Müeller-Hinton methylene blue media as an alternative to RPMI 1640 for determining the susceptibility of Cryptococcus neoformans and Cryptococcus gattii to posaconazole with Etest.

Mueller-Hinton modified agar (MH-GMB) was compared with RPMI + 2% glucose-agar to determine the MICs of 80 isolates of Cryptococcus neoformans and C. gattii to posaconazole with Etest. MH-GMB minimised trailing and agreement between both media was 94%. Agreement of M27-A2 microbroth reference method was 98% with RPMI and 94% with MB-GMB.

MICs and minimum fungicidal concentrations of amphotericin B, itraconazole, posaconazole and terbinafine in Sporothrix schenckii.

The in vitro susceptibility of 62 isolates of Sporothrix schenckii in its mycelial form, from Latin-American countries (Peru, Venezuela, Brazil and Uruguay) and Spain, to amphotericin B (AB), itraconazole (IZ), posaconazole (PZ) and terbinafine (TB) was determined by measuring the MICs and minimum fungicidal concentrations (MFCs) using a standardized Clinical and Laboratory Standards Institute method. In general, TB was the most active drug, with the lowest geometric mean (GM) MIC and MFC values amongst isolates from the five countries tested. IZ and PZ showed almost the same activity against all strains tested, except for isolates from Uruguay where IZ gave the highest GM MIC (10.68 mg l(-1)). AB showed the widest MIC range (0.03-16.0 mg l(-1)); however, this drug was less active against 79 % of isolates (MICs above 1 mg l(-1)). MFCs were 5 to 20 times higher than the MICs, but the lowest GM MFC and range values were found for TB. IZ and PZ gave the highest GM MFC. MFC may be a better predictor of therapeutic response than MIC, especially in immunosuppressed patients, making the use of IZ and PZ an inappropriate treatment. There were some differences in susceptibility according to the geographical source of the isolates, with the MIC being lower for TB in Venezuelan strains (P=0.066) and the MFC higher for PZ in Peruvian strains (P=0.02). Thus, geographical origin may be important for appropriate treatment, and may relate to the identification of species of the S. schenckii complex.

Laccase activity in Cryptococcus gattii strains isolated from goats.

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.

[Urease activity in Cryptococcus neoformans and Cryptococcus gattii]. / Diferencias en la actividad de la enzima ureasa entre Cryptococcus neoformans y Cryptococcus gattii.

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.

Diferencias en la actividad de la enzima ureasa entre Cryptococcus neoformans y Cryptococcus gattii / Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Se considera que la capacidad de Cryptococcus neoformans para sintetizarureasa es uno de sus principales factores de virulencia. Desde 2002 se aceptaque la variedad gattii de C. neoformans reúne los atributos necesarios paraconsiderarse como una nueva especie, Cryptococcus gattii. No existenestudios experimentales controlados que comparen el grado de actividadureasa de ambas especies. El objetivo de este trabajo ha sido analizar laproducción de esta enzima en aislamientos de C. neoformans y C. gattii,considerando la distribución en serotipos de los aislamientos y su origen(clínico o ambiental), utilizando un nuevo método semicuantitativoestandarizado. Veinticinco aislamientos de C. neoformans y 19 de C. gattiifueron sembrados en medio líquido de urea de Christensen para determinar laproducción de ureasa por espectrofotometría. Como referencia se determinóla actividad de una unidad de ureasa (jack beans urease®, Sigma) sobre elmismo medio de cultivo y se observó que el 50% de la actividad enzimáticacorrespondía a una densidad óptica de A550 = 0,215. Esta absorbancia permitióclasificar la actividad ureasa para cada aislamiento de Cryptococcus.Los resultados mostraron que bajo las mismas condiciones, estas levaduraspodían agruparse en dos categorías, bajas productoras de ureasa(A550 < 0,215) y altas productoras (A550 >= 0,215). A las 72 horas de incubación,el 76% de los aislamientos de C. neoformans y el 15,8% de los de C. gattiiresultaron altos productores de ureasa. El análisis estadístico mostródiferencias significativas entre ambas especies (p = 0,016). Los resultadosobtenidos muestran que C. neoformans es mayor productor de ureasa queC. gattii(AU)

Urease is an enzyme considered one of the main virulence factors inCryptococcus neoformans. Quantitative differences in urease productionbetween C. neoformans and the new species Cryptococcus gattii have notbeen so far documented. Using a standardized method, 25 isolates ofC. neoformans and 19 of C. gattii were seeded in Christensen urea brothmedium for urease activity detection. Approximately, the 50% of activity ofone unit of commercial jack beans urease (A550 = 0.215) was considered as areference to classified the Cryptococcus in two cathegories, low (A550 < 0.215)or high (A550 >= 0.215) urease producers. After 72 hours of incubation, 76% ofC. neoformans and 15.8% of C. gattii strains were high urease producers(p = 0.016). Based on these results, the species C. neoformans appeared asthe highest urease producer. Other virulence factors should also beinvestigated to explain C. gattii pathogenicity(AU)

Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcus neoformans y Cryptococcus gattii son los principales agentesde criptococosis, una grave micosis del hombre y los animales. Entre losfactores de patogenicidad de estas especies cabe destacar la lacasa(fenoloxidasa), enzima producida por éstas y otras especies fúngicas, queinduce la síntesis de melanina a partir de compuestos di-hidroxifenólicos.La gran mayoría de los numerosos estudios sobre la lacasa se han efectuadocon C. neoformans, y la información específica en C. gattii es muy escasa.El objetivo de este estudio ha sido evaluar la actividad de la lacasa enaislamientos de C. gattii serotipo B procedentes de cabrasinmunocompetentes muertas por criptococosis durante varios brotesepidémicos desarrollados en Cáceres (Extremadura, España).La producción de lacasa de estos aislamientos se ha comparado con la deotros de la misma especie y también con cepas de C. neoformans.Se procedió a la ruptura de las levaduras por métodos físicos, y elsobrenadante de cada aislamiento se añadió a una solución 20 mM de L-dopa. La actividad enzimática se midió a través de la absorbancia como unidadesenzimáticas (EU) a 450 nm. Los valores máximos de EU se observaron en trescepas de C. gattii aisladas de cabras (EU >12), mientras que el valor mas bajose observó en una cepa ambiental de C. gattii serotipo C (EU = 0,7).Para C. neoformans la mayor actividad lacasa se obtuvo en una cepa delserotipo A aislada en un paciente con meningitis criptocócica.Todos los aislamientos de C. gattii procedentes de los animales muertos enbrotes epidémicos mostraron diferentes grados de actividad lacasa.Esta enzima parece representar un factor de patogenicidad importante,aunque no exclusivo, en esta especie(au)

Cryptococcosis is a life-threatening infection in humans and animals causedby encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformansand Cryptococcus gattii are the main agents of this mycosis Until 2002C. gattii was classified as a variety of C. neoformans but now is accepted asan independent species. The laccase (phenoloxydase) enzyme produced bythese yeasts is considered one of the main pathogenic factors for its ability toinduce melanin from dihydroxyphenolic compounds. The vast majority of thestudies in laccase and melanin synthesis have been developed using isolatesof C. neoformans. The main objective of this study was to evaluate laccaseactivity in strains of C. gattii, serotype B isolated from immunocompetentgoats that died of lung and disseminated cryptococcosis, in several outbreaksoccurring in Spain. The laccase activities of these isolates were compared withthose of other strains of C. gattii and C. neoformans. After fungal cell rupture,the supernatant of each isolate was analyzed for its laccase activity using assubstrate an L-dopa 20 mM solution. The degree of enzymatic activity wasassessed according to its absorbance at 450 nm and scored using EnzymaticUnits (EU). The maximum values were observed in three strains of C. gattiifrom goats (EU > 12). The smallest values were observed in one environmentalisolate of C. gattii serotype C (EU = 0.7). The highest recorded value forC. neoformans was 6.3 EU in a serotype A isolate from one human case ofmeningitis. C. gattii serotype B obtained from goats showed different degreesof laccase activity, being the highest in those isolated from severe outbreaksof cryptococcosis. This enzyme appears to represent a major, thoughnonexclusive, pathogenic factor for Cryptococcus gattii(AU)

[Nasal fungal microbiota in allergic and healthy subjects]. / Microbiota fúngica nasal en sujetos alérgicos y sanos.

Environmental fungi, moulds and yeasts could reach the nasal cavity with the inhaled air causing respiratory symptoms in atopic subjects, but little is known about the fungal flora of this site. In the present study samples of the nasal cavities of 135 subjects aged 18-35 years (48 allergic patients to fungi, mites and/or cat fur and from 87 normal subjects--healthy, control group) were cultured. All of them lived in the metropolitan area of Barcelona. Fungi were isolated from 41.5% of healthy people and in 14.8% of allergy patients (p = 0.011). Morphologically, 50.4% of the isolates were located within 4 genera: Cladosporium, Penicillium, Aspergillus and Alternaria, fungi which are considered the most allergenic. The most prevalent species were: Cladosporium herbarum and C. cladosporioides (23.6%). Alternaria alternata was isolated only in 8.8% of samples from the allergic group, although most subjects were sensitive to this species. There were not differences in the isolation rate between genera and smoking-no-smoking groups. The lower prevalence of nasal fungi from allergic patients could be related to the nasal insufficiency, the hypersecretion and the larger use of handkerchiefs.

In vitro susceptibilities to yeasts using the ATB FUNGUS 2 method, compared with Sensititre Yeast One and standard CLSI (NCCLS) M27-A2 methods.

OBJECTIVES: The microbroth ATB FUNGUS 2 (ATBF2) method (bioMérieux, La Balme-les Grottes, France), designed for in vitro determination of the susceptibility of Candida spp. and Cryptococcus neoformans to antifungal agents, was evaluated with 100 yeasts and compared with Sensititre Yeast One (SYO; Trek Diagnostic Systems, UK), considering CLSI M27-A2 as the reference method. METHODS: ATBF2 consists of ready-to-use strips including amphotericin B (AMB), 5-flucytosine, fluconazole and itraconazole for MIC determinations. Reproducibility of ATBF2 was determined. Two quality control strains and a panel of eight Candida isolates were tested five different times with the three methods. The essential agreements within +/-2 log2 dilution between the ATBF2, SYO and M27-A2 methods were assessed. The yeast clinical isolates included were nine species of Candida (n = 80) and C. neoformans (n = 20). RESULTS: Inter- and intra-laboratory reproducibility, tested with the Candida panel, was >or=99%. MICs for the ATCC strains were within the expected ranges with the three methods. Visual and automated readings of ATBF2 presented good concordance, being lower with itraconazole. The overall essential agreements with the M27-A2 method were 94% and 99% for automated ATBF2 and visual ATBF2 readings, respectively. For SYO, the agreement was 91%. Percentages of agreements by drugs (automated ATBF2/visual ATBF2/SYO) were: 5-flucytosine, 97/100/90; AMB, 97/100/85; fluconazole, 93/97/95; and itraconazole, 89/98/95. Disagreement was higher between M27-A2 and SYO than between M27-A2 and ATBF2. CONCLUSIONS: ATBF2 is an objective, reproducible and simple method for the accurate determination of MICs of the most common antifungal drugs in yeasts.

In vitro susceptibility of Sporothrix schenckii to six antifungal agents determined using three different methods.

The in vitro susceptibility of Sporothrix schenckii to antifungal drugs has been determined with three different methods. Nineteen Peruvian clinical isolates of S. schenckii were tested against amphotericin B (AB), flucytosine (FC), fluconazole (FZ), itraconazole (IZ), voriconazole (VZ), and ketoconazole (KZ). Modified NCCLS M38-A, Sensititre YeastOne (SYO), and ATB Fungus 2 (ATBF2) methods were used to determine the MICs. ATCC isolates of Candida parapsilosis, Candida krusei, and Aspergillus flavus were used for quality control. Sporothrix inocula were prepared with the mycelial form growing on potato dextrose agar at 28 +/- 2 degrees C. MICs of AB, FC, FZ, and IZ were determined with all three methods, VZ with M38-A and SYO, and KZ with only SYO. The three methods showed high MICs of FZ and FC (MIC(90) of 0.5 microg/ml), being homogeneously lower than those of IZ and KZ. The M38-A method showed a variable MIC range of VZ (4.0 to 16 microg/ml); the geometric mean (GM) was 9.3 mug/ml. The MIC range of AB was wide (0.06 to 16 microg/ml), but the GM was 1.2 microg/ml, suggesting that the MIC is strain dependent. Agreement (two log(2) dilutions) between commercial techniques and the modified M38-A method was very high with FZ, IZ, and FC. In AB and VZ, the agreement was lower, being related to the antifungal concentrations of each method. The highest activity against S. schenckii was found with IZ and KZ. Lack of activity was observed with FZ, VZ, and FC. When AB is indicated for sporotrichosis, the susceptibility of the strain must be analyzed. Commercial quantitative antifungal methods have a limited usefulness in S. schenckii.

Microbiota fúngica nasal en sujetos alérgicos y sanos / Nasal fungal microbiota in allergic and healthy subjects

Los hongos ambientales, mohos y levaduras penetran con el aire en lacavidad nasal y pueden desencadenar alergias respiratorias en sujetosatópicos. No obstante, la flora fúngica de las fosas nasales es muy pococonocida.En este estudio, se han cultivado muestras de la mucosa nasal de135 sujetos, de los cuales 48 eran alérgicos a ácaros y epitelios u hongos,y los restantes, sin antecedentes de alergia, se consideraron sanos. Todosellos residían en el área metropolitana de Barcelona, y su edad era de entre18 y 35 años.Los resultados obtenidos demuestran que el 41,5% de los sujetos sanos eranportadores de una o más especies fúngicas, mientras que los alérgicos quepresentaron hongos fueron el 14,8% (p = 0,011). El 50,4% de los aisladoscorrespondieron a los géneros Cladosporium, Penicillium, Aspergillus yAlternaria, los cuales se consideran altamente alergénicos.Las especies fúngicas más comunes fueron: Cladosporium herbarum yCladosporium cladosporioides (23,6%). A pesar de que casi la mitad depacientes eran alérgicos a Alternaria alternata, ésta tan sólo se aisló en el8,8% de las muestras de dicho grupo. Las levaduras se aislaronpredominantemente en sujetos sanos.No se observaron diferencias entre sexos ni entre fumadores y no fumadores.Destaca la menor prevalencia de hongos nasales en sujetos alérgicos, quepodría ser debida a la insuficiencia nasal, rinorrea y/o mayor uso de pañuelos(AU)

Environmental fungi, moulds and yeasts could reach the nasal cavity with theinhaled air causing respiratory symptoms in atopic subjects, but little is knownabout the fungal flora of this site.In the present study samples of the nasal cavities of 135 subjects aged18-35 years (48 allergic patients to fungi, mites and/or cat fur and from 87normal subjects - healthy, control group) were cultured. All of them lived in themetropolitan area of Barcelona.Fungi were isolated from 41.5% of healthy people and in 14.8% of allergypatients (p = 0.011).Morphologically, 50.4% of the isolates were located within 4 genera:Cladosporium, Penicillium, Aspergillus and Alternaria, fungi which areconsidered the most allergenic.The most prevalent species were: Cladosporium herbarum andC. cladosporioides (23.6%). Alternaria alternata was isolated only in 8.8% ofsamples from the allergic group, although most subjects were sensitive to thisspecies.There were not differences in the isolation rate between genera andsmoking-no-smoking groups.The lower prevalence of nasal fungi from allergic patients could be related tothe nasal insufficiency, the hypersecretion and the larger use of handkerchief(AU)