[CAP59 gene amplification in Cryptococcus neoformans and Cryptococcus gattii directly from a yeast suspension]. / Amplificación del gen CAP59 en Cryptococcus neoformans y Cryptococcus gattii directamente a partir de una suspensión de levaduras.

Cryptococcus is an encapsulated yeast of class Basidiomycetes, etiologic agent of cryptococcosis. Cryptococcosis is one of the most common opportunistic infections in immunosuppressed patients, although it can affect immunocompetent individuals. In recent years, the identification of medically important fungal species has been achieved through the amplification of specific regions or genes of fungal DNA by polymerase chain reaction (PCR). The CAP59 gene is involved in the synthesis of the capsule in Cryptococcus neoformans and Cryptococcus gattii, and is useful in the molecular identification of serotypes. In this research, we use yeasts of different serotypes from collection strains and C. neoformans isolates recovered from patients with cryptococcosis. A standardized yeast suspension from different Cryptococcus isolates as template allowed CAP59 gene amplification. This procedure was quick, simple, and inexpensive and required no PCR steps. This is important for taxonomic studies in laboratories with implemented molecular biology tools.

Phenotypic and genotypic identification of Candida dubliniensis from subgingival sites in immunocompetent subjects in Argentina.

INTRODUCTION: It is generally recognized that Candida dubliniensis is commonly found in immunocompromised patients, such as those with advanced human immunodeficiency virus infection, at sites of periodontal disease. Since there are no data available for Argentina, the aim of this study was to determine the prevalence of and to identify C. dubliniensis in periodontal pockets from immunocompetent subjects living in Buenos Aires, Argentina, through a comparison of phenotypic and molecular assays. METHODS: Yeasts recovered from subgingival plaque samples were studied for 180 immunocompetent non-smoking patients with periodontal disease. Yeasts were identified by conventional mycological methods and by specific polymerase chain reaction (PCR) assay. Fluconazole and voriconazole susceptibility studies were performed in keeping with the Clinical and Laboratory Standards Institute. RESULTS: Among 76 yeasts isolated, C. dubliniensis comprised 10.5% (n = 8; 95% confidence interval 4.7-19.7), which corresponded to 4.4% of patients studied (8/180). C. albicans was the most frequently isolated species of yeast. A great majority of C. dubliniensis isolates was susceptible with only one isolate resistant to both antifungals. CONCLUSION: Micromorphology on Staib agar was the phenotypic method that was most concordant with PCR and it was useful for selecting presumptive C. dubliniensis. This is the first report to use PCR to identify C. dubliniensis in subgingival fluid from immunocompetent individuals with periodontal disease in Argentina. On the basis of the findings presented here, we confirm that C. dubliniensis can colonize periodontal pockets of immunocompetent patients with periodontal disease.

[Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR]. / Comparación de métodos de extracción de ADN de sangre para detectar ADN fúngico por PCR.

Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR. Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS land ITS 4 as target.

[Useful phenotypic characteristics for presumptive identification of Candida guilliermondii]. / Características fenotípicas útiles para la identificación presuntiva de Candida guilliermondii.

Candida guilliermondii developed a pink-purplish colony on CHROMagar Candida. In the micromorphology in milk-tween 80 1% agar at 28 degrees C after 48 h of incubation C. guilliermondii showed small (3-5 microm), spherical yeasts without pseudohyphaes. This Candida species presented a characteristic cluster of blastospores with pseudohyphaes radiating from the centre at 96 h. The trehalose-sucrose assimilation assay was applied to the C. guilliermondii isolates which proved negative for trehalose and positive for sucrose. These results allowed for the presumptive identification of C. guilliermondii. The results were concordant in 100% of the isolates with the identification of the C. guilliermondii species by the ID 32C and Vitek YBC methods. Such automated methods offered Candida famata as a second option, with a reliability percentage of 10%. Micromorphological studies increase yeast identification reliability, especially among species presenting similar biochemical profiles.

[Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001]. / Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas. Período 1999-2001.

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method.

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2% dextrose. Overall agreement between both methods was 90.0%. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6%, 19.05% and 52.17%, respectively. No very major discrepancies, 1.03% major discrepancies, and 8.97% minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80% vs 50% endpoints, and trailing growth near the endpoints.

Comparación de métodos de extracción de ADN de sangre para detectar ADN fúngico por PCR / Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR

La infección fúngica invasora (IFI) está asociada a un alto índice de mortalidad, que alcanza el 50% debido a la frecuente falla en el tratamiento antifúngico. Existen dificultades para realizar un diagnóstico micológico rápido y certero dada la baja sensibilidad de los métodos convencionales, especialmente en pacientes neutropénicos y con SIDA. Numerosos métodos para diagnosticar infecciones micóticas basados en el estudio del ADN fúngico están actualmente en desarrollo. Nosotros evaluamos la utilidad de dos procedimientos de extracción y purificación del ADN fúngico presente en sangre para su posterior detección por PCR. Ambos métodos resultaron igualmente eficientes para obtener ADNs de óptima calidad y para realizar la técnica de PCR con los iniciadores universales para hongos ITS 1 e ITS 4.

Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS 1and ITS 4 as target.

Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas: Período 1999-2001 / Prevalence of Candida albicansand Candida no albicans inclinical samples during 1999-2001

Las levaduras implicadas en procesos patológicos son de indiscutible importancia debido al incremento experimentado por estas infecciones en las últimas décadas, a los cambios observados en las especies causales y al uso empírico de antifúngicos. En el Centro de Micología se estudiaron 1006 aislamientos provenientes de una amplia gama de muestras clínicas durante el periodo 1999-2001. Candida albicans con 40,3% resultó la especie de mayor frecuencia de aislamiento, pero las especies de Candida no albicans con 54,9% resultaron de mayor prevalencia y el 4,8% fueron otras levaduras. En los hemocultivos Candida parapsilosis con 34,9%, C. albicans con 30,2% y C. tropicalis con 25,6% resultaron las más recuperadas, mientras que C. glabrata se presentó con un 2,3%. En las secreciones mucosas C.albicans con 60%-80% fue la especie preponderante. Hemos detectado especies de Candida causantes de mediastinitis, lo que nos alerta sobre su importancia en estos procesos. Las infecciones del tracto urinario por levaduras se detectaron en mayor frecuencia en individuos hospitalizados, resultando C. albicans con 47,7% la especie más aislada, y dentro de Candida no albicans, C. glabrata con 24,8% y C. tropicalis con 20,0%. En las onixis candidiásicas C. parapsilosis con 37,7% desplazó a C.albicans con 22,0% de este lugar anatómico. Los estudios de sensiblidad al fluconazol de las especies de Candida nos permiten concluir que C.albicans es una especie sensible y que los mayores porcentajes de resistencia se observaron en C. glabrata (21,41%) y and C. krusei (69,23%).

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).

Nocardiosis cerebral debida a nocardia asteroides / Nocardiosis cerebral on occount of nocardia asteroid

Presentamos el caso de una neocardiosis cerebral en un paciente de 63 años con antecedentes de lesión supurada en el tercio del muslo derecho.Del material de los abscesos cerebrales se aisló N.asteroides y se lo trató con antibióticos: cotrimoxazol y ceftriaxona. Se instiló amikacina en el lecho quirúrgico y además en los pequeños abscesos no resecados. A los seis meses se realizó un control tomográfico y se observó la resolución de las lesiones. Los controles posteriores comprobaron la curación clínica

Nocardiosis cerebral debida a nocardia asteroides / Nocardiosis cerebral on occount of nocardia asteroid

Presentamos el caso de una neocardiosis cerebral en un paciente de 63 años con antecedentes de lesión supurada en el tercio del muslo derecho.Del material de los abscesos cerebrales se aisló N.asteroides y se lo trató con antibióticos: cotrimoxazol y ceftriaxona. Se instiló amikacina en el lecho quirúrgico y además en los pequeños abscesos no resecados. A los seis meses se realizó un control tomográfico y se observó la resolución de las lesiones. Los controles posteriores comprobaron la curación clínica

Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas. Período 1999-2001. / [Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001]

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3

) was the most isolated species, although, the Candida no albicans species with 54.9

showed the major prevalence. In blood cultures Candida parapsilosis (34.9

), C. albicans (30.2

) and C. tropicalis (25.6

) were recovered most frequently while C. glabrata represented only 2.3

. C. albicans with 60

-80

was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7

), the most commonly isolated, followed by C. glabrata (24.8

) and C. tropicalis (20.0

). In the candidal onychomycoses, C. parapsilosis (37.7

) outplaced C. albicans (22.0

). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41

) and C. krusei (69.23

).

Diagnóstico de 2 casos de mucormicosis rino-facio-cerebral en pacientes diabeticos / Diagnosis two cases de rhino-facio-cerebral mucomycosis in diabetic patients

Se presentan dos casos de mucormicosis rino-facio-cerebral de evolución lenta, en pacientes diabéticos. El diagnóstico se realizó por el hallazgo del hongo en el exámen histopatológico y exámenes microcópicos de los materiales drenados de los senos maxilares, que mostraron hifas no septadas y abundantes. En ninguno de los casos se pudo recuperar el agente etiológico por cultivo, debido a la labilidad de los mucorales y por la presencia de un material necrótico de fácil contaminación microbiana

Diagnóstico de 2 casos de mucormicosis rino-facio-cerebral en pacientes diabeticos / Diagnosis two cases de rhino-facio-cerebral mucomycosis in diabetic patients

Se presentan dos casos de mucormicosis rino-facio-cerebral de evolución lenta, en pacientes diabéticos. El diagnóstico se realizó por el hallazgo del hongo en el exámen histopatológico y exámenes microcópicos de los materiales drenados de los senos maxilares, que mostraron hifas no septadas y abundantes. En ninguno de los casos se pudo recuperar el agente etiológico por cultivo, debido a la labilidad de los mucorales y por la presencia de un material necrótico de fácil contaminación microbiana

Esporotricosis experimental en ratas / Sporotrichosis experimental on rats

Ratas endocriadas se infectaron por vía intraperitoneal y en la cola con una suspensión de conidios de Sporothrix schenckii. Los frotis coloreados con May Grunwald-Giemsa y Gram Nicolle, así como los cultivos de macerados de los órganos que se biopsiaron: hígado, bazo, pulmón y glándula suprarrenal y de la cola manifestaron a los l5 días de inoculación una diseminación del hongo; no así a los 30 días de infección, excepto el material correspondiente a la cola que presentó en ese tiempo elementos de S.schenckii. Los estudios inmunológicos realizados permitieron la estandarización del exoantígeno preparado, se detectaron anticuerpos en los sueros de las ratas a los 15 y 30 días posteriores a la inoculación. Las intradermorreacciones realizadas en los animales provocaron una buena respuesta frente al exoantígeno preparado. De esta forma, se comprueba que la rata es un animal adecuado para la infección experimental y la estandarización de antígenos de S.schenckii

Esporotricosis experimental en ratas / Sporotrichosis experimental on rats

Ratas endocriadas se infectaron por vía intraperitoneal y en la cola con una suspensión de conidios de Sporothrix schenckii. Los frotis coloreados con May Grunwald-Giemsa y Gram Nicolle, así como los cultivos de macerados de los órganos que se biopsiaron: hígado, bazo, pulmón y glándula suprarrenal y de la cola manifestaron a los l5 días de inoculación una diseminación del hongo; no así a los 30 días de infección, excepto el material correspondiente a la cola que presentó en ese tiempo elementos de S.schenckii. Los estudios inmunológicos realizados permitieron la estandarización del exoantígeno preparado, se detectaron anticuerpos en los sueros de las ratas a los 15 y 30 días posteriores a la inoculación. Las intradermorreacciones realizadas en los animales provocaron una buena respuesta frente al exoantígeno preparado. De esta forma, se comprueba que la rata es un animal adecuado para la infección experimental y la estandarización de antígenos de S.schenckii

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method.

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2

dextrose. Overall agreement between both methods was 90.0

. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6

, 19.05

and 52.17

, respectively. No very major discrepancies, 1.03

major discrepancies, and 8.97

minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80

vs 50

endpoints, and trailing growth near the endpoints.

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2

. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6

major discrepancies, and 8.97

minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80

endpoints, and trailing growth near the endpoints.