Cocoa mycobiota : fungi and mycotoxins from cocoa until chocolate

Orientador: Jose Luiz PereiraTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O Brasil se encontra entre os maiores produtores mundiais de cacau. Parte da sua produção é destinada à exportação, principalmente após passar por algum processamento industrial, e o restante se destina ao consumo interno. A qualidade obtida ao final do processamento do cacau depende de uma ampla variedade de fatores, dentre os quais os microrganismos desempenham um papel fundamental. A presença de fungos é observada em várias fases do processamento, o que além do aspecto deteriorativo representa um perigo toxicológico devido a possibilidade de síntese de micotoxinas. Na presente tese são apresentados os resultados sobre a presença de fungos, ocratoxina A e aflatoxinas em um total de 494 amostras de cacau analisadas em diferentes fases do processamento. A maior quantidade e diversidade de fungos foi encontrada em amostras coletadas durante o processamento na fazenda, especialmente durante a secagem e estocagem das amêndoas. Foram isolados 1132 espécies potencialmente toxigênicas do gênero Aspergillus: A. flavus, A. parasiticus, A. niger, A. carbonarius e grupo dos A. ochraceus. Com relação à presença de micotoxinas, as amostras da secagem e estocagem foram as que apresentaram maiores níveis de ocratoxina A e aflatoxinas na fazenda, enquanto que nas amostras coletadas em indústrias processadoras de cacau os maiores níveis de ocratoxina A foram detectados na casca, torta e cacau em pó e os de aflatoxina, nos nibs e liquor. Nas amostras de chocolate observou-se uma relação direta entre a quantidade de cacau no produto e os níveis de aflatoxinas e ocratoxina A, sendo estes maiores em chocolate em pó e chocolate meio amargo. Também foram conduzidos experimentos para avaliar as condições que afetam a produção de ocratoxina A no cacau por A. carbonarius. Foi observado que, diante da presença de inóculo, a produção de ocratoxina A já ocorre durante a fermentação. Os níveis de ocratoxina A foram menores quando sementes de cacau com polpa integral foram fermentadas durante 6 dias em caixas, se comparados aos obtidos com sementes parcialmente despolpadas e conduzidas à secagem sem prévia fermentação. Os resultados em geral mostraram a existência de uma grande diversidade de fungos, muitos dos quais toxigênicos, durante o processamento do cacau e a inter-relação de fatores intrínsecos, ambientais e de processamento pareceram determinar a ocorrência ou não de micotoxinasAbstract: Brazil is one of the biggest cocoa producing countries. Part of its production is exported, mainly after undergoing some industrial processing, and the other supplies the internal market. The quality of the final cocoa product depends on several factors, and microorganisms play an important role on this. Investigations have demonstrated the presence of filamentous fungi in many processing stages. Besides the spoilage potential, those are a cause for concern from the toxicological aspect, since mycotoxins may be produced. This thesis presents the results concerning the presence of fungi, ochratoxin A and aflatoxins in a total of 494 cocoa samples collected at different processing stages, from farm to market. The greatest occurrence and the major diversity of fungi were found in samples collected during the drying and storage of the beans on the farm. A total of 1132 potentially toxigenic fungi were isolated, belonging to the species A. flavus, A. parasiticus A. niger, A. ochraceus group and A. carbonarius. Regarding the presence of mycotoxins, samples collected during drying and storage presented the highest ochratoxin A and aflatoxin levels amongst samples collected at farm. During industrialization, the most contaminated products were the cocoa shell, cocoa cake and cocoa cake with respect to ochratoxin A. The aflatoxin levels were higher in the nibs and liquor. A direct correlation between the amount of cocoa in the product and the ochratoxin A level in the chocolate was observed. Dark chocolate and powdered commercial cocoa were the most contaminated samples. An additional experiment was carried out to evaluate the conditions affecting ochratoxin A production by A. carbonarius in cocoa on the farm. It was observed that ochratoxin A production could start during fermentation if toxigenic fungi were present, and lower ochratoxin levels were found when the integral beans were fermented during 6 days in box if compared to those with pulp partially removed and submitted to drying fermentation. In general, during cocoa processing, a wide variety of fungi was found, some of these toxigenic. Intrinsic, environmental and processing factors acting together seemed to determine the production of mycotoxins in cocoaDoutoradoDoutor em Ciência de Alimento

Avaliação micológica de rações comerciais para cães e gatos e potencial micotoxicogênico de espécies selecionadas

A eficácia dos meios ágar batata acidificado, ágar dicloram rosa de bengala e cloranfenicol e ágar dicloram glicerol 18% foi comparada para isolamento e quantificação de fungos a partir da análise de 54 amostras de rações comerciais secas para cães e gatos (34 para cães e 20 para gatos), produzidas por 9 empresas. A atividade de água das amostras foi quantificada, apresentando valores entre 0,45 e 0,82. Em 74% das amostras foi detectada a presença fúngica, onde, além de fungos com micélio estéril e leveduras, 23 gêneros de fungos foram identificados. As 40 amostras positivas apresentaram níveis de contaminação, com contagens variando entre 10 1 e 103 UFC/g. Não se verificou correlação entre atividade de água e contaminação fúngica e não se observou diferença significativa entre o número de colônias isoladas e os diferentes meios de cultivo utilizados. Apesar disto, o DG18 foi o meio que apresentou melhores resultados tanto na quantidade quanto na variedade de fungos isolados. Comparandose os resultados obtidos com diferentes meios observa-se que os microrganismos isolados dependem dos meios de cultivo empregados. O gênero Aspergillus e a espécie Aspergillus niger foram os mais freqüentemente isolados. Isolados pertencentes a espécies potencialmente produtoras de aflatoxinas e ocratoxina A foram avaliados através do método de ágar plug-TLC. Vinte por cento dos A. flavus isolados produziram aflatoxina B1, todos os isolados de A. ochraceus produziram ocratoxina A e nenhum isolado de A. niger foi detectado como produtor de ocratoxina através do método de screening utilizado. A avaliação fúngica realizada com o emprego de 3 meios de cultura tornou claro que a detecção de fungos é dependente do meio de cultura utilizado. A Aw do alimento e do meio também devem ser consideradas para que as análises microbiológicas possam detectar ou valorar a micobiota que, efetivamente, está contaminando o alimento

Expanding the clinical and mutational spectrum of B4GALT7-spondylodysplastic Ehlers-Danlos syndrome

Spondylodysplastic EDS (spEDS) is a rare connective tissue disorder that groups the phenotypes caused by biallelic B4GALT7, B3GALT6, and SLC39A13 mutations. In the 2017 EDS nosology, minimal criteria (general and gene-specific) for a clinical suspicion of spEDS have been proposed, but molecular analysis is required to reach a definite diagnosis. The majority of spEDS patients presented with short stature, skin hyperextensibility, facial dysmorphisms, peculiar radiological findings, muscle hypotonia and joint laxity and/or its complications. To date only 7 patients with β4GALT7-deficiency (spEDS-B4GALT7) have been described and their clinical data suggested that, in addition to short stature and muscle hypotonia, radioulnar synostosis, hypermetropia, and delayed cognitive development might be a hallmark of this specific type of spEDS. Additional 22 patients affected with an overlapping phenotype, i.e., Larsen of Reunion Island syndrome, all carrying a homozygous B4GALT7 mutation, are also recognized. Herein, we report on a 30-year-old Moroccan woman who fitted the minimal criteria to suspect spEDS, but lacked radioulnar synostosis and intellectual disability and presented with neurosensorial hearing loss and limb edema of lymphatic origin. Sanger sequencing of B4GALT7 was performed since the evaluation of the spEDS gene-specific minor criteria suggested this specific subtype. Mutational screening revealed the homozygous c.829G>T, p.Glu277* pathogenetic variant leading to aberrant splicing. Our findings expand both the clinical and mutational spectrum of this ultrarare connective tissue disorder. The comparison of the patient's features with those of the other spEDS and Larsen of Reunion Island syndrome patients reported up to now offers future perspectives for spEDS nosology and clinical research in this field

Comments on "Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China. Toxins 2015, 7, 3858-3875"-in Attributing Ochratoxin A Biosynthesis Within the Genus <i>Penicillium </i>Occurring on Natural Agricultural Produce

The unusual attribution of trace amounts of ochratoxin A in some Chinese food commodities to Penicillium polonicum is questioned by European experience in searches for ochratoxinogenic food-spoilage Penicillia, where mistaken attribution is now known to have been due to cryptic Penicillium verrucosum contamination. Consequently, selection of single-spore isolates is recommended as pre-requisite for attributing mycotoxin biosynthetic potential to fungi

Transcriptome-wide expression profiling in skin fibroblasts of patients with joint hypermobility syndrome/ehlers-danlos syndrome hypermobility type

Joint hypermobility syndrome/Ehlers-Danlos syndrome hypermobility type (JHS/EDS-HT), is likely the most common systemic heritable connective tissue disorder, and is mostly recognized by generalized joint hypermobility, joint instability complications, minor skin changes and a wide range of satellite features. JHS/EDS-HT is considered an autosomal dominant trait but is still without a defined molecular basis. The absence of (a) causative gene(s) for JHS/EDS-HT is likely attributable to marked genetic heterogeneity and/or interaction of multiple loci. In order to help in deciphering such a complex molecular background, we carried out a comprehensive immunofluorescence analysis and gene expression profiling in cultured skin fibroblasts from five women affected with JHS/EDS-HT. Protein study revealed disarray of several matrix structural components such as fibrillins, tenascins, elastin, collagens, fibronectin, and their integrin receptors. Transcriptome analysis indicated perturbation of different signaling cascades that are required for homeostatic regulation either during development or in adult tissues as well as altered expression of several genes involved in maintenance of extracellular matrix architecture and homeostasis (e.g., SPON2, TGM2, MMP16, GPC4, SULF1), cell-cell adhesion (e.g., CDH2, CHD10, PCDH9, CLDN11, FLG, DSP), immune/inflammatory/pain responses (e.g., CFD, AQP9, COLEC12, KCNQ5, PRLR), and essential for redox balance (e.g., ADH1C, AKR1C2, AKR1C3, MAOB, GSTM5). Our findings provide a picture of the gene expression profile and dysregulated pathways in JHS/EDS-HT skin fibroblasts that correlate well with the systemic phenotype of the patients

Comparison of different culture media for mycological evaluation of commercial pet food

The choice of a culture media to proceed a mycological analysis of food is extremely important to guarantee the reliability of the analysis. The medium should allow an excellent recovery of fungal species present in the food at the same time that it is necessary to avoid bacterial development. Thus the real microbial ecology will be reflected in the results. The efficacy of three media for fungal isolation and quantification (potato dextrose agar, dichloran rose of bengal and chloramphenicol agar and dichloran glycerol 18% agar) was compared for analysis of 54 samples of dry commercial pet food (34 for dogs and 20 for cats). Fungi were present in 74% of samples and 23 genera were isolated and identified. Aspergillus sp. and Aspergillus niger were respectively the most frequent genera and species isolated, whatever the culture medium selected to proceed the analysis. Dichloran glycerol 18% agar was the medium that presented the best results considering both the quantity and variety of isolated fungi. Comparing the results obtained in different media, it was observed that the fungi  recovered can vary according to the selected culture medium. Eurotium was the genus that presented the biggest difference in occurrence among the media where samples were cultivated to enumeration in this study. Therefore, the utilization of specific media, selected according to the characteristics of the food to be analyzed (specially water activity) allow a better visualization of the presence of food spoilage fungi

The realistic positioning of UVA1 phototherapy after 25 years of clinical experience and the availability of new biologics and small molecules: a retrospective clinical study

BackgroundSince the early 1990s, Ultraviolet (UV) A1 phototherapy has been described as an effective and safe treatment of a multitude of skin disorders. However, after 30 years, its use has remained limited to few dermatological centers.ObjectiveTo analyze the changes over the years and the current position of UVA1 phototherapy through a Real-World Evidence (RWE) study at a single tertiary referral center.MethodsWe reviewed the medical files of 740 patients treated between 1998 and 2022. Treatment results were collected, efficacy was assessed by a grading scale and acute adverse effects were registered.ResultsWe treated patients with 26 different diseases. We registered marked improvement (MI) or complete remission (CR) in 42.8% of patients with morphea, 50% with Urticaria Pigmentosa, 40.7% with Granuloma annulare and 85.7% with skin sarcoidosis. Good results were obtained also in the treatment of chronic Graft Versus Host Disease (GVHD), Eosinophilic Fasciitis, Sclero-atrophic Lichen, skin manifestations of systemic lupus erythematosus and psoriasis of HIV+ patients. Systemic Sclerosis, Romberg’s Syndrome, Bushke’s Scleredema, Nephrogenic Fibrosing Dermopathy, REM Syndrome, Follicular Mucinosis, Pretibial Myxedema, Scleromyxedema, pemphigus foliaceus, chronic cutaneous lupus erythematosus, erythroderma of Netherton Syndrome and Necrobiosis Lipoidica were no or poorly responsive. In clinical indications where UVA1 was used as a second line phototherapy after narrow-band (NB)-UVB, we saw good MI or CR rates in Mycosis Fungoides (57% of patients), Atopic Dermatitis (33.9%), Pitiryasis Lichenoides chronica (50%), Pityriasis Lichenoides et varioliformis acute (75%) and Lymphomatod Papulosis (62.5%). Short-term adverse events were uncommon and mild.ConclusionOver the past decade, the annual number of treated patients has progressively declined for several reasons. Firstly, UVA1 phototherapy has taken a backseat to the cheaper and more practical NB-UVB phototherapy, which has proven effective for common indications. Secondly, the emergence of new, safe, and effective drugs for conditions such as atopic dermatitis, GVHD, and connective tissue disorders. Finally, our research has shown that UVA1 therapy is often ineffective or minimally effective for some rare diseases, contrary to previous case reports and small case series. Nonetheless, UVA1 continues to be a valuable treatment option for patients with specific skin disorders

La mano visible del Estado y la mano invisible del mercado: ¿Por qué la salud es más que un contrato entre partes?

La pandemia del COViD-19 ha evidenciado para todxs algo que la epidemiología conoce desde su origen: la salud individual está en estrecha relación con la salud social o colectiva. su cuidado -eje de los sistemas sanitarios- es entonces el pilar del cuidado colectivo, que se organiza en torno a la salud pública (en el sentido poblacional).Es así que el funcionamiento del sistema de salud requiere regirse por algunas direcciones comunes sobre todo en Argentina, donde el sistema es sumamente segmentado, fragmentado y desigual. Estos rasgos se suman a la poco articulada y coordinada interdependencia entre los subsectores (público, privado y seguridad social). Los modelos que excluyen la posibilidad de comprender los límites difusos y muchas veces inexistentes entre los subsectores desconocen el real funcionamiento del sistema sanitario.Fil: Luzuriaga, Maria José. Universidad Nacional de Lanus. Departamento de Salud Comunitaria; ArgentinaFil: Zunino, Marina Gabriela. Universidad Nacional de Lanus. Departamento de Salud Comunitaria; ArgentinaFil: Almirón, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Lanús. Rectorado. Instituto de Salud Colectiva; ArgentinaFil: Iusef Venturini, Nasim. Universidad Nacional de La Plata; Argentin

Dermatophytes isolated from dogs and cats suspected of dermatophytosis in Southern Brazil

Dermatophytosis which is characterized by a superficial infection confined to keratinised tissues, is the most common fungal disease in small animal veterinary medicine. It is unreliable to diagnose dermatophytosis on the basis of clinical signs alone, not only for the variable nature of the dermatological findings but also because there are several other skin diseases which mimic the typical fungal lesion (circular lesions with alopecia). The present study reports laboratory results of an extensive survey evaluating fungal and parasitic aetiology of skin diseases through the analysis of 1,240 fur, nails and skin scraping specimens from dogs and cats with clinical suspicion of dermatophytosis. Samples collected in several veterinary clinics of the Santa Catarina, Paraná and Rio Grande d o Sul states, mainly of the Santa Maria city in Rio Grande do Sul, were processed at the Mycology Research Laboratory of the Federal University of Santa Maria, Southern Brazil, between 1998 and 2003. Among canine and feline samples, the percentages of positive dermatophyte specimens were 10.2% and 27.8%, respectively. The most prevalent fungal specie in both cats and dogs was Microsporum canis, which was isolated in 68.5% of the positive cultures for dermatophytes in dogs’ samples, being the only species recovered from cats’ cultures. Malassezia pachydermatis was the most commonly isolated yeast from the skin of dogs. Acari, mainly Demodex canis, were found in 5.0% of all samples with suspected diagnosis of dermatophytosis