Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

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.

Early Molecular Immune Responses of Turbot (Scophthalmus maximus L.) Following Infection with Aeromonas salmonicida subsp. salmonicida.

Turbot aquaculture production is an important economic activity in several countries around the world; nonetheless, the incidence of diseases, such furunculosis, caused by the etiological agent A. salmonicida subsp. salmonicida, is responsible for important losses to this industry worldwide. Given this perspective, this study aimed to evaluate early immune responses in turbot (S. maximus L.) following infection with A. salmonicida subsp. salmonicida. For this, 72 fish were individually weighed and randomly distributed into 6 tanks in a circulating seawater system. For the bacterial challenge, half of the individuals (3 tanks with 36 individuals) were infected using a peritoneal injection with the bacterial suspension, while the other half of individuals were injected with PBS and kept as a control group. Several factors linked to the innate immune response were studied, including not only haematological (white blood cells, red blood cells, haematocrit, haemoglobin, mean corpuscular volume, mean cell haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, monocytes, lymphocytes, thrombocytes) and oxidative stress parameters, but also the analyses of the expression of 13 key immune-related genes (tnf-α, il-1ß, il-8, pparα-1, acox1, tgf-ß1, nf-kB p65, srebp-1, il-10, c3, cpt1a, pcna, il-22). No significant differences were recorded in blood or innate humoral parameters (lysozyme, anti-protease, peroxidase) at the selected sampling points. There was neither any evidence of significant changes in the activity levels of the oxidative stress indicators (catalase, glutathione S-transferase, lipid peroxidation, superoxide dismutase). In contrast, pro-inflammatory (tnf-α, il-1ß), anti-inflammatory (il-10), and innate immune-related genes (c3) were up-regulated, while another gene linked with the lipid metabolism (acox1) was down-regulated. The results showed new insights about early responses of turbot following infection with A. salmonicida subsp. salmonicida.

Strongyloides stercoralis Dissemination and Hyperinfection Associated with Long-Term Steroid Treatment in a Neurosurgical Population.

Strongyloidiasis develops from the infection with Strongyloides stercoralis (Family: Strongylidae) and was recently considered a neglected tropical disease by the World Health Organization due to its global distribution and high burden of infection. Here, we present the cases of two patients under corticosteroid therapy after neurosurgical surgery who developed septic shock-like hyperinfection syndrome due to disseminated strongyloidiasis. The first case is a 77-year-old man from Cape Verde who was diagnosed with an extra-axial right parietal brain mass. He was given dexamethasone and was submitted to a biparietal craniotomy. His condition deteriorated and he was admitted to the intensive care unit (ICU), where he was diagnosed with disseminated strongyloidiasis with hyperinfection. Anthelmintic treatment and corticosteroid therapy were rapidly tapered and stopped. Neurological dysfunction persisted and the patient was transferred to the ward. The patient had died after complications of hospital-acquired pneumonia. The second case is a 47-year-old man from Guinea-Bissau who was diagnosed with a space-occupying lesion in the right temporal region and started treatment with dexamethasone. He underwent a craniectomy with partial excision of the lesion (high-grade glioma). Later his neurologic state worsened, and he was diagnosed with septic shock and hospital-acquired pneumonia. He was admitted to the ICU, the diagnosis of disseminated strongyloidiasis and hyperinfection syndrome was made and he initiated treatment with ivermectin and albendazole. Corticosteroid therapy was tapered. The patient's clinical status deteriorated, and multiple opportunistic infections were diagnosed during the ICU stay, which lead him to die. Clinicians should have a high index of suspicion when in the presence of corticosteroid-treated patients with sepsis. Preventive strategies and subsequent treatment should be considered in patients with a risk of acquisition or dissemination. Treating severe strongyloidiasis is still a clinical challenge and a delayed diagnosis can significantly worsen the outcomes of the patients affected, as seen in the presented cases.

Proximate Composition and Fatty Acid Profile of Gilthead Seabream (Sparus aurata) Fed with Pelvetia canaliculata-Supplemented Diets: An Insight towards the Valorization of Seaweed Biomass.

Seaweeds are a sustainable source of protein and lipids that may be used to replace fish by-products in aquaculture feed. This study aimed at using the macroalgae Pelvetia canaliculata as an ingredient in gilthead seabream (Sparus aurata) feed, either as freeze-dried powder or as algae residue (waste) that was obtained after the supplementation of sunflower oil. The formulated diets and the fish muscle were analyzed concerning the proximate composition and the fatty acid profile. The health lipid indices hypocholesterolemic/hypercholesterolemic (h/H), atherogenic (AI), thrombogenic (TI), as well as n-3/n-6 and polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratios were calculated. Additionally, the peroxidizability index (PI) was determined. No differences were observed in the proximate composition of fish muscle regardless of the diet used. Fish fed a diet supplemented with 10% of algae waste (W10) stand out for the highest content in oleic acid (C18:1 n-9), and the lowest in both linoleic (C18:2 n-6) and palmitic (C16:0) fatty acids. All fish samples showed values of health lipid indices within the limits recommend for a nutritional balanced diet. These results highlight that fish fed diets supplemented with P. canaliculata are sources of healthy lipids that might be consumed on a regular basis to prevent cardiovascular diseases.

Campylobacter fetus Cellulitis.

Campylobacter fetus, a bacteria of the Campylobacter genus that are a group of bacteria known to cause intestinal infections, is a particular microbial agent due to its most common presentation being as a non-intestinal systemic infection and rarely as a focal infection, most frequently cellulitis. C. fetus's main reservoirs are cattle and sheep. Humans are usually infected by consuming raw milk and/or meat. Infection in humans is rare and generally related to immune deficiencies, malignancy, chronic liver disease, diabetes mellitus and elderly age, among other factors. Diagnosis is usually achieved by blood cultures due to the lack of focalized signs/symptoms and the pathogen's endovascular tropism. The authors present a case of cellulitis due to Campylobacter fetus, a microbial agent that affects susceptible patients with a mortality rate of up to 14%. We aim to emphasize the importance of potential bacterial seeding sites secondary to bacteremia given the agent tropism for vascular tissue. The medical diagnosis was performed by the identification of bacteria in blood cultures. Campylobacter spp. infections are more frequently related to undercooked poultry or meat, but in this case, the consumption of fresh cheese was considered the most likely source of infection. A literature review showed that, in patients with previous antibiotic cycles, a combination of carbapenem and gentamicin had better outcomes and lower relapse rates. Due to typical surface antigenic variation, immune control may not be attainable and may account for relapsing infections, even after appropriate therapy. The duration of treatment has yet to be well established. Based on other reported cases, we considered a four-week treatment to be sufficient, given clinical improvement and absence of recurrence in the follow-up time.

Modulatory effect of Gracilaria gracilis on European seabass gut microbiota community and its functionality.

Seaweeds are an important source of nutrients and bioactive compounds and have a high potential as health boosters in aquaculture. This study evaluated the effect of dietary inclusion of Gracilaria gracilis biomass or its extract on the European seabass (Dicentrarchus labrax) gut microbial community. Juvenile fish were fed a commercial-like diet with 2.5% or 5% seaweed biomass or 0.35% seaweed extract for 47 days. The gut microbiome was assessed by 16S rRNA amplicon sequencing, and its diversity was not altered by the seaweed supplementation. However, a reduction in Proteobacteria abundance was observed. Random forest analysis highlighted the genera Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae. SparCC correlation network analysis suggested several mutualistic and other antagonistic relationships that could be related to the predicted altered functions. These pathways were mainly related to the metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae. This study shows the beneficial potential of Gracilaria as a functional ingredient through the modulation of the complex microbial network towards fish health improvement.

Functional and Molecular Immune Response of Rainbow Trout (Oncorhynchus mykiss) Following Challenge with Yersinia ruckeri.

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30-70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout's short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1ß, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.

Detection of mcr-1 Gene in Undefined Vibrio Species Isolated from Clams.

The increase of antimicrobial resistant strains is leading to an emerging threat to public health. Pathogenic Vibrio are responsible for human and animal illness. The Enterobacteriaceae family includes microorganisms that affect humans, causing several infections. One of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filter-feeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In the present study, Vibrionaceae and coliforms microorganisms were isolated from clams. A microbial susceptibility test was performed using the disk diffusion method. From 43 presumptive Vibrio spp. and 17 coliforms, three Vibrio spp. with MICs to colistin >512 mg L-1 were found. From the 23 antimicrobial resistance genes investigated, only the three isolates that showed phenotypic resistance to colistin contained the mcr-1 gene. Genotypic analysis for virulence genes in EB07V indicated chiA gene presence. The results from the plasmid cure and transformation showed that the resistance is chromosomally mediated. Biochemical analysis and MLSA, on the basis of four protein-coding gene sequences (recA, rpoB, groEL and dnaJ), grouped the isolates into the genus Vibrio but distinguished them as different from any known Vibrio spp.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Pegylated Interferon Alpha for Chronic Hepatitis B Virus Infection.

Chronic hepatitis B (CHB) is a potentially life-threatening and prevalent disease worldwide. Far from attaining the ultimate treatment goal, hepatitis B virus (HBV) infection eradication, the two current therapeutic options aim to prevent progression to end-stage liver disease, maintaining long-term suppression of HBV replication. Pegylated interferon-α (PEG-INFα) is often poorly tolerated and disregarded considering the orally administered nucleos(t)ide analogues. However, PEG-INFα may achieve similar treatment endpoints with a finite course of treatment. We report a case of PEG-INFα-treated CHB that attained sustained off-treatment virological response with only 16 weeks of treatment, with loss of both HBeAg and HBsAg (this latter the optimal treatment endpoint).

Management of HIV-2 resistance to antiretroviral therapy in a HIV-1/HIV-2/HBV co-infected patient.

BACKGROUND: The current standard of care is to start antiretroviral therapy in all patients diagnosed with HIV-1, as for HIV-2 current DHHS guideline suggests ART for HIV-2 as soon as diagnosis is established, although this practice is not universal, for instance, in Portugal there are specific criteria to start treatment. CASE PRESENTATION: We present a case of a man, chronically infected with HIV-1, HIV-2 and hepatitis B virus who developed resistance to HIV-2 while maintaining HIV-1 under control. 6 years after starting antiretroviral therapy he had his first virologic failure. We performed HIV-2 resistance tests that revealed high-grade resistance to all nucleoside reverse-transcriptase inhibitors except tenofovir and to all protease inhibitors except darunavir. After a decade of permanent poor adherence to therapy he developed resistance to both tenofovir and darunavir. We put together a new regiment with tenofovir alafenamide + emtricitabine + dolutegravir + maraviroc and nowadays he is with undetectable HIV-1 and HIV-2 viral loads. CONCLUSIONS: This shows the importance of having access to HIV-2 viral load determination and HIV-2 resistance testing.

Potential use of macroalgae Gracilaria gracilis in diets for European seabass (Dicentrarchus labrax): Health benefits from a sustainable source.

Seaweeds still possess a large undisclosed potential, mainly due to their constituent's richness, which may have several uses for society. In aquaculture, they may play a role as an ecological sustainable aquafeed supplement to increase overall health and fight pathogenic outbreaks. This study aimed to evaluate the general health modulation that the inclusion of Gracilaria gracilis could accomplish in the diet of Dicentrarchus labrax. Dried algae at 2.5% and 5% and algal extract at 0.35% inclusion levels were supplemented to seabass diet to evaluate possible growth, haematological, immunological, antioxidant, metabolic, and intestinal morphological modulations. The supplementations did not impact growth or feed utilization, and barely affected the haematological profile and some metabolic parameters. Nevertheless, it caused a marked outcome on lysozyme, some oxidative stress biomarkers, and intestine morphology, suggesting beneficial consequences from the algal inclusion. Dried algae powder, with a 2.5% inclusion, boosted immune response, with higher plasmatic lysozyme and intestinal acid goblet cells and protected against oxidative damages by improved enzymatic and non-enzymatic responses. Thus, we provide evidence that dietary seaweed application may be a path towards a more sustainable aquaculture industry.

Seasonal Changes in the Nutritional Composition of Agarophyton vermiculophyllum (Rhodophyta, Gracilariales) from the Center of Portugal.

Seaweeds exhibit high nutritional value due to a balanced concentration of proteins, vitamins and minerals, a high concentration of low digestibility polysaccharides, and reduced levels of lipids, many of which are n-3 and n-6 fatty acids. The species Agarophyton vermiculophyllum is no exception and, as such, a comprehensive study of the chemical and nutritional profile of this red seaweed was carried out for 1 year. Seasonal variations in moisture, ash, protein and amino acids content, crude fibers, ascorbic acid, agar, lipids, and the corresponding fatty acid profile, were analyzed. We found low levels of fatty acids and a high protein content, but also noticed interesting seasonal change patterns in these compounds. The present study gives insights on the environmental conditions that can lead to changes in the nutritional composition of this species, aiming, therefore, to bring new conclusions about the manipulation of environmental conditions that allow for maximizing the nutritional value of this seaweed.

A Patient with Severe Malaria and COVID-19: How Do You Tell the Difference between These Infections?

Coronavirus infection, known as COVID-19, is characterized by clinical, epidemiological and biological features similar to those of malaria. In each case, fever, myalgia, fatigue, headaches and gastrointestinal symptoms may be present. Both diseases can also induce a cytokine storm and pro-coagulant states. An appropriate epidemiological approach and differential diagnosis are very important so that the right clinical intervention can be selected. Malaria remains a serious global public health issue, especially in endemic countries. Elimination campaigns are helping to control the disease, but in many countries these programs are now at risk of failure due to logistic and economic problems caused by COVID-19. The authors describe the case of a patient with co-infection with malaria and COVID-19, reminding us that during this coronavirus pandemic it is critical to consider other diagnoses, particularly in people traveling between countries. LEARNING POINTS: As far as we know, this is one of the first case reports of co-infection with COVID-19 and Plasmodium falciparum malaria.It is important to be aware of the clinical challenges of diagnosing the cause of fever in returned travellers.

Optimization of phycobiliprotein pigments extraction from red algae Gracilaria gracilis for substitution of synthetic food colorants.

The extraction of phycobiliprotein (PBP) pigments from red algae Gracilaria gracilis was optimized using maceration, ultrasound-assisted extraction (ultrasonic water bath and ultrasonic probe), high pressure-assisted extraction, and freeze-thaw. The experimental conditions, namely homogenization time (t1), buffer concentration (C), treatment time (t2), biomass: buffer ratio (R), and pressure (P), were optimized using Response Surface Methodology (RSM). The yield of phycoerythrin (PE) extracted, determined spectroscopically, was used as the response variable. Maceration was the most efficient extraction method yielding 3.6 mg PE/g biomass under the optimal conditions (t1 = t2 = 10 min; C = 0.1 M; R = 1:50). Scanning Electron Microscopy (SEM) analysis of the biomass before and after the cell disruption treatments revealed a more efficient cell wall rupture with maceration.

Cutaneous leishmaniasis in non-endemic countries: An emerging yet neglected problem.

Cutaneous leishmaniasis (CL) is the most common leishmaniasis syndrome, yet a neglected disease in industrialized non-endemic countries, where it has become an emergent problem. The lack of clinical experience, evidence-based literature and availability of some treatments complicates its management. We report a CL case in a 30 year-old man returned from Brazil, with a cutaneous ulcerated lesion, where it was possible to isolate Leishmania braziliensis/guyanensis complex (subgenus Viannia). An initial course of treatment with miltefosine was attempted, but considering the lack of response, liposomal amphotericin B was used, with very good results. Our report highlights the obstacles faced in the diagnosis and treatment of New World CL in non-endemic countries and the need for more funding and research.

Detection of Betanodavirus in experimentally infected European seabass (Dicentrarchus labrax, Linnaeus 1758) using non-lethal sampling methods.

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non-lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50 /ml and 106 TCID50 /ml, respectively), and samples collected 7, 15 and 30 days post-infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non-lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT-qPCR). The presence of virus in non-lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular-infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme-linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus-specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.

Acute Pyelonephritis with Bacteremia Caused by Enterococcus hirae: A Rare Infection in Humans.

Enterococci are one of the usual residents of the microflora in humans. In the last decade this genus has been reported as the third most common cause of bacteremia. We present the case of a 78-year-old female who was admitted to the emergency room because of nausea, lipothymia, and weakness. She was diagnosed with a pyelonephritis with bacteremia, with the isolation in blood and urine cultures of Escherichia coli and Enterococcus hirae. This last microorganism is a rarely isolated pathogen in humans. Currently it is estimated to represent 1-3% of all enterococcal species isolated in clinical practice.

Cystic Pancreatic Lymphangioma - Diagnostic Role of Endoscopic Ultrasound.

Pancreatic cystic lymphangiomas are rare benign lesions that arise from lymphatic vessels, accounting for less than 0.2% of all pancreatic cysts. Typically it is asymptomatic and discovery occurs during imaging exams for non-pancreatic disease. In the past, a definite diagnosis was made through surgery, with complete resection of all tumoral tissue to prevent recurrence. Nowadays, the development of endoscopic ultrasound (EUS) made it possible to identify these cysts combining morphologic ultrasound features, macroscopic aspirated fluid appearance, biochemical and cytological evaluation of the sample. We report two cases of cystic pancreatic lymphangioma diagnosed through EUS, allowing conservative management without surgery. These cases show that cystic pancreatic lymphangioma should be considered in the differential diagnosis of cystic pancreatic lesions and that EUS is an important tool for their recognition.

Os linfangiomas quísticos pancreáticos são lesões benignas raras com origem em vasos linfáticos, correspondendo a menos de 0,2% da totalidade de quistos pancreáticos. Na maioria são assintomáticos sendo a sua descoberta incidental. Tradicionalmente o seu diagnóstico era cirúrgico, com completa ressecção de todo o tecido tumoral para prevenir recorrência. Actualmente, o desenvolvimento da ecoendoscopia (EUS) permitiu identificar estes quistos combinando as suas características ultrasonográficas, aparência macroscópica do fluido aspirado, e avaliação bioquímica e citológica da amostra. Os autores descrevem dois casos de linfangiomas quísticos pancreáticos diagnosticados por EUS, permitindo uma abordagem conservadora. Estes demonstram que os linfangiomas quísticos pancreáticos devem ser considerados no diagnóstico diferencial de lesões quísticas pancreáticas e que a EUS é importante no seu reconhecimento.