Multiplex PCR designed to differentiate species within the Candida glabrata complex / PCR multiplex diseñada para diferenciar las especies del complejo Candida glabrata

Background. No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. Aims. To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. Methods. The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. Results. The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). Conclusions. A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains (AU)

Antecedentes. No hay métodos fenotípicos disponibles para diferenciar las especies del complejo Candida glabrata. Objetivos. Diseñar un método de PCR multiplex para diferenciar las tres especies del complejo C. glabrata y usarlo para estudiar una colección de cepas identificadas anteriormente como C. glabrata. Métodos. El método fue desarrollado con base en las diferencias de la secuencia internal transcribed spacer (ITS) entre las especies. El método se validó mediante el uso de una colección de cepas incógnitas y se utilizó posteriormente para estudiar una colección de 192 cepas. Los resultados se compararon con las secuencias ITS. Resultados. El método propuesto mostró 100% de concordancia con la secuenciación de las regiones ITS y demostró ser eficaz clínica y epidemiológicamente. Se identificaron dos aislamientos de Candida bracarensis y tres de Candida nivariensis dentro de las 192 cepas identificadas fenotípicamente como C. glabrata (prevalencia de 0,93% y 1,40%, respectivamente). Conclusiones. Presentamos un método de PCR múltiplex rápido, económico y fiable. La utilidad de la metodología queda demostrada con el estudio de una gran colección de cepas de C. glabrata sensu lato (AU)

Multiplex PCR designed to differentiate species within the Candida glabrata complex.

BACKGROUND: No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. AIMS: To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. METHODS: The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. RESULTS: The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). CONCLUSIONS: A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains.

Design, characterization, and in vitro evaluation of antifungal polymeric films.

The objective of the present paper was the development and the full characterization of antifungal films. Econazole nitrate (ECN) was loaded in a polymeric matrix formed by chitosan (CH) and carbopol 971NF (CB). Polyethylene glycol 400 and sorbitol were used as plasticizing agents. The mechanical properties of films were poorer when the drug was loaded, probably because crystals of ENC produces network outages and therefore reduces the polymeric interactions between the polymers. Polymers-ECN and CH-CB interactions were analyzed by Fourier-transform infrared spectroscopy (FTIR), thermal gravimetry analysis, and differential thermal analysis (DTA-TGA). ECN did not show structure alterations when loaded into the films. In scanning electron microphotographs and atomic force microscopy analysis, films prepared with CB showed an evident wrinkle pattern probably due to the strong interactions between the polymers, which were observed by FTIR and DTA-TGA. The in vitro activity of the formulations against Candida krusei and Candida parapsilosis was twice as greater as the commercial cream, probably as a result of the antifungal combination of the drug with the CH activity. All these results suggest that these polymeric films containing ECN are potential candidates in view of alternatives dosages forms for the treatment of the yeast assayed.

Soybean sudden death syndrome species diversity within north and South america revealed by multilocus genotyping.

Sudden death syndrome (SDS) of soybean has become a serious constraint to the production of this crop in North and South America. Phenotypic and multilocus molecular phylogenetic analyses, as well as pathogenicity experiments, have demonstrated that four morphologically and phylogenetically distinct fusaria can induce soybean SDS. Published molecular diagnostic assays for the detection and identification of these pathogens have reported these pathogens as F. solani, F. solani f. sp. glycines, or F. solani f. sp. phaseoli, primarily because the species limits of these four pathogens were only recently resolved. In light of the recent discovery that soybean SDS and Phaseolus and mung bean root rot (BRR) are caused by four and two distinct species, respectively, multilocus DNA sequence analyses were conducted to assess whether any of the published molecular diagnostic assays were species-specific. Comparative DNA sequence analyses of the soybean SDS and BRR pathogens revealed that highly conserved regions of three loci were used in the design of these assays, and therefore none were species-specific based on our current understanding of species limits within the SDS-BRR clade. Prompted by this finding, we developed a high-throughput multilocus genotyping (MLGT) assay which accurately differentiated the soybean SDS and two closely related Phaseolus and mung BRR pathogens based on nucleotide polymorphism within the nuclear ribosomal intergenic spacer region rDNA and two anonymous intergenic regions designated locus 51 and 96. The single-well diagnostic assay, employing flow cytometry and a novel fluorescent microsphere array, was validated by independent multilocus molecular phylogenetic analysis of a 65 isolate design panel. The MLGT assay was used to reproducibly type a total of 262 soybean SDS and 9 BRR pathogens. The validated MLGT array provides a unique molecular diagnostic for the accurate identification and molecular surveillance of these economically important plant pathogens.

Adherence, colonization and dissemination of Candida dubliniensis and other Candida species.

Adherence to host cells is essential for yeasts to develop their full pathogenic potential since it triggers the process that leads to colonization and enables their persistence in the host. The aim of this work was to study the in vitro adherence of Candida dubliniensis and other Candida species, as well as the relation of adherence with the colonization and dissemination of these yeasts in an experimental mice model. Clinical isolates of Candida dubliniensis, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis were tested for their in vitro ability to adhere to buccal epithelial cells and in vivo to colonize and disseminate in an experimental infant mice model. Although C. dubliniensis isolates showed variable adherence values, their ability to colonize and disseminate in mice tissue was almost null. All C. albicans strains showed high levels of adherence and a prolonged gastrointestinal (GI) tract colonization. Both C. glabrata and C. krusei, showed a minor in vitro adherence and limited colonization time in infant mice GI tract. C. albicans and C. parapsilosis demonstrated a higher ability to disseminate, but the other non-C. albicans Candida strains showed a lower ability to disseminate. This study demonstrates that C. dubliniensis has a low GI tract colonization ability, as well as low dissemination ability in relation to C. albicans.

Systemic infection caused by Trichosporon asahii in a patient with liver transplant.

Trichosporon species are emerging pathogens capable of causing severe infections in immunocompromised patients. In this paper, we report a case of systemic infection in a liver transplant patient caused by Trichosporon asahii to show the etiologic agent's aggressiveness and poor therapeutic results with the different antifungals employed.

Oxidative stress response involving induction of protective enzymes in Candida dubliniensis.

Candida dubliniensis is a yeast species closely related to Candida albicans, but in contrast to C. albicans, limited information is available on the virulence factors of this important fungal pathogen. The objective of the present study was to determine if this species was able to evoke an adaptive response to oxidants. C. dubliniensis, treated with a low concentration of either H(2)O(2) or methyl viologen (a superoxide generating agent), mounts an adaptive response that results in increased survival against lethal doses of both oxidants. This response was characterized by the induction of enzymes with known antioxidant function. C. dubliniensis strains were less resistant to oxidants than C. albicans, displaying higher susceptibility to their toxic effects. The adaptive response described here might be responsible, among other factors, for the ability of this pathogen to cause infections in individuals with impaired immunity.

High prevalence of oral colonization by Candida dubliniensis in HIV-positive patients in Argentina.

Candida dubliniensis is a recently described yeast species, closely related to Candida albicans. This work represents the first general survey of the carriage of C. dubliniensis in the oral cavities of HIV-positive patients in Argentina. We studied 133 strains isolated from 162 HIV-positive patients, using the following identification tests: chlamydospore production on corn meal agar with Tween 80; colony color on CHROMagar Candida media; differential growth at 45 degrees C on potato dextrose agar; D-xylose assimilation; chlamydospore formation on sunflower seed agar (SSA); carbohydrate assimilation profiles using the API 20 C Aux commercial kit and PCR using primers that hybridize to the class IV intron of the ACT1 gene. Out of the 133 strains, 21 were identified as C. dubliniensis, representing approximately 13% of the 162 patients in this study. From these data, we conclude that although the PCR assay is the most reliable method, clamydospore formation on SSA is an easier and less expensive test for the screening of C. dubliniensis in the routine laboratory. Our results show that C. dubliniensis has a high prevalence among HIV-positive patients in Argentina.

[In vitro effect of carbohydrates and enteric bacteria on adherence of Candida albicans]. / Efecto in vitro de hidratos de carbono y bacterias entéricas en la adherencia de Candida albicans.

The adherence of Candida albicans to any cell is considered essential in the process that leads to colonization. Our objective in this study was to evaluate the effect of different carbohydrates and the presence of lactobacilli and Escherichia coli on the in vitro adherence of Candida albicans. The adherence to buccal epithelial cells was higher when growing at concentrations of galactose of 50, and 200 mM, as well as 50, 200, and 500 mM of sucrose, and 500 mM of mannose, compared with that obtained when growing in Sabouraud dextrose broth (p < 0.01). The presence of other microorganisms, such as Lactobacillus acidophilus and L. casei, caused a decrease in the in vitro adherence of C. albicans to buccal epithelial cells (p < 0.05), whereas E. coli did not modify this adherence at all.

Diecisiete casos de micetomas actino y eumicóticos en Rosario, Argentina (1959-1998) / Seventeen cases of actno and eumycotic mycetoma in Rosario, Argentina (1959-1998)

Se comunican diecisiete casos de micetomas estudiados en el período comprendido entre enero de 1958 y noviembre de 1998, 10 eumicóticos (59 porciento) y 7 actinomicóticos (41 porciento) madurella grisea fue el agente más aislado (29.5 porciento) en los primeros y nocardia asteroides (17.6 porciento) en los segundos. Los agentes restantes fueron pseudallescheria boidii, madurella mycetomatis, fusarium solani, mocardia brasiliensis y actinomadura madurae. A pesar que los micetomas son afecciones benignas, las dificultades terapéuticas los transforman en cuadros clínicos más serios