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- Sitosterolemia In iberoamerican countries: 16 new cases and phenotype genotype analysisPublication . Alves, Ana Catarina; Chora, Joana Rita; Miranda, Beatriz; Medeiros, Ana Margarida; Graça, Rafael; Bañares, Virginia G.; Araujo, Maria Beatriz; Vilagut, Ferrán Trías; Soler, Cristina; Meavilla, Silvia; Toledo, Maria J. Benitez; Volpe, Camila Garcia; Reyes, Ximena; Dell'Oca, Nicolás; Martins, Paula; Marado, Diana; Vilarinho, Laura; Dias, Aureliano Jorge; Ferreira, Ana Cristina; Padeira, Gonçalo; Casañas, Marta; Alegre-González, Diana; Lozano, José Mosquera; Aguiar, Patrício; Gonçalves, Filipa Sousa; Ernaga, Ander; Apellaniz-Ruiz, Maria; Rubi, Rodrigo; Figueroa, Nahún Muñoz; Vasquez, Norma Alejandra; Valdivielso, Pedro; Bourbon, Mafalda; ElsevierBackground: Sitosterolemia is a rare autosomal recessive lipid disorder caused by biallelic pathogenic variants in ABCG5 or ABCG8 genes. It is characterized by elevated plasma plant sterol concentrations, xanthomas, and an increased risk of premature cardiovascular disease. As happens with familial hypercholesterolemia (FH), sitosterolemia is subdiagnosed and is frequently confounded with FH, resulting in inappropriate management. This study aims to describe newly identified cases across Iberoamerican countries and to highlight the need for improved diagnostic strategies. Methods: We report 16 cases of molecularly confirmed sitosterolemia from 5 Iberoamerican countries (Argentina, Mexico, Portugal, Spain, and Uruguay), including 12 index cases and 4 relatives identified by cascade screening. Clinical, biochemical, and molecular data were collected and analyzed. β-sitosterol levels were measured when possible, and variant classification followed American College of Medical Genetics and Genomics (ACMG) guidelines with disease-specific adaptations. Results: Fifteen individuals had biallelic variants in ABCG8 and 1 had a homozygous frameshift variant in ABCG5. Ten distinct ABCG8 variants were identified, including 7 nonsense and 3 missense variants. Xanthomas were observed in 56% of cases. Most cases were initially diagnosed as FH, with a diagnostic delay of up to 30 years. Treatment with ezetimibe, alone or combined with statins, led to biochemical and clinical improvement, including xanthoma regression in some cases. Conclusion: Sitosterolemia remains underdiagnosed due to lack of systematic screening and clinical overlap with FH. Our findings highlight the importance of including ABCG5/8 in genetic testing panels and of recognizing clinical clues for early diagnosis, enabling targeted treatment and prevention of adverse outcomes. Adapted ACMG variant classification improves interpretability for ABCG5/8-related sitosterolemia.
- Hazard identification and characterization of leachable chemicals from plastic products – a new PARC projectPublication . Dirven, Hubert; Bogusz, Aleksandra; Hans Bouwmeester; Busch, Mathias; Duflos, Guillaume; Eriksen, Gunnar S.; Fardilha, Margarida; Flores-Gomez, Daniela; Franko, Nina; Gaté, Laurent; Guichard, Yves; Silva, Maria Joao; Kamstra, Jorke H.; Kasiotis, Konstantinos M.; Kim, Sunmi; Kim, Young Jun; Kim, Youngsam; van der Koogh, Elise; Loureiro, Susana; Louro, Henriqueta; Machera, Kyriaki; Pieters, Raymond H. H.; Spyropoulou, Anastasia; Tzanetou, Evangelia N.; Malheiro, Catarina; Ravnjak, Tim; Repetto, Guillermo; Rivière, Gilles; Ryu, Chang Seon; Papadopoulou, Evgenia Anna; Aliferis, Konstantinos A.; Solhaug, Anita; Sollner Dolenc, Marija; Štampar, Martina; Tavares, Ana M.; Tollefsen, Knut Erik; Ventura, Célia; Walkowiak, Radoslaw; Zobl, Walter; Žegura, Bojana; Snapkow, Igor; Herzke, DorteA recent study has suggested that plastics may contain more than 16,000 chemicals, including additives, processing aids, starting substances, intermediates and Non-Intentionally Added Substances. Plastic chemicals are released throughout the plastic life cycle, from production, use, disposal and recycling. Most of these chemicals have not been studied for potential hazardous properties for humans and in the environment. To refine the risk assessment of these leachable chemicals, additional hazard data are needed. The PlasticLeach project within the EU co-funded Partnership for the Assessment of Risks from Chemicals (PARC) aims to address this data gap by screening several plastic products in daily use. Leachates will be prepared from a number of these plastic items, and these chemical mixtures will be further tested using several test guideline compliant assays and New Approach Methodologies covering both human health and environmental endpoints. The most toxic leachates will be characterized using a non-targeted analysis pipeline to identify chemicals in the leachate. When single chemicals of concern are identified, these will be further tested to determine hazardous properties and identify the respective potency factors to better understand their specific hazard profiles. A tiered approach for hazard testing will be followed. The experimental work will be complemented by toxicological profiling, using publicly available toxicity databases and tools, including Artificial Intelligence tools that cover both human and environmental endpoints. A comprehensive array of endpoints, including cytotoxicity, endocrine disruption, genotoxicity, immunotoxicity, reproductive toxicity and effects related to ecotoxicity will be evaluated. In this paper, we outline the plastic products to be tested and the battery of assays that will be used to identify hazards relevant to both human health and the environment. Data generated from approaches will be reported using standardized formats, stored within a centralized repository, and harmonized to adhere to the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). This integrated strategy will not only advance our understanding of the risks associated with plastic-derived chemicals but will also provide critical support for regulatory decision-making and facilitate the development of safer, and more ecofriendly plastic materials in the future.
- Surveying genetic markers of antibiotic resistance and genomic background in Chlamydia trachomatis: insights from a multiplex NGS-based approach in clinical strains from PortugalPublication . Lodhia, Zohra; da Silva, Jorge Costa; Correia, Cristina; Cordeiro, Dora; João, Inês; Carreira, Teresa; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Gonçalves, Elsa; Matos, Susana; Dias, Ana Paula; Côrte-Real, Rita; Carpinteiro, Dina; Duarte, Sílvia; Vieira, Luís; Gomes, João Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: To survey genetic markers of potential antimicrobial resistance (AMR) to macrolides and fluoroquinolones among Chlamydia trachomatis–positive samples from the collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections (STIs), and explore a multiplex PCR approach coupled with NGS to provide complementary information regarding a strain’s genomic backbone. Methods: A total of 502 C. trachomatis–positive samples, mostly anorectal exudates, were subjected to PCR and sequencing of five targets, including loci potentially driving AMR (23S rRNA, gyrA and parC) and loci potentially informative about a strain’s genomic backbone with emphasis on differentiation of lymphogranuloma venereum (LGV)/non-LGV and L2/L2b (a 9 bp insertion in pmpH, a 74 bp insertion upstream from CT105 and the polymorphic CT442). Results: No samples evidenced 23S rRNA mutations recognizably linked to macrolide resistance. Three samples harboured the Ser83Ile mutation in GyrA putatively driving fluoroquinolone resistance: two recombinant L2-L2b/D-Da (0.4%) and one L2 (0.2%). The screened regions in pmpH, upstream CT105 and CT442 were fully concordant with LGV/non-LGV differentiation. As expected, the pmpH L2b-specific genetic trait locus was detected in all L2b and recombinant L2-L2b/D-Da ompA genotypes, but also in 96.0% of L2 specimens, which also likely possess an L2b genomic backbone. The insertion upstream from CT105 exhibited full LGV specificity, constituting a promising target for the development of rapid LGV diagnostic assays. Conclusions: This study contributes to enhancing the knowledge of C. trachomatis molecular epidemiology, suggesting that the known genetic determinants of AMR are not disseminated in clinical C. trachomatis strains, and presents an exploratory approach that can be suitable for LGV/non-LGV and L2/L2b genomic background differentiation.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990–2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Probe-based metagenomic pathogen detection: advancing laboratory capacity for complex diagnosisPublication . Ferreira, Rita; Coelho, Luís; Santos, João Dourado; Sobral, Daniel; Isidro, Joana; Mixão, Verónica; Pinto, Miguel; Nunes, Alexandra; Borrego, Maria José; Lopo, Sílvia; Oleastro, Mónica; Sousa, Rita; Palminha, Paula; Veríssimo, Cristina; Gargaté, Maria João; Guiomar, Raquel; Cordeiro, Rita; Macedo, Rita; Bajanca-Lavado, Paula; Paixão, Paulo; Duarte, Sílvia; Vieira, Luís; Borges, Vítor; Gomes, João PauloProbe-based pathogen enrichment, followed by NGS, is a promising tool for complex diagnosis, overcoming traditional challenges of shotgun metagenomics, namely small microbial/human genetic material ratio and demanding computational resources. Here, we assessed the combined detection performance of two Illumina probe-based panels, the Respiratory and the Urinary Pathogen ID panels (RPIP and UPIP), using 99 clinical samples of 15 different matrices (e.g., cerebrospinal fluid, plasma, serum, urine, swabs, biopsies, etc.) available from Portuguese National Reference Laboratories. This sample set involved 114 "PCR-positive hits" (Ct values range of 9.7-41.3; median of 28.4) for 52 non-redundant human pathogens. For a more detailed bioinformatics assessment, as a complement of the Illumina turnkey solution (Explify), we applied an extended version of our INSaFLU-TELEVIR(+) metagenomics pipeline. Whereas Explify analyses resulted in an initial detection frequency of 73.7% (84/114), the subsequent application of INSaFLU-TELEVIR(+), including taxonomic classification followed by confirmatory read mapping, enabled an overall detection proportion of 79.8% (91/114) of the PCR-positive hits. This translated into a detection rate increment from 54.3% (19/35) to 65.7% (23/35) for bacteria, and from 85.3% (58/68) to 89.7% (61/68) for viruses. The implemented workflow was also very satisfactory for samples with qPCR Ct values above 30, with an overall detection frequency of 71.8% (28/39) when compared with the 92.0% (46/50) observed for those with Ct ≤ 30. In summary, this study validated and established a pioneering approach at the Portuguese National Institute of Health to support clinicians in complex diagnosis, contributing to advance diagnostic capabilities toward a more informed clinical decision and potential improvement of infectious disease outcomes.
- Lymphogranuloma venereum (LGV) ompA-subvariants of the Portuguese collection of Chlamydia trachomatis, 2007–2023Publication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Gonçalves, Elsa; Matos, Susana; Dias, Ana Paula; Corte-Real, Rita; Vieira, Luís; Gomes, Joao Paulo; Borges, Vítor; Jose Borrego, MariaBackground: Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis ompA-genotypes L1–L3, with increasing numbers of detected cases across Europe. Here, we analysed diversity and temporal distribution of the LGV ompA-subvariants detected in Portugal between 2007 and 2023, in order to better understand the dissemination and diversification landscape of LGV strains. Methods: The collection of the Portuguese National Reference Laboratory includes 1188 LGV ompA-genotyped samples between 2007 and 2023. In-depth analysis of the diversity of LGV ompA-subvariants circulating in Portugal across the years was performed, identifying newly described subvariants and integrating this data in a comprehensive compilation with all representative LGV ompA-subvariants described globally. Results: L2 ompA-variant (L2/434/Bu) was consistently the most frequently detected in our collection, with annual proportions ranging from 34.0% to 82.9%, between 2016 and 2023. L2bV5 was the second most frequent followed by L2b, ranging from 5.0% to 27.9% and 2.6% to 23.7% across the years, respectively, from 2017 to 2023. We highlighted the emergence and considerable increase in circulation of L1-like ompA-subvariants in recent years, representing 13.7% of LGV sequences in 2023. We also identified 13 novel LGV ompA-subvariants that had not been described before, differing by up to three mutations from the respective genotype reference sequences. Conclusions: This study contributes to the worldwide picture of the LGV molecular epidemiology, highlighting the importance of long-term molecular surveillance to monitor the circulation and geographical spread of LGV and to timely identify and track new strains, such as the recently emerging L1-like ompA-subvariants.
- Hazard characterization of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human healthPublication . Behr, Anne-Cathrin; Fæste, Christiane Kruse; Azqueta, Amaya; Tavares, Ana M.; Spyropoulou, Anastasia; Solhaug, Anita; Olsen, Ann-Karin; Vettorazzi, Ariane; Mertens, Birgit; Zegura, Bojana; Streel, Camille; Ndiaye, Dieynaba; Spilioti, Eliana; Dubreil, Estelle; Buratti, Franca Maria; Crudo, Francesco; Eriksen, Gunnar Sundstøl; Snapkow, Igor; Teixeira, João Paulo; Rasinger, Josef D.; Sanders, Julie; Machera, Kyriaki; Ivanova, Lada; Gaté, Laurent; Le Hegarat, Ludovic; Novak, Matjaz; Smith, Nicola M.; Tait, Sabrina; Fraga, Sónia; Hager, Sonja; Marko, Doris; Braeuning, Albert; Louro, Henriqueta; Silva, Maria João; Dirven, Hubert; Dietrich, JessicaEnniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.
- Follow‐up of the re‐evaluation of silver (E 174) as a food additive (EFSA‐Q‐2023‐00169)Publication . EFSA Panel on Food Additives and Flavourings (FAF); Andreassen, Monica; Aquilina, Gabriele; Bastos, Maria Lourdes; Boon, Polly; Castle, Laurence; Fallico, Biagio; FitzGerald, Reginald; Frutos Fernandez, Maria Jose; Grasl‐Kraupp, Bettina; Gundert‐Remy, Ursula; Gürtler, Rainer; Kurek, Marcin Andrzej; Louro, Henriqueta; Morales, Patricia; Passamonti, Sabina; Oomen, Agnes; Corsini, Emanuela; Wright, Matthew; Furst, Peter; Gaffet, Eric; Loeschner, Katrin; Mast, Jan; Undas, Anna; Mech, Agnieszka; Rincon, Ana Maria; Ruggeri, Laura; Smeraldi, CamillaSilver (E 174) is a food colour that was re‐evaluated by the EFSA ANS Panel (2016). The ANS Panel concluded that the information available then, was insufficient to assess the safety of silver as food additive. The major issues included limited characterisation of silver E 174 (e.g. quantity of nanoparticles) and release of ionic silver. Following a European Commission call for further data to fill the data gap, the Panel on Food Additives and Flavourings (FAF) was requested to assess the safety of silver (E 174). One interested business operator (IBO) submitted limited data on particle size distribution and morphology, two genotoxicity studies and one subchronic study. The Panel concluded that the technical data submitted on physicochemical characterisation of all types of silver used as food additive E 174 were not adequate. As a result, the Panel was unable to propose changes to the EU specifications of E174 on particle size and morphology. As the additional information requested was not provided, the assessment was based solely on the submitted data. Nonetheless, given the data provided and silver insolubility in water, the Panel concluded that E174 requires risk assessment at the nanoscale following the EFSA Guidance on Risk assessment of nanomaterials to be applied in the food and feed chain, to complement the conventional risk assessment. The Panel considered that the genotoxicity data and sub‐chronic toxicity data were inadequate. Consequently, the Panel could not conclude on the safety of the food additive silver E 174.
- HBM4EU E-waste study – An untargeted metabolomics approach to characterize metabolic changes during E-waste recyclingPublication . Kozlowska, Lucyna; Viegas, Susana; Scheepers, Paul T.J.; Duca, Radu C.; Godderis, Lode; Martins, Carla; Krzesimir, Ciura; Jagiello, Karolina; Silva, Maria João; Mahiout, Selma; Mārtiņsone, Inese; Matisāne, Linda; van Nieuwenhuyse, An; Puzyn, Tomasz; Sijko-Szpanska, Monika; Verdonck, Jelle; Santonen, Tiina; HBM4EU E-waste Study TeamE-waste contains hazardous chemicals that may be a direct health risk for workers involved in recycling. We conducted an untargeted metabolomics analysis of urine samples collected from male e-waste processing workers to explore metabolic changes associated with chemical exposures in e-waste recycling in Belgium, Finland, Latvia, Luxembourg, the Netherlands, Poland, and Portugal. Questionnaire data and urine samples were obtained from workers involved in the processing of e-waste (sorting, dismantling, shredding, pre-processing, metal, and non-metal processing), as well as from controls with no known occupational exposure. Pre- and post-shift urine samples were collected and analysed using ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-MS). A total of 32 endogenous urinary metabolites were annotated with a Variable Importance in Projection (VIP) above 2, indicating that e-waste recycling is mainly associated with changes in steroid hormone and neurotransmitter metabolism, energy metabolism, bile acid biosynthesis, and inflammation. The highest VIP was observed for dopamine-o-quinone, which is linked to Parkinson’s disease. These and other changes in metabolism in workers employed in the processing of e-waste need further verification in targeted studies.
- HBM4EU e-waste study – Occupational exposure assessment to chromium, cadmium, mercury and lead during e-waste recyclingPublication . Leese, Elizabeth; Verdonck, Jelle; Porras, Simo P.; Airaksinen, Jaakko; Duca, Radu C.; Galea, Karen S.; Godderis, Lode; Janasik, Beata; Mahiout, Selma; Martins, Carla; Mārtiņsone, Inese; Ani, Maria Mirela; van Nieuwenhuyse, An; Scheepers, Paul T.J.; Silva, Maria João; Viegas, Susana; Santonen, Tiina; HBM4EU E-waste Study TeamProcessing of electronic waste (e-waste) causes the release of toxic substances which may lead to occupational exposure. The study aimed to gather information on potential occupational exposure during e-waste recycling, with a focus on biomonitoring of chromium, cadmium, mercury and lead. In eight European countries, 195 workers involved in the recycling of lead batteries, white goods, brown goods and metals and plastics were studied. These workers were compared to 73 controls with no direct involvement of e-waste recycling or other metal processing activities. The samples collected consisted of urine, blood and hair samples, along with personal air samples, hand wipes, settled dust samples and contextual information. Chromium, cadmium, mercury and lead was measured in urine, hair, air samples, hand wipes and settled dust; cadmium and lead in whole blood and chromium in red blood cells. Results showed that lead exposure is of concern, with workers from all five types of e-waste showing exposure, with elevated measurements in all matrices. Internal exposure markers were positively correlated with markers of external exposure, indicating workers are not adequately protected. Exposure to mercury and cadmium was also observed but to a much lesser extent with raised cadmium concentrations in urine and blood of all workers when compared to controls and raised mercury concentrations were found in brown goods workers when compared to controls. This study has highlighted exposure concerns when processing e-waste, particularly for lead across all waste categories studied, indicating a need for improved control measures in this sector.