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%).

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.