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Cryptococcosis is a fungal disease affecting more than one million people per yearworldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryp-tococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-definingcondition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test(LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. Theaim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supplythe NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performanceand its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY)(Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLISand LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictivevalues of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effectivereagent, especially useful in countries where the commercial kit is not generally available andmust be obtained at a high cost. National production of reagents is the best choice for a reliableaccess to the rapid diagnosis of CM in Argentina. Resumen La criptococosis es una enfermedad fúngica que afecta a más de un millón de personas por año en todo el mundo. Los principales agentes etiológicos pertenecen a los complejos de especies Cryptococcus neoformans y Cryptococcus gattii. La criptococosis meníngea (CM) se considera una enfermedad marcadora de sida. El diagnóstico rápido de esta enfermedad a través de la detección del antígeno de Cryptococcus, ya sea por aglutinación en partículas de látex o por inmunocromatografía, es clave para disminuir la mortalidad. El objetivo del presente estudio fue desarrollar un reactivo de aglutinación en partículas de látex para el diagnóstico rápido y certero de la CM en Argentina. Este reactivo (denominado en adelante LA-ANLIS) será producido para abastecer a la Red Nacional de Laboratorios de Micología. Se evaluó el desempeno del reactivo LA-ANLIS, y se realizó una comparación con el reactivo comercial Immuno-Mycologics, Inc. (en adelante, LA-IMMY) utilizando 94 muestras de líquido cefalorraquídeo. Hubo un 100% de acuerdo positivo y un 97% de acuerdo negativo entre los resultados obtenidos con los reactivos LA-ANLIS y LA-IMMY. El reactivo LA-ANLIS mostró una sensibilidad del 94% y una especificidad del 97%; los valores predictivos positivo y negativo fueron del 94 y del 97%, respectivamente. Se concluye que el LA-ANLIS es un reactivo confiable y rentable, que arroja resultados reproducibles, por lo que es especialmente útil en países donde los reactivos comerciales generalmente no están disponibles o sus costos son elevados. La producción nacional de reactivos es la mejor opción para asegurar el acceso de todos los hospitales al diagnóstico rápido de la CM en Argentina.

The National Quality Control Program in Mycology (PNCCM) of Argentina was establishedin 1996 to improve the quality of the mycological diagnosis, to help establish and to setup standardized procedures and continuous training of laboratory staff. The aim of this studywas to assess the effectiveness of the PNCCM in the 1996---2018 period. Data from the NationalMycology Laboratory Network (NMLN) and PNCCM database was used to estimate the increasein the number of controlled laboratories and jurisdictions, the percentage of participation, theimprovement in the quality of results and the adherence to the program. Satisfaction surveyswere performed to assess user satisfaction. The number of controlled laboratories increasedfrom 29 to 146; participation increased from 49% to 93% and general adherence was 72% inthe evaluated period (1996---2018). Improvement in the quality of the results was 15% for lowcomplexity samples; 7% for intermediate complexity samples and 14% for the identification ofhigh complexity strains. Up to 84% of the users consider the PNCCM to be ‘‘very good’’ and 16%‘‘satisfactory’’. These results show the importance of the PNCCM, which is widely accepted bymycological diagnostic laboratories from Argentina. Resumen En 1996 se creó el Programa Nacional de Control de Calidad en Micología (PNCCM)de Argentina con el objetivo de mejorar la calidad del diagnóstico micológico, colaborar enel establecimiento de procedimientos estandarizados en aquellos laboratorios que carecen deellos y contribuir a la capacitación continua del personal.El objetivo de este estudio fue evaluar la efectividad del PNCCM en el período 1996-2018.Se utilizaron los datos de la base de la Red Nacional de Laboratorios de Micología (RNLM) ydel PNCCM para estimar el aumento en el número de laboratorios controlados y el porcentajede participación, la mejora de la calidad de los resultados y la adhesión al programa. Paraevaluar el grado de satisfacción de los usuarios, se analizaron las encuestas de satisfacción delos participantes. En el período evaluado, el número de laboratorios controlados aumentó de 29a 146, la participación aumentó de 49% a 93% y la adherencia general de los participantes fue del72%. La mejora de la calidad de los resultados de los laboratorios fue del 15% para muestras debaja complejidad, 7% para muestras de complejidad intermedia y 14% para la identificación decepas de alta complejidad. El 84% de los usuarios considera que el PNCCM es muy bueno y el 16%que es satisfactorio. Estos resultados evidencian la importancia del PNCCM, que es ampliamenteaceptado por los laboratorios que realizan diagnóstico micológico en nuestro país.

Fil: Davel, Graciela O. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Mazza, Mariana. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Refojo, Nicolás. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Taverna, Constanza G. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Rivas, María C. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Fernández, Julián. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Vivot, Matías E. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Mazza, Marta G. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Mastromónaco, Gladys M. Instituto Nacional de Tecnología Industrial (INTI); Argentina Fil: Bueno, Nadia S. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Molina, Viviana. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Brero, María L. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina Fil: Kornblit, Fernando. Instituto Nacional de Tecnología Industrial (INTI); Argentina

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina. Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance. Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.

The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microorganisms in clinical laboratories because it is rapid, relatively simple to use, accurate, and can be used for a wide number of microorganisms. Several studies have demonstrated the utility of this technique in the identification of yeasts; however, its performance is usually improved by the extension of the database. Here we developed an in-house database of 143 strains belonging to 42 yeast species in the MALDI Biotyper platform, and we validated the extended database with 388 regional strains and 15 reference strains belonging to 55 yeast species. We also performed an intra- and interlaboratory study to assess reproducibility and analyzed the use of the cutoff values of 1.700 and 2.000 to correctly identify at species level. The creation of an in-house database that extended the manufacturer's database was successful in view of no incorrect identification was introduced. The best performance was observed by using the extended database and a cutoff value of 1.700 with a sensitivity of .94 and specificity of .96. A reproducibility study showed utility to detect deviations and could be used for external quality control. The extended database was able to differentiate closely related species and it has potential in distinguishing the molecular genotypes of Cryptococcus neoformans and Cryptococcus gattii.

We studied 23 clinical and environmental strains of Sporothrix schenckii sensu lato collected from 1984 to 2017 in Argentina. The molecular identification (partial sequencing of a fragment of the calmodulin gene) of the strains was performed. For the yeast and mycelial phases, the in vitro susceptibility testing by a microdilution reference method was determined against eight antifungal drugs. Strains studied were identified as S. schenckii sensu stricto 13 (56.5%), S. brasiliensis 8 (34.7%) and S. globosa 2 (8.7%). The most active antifungal drugs tested for the yeast and mycelial phases expressed as geometric mean (GM) value of the minimal inhibitory concentration (MIC) (μg mL-1 ) were terbinafine (0.07 and 0.24), posaconazole (0.13 and 0.58), itraconazole (0.38 and 1.10) and ketoconazole (0.22 and 0.89), while fluconazole (110.10 and 131.92) and flucytosine (2.96 and 79.03) were the less active. For voriconazole and amphotericin B the GM-MIC values were acceptably low for the yeast phase (0.39 and 0.72 μg mL-1 ), while the mycelial phase showed values ≥2-fold higher (8.76 and 1.88 μg mL-1 ), P < .05. Here, we described S. schenckii sensu stricto, S. brasiliensis and S. globosa, these species were isolated from humans, animals and soil and are circulating in Argentina since at least 1984.

The purpose of this study is to analyze the current knowledge about matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of clinical yeasts comparing with the ribosomal DNA sequencing identification. We also review the lasts taxonomical and nomenclature changes of clinical yeasts. In most studies, the two main commercially available, MALDI Biotyper and VITEK MS, correctly identified more than 90% of yeasts isolates. Most drawbacks are due to low discrimination between closely related species or due to a lack of the reference main spectra (MSP) in the database. The addition of MSPs in the databases improves the performance. MALDI-TOF MS has proven to be good for the identification of clinical yeasts and to differentiate some closely related species or species undifferentiated by conventional techniques. However, some species remain difficult to differentiate by this technique and should be identified by ribosomal DNA sequencing. Accurate and rapid yeasts identification could improve the management of patients with yeasts infections.

This review summarizes the information available of Candida haemulonii sensu lato, and updates the in vitro susceptibility profile of the isolates of these species from Argentina. C. haemulonii sensu lato causes fungemia in low frequency in adults and children; however, the knowledge about this emerging yeast is relevant since it is considered as a multi-resistant pathogen. The discovery of new antifungal molecules, the determination of epidemiological cutoff value and clinical breakpoints for some yeasts of clinical impact, and the update of techniques to determine the in vitro susceptibility profile to yeast have generated some available information, although, for C. haemulonii sensu lato, this information is not always useful for clinical application. We determined the susceptibility profile of C. haemulonii sensu lato strains isolated in Argentina, as a first step to establish the epidemiological cutoff values in our region. We also review the current situation in other countries.

Cryptococcal meningitis is the most common fungal infection of the central nervous system and the third most frequent neurological complication in AIDS patients. To understand the Argentinean epidemiology of cryptococcosis, several efforts have been made by the National Reference Laboratory in Clinical Mycology. In Argentina, reports of distribution and frequency of Cryptococcus neoformans and Cryptococcus gattii isolates are scarce and very little is known about its circulating genotypes and mating types. The National Mycology Laboratory Network and the National Reference Laboratory in Clinical Mycology joined forces to estimate the prevalence of cryptococcosis and to obtain and to analyse epidemiological data of this important fungal infection. Data presented here were recovered from 1998 to 2016 and represents an approximation to the actual situation of cryptococcosis in Argentina. These results could be useful to design future investigations.

We report the first case of cryptococcosis due to Cryptococcus decagattii in an immunocompetent pediatric patient from an indigenous community in Argentina with a successful outcome. Two isolates (blood, cerebrospinal fluid) were genotyped by restriction fragment length polymorphism of the orotidine monophosphate pyrophosphorylase (URA5) gene as VGIV and identified by multi-locus sequence typing as C. decagattii. Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification indicated genotype VGIII. The minimum inhibitory concentration of amphotericin B, fluconazole, itraconazole, and voriconazole was determined (cerebrospinal fluid: 0.25, 16, 0.12, and 0.12, blood: 0.25, 4, 0.12, and 0.06, respectively, all in mg/L).

Fil: Taverna, CG. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. Fil: Bosco-Borgeat, ME. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. Fil: Mazza, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. Fil: Vivot, ME. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. Fil: Davel, G. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. Fil: Canteros, CE. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina. The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATα) was the most frequently isolated (n=326, 87.6%), followed by VNII (MATα) (n=22, 5.9%), the recently described VNII-VNIV (aADα) hybrid (n=14, 3.8%), VNIV (MATα) (n=4, 1.1%), VNIII (αADa) hybrid (n=1, 0.3%), and VNIII (αADα) hybrid (n=1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATα) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADα) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATα) and 1 VGII (MATα) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those variations in frequencies are associated with the existence of regional ecological niches or any other regional factors. Genotipificación; Genotyping; Hybrids; Híbridos; Mating type; Tipo sexual

Background No reliable data are available in the province of Buenos Aires regarding the frequency of dermatophytoses and other fungal diseases. The distribution of the clinical forms and the species involved are also unknown. Aims To present the data collected by the laboratories participating in the Mycology Network of the province of Buenos Aires (MNPBA) from a retrospective epidemiological survey on dermatophytoses. Methods A descriptive and exploratory analysis was performed on the cases of dermatophytoses gathered between 2002 and 2007 by the Mycology Network of the province of Buenos Aires. Results Of the 3966 dermatophytosis cases reported by 41 laboratories in 31 municipalities, more than a half occurred in three highly populated urban municipalities. The male:female ratio was 1:1.5. The most frequent clinical form was tinea unguium, diagnosed in 904 cases (51.83%), followed by tinea capitis (19.32%), tinea corporis (15.19%), tinea pedis (6.77%), tinea cruris (3.73%), and tinea manuum (2.18%). The species involved was identified in 1368 (33.49%) cases. Trichophyton rubrum was the most common species, with a frequency of 42.03%. An association was found between urban municipalities and T. rubrum or the Trichophyton mentagrophytes complex. Conclusions Results from the MNPBA survey provide valuable information that should enable further interventions to be designed in order to prevent and control the disease., UIQA -Unidade de Investigação Química Ambiental, info:eu-repo/semantics/publishedVersion

Infections due to Cryptococcus neoformans cause severe disease, mostly in AIDS patients. The antifungal drug recommended for the initial treatment of these infections is amphotericin B with or without flucytosine, but treatment failure occurs, associated with high mortality. Thus, antifungal susceptibility testing is needed. However, the in vitro susceptibility tests available for C. neoformans are not useful to detect isolates that are not susceptible to antifungal agents such as amphotericin B. The aims of the present study were: (1) to determine and compare the in vitro activity of amphotericin B against C. neoformans clinical isolates by using different dilution and diffusion methods; (2) to evaluate the concordance among the methods used and the reference method; (3) to evaluate which method could be the best to correlate with the clinical outcome. The reference method EDef 7.2 from the European Committee on Antimicrobial Susceptibility Testing and commercial Etest strips were used to determine the minimal inhibitory concentration against amphotericin B. curves, minimal fungicidal concentration, and a disk diffusion method were also developed to evaluate the cidal activity of amphotericin B. The time-kill curve assay showed correlation (p0.05) with clinical outcome, whereas EDef 7.2, minimal fungicidal concentration, Etest, and disk diffusion showed no correlation (p0.05). Thus, the time-kill curve assay could be a potential tool to guide a more efficient treatment when amphotericin B is used.

Resumen Antecedentes Durante un periodo de 4 meses, y mientras se llevaba a cabo un muestreo ambiental de aire, se diagnosticaron 2 casos de aspergilosis por Aspergillus flavus en un centro oncohematologico de Buenos Aires, Argentina. Objetivos Conocer la variabilidad y la relacion genetica entre los aislamientos clinicos y los ambientales obtenidos en el centro oncohematologico. Metodos Se utilizaron 2 tecnicas de genotipificacion con diferente poder discriminatorio (RAPD y AFLP). Una matriz de similitud genetica fue calculada usando el metodo de Jaccard y fue la base para la construccion de un dendrograma por el metodo de UPGMA. Se estimo el nivel de variabilidad genetica por medio del porcentaje de loci polimorficos, numero de alelos efectivos y heterocigosidad esperada, y el indice de asociacion (IA). Resultados El dendrograma mostro que los aislamientos de A. flavus recuperados de los pacientes no se relacionaron geneticamente con los del ambiente nosocomial. Los valores mas altos de diversidad genetica correspondieron a los aislamientos ambientales. El IA estimado para todos los aislamientos sugiere eventos de recombinacion. Conclusiones Los pacientes 1 y 2 no fueron infectados con los aislamientos obtenidos del ambiente hospitalario. Los aislamientos clinicos y ambientales de A. flavus mostraron alta variabilidad genetica entre ellos.

During the last decades, there has been an increasing proportion of patients susceptible to invasive fungal disease (IFD). The epidemiology of IFD varies mainly due to geography, antifungal exposure, and nosocomial reservoirs. We reviewed the Argentinean epidemiology of invasive mold disease (IMD) by analyzing laboratory and clinical data. Invasive mold disease was the second most prevalent IFD following the yeasts, with a prevalence that ranged from 0.98 to 1.31/100,000 population. The majority (60–85 %) of IMD was caused by hyalohyphomycetes followed by Mucorales (6–21 %) and phaeohyphomycetes (7–13 %). The most prevalent genera were Aspergillus (40–67 % of IMD) followed by Fusarium (10–14 %). The most prevalent species were A. fumigatus (38–50 %) followed by A. flavus (27–43 %). In immunocompromised patients in Argentina the most prevalent agents of IMD are Aspergillus, followed by Fusarium and Mucorales, while the most prevalent Aspergillus species are A. fumigatus followed by A. flavus.

Candida pseudorugosa is a novel species closely related to Candida rugosa for which only one case has been reported. We report the first case of a bloodstream infection in humans caused by a Candida sp. closely related to C. pseudorugosa . We contribute evidence to show this organism as a potential human pathogen that may be misidentified by conventional methods, also pointing out its lower sensitivity to azoles and other antifungal agents.

Resumen Antecedentes Histoplasma capsulatum es el agente causal de la histoplasmosis, micosis asociada principalmente a pacientes inmunocomprometidos. La rapida identificacion del hongo a partir del cultivo permite el tratamiento temprano. Objetivo Evaluar un sistema de PCR para dos dianas especificas de H. capsulatum en lisados acuosos de cultivos. Metodos Se utilizaron dos tecnicas de PCR previamente descritas que, en reacciones independientes, amplifican fragmentos especificos de 111 y 279 pb del gen AgM de H. capsulatum. Se analizaron 248 cepas de H. capsulatum y 68 de otras especies fungicas.Para la amplificacion se partio de un lisado acuoso (que contenia el ADN), obtenido por tres ciclos de hervido/enfriamiento rapido a 0 °C. En casos particulares se obtuvo ADN purificado y/o se secuencio el producto de la amplificacion. Resultados Las tecnicas de PCR amplificaron las dos bandas a partir del lisado acuoso de 239 cepas de H. capsulatum ; las 9 restantes solo mostraron bandas de amplificacion a partir de ADN purificado. No se observo amplificacion especifica a partir de lisado acuoso ni de ADN purificado de 66 cepas de especies distintas de H. capsulatum. Dos cepas de Emmonsia crescens presentaron ambas bandas de amplificacion cuyas secuencias resultaron tener una homologia superior al 97% con secuencias de H. capsulatum . El tiempo total de la prueba no supero las 7 h con un 96% de sensibilidad, 97% de especificidad y un valor predictivo positivo de 99%. Conclusiones El metodo es rapido, economico y puede ser utilizado como una alternativa para identificar presuntivamente H. capsulatum en lisados de cultivo sin purificar.

Resumen Antecedentes Los hongos Paracoccidioides brasiliensis , Histoplasma capsulatum y Coccidioides sp. son los agentes etiologicos de paracoccidioidomicosis, histoplasmosis y coccidioidomicosis, respectivamente. Estas 3 micosis son endemicas en distintas areas geograficas de Argentina. En Latinoamerica existen antecedentes de investigacion de areas endemicas que utilizan perros domesticos como indicadores epidemiologicos. Objetivo Determinar la presencia de P. brasiliensis , H. capsulatum y Coccidioides sp. en una zona rural del noreste de Argentina, denominada interfluvio Teuco-Bermejito, provincia del Chaco. Metodos Se evaluaron 89 sueros de igual numero de perros domesticos que habitaban 10 comunidades del area. Mediante la tecnica de western blot se buscaron anticuerpos especificos contra los antigenos extracelulares fungicos especificos: anti-gP43 de P. brasiliensis , anti-H/M de H. capsulatum y anti-120, 82 y 48 kDa de Coccidioides sp. Resultados Nueve de los 89 sueros (10%) mostraron anticuerpos especificos contra uno o mas de los antigenos probados. Ocho sueros tuvieron anticuerpos anti-H/M y uno solamente tuvo anticuerpos anti-M. Uno de estos 9 sueros revelo, ademas, anti-gp43 y otro reacciono contra los 3 antigenos probados. Conclusiones Este es el primer estudio realizado en Argentina que utiliza perros domesticos para detectar areas endemicas de paracoccidioidomicosis, histoplasmosis y coccidioidomicosis. Nuestros resultados indican que H. capsulatum es el principal agente de micosis endemicas en el interfluvio Teuco-Bermejito. Probablemente, los humanos que habitan esta zona tengan una exposicion similar a este hongo y, por tanto, el diagnostico de histoplasmosis debe considerarse cuando los pacientes que habitan esta area geografica presenten manifestaciones clinicas pulmonares o mucocutaneas compatibles con la enfermedad.

En 1892, Alejandro Posadas documentó el primer caso mundial de coccidioidomicosis en un paciente argentino de nombre Domingo Escurra. Con el objetivo de identificar la especie de Coccidioides involucrado en ese caso, analizamos una pieza de necropsia del paciente, conservada en el Museo de Patología de la Facultad de Medicina de la Universidad de Buenos Aires, Argentina. La porción del tejido con mayor número de endosporas del hongo libres e integras fue elegida utilizando una coloración inmunohistoquímica específica. El ADN fúngico fue amplificado usando una PCR anidada que reconoce un fragmento del gen Ag2/PRA cuyo polimorfismo diferencia Coccidioides immitis y C. posadasii. Se amplificó además, el ADN de dos cepas de referencia: C. immitis (M38-05) y C. posadasii (1-NL) y de cuatro aislamientos de Coccidioides de pacientes argentinos. Los fragmentos amplificados fueron secuenciados en ambas hebras. Las secuencias fueron editadas, alineadas y comparadas con las depositadas en GenBank C. posadasii (Acceso N° AY536446, cepa Silveira) y C. immitis (Acceso N° AY536445). Las secuencias del Coccidioides del caso Escurra, de los aislamientos argentinos y de la cepa 1-NL fueron idénticos entre sí y mostraron una mutación puntual de C→G en la posición 1228 en comparación con la secuencia de C. posadasii, cepa Silveira. Este es el primer trabajo donde se busca ADN de Coccidioides en una pieza anatómica de museo con más de 100 años de antigüedad. Los resultados confirman que el primer caso de coccidioidomicosis o enfermedad de Posadas documentado mundialmente fue producido por el recientemente descripto C. posadasii.In 1892 Alejandro Posadas described the first worldwide case of coccidioidomycosis in a patient named Domingo Escurra. A preserved necropsy piece from the patient's remains is conserved in the Museum of Pathology of the Medical School, Buenos Aires University. Paraffin-embedded specimens obtained from this piece served to identify the fungus involved in the case. Histological slices from different lesion sites were submitted to a genus-specific immunohistochemical staining in order to select the more suited areas in terms of abundance/integrity of fungal esporangia and endospora. Fungal DNA was amplified from selected deparaffinated slices using a nested PCR designed to amplify a segment of the gen Ag2/PRA and differentiate C. immitis from C. posadasii. This PCR was also applied to two reference strains (C. immitis M38-05, C. posadasii 1-NL) and isolates obtained from four recent coccidioidomycosis cases occurred in Argentina. Amplified products were submitted to sequencing of both DNA strands. The obtained sequences were edited, aligned and compared with C. posadasii (Access N° AY536446, strain Silveira) and C. immitis (Access N° AY536445) deposited in GenBank. DNA sequences from Escurra's lesions were 100% homologous to the recent Argentinean cases and the reference strain 1-NL. A single point C→G difference in position 1228 was observed with respect to sequence of strain C. posadasii Silveira. For the first time, Coccidioides DNA is recovered from a museum piece which is more than 100-year-old. Our results confirm that the original case of Posadas's disease was caused by the recently described C. posadasii.

Resumen El genero Malassezia ha sido revisado recientemente y, hasta el momento, se acepta que incluye 11 especies, que no siempre pueden ser diferenciadas mediante pruebas fisiologicas y morfologicas. En este trabajo se investigo la concordancia entre las pruebas fenotipicas y un metodo molecular rapido en la identificacion de especies de ese genero . Se analizaron 92 aislamientos clinicos de Malassezia spp. obtenidos de pacientes argentinos entre 2001 y 2005 y tres cepas de referencia ( Malassezia furfur CBS-7019, Malassezia sympodialis CBS-7222 y Malassezia slooffiae CBS-7956). El metodo molecular, basado en amplificacion del ADN por PCR y su digestion con tres diferentes endonucleasas (PCR-REA), permitio identificar inequivocamente los 92 aislamientos clinicos (63 M. sympodialis , 18 M. furfur , 10 Malassezia globosa y una Malassezia obtusa ) y las tres cepas de referencia. Mediante las pruebas fenotipicas se logro tipificar 85 aislamientos y dos de las cepas de referencia (concordancia total > 91%). Para las especies mas frecuentemente involucradas en patologia humana, tales como M. sympodialis , M. furfur y M. globosa , la concordancia oscilo entre 84-96%, lo que indicaria que los estudios fenotipicos son utiles para la identificacion presuntiva de estos miembros del genero en laboratorios que aun no tienen acceso a la metodologia molecular.

Epidemiological cut-off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild-type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC50 , MIC90 and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml(-1) each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml(-1) respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.

The molecular basis of fluconazole resistance in Cryptococcus neoformans has been poorly studied. A common azole resistance mechanism in Candida species is the acquisition of point mutations in the ERG11 gene encoding the enzyme lanosterol 14-α-demethylase, target of the azole class of drugs. In C. neoformans only two mutations were described in this gene. In order to evaluate other mutations that could be implicated in fluconazole resistance in C. neoformans we studied the genomic sequence of the ERG11 gene in 11 clinical isolates with minimal inhibitory concentration (MIC) values to fluconazole of ≥16 μg/ml. The sequencing revealed the G1855A mutation in 3 isolates, resulting in the enzyme amino acid substitution G484S. These strains were isolated from two fluconazole-treated patients. This mutation would not intervene in the susceptibility to itraconazole and voriconazole., Las bases moleculares de la resistencia al fluconazol en Cryptococcus neoformans han sido poco estudiadas. Un mecanismo de resistencia a los azoles en Candida albicans es la adquisición de mutaciones puntuales en el gen ERG11, que codifica la enzima lanosterol 14 α-demetilasa, blanco de las drogas azólicas. En C. neoformans solo 2 mutaciones en este gen han sido descriptas. Con el objetivo de estudiar otras mutaciones que podrían estar implicadas en la resistencia al fluconazol en C. neoformans, realizamos la secuenciación del gen ERG11 de 11 aislamientos clínicos con valores de concentración inhibitoria mínima (CIM) de fluconazol ≥16 μg/ml. En 3 aislamientos, la secuenciación reveló la mutación G1855A, que da como resultado la sustitución aminoacídica G484S. Estos aislamientos fueron recuperados de 2 pacientes tratados con fluconazol. Esta mutación no intervendría en la sensibilidad al itraconazol y al voriconazol., Facultad de Ciencias Veterinarias

Background Mycotic keratitis by moulds (MKM) is an important cause for corneal blindness and usually carries an unfavorable prognosis. Aims This study describes the risk factors and demographic and microbiological features of all MKM cases in Santa Lucia Ophthalmology Hospital during a period of 6 years. Methods A prospective study was performed for all MKM cases diagnosed between October 2007 and September 2013. Results Among 157 diagnosed cases, direct microscopic examination and culture were positive in 97 and 96% of the cases respectively. MKM represents 17% of all microbiologically confirmed corneal abscesses. No significant differences were detected in annual MKM frequencies across the study period, suggesting that MKM incidence remains constant over time. A male-to-female ratio was observed (2.8:1); the most affected age groups ranged from 31 to 40 years old (males) and 61–70 years old (females). The most frequent predisposing factor was trauma (40%) followed by the use of contact lenses (9%), herpetic abscesses (5%) and diabetes (4%). The predominant genera were Fusarium (66%), Aspergillus (10%), Curvularia (6%) and Alternaria (4%). The most frequent agent was Fusarium solani species complex (52%). More than two-thirds of the cases were produced by only 3 species or complexes. However, at least 29 different species were detected in the remaining cases. This is the first report of Pholiota sp. as causative agent of human MKM. Conclusions Argentina lacks extensive epidemiological and clinical data on MKM. This six-year study performed in Argentina is a first step leading to a better understanding of MKM epidemiology in our country.

Intact chromosomes of 19 clinical isolates of Histoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala and the laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal banding patterns of the human isolates revealed 5-7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsulatum varies widely in nature, as observed previously in laboratory strains. No definite association was found between electrokaryotype and geographical or clinical source.

Trichosporon species are emerging causative agents of mycoses; most are documented in immunocompromised patients. Species identification is important for epidemiological purposes in order to better define species clinical associations and to improve antifungal treatment. Here, we studied a collection of 41 Trichosporon strains recovered from hospitalized patients in Argentina. All strains were identified by sequencing the D1/D2 domain of 26S, internal transcribed spacer (ITS) regions, and intergenic spacer 1 (IGS1) region. In addition, we determined the IGS1 region genotypes of the suspected T. asahii strains. Antifungal susceptibility of all strains was investigated. Thirty-eight of the 41 strains in this study were identified as follows: 29 T. asahii, 3 T. inkin, 3 T. montevideense, 2 T. faecale, and 1 T. dermatis. The identity of the three remaining strains could not be confirmed. Strain DMic 114126 (Culture collection of the Mycology Department (DMic), National Institute of Infectious Diseases "Dr. Carlos G. Malbrán".) may represent a T. asahii subspecies or a new Trichosporon species, strain DMic 94750 was identified as T. cf. guehoae and strain DMic 114132 as T. cf. akiyoshidainum. The distribution of T. asahii genotypes was as follows: 12 genotype 3, 9 genotype 1, 4 genotype 4, 2 genotype 5, and 2 genotype 7. Amphotericin B minimal inhibitory concentrations (MICs) were ≤1 mg/l for 78% (32/41) of the strains. Fluconazole MICs were ≥2 mg/l for 90% of the strains. However, itraconazole, voriconazole, ketoconazole, and posaconazole MICs were ≤1 mg/l for 100% of the strains. Terbinafine MICs were ≤1 mg/l for 98% 40/41 of the strains.

Infection with Cryptococcus neoformans is an increasing problem in immunocompromised patients, particularly those with acquired immune deficiency syndrome (AIDS). Amphotericin B and fluconazole are currently acceptable therapies for cryptococcal meningitis; however, their effects remain suboptimal and recurrence or treatment failure is still a problem. Antifungal susceptibility testing may be an important tool for guiding therapy, but for C. neoformans, a reliable method is still not available. This retrospective study evaluated minimal inhibitory concentration (MIC) for amphotericin B and fluconazole, and minimal fungicidal concentration (MFC) and timed-kill curves for amphotericin B against 16 clinical isolates of C. neoformans obtained from AIDS patients with cryptococcal meningitis. No correlation between clinical outcome and MIC was observed for amphotericin B. In selected cases, the MFC seemed to be a better predictor of outcome than MIC. In this study, amphotericin B timed-kill curves appeared to show a correlation with clinical outcome of the 16 patients with AIDS-associated cryptococcal meningitis. These in vitro tests must be further evaluated in prospective studies to confirm their potential usefulness for guiding cryptococcal meningitis therapy.

Summary In the city of Buenos Aires, Argentina, Cryptococcus gattii genotype AFLP4/VGI was found to be associated with decaying wood in hollows of different tree species. The aim of this study was to investigate the presence of C. gattii in the environment of riverside cities of the river Parana, and to describe its serotypes and molecular types. Five hundred samples were collected in 50 parks by swabbing tree hollows. The samples were inoculated on caffeic acid agar supplemented with chloramphenicol, and incubated at 28 °C for 1 week with a daily observation. The isolates were identified by conventional methods. The serotype was determined by slide agglutination with specific antisera. Molecular typing was carried out by PCR-RFLP of the URA5 gene. Four isolates of C. gattii were recovered: Cryptococcus gattii serotype B, genotype AFLP4/VGI, isolated from Eucalyptus sp. in the city of Rosario and from Grevillea robusta in the city of La Paz; and C. gattii serotype C, genotype AFLP5/VGIII, isolated from two different Tipuana tipu trees in the city of Resistencia. Here, we report for the first time the isolation of C. gattii serotype C, genotype AFLP5/VGIII, from environmental samples in Argentina.

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

During 4 months, and while conducting an environmental sampling of air, 2 cases of aspergillosis by Aspergillus flavus (A. flavus) were diagnosed at an oncohematological center in Buenos Aires, Argentina.The aim of this study was to know the variability and the genetic relationship between the clinical and environmental isolates, obtained in the oncohematological center.Two genotyping techniques of different discriminatory power (RAPD and AFLP) were used. A genetic similarity matrix was calculated using Jaccard method and was the basis for the construction of a dendrogram by UPGMA. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective allele, expected heterocygozity and association index test (I(A)).The dendrogram reveals that the A. flavus isolates recovered from the patients were not genetically related to those gotten from the rooms occupied by the patients. The environmental isolates had higher values of genetic diversity than the clinical isolates. The I(A) estimated for all the isolates suggest that recombination events occurred.Patients 1 and 2 were not infected with isolates from the nosocomial environment. Clinical and environmental isolates of A. flavus showed high genetic variability among them.

Candida dubliniensis is an emerging pathogen that can cause invasive disease in patients who have a variety of clinical conditions. C. dubliniensis is often misidentified as Candida albicans by clinical laboratories. In Argentina, incidence data are still scarce, and only one systemic infection has been reported. This study aims to determine the prevalence of C. dubliniensis in blood samples in Argentina, to evaluate a novel PCR multiplex as well as several phenotypic methods for the identification of this yeast, and to know the susceptibility profile of isolates against seven antifungal drugs. We have found that prevalence in Argentina appears to be lower than that reported in other countries, occurring only in 0.96% of the Candidemia cases recovered in 47 hospitals during a 1-year period. All C. dubliniensis clinical isolates included in this study were genetically identical when comparing ITS genes sequences. This is in agreement with the previous studies suggesting little genetic variation within this species. The novel multiplex PCR proved to be 100% sensitive and specific for the identification of C. dubliniensis. Therefore, we propose its use as a rapid and inexpensive method for laboratories having access to molecular techniques. Although no single phenotypic test has proved to be infallible, both colony morphology on tobacco agar, as well as abundant chlamydospore formation on both tobacco agar and on sunflower seed agar, may be used as a presumptive differentiation method in routine mycology laboratories. It has been suggested that C. dubliniensis may have higher propensity to develop azole antifungal drug resistance than C. albicans. In this study, one of the five clinical isolates of C. dubliniensis was resistant to fluconazole.

Reference methods E.Def 7.1 and M27-A3 detect in vitro resistance; however, they are expensive and very laborious. Thus, their actual use in hospital laboratories is limited. There are commercial techniques available, having easier accessibility and development, which would yield results comparable to those of the reference methods. The objectives of this study were: a) to compare the results of minimal inhibitory concentration of 82 Candida spp. clinic isolates according to reference method E.Def 7.1 and ATB® Fungus 3; b) to compare the results of fluconazole susceptibility testing by disk diffusion in agar with Neo-SensitabsTM tablets and Malbrán disks with those of the reference methods. Minimal inhibitory concentration for amphotericin B, 5-flucytosine, fluconazole and itraconazole was performed according to the E.Def 7.1 and the ATB Fungus 3 methods and diffusion was carried out with fluconazole disks and tablets. General concordance between the reference method and ATB Fungus 3 was 90.2 % and 96.3 and 92.7% for diffusion with disks and tablets. The ATB Fungus 3 method was effective to determine susceptibility against amphotericin B and 5-flucytosine; however, discrepancies were observed with azole drugs. Disk diffusion methods are useful to determine susceptibility to fluconazole; however, 3 very major, 1 major and 2 minor errors were observed with the tablets, whereas only 3 minor errors were observed with the disks.

Histoplasma capsulatum is the agent of histoplasmosis, a deep mycosis mainly afflicting immunocompromised patients. Rapid identification of the fungus isolated from clinical specimens allows timely administration of specific treatment.To assess the ability of a dual PCR system targeting specific H. capsulatum DNA sites to identify fungal species in simple aqueous lysates from cultured fungi.We analysed the performance of two independent PCR reactions designed to amplify fragments of 111 and 279 bp included in H. capsulatum-specific gene AgM. We used 248 H. capsulatum strains and 68 isolates of other fungal species. Reaction templates consisted of aqueous lysates of cultured fungi (either in mycelial or yeast phase) obtained after three cycles of boiling and immediate cooling at 0°C. Selected strains were submitted to conventional DNA extraction and/or sequencing.Both PCR systems performed identically. Amplification from aqueous lysates was achieved from 239 H. capsulatum strains; the remaining 9 strains only showed specific bands when purified DNA was used as template. Of all other fungal species tested, only 2 Emmonsia crescens strains amplified H. capsulatum-specific bands and sequences of their amplified PCR products matched97% with H. capsulatum sequences. Total test time did not exceed 7h with 96% sensitivity, 97% specificity and 99% positive predictive value.The assay is fast, accurate and economical, and can be an alternative method for presumptive identification of H. capsulatum in simple culture lysates.

Three fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.To investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.We applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48kDa antigen bands of Coccidioides sp.Specific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.This is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.

In 1892 Alejandro Posadas described the first worldwide case of coccidioidomycosis in a patient named Domingo Escurra. A preserved necropsy piece from the patient's remains is conserved in the Museum of Pathology of the Medical School, Buenos Aires University. Paraffin-embedded specimens obtained from this piece served to identify the fungus involved in the case. Histological slices from different lesion sites were submitted to a genus-specific immunohistochemical staining in order to select the more suited areas in terms of abundance/integrity of fungal esporangia and endospora. Fungal DNA was amplified from selected deparaffinated slices using a nested PCR designed to amplify a segment of the gen Ag2/PRA and differentiate C. immitis from C. posadasii. This PCR was also applied to two reference strains (C. immitis M38-05, C. posadasii 1-NL) and isolates obtained from four recent coccidioidomycosis cases occurred in Argentina. Amplified products were submitted to sequencing of both DNA strands. The obtained sequences were edited, aligned and compared with C. posadasii (Access N degrees AY536446, strain Silveira) and C. immitis (Access N degrees AY536445) deposited in GenBank. DNA sequences from Escurra's lesions were 100% homologous to the recent Argentinean cases and the reference strain 1-NL. A single point C(R)G difference in position 1228 was observed with respect to sequence of strain C. posadasii Silveira. For the first time, Coccidioides DNA is recovered from a museum piece which is more than 100-year-old. Our results confirm that the original case of Posadas's disease was caused by the recently described C. posadasii.

The aim of this work was to describe the distribution of the members of the Cryptococcus species complex (Cryptococcus neoformans and C. gattii) in hollows of trees from seven parks in Buenos Aires City, to determine the serotypes and genotypes of these environmental isolates and to compare them with the ones reported in the 2001 survey. Four hundred and eighty nine samples were collected by swabbing all trees which had hollows or fissures in the seven parks studied. Each tree was sampled once during the study period and one or more isolates were recovered from each swab. Eight isolates of C. neoformans and 18 isolates of C. gattii were recovered from 15 out of 489 tree samples. C. neoformans was isolated from Tipuana tipu, Eucalyptus spp., and Phoenix sp. All isolates were serotype A and belonged to genotype VNI. C. gattii was isolated from Tipuana tipu, Cedrus deodara, Eucalyptus spp., Acacia visca, Cupresus sempervirens and Ulmus campestrus. All isolates were serotype B and genotype VGI, like both C. gattii strains isolated in 2001. On two occasions, both species were isolated from the same tree sample. These results reinforce and extend our previous findings especially about the presence of C. gattii serotype B, genotype VGI, in Argentina.

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.

The in vitro activities of amphotericin B (AMB), itraconazole (ITC), voriconazole (VCZ) and terbinafine (TBF) alone and in the combinations AMB + VCZ, TBF + ITC and TBF + VCZ were evaluated against 29 clinical isolates of Fusarium spp. (15 Fusarium solani, 7 Fusarium oxysporum, 2 Fusarium decemcellulare, 2 Fusarium dimerum and 3 other Fusarium spp.). Minimum inhibitory concentrations were determined using the method of the Clinical and Laboratory Standards Institute and the interaction activity was calculated using the fractional inhibitory concentration index. The four antifungal drugs tested alone showed very limited activity against most of the isolates. In contrast, the combination TBF + VCZ showed synergy for 21 isolates. The combination AMB + VCZ showed synergism for only five strains. No interaction or antagonism was observed among the remaining strains. TBF + ITC showed no interaction for 18 strains. The in vitro antifungal activity of the drugs alone and in combination varied for different species. These results corroborate previous in vitro studies in which the combination TBF + VCZ showed synergy against Fusarium spp., although further studies are needed to elucidate its potential usefulness for therapy.

Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-alpha lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S. We thank the Biopolymers Unit at Instituto de Salud Carlos III, Majadahonda, Madrid, Spain, for DNA sequencing and Sylvia Spitzer for providing the CNRE-1 repetitive probe. E.M. held a contract Ramon y Cajal from Ministry of Science and Technology. L.R. held a grant from PAHO and ISCIII. L.R. and E.M. contributed equally to this work.

A bloodstream infection due to Candida haemulonii afflicting a patient with fever and a medical history of megaloblastic anemia is reported. The clinical isolate was misidentified by the API 20C and VITEK identification systems. The results of susceptibility tests showed that the MIC of amphotericin B for C. haemulonii was 4 g/ml. Additional susceptibility testing procedures based on the use of antibiotic medium 3 and Iso-Sensitest broth were performed, and killing curves were determined. Two collection strains of C. haemulonii were employed as controls. The three isolates exhibited resistance to amphotericin B in vitro regardless of the antifungal susceptibility testing method employed. In addition, the MICs of fluconazole for the three isolates were high. Further studies are needed in order to ascertain whether this species exhibits innate or acquired resistance to amphotericin B and other antifungal agents. Fil: Rodero, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Cuenca-Estrella, Manuel. Instituto de Salud Carlos III. Servicio de Micología; España. Fil: Córdoba, Susana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Cahn, Pedro. Hospital Fernández. Servicio de Infectología; Argentina. Fil: Davel, Graciela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Kaufman, Sara. Hospital Fernández. Servicio de Microbiología; Argentina. Fil: Guelfand, Liliana. Hospital Fernández. Servicio de Microbiología; Argentina. Fil: Rodríguez-Tudela, Juan L. Instituto de Salud Carlos III. Servicio de Micología; España.

We report the first case of maxillary sinusitis caused by Actinomucor elegans in an 11-year-old patient. Histopathological and mycological examinations of surgical maxillary sinuses samples showed coenocytic hyphae characteristic of mucoraceous fungi. The fungi recovered had stolons and rhizoids, nonapophyseal and globose sporangia, and whorled branched sporangiophores and was identified as A. elegans. After surgical cleaning and chemotherapy with amphotericin B administered intravenously and by irrigation, the patient became asymptomatic and the mycological study results were negative. Fil: Davel, Graciela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Featherston, Patricia. Hospital San Juan de Dios; Argentina. Fil: Fernández, Anibal. Hospital San Juan de Dios; Argentina. Fil: Abrantes, Ruben. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Canteros, Cristina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Rodero, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina. Fil: Sztern, Carlos. Centro Oncológico. Fundación José María Mainetti; Argentina. Fil: Perrotta, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.