Sporotrichosis caused by Sporothrix brasiliensis in Argentina: Case report, molecular identification and in vitro susceptibility pattern to antifungal drugs.

Sporotrichosis is considered a neglected disease of humans and animals in many regions of the world and is the most frequent implantation mycosis in Latin America. OBJECTIVES: To illustrate the zoonotic importance of the disease, describing a case involving a veterinarian and an infant that acquired the disease from a domestic cat and to describe, genotype and characterize these new isolates. METHODS: Direct examination of tissue samples from the two patients and feline lesions revealed the presence of Sporothrix yeast-like organisms. Fungal cultures and molecular identification of the strains were performed. Since antifungal susceptibility data of animal-borne isolates are scarce, the in vitro susceptibility testing by a microdilution reference method was determined against azoles, amphotericin B and terbinafine. RESULTS: Fungal culture and sequence analysis of the ITS region of rDNA and calmodulin and ß-tubulin genes confirmed the diagnosis and the causative agent as Sporothrix brasiliensis. In all cases, terbinafine was the most active drug, followed by posaconazole, itraconazole and voriconazole; the least active drugs were amphotericine B and fluconazole. Lack of clinical response in the veterinarian and in the infant to itraconazole and potassium iodide, respectively was observed. CONCLUSIONS: This study contributed to the molecular epidemiology of Sporothrix species in Argentina and the characterization of the in vitro susceptibility pattern of S. brasiliensis isolates recovered from a cat and two humans involved in this case of zoonotic sporotrichosis. Bearing in mind the "One Health" concept, the experience described in the present study highlights the need for future strategies for sporotrichosis treatment, control and prevention.

Phenotypic and genotypic identification of Candida dubliniensis from subgingival sites in immunocompetent subjects in Argentina.

INTRODUCTION: It is generally recognized that Candida dubliniensis is commonly found in immunocompromised patients, such as those with advanced human immunodeficiency virus infection, at sites of periodontal disease. Since there are no data available for Argentina, the aim of this study was to determine the prevalence of and to identify C. dubliniensis in periodontal pockets from immunocompetent subjects living in Buenos Aires, Argentina, through a comparison of phenotypic and molecular assays. METHODS: Yeasts recovered from subgingival plaque samples were studied for 180 immunocompetent non-smoking patients with periodontal disease. Yeasts were identified by conventional mycological methods and by specific polymerase chain reaction (PCR) assay. Fluconazole and voriconazole susceptibility studies were performed in keeping with the Clinical and Laboratory Standards Institute. RESULTS: Among 76 yeasts isolated, C. dubliniensis comprised 10.5% (n = 8; 95% confidence interval 4.7-19.7), which corresponded to 4.4% of patients studied (8/180). C. albicans was the most frequently isolated species of yeast. A great majority of C. dubliniensis isolates was susceptible with only one isolate resistant to both antifungals. CONCLUSION: Micromorphology on Staib agar was the phenotypic method that was most concordant with PCR and it was useful for selecting presumptive C. dubliniensis. This is the first report to use PCR to identify C. dubliniensis in subgingival fluid from immunocompetent individuals with periodontal disease in Argentina. On the basis of the findings presented here, we confirm that C. dubliniensis can colonize periodontal pockets of immunocompetent patients with periodontal disease.

[Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR]. / Comparación de métodos de extracción de ADN de sangre para detectar ADN fúngico por PCR.

Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR. Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS land ITS 4 as target.

Comparación de métodos de extracción de ADN de sangre para detectar ADN fúngico por PCR / Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR

La infección fúngica invasora (IFI) está asociada a un alto índice de mortalidad, que alcanza el 50% debido a la frecuente falla en el tratamiento antifúngico. Existen dificultades para realizar un diagnóstico micológico rápido y certero dada la baja sensibilidad de los métodos convencionales, especialmente en pacientes neutropénicos y con SIDA. Numerosos métodos para diagnosticar infecciones micóticas basados en el estudio del ADN fúngico están actualmente en desarrollo. Nosotros evaluamos la utilidad de dos procedimientos de extracción y purificación del ADN fúngico presente en sangre para su posterior detección por PCR. Ambos métodos resultaron igualmente eficientes para obtener ADNs de óptima calidad y para realizar la técnica de PCR con los iniciadores universales para hongos ITS 1 e ITS 4.

Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS 1and ITS 4 as target.

[Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001]. / Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas. Período 1999-2001.

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).

Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas: Período 1999-2001 / Prevalence of Candida albicansand Candida no albicans inclinical samples during 1999-2001

Las levaduras implicadas en procesos patológicos son de indiscutible importancia debido al incremento experimentado por estas infecciones en las últimas décadas, a los cambios observados en las especies causales y al uso empírico de antifúngicos. En el Centro de Micología se estudiaron 1006 aislamientos provenientes de una amplia gama de muestras clínicas durante el periodo 1999-2001. Candida albicans con 40,3% resultó la especie de mayor frecuencia de aislamiento, pero las especies de Candida no albicans con 54,9% resultaron de mayor prevalencia y el 4,8% fueron otras levaduras. En los hemocultivos Candida parapsilosis con 34,9%, C. albicans con 30,2% y C. tropicalis con 25,6% resultaron las más recuperadas, mientras que C. glabrata se presentó con un 2,3%. En las secreciones mucosas C.albicans con 60%-80% fue la especie preponderante. Hemos detectado especies de Candida causantes de mediastinitis, lo que nos alerta sobre su importancia en estos procesos. Las infecciones del tracto urinario por levaduras se detectaron en mayor frecuencia en individuos hospitalizados, resultando C. albicans con 47,7% la especie más aislada, y dentro de Candida no albicans, C. glabrata con 24,8% y C. tropicalis con 20,0%. En las onixis candidiásicas C. parapsilosis con 37,7% desplazó a C.albicans con 22,0% de este lugar anatómico. Los estudios de sensiblidad al fluconazol de las especies de Candida nos permiten concluir que C.albicans es una especie sensible y que los mayores porcentajes de resistencia se observaron en C. glabrata (21,41%) y and C. krusei (69,23%).

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).

Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas. Período 1999-2001. / [Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001]

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3

) was the most isolated species, although, the Candida no albicans species with 54.9

showed the major prevalence. In blood cultures Candida parapsilosis (34.9

), C. albicans (30.2

) and C. tropicalis (25.6

) were recovered most frequently while C. glabrata represented only 2.3

. C. albicans with 60

-80

was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7

), the most commonly isolated, followed by C. glabrata (24.8

) and C. tropicalis (20.0

). In the candidal onychomycoses, C. parapsilosis (37.7

) outplaced C. albicans (22.0

). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41

) and C. krusei (69.23

).

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method.

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2% dextrose. Overall agreement between both methods was 90.0%. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6%, 19.05% and 52.17%, respectively. No very major discrepancies, 1.03% major discrepancies, and 8.97% minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80% vs 50% endpoints, and trailing growth near the endpoints.

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2

. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6

major discrepancies, and 8.97

minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80

endpoints, and trailing growth near the endpoints.

Susceptibility of Candida species to fluconazole assayed by a disk diffusion method with automated reading versus a microdilution method.

In vitro susceptibilities of 290 isolates of Candida spp to fluconazole were evaluated by a new NCCLS M44-P agar disk diffusion method that was read and interpreted automatically by the BIOMIC image-analysis plate reader system. Disk test results were compared to results obtained by a modified NCCLS M27-A broth microdilution method using RPMI-1640 supplemented with 2

dextrose. Overall agreement between both methods was 90.0

. Category agreement between the broth and disk test results for susceptible, susceptible dose-dependent and resistant disk results were 99.6

, 19.05

and 52.17

, respectively. No very major discrepancies, 1.03

major discrepancies, and 8.97

minor discrepancies were observed between results of the two test methods. This analysis suggests that results from both methods correlate highly for Candida strains susceptible to fluconazole. The lower agreement between the two methods for resistant and susceptible dose-dependent isolates was due to strains near or on that breakpoint, different media (Mueller-Hinton vs RPMI), agar vs broth, 80

vs 50

endpoints, and trailing growth near the endpoints.

Histopathological evolution of experimental paracoccidioidomycosis in Wistar rats.

Twenty Wistar rats were inoculated, by the intracardiac route, with 0.5 ml each of a yeast phase suspension of Paracoccidioides brasiliensis Utero strain. The rats were sacrificed at regular intervals post-infection, at which time their lungs, heart, liver, spleen and kidneys were removed, fixed and stained for study. The parameters of interest for the lung specimens were: (a) extent of the lesions; (b) number of fungi; (c) presence of a lymphomononuclear halo. Extrapulmonary lesions were also sought. Until the fourth month post-infection, the lesions were progressive in nature, contained great numbers of viable fungi, and were surrounded by an important lymphomononuclear halo which tended to be confluent. At four and a half months p.i., the extent of the pulmonary lesions was reduced, the granulomas were less compact with fewer viable fungi, macrophages showed microvacuolation, and the lymphomononuclear halo was less pronounced. Extrapulmonary lesions, which were frequently identified in the first months post-infection, diminished from the seventh month onwards. The histological characteristics of extrapulmonary lesions were always the same as those found in the lungs. Infection tended to be controlled by the animals from the fourth month, but without complete resolution of the lesions.

Estudio comparativo de la patogenicidad y la antigenicidad de 6 cepas de Paracoccidioides brasiliensis / Comparative study of pathogenicity and antigenicity of six Paracoccidioides brasiliensis strains

Fueron estudiadas en forma comparativa 6 cepas de P. brasiliensis con el proposito de determinar su patogenicidad para la rata y su antigenicidad. Las mismas fueron aisladas de: 1) biopsia de cuello uterino en 1989 (U), 2) biopsia de mucosa bucal en 1988 (V), 3) aspiracion osea en 1991 (63265), 4) testiculo de cobayo 1984 (C24), 5) puncion-aspiracion ganglionar en 1986 (G) y 6) cepa proveniente de la Escola Paulista de Medicina (339). Se prepararon antigenos citoplasmaticos liofilizados de cada una de ellas, en la concentracion final de 100mg/ml y se realizaron pruebas de inmunodifusion frente a 6 sueros patrones positivos de ratas....

[Comparative study of the pathogenicity and antigenicity of 6 Paracoccidioides brasiliensis strains]. / Estudio comparativo de la patogenicidad y la antigenicidad de 6 cepas de Paracoccidioides brasiliensis.

A comparative study of antigenicity and pathogenicity for rats of six Paracoccidioides brasiliensis strains was carried out. The antigenic capacity "in vitro" of cytoplasmic extract from each strain was determined by immunodiffusion test against 6 serum samples obtained from rats experimentally infected with Paracoccidioides brasiliensis, that had presented positive reactions with a metabolic control antigen. The cytoplasmic extracts were used at final concentration of 100 mg/ml. All of them showed 2 or 3 precipitation bands in this assay. One hundred twenty Wistar rats both sexes weighing approximately 200 g, were inoculated intracardiacally with suspensions of the yeast phase of different P. brasiliensis strains. Two concentrations containing 3 x 10(7) and 5 x 10(7) cells/ml of each isolate were prepared. The inoculated animals were divided in two groups, one was left to its spontaneous outcome and the percentages of deaths were registered and the other rats were sacrificed at 14, 28, 56 and 70 days post-infection. The following parameters were taken into account for evaluation: A) presence of macroscopic granulomas in lung, liver, spleen and kidney; B) presence of P. brasiliensis in microscopic exams of the same organs, in wet preparations and in histologic sections stained by H&E; C) culture of lung and D) immunodiffusion test using pre-mortem serum samples and the homologous antigen. The correlation between the most important parameters studied in each strain are summarized as follow: As no significant differences between the two inocula employed for each strain was observed, the before mention results are the average of those obtained with each inoculation doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Hongos anemófilos de la ciudad de La Plata y preparación de sus antígenos / Anemophilic fungi from La Plata city and preparation of their antigens

Se aislaron hongos anemófilos de la ciudad de La Plata por exposición simple de placas de Petri con medio de Sabouraud adicionados de antibióticos. Los microorganismos fueron obtenidos puros en los cultivos donde se examinaron los caracteres macro y micromorfológicos con el objeto de clasificarlos. Se comprobó un predominio franco de Alternaria, Penicillium, Acremonium, Drechslera y algunas especies de Aspergillus. Con los hongos más frecuentemente aislados se elaboraron preparaciones antigénicas obtenidas del sobrenadante del micelio roto. Simultáneamente fueron inmunizados dos cobayos para cada especie con una suspención de micelio muerto por tindalización. Finalizado el plan de inmunización, estos animales fueron sangrados y se les efectuó pruebas de contrainmunoelectroforesis e inmunodifusión y más tarde se les practicó intradermorreacción con biopsia cutánea a las 2 y 4 horas. No pudo demostrarse la presencia de anticuerpos precipitantes por las pruebas de inmunoprecipitación en medio gelosado, pero los estudios histológicos de las pruebas cutáneas de lectura rápida permitieron comprobar la presencia de un fenómeno de Arthus

Hongos anemófilos de la ciudad de La Plata y preparación de sus antígenos / Anemophilic fungi from La Plata city and preparation of their antigens

Se aislaron hongos anemófilos de la ciudad de La Plata por exposición simple de placas de Petri con medio de Sabouraud adicionados de antibióticos. Los microorganismos fueron obtenidos puros en los cultivos donde se examinaron los caracteres macro y micromorfológicos con el objeto de clasificarlos. Se comprobó un predominio franco de Alternaria, Penicillium, Acremonium, Drechslera y algunas especies de Aspergillus. Con los hongos más frecuentemente aislados se elaboraron preparaciones antigénicas obtenidas del sobrenadante del micelio roto. Simultáneamente fueron inmunizados dos cobayos para cada especie con una suspención de micelio muerto por tindalización. Finalizado el plan de inmunización, estos animales fueron sangrados y se les efectuó pruebas de contrainmunoelectroforesis e inmunodifusión y más tarde se les practicó intradermorreacción con biopsia cutánea a las 2 y 4 horas. No pudo demostrarse la presencia de anticuerpos precipitantes por las pruebas de inmunoprecipitación en medio gelosado, pero los estudios histológicos de las pruebas cutáneas de lectura rápida permitieron comprobar la presencia de un fenómeno de Arthus (AU)

Estudio micológico en muestras de tierra de la ciudad de La Plata / Mycological study in soil samples of La Plata city

Damos a canocer el estudio realizado sobre 100 muestras de tierra de la ciudad de La Plata. De la flora queratofítica, el género más frecuente fue el Microsporum gypseum (89%) y de la flora indígena la prevalencia del gênero Alternaria alcanzó un 79%, se encontraron altos porcentajes de Penicillium 34%, Aspergillus 45%, Absidia 37%, Chrysosporium 29%, Clodosporium 23%. Estos hongos, en circunstancias especiales, pueden provocar micosis viscerais oportunistas graves

Estudio micológico en muestras de tierra de la ciudad de La Plata / Mycological study in soil samples of La Plata city

Damos a canocer el estudio realizado sobre 100 muestras de tierra de la ciudad de La Plata. De la flora queratofítica, el género más frecuente fue el Microsporum gypseum (89%) y de la flora indígena la prevalencia del gÛnero Alternaria alcanzó un 79%, se encontraron altos porcentajes de Penicillium 34%, Aspergillus 45%, Absidia 37%, Chrysosporium 29%, Clodosporium 23%. Estos hongos, en circunstancias especiales, pueden provocar micosis viscerais oportunistas graves (AU)