Sydney Hartz Alves

Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul

In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by Cryptococcus neoformans. This pathogen is a ubiquitous environmental basidiomycetous encapsulated yeast, commonly found in soil and avian excreta. The present study investigates further the population structure of clinical and environmental C. neoformans isolates from south Brazil. One hundred five clinical and 19 environmental (pigeon excreta and Eucalyptus spp.) isolates from the Brazilian state Rio Grande do Sul were characterized based on morphological, biochemical, molecular and serological data. The majority of the clinical and environmental isolates analyzed belonged to C. neoformans var. grubii serotype A (89.5 and 52.6%, respectively), were mating type alpha (98.1 and 94.7%, respectively) and were phospholipase-positive (94.3 and 73.7%, respectively). PCR-fingerprinting with the microsatellite-specific primer M13 and the minisatellite-specific primer (GACA)(4) grouped the majority of the isolates into the molecular type VNI (89.5 of the clinical and 52.6% of the environmental isolates). Our results add considerable new information to the few available data on ecology, molecular biology and epidemiology of C. neoformans in the southern region of Brazil.

Hypertonic sabouraud broth as a simple and powerful test for Candida dubliniensis screening

We developed a new screening test for C. dubliniensis based on its inability to grow on Sabouraud dextrose broth with 6.5% NaCl. A total of 266 clinical yeast isolates and 3 reference strains were tested, including 250 C. albicans and 19 C. dubliniensis strains. All C. albicans isolates tested exhibited significant growth on hypertonic Sabouraud broth up to 96 h, while, all C. dubliniensis isolates did not exhibit any visually detectable growth during the same period.

Atividade antimicrobiana dos óleos essenciais de orégano, tomilho e canela frente a sorovares de Salmonella enterica de origem avícola

Essential oils from oregano (Origanum vulgare), thyme (Thymus vulgaris) and cinnamom (Cinnamomum zeylanicum) were tested for their inhibitory activity against sixty Salmonella sp. isolates. These microorganisms were distributed in 20 serovars, all of them isolated from broiler chickens. Based on microdilution technique, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined. The strongest antibacterial activity was obtained for oregano (MIC mean = 529mg ml-1 and MBCmean = 661mg ml-1), followed by a moderate activity for thyme (MICmean=961mg ml-1 and MBCmean=1074mg ml-1) and the lowest obtained activity observed for cinnamon (MICmean= 1335mg ml-1 and MBCmean= 1979mg ml-1). Despite of this activity pattern, the serovars of Salmonella enterica showed significant susceptibility variations. However none of them could be classified as sensitive or resistant to the three essential oils tested. At the moment, when the antibiotics, as growth promoters, are being replaced, the essential oils from spices could be a very reasonable choice and the present results show us two points: the essential oils from oregano and thyme are effective against Salmonella isolates but the variations among the serovars must be considered.

Three types of immunotherapics against pythiosis insidiosi developed and evaluated

Pythiosis is a granulomatous disease of horses, cattle, dogs, cats and humans identified in tropical and subtropical areas and caused by Pythium insidiosum, a zoosporic fungus. Experimental models of pythiosis in naturally infected species have not yet been reported but, rabbits may be inoculated with zoospores as an experimental model for studying the disease. The present study evaluates the efficacy of three different of immunotherapics in the rabbit model. Approximately 17500 zoospores of oomycete P. insidiosum (CBS 101555 strain) were inoculated in each animal to generate the disease. Immunotherapics were produced from vortexed or sonicated cultures of the same strain. Four groups of five animals were employed: group 1, placebo; group 2, sonicated immunotherapic; group 3, mixed immunotherapic; and group 4, vortexed immunotherapic. All rabbits were inoculated with viable zoospores one month before administration of the immunotherapics. Eight doses of immunotherapic or placebo were used in each animal with a 14 day interval between injections. Rabbits receiving the vortexed immunotherapic were most effectively protected (P<0.05), showing a decrease in the area of coastal nodules due to Pythiosis insidiosum by 71.8% after 26 weeks of evaluation. Moreover, two animals in this group showed complete remission of the infection at the end of the 26 weeks. In contrast to these findings, rabbits given the sonicated immunotherapic did not show any protection and had an increase of 211.8% in the size of lesions. This failure of sonicated immunotherapic may reflect denaturation of protective antigens due to the sonication method.

Two Cases of Subcutaneous Infection Due to Phaeoacremonium spp.

ABSTRACT We describe two cases in Brazil of human subcutaneous infections due to Phaeoacremonium spp. The first case was caused by Phaeoacremoniumaleophilum. The patient presented with a unique fistulized nodule on the left ankle. The fungus was detected by direct microscopic examination and was isolated repeatedly from material collected from the lesion. This is the first reported case of human infection caused by this fungus. The second case was caused by Phaeoacremoniumrubrigenum. The patient presented with multiple nodules around the left ankle and foot. The fungus was detected by direct examination of pus and histological sections of the nodules. It was repeatedly isolated from the clinical specimens. This is the second reported case of human infection caused by this species.

In Vitro Activities of Voriconazole, Itraconazole, and Terbinafine Alone or in Combination against Pythium insidiosum Isolates from Brazil

ABSTRACT We evaluated the in vitro activities of voriconazole, itraconazole, and terbinafine against 30 clinical isolates of Pythium insidiosum using a checkerboard macrodilution method. The combined activity of terbinafine plus itraconazole or plus voriconazole was synergic against 17% of the strains. Antagonism was not observed.

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.

Candida dubliniensis: Epidemiology and Phenotypic Methods for Identification

Candida dubliniensis is an emerging pathogen first described in 1995, which shares many phenotypic features with Candida albicans and therefore may be misidentified in microbial laboratories. Despite various phenotypic techniques described in the literature to differentiate the two species, the correct identification of C. dubliniensis remains problematic due to phenotypic similarities between these species. Thus, as the differences between both are best characterized at genetic levels, several molecular methods have been proposed to provide a specific and rapid identification of this species. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and it is primarily associated with oral carriage and oropharyngeal infections in patients infected with human immunodeficiency virus (HIV). However, data acquired from its isolation from other healthy and immunocompromised patients are variable, and there is still no real consensus on the epidemiological relevance of this species. In this article, we review the various phenotypic methods used in the identification of C. dubliniensis and the epidemiological impact of this new species.

Antifungal Susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii Complex Identified in Brazil

ABSTRACT We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.

Epidemiology of sporotrichosis in the central region of Rio Grande do Sul

Thirty-one cases of sporotrichosis diagnosed in the central region of Rio Grande do Sul from 1988 to 1997 were studied. Clinical data were compared with a study concerning three past decades, clearly showing a decrease in the incidence of the mycosis, and an alteration in the profile of the infection, with a decrease of sporotrichosis in rural patients, children, women and farmers. In the past decade the mycosis was most frequent among urban adults with different professions, with the onset of the disease being associated with rural leisure activities such as fishing and hunting.