Fungal Transformation and Schistosomicidal Effects of Pimaradienoic Acid

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100 mu M. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100 mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO-/- mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO-/- mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

The fungal metabolite eugenitin as additive for Aspergillus niveus glucoamylase activation

Endophytic microorganisms live inside tissues of host plants apparently do not causing warning to them, and area promising source of bioactive molecules as antimicrobial and antitumoral drugs. In this work, we report the isolation of eugenitin from cultures of the endophyte Mycoleptodiscus indicus and its potential as additive for Aspergillus niveus glucoamylase activation. The glucoamylase hydrolytic activity increased twofold using 5 mM of eugenitin and this activation could be explained by the binding mode of eugenitin with the three-dimensional structure of glucoamylase. The in silica prediction of ligand binding sites revealed at least 9 possible interaction sites able to accommodate eugenitin on glucoamylase from Hypocrea jecorina. Besides, we evaluated the effect of pH and temperature on activity and stability, as well as in the hydrolysis of different substrates and kinetic parameters either in presence or absence of eugenitin. The results displayed by eugenitin as additive to glucoamylase activation are promising and provide novel perspectives for applications of fungal metabolites. (C) 2011 Elsevier B.V. All rights reserved.

Nursing Workload as a Risk Factor for Healthcare Associated Infections in ICU: A Prospective Study

Introduction: Nurse understaffing is frequently hypothesized as a potential risk factor for healthcare-associated infections (HAI). This study aimed to evaluate the role of nursing workload in the occurrence of HAI, using Nursing Activities Score (NAS). Methods: This prospective cohort study enrolled all patients admitted to 3 Medical ICUs and one step-down unit during 3 months (2009). Patients were followed-up until HAI, discharge or death. Information was obtained from direct daily observation of medical and nursing rounds, chart review and monitoring of laboratory system. Nursing workload was determined using NAS. Non-compliance to the nurses' patient care plans (NPC) was identified. Demographic data, clinical severity, invasive procedures, hospital interventions, and the occurrence of other adverse events were also recorded. Patients who developed HAI were compared with those who did not. Results: 195 patients were included and 43 (22%) developed HAI: 16 pneumonia, 12 urinary-tract, 8 bloodstream, 2 surgical site, 2 other respiratory infections and 3 other. Average NAS and average proportion of non compliance with NPC were significantly higher in HAI patients. They were also more likely to suffer other adverse events. Only excessive nursing workload (OR: 11.41; p: 0.019) and severity of patient's clinical condition (OR: 1.13; p: 0.015) remained as risk factors to HAI. Conclusions: Excessive nursing workload was the main risk factor for HAI, when evaluated together with other invasive devices except mechanical ventilation. To our knowledge, this study is the first to evaluate prospectively the nursing workload as a potential risk factor for HAI, using NAS.

Endo-xylanase GH11 activation by the fungal metabolite eugenitin

Eugenitin, a chromone derivative and a metabolite of the endophyte Mycoleptodiscus indicus, at 5 mM activated a recombinant GH11 endo-xylanase by 40 %. The in silico prediction of ligand-binding sites on the three-dimensional structure of the endo-xylanase revealed that eugenitin interacts mainly by a hydrogen bond with a serine residue and a stacking interaction of the heterocyclic aromatic ring system with a tryptophan residue. Eugenitin improved the GH11 endo-xylanase activity on different substrates, modified the optimal pH and temperature activities and slightly affected the kinetic parameters of the enzyme.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

Production of xylanase and beta-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and beta-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 A degrees C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of beta-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of beta-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of beta-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of beta-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Opportunistic and Other Infections in HIV-Infected Children in Latin America Compared to a Similar Cohort in the United States

Opportunistic and other infections have declined since the introduction of highly active antiretroviral therapy (HAART) in developed countries but few studies have addressed the impact of HAART in HIV-infected children from developing countries. This study examines the prevalence and incidence of opportunistic and other infections in Latin America during the HAART era. Vertically HIV-infected children enrolled in a cohort study between 2002 and 2007 were followed for the occurrence of 29 targeted infections. Cross-sectional and longitudinal analyses were performed to calculate the prevalence of infections before enrollment and the incidence rates of opportunistic and other infections after enrollment. Comparisons were made with data from a U. S. cohort (PACTG 219C). Of the 731 vertically HIV-infected children 568 (78%) had at least one opportunistic or other infection prior to enrollment. The most prevalent infections were bacterial pneumonia, oral candidiasis, varicella, tuberculosis, herpes zoster, and Pneumocystis jiroveci pneumonia. After enrollment, the overall incidence was 23.5 per 100 person-years; the most common infections (per 100 person-years) were bacterial pneumonia (7.8), varicella (3.0), dermatophyte infections (2.9), herpes simplex (2.5), and herpes zoster (1.8). All of these incidence rates were higher than those reported in PACTG 219C. The types and relative distribution of infections among HIV-infected children in Latin America in this study are similar to those seen in the United States but the incidence rates are higher. Further research is necessary to determine the reasons for these higher rates.

Healthcare-associated infection in hematopoietic stem cell transplantation patients: risk factors and impact on outcome

Objective: The objective of this study was to analyze the incidence of and risk factors for healthcare-associated infections (HAI) among hematopoietic stem cell transplantation (HSCT) patients, and the impact of such infections on mortality during hospitalization. Methods: We conducted a 9-year (2001-2009) retrospective cohort study including patients submitted to HSCT at a reference center in Sao Paulo, Brazil. The incidence of HAI was calculated using days of neutropenia as the denominator. Data were analyzed using EpiInfo 3.5.1. Results: Over the 9-year period there were 429 neutropenic HSCT patients, with a total of 6816 days of neutropenia. Bloodstream infections (BSI) were the most frequent infection, presenting in 80 (18.6%) patients, with an incidence of 11.7 per 1000 days of neutropenia. Most bacteremia was due to Gram-negative bacteria: 43 (53.8%) cases were caused by Gram-negative species, while 33 (41.2%) were caused by Gram-positive species, and four (5%) by fungal species. Independent risk factors associated with HAI were prolonged neutropenia (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.04-1.10) and duration of fever (OR 1.20, 95% CI 1.12-1.30). Risk factors associated with death in multivariate analyses were age (OR 1.02, 95% CI 1.01-1.43), being submitted to an allogeneic transplant (OR 3.08, 95% CI 1.68-5.56), a microbiologically documented infection (OR 2.96, 95% CI 1.87-4.6), invasive aspergillosis disease (OR 2.21, 95% CI 1.1-4.3), and acute leukemias (OR 2.24, 95% CI 1.3-3.6). Conclusions: BSI was the most frequent HAI, and there was a predominance of Gram-negative microorganisms. Independent risk factors associated with HAI were duration of neutropenia and fever, and the risk factors for a poor outcome were older age, type of transplant (allogeneic), the presence of a microbiologically documented infection, invasive aspergillosis, and acute leukemia. Further prospective studies with larger numbers of patients may confirm the role of these risk factors for a poor clinical outcome and death in this transplant population. (C) 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Background: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe3O4 MNPs in order to provoke cell death remotely using TVMFs. Methods: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO4 in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. Results: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Conclusion: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

Orbivirus Infections in Collared Peccaries (Tayassu tajacu) in Southeastern Brazil

We surveyed 49 free-living collared peccaries (Pecan tajacu) in Brazil for antibodies against bluetongue virus (BTV) and porcine circovirus 2 (PCV2). Antibodies against BTV were detected in 19/49 (39%) samples. All samples were negative for PCV2. The importance of antibodies to BTV in collared peccaries remains to be determined.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Highly debilitating natural Trypanosoma vivax infections in Brazilian calves: epidemiology, pathology, and probable transplacental transmission

Clinical, epidemiological, and pathological aspects of trypanosomiasis caused by Trypanosoma vivax in calves were reported for the first time in northeast Brazil. Clinical and epidemiological data, packed cell volumes (PCV), and parasitemia were assessed in 150 calves in May 2009 (rainy season-survey 1) and in 153 calves in November 2009 (dry season-survey 2) in three farms (A, B, and C). Prevalence of T. vivax in calves examined in the survey 1 was 63.3%, 65.0%, and 80.0% in farms A, B, and C, respectively. Morbidity varied from 63.3% to 80%, mortality from 15% to 30% and lethality from 23% to 37.5%. In survey 1, for all farms, high parasitemia (from 30.3 to 26.2x10(6) parasites/mL), fever (from 39.8 to 40.3 degrees C), low PCV (from 15.7% to 18.1%), and body score (from 2.5 to 3.5) were detected. Calves showed depression, weight loss, pale mucous membranes, enlarged lymph nodes, edema of the dewlap, cough, coryza, and diarrhea. The animals from farms A and B were treated with diminazene aceturate. Six months after, in survey 2, non-treated calves from farm C showed values for prevalence (81.82), morbidity (81.82), mortality (12.73), and lethality (15.55) similar to those in survey 1 (P>0.05). Also in survey 2, four calves aging merely 1-3 days old presented high parasitemia levels (from 32x10(6) to 74x10(6) parasites/mL), suggesting transplacental transmission. In conclusion, trypanosomiasis by T. vivax constitutes high prevalent disease for calves raised in Brazilian semiarid and may have transplacental transmission.

Assessing the effects of multiple infections and long latency in the dynamics of tuberculosis

In order to achieve a better understanding of multiple infections and long latency in the dynamics of Mycobacterium tuberculosis infection, we analyze a simple model. Since backward bifurcation is well documented in the literature with respect to the model we are considering, our aim is to illustrate this behavior in terms of the range of variations of the model's parameters. We show that backward bifurcation disappears (and forward bifurcation occurs) if: (a) the latent period is shortened below a critical value; and (b) the rates of super-infection and re-infection are decreased. This result shows that among immunosuppressed individuals, super-infection and/or changes in the latent period could act to facilitate the onset of tuberculosis. When we decrease the incubation period below the critical value, we obtain the curve of the incidence of tuberculosis following forward bifurcation; however, this curve envelops that obtained from the backward bifurcation diagram.