National production of certified reference fungal cultures / Producción nacional de cultivos fúngicos de referencia certificados

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short-and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20 ±5 °C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.

Los cultivos microbianos de referencia (CR) son imprescindibles para el control de calidad interno de los laboratorios. Asegurar su producción requiere de procedimientos estandarizados (IRAM 14950:2016 e ISO 17034:2016) realizados en un laboratorio reconocido y acreditado. El objetivo de este estudio fue producir cultivos fúngicos de referencia en discos de papel, a partir de un panel de cultivos autóctonos caracterizados por dos métodos de referencia, trazables a nivel taxonómico de especie, homogéneos y estables. Se produjeron CR de 14 especies circulantes en Argentina (7 de levaduras y 7 de hongos miceliales), depositadas en la colección de hongos de interés médico (DMic). Los discos de papel fueron embebidos con una suspensión del cultivo por producir, secados y envasados. Se verificó la homogeneidad, viabilidad, identidad y pureza de cada lote. Se evaluó la estabilidad a corto y largo plazo a distintas temperaturas y tiempos de almacenamiento. La caracterización de cada CR nos permitió confirmar su identidad y asegurar su trazabilidad a nivel internacional. Los lotes producidos fueron homogéneos y estables durante 30 meses conservados a -20 ±5 °C. Este método resultó adecuado para producir CR homogéneos y estables, con características fenotípicas y genotípicas correctamente definidas y trazables a nivel internacional. Los procedimientos estandarizados desarrollados en este trabajo pueden ser transferidos para producir CR certificados de otros microorganismos. La provisión de CR que represente cepas regionales permite a los laboratorios producir resultados más confiables con un impacto favorable en el diagnóstico médico, los estudios ambientales y la industria alimenticia.

Influencia del campo magnético sobre el crecimiento de microorganismos patógenos ambientales aislados en el Archivo Nacional de la República de Cuba / Influence of magnetic field on the growth of pathogen microorganisms isolated from the indoor environment at the Archivo Nacional de la República de Cuba

Introducción. En el Archivo Nacional de la República de Cuba, existe contaminación electromagnética y la influencia del campo magnético oscilante de frecuencia extremadamente baja podría cuantificarse con microorganismos patógenos aislados de su ambiente interior. Objetivo. Cuantificar la influencia de este tipo de campo magnético sobre el crecimiento de microorganismos patógenos aislados del ambiente en el Archivo Nacional de la República de Cuba. Materiales y métodos. Se emplearon cinco microorganismos: Streptococcus sp. (1), Listeria sp. (2) y Candida guillermondii (3), aislados en el Archivo, así como Escherichia coli ATCC 25922 (4) y Saccharomyces cerevisiae (5), como referencia. Se les aplicó un campo magnético oscilante de frecuencia extremadamente baja de 60 Hz/220 V de 3 mT durante dos horas, en tres tubos de cultivo con agua destilada y con caldo nutriente. Después se inocularon 0,1 ml en placas de Petri con los medios de cultivo agar CromoCen SC (1 y 2), agar de dextrosa y papa (3), agar CromoCen CC 4227 (4) y agar con extracto de malta (5). Las colonias se contaron (log UFC/ml) mediante el procesamiento digital de las imágenes de las placas de Petri empleando el programa MatLab ® . Resultados. Se observó una estimulación significativa (p=0,05) de la cantidad de colonias tratadas con respecto a los controles, siendo mayor en el caldo nutriente que en el agua destilada y más en las bacterias (caldo nutriente-colonias tratadas: 9,43 a 10,62 UFC/ml) que en las levaduras (caldo nutriente-colonias tratadas: 8,31 a 8,79 UFC/ml). La estimulación se produjo en orden decreciente así: Listeria sp., E. coli ATCC 25922, Streptococcus sp., C. guillermondii y S. cerevisiae . Conclusión. Se concluyó que el campo magnético aplicado tuvo un efecto estimulante sobre los microorganismos estudiados, lo cual potencia el riesgo para la salud del personal y los visitantes del Archivo Nacional de la República de Cuba.

Introduction: Electromagnetic pollution has been detected at the Archivo Nacional de la República de Cuba and the influence of extremely low frequency magnetic fields could be quantified with pathogenic microorganisms isolated from the indoor environment. Objective: To quantify the influence of an extremely low frequency magnetic field on the growth of pathogenic microorganisms isolated from the environment at the Archivo Nacional. Materials and methods: We used five microorganisms isolated at the Archivo Nacional: Streptococcus sp. (1), Listeria sp. (2) and Candida guillermondii (3), and Escherichia coli ATCC 25922 (4) and Saccharomyces cerevisiae (5) as references. We applied this magnetic field of extremely low frequency, 60 Hz/220 V (3 mT), for two hours to these microorganisms on three culture tubes with distilled water and nutrient broth. Then we inoculated 0.1 mL in the following solid culture mediums on Petri dishes: CromoCen SC Agar (1 and 2), Potato Dextrose Agar (3), CromoCen DC 4227 (4) and Malt Extract Agar (5). The colonies were counted (log CFU/mL) by digital processing of the images of Petri dishes using the MatLab ® tool. Results: We observed a statistically significant stimulation (p=0.05) in the quantity of treated colonies as compared to controls, which was higher in nutrient broth than in distilled water, and in bacteria (nutrient broth and treated colonies: 9.43 to 10.62 CFU/mL) as compared with yeasts (nutrient broth-treated colonies: 8.31 to 8.79 CFU/mL). In decreasing order, stimulation was as follows: Listeria sp., E. coli ATCC 25922, Streptococcus sp., C. guillermondii and S. cerevisiae . Conclusion: We concluded that the magnetic field applied had a stimulating effect on the microorganisms under study, which increases the risk to the health of staff and visitors at the Archivo Nacional .

Concordancia entre los métodos del diagnóstico micológico de la paracoccidioidomicosis / Concordance between methods of mycological diagnosis of paracoccidioidomycosis

La paracoccidioidomicosis (PCM) es una enfermedad crónica, sistémica, granulomatosa, endémica en nuestro país, producida por un hongo dimorfo denominado Paracoccidioides brasiliensis. Existen numerosas técnicas para realizar el diagnóstico de esta entidad. Nos planteamos la posibilidad de realizar un estudio para determinar la concordancia que pudiese existir entre las diferentes técnicas que se utilizan para el diagnóstico de la PCM. Se realizó un registro de historias clínicas. Se evaluaron 251 historias clínicas de pacientes con diagnóstico de PCM, de la consulta externa de la Sección de Micología Médica “Dr. Dante Borelli” del IMT-UCV, entre los años 2000 y 2010. Se determinó la concordancia entre los métodos diagnósticos por medio del análisis de concordancia de atributos para datos binarios. Entre el examen directo y el cultivo, no hubo acuerdo. Entre la serología y el examen directo, se encontró que hubo equivalencia, así como entre el cultivo y la serología. No se pudo calcular correlación alguna con la histopatología, ya que no hubo datos negativos, en vista de que todas las muestras procesadas fueron positivas. El diagnóstico de la PCM se basa en la identificación y el aislamiento del hongo. Es obligatoria la realización del examen directo en fresco de toda muestra clínica. Nuestro estudio de muestra que deben realizarse todos los métodos que estén al alcance (examen directo en fresco, cultivo, serología, histopatología) a fin de aumentar la probabilidad de llegar a un diagnóstico certero de esta patología

Paracoccidioidomycosis (PCM) is a chronic, granulomatous disease, endemic in our country, produced by a dimorphic fungus, Paracoccidioides brasiliensis. Several techniques are used for the diagnosis. A study was performed to determine the agreement that could exist between the different techniques, used for the diagnosis of PCM. Clinical records of patients with diagnosis of PCM was made. 251 clinical records were reviewed, from the Sección de Micología Médica “Dr. Dante Borelli”, IMT- UCV, between 2000 and 2010. The agreement between the methods was determined by means of the analysis of agreement of attributes for binary data. Between direct examination and culture, there was no agreement. Between serology and direct examination, there was equivalence, as well as between culture and serology. Correlation could not be calculated with histopathology, since there were no negative data, due to the fact that all the processed samples were positive. The diagnosis of the PCM is based on the identification and isolation of the fungus. Direct examination is mandatory in all clinical samples. Our study demonstrates that all the methods must be performed (direct examination, culture, serology, histopathology) in order to increase the probability of reaching an accurate diagnosis

Aeromicología y salud humana / Aeromycology and human health

Introducción: la aeromicología estudia la variación temporal y espacial del contenido fúngico de la atmósfera, así como la influencia de los factores meteorológicos sobre dichas variaciones. En países tropicales como Cuba, la elevada temperatura y la humedad relativa favorecen el crecimiento de los hongos, así como la formación y liberación de sus esporas, las cuales pueden afectar la salud humana. Objetivo: destacar el impacto de los estudios aeromicológicos para la salud humana. Métodos: se realizó una revisión de la literatura científica sobre aspectos generales de la aeromicología, las principales especies fúngicas presentes en ambientes exteriores e interiores, su impacto en la salud humana y las medidas para disminuir el riesgo de afectación a la salud por dichos hongos. Resultados: se expone información actualizada y valiosa sobre la aeromicología, útil para la prevención de enfermedades ocasionadas por hongos presentes en el aire. Además se destacan los estudios realizados en Cuba hasta la actualidad. Conclusiones: la determinación ambiental de propágulos fúngicos así como sus variaciones estacionales es un parámetro relevante a evaluar dentro de la salud preventiva(AU)

Introduction: aeromycology studies the time and space variation of the air fungal content, as well as the influence of weather factors on these variations. In tropical countries like Cuba, high temperatures and relative humidity favor fungal growth and the formation and release of its spores, which can have an impact on human health. Objective: to highlight the impact of Aeromycology in the human health. Methods: Scientific literature addressing the general aspects of aeromycology, the main indoor and outdoor fungal species, their impact on human health and the actions aimed at decreasing the risk for human health was reviewed. Results: updated and valuable information on aeromycology was presented which can be used to prevent diseases caused by airborne fungi. Additionally, this review highlighted the studies conducted in Cuba up to the present. Conclusions: the environmental determination of fungal propagules and their seasonal variations is a relevant parameter to be evaluated in preventive health care systems(AU)

Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid detection of phenol constituents.

Most of the phenol compounds are toxic and have been considered as hazardous pollutants. Several physicochemical and biological methods are available to detect and monitor the phenol pollutants in water and soil. In the present study, phenol constituents of winery, paper and plastic industrial effluents were successfully detected employing tyrosinase-gold nanoparticles bioconjugate. The synthesis of extracellular tyrosinase and gold nanoparticles was achieved by a single isolate of Streptomyces sp. DBZ-39. Enhanced production (369.41 IU) of tyrosinase was produced in submerged bioprocess employing response surface method with central composite design. Extracellular gold nanoparticles synthesized (12-18 nm) by Streptomyces sp. DBZ-39 were characterized with TEM, EDAX and FTIR analysis. A rapid detection (within 10 min) of phenol constituents from winery effluents was achieved by bioconjugate, when compared to tyrosinases and gold nanoparticles independently. Streptomyces tyrosinase could exhibit relatively a better performance than commercially available mushroom tyrosinase in the detection of phenol constituents. Winery effluent has shown much higher content (0.98 O.D) of phenol constituents than paper and plastic effluents based on the intensity of color and U.V absorption spectra.

Production, purification and properties of fungal chitinases—A review.

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito-oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

Evaluación de las tarjetas AST-YSO1 del sistema Vitek 2 para determinar la sensibilidad a antifúngicos de levaduras del género Candida / Evaluation of the VITEK 2 system (AST-YSO1 cards) for antifungal susceptibility testing against different Candida species

El objetivo de este trabajo fue evaluar los resultados de sensibilidad a los antifúngicos de diversas especies de Candida utilizando el sistema semiautomatizado Vitek 2 (tarjetas AST-YSO1; bioMérieux), y compararlos con los obtenidos por el método de referencia del Clinical and Laboratory Standards Institute (CLSI), la microdilución en caldo (Documento M27-A3, 2008). La concordancia esencial fue > 90 %, excepto en el caso de Candida glabrata frente al voriconazol (VCZ) y de Candida krusei frente al fluconazol (FCZ). La concordancia global por categoría (variación no mayor que 2 diluciones, sin discriminar por especie) fue > 90 % cuando se evaluó el FCZ, y 89,5 % a las 24 h y 80,7 % a las 48 h con el VCZ. El tiempo promedio para obtener los resultados fue de 15,5 h. Los errores menores (sensible o resistente por un método y dosis dependiente por el otro) para FCZ fueron de 7,8 % a las 24 h y 6,1 % a las 48 h; para VCZ, 10,5 % a las 24 h y 19,3 % a las 48 h. Solo se detectó 1 error muy mayor (resistente por el método de referencia y sensible por Vitek 2) con Candida parapsilosis frente a FCZ a las 48 h. No se observaron errores mayores (sensibles por el método de referencia y resistentes por Vitek 2). Con respecto a la anfotericina B, solo 3 cepas presentaron una CIM = 2 ?g/ml. El sistema Vitek 2 detectó correctamente el valor de CIM para diversas especies de Candida y presentó una excelente concordancia con el método de referencia propuesto por el CLSI.

The aim of this investigation was to evaluate the results of antifungal susceptibility for various Candida species using the Vitek 2 semi-automated system (AST-YSO1 cards, bioMérieux), and to compare them with those obtained by the CLSI (Clinical and Laboratory Standards Institute) broth microdilution reference method (Document M27-A3,2008). The essential agreement (EA) was > 90%, except for Candida glabrata against voriconazole (VCZ); and for Candida krusei against fluconazole (FCZ). The overall categorical agreement (CA) was > 90% when FCZ was evaluated and 89.5% at 24 h and 80.7% at 48 h for VCZ. The average time for obtaining results was 15.5 h. Minor errors were 7.8% at 24 h and 6.1% at 48 h for FCZ, and 10.5% at 24 h and 19.3% at 48 h for VCZ. There was only one very major error for FCZ against Candida parapsilosis and no major errors were observed. For amphotericin B, only three isolates showed MICs = 2 ?g/ml. The Vitek 2 system detected the MIC value for various Candida species and showed excellent agreement with the reference method proposed by the CLSI.

Keratinophilic fungi recovered from feathers of different species of birds in St Kitts and Nevis / Hongos queratinofílicos obtenidos de las plumas de diferentes especies de aves en St Kitts y Nevis

OBJECTIVE: The aim of the present study was to investigate the occurrence of keratinophilic fungi including dermatophytes on feathers of domestic and wild birds in the islands of St Kitts and Nevis. METHOD: During 2010-2011, samples of feathers from ninety-four birds were examined by hair-baiting technique in Petri-dishes containing sterilized soil. Fungal growths appearing on the feathers and the hair-baits were microscopically examined and the cultures obtained were identified on the basis of their microscopic and colonial morphology. RESULTS: Chrysosporium constituted the majority (86.9%) of the 72 isolates of keratinophilic fungi, represented by mainly C tropicum and C indicum. Sepedonium spp isolates were recovered from nine of the feather samples; two of these were identified as Sepedonium chrysospermum, and the other two as S ampullosporum. CONCLUSION: Recovery of four isolates of the dermatophyte, Microsporum gypseum complex (two each of M gyspeum and M fulvum) from feathers of birds is a finding of public health significance.

OBJETIVO: El objetivo del presente estudio fue investigar la presencia de hongos queratinofílicos, incluyendo dermatofitos, en las plumas de aves domésticas y silvestres en las islas de St Kitts y Nieves. MÉTODOS: Durante 2010-2011, se examinaron muestras de plumas de noventa y cuatro aves, utilizando la técnica de anzuelo queratínico (técnica de Vanbreuseghem) en placas de Petri con tierra esterilizada. Los crecimientos fúngicos que aparecieron sobre las plumas y los anzuelos de queratina de pelos (hair baits) fueron examinados bajo el microscopio, y los cultivos obtenidos fueron identificados sobre la base de su morfología microscópica y colonial. RESULTADOS: Chrysosporium constituyó la mayor parte (86.9%) de los 72 aislados de hongos queratinofílicos, representados principalmente por el C tropicum y el C indicum. Aislados de Sepedonium spp fueron obtenidos de nueve muestras de plumas. Dos de ellos fueron identificados como Sepedonium chrysospermum y los otros dos como S ampullosporum. CONCLUSIÓN: La recuperación de cuatro aislados del complejo M gypseum dermatofito (formado por dos M gyspeum y dos M fulvum respectivamente) de las plumas de aves, es un hallazgo de importancia para la salud pública.

Invasive candidiasis: A review of nonculture-based laboratory diagnostic methods.

Invasive candidiasis is a life-threatening complication of critically ill immunocompromised patients with high attributable mortality. Due to non-specific clinical presentation, early detection of candidemia and accurate identification of Candida species are essential pre-requisites for improved therapeutic outcome. Since blood culture-based methods lack sensitivity and species-specific identification by conventional methods is time-consuming, detection of immunological and molecular markers has provided an alternative for early diagnosis of invasive candidiasis. Additionally, serial estimations of these biomarkers have provided opportunities to monitor response to therapy and initiate pre-emptive therapy in suspected patients before clinical signs appear. Antigen-based methods include detection of β-D-glucan, a panfungal marker, and Candida mannan, a genus-specific marker. Although both these markers have moderate sensitivity, they provide a useful adjunct to the diagnosis if performed in select patient population in parallel for exclusion of false positive/negative results. A negative β-D-glucan test on at least two occasions has a high negative predictive value. Concomitant detection of Candida mannan and anti-mannan antibodies has sensitivity of ~70% before blood cultures become positive. Significant advances have also been made in nucleic acid-based detection methods, including a commercial real-time PCR assay (SeptiFast) for detection of five major clinically important Candida spp. in blood specimens within 6 h. Furthermore, matrix-assisted laser desorption ionization time-of-flight mass spectrometry enables species-specific identification of yeast isolates within an hour. While these immunological and molecular tools mark a significant advance towards early and specific diagnosis of candidemia and invasive candidiasis, further evaluation of these approaches in different clinical settings is warranted.

Keratomycosis caused by Exserohilum rostratum.

We report a case of keratomycosis caused by Exserohilum rostratum. A 46-year-old farmer presented with history of pain, watery discharge and redness of the right eye for the past 2 weeks following trauma with vegetable matter. On ocular examination, a central corneal ulcer of about 8 mm with a greyish-white slough, feathery edges and diffuse corneal edema was seen in the right eye. KOH examination of corneal scrapings revealed thick, brown, branched, septate hyphae. Culture of corneal scrapings on Sabouraud dextrose agar showed velvety greenish-black colony with a black pigment on the reverse. The culture was identified as E. rostratum on the basis of microscopic morphology. The patient responded well to treatment with topical natamycin and oral itraconazole.

Conservación de cultivos fúngicos de alto riesgo de Histoplasma y Cryptococcus / Preservation of high risk fungal cultures of Histoplasma and Cryptococcus

Introducción: las colecciones de cultivos microbianos son las encargadas de garantizar el material biológico requerido para el desarrollo de las ciencias biológicas. Entre los métodos de conservación de cultivos fúngicos, la inmersión en agua destilada, por su bajo costo y sencillez, constituye una ventajosa alternativa. Objetivo: evaluar la utilidad de este método de conservación en los cultivos fúngicos de Histoplasma y Cryptococcus. Métodos: se realizó una evaluación del estado de conservación de las especies de mayor riesgo biológico, pertenecientes a los géneros Histoplasma y Cryptococcus de la colección de cultivos de hongos del Instituto de Medicina Tropical "Pedro Kourí". Se analizaron 102 cepas conservadas en agua destilada, de las cuales 92 por ciento estaba preservado por más de 10 años. Resultados: los porcentajes de recuperación para H. capsulatum, C. neoformans y C. gattii fueron 64,3; 79,1 y 100 por ciento, respectivamente. Se demostró que este método de conservación resulta satisfactorio para cultivos fúngicos en laboratorios de recursos limitados. Se implementó sobre plataforma web una base de datos digital con la información de interés de la colección. Se hizo una valoración de la importancia del estricto cumplimiento de las medidas de bioseguridad para el trabajo de las colecciones, especialmente frente a patógenos de alto riesgo. Conclusiones: la conservación de cultivos de hongos en agua destilada es un método de gran utilidad en laboratorios de recursos limitados. El trabajo de las colecciones de cultivos debe considerarse una actividad imprescindible para enfrentar los nuevos retos del desarrollo de las ciencias biomédicas.

Introduction: culture collections are responsible for providing the microbial resources for development of biological sciences. Storage in distilled water is one of the easiest and least expensive method for long-term fungal preservation. Objective: to evaluate the usefulness of this preservation method in fungal culture of Histoplasma and Cryptococcus. Methods: the preservation condition of the highest biological risk species from Histoplasma y Cryptococcus genera, included in the fungal culture collection of "Pedro Kourí" Institute of Tropical Medicine in Havana, was evaluated in this study. One hundred and two strains stored in distilled water, 92 percent of which had been preserved for more than 10 years, were analyzed. Results: the percentages of recovered strains from H. capsulatum, C. neoformans and C. gattii were 64.3 percent; 79.1 percent and 100 percent respectively. This method of preservation proved to be satisfactory for fungal culture in labs with limited financial resources. A web-based database with interesting information about the collection was made. The importance of strict compliance with the biosafety measures in these collections, particularly with high risk pathogens. Conclusions: preservation of fungal cultures in distilled water is a very useful method for laboratories with limited resources. Culture collections should be assumed as an essential activity in order to solve increasing challenges in the development of biomedical sciences.

Onicomicosis: epidemiología, agentes causales y evaluación de los métodos diagnósticos de laboratorio / Onychomycoses: epidemiology, causative agents and assessment of diagnostic laboratory methods

Desde marzo de 2007 hasta marzo de 2011 se estudiaron prospectivamente 414 pacientes con onicodistrofias en un laboratorio privado de Esquel. La prevalencia de onicomicosis de pie fue del 78 %; la de mano, del 58 %. Los principales agentes etiológicos fueron Trichophyton rubrum, Candida spp. y Trichophyton mentagrophytes. El desarrollo de dermatofitos prevaleció en las onicopatías de pie y el de Candida spp. en las de uñas de mano (ambos, p < 0,05). En las onicomicosis candidiásicas predominaron especies diferentes a Candida albicans. Las onicomicosis fueron más frecuentes en los hombres que en las mujeres. A su vez, en los hombres hubo más aislamientos de T. rubrum en pies (p < 0,05) y mayor proporción de exámenes directos (ED) y cultivos positivos (ambos, p < 0,05). La correlación entre los resultados del ED y del cultivo fue del 68 %. El rédito de ambos métodos se asoció a un mayor tamaño de la lesión ungueal. El ED fue más efectivo en onicodistrofias que superaban los 5 años de evolución. La positividad del cultivo fue independiente de la cronicidad de la onicodistrofia.

Since March 2007 to March 2011, 414 patients with onychopathies were prospectively analyzed. Prevalence of the toenail and fingernail mycoses was 78 % and 58 %, respectively. The major etiological agents were Trichophyton rubrum, Candida spp. and Trichophyton mentagrophytes. Dermatophytes were more frequently cultured from toenails, whereas Candida spp. from fingernails (both, p < 0.05). In candidal onychomycosis, species different from C. albicans were prevalent. A higher prevalence of toenail and fingernail mycoses, a predominance of T. rubrum in toenails (p < 0.05), and greater positivity in the direct examination (DE) and in culture (both, p < 0.05) were more frequently observed in men than in women. The correlation between DE and culture was 68 %. DE and culture yields were associated with a greater size lesion. DE was more effective in onycodystrophies with duration of more than 5 years. Culture positivity was independent of nail affection chronicity.

Schizophyllum commune sinusitis in an immunocompetent host.

Schizophyllum commune is widely distributed in the nature, but it rarely causes human infection. We have isolated this mould in a 46-year-old immunocompetent, non-diabetic patient with chronic sinusitis, previously treated with multiple antibiotics and topical steroid nasal drops with no response. Materials obtained from the nasal sinus during the endoscopic surgery, on KOH mount and histopathological study revealed broad septed hyaline hyphae. Growth on the Sabouraud's dextrose agar and potato dextrose agar produced white moulds with microscopic and macroscopic characters of S. commune. Till date there are few reports of S. commune sinusitis in immunocompetent individuals Worldwide. This is the first reported case in India to the best of our knowledge.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

A rare case of Histoplasma fungemia in an AIDS patient.

Disseminated cases of histoplasmosis in acquired immune deficiency syndrome (AIDS) are rarely reported from India. Most of these cases report isolation of this fungus from the bone marrow, lymph node aspirate, spleenic aspirate, and biopsies. We report isolation of Histoplasma capsulatum from the blood of an AIDS patient. A 30-year-old male from Utter Pradesh was admitted with fever, loss of appetite, and nausea since two months. Few intracellular and extracellular budding cells were observed on bone marrow examination on the fifth day of admission. Diagnosis was confirmed by blood cultures taken on the 11th day of admission. Amphotericin B was started, but the patient's condition deteriorated and he died.

Comparison of the activities of amphotericin B, itraconazole, and voriconazole against clinical and environmental isolates of Aspergillus species.

Background: Invasive fungal infections are a significant cause of morbidity and mortality in immunocompromised populations. Aims: To evaluate the susceptibility pattern of our isolates against amphotericin B, itraconazole, and voriconazole and to compare the antifungal activities of these agents with each other against the Aspergillus species tested. Settings and Design: A prospective study was designed to include clinical and environmental isolates of Aspergillus species. Materials and Methods: 420 sputum samples, 70 bronchoalveolar lavage fluids, 160 oral washings, and 47 environmental samples were collected. Direct microscopy by potassium hydroxide and lactophenol cotton blue mounts followed by culture on Sabourad`s dextrose agar (SDA) was done. Susceptibility testing was performed by the broth microdilution technique as per Clinical Laboratory Standards Institute standards (M-38A). Additionally, all the isolates were also tested by the colorimetric microdilution technique using Alamar Blue dye. Statistical Analysis: It was done by the Chi-square test and Z-test using SPSS statistical software version 12.0. Results and Conclusion: Twenty-seven isolates (47.3%) were recovered from patients with chronic bronchial asthma followed by fibrocavitary pulmonary tuberculosis in 9 (15.7%), allergic bronchopulmonary aspergillosis (ABPA) in 6 cases (10.5%), bronchiectasis in 3 (5.2%), bronchogenic carcinoma in 5 (8.7%) and those receiving radiotherapy for head and neck cancer 7 (12.2%). Thirteen environmental isolates were also included in the study. The most common isolate was A. fumigatus 28 (40%), followed by A. niger 22 (31%), A. flavus 13 (19%), and A. terreus 7(10%). All isolates were susceptible to amphotericin B, itraconazole, and voriconazole. Among the three agents tested, voriconazole exhibited lowest MICs (≤1 μg/ml) against all Aspergillus species.

In Vitro effect of low-level laser therapy on typical oral microbial biofilms

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms - SSB and DSB - were exposed to laser doses of 5, 10 or 20 J/cm2 from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.

O objetivo deste estudo foi avaliar o efeito de parâmetros específicos de irradiação com laser de baixa intensidade sobre biofilmes formados por Streptococcus mutans (S. mutans), Candida albicans (C. albicans) ou associação de ambas as espécies. Biofilmes isolados ou associados destes microrganismos foram irradiados com um dispositivo laser infra-vermelho próximo de diodos InGaAsP (LaserTABLE 780 ±3 nm, 0,04W), utilizando-se para isto o dispositivo LASERTable. Quinze horas após a irradiação, foi demonstrado, por meio da avaliação da viabilidade celular (Teste de MTT), da morfologia das células (MEV) e do crescimento do biofilme (UFC/mL), que esta terapia foi capaz de reduzir o metabolismo celular, número de microrganismos presentes no biofilme, bem como seu crescimento no local. Quanto à viabilidade celular, a resposta à irradiação do biofilme de S. mutans (SSB) foi semelhante para todas as doses de energia, sendo que o crescimento do biofilme foi dose dependente. Porém, quando associado à C. albicans, este microrganismo apresentou resistência à fototerapia. Já a C. albicans associada ao S. mutans apresentou redução de crescimento significativa, sendo este resultado também foi dose dependente. A morfologia dos microrganismos não foi alterada pelas irradiações realizadas quando em biofilmes isolados. A associação entre os microrganismos promoveu redução na formação de hifas pela C. albicans. A laserterapia de baixa intensidade apresentou efeito inibitório sobre microrganismos, sendo que esta capacidade pode ser alterada de acordo com a interação entre diferentes microrganismos.