Autochthonous and dormant Cryptococcus gattii infections in Europe.

Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe (57 from 40 human patients, 22 from the environment, and 21 from animals) with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.

Respiratory allergy to fungi in Barcelona, Spain: clinical aspects, diagnosis and specific treatment in a general allergy unit.

BACKGROUND: The importance of hypersensitivity to fungal allergens is a relatively unknown and somewhat controversial subject. METHODS: An open prospective study was carried out in just one centre to determine the clinical and epidemiological characteristics as well as the diagnostic usefulness of skin prick and conjunctiva provocation tests, associated with total and specific IgE determination in two groups of patients, one of which was monosensitised to fungi and the other of which had multiple sensitisations, including fungi. RESULTS: Rhinitis, exclusive or associated with asthma, was the main consultation cause (88% in monosensitised patients). Severe asthma was rarely found. In the polysensitised group, 64% were simultaneously allergic to moulds and mites. Alternaria alternata was the most common sensitising fungus, although a considerable number of cases were associated with other species such as Cladosporium, Penicillium and/or Aspergillus. The skin prick test gave the highest sensitivity and specificity. In 67% of the cases, the specific IgE was found between classes 3 and 4. The conjunctival provocation test was an innocuous and highly useful method for verifying the diagnosis and determining the degree of clinical sensitisation. A large number of patients exclusively allergic to fungi received specific immunotherapy, and it was generally well tolerated. CONCLUSIONS: This protocolised study shows the importance of Alternaria and other fungi sensitisations in rhinitis alone or associated with asthma. Combined diagnosis of prick test, specific IgE and conjunctiva provocation test is very useful for deciding specific immunotherapy.

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.

[Histoplasmosis 45 years after infection in an immunocompetent man]. / Histoplasmosis en un varón inmunocompetente manifestada 45 años después de la infección.

We report a case of a lung reactivation of a latent histoplasmosis in a 68-year-old patient without immunologic dysfunction living in Barcelona (Spain). The Histoplasma capsulatum var. capsulatum infection was probably acquired in a previous stay in Equatorial Guinea 45 years before. Diagnosis of mild chronic pulmonary histoplasmosis was performed by histopathology of lung biopsy plus antibodies detection against H. capsulatum. Treatment with oral itraconazol during three months leaded to a complete clinical cure, and antibodies disappeared after four years of follow up.

[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.

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.

[Candiduria in type 2 diabetes mellitus patients and its clinical significance. Candida spp. antifungal susceptibility]. / Candiduria en pacientes con diabetes mellitus tipo 2. Sensibilidad antifúngica in vitro.

BACKGROUND: Candida is frequently seen in urinalysis studies in patients with diabetes mellitus. The objective was to determine the presence and clinical significance of candiduria, and to identify the different isolated Candida species and their in vitro susceptibility pattern to different antifungal agents by means of the broth microdilution method. METHODS: We studied the urine from 50 type 2 diabetes mellitus (DMT2) patients. 24 patients had controlled DMT2 and 26 non-controlled DMT2. RESULTS: Twenty-three Candida spp. positive cultures were obtained, of which 17 were obtained from the non-controlled DMT2 patients; 30.7% of the isolates were caused by Candida infection. The main isolated species were C. glabrata (48%) and C. albicans (35%). Itraconazole, amphotericine B, and ketoconazole showed less antifungal activity in C. glabrata isolates, whereas fluconazole and voriconazole displayed higher antifungal activity. CONCLUSIONS: It is important to search routinely for yeast in the urine of DMT2 patients to detect candidiasis, and to perform antifungal susceptibility tests to Candida isolates in order to establish antifungal therapy for these patients.

[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.

[Bilateral proximal cellulitis and onychomycosis in both big toes due to Fusarium solani]. / Celulitis y onicomicosis proximal de ambos ortejos mayores producida por Fusarium solani.

We report a case of proximal fold cellulitis in both big toes, associated with a bilateral proximal onychomycosis and an intertrigo of the fourth space due to Fusarium solani. The infection occurred in an immunocompetent man with diabetes mellitus type II. Apparently, the infection was acquired in a tropical country and once the patient was in Spain the infection progressed causing nail detachment (onychomadesis). Seven months later a relapse that affected the left toenail occurred. The patient was treated topically with chemical toenail avulsion contained 40% urea associated with bifonazole followed by ciclopirox-olamine nail lacquer for 12 months. Complete cure without relapse was observed after 10 years of follow-up. In vitro antifungal susceptibility study demonstrated that two of the recovered isolates were both resistant to itraconazole and voriconazole.

[Onychomycosis-causing yeasts in four Mexican dermatology centers and their antifungal susceptibility to azolic compounds]. / Levaduras causantes de onicomicosis en cuatro centros dermatológicos mexicanos y su sensibilidad antifúngica a compuestos azólicos.

BACKGROUND: Yeasts represent the second cause of nail fungal infection in the world, and Candida albicans and Candida parapsilosis are the two most common species. OBJECTIVES: To determine the yeast species frequency and their in vitro antifungal susceptibility, obtained from patients with clinical features suggestive of onychomycosis. METHODS: A prospective study was carried out in four dermatological care centers in Mexico from 2004 to 2007. Clinical diagnosis was corroborated by direct examination and culture. The yeast species was determined by morphological and biochemical tests. An antifungal susceptibility test to ketoconazole, itraconazole and fluconazole by the broth microdilution method was performed on each isolate (document M27-A2). RESULTS: One hundred sixty-six yeast isolates were obtained; the most frequently found species were C. parapsilosis (31.9%), C. albicans (22.4%) and Candida guilliermondii (12.7%). Of all isolates, 51 showed resistance to one or several of the azole compounds: 33 to itraconazole, 12 to ketoconazole and 6 to fluconazole. It was remarkable that the four Candida glabrata isolates were resistant to the three azole compounds; C. guilliermondii and Candida famata were resistant to itraconazole in 42.9% and 54.5%, respectively. CONCLUSION: The results obtained show the importance of identifying the aetiological agent and antifungal susceptibility testing in order to avoid therapeutic failures in onychomycosis.

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.

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.

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.

Cryptococcus gattii: in vitro susceptibility to the new antifungal albaconazole versus fluconazole and voriconazole.

Minimal inhibitory concentrations (MIC) and minimal fungicidal activity of albaconazole, voriconazole and fluconazole against 55 strains of Cryptococcus gattii, clinically or environmentally isolated in Spain and some Latin American countries, were assessed. By means of the microbroth method (National Committee for Clinical Laboratory Standards; document M27-A2), the geometric mean value for fluconazole was 5.01 microg/ml; however, MIC for 12.7% of isolates ranged from 16 to 32 microg/ml, suggesting increased resistance against fluconazole. Geometric mean values of 0.02 and 0.03 microg/ml for albaconazole and voriconazole, respectively, were found, indicating not only a higher susceptibility to these new azoles but also a better performance of albaconazole (P = 0.003). Minimal fungicidal concentrations were also very low for albaconazole and voriconazole (P<0.001; geometric mean values of 0.023 microg/ml and 0.07 microg/ml, respectively). Both azoles may be good alternatives for the treatment of C. gattii cryptococcosis.

First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain.

We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection.

Utilidad de las muestras de sangre total desecada en papel de filtro en la detección de anticuerpos antitoxoplasma / Usefulness of total dry blood samples on filter paper in the detection of antitoxoplasma antibodies

Se compara la técnica de Aglutinación Directa (AD) utilizando muestras de sangre total desecada en papel de filtro, con la técnica de ELISA y la misma AD utilizando muestras de suero de los mismos pacientes, para la detección de anti-cuerpos antitoxoplasmas. Los rsultados muestran la validez del método de la sangre desecada en papel de filtro para la detección de anticuerpos antitoxoplasma con la técnica de AD, y se considera su utilidad en los estudios epidemiológicos de campo

Sensibilidad de hongos miceliares dematiaceos a diez antifungicos empleando un metodo de difusion en agar / In vitro susceptibiblity of dematiaceous fungi to ten antifungal drugs using an agar difussion test

Se ha evaluado la utilidad del metodo de difusion en agar NeoSensitabs para determinar la sensibilidad in vitro de 52 aislamientos de hongos filamentosos dematiaceos a diez antifungicos: anfotericina B.5-fluorocitosina, Ketoconasol, fluconazol, itraconazol, terbinafina, bifonazol, miconazol, clotrimazol, y griseofulvina. Para la preparacion del inoculo se ultilizo un metodo espectrofotometrico empleandose los medios de Shadomy y Casitone agar (CAS), simultaneamente. Todos los aislamientos fueron sensibles, al itraconazol, terbinafina y bifonazol. Al ketonazol el 90,4% resultaron sensibles al miconazol el 71% y al clotrimazol el 46%. El 63% de las cepas fueron sensibles a la anfotericina B y el 28,8% resistente. Por el contrario, el 94,2% de los aislamientos resultaron resistentes a la griseofulvina y el 96% al flucanazol. El 100% de las cepas fueron resistentes a la 5-fluorocitosina. Las zonas de inhibicion no mostraron variaciones en cuanto a la sensibilidad dependiendo del medio; sin embargo, hubo un mejor desarrollo fungico en el medio CAS. Las variaciones en la sensibiblidad observadas con especies como Exophiala spinifera y Fonsecaea pedrosi justificarian el estudio de la sensibiblidad in vitro para valorar el tratamiento clinico con antifungicos. Estos resultados demuestran que el metodo de difusion en agar NeoSensitabs es facil de realizar, rapido y economico por lo que esta al alcance de muchos laboratorios clinicos para el estudio de la sensibilidad in vitro en mochos dematiaceos