Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Evaluation of an alternative spectroscopic approach for aflatoxin analysis: Comparative analysis of food and feed samples with UPLC-MS/MS

Increasing research has highlighted the effects of changing climates on the occurrence and prevalence of toxigenic Aspergillus species producing aflatoxins. There is concern of the toxicological effects to human health and animal productivity following acute and chronic exposure that may affect the future ability to provide safe and sufficient food globally. Considerable research has focused on the detection of these toxins, based on the physicochemical and biochemical properties of the aflatoxin compounds, in agricultural products for human and animal consumption. As improvements in food security continue more regulations for acceptable levels of aflatoxins have arisen globally; the most stringent in Europe. These regulations are important for developing countries as aflatoxin occurrence is high significantly effecting international trade and the economy. In developed countries analytical approaches have become highly sophisticated, capable of attaining results with high precision and accuracy, suitable for regulatory laboratories. Regrettably, many countries that are affected by aflatoxin contamination do not have resources for high tech HPLC and MS instrumentation and require more affordable, yet robust equally accurate alternatives that may be used by producers, processors and traders in emerging economies. It is especially important that those companies wishing to exploit the opportunities offered by lucrative but highly regulated markets in the developed world, have access to analytical methods that will ensure that their exports meet their customers quality and safety requirements.

This work evaluates the ToxiMet system as an alternative approach to UPLC–MS/MS for the detection and determination of aflatoxins relative to current European regulatory standards. Four commodities: rice grain, maize cracked and flour, peanut paste and dried distillers grains were analysed for natural aflatoxin contamination. For B1 and total aflatoxins determination the qualitative correlation, above or below the regulatory limit, was good for all commodities with the exception of the dried distillers grain samples for B1 for which no calibration existed. For B1 the quantitative R² correlations were 0.92, 0.92, 0.88 (<250 μg/kg) and 0.7 for rice, maize, peanuts and dried distillers grain samples respectively whereas for total aflatoxins the quantitative correlation was 0.92, 0.94, 0.88 and 0.91. The ToxiMet system could be used as an alternative for aflatoxin analysis for current legislation but some consideration should be given to aflatoxin M1 regulatory levels for these commodities considering the high levels detected in this study especially for maize and peanuts

Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Occupational exposure to fungi and particles in animal feed industry

Background: Very few studies regarding fungal and particulate matter (PM) exposure in feed industry have been reported, although such contaminants are likely to be a significant contributing factor to several symptoms reported among workers. The purpose of this study has been to characterize fungal and dust exposure in one Portuguese feed industry. Material and Methods: Air and surface samples were collected and subject to further macro- and microscopic observations. In addition we collected other air samples in order to perform real-time quantitative polymerase chain reaction (PCR) amplification of genes from Aspergillus fumigatus and Aspergillus flavus complexes as well as Stachybotrys chartarum. Additionally, two exposure metrics were considered – particle mass concentration (PMC), measured in 5 different sizes (PM0.5, PM1, PM2.5, PM5, PM10), and particle number concentration (PNC) based on results given in 6 different sizes in terms of diameter (0.3 μm, 0.5 μm, 1 μm, 2.5 μm, 5 μm and 10 μm). Results: Species from the Aspergillus fumigatus complex were the most abundant in air (46.6%) and in surfaces, Penicillium genus was the most frequently found (32%). The only DNA was detected from A. fumigatus complex. The most prevalent in dust samples were smaller particles which may reach deep into the respiratory system and trigger not only local effects but also the systemic ones. Conclusions: Future research work must be developed aiming at assessing the real health effects of these co-exposures.

Assessment of toxigenic fungi in poultry feed

Introduction - Feed supplies the necessary nutrients for the growth of healthy animals, which are a part of the human diet. The presence of toxigenic fungi in animal feed such as Aspergillus spp. may contribute to 1) the loss of nutritional value of feedstuff, since fungi will assimilate the most readily available nutrients present in the feed, and 2) the development of mycotoxicoses and chronic conditions, which can raise economic issues due to animal disease and contamination of animal derived products. Aim of the study - The goal of this work was to evaluate the incidence of Aspergilli, particularly from the Circumdati, Flavi and Fumigati sections, through real-time quantitative PCR (qPCR) in 11 feed samples.

Fungi distribution in poultry feed

Feed can easily be contaminated and colonized by fungi that use up the nutrients for their own metabolism and growth, producing secondary metabolites such as mycotoxins that are not eliminated throughout the feed processing. The major problems associated with mycotoxin contaminated animal feed are metabolic disturbances resulting in poor animal productivity. In addition, handling contaminated animal feed can also raise health issues regarding workers exposure to fungi and mycotoxins. The scope of this work was to characterize fungal distribution in 11 poultry feed samples. Twenty grams of feed were suspended in 180 mL of distilled water and homogenized during 20 minutes at 200 rpm. The washed supernatant was plated in malt extract agar (MEA) and dichloran glycerol agar base (DG18) media for morphological identification of the mycobiota present. Using macro- and microscopic analysis of the colonies, fungal contamination was evident in 72.7% of the analyzed poultry feed samples. Fungal load ranged from 0 to 13140 CFU/g, and the most prevalent species/genera were F. graminearum complex (71.1%), Penicillium sp. (11.6%), Cladosporium sp. (8.8%), and Fusarium poae (3.6%). In addition to these species, we also isolated Aspergillus sections Circumdati, Nigri and Aspergilli, and Mucor and Rhizopus genus albeit at a lower abundance. The data obtained showed that, besides high fungal contamination, mycotoxins contamination is probably a reality, particularly in the final product since mycotoxins resist to all the processing operations including thermal treatment. Additionally, data claimed attention for the probable co-exposure to fungi and mycotoxins of the workers in feed industries.

Toxigenic fungi in coffee samples: a menace to public health

Introduction - Mycotoxin contamination was reported to occur in some food and commodities, such as coffee, particularly due to the presence of toxigenic fungi such as Aspergillus, Penicillium and Fusarium spp. Aspergilli are known to produce high levels of mycotoxins, such as ochratoxin and aflatoxin. Aspergillus ochraceus has been proposed as the major cause of ochratoxin A contamination in coffee beans. Aim of the study - The aim of this work was to evaluate the prevalence of Aspergillus sections Circumdati, Flavi and Fumigati in 28 green coffee samples to be used by Portuguese coffee industry, from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) species from different origins.

Fungal contamination in coffee samples: a public health concern

Introduction - Fungi are natural coffee contaminants and under certain environmental conditions have the potential to produce toxins. Many studies revealed that the important toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants, and are present from the field to storage. Aspergilli from the Circumdati and Nigri sections are known to produce high levels of ochratoxin A, a mycotoxin known as nephrotoxic for animals and humans. This work aimed to evaluate fungal distribution and also the prevalence of Aspergillus sections Fumigati, Flavi, Nigri and Circumdati from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) green samples.

Emergency emicolectomy for intestinal primary Aspergillosis in acute myeloid leukemia

ntestinal aspergillosis is an infection with a very high death rate especially in leukemic patients. Here we describe a case of a 46 years old woman with acute myeloid leukemia (LAM M5) who developed intestinal primary aspergillosis. This patient was diagnosed with LAM M5 through bone marrow aspiration and bone biopsy in March 2004. Symptoms of the disease were slight persistent fever, weight loss, asthenia, anemia, thrombocytopenia,and leukocytosis with high number of blasts in peripheral blood. After induction chemotherapy with ICE (Ifosfamide, Carboplatin, Etoposide), she developed neutropenia and high fever without apparent infective foci. She was treated with empiric antibiotic therapy, nevertheless she developed an intense diarrhea and ileo-cecal distention. Diagnostic exams didn’t show signs of a focal lesion. Despite the change in antibiotic treatment and the transfusions of granulocytes and blood cells, the patient developed extremely critical conditions with persistence of neutropenia and abdominal distention. A surgical treatment was decided at the time. We treated the patient with a two steps surgical procedure. The first step was a right abdominal ileostomy followed by improvement of general conditions and then the second step a right colectomy. The histological morphology confirmed necrotizing colitis with Aspergillus ife. At that time , treatment with voriconazole was started. The general conditions of the patient improved rapidly and we were able to treat the patient with other medical anti-leukemic therapies. The patient is now cured and in healthy state. We obtained a good clinical result as only in other few cases described in literature.

Climate changes influence in occupational exposure to fungi and mycotoxins

Climate changes and their effects on fungal distribution and activity are aspects of concern regarding the human exposure to mycotoxins. An exhaustive search was made for papers available in scientific databases reposrting the influence that climate cchange has on fungi and mycotoxins. Also a review regarding fungal burden, collected between 2010 and 2015 in different settings, was done to support the discussion about future fungi and mycotoxins ocuupational exposure. A. flavus complex, E. graminerarum complex and F. verticilliodes were the most reported to be influenced by climate changes. We noted also that the analyzed Portuguese settings presented already an occupational problem due to their fungal burden. It will be important to know future climate changes to select what complexes/species and strains, and consequently the mycotoxins, we should consider as indicators of an occupational problem. In addition, epidemiologic studies are needed to increase knowledge about potential health effects related with the exposure to both risk factors.

Bibliotecas na saúde… e a saúde nas bibliotecas?

Em arquivos e bibliotecas a presença de fungos é considerada nefasta pelas suas implicações na conservação e leitura de documentos históricos e pela sua associação a problemas de saúde sentidos pelos funcionários e utentes que frequentam estes locais. De acordo com alguns autores, os problemas de saúde mais reportados por funcionários em Bibliotecas e Arquivos são dermatite, rinite, alergias e asma. Embora revestida de inegável importância, existem poucos estudos internacionais sobre a temática e, em Portugal, a contaminação fúngica em ambiente arquivístico e em bibliotecas é ainda muito pouco conhecida. O estudo realizado em quatro Arquivos Portugueses teve como objectivo conhecer a contaminação fúngica, contribuindo para a análise da qualidade do ar interior desses espaços e sua comparação com estudos internacionais. Para isso foram recolhidas amostras de ar e de superfícies e estas foram analisadas por métodos clássicos de cultura e, quando necessário, por métodos de biologia molecular. A avaliação foi feita quantitativa e qualitativamente, considerando os requisitos legais em vigor. No que respeita à análise do ar, o número de unidades formadoras de colónias (UFC)/m3 nunca excedeu as 500 (limite legislado), tendo sido verificada contaminação interior em todos os locais estudados. Comparativamente aos estudos realizados anteriormente em contextos semelhantes foram encontrados níveis elevados de contaminação por leveduras nas amostras de ar analisadas em Arquivos Portugueses. Não foi identificado nenhum fungo patogénico neste estudo, mas em quase todas as amostras estavam presentes fungos potencialmente toxinogénicos. Dentro do grupo dos Aspergillus, o A.versicolor mostrou predominância, tendo este fungo reconhecidas capacidades de emissão de micotoxinas em ambiente de interior. A inclusão de amostras de superfície revelou-se vital para conhecer todo o espectro fúngico existente em cada um dos locais estudados, incluindo a detecção de Stachybotrys chartarum e a do fungo potencialmente queratinofílico, Chrysosporium carmichaelli. Tanto para a saúde como para a conservação, o recente estudo realizado em quatro arquivos permitiu retirar importantes conclusões e reforçar a necessidade de vigilância, sendo também útil para a definição de padrões de qualidade no campo do património cultural.

Determinación de la flora micológica del maíz seco y su harina en la parroquía San Juan-cantón Gualaceo

El presente trabajo de tesis tuvo como finalidad evaluar la flora micológica del maíz seco y su harina producidos en la parroquia San Juan - Cantón Gualaceo con el fin de aportar con información sobre la calidad de estos alimentos. Además se evaluaron los siguientes factores para el desarrollo de la flora micológica: temperatura, actividad acuosa, almacenamiento; así como también el crecimiento en distintos medios de cultivos y el uso de la desinfección del grano como parte de la técnica de siembra. La detección, recuento y aislamiento de hongos se realizó de acuerdo a la técnica de recuento en placa por siembra en profundidad para la harina y en el caso del grano por la técnica de siembra directa, con y sin desinfección de la superficie del grano utilizándose el agar MEA (Agar Extracto de Malta), PDA (Agar Papa Dextrosa) y DRBC (Agar Rosa de Bengala Diclorán) y sometiendo a dos tratamientos térmicos (18°C vs 25°C). Se encontró que la micoflora presente en las muestras analizadas corresponde a los géneros Penicillium, Fusarium, Rhizopus, Aspergillus y levaduras, siendo el género predominante Penicillium tanto en el grano como en la harina. La temperatura óptima para el crecimiento fue 25°C; y a su vez el crecimiento fue mejor sin desinfectar las superficies del grano. El crecimiento micológico varió dependiendo del medio, siendo PDA el más óptimo para Penicillium y Rhizopus, mientras que para Fusarium y Aspergillus no hubo diferencia. Igualmente se encontró que la actividad acuosa influyó en forma proporcional al crecimiento micológico y que el almacenamiento controlado influyó positivamente en su disminución.