Método de difusión con discos para la determinación de sensibilidad a fluconazol en aislamientos de Candida spp / Disk diffusion method for fluconazole susceptibility testing of Candida spp. isolates

Se estudiaron 1193 aislamientos clínicos para estandarizar y evaluar un método de difusión con discos de fluconazol de lectura visual, que permita detectar levaduras sensibles al antifúngico. Las especies analizadas fueron: Candida albicans (n=584), Candida parapsilosis (n=196), Candida tropicalis (n=200), Candida glabrata (n=113), Candida krusei (n=50), Candida spp. y otras levaduras oportunistas (n=50). Los discos fueron manufacturados en el INEI-ANLIS "Dr. Carlos G. Malbrán". Se midieron los halos de inhibición del crecimiento producidos por fluconazol y la concentración inhibitoria mínima (CIM) por el método de referencia M27-A2 modificado por EUCAST. Se establecieron los valores de corte del método de difusión en: ≥16 mm para levaduras sensibles a fluconazol (CIM ≤ 8 µg/ml), entre 9 y 15 mm para sensibles dependientes de la dosis (CIM = 16-32 mg/ml) y ≤ 8 mm para resistentes (CIM ≥ 64 µg/ml). El método de difusión tuvo 94,7% de concordancia con el de referencia, con 0,2% de errores very major y 0,3% de errores major. La reproducibilidad inter e intralaboratorio fue muy buena. Para detectar aislamientos sensibles a fluconazol, este método resulta confiable y de bajo costo; sin embargo, es conveniente que los aislamientos con halos ≤ 15 mm sean reevaluados por el método de referencia.

In order to standardize and evaluate a disk diffusion method with visual reading to detect in vitro fluconazole susceptibility of yeast, 1193 clinical isolates were tested. These included 584 Candida albicans, 196 Candida parapsilosis, 200 Candida tropicalis, 113 Candida glabrata, 50 Candida krusei and 50 Candida spp. and other opportunistic yeasts. The disks were manufactured in the INEI-ANLIS "Dr. Carlos G. Malbrán". The disk diffusion method results were compared to MIC results obtained by the reference CLSI M27-A2 broth microdilution method modified by EUCAST. The interpretative breakpoints for in vitro susceptibility testing of fluconazole were established at: zone diameter ≥ 16 mm for MIC ≤ 8 µg/ml (susceptible isolates), between 9 and 15 mm for MIC = 16-32 mg/ml (susceptible dose-dependent isolates), and ≤ 8 mm for MIC ≥ 64 µg/ml (resistant isolates). Overall agreement between the two methods was 94.7%, with 0.2% very major errors, and 0.3% major errors. Inter - and intralaboratory agreement was good. The disk diffusion method for drug susceptibility testing of Candida spp. isolates is inexpensive, reliable and reproducible. However, when the inhibition zone diameter is ≤ 15 mm, it is advisable to test the isolate by the reference microdilution method.

Estudio multicéntrico de fungemias por levaduras en la República Argentina / Multicenter study of bloodstream infections due to yeasts in Argentina

La incidencia de candidemias aumentó aproximadamente en un 500% en hospitales de alta complejidad y se observó un cambio en la distribución de especies del género Candida, con un incremento de las levaduras no Candida albicans. Con el objeto de conocer la distribución de especies asociadas a fungemias por levaduras en Argentina y determinar su sensibilidad a los antifúngicos de uso convencional, se realizó un estudio multicéntrico durante el período abril 1999 a abril 2000. Participaron 36 instituciones del país. Se colectaron 265 aislamientos de levaduras provenientes de hemocultivos, que se identificaron utilizando pruebas morfológicas, fisiológicas y bioquímicas y la determinación de la concentración inhibitoria mínima se realizó en base al estándar del NCCLS. La distribución de especies fue: Candida albicans (40,75%), Candida parapsilosis (28,67%), Candida tropicalis (15,84%), Candida famata (3,77%), Cryptococcus neoformans (3,77%), Candida glabrata (2,64%) y otras (4,53%). La mayoría de los aislamientos fueron sensibles a anfotericina B, fluconazol e itraconazol. La mortalidad asociada a las fungemias por levaduras estudiadas (n=265) fue del 30%, siendo más baja a lo descrito (33-54%) y fue menor en los pacientes que recibieron tratamiento antifúngico (26,3%), que en los no tratados (47%).

The incidence of candidemia has increased approximately 500% in high-complexity hospitals. A change in the spectrum of Candida infections due to species other than Candida albicans has also been detected. Between April 1999 and April 2000 a multicenter study was performed in order to determine the species distribution associated to candidemias in Argentina and the susceptibility profile of the isolates to the current antifungal drugs. Thirty six institutions have participated. All the 265 yeast strains isolated from blood cultures were identified by morphological, physiological, and biochemical tests. The antifungal susceptibility testing of isolates was performed based on the reference NCCLS procedure. The distribution of species was: Candida albicans (40.75%), Candida parapsilosis (28.67%), Candida tropicalis (15.84%), Candida famata (3.77%), Cryptococcus neoformans (3.77%), Candida glabrata (2.64%), and others (4.53%). Most of the isolates were susceptible to amphotericin B, fluconazole and itraconazole. Mortality associated to the fungemia by yeasts episodes (n=265) was 30%, lower than results previously determined (33-54%). The mortality percentage in patients who received antifungal therapy versus patients without treatment was 26.3% and 47%, respectively.

Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina / Firs environmental detection of Cryptococcus neoformans var. gattii in Argentina

Dos cepas de C.neoformans var. gattii serotipo B, fueron aisladas en el Parque de los Patricios de la ciudad de Buenos Aires, Argentina. Las muestras se recogieron en primavera, mediante el hisopado de la superficie interior de huecos y grietas de Eucalyptus spp. y otros árboles que, lamentablemente, no fueron identificados en el momento de la recolección. Los hisopos se inocularon en placas de agar ácido cafeico suplementado con antibióticos que se incubaron a 28§C durante una semana con observación diaria. Los aislamientos produjeron colonias características de la especie, lo que permitió detectarlos y separarlos de otros microorganismos presentes en la muestra. La identificación se realizó mediante estudios de fermentación de carbohidratos, asimilación de fuentes de carbono y nitrógeno y detección de ureasa y fenoloxidasa. La variedad se determinó utilizando el medio agarizado de canavanina-glicina-azul de bromotimol (CGB). La caracterización genotípica por reacción en cadena de la polimerasa con primer aleatorio (AP-PCR con el primer (GACA)4)mostró que el patrón de bandas de ambos aislamientos se diferenciaba en una única banda. En ambos casos el material provino de especies arbóreas diferentes de Eucalyptus spp. Estos dos aislamientos de fuentes naturales demuestran que C.neoformans var. gattii está presente en el medio ambiente de la ciudad de Buenos Aires, no necesariamente asociado a Eucalyptus spp. Este hallazgo podría explicar su asociación esporádica a infecciones humanas en la población local.

Transmisión nosocomial de Candida albicans en recién nacidos / Nosocomial transmission of Candida albicans in newborn infants

Candida spp. colonization in neonates occurs due to vertical or horizontal transmission. Preliminary studies determined that Candida albicans is the principal agent of these infections. In order to establish nosocomial transmission, 26 Candida albicans strains isolated from patients with candidosis hospitalized during a 18-month period in 2 neonatal intensive care units (NICU) from a pediatric hospital were studied. Fourteen isolates from patients and health care workers, involved in possible outbreaks of an intensive care unit (UCI) and a NICU from another pediatric hospital were also studied. All Candida albicans strains were genotyped by Southern blot hybridization with 27A. Isolates for outbreak confirmation were also hybridized with another specific Candida albicans probe, Ca3. Hybridization patterns demonstrated horizontal transmission in all the units studied. In a NICU, transmission among 4 patients during a 10-month period could be established and in the other NICU, 3 cases of transmission among 2 patients each were demonstrated in periods of 2 to 20 days. The outbreak studies showed the same strain isolated from 2 nurses and from one patient at the NICU and at the ICU identical strains were found in 3 patients. In this study, hybridization with Ca3 in addition to 27A probe did not increase discrimination power among isolates. Genotypic analysis allows, not only, determination of transmission and persistence of strains during prolonged periods or in sporadic outbreaks, but also facilitates necessary epidemiological decisions for optimizing nosocomial fungal infection control measures.

Estudio multicéntrico de micosis superficiales en Argentina. Grupo EMMS / Multicenter study of superficial mycoses in Argentina. EMMS Group

Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58 female, 42 male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6 of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3 of the diagnosticated infections, yeast 12.2 and Malassezia furfur 13.3. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7), T. mentagrophytes (19.2) and M. canis (14.2). C. albicans was the predominant causative agent of yeast infections (44.4). In children below 11 years old, the most frequent infections were tinea capitis 55.4 (M. canis 60, T. tonsurans and M. gypseum 8.3) and tinea corporis 20 (M. canis 46, T. mentagrophytes 30). The most frequent localizations in adult were toenail and feet (63) (T. rubrum 47.3 and 45.7, T. mentagrophytes 9.5 and 23.4, respectively), fingernail 15.6 (C. albicans 25 and T. rubrum 8.3) and trunk and extremities 12.2 (M. furfur 57.5 and T. mentagrophytes 14.9). Direct examination allowed the diagnosis of 94.66 of all positive samples and 91.74 of the cases with positive isolation.

Estudio multicéntrico sobre candidiasis nosocomial en la República Argentina. Groupo EMIFN / Multicenter study on nosocomial candidiasis in the Republic of Argentina

A multicenter study was performed between April and September 1998 with the participation of 12 medical centers located in 8 different provinces and in the capital city of Argentina. The aim of this study was to determine the species distribution and the antifungal susceptibility profile of isolates causing nosocomial fungal infections. All the fungal isolates obtained were sent to the Mycology Department for reference identification and antifungal susceptibility testing. Eighty-nine isolates were received from different clinical specimens. The distribution of species obtained was C. albicans (50.6), C. tropicalis (22.5), C. parapsilosis (20.2), C. krusei (3.4), C. glabrata (2.2) and Debaryomyces hansenii (1.1). Most of the isolates (85/88) had MICs for amphotericin B < or = 1 microgram/ml, C. krusei showed resistance to fluconazole but was dose dependent susceptible to itraconazole, C. glabrata (2/2) were resistant against both drugs, most of the isolates of C. albicans, C. tropicalis and C. parapsilosis were susceptible to these triazole drugs. These data showed a different distribution of Candida species compared with results obtained in other countries. The low frequency of appearance of C. krusei and C. glabrata in our country suggests a reduced selective pressure by triazoles.