La coccidioidomicosis en Argentina, 1892-2009 / Coccidioidomycosis in Argentina, 1892-2009

Los casos clínicos de coccidioidomicosis en Argentina son pocos y han tenido lugar fundamentalmente en la extensa región árida precordillerana. Este trabajo tiene como objetivos realizar una revisión retrospectiva del total de casos de coccidioidomicosis documentados en Argentina desde el año 1892 hasta 2009 y describir una serie de casos ocurridos en los últimos 4 años. En 117 años se documentaron 128 casos. Desde la primera descripción de la enfermedad en 1892 hasta 1939 se registraron 6 casos; desde 1940 hasta 1999, 59 casos (6-14 casos cada 10 años); y los 63 casos restantes (49% del total histórico) se produjeron en el último decenio. La mediana de edad de los 34 pacientes registrados en el período 2006-2009 fue de 31 años (rango: 7-89), la relación hombre:mujer fue 1,3:1; 12 de estos individuos eran inmunocomprometidos. Veintiséis casos se confirmaron por examen microscópico, por cultivo o por ambos procedimientos; los casos restantes se confirmaron por serología. Todos los aislamientos recuperados fueron identificados como Coccidioides posadasii. Treinta pacientes residían en una amplia área geográfica con epicentro en el valle de Catamarca. Entre 2006 y 2009, la tasa de incidencia en la provincia de Catamarca se incrementó desde valores históricos inferiores a 0,5 casos cada 100 000 habitantes hasta 2,0 casos cada 100 000 habitantes. Este aumento sugiere una emergencia de la coccidioidomicosis en el área.

Clinical cases of coccidioidomycosis are rare in Argentina and are generally found in the large arid precordilleran area of the country. This study aims to perform a retrospective review of all coccidioidomycosis cases documented in the country from 1892 to 2009, and to describe those occurring in the last 4 years. One hundred and twenty eight cases were documented in the 117 year-period. Since the original description of the disease in 1892 until 1939, only 6 cases were registered; between 1940 and 1999, 59 (6-14/10 yrs) and the remaining 63 (49% of total cases) occurred in the last decade. The median age of 34 patients registered in 2006-2009 was 31 years (range: 7-89), male/female ratio was 1.3:1 and 12 patients were immunocompromised. Twenty-six cases were confirmed by direct microscopy and/or culture whereas the remaining ones by serology. All isolates were identified as Coccidioides posadasii. Thirty patients lived in a vast geographic region with epicenter in Catamarca Valley. Between 2006 and 2009, annual disease incidence rates in Catamarca Province increased from historical values below 0.5/100,000 to 2/100,000 inhabitants. Such increase suggests an emergency of coccidioidomycosis in that region.

Diagnóstico molecular de histoplasmosis humana en muestras de sangre entera / Molecular diagnosis of human histoplasmosis in whole blood samples

Se evaluó el uso de sangre entera para el diagnóstico molecular de histoplasmosis utilizando un método artesanal de extracción de ADN fúngico y una PCR anidada que amplifica una porción del gen HcP100 específica de Histoplasma capsulatum. La sangre entera se trató con liticasa, enzima lisante de Trichoderma harzianum y proteinasa K, seguido de una extracción fenólica. Este tratamiento permitió una lisis completa de las células, mostró buen rendimiento en la obtención de ADN y posibilitó la detección de la banda de 210 pb específica de H. capsulatum en la PCR anidada. El límite de detección fue de 0,25-1 levaduras/ml de sangre. El método se evaluó en 31 muestras de sangre de 19 pacientes con diagnóstico microbiológico de histoplasmosis, en 21 muestras de pacientes con otras micosis o infecciones por micobacterias y en 30 controles sanos. La PCR fue positiva en sangre para 17/19 pacientes con histoplasmosis (14/15 inmunocomprometidos y 3/4 sin inmunocompromiso aparente). Las muestras de sangre de los 30 controles sanos y de 20 pacientes con otras patologías fueron negativas, sólo hubo un falso positivo correspondiente a un paciente con infección por Mycobacterium avium-intracellulare. El método presentó 89% de sensibilidad y 96% de especificidad para el diagnóstico de histoplasmosis en sangre entera.

To assess the value of using whole blood samples for the molecular diagnosis of histoplasmosis, we applied an in-house DNA extraction method and a nested PCR targeting a 210 bp specific segment of the Histoplasma capsulatum HcP100 gene. A whole blood volume of 2.5-3 milliliters was centrifuged and the cellular pellet was treated with Trichoderma harzianum lyticase and proteinase K prior to applying a conventional phenol DNA extraction. This procedure allowed complete cell lysis, high DNA yield and specific amplification. The PCR detection limit was 0.25-1 yeast cells/ml of blood sample. The method was assessed on 31 blood samples from 19 patients with microbiological diagnosis of histoplasmosis, 30 healthy persons and 21 patients with other mycoses or mycobacterial diseases. Positive results were obtained in samples from 17/19 patients with histoplasmosis (14/15 immunocompromised and 3/4 without known immunological disorder). Blood samples from the 30 healthy controls and 20 patients with other conditions proved negative; the only false positive result was obtained from a patient with Mycobacterium avium-intracellulare infection. With 89% sensitivity and 98% specificity, this molecular method for detection of the agent in blood shows promising for the rapid diagnosis of human histoplasmosis.

Situación de las micosis en la República Argentina / Epidemiological status of mycoses in the Argentine Republic

Se presentan los resultados de una encuesta nacional sobre micosis diagnosticadas entre enero y diciembre de 2004, con datos provistos por 72 laboratorios de 19 provincias y la Ciudad Autónoma de Buenos Aires. De las 801.805 muestras microbiológicas procesadas ese año, sólo 62.681 (8%) fueron sometidas a estudios micológicos. Se diagnosticaron 23.600 casos de micosis: 11.107 (47%) superficiales, 10.830 (46%) candidiasis de las mucosas y 1.663 (7%) profundas. La frecuencia de agentes de micosis superficiales no sufrió cambios significativos (p>0,05) cuando se comparó con un estudio realizado en población de Provincia y Ciudad de Buenos Aires y dos estudios realizados en 1999 y 2002 por la Red Nacional de Laboratorios y el Programa Nacional de Control de Calidad en Micología (RNLM y PNCCM). Del total de micosis profundas, las más frecuentes fueron fungemia por levaduras (34%), criptococosis (20%), aspergilosis broncopulmonar (13%), histoplasmosis (11%), paracoccidioidomicosis (7%) y neumocistosis (5%). En contraste con los resultados de cuatro estudios previos sobre micosis broncopulmonares, incluyendo el realizado por RNLM y PNCCM en 2002, la histoplasmosis aumentó ubicándose como la micosis endémica más frecuente en Argentina, superando a la paracoccidioidomicosis.

We herein report the results of a retrospective nationwide survey on mycoses diagnosed between January and December, 2004. The study included data provided by 72 laboratories located in 19 provinces and in Buenos Aires City. Out of 801,805 microbiological specimens processed that year, only 62,681 (8%) were submitted to mycological studies. A total of 23,600 mycoses cases were diagnosed: 11,107 (47%) superficial mycoses, 10,830 (46%) mucosal candidiasis and 1,663 (7%) deep mycoses. Relative frequencies of superficial mycoses did not differ significantly (p>0.05) from frequencies observed in a previous study covering Buenos Aires City and Province (1993), and from two countrywide surveys conducted by the National Network of Mycology Laboratories and National Quality Control Program (NNML and NQCP) in 1999 and 2002. The most frequent deep mycoses were yeast fungaemia (34 %), cryptococcosis (20%), broncho-pulmonary aspergillosis (13%), histoplasmosis (11%), paracoccidioidomycosis (7%) and pneumocystosis (5%). In contrast with results of four previous nationwide studies on broncho-pulmonary mycoses including a survey performed by NNML and NQCP in 2002, our study revealed that histoplasmosis prevailed over paracoccidioidomycosis, thus ranking for the first time as the most frequent endemic mycosis in Argentina.

Método de difusión con discos para la determinación de sensibilidad a fluconazol en aislamientos de Candida spp / Disk diffusion method for fluconazole susceptibility testing of Candida spp. isolates

Se estudiaron 1193 aislamientos clínicos para estandarizar y evaluar un método de difusión con discos de fluconazol de lectura visual, que permita detectar levaduras sensibles al antifúngico. Las especies analizadas fueron: Candida albicans (n=584), Candida parapsilosis (n=196), Candida tropicalis (n=200), Candida glabrata (n=113), Candida krusei (n=50), Candida spp. y otras levaduras oportunistas (n=50). Los discos fueron manufacturados en el INEI-ANLIS "Dr. Carlos G. Malbrán". Se midieron los halos de inhibición del crecimiento producidos por fluconazol y la concentración inhibitoria mínima (CIM) por el método de referencia M27-A2 modificado por EUCAST. Se establecieron los valores de corte del método de difusión en: ≥16 mm para levaduras sensibles a fluconazol (CIM ≤ 8 µg/ml), entre 9 y 15 mm para sensibles dependientes de la dosis (CIM = 16-32 mg/ml) y ≤ 8 mm para resistentes (CIM ≥ 64 µg/ml). El método de difusión tuvo 94,7% de concordancia con el de referencia, con 0,2% de errores very major y 0,3% de errores major. La reproducibilidad inter e intralaboratorio fue muy buena. Para detectar aislamientos sensibles a fluconazol, este método resulta confiable y de bajo costo; sin embargo, es conveniente que los aislamientos con halos ≤ 15 mm sean reevaluados por el método de referencia.

In order to standardize and evaluate a disk diffusion method with visual reading to detect in vitro fluconazole susceptibility of yeast, 1193 clinical isolates were tested. These included 584 Candida albicans, 196 Candida parapsilosis, 200 Candida tropicalis, 113 Candida glabrata, 50 Candida krusei and 50 Candida spp. and other opportunistic yeasts. The disks were manufactured in the INEI-ANLIS "Dr. Carlos G. Malbrán". The disk diffusion method results were compared to MIC results obtained by the reference CLSI M27-A2 broth microdilution method modified by EUCAST. The interpretative breakpoints for in vitro susceptibility testing of fluconazole were established at: zone diameter ≥ 16 mm for MIC ≤ 8 µg/ml (susceptible isolates), between 9 and 15 mm for MIC = 16-32 mg/ml (susceptible dose-dependent isolates), and ≤ 8 mm for MIC ≥ 64 µg/ml (resistant isolates). Overall agreement between the two methods was 94.7%, with 0.2% very major errors, and 0.3% major errors. Inter - and intralaboratory agreement was good. The disk diffusion method for drug susceptibility testing of Candida spp. isolates is inexpensive, reliable and reproducible. However, when the inhibition zone diameter is ≤ 15 mm, it is advisable to test the isolate by the reference microdilution method.

Estudio multicéntrico de fungemias por levaduras en la República Argentina / Multicenter study of bloodstream infections due to yeasts in Argentina

La incidencia de candidemias aumentó aproximadamente en un 500% en hospitales de alta complejidad y se observó un cambio en la distribución de especies del género Candida, con un incremento de las levaduras no Candida albicans. Con el objeto de conocer la distribución de especies asociadas a fungemias por levaduras en Argentina y determinar su sensibilidad a los antifúngicos de uso convencional, se realizó un estudio multicéntrico durante el período abril 1999 a abril 2000. Participaron 36 instituciones del país. Se colectaron 265 aislamientos de levaduras provenientes de hemocultivos, que se identificaron utilizando pruebas morfológicas, fisiológicas y bioquímicas y la determinación de la concentración inhibitoria mínima se realizó en base al estándar del NCCLS. La distribución de especies fue: Candida albicans (40,75%), Candida parapsilosis (28,67%), Candida tropicalis (15,84%), Candida famata (3,77%), Cryptococcus neoformans (3,77%), Candida glabrata (2,64%) y otras (4,53%). La mayoría de los aislamientos fueron sensibles a anfotericina B, fluconazol e itraconazol. La mortalidad asociada a las fungemias por levaduras estudiadas (n=265) fue del 30%, siendo más baja a lo descrito (33-54%) y fue menor en los pacientes que recibieron tratamiento antifúngico (26,3%), que en los no tratados (47%).

The incidence of candidemia has increased approximately 500% in high-complexity hospitals. A change in the spectrum of Candida infections due to species other than Candida albicans has also been detected. Between April 1999 and April 2000 a multicenter study was performed in order to determine the species distribution associated to candidemias in Argentina and the susceptibility profile of the isolates to the current antifungal drugs. Thirty six institutions have participated. All the 265 yeast strains isolated from blood cultures were identified by morphological, physiological, and biochemical tests. The antifungal susceptibility testing of isolates was performed based on the reference NCCLS procedure. The distribution of species was: Candida albicans (40.75%), Candida parapsilosis (28.67%), Candida tropicalis (15.84%), Candida famata (3.77%), Cryptococcus neoformans (3.77%), Candida glabrata (2.64%), and others (4.53%). Most of the isolates were susceptible to amphotericin B, fluconazole and itraconazole. Mortality associated to the fungemia by yeasts episodes (n=265) was 30%, lower than results previously determined (33-54%). The mortality percentage in patients who received antifungal therapy versus patients without treatment was 26.3% and 47%, respectively.

Primer aislamiento de Histoplasma capsulatum de murciélago urbano Eumops bonariensis / First isolation of Histoplasma capsulatum from the urban bat Eumops bonariensis

Se comunica el primer aislamiento de Histoplasma capsulatum var. capsulatum de un murciélago macho de la especie Eumops bonariensis, capturado en la ciudad de Buenos Aires en 2003. Los aislamientos fueron recuperados de bazo e hígado e identificados fenotípicamente. Se los comparó por PCR, con 17 aislamientos clínicos, 12 de pacientes residentes en la ciudad de Buenos Aires y cinco de otros países de América, usando los iniciadores 1283, (GTG)5, (GACA)4 y M13. Con los cuatro iniciadores, los perfiles de los aislamientos de murciélago resultaron idénticos entre sí y más relacionados a los de pacientes de Buenos Aires que a los de otros países (porcentaje de similitud: 91-100% y 55-87%, respectivamente). La alta relación genética entre los aislamientos obtenidos del murciélago y de los humanos residentes en Buenos Aires sugiere una fuente común de infección. Este es el primer registro de E. bonariensis infectado con H. capsulatum en el mundo, y el primer aislamiento del hongo en la población de quirópteros de la Argentina. Así como estos mamíferos actúan como reservorio y dispersan el hongo en la naturaleza, la infección en murciélagos urbanos podría asociarse al elevado número de casos de histoplasmosis entre pacientes inmunodeprimidos en la ciudad de Buenos Aires.

We report the first isolation of Histoplasma capsulatum var. capsulatum from a male bat Eumops bonariensis captured in Buenos Aires city in 2003. The pathogen was recovered from spleen and liver specimens, and was identified by its phenotypic characteristics. PCR with primers 1283, (GTG)5, (GACA)4 and M13 was used to compare both bat isolates with 17 human isolates, 12 from patients residing in Buenos Aires city, and 5 from other countries of the Americas. The profiles obtained with the four primers showed that both bat isolates were identical to each other and closer to Buenos Aires patients than to the other isolates (similarity percentage: 91-100% and 55-97%, respectively). The high genetic relationship between bat isolates and those from patients living in Buenos Aires suggests a common source of infection. This is the first record of E. bonariensis infected with H. capsulatum in the world, and the first isolation of the fungus in the Argentinean Chiroptera population. In the same way as these wild mammals act as reservoir and spread the fungus in the natural environment, infection in urban bats could well be associated with the increase in histoplasmosis clinical cases among immunosuppressed hosts in Buenos Aires city.

Inmunodiagnóstico de micosis endémicas y aspergilosis broncopulmonar: Estudio multicéntrico en la República Argentina / Immunodiagnosis of endemic mycoses and bronchopulmonary aspergilosis: A multicenter study in Argentina

Se realizó entre 01-04-2000 y 30-03-2001, un estudio de corte transversal, para conocer la frecuencia relativa de las enfermedades por hongos dimorfos y Aspergillus spp. en la República Argentina y evaluar la certeza en el diagnóstico de los laboratorios de diferentes áreas geográficas. Participaron 25 centros de salud provenientes de 12 provincias y de la Ciudad Autónoma de Buenos Aires. Fueron analizados en el laboratorio de origen 965 sueros de pacientes con sospecha clínica de histoplasmosis (HP), paracoccidioidomicosis (PCM), coccidioidomicosis (CM) y aspergilosis. Todos los sueros positivos y el 35% de los negativos fueron reevaluados en el laboratorio de referencia por inmunodifusión doble en agar. La concordancia entre los resultados obtenidos en los centros de origen y el de referencia fue de 98,8%. Se detectaron anticuerpos específicos en 120 sueros correspondientes a 98 pacientes. El 71,4% (70 casos) de los diagnósticos correspondió a micosis endémicas (HP, PCM y CM) y el resto a aspergilosis. PCM fue diagnosticada en 47,9% (47 casos), aspergilosis en 28,6% (28 casos), HP en13,3% (13 casos) y CM en 10,2% (10 casos). La participación en este estudio fue voluntaria y no todos los centros del país estaban representados, sin embargo, las frecuencias de enfermedades fúngicas fueron las esperadas y coincidentes con estudios previos realizados a nivel nacional.

In order to contribute to the knowledge of the relative frequency of chronic fungal diseases and assess the performance of diagnostic laboratories in Argentina, a multicenter study was performed with the participation of 25 medical centers located in 12 different provinces and Buenos Aires City. Between 04-01-2000 and 03-30-2001, 965 serum specimens from patients clinically suspected of having histoplasmosis (HP), paracoccidioidomycosis (PCM), coccidioidomycosis (CM) or aspergilosis were analyzed. Agar immunodiffusion tests (IDD) were done locally. All positive and 35% of negative sera were retested in the reference center. Results of laboratories of origin showed 98.8% concordance with those of reference center. Antibodies against any of the etiological agents were detected in 120 specimens from 98 patients. Endemic mycoses (HP, PCM and CM) were diagnosed in 70 patients (71.4%) and aspergilosis in 28 (28.6%). The frequencies of the different mycoses in decreasing order were PCM 47 patients (47.9%), aspergilosis 28 patients (28.6%), HP 13 patients (13.3%) and CM 10 patients (10.2%). The study was carried out on a voluntary basis and some areas of the country were not represented. However, the frequencies were in range with the expected rates in the population under study.

Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina / Firs environmental detection of Cryptococcus neoformans var. gattii in Argentina

Dos cepas de C.neoformans var. gattii serotipo B, fueron aisladas en el Parque de los Patricios de la ciudad de Buenos Aires, Argentina. Las muestras se recogieron en primavera, mediante el hisopado de la superficie interior de huecos y grietas de Eucalyptus spp. y otros árboles que, lamentablemente, no fueron identificados en el momento de la recolección. Los hisopos se inocularon en placas de agar ácido cafeico suplementado con antibióticos que se incubaron a 28§C durante una semana con observación diaria. Los aislamientos produjeron colonias características de la especie, lo que permitió detectarlos y separarlos de otros microorganismos presentes en la muestra. La identificación se realizó mediante estudios de fermentación de carbohidratos, asimilación de fuentes de carbono y nitrógeno y detección de ureasa y fenoloxidasa. La variedad se determinó utilizando el medio agarizado de canavanina-glicina-azul de bromotimol (CGB). La caracterización genotípica por reacción en cadena de la polimerasa con primer aleatorio (AP-PCR con el primer (GACA)4)mostró que el patrón de bandas de ambos aislamientos se diferenciaba en una única banda. En ambos casos el material provino de especies arbóreas diferentes de Eucalyptus spp. Estos dos aislamientos de fuentes naturales demuestran que C.neoformans var. gattii está presente en el medio ambiente de la ciudad de Buenos Aires, no necesariamente asociado a Eucalyptus spp. Este hallazgo podría explicar su asociación esporádica a infecciones humanas en la población local.

Especies de Malassezia aisladas de patologías de piel en un centro asistencial de la ciudad de Buenos Aires, Argentina / [Malassezia species isolated from skin diseases in a care center in the city of Buenos Aires, Argentina]

The epidemiology of Malassezia genus is poorly understood; geographical and population factors might have influence on the species distribution. The aim of this work was to determine the frequency of Malassezia species involved in skin diseases in a diagnostic center at Buenos Aires City. From 02/01/1999 to 10/31/2001, 456 skin specimens from 447 patients diagnosed as pityriasis versicolor, seborrheic dermatitis, foliculitis and atopic dermatitis, were analyzed by microscopic examination and culture. Malassezia spp was evidenced in 255 skin samples (55.9

) by microscopic analysis but it could be recovered from only 99 (38.8

) scarring lesions that were negative by microscopy. Among 109 isolates, M. sympodialis (58.7

) was the most frequent, followed by M. globosa and M. furfur (18.3

) of M. obtusa, M. slooffiae and M. restricta were recovered. Eleven isolates (10.1

) could not be identified because they had lost viability in the culture. This is the first study on Malassezia species associated to skin diseases in Argentina; further screening including other geographic regions of the country is necessary in order to confirm these results.

Cariotipos electroforéticos y análisis de los fragmentos de restricción de ADN genómico: su utilidad como herramientas en estudios epidemiológicos de Candida parapsilosis / [Electrophoretic karyotypes and genomic DNA restriction fragment analysis: their usefulness as tools in the epidemiological study of Candid parapsilosis]

During the past decades, several studies have reported an increase in the incidence of nosocomial candidosis. In a prospective study, performed at the Departamento de Micología, INEI, ANLIS Dr. C. G. Malbrán and the Servicio de Neonatología and Microbiología, Hospital de Niños Sor María Ludovica, from October 1995 to December 1996, 167 patients with candidosis were detected. Candida species isolated were C. albicans (53.1

). The aim of this work was to characterize the clinical C. parapsilosis isolates from pediatric patients hospitalized in two neonatal intensive care units from the same hospital and to evaluate the usefulness of electrophoretic karyotype (EK) and restriction endonuclease analysis of genomic DNA (REAG) using a low frequency digestion enzyme. EK of all isolates disclosed 12 banding patterns and REAG with endonuclease Sfi I showed only 5 groups. However, isolates from the control group could not be separated from the clinical isolates. The isolates within each dendogram group for EK or REAG were apparently unrelated. Our results show that EK yields better results than REAG, but that it falls short of the desired discrimination, which suggests that these techniques do not seem to be useful for studying nosocomial C. parapsilosis outbreaks.

) and C. tropicalis (14.8

Distribución de los serotipos de Cryptococcus neoformans asociados a infecciones humanas en Argentina / [Distribution of Cryptococcus neoformans serotypes associated with human infections in Argentina]

C. neoformans (Cn) causes severe meningitis in immunocompromised patients, particularly those with AIDS. Little is known about Cn serotypes associated with human infections in Argentina due to the lack of antisera availability. The aim of this study was to produce these antisera, to serotype the Argentinean clinical isolates of Cn received at our laboratory from 1984 to 2001 and to evaluate the agreement of these results with the variety differentiation obtained with Canavanine-Glycine-Bromothymol Blue agar (CGB) medium. We studied 123 isolates recovered from first episodes of cryptococcosis from HIV-infected patients (85), non HIV-infected patients (6) and from other non specified patients (32). Of the isolates, 89

(5/123) corresponded to serotype D, 3

(3/129) were serotype AD, 3 isolates were untypable and 2 corresponded to serotype B. All the serotyped isolates agreed with the variety determined by CGB medium. These results indicate that, in our country, most cryptococcal infections in patients with AIDS are associated with serotype A (77/85), which agrees with international studies. In patients with other immunosuppressions, this serotype may be also predominant, although a low number of isolates were tested (4/6). Recovery of Cn serotype B in these clinical isolates suggests that studying the variety and their serotypes might be important to detect a probable epidemiological alteration.

Caracterización molecular de aislamientos clínicos autóctonos de Histoplasma capsulatum var. capsulatum mediante RAPD-PCR / Molecular characterization of autochthonous clinical isolates of Histoplasma capsulatum var. capsulatum using RAPD-PCR

The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.

Candidiasis vaginal: etiología y perfil de sensibilidad a agentes antifúngicos de uso clínico / [Vaginal candidiasis: etiology and sensitivity profile to antifungal agents in clinical use]

Candida vaginitis is one of the most frequent infection of the female genital tract with a high incidence. Approximately 75

of sexually active women suffer at least one episode of Candida vaginitis and 10

of them have recurrent episodes. Pregnancy, diabetes mellitus and antibiotic treatment are the most common predisposing factors, C. albicans is the etiologic agent most frequently found. The widespread reports of fluconazole resistance in Candida species and the selection of non Candida albicans prompted the study of species distribution of vulvovaginal candidiasis and their in vitro susceptibility against current antifungal agents. A total of 314 women with vaginal infection were studied. Yeasts were isolated from 104 patients with vulvovaginal candidiasis. The following species were identified: C. albicans 87.5

included C. krusei, C. famata, C. tropicalis and S. cerevisiae. The minimal inhibitory concentration (MIC) was determined for nystatin, isoconazole, fluconazole and ketoconazole, using a broth microdilution method based on NCCLS procedure. Although most of the isolates were C. albicans, the high percentage of C. glabrata recovered suggests the need to identify the yeasts isolated. Fluconazole resistant C. albicans were isolated in 13.46

of the cases. Thus, further studies are required to correlate the possible role of these strains in recurrent vulvovaginal candidiasis.

Transmisión nosocomial de Candida albicans en recién nacidos / Nosocomial transmission of Candida albicans in newborn infants

Candida spp. colonization in neonates occurs due to vertical or horizontal transmission. Preliminary studies determined that Candida albicans is the principal agent of these infections. In order to establish nosocomial transmission, 26 Candida albicans strains isolated from patients with candidosis hospitalized during a 18-month period in 2 neonatal intensive care units (NICU) from a pediatric hospital were studied. Fourteen isolates from patients and health care workers, involved in possible outbreaks of an intensive care unit (UCI) and a NICU from another pediatric hospital were also studied. All Candida albicans strains were genotyped by Southern blot hybridization with 27A. Isolates for outbreak confirmation were also hybridized with another specific Candida albicans probe, Ca3. Hybridization patterns demonstrated horizontal transmission in all the units studied. In a NICU, transmission among 4 patients during a 10-month period could be established and in the other NICU, 3 cases of transmission among 2 patients each were demonstrated in periods of 2 to 20 days. The outbreak studies showed the same strain isolated from 2 nurses and from one patient at the NICU and at the ICU identical strains were found in 3 patients. In this study, hybridization with Ca3 in addition to 27A probe did not increase discrimination power among isolates. Genotypic analysis allows, not only, determination of transmission and persistence of strains during prolonged periods or in sporadic outbreaks, but also facilitates necessary epidemiological decisions for optimizing nosocomial fungal infection control measures.

Infección in vitro por diferentes cepas de Paracoccidioides brasiliensis / In vitro infection by different strains of Paracoccidioides brasiliensis

We analyzed the in vitro infection process by P. brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines. The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent). Strains of intermediate and high virulence had higher adherence at first contact (about 16). Strain B339 had a slower adherence at first contact (8) than the others during the same period. The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains. We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro. We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents.

Estudio multicéntrico de micosis superficiales en Argentina. Grupo EMMS / Multicenter study of superficial mycoses in Argentina. EMMS Group

Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58 female, 42 male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6 of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3 of the diagnosticated infections, yeast 12.2 and Malassezia furfur 13.3. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7), T. mentagrophytes (19.2) and M. canis (14.2). C. albicans was the predominant causative agent of yeast infections (44.4). In children below 11 years old, the most frequent infections were tinea capitis 55.4 (M. canis 60, T. tonsurans and M. gypseum 8.3) and tinea corporis 20 (M. canis 46, T. mentagrophytes 30). The most frequent localizations in adult were toenail and feet (63) (T. rubrum 47.3 and 45.7, T. mentagrophytes 9.5 and 23.4, respectively), fingernail 15.6 (C. albicans 25 and T. rubrum 8.3) and trunk and extremities 12.2 (M. furfur 57.5 and T. mentagrophytes 14.9). Direct examination allowed the diagnosis of 94.66 of all positive samples and 91.74 of the cases with positive isolation.

Estudio multicéntrico sobre candidiasis nosocomial en la República Argentina. Groupo EMIFN / Multicenter study on nosocomial candidiasis in the Republic of Argentina

A multicenter study was performed between April and September 1998 with the participation of 12 medical centers located in 8 different provinces and in the capital city of Argentina. The aim of this study was to determine the species distribution and the antifungal susceptibility profile of isolates causing nosocomial fungal infections. All the fungal isolates obtained were sent to the Mycology Department for reference identification and antifungal susceptibility testing. Eighty-nine isolates were received from different clinical specimens. The distribution of species obtained was C. albicans (50.6), C. tropicalis (22.5), C. parapsilosis (20.2), C. krusei (3.4), C. glabrata (2.2) and Debaryomyces hansenii (1.1). Most of the isolates (85/88) had MICs for amphotericin B < or = 1 microgram/ml, C. krusei showed resistance to fluconazole but was dose dependent susceptible to itraconazole, C. glabrata (2/2) were resistant against both drugs, most of the isolates of C. albicans, C. tropicalis and C. parapsilosis were susceptible to these triazole drugs. These data showed a different distribution of Candida species compared with results obtained in other countries. The low frequency of appearance of C. krusei and C. glabrata in our country suggests a reduced selective pressure by triazoles.

Sensibilidad in vitro de Paracoccidioides brasiliensis frente a los antifúngicos de uso sistémico / In vitro sensitivity of Paracoccidioides brasiliensis to systemically used antifungal agents

In 1992, the NCCLS proposed a broth macrodilution method for determining the in vitro susceptibility of yeast. However, for dimorphic fungi no standardised method is available. The aim of our study was to evaluate the reference broth macrodilution method with isolates of Paracoccidioides brasiliensis. The in vitro susceptibility of two ATCC strains (32069 and 36324) and nine clinical isolates were determined against serial dilutions of amphotericin B (AMB), ketoconazole (KTZ), and itraconazole (ITZ) from 0.02 to 20 mg/l and fluconazole (FCZ) from 0.1 to 100 mg/l. The inoculum used was 0.5 x 10(4)-2.5 x 10(5) CFU/ml, employing the yeast phase in order to obtain homogeneous growth. The test was incubated with agitation in a rotating shaker at 35 degrees C, and 7 days was the optimal time for MIC final reading. Average MIC50/MIC90 (mg/l) for the eleven isolates tested were the following: AMB: < 0.02/0.16, FCZ: 1.6/6.2, KTZ: 0.08/0.31, ITZ: 0.31/1.3. One clinical isolate showed high MIC for azole drugs (KTZ: 1.3 mg/ml, FCZ: 100 mg/l and ITZ 5 mg/l). Although further studies are necessary to evaluate the usefulness of this methodology for P. brasiliensis, the latter may be clinically useful to predict the development of resistance.

Producción de antisueros fúngicos específicos en conejo / Specific fungi antisera production in rabbits

Se probó un método de inoculación en conejos, por vía intradérmica para la producción de antisueros fúngicos contra Aspergillus fumigatus (AF), Histoplasma capsulatum (HC), Paracoccidioides brasiliensis (PB) y Coccidioides immitis (CI), con el fin de ser utilizados como sueros control positivos para pruebas de inmunodifusión en gel de agar (ID). En este trabajo se utilizó como inmunógeno el antígeno en la dilución de uso (DU) para las pruebas de rutina de ID, lo que facilitó su estandarización. Los antígenos de AF, HC, CI se prepararon por los métodos descriptos por el Center for Disease Control (CDC), y el de PB según la técnica descripta por Camargo y col. Todos los conejos produjeron anticuerpos contra los diferentes antígenos específicos (bandas por ID) en el momento en que se alcanzaron los picos de respuesta primaria y luego de cada refuerzo. La respuesta secundaria fue igual o menor que la primaria, aunque se mantuvo la calidad y cantidad de bandas de precipitación específicas detectadas por ID. La inmunización primaria con concentraciones de antígeno mayores o menores a la DU, sólo analizadas en el caso de A. fumigatus, produjo bandas de precipitación menos definidas o en menor número respectivamente, aunque los títulos de anticuerpos fueron similares