Serotype and PCR-fingerprints of Clinical and Environmental isolates of Cryptococcus neoformans in Chiang Mai,Thailand

From May 1999 to April 2000, serotypes of clinical and environmental isolates of Cryptococcus neoformans were studied in Chiang Mai province, northern Thailand. Three hundred and eighty-five environmental samples, of which 100 were dove droppings, 55 pigeon droppings and 230 eucalyptus flower, were collected from 7 Amphoes in Chiang Mai. C. neoformans was isolated from 45 of 100 (45.0%) dove dropping samples, 9 of 55 (16.4%) pigeon dropping samples and 2 of 230 (0.9%) eucalyptus flower samples. Serotypes of 56 environmental isolates and 75 clinical isolates of C. neoformans,obtained during the same period, were determined by the slide agglutination test. Fifty-six environmental and 74 clinical isolates belonged to C. neoformans serotype A (C. neoformans var. grubii), and only one clinical isolate belonged to C. neoformans serotype AD. The isolation of C. neoformans var. grubii from eucalyptus flower samples suggests contamination of avian droppings. PCR-fingerprinting, using (GACA)4 as a primer, discriminated 131 clinical and environmental isolates into 2 groups (group I and II). Seventy-five clinical and 54 environmental isolates were of group I, which had two major specific bands of approximately 1,250 and 960 base pairs. Two environmental isolates, one from pigeon excreta and the other from a eucalyptus flower sample were of group II, which had two major specific bands of approximately 1,180 and 500 base pairs.     

Resistance of Asian Cryptococcus neoformans serotype A is confined to few microsatellite genotypes

Background

Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries.

Methodology/Principal Findings

Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17.

Conclusions

The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.     

Isolation of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> from a <Emphasis Type="Italic">Castanopsis argyrophylla</Emphasis> tree hollow (Mai-Kaw), Chiang Mai,Thailand

The pathogenic yeast Cryptococcus gattii was isolated from a tree hollow of a Castanopsis argyrophylla King ex Hook.f. (Fagaceae) in Chiang Mai, Thailand. Molecular characterization with amplified fragment length polymorphism analysis and multi-locus sequence typing showed that this isolate belonged to genotype AFLP4/VGI representing C. gattii sensu stricto. Subsequent comparison of the environmental isolate with those from clinical samples from Thailand showed that they grouped closely together in a single cluster.     

Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms

Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action. Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) (TCS), is a common antibacterial agent that is found in significant amounts in the aquatic environment. In this work, the possible effects and modes of action of TCS were investigated in the marine bivalve Mytilus galloprovincialis Lam. In mussel immune cells, the hemocytes, in vitro short-term exposure to TCS in the low microM range reduced lysosomal membrane stability (LMS) and induced extracellular release of lysosomal hydrolytic enzymes. The effects on LMS were mediated by activation of ERK MAPKs (Extracellularly Regulated Mitogen Activated Protein Kinases) and PKC (protein kinase C) alpha and betaII isoforms, as demonstrated by both specific kinase inhibitors and Western blotting with specific anti-phospho-antibodies. The effects of TCS were confirmed in vivo, in the hemocytes of mussels injected with different concentrations of TCS (corresponding to 0.29, 2.9 and 29 ng/g dry weight) and sampled at 24 h post-injection. The possible in vivo effects of TCS were also evaluated on the activity of different enzymes in the digestive gland, the tissue mainly involved in accumulation and metabolism of organic contaminants in mussels. Significant increases were observed in the activity of the glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), as well as of GST (GSH transferase) and GSR (GSSG reductase), whereas a decrease in catalase activity was observed. The results demonstrate that in mussels TCS can act on kinase-mediated cell signalling, lysosomal membranes and redox balance in different systems/organs. Although further studies are needed in order to evaluate possible consequences of environmental exposure to TCS on mussel health, the results represent the first data on the possible modes of action of this widespread antibacterial in aquatic invertebrates.     

Fasciola gigantica: Histology of the digestive tract and the expression of cathepsin L

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite’s body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.     

Detection of Environmental Sources of Histoplasma capsulatum in Chiang Mai, Thailand, by Nested PCR

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.     

Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, n-CeO(2)) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials.     

Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO(2), TiO(2), ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36 h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO(2) and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25 μg/ml and 50 μg/ml, respectively and for Lys-SiO(2) NM at 3.5 μg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50 μg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO(2) both in the absence or presence of serum up to 5 μg/ml. No toxicity was detected for TiO(2) and MWCNTs in both 10% and 0% serum, up to the dose of 250 μg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5 μg/ml of Lys-SiO(2) NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO(2) NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.