Human Toxocariasis in Portugal-An Overview of a Neglected Zoonosis over the Last Decade (2010-2020).

Toxocariasis is one of the most widespread and important zoonotic parasitic diseases, although neglected. Data regarding human Toxocara infection in Portugal are almost absent. This article gives an overview of the situation of toxocariasis in Portugal over the last decade based on casuistic data. A total of 846 serum samples from individuals suspected of toxocariasis, collected from 2010 to 2020, were analyzed at the Institute of Hygiene and Tropical Medicine. Sera were tested for IgG antibodies to Toxocara canis excreted-secreted larval antigens by enzyme-linked immunosorbent assay and counterimmunoelectrophoresis. Positivity was detected in 18.8% (159/846) [CI 95%: 16.3-21.6], with positives detected throughout continental Portugal. Overall, 59.7% of the positives were diagnosed in younger than 20 years (35.2% aged 0-9 years and 24.5% aged 10-19 years). Eosinophilia was the most frequent feature reported (27.7%). Pediatrics (41.5%) and Infectiology (25.8%) were the specialties with the highest number of positives. An average of 77 samples/year were received, recording a maximum positivity in 2012 (41.5%, n = 27/65) and a minimum in 2020 (6.4%, n = 3/47). These numbers may reflect the effectiveness of current preventive measures, highlighting the need to maintain public awareness to control this helminthozoonosis and promote a higher public health standard.

Impact of Annual Albendazole versus Four-Monthly Test-and-Treat Approach of Intestinal Parasites on Children Growth-A Longitudinal Four-Arm Randomized Parallel Trial during Two Years of a Community Follow-Up in Bengo, Angola.

Malnutrition and intestinal parasites continue to have serious impacts on growth and cognitive development of children in Angola. A longitudinal four-arm randomized parallel trial was conducted to investigate if deworming with a single annual dose of albendazole (annual-ALB) or a four-monthly test-and-treat (4TT) intestinal parasites approach at individual or household levels improve nutritional outcomes of pre-school children in Bengo province. Children with intestinal parasites (n = 121) were randomly assigned (1:1:1:1) to arm A1: annual-ALB*individual level; A2: annual-ALB*household level; A3: 4TT*individual; and A4: 4TT*household level. At baseline, 4, 8, 12, 16, 20, and 24 months of follow-up, growth was assessed by height, weight, height-for-age, weight-for-height, weight-for-age, and mid-upper arm circumference. Intention-to-treat analysis was done using non-parametric approach, mixed effect models, and generalized estimating equations (GEE). Initially, 57% and 26% of the children were infected by Giardia lamblia and Ascaris lumbricoides, respectively. This study did not show that a 4TT intestinal parasites approach results on better growth outcomes of children (height, weight, HAZ, WAZ, WHZ and MUACZ) when compared with annual ALB, with exception of height and WHZ using GEE model at 5% level. Positive temporal effects on most nutrition outcomes were observed. Implementing a longitudinal study in a poor setting is challenging and larger sample sizes and 'pure and clean' data are difficult to obtain. Nevertheless, learned lessons from this intensive study may contribute to future scientific research and to tailor multidisciplinary approaches to minimize malnutrition and infections in resource-poor countries.

Synergistic properties of mustard and cinnamon essential oils for the inactivation of foodborne moulds in vitro and on Spanish bread.

This work was performed to evaluate the antifungal effect of cinnamon and mustard essential oil (EO) alone and in combination against a range of mould strains. A wide range of resistance levels was observed among different mould species, being R. stolonifer the most resistant one. Mustard EO showed the biggest antifungal effect, and for this reason it was selected to study its effect in vapour phase on bread. The shelf life of bread inoculated with R. stolonifer and non- inoculated increased by 3-4 days at 25 °C, while at 4 °C the shelf-life increased beyond 50 days. However, the results from the acceptability test were negative. Based on this, the antifungal effect of cinnamon/mustard EO in combination was evaluated, with results mostly additive and synergistic. The ratio of the combination was defined taking into account the most resistant strain (100:8, cinnamon and mustard respectively) and their application was performed in vapour phase. This combination maintained the antifungal activity presented by mustard EO by itself and masked the mustard flavour, providing positive results in the acceptability tests. It was therefore suggested to be used at industrial scale.

Effect of an active label based on benzyl isothiocyanate on the morphology and ochratoxins production of Aspergillus ochraceus.

The aim of this work was the study of the main effects of benzyl isothiocyanate (BITC) on A. ochraceus morphology and on its production metabolism of ochratoxins. This compound was evaluated as active agent of an antimicrobial label in food packaging. Microbiological studies showed a slowdown in mould growth when the active material was applied to A. ochraceus and the presence of three different areas of growth. Scanning electron microscopy was successfully used to demonstrate the mode of action of BITC on this strain. A. ochraceus exhibited modifications in morphology compared to the control samples such as the disappearance of sclerotia or cleistothecia An extraction protocol and an analytical method by UPLC-MS/MS to determine ochratoxins (OTs) was developed. The results showed that all these morphological changes were related to a decrease on OTs production, both ochratoxin A (OTA) and ochratoxin B (OTB). The presence of BITC caused a great decrease on OTA that modified the OTA/OTB ratio, increasing the OTB proportion. Furthermore, the active packaging also modified the production of other secondary metabolites. The morphological and metabolic effects observed, as well as the relationship between them, are of great interest since they have not been reported before for A. ochraceus.

Raman spectroscopy, electronic microscopy and SPME-GC-MS to elucidate the mode of action of a new antimicrobial food packaging material.

One critical challenge when developing a new antimicrobial packaging material is to demonstrate the mode of action of the antimicrobials incorporated into the packaging. For this task, several analytical techniques as well as microbiology are required. In this work, the antimicrobial properties of benzyl isothiocyanate, allyl isothiocyanate and essential oils of cinnamon and oregano against several moulds and bacteria have been evaluated. Benzyl isothiocyanate showed the highest antimicrobial activity and it was selected for developing the new active packaging material. Scanning electron microscopy and Raman spectroscopy were successfully used to demonstrate the mode of action of benzyl isothiocyanate on Escherichia coli. Bacteria exhibited external modifications such as oval shape and the presence of septum surface, but they did not show any disruption or membrane damage. To provide data on the in vitro action of benzyl isothiocyanate and the presence of inhibition halos, the transfer mechanism to the cells was assessed using solid-phase microextraction-gas chromatography-mass spectrometry. Based on the transfer system, action mechanism and its stronger antimicrobial activity, benzyl isothiocyanate was incorporated to two kinds of antimicrobial labels. The labels were stable and active for 140 days against two mould producers of ochratoxin A; Penicillium verrucosum is more sensitive than Aspergillus ochraceus. Details about the analytical techniques and the results obtained are shown and discussed. Graphical Abstract Antimicrobial evaluation of pure compounds, incorporation in the packaging and study for mode of action on S. coli by Raman, SEM and SPME-GC-MS.

Comparing concentration methods: parasitrap® versus Kato-Katz for studying the prevalence of Helminths in Bengo province, Angola.

BACKGROUND: Helminth intestinal parasitoses are responsible for high levels of child mortality and morbidity. Hence, the capacity to diagnose these parasitoses and consequently ensure due treatment represents a factor of great importance. OBJECTIVES: The main objective of this study involves comparing two methods of concentration, parasitrap and Kato-Katz, for the diagnosis of intestinal parasitoses in faecal samples. METHODS: Sample processing made recourse to two different concentration methods: the commercial parasitrap® method and the Kato-Katz method. RESULTS: We correspondingly collected a total of 610 stool samples from pre-school and school age children. The results demonstrate the incidence of helminth parasites in 32.8% or 32.3% of the sample collected depending on whether the concentration method applied was either the parasitrap method or the Kato-Katz method. We detected a relatively high percentage of samples testing positive for two or more species of helminth parasites. We would highlight that in searching for larvae the Kato-Katz method does not prove as appropriate as the parasitrap method. CONCLUSION: Both techniques prove easily applicable even in field working conditions and returning mutually agreeing results. This study concludes in favour of the need for deworming programs and greater public awareness among the rural populations of Angola.

Raman Imaging Spectroscopy as a Tool To Investigate the Cell Damage on Aspergillus ochraceus Caused by an Antimicrobial Packaging Containing Benzyl Isothiocyanate.

Raman imaging spectroscopy is a nondestructive analytical method that can be a useful tool to obtain detailed information about the molecular composition and morphology of biological samples. Its high spatial resolution was used to collect spectra of Aspergillus ochraceus, a mold producer of ochratoxin A (OTA), in order to investigate the cell damage caused on it by the action of the antimicrobial benzyl isothiocyanate (BITC). The study was performed in both direct contact and vapor phase, in order to check the use of BITC as active agent in food packaging material. The results showed that there were morphologic alteration and a characteristic Raman spectrum on spore and hyphae exposed to BITC. BITC was accumulated in the mold cells where it caused an enormous amount of alterations in cellular components (lipids, proteins, saccharides, amino acids...) and cellular functions (cell cycle, respiration, metabolism, transcription of genes, fluidity of the cellular wall). All these structural, composition, and metabolic changes will affect the production of OTA. Pattern recognition with chemometrics using principal component analysis (PCA) demonstrated an excellent separation between control and BITC treated samples, both in spores and hyphae. PCA results also showed two different affection levels when samples were exposed to BITC in the vapor phase.

Migration from printing inks in multilayer food packaging materials by GC-MS analysis and pattern recognition with chemometrics.

Inks and varnishes used in food packaging multilayer materials can contain different substances that are potential migrants when packaging is in contact with food. Although printing inks are applied on the external layer, they can migrate due to set-off phenomena. In order to assess food safety, migration tests were performed from two materials sets: set A based on paper and set B based on PET; both contained inks. Migration was performed to four food simulants (EtOH 50%, isooctane, EtOH 95% and Tenax(®)) and the volatile compounds profile was analysed by GC-MS. The effect of presence/absence of inks and varnishes and also their position in the material was studied. A total of 149 volatile compounds were found in migration from set A and 156 from set B materials, some of them came from inks. Quantitative analysis and a principal component analysis were performed in order to identify patterns among sample groups.

Influence of oxygen and long term storage on the profile of volatile compounds released from polymeric multilayer food contact materials sterilized by gamma irradiation.

The profile of volatile compounds released from 13 different multilayer polymeric materials for food use, before and after their exposure to gamma radiation, has been assessed by solid-phase microextraction-gas chromatography-mass spectrometry. Thermosealed bags of different materials were filled with either air or nitrogen to evaluate the oxygen influence. One-third of the samples were analyzed without irradiation, whereas the rest were irradiated at 15 and 25 kGy. Half of the samples were processed just after preparation and the other half was stored for 8 months at room temperature prior to analysis. Very significant differences between unirradiated and irradiated bags were found. About 60-80 compounds were released and identified per sample. A huge peak of 1,3-ditertbutylbenzene was present in most of the irradiated samples. An outstanding reproducibility in all the variables evaluated (chromatograms, oxygen percentage, volume of bags) was noticed. Independently of filling gas, the results of unirradiated materials were almost identical. In contrast, the chromatographic profile and the odor of irradiated bags filled with nitrogen were completely different to those filled with air. Principal component analysis was performed and 86.9% of the accumulated variance was explained with the first two components. The migration of compounds from irradiated materials to the vapor phase was much lower than the limits established in the Commission Regulation (EU) No 10/2011.

Rational stabilization of complex proteins: a divide and combine approach.

Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones).

Prevención y tratamiento de las úlceras por presión

Programa de prevención y tratamiento de úlceras por presión iniciado por la Dirección de Enfermería del Hospital Universitario de Bellvitge, en 1991 (AU)