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We report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype. [ABSTRACT FROM AUTHOR]

This study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1–4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15–44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013–2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Information on vaccine effectiveness in a context of novel variants of concern (VOC) emergence is of key importance to inform public health policies. This study aimed to estimate a measure of comparative vaccine effectiveness between Omicron (BA.1) and Delta (B.1.617.2 and sub‐lineages) VOC according to vaccination exposure (primary or booster). Methods: We developed a case–case study using data on RT‐PCR SARS‐CoV‐2‐positive cases notified in Portugal during Weeks 49–51, 2021. To obtain measure of comparative vaccine effectiveness, we compared the odds of vaccination in Omicron cases versus Delta using logistic regression adjusted for age group, sex, region, week of diagnosis, and laboratory of origin. Results: Higher odds of vaccination were observed in cases infected by Omicron VOC compared with Delta VOC cases for both complete primary vaccination (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.8 to 2.4) and booster dose (OR = 5.2; 95% CI: 3.1 to 8.8), equivalent to reduction of vaccine effectiveness from 44.7% and 92.8%, observed against infection with Delta, to −6.0% (95% CI: 29.2% to 12.7%) and 62.7% (95% CI: 35.7% to 77.9%), observed against infection with Omicron, for complete primary vaccination and booster dose, respectively. Conclusion: Consistent reduction in vaccine‐induced protection against infection with Omicron was observed. Complete primary vaccination may not be protective against SARS‐CoV‐2 infection in regions where Omicron variant is dominant. [ABSTRACT FROM AUTHOR]

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April–June 2022, we implemented a case–case and cohort study and classified lineages using wholegenome sequencing or spike gene target failure. For the case–case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93–1.23]) or booster (aOR 0.96 [95% CI 0.84–1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%–90%] vs. VE 93% [95% CI 86%–97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease. [ABSTRACT FROM AUTHOR]

Haemophilus influenzae is an opportunistic pathogen adapted to the human respiratory tract. Non-typeable H. influenzae are highly heterogeneous, but few studies have analysed the genomic variability of capsulated strains. This study aims to examine the genetic diversity of 37 serotype f isolates from the Netherlands, Portugal, and Spain, and to compare all capsulated genomes available on public databases. Serotype f isolates belonged to CC124 and shared few single nucleotide polymorphisms (SNPs) (n = 10,999), but a high core genome (> 80%). Three main clades were identified by the presence of 75, 60 and 41 exclusive genes for each clade, respectively. Multi-locus sequence type analysis of all capsulated genomes revealed a reduced number of clonal complexes associated with each serotype. Pangenome analysis showed a large pool of genes (n = 6360), many of which were accessory genome (n = 5323). Phylogenetic analysis revealed that serotypes a, b, and f had greater diversity. The total number of SNPs in serotype f was significantly lower than in serotypes a, b, and e (p < 0.0001), indicating low variability within the serotype f clonal complexes. Capsulated H. influenzae are genetically homogeneous, with few lineages in each serotype. Serotype f has high genetic stability regardless of time and country of isolation. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae antimicrobial resistance (AMR) and gonorrhea disease burden remain major public health concerns worldwide. To contribute to the supranational demands to monitor and manage the spread of antimicrobial-resistant N. gonorrhoeae, the Portuguese NIH promoted the creation of the National Laboratory Network for Neisseria gonorrhoeae Collection (PTGonoNet). The present study reports the N. gonorrhoeae major AMR trends observed from 2003 up to 2018. All isolates described in the present study constitute the opportunistic ongoing N. gonorrhoeae isolate collection supported by the National Reference Laboratory for Sexually Transmitted Infections of the Portuguese NIH, enrolling strains isolated in 35 different public and private laboratories. Minimum inhibitory concentrations were determined using E-tests for azithromycin, benzylpenicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, spectinomycin and tetracycline. Molecular typing was determined using NG-MAST. AMR data of 2596 country-spread isolates show that 87.67% of all N. gonorrhoeae isolates presented decreased susceptibility to at least one antimicrobial. A continuous decreased susceptibility and resistance to penicillin, tetracycline and ciprofloxacin can be observed along the years. However, no decreased susceptibility to cephalosporins was observed until 2018, while for azithromycin, this was always low. The most common observed NG-MAST genogroups were G1407, G7445, G225, G2, and G1034. This study evidences the advantages of a nationwide collection of isolates and of centralized AMR testing to respond to supranational (EURO-GASP) requirements while providing unprecedented data on AMR in the context of 15 years of surveillance. [ABSTRACT FROM AUTHOR]

Gastrointestinal infections caused by nontyphoidal Salmonella (NTS) remain one of the main causes of foodborne illness worldwide. Within the multiple existing Salmonella enterica serovars, the serovar Rissen is rarely reported, particularly as a cause of human salmonellosis. Between 2015 and 2017, the Portuguese National Reference Laboratory of Gastrointestinal Infections observed an increase in the number of clinical cases caused by multidrug-resistant (MDR) S. enterica serovar Rissen, particularly from the Azores archipelago. In the present study, we analyzed by whole genome sequencing (WGS) all clinical, animal, food, and environmental isolates received up to 2017 in the Portuguese Reference Laboratories. As such, through a wgMLST-based gene-by-gene analysis, we aimed to identify potential epidemiological clusters linking clinical and samples from multiple sources, while gaining insight into the genetic diversity of S. enterica serovar Rissen. We also investigated the genetic basis driving the observed multidrug resistance. By integrating 60 novel genomes with all publicly available serovar Rissen genomes, we observed a low degree of genetic diversity within this serovar. Nevertheless, the majority of Portuguese isolates showed high degree of genetic relatedness and a potential link to pork production. An in-depth analysis of these isolates revealed the existence of two major clusters from the Azores archipelago composed of MDR isolates, most of which were resistant to at least five antimicrobials. Considering the well-known spread of MDR between gastrointestinal bacteria, the identification of MDR circulating clones should constitute an alert to public health authorities. Finally, this study constitutes the starting point for the implementation of the "One Health" approach for Salmonella surveillance in Portugal. [ABSTRACT FROM AUTHOR]

Aims: To evaluate prescription of prophylactic treatment before and after consultation in a neurology headache clinic and to determine predictors for prophylactic treatment and clinical improvement. Methods: Clinical records of consecutive patients assessed in a neurologic headache clinic in Portugal and diagnosed with acute or chronic migraine and/or tension-type headache were assessed. Prescription of prophylaxis before and after the first visit to the clinic were compared. Logistic regression was used to evaluate predictors of the need for therapeutic intervention and clinical improvement. Results: Among 409 patients (86.8% women; mean age 41.6 years), 315 (77%) had indication for prophylaxis, and 70 (22%) of these patients were already on prophylactic treatment. Among the 265 patients with information for follow-up, prophylactic treatment was added in 178 (67.2%), and there was a significant change in the number of treated patients between the first and second visits. Ongoing treatment was switched or the dose increased in 21 patients. Multivariate logistic regression revealed that women (odds ratio [OR] = 2.09, 95% confidence interval [CI] 1.1 to 3.97] and patients with medication overuse headache (MOH) (OR = 6.97, 95% CI 1.60 to 30.39) were more likely to need therapeutic intervention, whereas patients referred from the emergency room were less likely to need it (OR = 0.44, 95% CI 0.22 to 0.89). Of the 265 patients, 185 (69.8%) had improved at a follow-up. Having prophylactic treatment at the time of the second visit was associated with improvement (OR = 2.39, 95% CI 1.23 to 4.63; P = .01). Conclusion: Women and medication overuse headache patients were more likely to need therapeutic intervention. However, only a minority of patients with treatment indication were treated before their first visit to the headache clinic. Prophylaxis prescription was associated with clinical improvement at follow-up. [ABSTRACT FROM AUTHOR]

We report a multidrug-resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Because of its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain. [ABSTRACT FROM AUTHOR]

Mapping burned areas using satellite imagery has become a subject of extensive research over the past decades. The availability of high-resolution satellite data allows burned area maps to be produced with great detail. However, their increasing spatial resolution is usually not matched by a similar increase in the temporal domain. Moreover, high-resolution data can be a computational challenge. Existing methods usually require downloading and processing massive volumes of data in order to produce the resulting maps. In this work we propose a method to make this procedure fast and yet accurate by leveraging the use of a coarse resolution burned area product, the computation capabilities of Google Earth Engine to pre-process and download Sentinel-2 10-m resolution data, and a deep learning model trained to map the multispectral satellite data into the burned area maps. For a 1500 ha fire our method can generate a 10-m resolution map in about 5 min, using a computer with an 8-core processor and 8 GB of RAM. An analysis of six important case studies located in Portugal, southern France and Greece shows the detailed computation time for each process and how the resulting maps compare to the input satellite data as well as to independent reference maps produced by Copernicus Emergency Management System. We also analyze the feature importance of each input band to the final burned area map, giving further insight about the differences among these events. [ABSTRACT FROM AUTHOR]

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).)

The increase in frequency and intensity of extreme climate events over the last few decades has been leading to profound changes in estuarine and marine ecosystems worldwide, with strong implications for the species inhabiting these ecosystems as well as for the services provided by them. In this study, we analysed the effects of climate variability on the temporal and spatial variations in population dynamics of the green crab Carcinus maenas in the Mondego estuary (Portugal), between 2003 and 2018. In this 15-year period, a greater recruitment of C. maenas was observed during drought periods, periods which was matched by an increase in secondary production. Ontogenic stage segregation was also observed, with juveniles being found mainly in the further upriver areas of the estuary. The estuarine population was mainly composed of the green morphotype, with the orange and red morphotypes present in more downstream areas of the estuary. Redundancy analysis (RDA) showed high spatial and temporal variability of C. maenas in the estuary which was related with environmental changes over the 15-year period. A correlation between C. maenas biological features and several local-scale (salinity and river runoff) and large-scale (North Atlantic Oscillation index and Eastern Atlantic pattern) environmental variables was identified through cumulative sums analysis (CUSUM), indicating a strong environmental control on C. maenas population dynamics. This paper shows the importance of relatively long-term datasets to unravel the effects of extreme weather events due to climate change on key epibenthic estuarine species, and also how they might cope with a changing marine environment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Pregnancy among patients with congenital myasthenic syndrome (CMS) is a rare occurrence. Since most of the patients with CMS reach adulthood, questions regarding clinical outcome with pregnancy arise., Case Report: We describe a 38-year-old Portuguese female who presented in the second trimester of pregnancy with proximal fluctuating limb-girdle weakness, hyperlordosis, waddling gait, dysphagia, dysphonia and ptosis, with no ophthalmoparesis. Initial diagnosis of seronegative myasthenia, supported by neurophysiology findings, led to unsuccessful treatment with intravenous immunoglobulin, pyridostigmine, prednisolone and plasmapheresis, and the patient slowly progressed to a severe tetraparesis with facial and bulbar involvement. Genetic testing for CMS identified a novel compound heterozygous mutation (c.1124&#95;1127dupTGCC and c.935&#95;936del) in the DOK7 gene. Subsequent treatment with salbutamol resulted in substantial clinical benefit., Conclusions: This case underlines the importance of considering the diagnosis of CMS in patients with fluctuating weakness during pregnancy. Patients of child-bearing potential diagnosed with CMS, particularly due to DOK7 mutations, should be counseled in advance and closely followed during pregnancy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Whole-genome sequencing (WGS) offers unprecedented resolution for tracking Mycobacterium tuberculosis transmission and antibiotic-resistance spread. Still, the establishment of standardized WGS-based pipelines and the definition of epidemiological clusters based on genetic relatedness are under discussion. We aimed to implement a dynamic gene-by-gene approach, fully relying on freely available software, for prospective WGS-based tuberculosis surveillance, demonstrating its application for detecting transmission chains by retrospectively analysing all M/XDR strains isolated in 2013-2017 in Portugal. We observed a good correlation between genetic relatedness and epidemiological links, with strongly epilinked clusters displaying mean pairwise allele differences (AD) always below 0.3% (ratio of mean AD over the total number of shared loci between same-cluster strains). This data parallels the genetic distances acquired by the core-SNV analysis, while providing higher resolution and epidemiological concordance than MIRU-VNTR genotyping. The dynamic analysis of strain sub-sets (i.e., increasing the number of shared loci within each sub-set) also strengthens the confidence in detecting epilinked clusters. This gene-by-gene strategy also offers several practical benefits (e.g., reliance on freely-available software, scalability and low computational requirements) that further consolidated its suitability for a timely and robust prospective WGS-based laboratory surveillance of M/XDR-TB cases., (Copyright © 2019. Published by Elsevier Ltd.)

Estuaries may be reservoirs of a wide variety of pollutants, including mutagenic and carcinogenic substances that may impact on the ecosystem and human health. A previous study showed that exposure of human hepatoma (HepG2) cells to extracts from sediment samples collected in two areas (urban/industrial and riverine/agricultural) of an impacted estuary (Sado, Portugal), produced differential cytotoxic and genotoxic effects. Those effects were found to be consistent with levels and nature of sediment contamination. The present study aimed at evaluating whether the mixtures of contaminants contained in those extracts were able to modulate DNA repair capacity of HepG2 cells. The residual level of DNA damage was measured by the comet assay in cells exposed for 24 or 48 h to different extracts, after a short preexposure to a challenging concentration range of ethyl methanesulfonate (EMS), as a model alkylating agent. The results suggested that the mixture of contaminants present in the tested samples, besides a potential direct effect on the DNA molecule, may also interfere with DNA repair mechanisms in HepG2 cells, thus impairing their ability to deal with genotoxic stress and, possibly, facilitating accumulation of mutations. Humans are environmentally/occupationally exposed to mixtures rather than to single chemicals. Thus, the observation that estuarine contaminants induce direct and indirect DNA strand breakage in human cells, the latter through the impairment of DNA repair, raises additional concerns regarding potential hazards from exposure and the need to further explore these endpoints in the context of environmental risk assessment.

Introduction: The estimated prevalence of arterial hypertension (HT) in Portugal in 2003 was 43%, with only 28.8% of patients undergoing antihypertensive treatment having their blood pressure controlled. In the light of this fact, if HT is detected early and adequately controlled, there is great potential for reducing cardiovascular morbidity and mortality., Objectives: This study aimed to analyze patterns of antihypertensive drug utilization in Portugal at the regional and national level, to compare them with other countries, and to evaluate trends in certain indicators regarding use of these drugs., Methods: Data on antihypertensive drug utilization refer to drugs prescribed and dispensed in the ambulatory population covered by the Portuguese national health service, from January 1, 1999 to June 30, 2004. Data were expressed in defined daily doses (DDD) per 1000 inhabitants per day (DID)., Results: Use of antihypertensives increased from 183 DID in the first half of 1999 to 256 DID in the first half of 2004, an increase of 40%. Angiotensin-converting enzyme inhibitors were the most frequently prescribed drug class, being used by 31% of the study population, followed by diuretics (16%) and calcium channel blockers (14%). Angiotensin receptor blockers (ARBs) accounted for most of the growth in antihypertensive use, although they represent the most expensive therapeutic option. This pattern of use had a strong impact on antihypertensive drug expenditure, which increased 60% over the study period. ARBs, plain or in combination formulations, accounted for 55% and 71% of the increase in utilization and expenditure respectively., Discussion: From the standpoint of health gains, the significant rise in antihypertensive drug utilization represents a potentially favorable development in terms of increased numbers of patients undergoing drug treatment. Compared to other European countries, the data showed greater use of drugs that act on the renin-angiotensin system in Portugal and less use of diuretics and beta-blockers. Most national and international guidelines recommend diuretics as first-line agents in HT treatment. Considering that these drugs have the lowest daily treatment cost, a high, and increasing, rate of utilization would be expected, but this is not the case in Portugal., Conclusions: This study reveals not only significant differences in antihypertensive drug utilization at the regional level in Portugal but also a lack of adherence to national and international guidelines, and therefore more attention should be paid to their implementation in order to improve health gains.