Concordance analysis between different methodologies used for identification of oral isolates of Candida species / Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de Candida

Abstract Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

Resumen Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.

Comparison between four usual methods of identification of candida species / Comparação entre quatro métodos usuais de identificação de espécies de Candida

SUMMARYInfection by Candidaspp. is associated with high mortality rates, especially when treatment is not appropriate and/or not immediate. Therefore, it is necessary to correctly identify the genus and species of Candida. The aim of this study was to compare the identification of 89 samples of Candidaspp. by the manual methods germ tube test, auxanogram and chromogenic medium in relation to the ID 32C automated method. The concordances between the methods in ascending order, measured by the Kappa index were: ID 32C with CHROMagar Candida(κ = 0.38), ID 32C with auxanogram (κ = 0.59) and ID 32C with germ tube (κ = 0.9). One of the species identified in this study was C. tropicalis,which demonstrated a sensitivity of 46.2%, a specificity of 95.2%, PPV of 80%, NPV of 81.1%, and an accuracy of 80.9% in tests performed with CHROMagar Candida;and a sensitivity of 76.9%, a specificity of 96.8%, PPV of 90.9%, NPV of 91%, and an accuracy of 91% in the auxanogram tests. Therefore, it is necessary to know the advantages and limitations of methods to choose the best combination between them for a fast and correct identification of Candidaspecies.

RESUMOA infecção por Candidaspp. está associada com alta mortalidade, principalmente quando o tratamento não é adequado, nem imediato. Assim, a correta identificação do gênero e espécie é necessária. O objetivo deste trabalho foi comparar 89 amostras de Candidaspp. pelos métodos manuais prova do tubo germinativo, auxanograma e CHROMagar em relação ao método automatizado ID 32C. As concordâncias entre os métodos em ordem crescente, medidas pelo coeficiente de Kappa, foram: ID 32C com CHROMagar Candida(κ = 0,38), ID 32C com auxanograma (κ = 0,59) e ID 32C com tubo germinativo (κ = 0,9). Uma das espécies identificadas neste trabalho foi a C. tropicalis, que demonstrou uma sensibilidade de 46,2%, especificidade de 95,2%, VPP de 80%, VPN de 81,1% e acurácia de 80,9% nos testes com CHROMagar Candidae uma sensibilidade de 76,9%, especificidade de 96,8%, VPP de 90,9%, VPN de 91% e acurácia de 91% nos testes de auxanograma. Portanto, o conhecimento das vantagens e limitações dos métodos é necessário para a escolha da melhor combinação entre os mesmos visando uma rápida e correta identificação das espécies de Candida.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.

Use of mueller-iinton broth and agar in the germ tue test / Utilização de ágar e caldo Mueller-Hinton na prova do tubo germinativo

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

Candida albicans é frequentemente isolada em amostras clínicas, assim a sua diferenciação presuntiva de outras espécies do gênero pode ser baseada na habilidade em formar o tubo germinativo em soro humano. Entretanto, existem outras duas espécies que também possuem essa característica, C. dubliniensis e C. africana. O objetivo foi comparar quatro diferentes substratos para a realização da prova do tubo germinativo (TG). Utilizou-se isolados de Candida spp. identificados através de meio manual (135 C. albicans, 24 C. tropicalis e um C. dubliniensis). A prova do tubo germinativo foi realizada utilizando soro previamente congelado e fresco, caldo e ágar Mueller-Hinton (MH). O TG através da técnica do soro a fresco foi observado em 96% (130/136), 94% (128/136) através do soro previamente congelado, 92% (125/136) no ágar e 90% (122/136) no caldo MH. A sensibilidade de cada teste foi maior que 90% e especificidade de 100%. Tanto o caldo quanto o ágar MH foram capazes de identificar apenas os verdadeiros positivos e não ocorrendo falsos positivos, porém deixaram de identificar alguns isolados de C. albicans. O ágar e o caldo MH podem ser utilizados na rápida e presuntiva identificação laboratorial de C. albicans, como uma alternativa para o teste do tubo germinativo.

Estudio comparativo entre los sistemas automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales para la identificación de levaduras de interés clínico / A comparative study between the Vitek YBC® and Microscan Walk Away RYID® automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest

El objetivo de este trabajo fue comparar la identificación de levaduras de interés clínico por los métodos automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales. Se utilizaron 193 aislamientos de levaduras provenientes de muestras clínicas y cinco cepas controles. Todas las levaduras fueron identificadas por los métodos automatizados antes nombrados y los métodos fenotípicos convencionales de asimilación de carbohidratos, visualización de la morfología microscópica con agar harina de maíz y el uso de agar cromogénico. Para evaluar las variables se utilizaron tablas de contingencia de 2×2, Chi cuadrado de Mc Nemar, el índice Kappa, y se calcularon los valores de concordancia, así como los errores mayores y menores de los métodos automatizados. Las levaduras se dividieron en dos grupos: 1) de aislamiento frecuente y 2) de aislamiento poco frecuente. Los sistemas Vitek YBC® y Microscan Walk Away RYID® fueron concordantes en un 88,4 y 85,9% respectivamente, cuando se compararon con los métodos fenotípicos convencionales. Aunque ambos sistemas automatizados se pueden utilizar para la identificación de levaduras, la presencia de errores mayores y menores indica la posibilidad de identificaciones incorrectas, por lo tanto, el operador de estos equipos debe utilizar paralelamente pruebas fenotípicas como la visualización de la morfología microscópica en agar harina de maíz y el agar cromogénico, sobre todo frente a levaduras de aislamiento poco frecuente. Los sistemas automatizados son una herramienta valiosa, sin embargo, la experiencia y el criterio del microbiólogo son una fortaleza importante para asegurar la calidad de los resultados.

The aim of this study was to compare the identification of clinically relevant yeasts by the Vitek YBC® and Microscan Walk Away RYID® automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2×2 contingency tables, McNemar’s Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: 1) frequent isolation and 2) rare isolation. The Vitek YBC® and Microscan Walk Away RYID® systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.

Evaluación del sistema Vitek 2 para la identificación de las principales especies de levaduras del género Candida / Avaluation of Vitek 2 for the identification of Candida yeasts

El objetivo del trabajo fue evaluar el desempeño de las tarjetas YST del sistema Vitek 2 para la identificación de levaduras del género Candida. Se analizaron 168 aislamientos; los resultados fueron comparados con los obtenidos por los equipos API 20C AUX (24 %) o API ID 32C (76 %). Cada cepa se subcultivó en agar cromogénico para levaduras y se observó la micromorfología. C. albicans y C. dubliniensis fueron identificadas a través de pruebas bioquímicas y moleculares adicionales. La concordancia observada fue del 98,3 %. Solo tres cepas no fueron identificadas correctamente por el sistema Vitek 2: una cepa de C. tropicalis y una de C. krusei fueron identificadas erróneamente como C. parapsilosis y otra cepa de C. krusei fue identificada de manera incompleta por el software del equipo. El tiempo promedio de identificación con las tarjetas YST fue de 18,25 h. El sistema Vitek 2 surge como un método confiable, simple y efectivo para la identificación de las principales especies del género Candida

The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20C AUX (24%) o API ID 32C (76%) kits (bioMérieux, Marcy L'Etoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3%. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species

Estudio taxonómico de aislamientos clínicos de Trichophyton en Rosario, Argentina / Taxonomic study of clinical isolates of Trichophyton in Rosario, Argentina

Debido al pleomorfismo y la variabilidad cultural que presentan las especies del género Trichophyton, los métodos de identificación basados exclusivamente en caracteres morfológicos a menudo no son suficientes para su clasificación. El objetivo de este estudio fue probar un conjunto de métodos fenotípicos para identificar aislamientos fúngicos pertenecientes al género Trichophyton empleando una técnica de taxonomía molecular como método de referencia. Se utilizaron 56 aislamientos clínicos y 6 cepas de referencia, con los que se realizaron estudios macro y micromorfológicos, así como las siguientes pruebas bioquímicas y fisiológicas: perforación del pelo in vitro, requerimientos nutricionales en medios agar Trichophyton, producción de ureasa y crecimiento en el medio agar púrpura de bromocresol-leche-glucosa (APBC-L-G). A su vez se realizó una PCR fingerprinting utilizando el cebador simple (GACA)4. Como resultado de la reacción de PCR se observaron perfiles específicos bien diferenciables para Microsporum canis, Epidermophyton fl occosum, Trichophyton rubrum y Trichophyton interdigitale, pero un mismo perfil para Arthroderma benhamiae y Trichophyton erinacei. Del total de los aislamientos clínicos evaluados, 39 coincidieron con el perfil de T. rubrum, 13 con el de A. benhamiae y 4 con el de T. interdigitale. El ensayo fenotípico más útil para diferenciar T. rubrum del complejo T. mentagrophytes fue la alcalinización del medio APBC-L-G. Con las pruebas fenotípicas se pudo diferenciar T. rubrum de los aislamientos del complejo T. mentagrophytes, pero no las especies pertenecientes a dicho complejo. La técnica molecular permitió caracterizar tanto los aislamientos de T. rubrum como los pertenecientes al complejo T. mentagrophytes, que en nuestro estudio correspondieron a T. interdigitale y Trichophyton sp. anamorfo de A. benhamiae.

Due to the pleomorphism and cultural variability displayed by species of the genus Trichophyton, the identification methods based solely on morphological features are usually insufficient for their classification. The goal of the present work was to test a set of phenotypic methods in order to identify fungal isolates that belong to the aforementioned genus. These methods were based on a molecular taxonomic technique used as standard. Clinical isolates (56) were used as samples along with 6 reference strains. Macro and micromorphological studies were performed as well as biochemical and physiological tests such as in vitro hair perforation, nutritional requirements in Trichophyton agar media, urease production and growth on bromocresol purple-milk solids-glucose (BCP-MS-G) agar. Additionally, PCR fingerprinting using the (GACA)4 primer was employed. As a result of the PCR method, specific profiles were observed for Microsporum canis, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton interdigitale. Identical profiles were obtained for Arthroderma benhamiae y Trichophyton erinacei. Of the total number of clinical isolates, 39 matched the T. rubrum profile while 13 corresponded to A. benhamiae and 4 to T. interdigitale. The most useful phenotypic test to differentiate between T. rubrum and T. mentagrophytes complex strains was alkalinization of the BCP-MS-G medium. Phenotypic tests did not allow differentiation among the T. mentagrophytes complex species. On the other hand, the molecular technique allowed characterization of T. rubrum isolates as well as of those observed in our study and included in the T. mentagrophytes complex: T. interdigitale and Trichophyton sp. , the anamorph of A. benhamiae.

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar / Diferenciação entre Candida albicans e Candida dubliniensis usando caldo Sabouraud hipertônico e ágar Tabaco

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

INTRODUÇÃO: Infecções fúngicas oportunistas em hospedeiros imunocomprometidos são causadas por espécies de Candida, cuja maioria das infecções se deve a Candida albicans. Entretanto, o patógeno emergente Candida dubliniensis demonstra várias características fenotípicas em comum com C. albicans, tais como produção de tubo germinativo e clamidósporos, solicitando atenção por desenvolver resistência in vitro estável ao fluconazol. O objetivo do presente estudo foi avaliar a performance da identificação bioquímica na diferenciação entre C. albicans e Candida dubliniensis, analisando fenotipicamente leveduras previamente identificadas como C. albicans. MÉTODOS: Setenta e oito isolados identificados como C. albicans pelo sistema API ID 32C foram cultivados em ágar Sabouraud dextrose a 30°C por 24-48h e em seguida inoculados em caldo hipertônico Sabouraud e agar tabaco. RESULTADOS: Nossos resultados mostraram que 17 (21,5%) isolados tiveram o crescimento inibido no caldo hipertônico Sabouraud, característica fenotípica inconsistente para C. albicans neste meio de cultura. Entretanto, os resultados observados em ágar tabaco mostraram que somente 9 (11,4%) dos isolados inibidos produziram colônias características de C. dubliniensis (colônias rugosas, marrom-amarelada com fragmentos de hifas e abundantes clamidósporos). CONCLUSÕES: Os resultados obtidos sugerem que este é um instrumento simples para triagem entre leveduras de C. albicans e não-albicans, bem como confirmação de identificação automatizada.

Virus del papiloma humano (VPH) detección y tipificación en la consulta privada / Human papilloma virus (HPV) detection and typification in private practice

Determinar cuáles son los tipos de virus del papiloma humano en mujeres y hombres que asisten a una consulta privada de ginecología. Se estudiaron 200 pacientes: 175 mujeres y 25 hombres, con diagnóstico de virus del papiloma humano a simple vista, colposcopia, citología y/o biopsia. A todos se les realizó tipificación de virus del papiloma humano por reacción en cadena de la polimerasa. En la Unidad de Ginecología Reproducción y Salud Integral. Valencia. La edad de los pacientes se ubicó en más del 70 por ciento en los grupos etarios de 15 a 34 años. Las pacientes nuligestas y nulíparas estaban alrededor del 60 por ciento. La imagen colposcópica dominante en las mujeres fue epitelio aceto-blanco (77 por ciento), mientras que en la totalidad de los hombres correspondió a verrugas. La citología cervical reportó virus del papiloma humano (con o sin NIC) en solo 40 por ciento, mientras que la biopsia lo demostró cerca del 80 por ciento. Se realizó detección/tipificación de virus del papiloma humano por el método de reacción en cadena de la polimerasa en todos los casos, encontrándose cerca del 10 por ciento de resultados negativos en mujeres y 12 por ciento en hombres. Fueron hallados 15 tipos de virus del papiloma humano en mujeres, de los cuales más de la mitad eran de alto riesgo. En los hombres solo encontramos 5 tipos diferentes, de los cuales solo la cuarta parte aproximadamente eran de alto riesgo. En nuestra serie de mujeres los tipos 6 y 11 fueron los más frecuentes de bajo riesgo, lo cual coincide a lo publicado en la literatura. En cuanto a los de alto riesgo, los de mayor frecuencia fueron los tipos 16, 31 y 33, lo cual no concuerda totalmente con las publicaciones. En nuestra serie de pacientes masculinos, el 64 por ciento de tipos de bajo riesgo fueron el 6 y el 11, lo cual es similar a lo publicado en la bibliografía estudiada. A esta diferencia entre los tipos encontrados....

To assess which are the HPV types in both male and female patients attending a private gynecological outpatient clinic. Two hundred patients were assessed: 175 women and 25 men who were diagnosed positive with HPV by visual examination, colposcopy, cytology and/or biopsy. Cervical cytology reported HPV (with or without CIN) in approximately only 40 percent of the cases, while biopsy showed this in nearly 80 percent of the cases. The typification method used for all the cases was PCR. Unidad de Ginecologia Reproduccion y Salud Integral. Valencia. More than 70 percent patients were within 15-34 years old. 60 percent of the women were nuligravidas and nulliparous. The more frequent colposcopic picture in women was acetowhite epithelium (77 percent) while all the cases of men showed warts. Detection/typification of HPV was performed by PCR in all cases with negative results in close to 10 percent women and 12 percent men. Fifteen types of HPV were found in women, in which more than half were high risk. In men only 5 different HPV types were found and only around one fourth of these cases were high risk. In our series, HPV types 6 and 11 were the low risk HPV in women, which coincides with publications in the literature. In relation to high risk HPVs, the more frequent types were 16, 31 and 33, which doesn’t coincide totally with publications. In our series of men, 64 percent of low risk were 6 and 11, which is similar to the data published in world literature. This difference between HPV types found in men and women suggests an incomplete understanding of all the factors involved in sexual transmission

Differentiation of Candida dubliniensis from Candida albicans with the use of killer toxins / Avaliação das toxinas killer na diferenciação entre Candida albicans e Candida dubliniensis

The aim of this study was to report the ability of killer toxins, previously used as biotyping techniques, as a new tool to differentiate C. albicans from C. dubliniensis. The susceptibility of C. albicans and C. dubliniensis to killer toxins ranged from 33.9 to 93.3 percent and from 6.67 to 93.3 percent, respectively.

Avaliou-se a capacidade das toxinas killer, previamente utilizadas na biotipagem de C. albicans, como método para diferenciar C. albicans de C. dubliniensis. A susceptibilidade de C. albicans e C. dubliniensis às toxinas killer variou de 33,9 por cento a 93,3 por cento para C. albicans e de 6,67 por cento a 93,3 por cento para C. dubliniensis.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50 percent), Cryptococcus gattii VGII (25/56, 44.64 percent) and C. gattii VGI (3/56, 5.26 percent). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95 percent 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

Molecular investigation of a fungemia outbreak due to Candida parapsilosis in an intensive care unit

We investigated a nosocomial cluster of four Candida parapsilosis fungemia episodes that occurred in a neurological intensive care unit over a two-week period. The four infected patients had received parenteral nutrition through central lines, and all four had catheter-related candidemia. All of the isolates were susceptible to all of the antifungals tested, including amphotericin B, fluconazole, voriconazole, and caspofungin. They had strictly related fingerprints, based on randomly amplified polymorphic DNA analysis. Additional DNA sequencing data revealed that they were same strain. Although no isolate of Candida parapsilosis was recovered from other clinical, surveillance, or environmental samples, nosocomial spread of this yeast ceased, following the reinforcement of infection-control measures. Candida parapsilosis may require an intravascular foreign body to cause fungemia, but this outbreak shows that it can be transmitted nosocomially and can cause epidemics.

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95 percent of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64 percent), followed by VGII (21 percent), VNII (5 percent), VGIII (4 percent), VGI and VNIV (3 percent each), and VNIII (< 1 percent). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Evaluación de la técnica de MSP-PCR para la caracterización molecular de aislamientos de Rhodotorula mucilaginosa provenientes de la Patagonia noroccidental / Assessment of the MSP-PCR technique for the molecular characterization of Rhodotorula mucilaginosa isolates from northwestern Patagonia

La identificación rápida de levaduras de origen ambiental o clínico es de importancia para el estudio de la biodiversidad de estos microorganismos y para la detección de posibles patógenos. Rhodotorula mucilaginosa es una levadura ubicua y pigmentada, capaz de producir infecciones en pacientes inmunocomprometidos. En este trabajo se evaluó la utilidad de la técnica de fingerprinting conocida como MSP-PCR (Micro/Minisatellite-Primed PCR) en la caracterización e identificación de aislamientos ambientales de R. mucilaginosa provenientes de la Patagonia noroccidental. Sobre la base de sus caracteres fenotípicos, de un total de 200 levaduras pigmentadas se seleccionaron 110 aislamientos que presuntamente corresponderían a la especie R. mucilaginosa. Se evaluaron los iniciadores (GTG)5, (GAC)5 y M13 en aislamientos representativos, y se seleccionó el iniciador (GTG)5 por ser el que permitió una mejor agrupación de los aislamientos pertenecientes a R. mucilaginosa y una mejor diferenciación de éstos con los de especies filogenéticamente próximas. Utilizando dicho iniciador, el 87% de los aislamientos de R. mucilaginosa presentó un perfil de MSP-PCR similar (> 60%) al de la cepa de referencia CBS 316T de R. mucilaginosa. La técnica de MSP-PCR resultó efectiva, tanto para caracterizar e identificar un número elevado de aislamientos ambientales de R. mucilaginosa como para detectar polimorfismos en la especie.

The rapid identification of environmental or clinical yeast isolates is important for biodiversity studies and the detection of probable pathogens. Rhodotorula mucilaginosa is a ubiquitous and pigmented yeast capable of infecting immunocompromised patients. In this study, we evaluated the Micro/mini satellite-primed PCR (MSP-PCR) fingerprinting method for the characterization and identification of R. mucilaginosa isolates from natural environments in northwestern Patagonia. There were selected 110 putative R. mucilaginosa isolates from 200 environmental pigmented yeast isolates on the basis of phenotypic criteria. (GTG)5, (GAC)5 and M13 primers were initially evaluated in representative R. mucilaginosa isolates. (GTG)5 allowed a good grouping of these isolates and, at the same time, a good differentiation among closely related species, and thus was selected for subsequent studies. R. mucilaginosa isolates (87%) presented similar (> 60%) MSP-PCR profiles to those of the reference strain CBS 316T. The MSP-PCR technique was effective, both, for the characterization and identification of a large number of R. mucilaginosa environmental isolates as well as for the detection of polymorphisms within the species.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Cryptococcus neoformans, pertencente à classe dos basidiomicetos, é um importante patógeno, principalmente em pacientes imunocomprometidos. Neste estudo, 47 isolados clínicos de C. neoformans de várias regiões do Estado de São Paulo foram avaliados quanto aos sorotipos e ao mating-type por PCR. A diversidade genética foi analisada por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, RAPD com o iniciador 6 (Amersham Pharmacia Biotech) e por RFLP do gene PLB1 digerido com AvaI. Todos os isolados foram obtidos de pacientes HIV positivos e identificados como sorotipo A e MATalfa. A maioria dos isolados pertencia ao tipo molecular VNI (45/47) e apenas dois foram VNII quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação alta (> 0.9). Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. Por PCR-RFLP, nenhum tipo molecular novo foi encontrado, realçando a idéia de que em genes conservados como PLB1, as diferenças podem ser resultantes de divergências evolutivas dentro do complexo C. neoformans, separando os isolados nos oito subtipos moleculares já estabelecidos. Nossos resultados fornecem novas informações sobre a epidemiologia molecular de C. neoformans na região sudeste do Brasil.

An evaluation of manual and mechanical methods to identify Candida spp. from human and animal sources

To compare two yeast identification methods, i. e, the manual and the VITEK mechanical methods, 62 clinical samples from hemocultures and animal sources were analyzed. After identification as Candida yeasts by the VITEK method, the strains were recharacterized using manual assimilation methods and sugar fermentation tests. Our findings reveal 58 percent concurrent identification between the two methods for animal strains, and 51 percent for human hemoculture strains.

Objetivando comparar os resultados de dois métodos de identificação, o manual e o método automatizado VITEK, foram analisadas 62 amostras clínicas isoladas de hemoculturas humanas e de origem animal. Estas amostras, após serem identificadas como Candida spp. pelo método automatizado VITEK, foram recaracterizadas com a utilização de métodos manuais de assimilação e fermentação de fontes carbonadas. Os resultados demonstraram uma relação de 58 por cento de identificações coincidentes entre os dois métodos para as amostras de origem animal e de 51 por cento para as amostras de hemoculturas humanas.

Comparison among tomato juice agar with other three media for differentiation of Candida dubliniensis from Candida albicans

O presente estudo teve como objetivo comparar o ágar suco de tomate, um tradicional meio utilizado para observação de ascósporos em leveduras, com o ágar semente de niger, ágar caseína e ágar semente de girassol, na diferenciação fenotípica entre C. albicans e C. dubliniensis. Após 48 h de incubação a 30 ºC, os 26 isolados de C. dublinienis (100%) evidenciaram a formação de clamidoconídios igualmente em todos os meios comparados. Entretanto, quando semeados com C. albicans, a formação de clamidoconídios foi raramente observada, resultando nos seguintes percentuais de ausência destas estruturas: ágar suco de tomate (92,47%), ágar niger (96,7%), ágar caseína (91,39%), ágar semente de girassol (96,7%). Estes resultados permitem-nos sugerir a utilização do ágar suco de tomate como mais um meio que, já no primo-isolamento, é capaz de, presuntivamente, diferenciar C. albicans de C. dubliniensis.