Indoor and outdoor atmospheric fungal spores in the So Paulo metropolitan area (Brazil): species and numeric concentrations

The aim of this study was to estimate the indoor and outdoor concentrations of fungal spores in the Metropolitan Area of Sao Paulo (MASP), collected at different sites in winter/spring and summer seasons. The techniques adopted included cultivation (samples collected with impactors) and microscopic enumeration (samples collected with impingers). The overall results showed total concentrations of fungal spores as high as 36,000 per cubic meter, with a large proportion of non culturable spores (around 91% of the total). Penicillium sp. and Aspergillus sp. were the dominant species both indoors and outdoors, in all seasons tested, occurring in more than 30% of homes at very high concentrations of culturable airborne fungi [colony forming units(CFU) m(-3)]. There was no significant difference between indoor and outdoor concentrations. The total fungal spore concentration found in winter was 19% higher than that in summer. Heat and humidity were the main factors affecting fungal growth; however, a non-linear response to these factors was found. Thus, temperatures below 16A degrees C and above 25A degrees C caused a reduction in the concentration (CFU m(-3)) of airborne fungi, which fits with MASP climatalogy. The same pattern was observed for humidity, although not as clearly as with temperature given the usual high relative humidity (above 70%) in the study area. These results are relevant for public health interventions that aim to reduce respiratory morbidity among susceptible populations.

Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells

Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-alpha), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.

Influence of macro- and micronutrient fertilization on fungal contamination and fumonisin production in corn grains

The aim of this study was to investigate the effects of nutrients (nitrogen, zinc and boron) on fungal growth and fumonisins production in corn samples obtained at the beginning of grain formation and at harvest. Three nitrogen doses were applied to the corn plants through soil in combination with three zinc doses and two boron doses during sowing. Mycological analysis of grains, using Dichloran Rose-Bengal Chloramphenicol Agar, collected at the beginning of formation demonstrated a fungal population predominantly of yeasts. Analysis of freshly harvested corn revealed a higher frequency of Penicillium spp. (72%) and F verticillioides (27%). High Performance Liquid Chromatography analysis revealed that 100% of grains were contaminated with fumonisins B, at levels ranging from 0.3 to 24.3 mg/kg and 93% contaminated with fumonisin B(2) at levels ranging from 0.05 to 5.42 mg/kg. Nitrogen (50 kg/ha) in combination with boron (0.5 kg/ha) resulted in an increased fumonisin B2 production. (c) 2007 Elsevier Ltd. All rights reserved.

Trophallaxis in Dehydrated Leaf Cutting Colonies of Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Trophallaxis, the transfer of liquid among individuals by oral regurgitation or anal deposition, occurs in many insect groups including ants. The first indication that trophallaxis could occur in leaf cutting ants (Atta sexdens rubropilosa) was made by Autuori in 1942. He reported water collection by this ant species, and highlighted what in those days was an undescribed behavior for this species. In 2005, Da-Silva and Ribeiro presented preliminary results suggesting the existence of trophallaxis in A. sexdens rubropilosa. Here we report on a formal test of the hypothesis of trophallaxis in that species. Our approach was to test ant pairs in which only one individual (Group I) had access to blue-dyed water and the other individual (Group II), a nest-mate, came from a colony dehydrated by offering dry crushed corn for fungal growth. Positive results for trophallaxis were obtained in ants from four colonies and accounted for 33%-46% of all tests in which ants from Group I drank dyed water. These results indicate that trophallaxis occurs in this species.

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28(-/-)) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28(-/-) mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro-and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.

Use of PCR for detecting Aspergillus flavus in maize treated by gamma radiation process

The aim of this study was to verify the effects of gamma radiation process on the fungal DNA and the application of PCR in the detection of Aspergillus flavus in irradiated maize grains. The samples were inoculated with a toxigenic strain and incubated under controlled conditions of relative humidity, water activity, and temperature for 15 days. After incubation, the samples were treated with gamma radiation with doses of 5 and 10 kGy and individually analyzed. The use of PCR technique showed the presence of DNA bands of Aspergillus flavus in all irradiated samples that showed no fungal growth in agar medium.

Effects of gamma-radiation on the fungus Alternaria alternata in artificially inoculated cereal samples

The objective of this study was to evaluate the effects of different gamma-radiation doses on the growth of Alternaria alternata in artificially inoculated cereal samples. Seeds and grains were divided into four groups: Control Group (not irradiated), and Groups 1, 2 and 3, inoculated with an A. alternata spore suspension (1 x 10(6) spores/mL) and exposed to 2, 5 and 10 kGy, respectively. Serial dilutions of the samples were prepared and seeded on DRBC (dichloran rose bengal chloramphenicol agar) and DCMA (dichloran chloramphenicol malt extract agar) media, after which the number of colony-forming units per gram was determined in each group. In addition, fungal morphology after irradiation was analyzed by scanning electron microscopy (SEM). The results showed that ionizing radiation at a dose of 5 kGy was effective in reducing the growth of A. alternata. However, a dose of 10 kGy was necessary to inhibit fungal growth completely. SEM made it possible to visualize structural alterations induced by the different gamma-radiation doses used. (C) 2009 Elsevier Ltd. All rights reserved.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

Evaluation of Fungal Burden and Aflatoxin Presence in Packed Medicinal Plants Treated by Gamma Radiation

This study was developed to evaluate the fungal burden, toxigenic molds, and mycotoxin contamination and to verify the effects of gamma radiation in four kinds of medicinal plants stored before and after 30 days of irradiation treatment. Eighty samples of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia. and Cassia angustifolia) purchased from drugstores, wholesale, and open-air markets in Sao Paulo city, Brazil, were analyzed. The samples were treated using a (60)Co gamma ray source (Gammacell) with doses of 5 and 10 kGy. Nonirradiated samples were used as controls of fungal isolates. For enumeration of fungi on medicinal plants, serial dilutions of the samples were plated in duplicate onto dichloran 18% glycerol agar. The control samples revealed a high burden of molds, including toxigenic fungi. The process of gamma radiation was effective in reducing the number of CFU per gram in all irradiated samples of medicinal plants after 30 days of storage, using a dose of 10 kGy and maintaining samples in a protective package. No aflatoxins were detected. Gamma radiation treatment can be used as an effective method for preventing fungal deterioration of medicinal plants subject to long-term storage.

A Survey of Fungal Contamination on Books in Public Libraries with Mechanical and Natural Ventilation

Libraries are very propitious environments for the growth of fungi. The great concentration of organic material available for these microorganisms, and often with the lack of adequate ventilation or climate control, would favour this situation. This study was conducted in 2003 to determine the predominant genera of fungi in public libraries by a survey of fungi contaminating the upper surface of books, with and without air conditioning in the city of Sao Paulo, Brazil, in the winter and summer, during the respective periods with high and low levels of airborne fungi in that city. Six libraries were chosen, located on the campus of the University of Sao Paulo, three of them with air conditioning and the other three with natural ventilation. In these six libraries, 31 genera of fungi were identified in total. The genera and frequency of contaminant fungi recovered differed significantly between the libraries with and without air conditioning and in the samples collected in the summer as opposed to the winter. Cladosporium was the most frequent in the libraries with and without air conditioning, and in the winter. Aspergillus was isolated more often in the summer.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Influence of culture conditions on mycelial growth and bioluminescence of Gerronema viridilucens

Herein we describe a procedure for measuring the total light emission of the naturally bioluminescent tropical fungus Gerronema viridilucens and the optimization of culture conditions using multivariate factorial ANOVA. Cultures growing on an agar surface in 35 mm Petri dishes at 90% humidity show optimal bioluminescence emission at 25 degrees C in the presence of 1.0% sugar cane molasses, 0.10% yeast extract and pH 6.0 (nonbuffered). Temperature and pH are the most important factors for both mycelial growth and bioluminescence.