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The aim of this study was to perform a retrospective evaluation of the success of patient referral for partner notification of chlamydial infections to the major Portuguese sexually transmitted infection (STI) clinic. A total of 8277 patients were screened for Chlamydia trachomatis during 2000-2007, and 695 (8.4%) tested positive. The sexual partners of 34% of these index cases attended the clinic as contacts following partner referral. In univariate analysis, heterosexual men referred partners more frequently than men who have sex with men (MSM) and HIV-negative index cases referred partners four-fold more frequently than HIV-positives; however, these associations were non-significant after multivariate analysis (adjusted odds ratio [OR] 1.4, 95% confidence interval [CI] 0.7-2.6; adjusted OR 4.2, 95% CI 0.9-18.7, respectively). Index patients who reported lower numbers of partners referred more frequently, and this association remained significant after multivariate analysis. One-third of the referred individuals tested C. trachomatis-positive; the risk for infection was three-fold higher for referred partners from symptomatic index cases (P < 0.001, adjusted OR, 95% CI 1.8-6.3). In conclusion, the results of the present study concerning sexual behaviour, HIV status and clinical signs highlight the need for further evaluations that may shape future partner notification strategies in order to reduce the chlamydial disease burden. [ABSTRACT FROM AUTHOR]

Objectives: 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., (© The Author(s) 2025. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: 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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Mpox is a zoonotic disease caused by the Monkeypox virus (MPXV), and since May 2022, tens of thousands of cases have been reported in non-endemic countries. We aimed to evaluate the suitability of different sample types for mpox diagnostic and assess the temporal dynamics of viral load. We evaluated 1914 samples from 953 laboratory-confirmed cases. The positivity rate was higher for lesion (91.3%) and rectal swabs (86.1%) when compared with oropharyngeal swabs (69.5%) and urines (41.2%), indicating higher viral loads for the former. Supporting this, lesion and rectal swabs showed lower median PCR C t values (C t  = 23 and C t  = 24), compared to oropharyngeal swabs and urines (C t  = 31). Stable MPXV loads were observed in swabs from lesions up to 30 days after symptoms onset, contrasting with a considerable decrease in viral load in rectal and oropharyngeal swabs. Overall, these results point to lesion swabs as the most suitable samples for detecting MPXV in the 2022-2023 multicountry outbreak and show comparable accuracy to rectal swabs up to 8 days after symptoms onset. These findings, together with the observation that about 5% of patients were diagnosed through oropharyngeal swabs while having negative lesions, suggest that multisite testing should be performed to increase diagnostic sensitivity., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

In 2023, a second wave of the global mpox epidemic, which is mainly affecting men who have sex with men (MSM), was observed in some countries. Herein, we benefited from a large viral sequence sampling (76/121; 63%) and vast epidemiological data to characterise the re-emergence and circulation of the Monkeypox virus (MPXV) in Portugal during 2023. We also modelled transmission and forecasted public health scenarios through a compartmental susceptible-exposed-infectious-recovered (SEIR) model. Our results suggest that the 2023 mpox wave in Portugal resulted from limited introduction(s) of MPXV belonging to C.1.1 sublineage, hypothetically from Asia, followed by sustained viral transmission and potential exportation to other countries. We estimated that the contribution of the MSM high sexual activity group to mpox transmission was 120 (95% CrI: 30-3553) times higher than that of the low sexual activity group. However, among the high sexual activity group, vaccinated individuals likely contributed approximately eight times less [0.123 (95% CrI: 0.068-0.208)] than the unvaccinated ones. Vaccination was also linked to potential reduced disease severity, with a Mpox Severity Score of 6.0 in the vaccinated group compared to 7.0 in unvaccinated individuals. Scenario analysis indicated that transmission is highly sensitive to sexual behaviour, projecting that a slight increase in the MSM sub-population with high sexual activity can trigger new mpox waves. This study strongly supports that continued vaccination, targeted awareness among risk groups and routine genomic epidemiology is needed to anticipate and respond to novel MPXV threats (e.g. global dissemination of clade I viruses).

Opportunist infections caused by nontuberculous mycobacteria (NTM) have emerged as a significant public health problem. Among these, species of the Mycobacterium avium complex (MAC) are the main responsible for the increase in the number of human disease cases. In order to address the current needs in the detection and surveillance of MAC disease cases, we evaluated different species classification methodologies (BLASTn-based marker-gene approach, Kraken v2, rMLST and MLST databases) and their congruence with a core-SNP phylogenetic approach, based on whole genome sequencing (WGS) data. For this purpose, we used a collection of 142 MAC isolates from Portuguese patients diagnosed between 2014 and 2022. The marker-gene approach (based on the rpoB, hsp65 and groEL genes), showed the best results, allowing the identification of the 142 MAC isolates to the species/subspecies level (M. avium subsp. hominissuis, M. intracellulare, M. intracellulare subsp. chimaera, M. intracellulare subsp. yongonense, M. marseillence and M. colombiense). Additionally, we performed drug susceptibility testing that confirmed clarithromycin efficacy as a first-line treatment for MAC disease, as 93 % of the Portuguese isolates were susceptible. Using a core-SNP approach we also performed an in-depth phylogenetic analysis within each identified species group, and despite the high genetic diversity within the MAC species, we were able to clearly distinguish all the species/subspecies and identify genetic clusters with epidemiological potential. We highlight not only the need for the standardization of an appropriate genotyping approach for species identification and management of MAC disease, but also a more robust large-scale WGS data analysis, in a One Health perspective, in order to identify potential routes of transmission., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: 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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: By March, 2023, 54 countries, areas, and territories (hereafter CAT) in the WHO European Region had reported more than 2·2 million COVID-19-related deaths to the WHO Regional Office for Europe. Here, we estimated how many lives were directly saved by vaccinating adults in the WHO European Region from December, 2020, to March, 2023., Methods: In this retrospective surveillance study, we estimated the number of lives directly saved by age group, vaccine dose, and circulating variant-of-concern (VOC) period, regionally and nationally, using weekly data on COVID-19 mortality and infection, COVID-19 vaccination uptake, and SARS-CoV-2 virus characterisations by lineage downloaded from The European Surveillance System on June 11, 2023, as well as vaccine effectiveness data from the literature. We included data for six age groups (25-49 years, 50-59 years, ≥60 years, 60-69 years, 70-79 years, and ≥80 years). To be included in the analysis, CAT needed to have reported both COVID-19 vaccination and mortality data for at least one of the four older age groups. Only CAT that reported weekly data for both COVID-19 vaccination and mortality by age group for 90% of study weeks or more in the full study period were included. We calculated the percentage reduction in the number of expected and reported deaths., Findings: Between December, 2020, and March, 2023, in 34 of 54 CAT included in the analysis, COVID-19 vaccines reduced deaths by 59% overall (CAT range 17-82%), representing approximately 1·6 million lives saved (range 1·5-1·7 million) in those aged 25 years or older: 96% of lives saved were aged 60 years or older and 52% were aged 80 years or older; first boosters saved 51% of lives, and 60% were saved during the Omicron period., Interpretation: Over nearly 2·5 years, most lives saved by COVID-19 vaccination were in older adults by first booster dose and during the Omicron period, reinforcing the importance of up-to-date vaccination among the most at-risk individuals. Further modelling work should evaluate indirect effects of vaccination and public health and social measures., Funding: US Centers for Disease Control and Prevention., Competing Interests: Declaration of interests GD reports that the predecessor of the organisation he works for, Public Health England, received an unrestricted grant from GSK to undertake a study on the outcome of patients who received parenteral zanamavir. The funder received data and interim reports from Public Health England but did not influence analysis and reporting of the study. GD had no involvement in the GSK-funded study on parenteral zanamavir. Furthermore, the currently submitted work was part of the public health response activities to COVID-19 and had no relationship to GSK or the study on parenteral zanamivir. EP has received a personal grant from the Finnish Medical Foundation for PhD studies. JM declares that Public Health Scotland received funding from the EU Horizon 2020 programme for work in describing the epidemiology of COVID-19 and its impact on primary and secondary care as a partner in the IMOVE-COVID-19 project. MK declares having received consulting fees from Gilead Sciences for advising on development of a clinical module for collection of patient-reported outcome data from people living with HIV, and having received an honoraria from GESIDA for speaking at an annual conference on patient-reported outcome measures for people with HIV. All other authors declare no competing interests., (Copyright © 2024 World Health Organization. Published by Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Invasive meningococcal disease (IMD) continues to be a public health problem due to its epidemic potential, affecting mostly children. We aimed to present a detailed description of the epidemiology of IMD in Portugal, including insights into the genetic diversity of Neisseria meningitidis strains. Epidemiological analysis included data from the Portuguese National Reference Laboratory of Neisseria meningitidis during 2003 to 2020. Since 2012, N. meningitidis isolates have also been assessed for their susceptibility to antibiotics and were characterized by whole genome sequencing. During 2003-2020, 1392 confirmed cases of IMD were analyzed. A decrease in the annual incidence rate was observed, ranging from 1.99 (2003) to 0.39 (2020), with an average case fatality rate of 7.1%. Serogroup B was the most frequent (69.7%), followed by serogroups C (9.7%), Y (5.7%), and W (2.6%). Genomic characterization of 329 isolates identified 20 clonal complexes (cc), with the most prevalent belonging to serogroup B cc41/44 (26.3%) and cc213 (16.3%). Isolates belonging to cc11 were predominantly from serogroups W (77.3%) and C (76.5%), whereas cc23 was dominant from serogroup Y (65.7%). Over the past 4 years (2017-2020), we observed an increasing trend of cases assigned to cc213, cc32, and cc11. Regarding antimicrobial susceptibility, all isolates were susceptible to ceftriaxone and 61.8% were penicillin-nonsusceptible, whereas 1.4% and 1.0% were resistant to ciprofloxacin and rifampicin. This is the first detailed study on the epidemiology and genomics of invasive N. meningitidis infections in Portugal, providing relevant data to public health policy makers for a more effective control of this disease., (© 2023. The Author(s).)

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined., (© 2024. The Author(s).)

Introduction and Objectives: Nontuberculous mycobacteria (NTM) are opportunistic human pathogens found in the environment. The transmission seems to be associated with inhalation of aerosol droplets, ingestion or trauma events. Recent studies indicate that NTM disease is increasing worldwide, however, the true clinical impact of NTM infections is difficult to determine due to challenges in discriminating between disease and colonization as they are ubiquitous in the environment. In addition, understanding the epidemiology of NTM is difficult and has not yet been established. In this work, we used a country NTM representative collection from the National Reference Laboratory for Tuberculosis (NRL-TB) of the National Institute of Health (INSA), to characterize the circulation trends of NTM species in Portugal and the most affected regions, contributing to a better understanding of the NTM epidemiology., Material and Methods: We conducted a nationwide retrospective study where all individuals with positive NTM cultures at the NRL-TB of the INSA from 2014 to December 2020 were included. Positive cultures were identified using GenoType Mycobacterium CM/AS® (Hain Lifescience) according to manufacturer's instructions, or hsp65 DNA sequencing as previously described. Social-demographic data from patients were also analyzed and patients classified into 3 groups according only to microbiological data, "definite NTM disease", "NTM colonization" and, "possible NTM disease"., Results: In the period 2014-2020, the NRL-TB performed 50397 cultures. Among these, 1118 cultures were NTM positive retrieved from 944. Most of our cases were in patients whose mean age was 64±15.9 years, and no significant differences between gender was observed, although more frequent in male patients. Overall, from the 944 cases, we were able to identified 93 "definite NTM disease" cases and 79 "possible NTM disease". Mycobacterium avium complex (MAC) (40,8%), Mycobacterium abscessus-chelonae complex (MABC) (9,6%) and Mycobacterium fortuitum (6,3%) were responsible for most of the infections. The geographical distribution of NTM cases varied significantly and was possible to observe that was independent of population density. The region were most cases occurred was Lisbon Metropolitan Area (31,9%), followed by North (25,3%) and Centre (24,4%), however North region has the highest number of "definite NTM disease" cases (n=33)., Conclusions: This is the first national wide epidemiological study on this subject, contributing to a better understanding of NTM dynamics in Portugal. MAC was the NTM species responsible for the majority of infections and, LMA the region with the highest number of cases. It was also possible to conclude that the number of NTM isolates is independent of the demography of the region., (Copyright © 2022 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved.)

Technology-facilitated sexual abuse refers to the use of information and communication technologies to facilitate both virtual and in-person sexual crimes. Research on this topic has focused on rates, risk factors, and consequences. This scoping review aims to understand whether and how forensic psychological procedures are adapted to assess adolescent victims and how Internet-based information might be useful as complementary data. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extensions for Scoping Reviews guidelines, searches were conducted in April 2023 in five electronic databases to include Portuguese, Spanish, or English quantitative, qualitative, or mixed-method peer-reviewed studies. Of the 2523 studies, six were considered eligible. Identified procedures include forensic interviews following the National Institute for Child Health and Human Development Protocol, and risk and trauma assessments. While discussing technology's role in abuse during interviews was informative, confronting adolescents with evidence of their abuse had adverse effects on their testimony and recovery. The assessment tools often had a narrow focus or overlooked the abuse unless explicitly disclosed, implied a referral, or when safeguarding concerns were raised. Clinical, forensic, and criminal implications are elaborated., (© 2024 The Authors. Behavioral Sciences & The Law published by John Wiley & Sons Ltd.)

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 10 4  cfu g -1 , Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 10 4  cfu g -1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production., (© 2024. The Author(s).)

Listeria monocytogenes , a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin ( fosX ), lincosamides ( lin ), fluoroquinolones ( norB ), and tetracycline ( tetM ). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.

We investigated molecular evolution and spatiotemporal dynamics of atypical Legionella pneumophila serogroup 1 sequence type 1905 and determined its long-term persistence and linkage to human disease in dispersed locations, far beyond the large 2014 outbreak epicenter in Portugal. Our finding highlights the need for public health interventions to prevent further disease spread.

Background: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification., Results: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis., Conclusions: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code)., (© 2024. The Author(s).)

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene ( dfrD ), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM , fosX , lin , norB , lmrB , sul , and mprF . The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

The aim of the current study was to evaluate the influence of increasing contents (5%-25%) of avocado pulp powder (APP) produced by foam-mat drying (FMD) as a substitute for hydrogenated vegetable fat in bread on its nutritional composition, physical properties, α-amylase, α-glucosidase, and lipase inhibition, total phenolic content, antioxidant activity, color, structure, and x-ray diffraction patterns. The increase in the APP content decreased the values of lipids, carbohydrates, energy, firmness, and specific volume of breads. The inhibition of lipase activity showed a pronounced increase, while the total phenolic content and antioxidant activity were significantly elevated. The color parameters a* and b* were higher in the breads with added APP. The crystalline structure transitioned from type A to type V with 15% APP incorporation. Taken together, these results suggest that APP has potential to act as a healthier substitute for saturated fats in breads, paving the way to develop creative and innovative solutions for the functionalization of bakery food products., (© 2024 Institute of Food Technologists.)

Child sexual abuse (CSA) is widely recognized as a global public health problem with negative consequences for victims, their families, and society. The child's testimony is essential to the case outcome, given the frequent absence of physical or biological evidence of the abusive acts. Thus, the child forensic interview plays a decisive role in criminal investigation. The present scoping review aims to identify and describe the judicial procedures for collecting CSA victims' testimony using an evidence-based approach and a structured methodology. The review followed Preferred Reporting Items of Systematic Reviews and Meta-Analysis-Scoping Review guidelines. Studies were identified through manual reference checking and in four electronic databases: PsycARTICLES, PubMed, SCOPUS, and Web of Science. In all, 146 studies were identified according to the defined inclusion criteria, that is, empirical studies identifying judicial procedures to collect CSA victims' testimony, published in English or Portuguese. In total, 30 different forensic interview procedures to collect the child victim's testimony were found. The National Institute for Child Health and Human Development investigative interview protocol was the most frequently mentioned. Despite the variety of protocols, it was possible to conclude that they have a similar general structure. This review also identified gaps in interviewing practices with CSA victims. The scoping review corroborates the importance of forensic interviews with CSA victims, stating its implications for criminal investigation, the legal system, and the child's recovery process., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Increasingly, scientific collections play a fundamental role in the conservation and analysis of biodiversity as a witness and repository of ecosystem organisms at risk of losing their species. The Invertebrate Collection of the Instituto Nacional de Pesquisas da Amaznia (INPA) safeguards biological information, mainly from the Amazon biome, one of the most diverse and threatened ecosystems on the planet. Mantodea specimens deposited dry in the INPA Collection were reviewed. A total of 2,721 specimens were found deposited until the year 2019. A total of 194 species and 81 genera were found with representatives of all the neotropical and several "Old-World" families. Families such as Mantidae, Photinaidae, and Thespidae represented the highest percentages of specimens collected within the sample, with 30%, 11%, and 9%, respectively. Four genera and fourteen species are recorded for the first time for Brazil, bringing the number of species to 247. This officially makes Brazil the most diverse country in terms of Mantodea fauna on the planet. The Collection stands out for being the most diverse reported in Latin America, even with several species yet to be described. Presently it has seven primary type specimens and 26 secondary type specimens. The excellent state of conservation of the specimens, the number of specimens, and the diversity represented have made the INPA Collection an international benchmark for Amazonian and Neotropical studies on Mantodea.

Introduction: Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited., Methods: To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity., Results: We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries., Discussion: Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis , being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Isidro, Escudero, Luque-Larena, Pinto, Borges, González-Martín-Niño, Duarte, Vieira, Mougeot, Vidal, Herrera-Rodríguez, Rodríguez-Pastor, Herrero-Cófreces, Jubete-Tazo, Gomes and Lopes de Carvalho.)

Mosquitoes are the vector of diseases that kill more than one million people per year worldwide. Surveillance systems are essential for understanding their complex ecology and behaviour. This is fundamental for predicting disease risk caused by mosquitoes and formulating effective control strategies against mosquito-borne diseases such as malaria, dengue, and Zika. Mosquito populations vary heterogeneously in urban and rural landscapes, fluctuating with seasonal and climatic trends and human activity. Several approaches provide environmental data for mosquito mapping and risk prediction. However, they rely traditionally upon labour-intensive techniques such as manual traps. This paper presents the optimal audio features for mosquito identification using ecoacoustics signals to automatically identify different mosquito species from their wingbeat sounds based on popular audio features. The audio selection method uses Density-Based Spatial Clustering of Applications with Noise (DBSCAN) and Silhouette coefficient to evaluate the clusters in the data through the optimal-combined audio features. To classify the mosquito species and distinguish them from environmental-urban noise, the method comprises the Gaussian Mixture Model (GMM) and Gibbs approach for Aedes aegypti, and Culex quinquefasciatus, using the acoustic recordings of their wingbeat signals. Finally, comparing GMM and Gibbs, the two have very similar accuracy, but the classification time is much faster for Gibbs sampling, making it a good candidate for a lightweight solution. These are essential when deploying the described models to monitor mosquito vectors in the wild with Internet of Things (IoT) technologies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dinarte Goncalves Vasconcelos reports financial support and article publishing charges were provided by Universidade de Lisboa - Instituto Superior Tecnico. Dinarte Goncalves Vasconcelos reports financial support, article publishing charges, and equipment, drugs, or supplies were provided by Interactive Technologies Institute - Larsys. We would like to acknowledge the Natural History Museum of the Municipality of Funchal and IASaude, the regional health authority of Madeira Islands, for facilitating their infrastructures and materials. This research was funded by FCT - Fundação para a Ciência e Tecnologia under the scope of the LARSyS project UID/50009/2020., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population., (© 2023. The Author(s).)

Guava, pitanga and acerola are known for their vitamin content and high levels of bioactive compounds. Thus, the preparation of combinations of these fruits comprises a blend with high nutraceutical potential, yielding a strong and attractive pigmentation material. In this study, the influence of different proportions of maltodextrin on the lyophilization of a blend of guava, acerola and pitanga was evaluated considering not only the physicochemical, physical and colorimetric parameters but also the bioactive compounds in the obtained powders. The blend was formulated from the mixture and homogenization of the three pulps in a ratio of 1:1:1 (m/m), then maltodextrin was added to the blend, resulting in four formulations: blend without adjuvant (BL0), and the others containing 10% (BL10), 20% (BL20) and 30% (BL30) maltodextrin. The formulations were lyophilized and disintegrated to obtain powders. The powders were characterized in terms of water content, water activity, pH, total titratable acidity, ash, total and reducing sugars, ascorbic acid, total phenolic content, flavonoids, anthocyanins, carotenoids, lycopene, color parameters, Hausner factor, Carr index, angle of repose, solubility, wettability and porosity. All evaluated powders showed high levels of bioactive compounds and the increase in maltodextrin concentration promoted positive effects, such as reductions in water content, water activity and porosity and improved flow, cohesiveness and solubility characteristics.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects β-strands 5 and 6 and forms a constriction zone involved in Ca 2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within por A. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp., Competing Interests: The authors declare no conflict of interest.

Candida auris is an opportunistic human pathogen that has rapidly spread to multiple countries and continents and has been associated with a high number of nosocomial outbreaks. Herein, we report the first case of C. auris in Portugal, which was associated with a patient transferred from Angola to an ICU in Portugal for liver transplantation after a SARS-CoV-2 infection. C. auris was isolated during the course of bronchoalveolar lavage, and it was subjected to antifungal susceptibility testing and whole-genome sequence analysis. This isolate presents low susceptibility to azoles and belongs to the genetic clade III with a phylogenetic placement close to African isolates. Although clade III has already been reported in Europe, taking into account the patient's clinical history, we cannot discard the possibility that the patient's colonization/infection occurred in Angola, prior to admission in the Portuguese hospital. Considering that C. auris is a fungal pathogen referenced by WHO as a critical priority, this case reinforces the need for continuous surveillance in a hospital setting.

Mycobacteroides abscessus complex (MAB), a fast-growing nontuberculous mycobacterium, is emerging as a significant infectious disease threat, due to both intrinsic and acquired resistance mechanisms to antibiotics and disinfectants and the need for extensive and multidrug regimens for treatment. Despite the prolonged regimens, outcomes are poor and persistence cases have been reported. Here, we describe clinical, microbiologic and genomic features of a M. abscessus subsp. bolletii (M. bolletii) strain consecutively isolated from a patient within an eight-year infection period. From April 2014 to September 2021, the National Reference Laboratory for Mycobacteria received eight strains isolated from a male patient. Species identification, molecular resistance profile and phenotypic drug susceptibility were determined. Five of these isolates were recovered for further in-depth genomic analysis. Genomic analysis confirmed the multidrug resistant pattern of the strain and also other genetic changes associated with adaptation to environment and defence mechanisms. We highlight the identification of new mutations in locus MAB&#95;1881c and in locus MAB&#95;4099c (mps1 gene), already described as associated with macrolides resistance and morphotype switching, respectively. Additionally, we also observed the emergence and fixation of a mutation in locus MAB&#95;0364c that appeared at a frequency of 36% for the 2014 isolate, 57% for the 2015 isolate and 100% for the 2017 and 2021 isolates, clearly illustrating a fixation process underlying a microevolution of the MAB strain within the patient. Altogether these results suggest that the observed genetic alterations are a reflection of the bacterial population's continuous adaptation and survival to the host environment during infection, contributing to persistence and treatment failure., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Foam mat drying is a widely used technique for liquid products because it has a number of advantages; however, for an efficient process, the choice of additives and temperatures is extremely important. The objective of this study was to evaluate the effect of additives and drying temperatures on the powders obtained from the blend of tropical red fruits, such as acerola, guava, and pitanga. The foam formulations were prepared by mixing the pulps of the three fruits in equal proportions (1:1:1), all added with 6% albumin and 1% stabilizing agent: E1, gum Arabic; E2, guar gum; E3, gelatin. The combinations were subjected to beating, and subsequently, they were dried in an oven with forced air circulation at four temperatures (50 to 80 °C), with a mat thickness of 0.5 cm. The obtained powders showed low levels of water and water activity and high levels of bioactive compounds, colors with a predominance of yellow, intermediate cohesiveness, poor fluidity, and solubility above 50%. The best temperature for obtaining the powders was 60 °C. The formulation that produced the best results for the production of the tropical red fruit blend powder was the combination of albumin and gelatin.

The combination of fruit pulps from different species, in addition to multiplying the offer of flavors, aromas and textures, favors the nutritional spectrum and the diversity of bioactive principles. The objective was to evaluate and compare the physicochemical characteristics, bioactive compounds, profile of phenolic compounds and in vitro antioxidant activity of pulps of three species of tropical red fruits (acerola, guava and pitanga) and of the blend produced from the combination. The pulps showed significant values of bioactive compounds, with emphasis on acerola, which had the highest levels in all parameters, except for lycopene, with the highest content in pitanga pulp. Nineteen phenolic compounds were identified, being phenolic acids, flavanols, anthocyanin and stilbene; of these, eighteen were quantified in acerola, nine in guava, twelve in pitanga and fourteen in the blend. The blend combined positive characteristics conferred by the individual pulps, with low pH favorable for conservation, high levels of total soluble solids and sugars, greater diversity of phenolic compounds and antioxidant activity close to that of acerola pulp. Pearson's correlation between antioxidant activity and ascorbic acid content, total phenolic compounds, flavonoids, anthocyanins and carotenoids for the samples were positive, indicating their use as a source of bioactive compounds.

Background: Genomics-informed pathogen surveillance strengthens public health decision-making, playing an important role in infectious diseases' prevention and control. A pivotal outcome of genomics surveillance is the identification of pathogen genetic clusters and their characterization in terms of geotemporal spread or linkage to clinical and demographic data. This task often consists of the visual exploration of (large) phylogenetic trees and associated metadata, being time-consuming and difficult to reproduce., Results: We developed ReporTree, a flexible bioinformatics pipeline that allows diving into the complexity of pathogen diversity to rapidly identify genetic clusters at any (or all) distance threshold(s) or cluster stability regions and to generate surveillance-oriented reports based on the available metadata, such as timespan, geography, or vaccination/clinical status. ReporTree is able to maintain cluster nomenclature in subsequent analyses and to generate a nomenclature code combining cluster information at different hierarchical levels, thus facilitating the active surveillance of clusters of interest. By handling several input formats and clustering methods, ReporTree is applicable to multiple pathogens, constituting a flexible resource that can be smoothly deployed in routine surveillance bioinformatics workflows with negligible computational and time costs. This is demonstrated through a comprehensive benchmarking of (i) the cg/wgMLST workflow with large datasets of four foodborne bacterial pathogens and (ii) the alignment-based SNP workflow with a large dataset of Mycobacterium tuberculosis. To further validate this tool, we reproduced a previous large-scale study on Neisseria gonorrhoeae, demonstrating how ReporTree is able to rapidly identify the main species genogroups and characterize them with key surveillance metadata, such as antibiotic resistance data. By providing examples for SARS-CoV-2 and the foodborne bacterial pathogen Listeria monocytogenes, we show how this tool is currently a useful asset in genomics-informed routine surveillance and outbreak detection of a wide variety of species., Conclusions: In summary, ReporTree is a pan-pathogen tool for automated and reproducible identification and characterization of genetic clusters that contributes to a sustainable and efficient public health genomics-informed pathogen surveillance. ReporTree is implemented in python 3.8 and is freely available at https://github.com/insapathogenomics/ReporTree ., (© 2023. The Author(s).)

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: NDG is a consultant for Tempus Labs and the National Basketball Association for work related to COVID-19. All other authors have declared that no competing interests exist., (Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pepper ( Capsicum spp.) is among the oldest and most cultivated crops on the planet. Its fruits are widely used as natural condiments in the food industry for their color, flavor, and pungency properties. Peppers have abundant production; on the other hand, their fruits are perishable, deteriorating within a few days after harvesting. Therefore, they need adequate conservation methods to increase their useful life. This study aimed to mathematically model the drying kinetics of smelling peppers ( Capsicum chinense ) and pout peppers ( Capsicum chinense Jacq.) to obtain the thermodynamic properties involved in the process and to determine the influence of drying on the proximal composition of these peppers. Whole peppers, containing the seeds, were dried in an oven with forced air circulation, at temperatures of 50, 60, 70, and 80 °C, with an air speed of 1.0 m/s. Ten models were adjusted to the experimental data, but the Midilli model was the one that provided the best values of coefficient of determination and lowest values of the mean squared deviation and chi-square value in most of the temperatures under study. The effective diffusivities were well represented by an Arrhenius equation, appearing in the order of 10 -10 m 2 ·s -1 for both materials under study, since the activation energy of the smelling pepper was 31.01 kJ·mol -1 and was 30.11 kJ·mol -1 in the pout pepper, respectively. Thermodynamic properties in both processes of drying the peppers pointed to a non-spontaneous process, with positive values of enthalpy and Gibbs free energy and negative values of entropy. Regarding the influence of drying on the proximal composition, it was observed that, with the increase in temperature, there was a decrease in the water content and the concentration of macronutrients (lipids, proteins, and carbohydrates), providing an increase in the energy value. The powders obtained in the study were presented as an alternative for the technological and industrial use of peppers, favoring obtaining a new condiment, rich in bioactives, providing the market with a new option of powdered product that can be consumed directly and even adopted by the industry as a raw material in the preparation of mixed seasonings and in the formulation of various food products.

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.

The aim of this study was to determine the thermophysical properties and process parameters of cylindrical carrot pieces during their chilling. For this, the temperature of the central point of the product, initially at 19.9 °C, was recorded during chilling under natural convection, with the refrigerator air temperature maintained at 3.5 °C. A solver was created for the two-dimensional analytical solution of the heat conduction equation in cylindrical coordinates. This solver and the experimental data set were coupled to the LS Optimizer (V. 7.2) optimization software to simultaneously determine not only the values of thermal diffusivity (α) and heat transfer coefficient (hH), but also the uncertainties of these values. These values were consistent with those reported in the literature for carrots; in this study, the precision of these values and the confidence level of the results (95.4%) were also presented. Furthermore, the Biot numbers were greater than 0.1 and less than 40, indicating that the mathematical model presented in this study can be used to simultaneously estimate α and hH. A simulation of the chilling kinetics using the values obtained for α and hH showed good agreement with the experimental results, with a root mean square error RMSE = 9.651 × 10 -3 and a chi-square χ 2 = 4.378 × 10 -3 .

Brazil stands out as the largest producer of sour passion fruit; however, the water available for irrigation is mostly saline, which can limit its cultivation. This study was carried out with the objective of evaluating the effects of salicylic acid in the induction of tolerance in sour passion fruit to salt stress. The assay was conducted in a protected environment, using a completely randomized design in a split-plot scheme, with the levels of electrical conductivity of the irrigation water (0.8, 1.6, 2.4, 3.2, and 4.0 dS m -1 ) considering the plots and concentrations of salicylic acid (0, 1.2, 2.4, and 3.6 mM) the subplots, with three replications. The physiological indices, production components, and postharvest quality of sour passion fruit were negatively affected by the increase in the electrical conductivity of irrigation water, and the effects of salt stress were intensified in the second cycle. In the first cycle, the foliar application of salicylic acid at concentrations between 1.0 and 1.4 mM partially reduced the harmful effects of salt stress on the relative water content of leaves, electrolyte leakage, gas exchange, and synthesis of photosynthetic pigments, in addition to promoting an increase in the yield and quality parameters of sour passion fruit.

Among the vegetables that stand out for their high concentration of anthocyanins, red cabbage appears as one of the most-used sources of these pigments in food production and it is considered a suitable raw material for the extraction of natural dye. Therefore, the objective was to carry out the production of natural extracts from red cabbage, under different conditions, varying the solvent, type of pre-treatment, pH range, and processing temperature during the concentration of the extracts. The anthocyanins were extracted from red cabbage using the following solvents: distilled water, 25% ethyl alcohol, and 70% ethyl alcohol. The raw material was divided into two groups, the first was subjected to a drying pre-treatment at 70 °C for 1 h and for the second group, the extraction was performed with the raw material in natura. Two pH ranges of 4.0 and 6.0 and extraction temperatures of 25 °C and 75 °C were used in the extracts, resulting in 24 formulations. The extracts obtained were analyzed for colorimetric parameters and anthocyanins. The results of anthocyanins show that the methodology that uses 25% alcohol, pH 4.0, and processing temperature of 25 °C produces a reddish extract and better results in the extraction, presenting average values of 191.37 mg/100 g of anthocyanins, being 74% higher compared to the highest values obtained in the other extracts where the same raw material was used and the solvents differed.