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.

Ascochyta blight: isolation, characterization, and development of a rapid method to detect inhibitors of the chickpea fungal pathogen Ascochyta rabiei.

Ascochyta blight is the major disease attacking chickpea (Cicer arietinum) around the world. Since its first time report of isolation in Argentina in 2012, the pathogen has caused severe economic losses and has acquired a great importance. We report here the isolation of Ascochyta rabiei from infected chickpea beans cultivated in Santa Fe, Argentina; its identification by morphological analysis and molecular biology techniques based on internal transcribed spacer (ITS) sequence alignment, its biochemical characterization regarding the capacity to produce proteinase and phospholipase enzymes, and its antifungal susceptibility to common used antifungal agents. In order to detect new inhibitors for A. rabiei from natural sources, a bioautographic method was developed. From the screening method developed, we found that extracts from cultures of Aspergillus parasiticus are active against A. rabiei.

Relaxation versus fractionation as hypnotic deepening: do they differ in physiological changes?

After rapid hypnotic induction, 12 healthy volunteers underwent hypnotic deepening with relaxation or with fractionation (without relaxation) in a random latin-square protocol. Electroencephalographic occipital alpha activity was measured, low-resolution brain electromagnetic tomography was performed, and hemodynamics (stroke volume, heart rate, cardiac output, mean arterial blood pressure, forearm arterial flow and resistance) were monitored in basal conditions and after deepening. After relaxation, both forearm flow (-18%) and blood pressure (-4%) decreased; forearm resistance remained unchanged. After fractionation, a forearm flow decrease comparable to that recorded after relaxation was observed, but blood pressure remained unchanged, leading to an increase of forearm resistance (+51%). Central hemodynamics did not change. Alpha activity increased in the precuneus after fractionation only. In conclusion, both relaxation and fractionation have vasoconstrictor effects, but fractionation is also associated with an increase in peripheral resistance.

Measured outcomes with hypnosis as an experimental tool in a cardiovascular physiology laboratory.

The authors detail their multidisciplinary collaboration of cardiologists, physiologists, neurologists, psychologists, engineers, and statisticians in researching the effects of hypnosis on the cardiovascular system and their additions to that incomplete literature. The article details their results and provides guidelines for researchers interested in replicating their research on hypnosis' effect on the cardiovascular system.

Early complications and symptoms of cerebellopontine angle tumor surgery: a prospective analysis.

UNLABELLED: Our objective is to prospectively report very early complications and outcomes of cerebellopontine angle (CPA) surgery. Between January and August 2007, 72 patients were operated on by different transpetrosal approaches in a tertiary referral center. During preoperative assessment, facial nerve function (House-Brackmann grading system), the presence of vertigo or tinnitus and caloric test results were recorded and correlated with complications and symptoms occurring daily from day (D) 1 to D 8. The overall number of complications did not differ from those of former retrospective studies; nevertheless, the prospective feature of this study prompts several comments. Even slight (grade II) preoperative facial impairments increased the risk of severe postoperative facial dysfunction. Keratitis was frequent (42%) even in patients with normal facial function. Thrombo-embolic complications only occurred after long air-travel (≥5 h). Preoperative caloric test status was predictive of postoperative vestibular disturbance occurrence. With respect to the activity recovery; younger patients (<40 years old) displayed faster central compensations than the older (>60 years old) patients. This study highlights several features that may be used for preoperative patient counseling and complication management. In particular, the practitioner has to pay attention to even minor preoperative clinical signs of facial dysfunction to properly inform the patient of facial outcome. Routine ophthalmologic evaluation should be practiced, even when facial function is normal or subnormal. LEVEL OF EVIDENCE: 1b.

Neurophysiological correlates of post-hypnotic alexia: a controlled study with Stroop test.

To clarify whether hypnotically-induced alexia was able to reduce the Stroop effect due to color/word interference, 12 volunteers (6 with high and 6 with low hypnotizability according to Stanford Hypnotic Susceptibility Scale Form C) underwent a Stroop test consisting of measuring, both in basal conditions and during post-hypnotic alexia, the reaction times (RT) at appearance of a colored word indicating a color. In basal conditions, RT were greater in case of incongruence. In highly hypnotizable participants, the interference was less pronounced during post-hypnotic alexia (-34%, p = 0.03). During alexia, late positive complexamplitude was also greater for congruent than incongruent conditions (p < 0.03), and cardiovascular response to stress was less pronounced as well. In participants showing low hypnotizability, no reduction of Stroop effect was detected during post-hypnotic alexia. Posthypnotic alexia is therefore a real and measurable phenomenon, capable of reducing the color-word interference and the haemodynamic effects of the Stroop test.

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.

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples.

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.