Antimicrobial and Cytotoxic Activity of Fruit Extract from Syzygium cumini (L.) Skeels

The aim of the present study was to evaluate the antimicrobial and cytotoxic activity of the ethanolic extract of S. cumini according to the Clinical and Laboratory Standards Institute reference method (with modifications), determining the minimal inhibitory and lethal concentration. Activity against Gram-positive (Staphylococcus aureus and S. epidermidis), Gram-negative (Pseudomonas aeruginosa) and yeast of Candida sp and Cryptococcus neoformans was evaluated. The effects of the fruit extract were examined in hamster cells ovaries in concentrations ranging from 1250.0 a 4.9 mu g/ml, measuring the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium. The extract showed both bactericidal and fungicidal activity among the various microorganisms tested and the MIC ranging from 7.8 to 250 mu g/ml. The MIC, MBC and MFC should values that were similar for all the microorganisms. Cytotoxicity index of the dried extract corresponded to the concentration of 400 mu g/ml. The extract could potentially be used in topical antimicrobial products. Thus, the activity of extract was potent to bacteria and mainly to non-albicans species and C. neoformans.

Phylogeny and phenotypic characterization of pathogenic cryptococcus species and closely related saprobic taxa in the tremellales

Eukaryotic Cell, Vol.8, Nº3

Asociacion de leveduras del genero Cryptococcus con especies de Eucalyptus en Santafe de Bogota

El aislamiento de Cryptococcus neoformans var. gattii, serotipo B, a partir del medio ambiente se estableció inicialmente en Australia en 1989, en asocio con el Eucalyptus camaldulensis y posteriormente con E. tereticornis. Con estos hallazgos se postuló que desde allí, el hongo se ha podido exportar, por medio de las semillas contaminadas, a otras regiones geográficas, incluyendo Colombia. El objetivo de éste estudio fue identificar las levaduras del género Cryptococcus asociadas con especies de Eucalyptus sp., como primera evaluación en la ecología de C. neoformans var. gattii en nuestro país. Se realizó en Santafé de Bogotá, con una población de 100 árboles ubicados al centro, nororiente, oriente y occidente de la ciudad, recolectando de cada uno de ellos flores, frutos, hojas, cortezas y detritos; el procesamiento de las muestras incluyó extracción del material con una solución salina con antibióticos, siembra en medios selectivos e identificación de las especies con base en las características morfológicas, macro y microscópicas y bioquímicas. Se aislaron 27 cepas de Cryptococcus pertenecientes a 9 especies de Cryptococcus, a partir de 21 árboles ubicados en 5 zonas diferentes de la ciudad. Se aisló C. neoformans y se identificó como C. neoformans var. neoformans serotipo A. Estos datos iniciales son importantes como primera evaluación de la asociación de Cryptococcus sp. con los Eucalyptus en nuestro país.

Interferon gamma increases survival in urine experimental cryptococcosis

Systemic disease by Cryptococcus neoformans (C. neoformans) is a common opportunistic infection in immunodeficient patients. Cellular immunity seems to be the most important determinant of resistance. The aim of this study was to assess the effect of recombinant rat interferon gamma (IFN-gamma) in murine cryptococcosis (Balb/c mice infected by IP route with the Rivas strain of C. neoformans), evaluating survival time, macroscopic and microscopic examination of the organs, and massive seeding of brain homogenate. IFN-gamma treatment, at a daily dose of 10,000 IU, did not modify significantly these variables when mice were challenged with a high inoculum (10(7) yeasts) and treatment was delayed to 5 days after infection (median survival 21 days in control mice vs. 23 days in IFN-treated). Another set of experiments suggested that IFN-gamma treatment, at a dose of 10,000 IU/day, begun at the moment of infection could be useful (it prolonged survival from 20 to 28 days, although the difference did not achieve statistical signification). When used simultaneously with infection by 3.5 x 10(5) yeasts, IFN-gamma at 10,000 IU/day for 15 days significantly prolonged survival of mice (p = 0.004). These results suggest that, depending on the experimental conditions, IFN-gamma can improve survival of mice infected with a lethal dose of C. neoformans.

Cryptococcus gattii fungemia: report of a case with lung and brain lesions mimicking radiological features of malignancy

A 64-year-old apparently immunocompetent white man developed lung and brain lesions of disseminated cryptococcosis. The radiologic features mimicked those of lung cancer metastatic to the central nervous system. C. gattii was recovered from cultures of bronchoalveolar lavage fluid, brain biopsy, and blood. The same fungus was recovered from pulmonary and brain specimens at autopsy. Serum and cerebrospinal fluid cryptococcal antigen tests were diagnostic in our case and should be included in the diagnostic evaluation of unexplained pulmonary and cerebral lesions. A literature search showed few reports of fungemia by this species of Cryptococcus, contrasting to C. neoformans.

Clinical and epidemiological features of 123 cases of cryptococcosis in Mato Grosso do Sul, Brazil

To identify the clinical and epidemiological profile of cryptococcosis diagnosed at the University Hospital of the Federal University of Mato Grosso do Sul, Brazil, medical records of 123 patients admitted from January 1995 to December 2005 were analyzed. One hundred and four cases (84.5%) had HIV infection, six (4.9%) had other predisposing conditions and 13 (10.6%) were immunocompetent. Male patients predominated (68.3%) and their age ranged from 19 to 69 years (mean: 35.9). Most patients (73.2%) were born and lived lifelong in the state of Mato Grosso do Sul. Involvement of the central nervous system occurred in 103 patients (83.7%) and headache and vomiting were the most frequent symptoms. In 77 cases it was possible to identify the Cryptococcus species: 69 (89.6%) C. neoformans and eight (10.4%) C. gattii. Amphotericin B was the drug of choice for treatment (106/123), followed by fluconazole in 60% of cases. The overall lethality rate was 49.6%, being 51% among the HIV infected patients and 41.2% among the non-HIV infected (p > 0.05). Although cryptococcosis exhibited in our region a similar behavior to that described in the literature, the detection of an important rate of immunocompetent individuals and five C. gattii cryptococcosis in HIV-infected patients is noteworthy.

Unusual morphologies of Cryptococcus spp. in tissue specimens: report of 10 cases

Ten cases of cryptococcosis due to unusual microscopic forms of Cryptococcus sp. observed over a twenty-eight year period (1981-2009) are presented. The most important clinicopathological and laboratory data are tabulated. The uncommon forms of cryptococcal cells given are: structures resembling germ tube (one case), chains of budding yeasts (one case), pseudohyphae (two cases) and nonencapsulated yeast-like organisms (eight cases). The diagnosis was based on the histopathological findings. The causative organism was isolated and identified in seven cases; five were due to C. neoformans, and two to C. gattii. In addition, the importance of using staining histochemical techniques - Grocott's silver stain (GMS), Mayer's mucicarmine stain (MM) and Fontana-Masson stain (FM) - in the diagnosis of cryptococcosis is argued.

Unveiling the mating system and genetic variability in the yeast Kwoniella mangroviensis using molecular approaches

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.

Clinical and microbiological features of cryptococcal meningitis

Introduction In this study, the clinical features, underlying diseases and clinical outcomes of patients with cryptococcosis were investigated. In addition, a molecular analysis of the Cryptococcus neoformans species complex isolated from these patients was performed. Methods A prospective study of 62 cases of patients with cryptococcal infection was conducted at the Hospital de Doenças Tropicais de Goiás Dr. Anuar Auad from 2009-2010. Cryptococcal meningitis cases were diagnosed by direct examination and cerebrospinal fluid (CSF) sample culture. The profiling of these patients was assessed. The CSF samples were submitted to India ink preparation and cultured on Sabouraud dextrose agar, and C. neoformans was identified by the production of urease, a positive phenoloxidase test and assimilation of carbohydrates. C. neoformans and C. gattii isolates were distinguished by growth on L-canavanine-glycine-bromothymol blue medium, and molecular analysis was conducted via PCR fingerprinting reactions using M13 and (GACA)4 primers. Results From the 62 patients with cryptococcosis, 71 isolates of CSF were obtained; 67 (94.4%) isolates were identified as C. neoformans var. grubii/VNI, and 4 (5.6%) were identified as C. gattii/VGII. Of these patients, 53 had an HIV diagnosis. The incidence of cryptococcosis was higher among patients 20-40 years of age, with 74.2% of the cases reported in males. Cryptococcus-related mortality was noted in 48.4% of the patients, and the symptoms were altered sensorium, headache, fever and stiff neck. Conclusions The high morbidity and mortality observed among patients with cryptococcosis demonstrate the importance of obtaining information regarding the epidemiological profile and clinical course of the disease in the State of Goiás, Brazil.

Cryptococcosis in Atlántico, Colombia: an approximation of the prevalence of this mycosis and the distribution of the etiological agent in the environment

ABSTRACTINTRODUCTION:Cryptococcosis is an invasive disease acquired by inhalation of infectious propagules from the environment. Currently, compulsory notification of the spread of this disease is not required in Colombia. However, reporting of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome cases to the National Surveillance System has suggested that there is a growing population at risk of contracting cryptococcosis. Few studies have described the occurrence of cryptococcosis in Colombia. Therefore, in this study, we examined the pathology of this disease in Atlántico, Colombia and determined the distributions of Cryptococcus neoformans and Cryptococcus gattii in the environment.METHODS:Clinical samples/isolates were gathered from cases of cryptococcosis previously diagnosed at health institutions in Atlántico, and surveys were completed by clinicians. The environmental study considered 32 sampling points and three tree species, i.e., Quickstick ( Gliricidia sepium ), Almond ( Terminalia catappa ), and Pink trumpet ( Tabebuia rosea ). Environmental and clinical samples/isolates were analyzed for phenotypic and genotypic confirmation.RESULTS:From 1997-2014, 41 cases of cryptococcosis were reported. The mean patient age was 40.5 years (range: 18-63 years); 76% were men, and 78% were HIV positive. Isolation was possible in 38 cases ( C. neoformans , molecular type VNI in 37 cases and C. gattii , molecular type VGI in one case). In 2012-2014, 2,068 environmental samples were analyzed with a positivity of 0.4% ( C. neoformans , molecular type VNI) in Almond and Pink trumpet trees.CONCLUSIONS:Cryptococcus neoformans , molecular type VNI had a higher prevalence than C. gattii and was associated with human exposure and the pathogenesis of cryptococcosis in this geographical region.

In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil

Infections by Cryptococcus strains other than C. neoformans have been detected in immunocompromised patients. Of these strains, three are considered human pathogens: C. albidus, C. laurenttii, and C. uniguttulatus. This study deals with the in vitro susceptibility of Cryptococcus to drugs such as amphotericin B, itraconazole, fluconazole, and 5-fluorocytosine. Environmental Cryptococcus isolates (50) distributed as follows: C. neoformans var. neoformans (16), C. albidus (17), C. laurentii (14), and C. uniguttulatus (3) were evaluated by the micro and macrodilution techniques, according to EUCAST and NCCLS recommendations, respectively. Considering both methodologies the respective minimal inhibitory concentrations (MIC) were 0.125 and 2 µg/ml for amphotericin B, 0.06 and 8 µg/ml for itraconazole, and 0.5 and more than 64 µg/ml for fluconazole and 5-fluorocytosine. Agreement percentages for the two methodologies were 100% for amphotericin B and fluconazole for all the strains tested. For itraconazole, the agreement percentage was 81.3% in the C. neoformans strain and 100% for all the others. All species had a agreement percentage of 94.1 to 100% when susceptibility to 5-fluorocytosine was tested. It is concluded that environmental isolates of C. neoformans var. neoformans, C. albidus, C. laurentii, and C. uniguttulatus may show high MICs against certain drugs, suggesting in vitro primary resistance to the antifungals tested.

Occurrence and molecular characterization of cryptococcosis in dogs and cats in Mato Grosso, Brazil

Cryptococcosis is an infection that affects humans and animals, the etiology is attributed to Cryptococcus neoformans variety neoformans, C. neoformans var. grubii and Cryptococcus gattii. The infection is common in dogs and cats, causing respiratory, neurological, cutaneous and ocular infections. Aiming to better understand the epidemiology of cryptococcosis in animals in the region, this paper describe the occurrence and characterization of the Cryptococcus species involved in this illness in pet animals at Mato Grosso State, Brazil. Clinical samples of four cases, two in cats and two dogs, were submitted for pathological, microbiological and molecular analysis. Microscopically, in three cases, tissue sections stained with hematoxylin and eosin had absence to severe granulomatous reaction composed by histiocytes, multinucleated cells and lymphocytes infiltration. In one case, citological imprint analysis showed similar inflammatory mainly mononuclear and lymphocyte cells infiltration. All cases had variable amounts of intracellular and extracellular fungal structures compatible with Cryptococcus sp. on Periodic Acid-Schiff (PAS) stain. All clinical samples were positive for culture on Sabouraud Dextrose Agar (SDA) and morphologically classified as Cryptococcus sp. The isolates were PCR positive for C. gatti, being confirmed by sequencing technique. The findings characterize the molecular species involved in animal infections in the region, and may contribute to future studies of the epidemiology of C. gattii.

Étude de l’infection au Cryptococcus chez la souris transgénique exprimant le génome du VIH-1

Cryptococcus neoformans var. grubii est responsable de la majeure partie des infections au Cryptococcus chez les individus infectés au VIH-1. Cryptococcus gattii infecte généralement les personnes immunocompétentes. Afin de comprendre les mécanismes responsables de la susceptibilité différentielle de ces espèces lors de l’infection au VIH-1, nous avons établi et caractérisé un modèle novateur de la cryptococcose chez des souris transgéniques (Tg) CD4C/HIVMutA exprimant des gènes du VIH-1, et qui développent une maladie similaire au SIDA. Les objectifs sont de démontrer une différence significative au niveau de la survie, de la réponse inflammatoire et du recrutement cellulaire pulmonaire en fonction de la présence du transgène et de l’espèce de Cryptococcus inoculée. Des analyses de survie, d’histopathologie et de cytométrie en flux sur les populations cellulaires pulmonaires ont été effectuées. Les souris Tg infectées avec C. neoformans H99 ou C23 ont démontré une survie réduite et une augmentation de la dissémination comparativement aux souris non-Tg, contrairement aux souris Tg infectées au C. gattii R265 ou R272. L’examen histopathologique des poumons de souris Tg infectées au H99 a montré une faible réponse inflammatoire, contrairement aux souris non-Tg. Pour la souche R265, il y avait une très faible réponse inflammatoire chez les deux types de souris. Enfin, l’étude des populations cellulaires du poumon a révélé chez les souris Tg une augmentation des pourcentages de macrophages interstitiels et de cellules polymorphonucléaires, ainsi qu’une diminution des lymphocytes T CD4+ et CD8+, indépendamment de l’infection au Cryptococcus. Ce modèle novateur représente donc un outil très pertinent pour l’étude de l’immunopathogenèse de la cryptococcose dans le contexte du VIH.

Immunopathogenèse de la cryptococcose chez la souris transgénique exprimant le génome du VIH-1

La cryptococcose chez les patients atteints du VIH-1 est principalement causée par Cryptococcus neoformans var. grubii tandis que Cryptococcus gattii infecte surtout les personnes immunocompétentes. Afin d’élucider les mécanismes causant la susceptibilité différentielle à l’égard de ces deux espèces de Cryptococcus dans le contexte de l’infection au VIH-1, nous avons utilisé un modèle novateur de la cryptococcose chez la souris transgénique CD4C/HIVMutA, qui exprime les gènes nef, env et rev du VIH-1. L’expression du transgène VIH-1 a augmenté le recrutement pulmonaire des macrophages alvéolaires mais a diminué celui des lymphocytes T CD4+ et CD8+ en réponse à l’infection par le C. neoformans ou le C. gattii. La production pulmonaire des chimiokines MCP-1 (CCL2) et RANTES (CCL5) était également réduite chez les souris transgéniques infectées par l’une ou l’autre de ces espèces de Cryptococcus. La production pulmonaire de MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 et IL-13 était augmentée chez la souris infectée au C. neoformans comparativement à C. gattii. In vitro, les macrophages alvéolaires prélevés chez la souris Tg et stimulés par des agonistes ont produit davantage de MIP-1β, alors que les chimiokines MCP-1 et RANTES n’ont pas été détectées.