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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).

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

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.

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.

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.

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.

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 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.