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Introduction Urinary symptoms constitute the primary reason for female patients to consult their general practitioner. The urinary dipstick test serves as a cornerstone for diagnosing urinary tract infections (UTIs), yet traditional visual interpretation may be subject to variability. Automated devices for dipstick urinalysis are routinely used as alternatives, yet the evidence regarding their accuracy remains limited. Therefore we aimed to compare concordance between visual and automated urinary dipstick interpretation and determine their test characteristics for the prediction of bacteriuria.Material and methods We conducted a prospective validation study including urine samples originating from adult patients in general practice that were sent to the Maastricht Medical Centre + for urinary culture. Urinary dipstick tests were performed on each sample, which were interpreted visually and automatically. We calculated Cohen’s κ and percentage agreement and used 2 × 2 tables to calculate test characteristics.Results We included 302 urine samples. Visual and automated analysis showed almost perfect agreement (κ = 0.82 and κ = 0.86, respectively) for both nitrite and leukocyte esterase, but moderate agreement for erythrocytes (κ = 0.51). Interpretation of clinically relevant (nitrite and/or leukocyte esterase positive) samples showed almost perfect agreement (κ = 0.88). Urinary dipsticks show similar test characteristics with urinary culture as gold standard, with sensitivities of 0.92 and 0.91 and specificities of 0.37 and 0.41 for visual and automated interpretation respectively.Conclusion Automated and visual dipstick analysis show near perfect agreement and perform similarly in predicting bacteriuria. However, automated analysis requires maintenance and occasionally measurement errors can occur.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness.IMPORTANCEThis study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

Abstract Background Vaginal microbiota composition is associated with spontaneous preterm birth (sPTB), depending on ethnicity. Host-microbiota interactions are thought to play an important underlying role in this association between ethnicity, vaginal microbiota and sPTB. Methods In a prospective cohort of nulliparous pregnant women, we assessed vaginal microbiota composition, vaginal immunoglobulins (Igs), and local inflammatory markers. We performed a nested case–control study with 19 sPTB cases, matched based on ethnicity and midwifery practice to 19 term controls. Results Of the 294 included participants, 23 pregnancies ended in sPTB. We demonstrated that Lactobacillus iners-dominated microbiota, diverse microbiota, and ethnicity were all independently associated with sPTB. Microbial Ig coating was associated with both microbiota composition and ethnicity, but a direct association with sPTB was lacking. Microbial IgA and IgG coating were lowest in diverse microbiota, especially in women of any ethnic minority. When correcting for microbiota composition, increased microbial Ig coating correlated with increased inflammation. Conclusion In these nulliparous pregnant women, vaginal microbiota composition is strongly associated with sPTB. Our results support that vaginal mucosal Igs might play a pivotal role in microbiota composition, microbiota-related inflammation, and vaginal community disparity within and between ethnicities. This study provides insight in host-microbe interaction, suggesting that vaginal mucosal Igs play an immunomodulatory role similar to that in the intestinal tract. Video Abstract

Abstract Purpose The addition of intrathecal morphine to general anesthesia for laparoscopic colorectal surgery is an effective method of providing analgesia. However, the analgesia duration of approximately 24 hours poses the risk of rebound pain on the second postoperative day. Therefore, this study aimed to investigate the patients' perception on postoperative pain management using the International Pain Outcomes questionnaire on both the first and second postoperative day. Methods This prospective single-center cohort study was conducted between November 2020 and March 2021 and included forty patients. The primary outcome was a difference in postoperative pain on the first and second postoperative day. Secondary outcomes included opioid consumption, interference of pain with activities, side effects, and patients’ perception and satisfaction with pain treatment. Results The intensity of postoperative pain did not increase on the second postoperative day NRS [numeric rating scale] 5 (2–7 [0–10]) vs 5 (3–7 [1–10]), p=0.414), but the percentage of time spent in severe pain increased (20% (10-40 [0-90]) vs 30% (20-50 [0-80]), p=0.010). There was no difference in opioid consumption (6 mg (0–12) [0–42] vs 6 mg (0–12) [0–29], p=0.914). Pruritis (NRS 2 (0–6 [0–10]) vs 0 (0–3 [0–8]), p=0.001) and dizziness (NRS 2 (0–7 [0–10]) vs 0 (0–2 [0–9]), p=0.002) decreased on the second postoperative day. Patients reported high satisfaction during the first two days after surgery (NRS 8 (7–9) [0–10] vs 8 (7–9) [0–10], p=0.395). Conclusion Intrathecal morphine is a suitable analgesic modality in laparoscopic colorectal surgery within an enhanced recovery after surgery program, without causing important rebound pain. Pain scores, however, may be further reduced by adding non-opioid analgesics.

Abstract Strain-specific probiotics can present antioxidant activity and reduce damage caused by oxidation. Streptococcus alactolyticus strain FGM (S. alactolyticus strain FGM) isolated from the chicken cecum shows potential probiotic properties which have been previously demonstrated. However, the antioxidant properties of S. alactolyticus strain FGM remain unknown. In this view, cell-free supernatant (CFS), intact cells (IC) and intracellular extracts (CFE) of strain FGM and 3 strains of Lactobacillus (LAB) were prepared, and their scavenging capacities against DPPH, hydroxyl radicals and linoleic acid peroxidation inhibitory were compared in this study. The effects of strain FGM cell-free supernatant (FCFS) on NO production, activity of SOD and GSH-Px in RAW264.7 cells and LPS-induced RAW264.7 cells were analyzed. The metabolites in the supernatant were quantitated by N300 Quantitative Metabolome. It was shown that the physicochemical characteristics of CFS to scavenge DPPH, hydroxyl radicals, and linoleic acid peroxidation inhibitory were significantly stronger than that of IC and CFE in the strain FGM (P

Objectives: The COVID-19 pandemic highlights the importance of understanding facilitators for disease transmission. Events such as Carnival, characterized by large gatherings and extensive social interactions, have the potential to become ‘super spreading events' for respiratory infections. This paper aims to assess the impact of large gatherings on virus transmission, providing crucial insights for the development of effective public health strategies. Study design: An ecological study was performed. Methods: The age-standardized number of COVID-19 cases reported in 2022, stratified by age (under 60 and 60+ years) was compared countrywide for Dutch provinces where Carnival was celebrated versus those where it was not. Additionally, we compared standardized hospitalization rates in 2022 and 2023 for both areas. Results: Countrywide, 2,278,431 COVID-19 cases were reported between 06-02-2022 and 10-04-2022. Daily incidence increased after Carnival, peaking at 803 per 100,000 inhabitants for under 60s in carnival provinces and 368 in non-carnival provinces. For individuals 60+ daily incidence peaked at 396 in carnival provinces and 247 in non-carnival provinces. Over the 10 weeks following the start of Carnival, the carnival provinces demonstrated a 15 % (2022) 17 % (2023) higher hospitalization rate compared to non-carnival provinces. Conclusions: The peak in cases and hospitalizations in regions actively celebrating Carnival compared to the rest of the Netherlands qualifies Carnival as a ‘super-spreading’ event. Our findings underscore the elevated risk of respiratory infections associated with large gatherings, advocating guided policies, including transparent risk communication and healthcare preparedness.

The intestinal microbiota, consisting of an estimated 10^10–10^11 organisms, regulate physiological processes involved in digestion, metabolism, and immunity. Surprisingly, these intestinal microorganisms have been found to influence tissues that are not directly in contact with the gut, such as adipose tissue, the liver, skeletal muscle, and the brain. This interaction takes place even when intestinal barrier function is uncompromised. An increasing body of evidence suggests that bacterial membrane vesicles (bMVs), in addition to bacterial metabolites such as short-chain fatty acids, are able to mediate effects of the microbiota on these host tissues. The ability of bMVs to dissipate from the intestinal lumen into systemic circulation hereby facilitates the transport and presentation of bacterial components and metabolites to host organs. Importantly, there are indications that the interaction between bMVs and tissues or immune cells may play a role in the etiology of (chronic metabolic) disease. For example, the gut-derived bMV-mediated induction of insulin resistance in skeletal muscle cells and pro-inflammatory signaling by adipocytes possibly underlies diseases such as type 2 diabetes and obesity. Here, we review the current knowledge on bMVs in the microbiota’s effects on host energy/substrate metabolism with a focus on etiological roles in the onset and progression of metabolic disease. We furthermore illustrate that vesicle production by bacterial microbiota could potentially be modulated through lifestyle intervention to improve host metabolism.

ABSTRACT With the emergence of highly transmissible variants of concern, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a global threat of coronavirus disease 2019 (COVID-19) resurgence. Cellular responses to novel variants are more robustly maintained than humoral responses, and therefore, cellular responses are of interest in assessing immune protection against severe disease in the population. We aimed to assess cellular responses to SARS-CoV-2 at the population level. IFNγ (interferon γ) responses to wild-type SARS-CoV-2 were analyzed using an ELISpot assay in vaccine-naive individuals with different humoral responses: Ig (IgM and/or IgG) seronegative (n = 90) and seropositive (n = 181) with low (

We describe vaginal microbiota, including Gardnerella species and sexually transmitted infections (STIs), during pregnancy and their associations with recurrent spontaneous preterm birth (sPTB). We performed a prospective cohort study in a tertiary referral centre in the Netherlands, among pregnant women with previous sPTB

IntroductionCross-border mobility (CBM) to visit social network members or for everyday activities is an important part of daily life for citizens in border regions, including the Meuse-Rhine Euroregion (EMR: neighboring regions from the Netherlands, Belgium, and Germany). We assessed changes in CBM during the COVID-19 pandemic and how participants experienced border restrictions.MethodsImpact of COVID-19 on the EMR’ is a longitudinal study using comparative cross-border data collection. In 2021, a random sample of the EMR-population was invited for participation in online surveys to assess current and pre-pandemic CBM. Changes in CBM, experience of border restrictions, and associated factors were analyzed using multinomial and multivariable logistic regression analysis.ResultsPre-pandemic, 82% of all 3,543 participants reported any CBM: 31% for social contacts and 79% for everyday activities. Among these, 26% decreased social CBM and 35% decreased CBM for everyday activities by autumn 2021. Negative experience of border restrictions was reported by 45% of participants with pre-pandemic CBM, and was higher (p < 0.05) in Dutch participants (compared to Belgian; aOR= 1.4), cross-border [work] commuters (aOR= 2.2), participants with cross-border social networks of friends, family or acquaintances (aOR= 1.3), and those finding the measures ‘limit group size’ (aOR= 1.5) and ‘minimalize travel’ (aOR= 2.0) difficult to adhere to and finding ‘minimalize travel’ (aOR= 1.6) useless.DiscussionCBM for social contacts and everyday activities was substantial in EMR-citizens, but decreased during the pandemic. Border restrictions were valued as negative by a considerable portion of EMR-citizens, especially when having family or friends across the border. When designing future pandemic control strategies, policy makers should account for the negative impact of CBM restrictions on their citizens.

Summary: Two SARS-CoV-2 nosocomial outbreaks occurred on the haematology ward of our hospital. Patients on the ward were at high risk for severe infection because of their immunocompromised status. Whole Genome Sequencing proved transmission of a particular SARS-CoV-2 variant in each outbreak. The first outbreak (20 patients/31 healthcare workers (HCW)) occurred in November 2020 and was caused by a variant belonging to lineage B.1.221. At that time, there were still uncertainties on mode of transmission of SARS-CoV-2, and vaccines nor therapy were available. Despite HCW wearing II-R masks in all patient contacts and FFP-2 masks during aerosol generating procedures (AGP), the outbreak continued. Therefore, extra measures were introduced. Firstly, regular PCR-screening of asymptomatic patients and HCW; positive patients were isolated and positive HCW were excluded from work as a rule and they were only allowed to resume their work if a follow-up PCR CT-value was ≥30 and were asymptomatic or having only mild symptoms. Secondly, the use of FFP-2 masks was expanded to some long-lasting, close-contact, non-AGPs. After implementing these measures, the incidence of new cases declined gradually. Thirty-seven percent of patients died due to COVID-19.The second outbreak (10 patients/2 HCW) was caused by the highly transmissible omicron BA.1 variant and occurred in February 2022, where transmission occurred on shared rooms despite the extra infection control measures. It was controlled much faster, and the clinical impact was low as the majority of patients was vaccinated; no patients died and symptoms were relatively mild in both patients and HCW.

The urogenital microbiota is increasingly gaining recognition as a significant contributor to reproductive health. Recent studies suggest that microbiota can serve as predictors for fertility treatment outcomes. Our objective was to investigate the degree of similarity in microbial composition between patient-collected urine and vaginal samples in a subfertile population. We enrolled women of reproductive age (20–44 years) diagnosed with subfertility and requiring in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment. They self-collected both mid-stream urine samples and vaginal swabs before commencing the IVF or IVF-ICSI procedure. All samples were analysed using the intergenic spacer profiling (IS-pro) technique, a rapid clinical microbiota analysis tool. The main outcome measures were the degree of similarity of microbial composition between the two different, but simultaneously collected, samples. Our findings revealed a high correlation (R squared of 0.78) in microbiota profiles between paired urine and vaginal samples from individual patients. Nevertheless, the urinary microbiota profiles contained fewer species compared to the vaginal microbiota, resulting in minor but distinguishable differences. Furthermore, different subfertility diagnoses appeared to be associated with differences in microbial profiles. A noteworthy observation was the exclusive presence of Escherichia coli (E. coli) in both samples of women diagnosed with male factor subfertility. In conclusion, since urinary microbiota profiles seem to represent a diluted version of the vaginal microbiota, vaginal microbiome sampling to predict fertility treatment outcome seems preferable. To enhance the success of fertility treatments, further research is needed to gain deeper insights into a putative causal role of microbiota in the mechanisms of subfertility.

In recent decades, minimally invasive surgery has become the favoured surgical technique, with increasing utilisation of robotic surgery to enhance patient outcomes. However, the design complexity of surgical robotic instruments can pose challenges in maintaining adequate cleaning, disinfection and sterilisation-particularly of the device's interior. In our hospital, robotic instruments are reused for a maximum of ten successive patients, following the manufacturer's guidelines. To the best of our knowledge, neither the manufacturer nor ISO standards have specified any methods to determine the sterility of robotic instruments after cleaning, disinfection and sterilisation procedures. In a small pilot study, we used a locally developed protocol to evaluate the sterility of 20 da Vinci SI robotic instruments, with the aim of determining whether the recommended cleaning, disinfection and sterilisation process is adequate to achieve safe usage in subsequent patients. None of the 20 instruments showed viable micro-organisms, therefore the robotic instruments were considered sterile, and suitable for re-use. We recommend our protocol to other hospitals, to be used as an essential control element in the assessment of their unique reprocessing technique for robotic instruments.

Abstract Background Comparative data collection in transborder areas can contribute to informed decision making processes when dealing with borderless health threats such as pandemics, and thus help minimize the negative health effects for its citizens. To examine the pandemic response over time and the impact of infectious disease control in a cross-border setting, a prospective longitudinal study was conducted in the border area between Germany, Belgium and the Netherlands. In the spring of 2021, a random sample of 26,925 adult citizens selected from governmental registries was invited to collect a blood sample at home for SARS-CoV-2 antibody testing and to fill in an online questionnaire on attitudes and behaviour towards infection prevention measures, cross-border mobility, social network and support, COVID-19 self-reported infection(s) and symptoms, vaccination, general self-reported health and socio-demographics. In autumn 2021, participants were invited for a follow-up round. An online tool was developed to coordinate fieldwork procedures, real-time monitoring of participation and consultation of antibody test results. Furthermore, a helpdesk in all three languages for participants’ support was set up. Results In the first round, 6,006 citizens in the Meuse-Rhine Euroregion participated. 15.3% of the invited citizens on the Belgian side of the border participated. In the Netherlands and Germany this was respectively 27% and 23.7%. In the follow-up round 4,286 (71.4%) citizens participated for the second time. The participation rate was highest in the age group 50–69 years and lowest in > 80 in all sub regions of the Meuse-Rhine Euroregion. More women participated than men. Overall, more blood samples were returned than completed questionnaires. In total, 3,344 citizens in the Meuse-Rhine Euroregion completed all components of participation in both rounds. Conclusions The collection of comparative data can help better assess the pandemic response and the impact of infectious disease control in a cross-border area. Recommendations for a longitudinal cross-border study include a centralized online environment, mapping out potential challenges related to national regulations in the preparation phase and organizing regional coordination centres to create more familiarity and trust towards the involved organisations.

Abstract Background Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens—on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. Results The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. Conclusions Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Introduction: Higher antibody levels, in particular antibodies directed against the receptor-binding domain (RBD) of the spike protein, protect against re-infection with SARS-CoV-2. Quantitative antibody response data provide insight into population immunity and are essential for decision-making on booster-vaccination strategies. We aimed to identify predictors for higher antibody responses such as gender, age, experienced COVID-19-compatible symptoms, disease severity and exposure to pre-determined risk factors associated with SARS-CoV-2 seropositivity. Materials and methods: Quantitative anti-S-RBD responses were analysed in seropositive vaccine-naive individuals (n = 1,857) from a study population of 10,001 adults, including healthcare workers (n = 211) and individuals with a known date of a positive PCR (n = 214). Regression models tested associations between age, gender, the period of symptoms, disease severity, pre-identified exposure factors associated with SARS-CoV-2 seropositivity, and anti-S-RBD responses. Results: Symptoms of more severe disease (fever and/or dyspnoea: OR 2.42 [95%CI 1.76–3.34], and hospital admission: OR 11.41 [95%CI 4.66–27.97]), and a longer interval between COVID-19-compatible symptoms and serum collection (OR 3.17 [95%CI 1.32–7.63]) were predictive for anti-S-RBD levels ≥300 U/mL. Working in healthcare was inversely associated with anti-S-RBD levels ≥300 U/mL (OR 0.51 [95%CI 0.31–0.82]). None of the pre-identified exposure factors associated with SARS-CoV-2 seropositivity could be identified as predictive of higher anti-S-RBD responses. Conclusion: No exposure factors were identified as predictors of higher neutralising antibody responses. Nevertheless, higher neutralising antibody levels in individuals with more severe symptoms suggest better immunological protection against SARS-CoV-2 re-infection. In seroprevalence studies, that mainly include asymptomatic or mildly infected individuals, the determination of quantitative antibody responses may help in the interpretation of population immunity.

BackgroundLichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.ResultsA) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting.ConclusionsThe surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

COVID-19 booster vaccination has shown to add to the protection against infection with SARS-CoV2 and subsequent severe disease. This longitudinal cross-border study aimed to identify factors associated with COVID-19 booster vaccine intentions in an initially vaccinated adult population living in the Meuse-Rhine Euroregion (EMR; including the Netherlands, Belgium, and Germany) and differences between countries.Data collection took place in autumn of 2021 and consisted of online questionnaires sent to a random sample of the population based on governmental registries. Data from 3,319 fully and partially vaccinated adults were used to examine determinants of non-positive intention for a booster vaccination (i.e., uncertain or do not want), using multivariable logistic regression analyses weighted by age group, sex, and country.Compared to German residents, Dutch residents (OR = 2.4) and Belgian residents (OR = 1.4) were more likely to be uncertain or not want to receive a booster vaccine in September-October 2021. Factors independently associated with non-positive intention were female sex (OR = 1.6), absence of comorbidities (OR = 1.3), time since last vaccination less than 3 months ago for those fully vaccinated (OR = 1.6), being partially vaccinated (OR = 3.6), a negative experience with communication of COVID-19 measures (OR = 2.2), and regarding measures as ineffective (OR = 1.1).Results indicate that booster vaccine intentions differ between countries in the cross border Meuse-Rhine Euroregion. Non-positive intention for the booster vaccine is prevalent in all three countries of the EMR, but to a different extent, as shown in this study. Cross-border collaboration and sharing information and knowledge about vaccination strategies could play a role in limiting the impact of COVID-19.

Porcine epidemic diarrhea virus (PEDV) infection poses a significant threat to the global pig industry. Current prevention and control strategies are inadequate in protecting pigs from new PEDV variants. This review aims to examine the relationship between PEDV and intestinal microbes, and investigate whether modulating intestinal microbes could affect PEDV infection. The mechanisms by which various intestinal microbes affect viral infection were initially introduced. Intestinal microbes can influence enteric viral infection through direct contact, such as binding, or by affecting interferons (IFNs) production and the intestinal barrier. Influencing the intestinal barrier by microbes can impact PEDV infection in young piglets. To narrow down the range of microbes that may influence PEDV infection, this review summarized microbes that change after infection. Short chain fatty acids (SCFAs), bacterial cell components, and toxins from microbes were identified as important mediators affecting PEDV infection. SCFAs primarily strengthen the intestinal barrier and inhibit intestinal inflammation, while bacterial cell components and toxins are more likely to damage the intestinal barrier. Therefore, this review hypothesizes that fecal transplantation, which allows the host to colonize more SCFAs-producing microbes, may prevent PEDV infection. However, these hypotheses require further proof, and the transplantation of intestinal microbes in pigs requires more exploration.

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants.

Abstract There has been a growing body of evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) shows enhanced transmissibility and increased viral loads compared to other variants. A recent study has even suggested that respiratory samples from people infected with the Delta variant can harbor up to 1000 times higher viral loads compared to samples with variants that are more closely related to the original Wuhan strain, although the sample size of this study (n = 125) was very limited. Here, we have compared the viral load in 16,185 samples that were obtained in periods during which non-VOC, the Alpha (B.1.1.7) or Delta variant (B.1.617.2) were dominant as evidenced by genomic surveillance. We found that the Delta variant contained about fourfold higher viral loads across all age groups compared to the non-VOC or Alpha variants, which is significantly lower than reported earlier. Interestingly, the increased viral load for the Delta variant seemed to be age-dependent, regardless of sex, as the viral load was about 14-fold higher for Delta compared to the non-VOC or Alpha variant in age group 0–20 years and fourfold higher in age group 21–40 years, while there was no difference in viral load between variants in age groups 41–60 and 61+ years, most likely as a consequence of a higher degree of vaccination in the older age groups.

Abstract Background Variant of concern (VOC) SARS-CoV-2 alpha variant (B.1.1.7) was the dominant strain in the Netherlands between March 2021–June 2021. We describe three primary school outbreaks due to the alpha variant using whole genome sequencing with evidence of large-scale transmission among children, teachers and their household contacts. Method All outbreaks described were investigated by the South Limburg Public Health Service, the Netherlands. A case was defined as an individual with a real-time polymerase chain reaction test or antigen test positive for SARS-CoV-2. Whole genome sequencing was performed on random samples from at least one child and one teacher of each affected class. Results Peak attack rates in classes were 53%, 33% and 39%, respectively. Specific genotypes were identified for each school across a majority of affected classes. Attack rates were high among staff members, likely to promote staff-to-children transmission. Cases in some classes were limited to children, indicating child-to-child transmission. At 39%, the secondary attack rate (SAR) in household contacts of infected children was remarkably high, similar to SAR in household contacts of staff members (42%). SAR of household contacts of asymptomatic children was only 9%. Conclusion Our findings suggest increased transmissibility of the alpha variant in children compared to preceding non-VOC variants, consistent with a substantial rise in the incidence of cases observed in primary schools and children aged 5–12 since the alpha variant became dominant in March 2021. Lack of mandatory masking, insufficient ventilation and lack of physical distancing also probably contributed to the school outbreaks. The rise of the delta variant (B.1.617.2) since July 2021 which is estimated to be 55% more transmissible than the alpha variant, provides additional urgency to adequate infection prevention in school settings.

Abstract Background Patients still receive non-beneficial treatments when nearing the end of life. Advance care planning (ACP) interventions have shown to positively influence compliance with end of life wishes. Hospital physicians seem to miss opportunities to engage in ACP, whereas patients visiting the outpatient clinic usually have one or more chronic conditions and are at risk for medical emergencies. So far, implemented ACP interventions have had limited impact. Structural implementation of ACP may be beneficial. We hypothesize that having ACP conversations more towards the end of life and involving the treating physician in the ACP conversation may help patient wishes and goals to become more concrete and more often documented, thus facilitating goal-concordant care. Aim To facilitate timely shared decision making and increase patient autonomy we aim to develop an ACP intervention at the outpatient clinic for frail patients and determine the feasibility of the intervention. Methods The United Kingdom’s Medical Research Council framework was used to structure the development of the ACP intervention. Key elements of the ACP intervention were determined by reviewing existing literature and an iterative process with stakeholders. The feasibility of the developed intervention was evaluated by a feasibility study of 20 ACP conversations at the geriatrics and pulmonology department of a non-academic hospital. Feasibility was assessed by analysing evaluation forms by patients, nurses and physicians and by evaluating with stakeholders. A general inductive approach was used for analysing comments. The developed intervention was described using the template for intervention description and replication (TIDieR). Results We developed a multidisciplinary timely undertaken ACP intervention at the outpatient clinic. Key components of the developed intervention consist of 1) timely patient selection 2) preparation of patient and healthcare professional 3) a scripted ACP conversation in a multidisciplinary setting and 4) documentation. 94.7% of the patients, 60.0% of the nurses and 68.8% of the physicians agreed that the benefits of the ACP conversation outweighed the potential burdens. Conclusion This study showed that the developed ACP intervention is feasible and considered valuable by patients and healthcare professionals.

Background Recently, the Rome classification was proposed in which objective and readily measurable variables were integrated to mark exacerbations of COPD (ECOPD) severity. The aim of this study is to investigate the distribution of a real-world patient population with hospitalised ECOPD according to the current classification across the newly proposed severity classification. We assume that a significant proportion of hospitalised patients will have a mild or moderate event. Methods The Rome classification was applied to a cohort of 364 COPD patients hospitalised at the Department of Respiratory Medicine of Maastricht University Medical Center (MUMC) with a severe ECOPD. Differences in in-hospital, 30- and 90-day mortality were compared between mild, moderate and severe ECOPD according to the new classification. Moreover, data were stratified by the different severity classes and compared regarding general disease characteristics and clinical parameters. Results According to the Rome proposal, 52 (14.3%) patients had a mild ECOPD, 204 (56.0%) a moderate and 108 (29.7%) a severe ECOPD. In-hospital mortality in mild, moderate and severe events was 3.8%, 6.9% and 13.9%, respectively. Most clinical parameters indicated a significantly worse condition in patients classified in the severe group, compared to those in mild or moderate groups. Conclusion Most of the events, traditionally all classified as severe because of the hospitalisation, were classified as moderate, while almost 15% were mild. The results of this study provide insight into the heterogeneity of hospitalised ECOPD and show that the newly proposed Rome criteria can differentiate between events with different short-term mortality rates.

BackgroundIn an attempt to control the spread of SARS-CoV-2, many governments decided to close public venues including bars, restaurants, and nightclubs during the pandemic, making it difficult to study how transmission occurs in these environments. In this study, we were able to gain insight into the transmission dynamics of SARS-CoV-2 in 16 venues in the city of Maastricht using a combination of epidemiological and whole-genome sequencing (WGS) data during a period of 2 weeks in 2021, when bars, restaurants, and nightclubs were temporarily reopened in the Netherlands. This led to a subsequent rise of SARS-CoV-2 cases in the community following the reopening.MethodsWGS was performed on samples from 154/348 of selected cases and combined with epidemiological investigation (e.g., contact tracing and linking cases to specific venues) to identify SARS-CoV-2 transmission clusters. In addition, genomic surveillance data were used to investigate spillover of outbreak-associated genotypes into the community.ResultsClustering was observed in 129/136 (95%) successfully genotyped samples. We established that most cases were linked to venues with dancing facilities and that specific genotypes of the Delta variant were more frequently spread within and from these venues compared to venues without dancing facilities. In addition, we show indications of spillover of certain genotypes from the bar and restaurant industry into the community, with the number of hospital admissions increasing in the weeks following peak cases in the community.ConclusionLifting restrictions on bar and restaurant industry venues with a corona entree ticket in a largely unvaccinated population led to a surge in COVID-19 cases and promoted the spread of new (sub)variants. Nightclubs were identified as potential super-spreading locations.

Presently, many studies assess allergenicity via IgE immunoblotting and IgE binding tests; however, IgE detection does not always signal the manifestation of a clinical allergy. However, the capacity of food allergens to trigger basophils makes it possible to use the in vitro functional basophil activation test (BAT) to assess allergenicity. The effect of the Maillard reaction (MR) on the allergenic potential of processed soy proteins was evaluated by two IgE binding tests (Competitive ELISA and Inhibition ImmunoCAP), a Western Blot and a functional BAT; with the aim to analyze whether the sIgE binding results correspond to the functional assay results. The results between the IgE binding tests and the functional assay were in-line for 2 of the 6 studied patients. For one patient there was no correlation between any of the results. For the raw soy protein extract heated with glucose for 10 and 30 min (SH SPE + Glu and LH SPE + Glu, respectively), the results were in-line for only 3 out of the 6 patients. Thus, the present study shows a discrepancy between IgE binding tests and basophil stimulation when assessing the effect of soy processing on its allergenicity. Since IgE-binding capacity does not always correlate to IgE cross-linking capacity, the conclusions of the allergenic potential based on the IgE binding tests alone should be drawn with care and further studies on this matter would benefit from the inclusion of a functional assay such as the BAT.

Background: The intestinal microbiota plays an important role in the etiology of obesity. Sleeve gastrectomy (SG) is a frequently performed and effective therapy for morbid obesity. Objective: To investigate the effect of sleeve gastrectomy on the fecal microbiota of individuals with morbid obesity and to examine whether shifts in microbiota composition are associated with markers of inflammation and intestinal barrier function. Methods: Fecal and blood samples of healthy individuals (n = 27) and morbidly obese individuals pre-SG (n = 24), and at 2 months (n = 13) and 6 months post-SG (n = 9) were collected. The 16SrRNA gene was sequenced to assess microbiota composition. Fecal calprotectin, plasma inflammatory markers and intestinal permeability markers (multi-sugar test) were determined. Results: Fecal microbiota composition between morbidly obese and lean individuals was significantly different. The fecal microbiota composition changed significantly 2 and 6 months post-SG (p = 0.008) compared to pre-SG but not towards a more lean profile. The post-SG microbiota profile was characterized by an increase in facultative anaerobic bacteria, characteristic for the upper gastrointestinal tract. No correlations were found between inflammatory markers, intestinal permeability and microbial profile changes. Conclusions: Fecal microbiota composition in morbidly obese individuals changed significantly following SG. This change might be explained by functional changes induced by the SG procedure.

Abstract Background Individuals with intellectual and developmental disabilities (IDD) living in congregated settings have increased risk of COVID-19 infection and mortality. Little is known about variant B.1.1.519 with spike mutation T478K, dominant in Mexico. We describe a linked SARS-CoV-2 B.1.1.519 outbreak in three IDD facilities in the Netherlands. Methods Following notification of the index, subsequent cases were identified through serial PCR group testing. Positive specimens were submitted for whole-genome-sequencing. Clinical information was gathered through interviews with staff members of the three facilities. Results Attack rate (AR) in clients of the index facility was 92% (23/25), total AR in clients 45% (33/73) and in staff members 24% (8/34). 55% (18/33) of client cases were asymptomatic, versus 25% (2/8) of staff members. Five client cases (15%) were hospitalized, two died (6%). Sequencing yielded the same specific B.1.1.519 genotype in all three facilities. No significant difference in median viral load was established comparing the B.1.1.519 variant with other circulating variants. The index of the linked outbreak reported no travel history or link to suspected or confirmed cases suggesting regional surveillance. Observed peak regional prevalence of B.1.1.519 during the outbreak supports this. Conclusion AR, morbidity and mortality prior to control measures taking effect were high, probably related to the specific characteristics of the IDD setting and its clients. We assessed no evidence for intrinsic contributing properties of variant B.1.1.519. Our study argues for enhanced infection prevention protocols in the IDD setting, and prioritization of this group for vaccination against COVID-19.

Abstract Background Immunoglobulin A (IgA) plays an important role in maintaining a healthy intestinal microbiome, but little is known about the interaction between local immunoglobulins and the vaginal microbiome. We assessed immunoglobulins (unbound and bound to bacteria), their association with vaginal microbiota composition and the changes over time in 25 healthy women of reproductive age. Results In both Lactobacillus crispatus-dominated and non-L. crispatus-dominated microbiota, IgA and IgG (unbound and bound to bacteria) were higher during menses (T = 1) compared to day 7‑11 (T = 2) and day 17‑25 (T = 3) after menses onset. The majority of vaginal bacteria are coated with IgA and/or IgG. Women with L. crispatus-dominated microbiota have increased IgA coating of vaginal bacteria compared to women with other microbiota compositions, but contained less IgA per bacterium. Presence of a dominantly IgA-coated population at T = 2 and/or T = 3 was also strongly associated with L. crispatus-dominated microbiota. In women with non-L. crispatus-dominated microbiota, more bacteria were uncoated. Unbound IgA, unbound IgG, and bound IgG levels were not associated with microbiota composition. Conclusions In conclusion, L. crispatus-dominated vaginal microbiota have higher levels of bacterial IgA coating compared to non-L. crispatus-dominated vaginal microbiota. Similar to its regulating function in the intestinal tract, we hypothesize that IgA is involved in maintaining L. crispatus-dominated microbiota in the female genital tract. This may play a role in L. crispatus-associated health benefits. Video abstract

Background: While positive blood cultures are the gold standard for late-onset sepsis (LOS) diagnosis in premature and very low birth weight (VLBW) newborns, these results can take days, and early markers of possible treatment efficacy are lacking. The objective of the present study was to investigate whether the response to vancomycin could be quantified using bacterial DNA loads (BDLs) determined by real-time quantitative polymerase chain reaction (RT-qPCR).Methods: VLBW and premature neonates with suspected LOS were included in a prospective observational study. Serial blood samples were collected to measure BDL and vancomycin concentrations. BDLs were measured with RT-qPCR, whereas vancomycin concentrations were measured by LC-MS/MS. Population pharmacokinetic–pharmacodynamic modeling was performed with NONMEM.Results: Twenty-eight patients with LOS treated with vancomycin were included. A one-compartment model with post-menstrual age (PMA) and weight as covariates was used to describe the time PK profile of vancomycin concentrations. In 16 of these patients, time profiles of BDL could be described with a pharmacodynamic turnover model. The relationship between vancomycin concentration and first-order BDL elimination was described with a linear-effect model. Slope S increased with increasing PMA. In 12 patients, no decrease in BDL over time was observed, which corresponded with clinical non-response.Discussion: BDLs determined through RT-qPCR were adequately described with the developed population PKPD model, and treatment response to vancomycin using BDL in LOS can be assessed as early as 8 h after treatment initiation.

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses.

Daniela Briceno Noriega,1 Malgorzata Teodorowicz,1 Huub Savelkoul,1 Janneke Ruinemans-Koerts1,2 1Cell Biology and Immunology Group, Wageningen University & Research, Wageningen, the Netherlands; 2Laboratory of Clinical Chemistry and Hematology, Rijnstate Hospital, Arnhem, the NetherlandsCorrespondence: Janneke Ruinemans-KoertsCell Biology and Immunology Group, Wageningen University & Research, Wageningen, the NetherlandsEmail j.ruinemans-koerts@rijnstate.nlAbstract: The basophil activation test (BAT) is an ex vivo functional assay that measures by flow cytometry the degree of basophil degranulation after stimulation with an allergen. In recent years, there has been an increased interest in the diagnostic value of the BAT as it has the potential to mimic the clinical phenotype of sIgE sensitized patients, in contrast to allergen-specific IgE levels. This diagnostic potential would be of particular interest for food allergies present early in life such as peanut, cow’s milk and eggs, which require an expensive, time-consuming and patient unfriendly oral food challenge (OFC) for diagnosis. However, routine applications of the BAT for clinical use are not yet feasible due to the lack of standardized protocols and large clinical validation studies. This review will summarize the current data regarding the application of the BAT in food allergy (FA) for cow’s milk, egg and peanut, being the most common causes of FA in children. Additionally, it will discuss the hurdles for widespread clinical use of the BAT and possible future directions for this diagnostic procedure.Keywords: basophil activation test, food allergy, diagnosis, peanut, cow’s milk, chicken egg

Abstract Background The role of serotonin and its metabolic pathway in proper functioning of the pancreas has not been thoroughly investigated yet in acute pancreatitis (AP) patients. Tryptophan hydroxylase (TPH) as the rate-limiting enzyme of serotonin synthesis has been considered for possible associations in various diseases. Single-nucleotide polymorphisms (SNPs) in TPH genes have been already described in associations with psychiatric and digestive system disorders. This study aimed to explore the association of a rs211105 (T/G) polymorphism in TPH1 gene with tryptophan hydroxylase 1 concentrations in blood serum in a population of acute pancreatitis patients, and to investigate this association with acute pancreatitis susceptibility. Results Our data showed an association between the presence of the T allele at the position rs211105 (OR = 2.47, 95 % CI 0.94–6.50, p = 0.06) under conditions of a decreased AP incidence. For TT and GT genotypes in the control group, the lowest concentration of TPH was associated with higher serotonin levels (TT: Rs = − 0.415, p = 0.0018; GT: Rs = − 0.457, p = 0.0066), while for the AP group the highest levels of TPH among the TT genotype were associated with lower levels of serotonin (TT: Rs = − 0.749, p

Breakthrough SARS-CoV-2 infections have been reported in fully vaccinated individuals, in spite of the high efficacy of the currently available vaccines, proven in trials and real-world studies. Several variants of concern (VOC) have been proffered to be associated with breakthrough infections following immunization. In this study, we investigated 378 breakthrough infections recorded between January and July 2021 and compared the distribution of SARS-CoV-2 genotypes identified in 225 fully vaccinated individuals to the frequency of circulating community lineages in the region of South Limburg (The Netherlands) in a week-by-week comparison. Although the proportion of breakthrough infections was relatively low and stable when the Alpha variant was predominant, the rapid emergence of the Delta variant lead to a strong increase in breakthrough infections, with a higher relative proportion of individuals vaccinated with Vaxzevria or Jcovden being infected compared to those immunized with mRNA-based vaccines. A significant difference in median age was observed when comparing fully vaccinated individuals with severe symptoms (83 years) to asymptomatic cases (46.5 years) or individuals with mild-to-moderate symptoms (42 years). There was no association between SARS-CoV-2 genotype or vaccine type and disease symptoms. Furthermore, the majority of adaptive mutations were concentrated in the N-terminal domain of the Spike protein, highlighting its role in immune evasion. Interestingly, symptomatic individuals harbored significantly higher SARS-CoV-2 loads than asymptomatic vaccinated individuals and breakthrough infections caused by the Delta variant were associated with increased viral loads compared to those caused by the Alpha variant. In addition, we investigated the role of the Omicron variant in causing breakthrough infections by analyzing 135 samples that were randomly selected for genomic surveillance during the transition period from Delta to Omicron. We found that the proportion of Omicron vs. Delta infections was significantly higher in individuals who received a booster vaccine compared to both unvaccinated and fully vaccinated individuals. Altogether, these results indicate that the emergence of the Delta variant and in particular Omicron has lowered the efficiency of particular vaccine types to prevent SARS-CoV-2 infections and that, although rare, the elderly are particularly at risk of becoming severely infected as the consequence of a breakthrough infection.

Objectives: To evaluate the effect of French Maritime Pine Bark Extract (PBE; Pycnogenol®) on immune, oxidative stress and neurochemical biomarkers in paediatric Attention-Deficit Hyperactivity Disorder (ADHD) as compared to methylphenidate (MPH) and placebo. Results: Paediatric ADHD patients (n = 88, 70 % male, mean age 10.1 years) were randomised (placebo (n = 30), PBE (n = 32) and MPH (n = 26)) receiving 20 mg/day if