected a good concordance with the IDD. No correspondence between the absorbance detected by the EIA and the title of specific antibodies obtained through IDD was observed in the follow-up of CDM patients. Cross reactions with serum samples from paracoccidioidomycosis and histoplasmosis patients was observed. It was concluded that the EIA can be a useful technique for the diagnosis but not for the follow-up of CDM patients.

Clasificación e identificación de especies de actinomicetos: Un estudio fenotípico comparativo / Classification and identification of Actinomyces species: A comparative phenotype study

Las pruebas fisiológicas y bioquímicas constituyen dos de las principales metodologías utilizadas, principalmente en los laboratorios de microbiología, para la identificación y diferenciación de los actinomicetos. La finalidad de este trabajo fue comparar y evaluar los métodos fenotípicos que son utilizados de manera rutinaria en la identificación de estos microorganismos. Se estudiaron setenta y tres cepas de actinomicetos provenientes de tres laboratorios de microbiología de Venezuela. El comportamiento fisiológico y bioquímico de las cepas en estudio fue evaluado mediante pruebas de descomposición de diferentes sustratos. Los resultados obtenidos permitieron observar diferencias en la identificación preliminar de las cepas realizada por estos laboratorios, conduciendo algunas veces a una nueva identificación de las mismas, gracias a la utilización de estos sustratos. Se pudo observar que la metodología empleada permitió la reclasificación taxonómica de casi todas las cepas estudiadas, lo que sugiere que es necesario estandarizar la metodología de identificación para los actinomicetos.

Physiologic and biochemical tests constitute two of the main methodologies, mainly used in microbiology laboratories, for the identification and differentiation of actinomyces. The purpose of this work was to compare and evaluate the phenotypic methods used routinely for the identification of these microorganisms. The study included seventy three actinomyces strains from three microbiology laboratories in Venezuela. The physiologic and biochemical behavior of the strains was evaluated through decomposition tests using different substrates. The results obtained showed differences in the preliminary identification of the strains done in the various laboratories, leading occasionally to a new identification arisen from the use of these substrates. It was seen that with the methods used it was possible to taxonomically reclassify almost all the strains studied, suggesting that it is necessary to standardize the methods for the identification of actinomyces.

Especies de Candida en cavidad oral de niños desnutrición: a proposito de un caso con Candida dubliniensis / Species of Candida in oral cavity in underfed children: a case of Candida dubliniesis

En este estudio se evaluó la incidencia de las especies de Candida de la cavidad oral sana de una población infantil desnutrida. Diversas especies de Candida fueron encontradas, C. albicans fue aislada en 29.4%, posteriormente se identificó una como C dubliniensis por PCR usando cebadores específicos para esta especie.