Well diffusion for antifungal susceptibility testing.

INTRODUCTION: The increasing clinical and microbiologic resistance of Candida spp. isolates to several antifungal agents is becoming a serious problem. It is now reasonable to propose the use of antifungal susceptibility testing in Candida spp. isolates from patients who have failed conventional therapy, before the selection of an empirical therapy. METHODS: One hundred and fifty eight isolates of Candida spp. were evaluated simultaneously by broth microdilution (NCCLS standard) and well diffusion testing (WD), a diffusion method similar to disc diffusion. RESULTS: According to the Wilcoxon Signed Ranks test performed, there was no significant difference (p>0.05) between both methodologies for all antifungal agents tested (fluconazole, itraconazole, posaconazole, caspofungin and amphotericin B, with C. tropicalis, C. krusei, C. dubliniensis, C. guillermondii, C. parapsilosis, C. albicans and C. glabrata). A significant difference was observed when comparing well diffusion with NCCLS for fluconazole WD 80% (p=0.008) in C. glabrata, as well as WD 80% (p=0.002) and WD 50% (p=0.002) in C. albicans. CONCLUSIONS: The well diffusion test is simple, easy to reproduce, inexpensive, easy both to read and interpret, and has a good correlation to the reference NCCLS microdilution test and may represent an alternative method for antifungal drug susceptibility testing of Candida spp., mainly in laboratories with few resources.

Mantenimiento de crytococcus sp con el método de castellani / Preservation of cryptococcus sp with the castellani method

Las dificultades para mantener cepas de bacterias y de hongos viables durante diferentes períodos de tiempo, sin cambios morfológicos ni fisiológicos, son bien conocidas. Existen métodos de conservación con diferentes niveles de eficacia, tales como el subcultivo periódico, la liofilización, la inmersión en aceite mineral y el método de Castellani [1-3]. En 1989, Hartung y colaboradores lograron demostrar la eficiencia de este método para la conservación de hongos, mantenidos en la Sección de Micología Médica del Instituto de Medicina Tropical de la Universidad Central de Venezuela desde 1962 [4,5]. En la literatura existen pocos trabajos que reportan la efectividad de dicho método [5-8]. El presente estudio evalúa la estabilidad morfológica, viabilidad y pureza de 26 cepas de cryptococcus sp. provenientes de la micoteca de esta sección, las cuales fueron aisladas de diversas muestras clínicas y conservadas utilizando el método de Castellani, desde 1962 hasta 2002

Identificación de cryptococcus neoformans var. gattii mediante el uso del medio canavaninaglicina-azul de bromotinol (CGB) / Identification of cryptococcus neoformans var. gattii by canavanine glycine bromothymol blue medium (CGB)

Se conocen 2 variedades de cryptococcus neoformans y 5 serotipos: cryptococcus neoformans var. neoformans (serotipos A,D y AD), usualmente aislados a partir de muestras clínicas, encontrándose cryptococcus neoformans var. neoformans serotipo A, con mayor incidencia en pacientes con SIDA; cryptococcus neoformans var. gattii (serotipos B y C), los cuales son aislados menos frecuentemente principalmente en pacientes VIH seronegativos y excepcionalmente han sido asociados con SIDA. Se estudiaron 43 cepas de cryptococcus neoformans. El medio de canavanina-glicina-azul de bromotinol (CGB) permite diferenciar en crytococcus la variedad gattii de la variedad neoformans. Nuestro estudio refleja que las 2 cepas (4,65 por ciento) de cryptococcus neoformans var. gattii, obtenidas a partir del aislamiento de muestras de líquido cefalorraquídeo, procedían de una paciente con inmunodeficiencia celular y humoral catalogada como ideopática, y de un paciente con SIDA, asociación rara vez reportada. Son escasos los estudios epidemiológicos de esta micosis en Venezuela, y pocos hacen identificación más allá de la especie, por lo cual es dificil comparar nuestros resultados

"In vitro" antifungal activity of protease inhibitors.

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept, Roche), Indinavir (Crixivan Merck), Ritonavir (Norvir, Abbott), Saquinavir (Fortovase, Roche). Recently, we observed that recurrent candidiasis was less frequent and no Candida could be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the "in vitro" antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test, while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micromethod and 16 by the well diffusion test. Our results showed anticandidal activity "in vitro" of PIs, mainly Saquinavir.

Viability studies on actinomycetes.

Eighty-nine Actinomycetes strains were tested for their viability, morphological and physiological characteristics after being kept under paraffin oil overlay and distilled water for a period between 10-30 years. Most of the studied strains belong to the "Lorenzo De Montemayor" collection. Almost all the recovered strains were 28-30 years old and had never been subcultured since the paraffin oil was overlaid. 71.4% of viable Streptomycetes strains had been kept on Sabouraud-dextrose agar and 28.6% were kept on Negroni and Bonfiglioli-medium. Streptomyces violaceusruber produced its characteristic pigment even after 28 years under these conditions. All of the recovered strains were tested for their biological activity, but only Streptomyces lavendulae showed growth-inhibition against Staphylococcus aureus and Bacillus subtilis.

An atypical Microsporum canis isolate.

An atypical strain of Microsporum canis isolated from a two-year-old boy with tinea corporis is described. When cultured on Lactritmel agar the strain presented the typical pigment of M. canis without developing characteristic macroconidia. After 6 weeks, scarce, rudimentary, fusiform macroconidia 120-150 microns long developed on Lactritmel and Sablac microcultures. In vitro, the strain developed hair perforating organs. Experimental inoculation of a healthy volunteer produced a tinea with typical fluorescence and endoectothrix attack. After isolation the strain remained stable. These results differ from the results reported by English and Tucker. This is the first atypical strain of M. canis reported in Venezuela.

Preservation of fungi in water (Castellani): 20 years.

Five-hundred ninety-four strains of fungi were studied. They were found being preserved with Castellani's method with distilled water during 1 to 20 years. 62% of the strains (n=368) did grow when subcultured and maintained their main morphological features. 90% of the 20 years old strains of different species were viable. It is argued that the technique of introduction of the strains into the water and their optimal condition will determine survival. The Castellani's method is recommended as easy, cheap and satisfactory for preservation of most species of fungi.