Necrosome-positive granulovacuolar degeneration is associated with TDP-43 pathological lesions in the hippocampus of ALS/FTLD cases.

AIM: Granulovacuolar degeneration (GVD) in Alzheimer's disease (AD) involves the necrosome, which is a protein complex consisting of phosphorylated receptor-interacting protein kinase 1 (pRIPK1), pRIPK3 and phosphorylated mixed lineage kinase domain-like protein (pMLKL). Necrosome-positive GVD was associated with neuron loss in AD. GVD was recently linked to the C9ORF72 mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with transactive response DNA-binding protein (TDP-43) pathology (FTLD-TDP). Therefore, we investigated whether GVD in cases of the ALS-FTLD-TDP spectrum (ALS/FTLD) shows a similar involvement of the necrosome as in AD, and whether it correlates with diagnosis, presence of protein aggregates and cell death in ALS/FTLD. METHODS: We analysed the presence and distribution of the necrosome in post-mortem brain and spinal cord of ALS and FTLD-TDP patients (n = 30) with and without the C9ORF72 mutation, and controls (n = 22). We investigated the association of the necrosome with diagnosis, the presence of pathological protein aggregates and neuronal loss. RESULTS: Necrosome-positive GVD was primarily observed in hippocampal regions of ALS/FTLD cases and was associated with hippocampal TDP-43 inclusions as the main predictor of the pMLKL-GVD stage, as well as with the Braak stage of neurofibrillary tangle pathology. The central cortex and spinal cord, showing motor neuron loss in ALS, were devoid of any accumulation of pRIPK1, pRIPK3 or pMLKL. CONCLUSIONS: Our findings suggest a role for hippocampal TDP-43 pathology as a contributor to necrosome-positive GVD in ALS/FTLD. The absence of necroptosis-related proteins in motor neurons in ALS argues against a role for necroptosis in ALS-related motor neuron death.

Animal experiments show impact of vaccination on reduction of SARS-CoV-2 virus circulation: A model for vaccine development?

BACKGROUND: In the pre-clinical phase, SARS-CoV-2 vaccines were tested in animal models, including exposure trials, to investigate protection against SARS-CoV-2. These studies paved the way for clinical development. The objective of our review was to provide an overview of published animal exposure results, focussing on the capacity of vaccines to reduce/prevent viral shedding. METHOD: Using Medline, we retrieved eighteen papers on eight different vaccine platforms in four animal models. Data were extracted on presence/absence of viral RNA in nose, throat, or lungs, and neutralizing antibody levels in the blood. RESULTS: All vaccines showed a tendency of reduced viral load after exposure. Particularly nasal swab results are likely to give an indication about the impact on virus excretion in the environment. Similarly, the reduction or prevention of viral replication in the bronchoalveolar environment might be related with disease prevention, explaining the high efficacy in clinical trials. DISCUSSION: Although it remains difficult to compare the results directly, the potential for a strong reduction of transmission was shown, indicating that the animal models predicted what is observed in the field after large scale human vaccination. This merits further attention for standardization of exposure experiments, with the intention to speed up future vaccine development.

Amyotrophic lateral sclerosis: a clinical review.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily the motor system, but in which extra-motor manifestations are increasingly recognized. The loss of upper and lower motor neurons in the motor cortex, the brain stem nuclei and the anterior horn of the spinal cord gives rise to progressive muscle weakness and wasting. ALS often has a focal onset but subsequently spreads to different body regions, where failure of respiratory muscles typically limits survival to 2-5 years after disease onset. In up to 50% of cases, there are extra-motor manifestations such as changes in behaviour, executive dysfunction and language problems. In 10%-15% of patients, these problems are severe enough to meet the clinical criteria of frontotemporal dementia (FTD). In 10% of ALS patients, the family history suggests an autosomal dominant inheritance pattern. The remaining 90% have no affected family members and are classified as sporadic ALS. The causes of ALS appear to be heterogeneous and are only partially understood. To date, more than 20 genes have been associated with ALS. The most common genetic cause is a hexanucleotide repeat expansion in the C9orf72 gene, responsible for 30%-50% of familial ALS and 7% of sporadic ALS. These expansions are also a frequent cause of frontotemporal dementia, emphasizing the molecular overlap between ALS and FTD. To this day there is no cure or effective treatment for ALS and the cornerstone of treatment remains multidisciplinary care, including nutritional and respiratory support and symptom management. In this review, different aspects of ALS are discussed, including epidemiology, aetiology, pathogenesis, clinical features, differential diagnosis, investigations, treatment and future prospects.

In vitro antimicrobial combinatory effect of Cinnamomum cassia essential oil with 8-hydroxyquinoline against Staphylococcus aureus in liquid and vapour phase.

AIMS: The objective of the study was to evaluate the antimicrobial interactions between two volatile agents, Cinnamomum cassia essential oil (CCEO) and 8-hydroxyquinoline (8-HQ) against Staphylococcus aureus strains in liquid and vapour phases. METHODS AND RESULTS: In vitro antimicrobial effect of CCEO in combination with 8-HQ was evaluated against 12 strains of S. aureus by broth volatilization chequerboard method. Results show additive effects against all S. aureus strains for both phases. In several cases, sums of fractional inhibitory concentration values of our test combinations were lower than 0·6, which can be considered as a strong additive interaction. Moreover, composition of CCEO was analysed by gas chromatography-mass spectrometry analysis. In the CCEO, 26 compounds in total were identified, where (E)-cinnamaldehyde was the predominant compound, followed by cinnamyl acetate, α-copaene, bornyl acetate and caryophyllene. CONCLUSIONS: Results showed additive in vitro growth-inhibitory effect of CCEO and 8-HQ combination against various standard strains and clinical isolates of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on antibacterial effect of 8-HQ and CCEO combination in liquid and vapour phases. Results of the study suggest these agents as potential candidates for development of new anti-staphylococcal applications that can be used in the inhalation therapy against respiratory infections.

Serum neurofilament light chain levels as a marker of upper motor neuron degeneration in patients with Amyotrophic Lateral Sclerosis.

AIMS: Amyotrophic lateral sclerosis (ALS) is the most common motor neuron degeneration disease with a diagnostic delay of about 1 year after symptoms onset. In ALS, blood neurofilament light chain (NfL) levels are elevated, but it is not entirely clear what drives this increase and what the diagnostic performance of serum NfL is in terms of predictive values and likelihood ratios. The aims of this study were to further explore the prognostic and diagnostic performances of serum NfL to discriminate between patients with ALS and ALS mimics, and to investigate the relationship between serum NfL with motor neuron degeneration. METHODS: The diagnostic performances of serum NfL were based on a cohort of 149 serum samples of patients with ALS, 19 serum samples of patients with a disease mimicking ALS and 82 serum samples of disease control patients. The serum NfL levels were correlated with the number of regions (thoracic, bulbar, upper limb and lower limb) displaying upper and/or lower motor neuron degeneration. The prognostic performances of serum NfL were investigated based on a Cox regression analysis. RESULTS: The associated predictive values and likelihood ratio to discriminate patients with ALS and ALS mimics were established. Serum NfL was associated with motor neuron degeneration driven by upper motor neuron (UMN) degeneration and was independently associated with survival in patients with ALS. CONCLUSIONS: Altogether, these findings suggest that elevated serum NfL levels in ALS are driven by UMN degeneration and the disease progression rate and are independently associated with survival at time of diagnosis.

Tracking the sources of psychrotrophic bacteria contaminating chicken cuts during processing.

The major aim of the study was to establish the routes via which spoilage associated psychrotrophic bacteria contaminate poultry products at a large processing plant located in Belgium. Environmental samples were collected consisting of samples of air and swabs of food contact surfaces. Product samples were also collected consisting of modified atmosphere packaged (MAP) chicken wings and legs, which were analyzed microbiologically on the same day they were produced as well as after their sell-by date. Psychrotrophic bacteria from these samples were subsequently clustered and identified by means of MALDI-TOF MS and 16S rRNA gene sequencing. Carnobacterium maltaromaticum was determined to dominate the spoilage flora of both wings and legs. Other psychrotrophic bacteria able to grow on MRS which were identified on expired wings and legs included Carnobacterium divergens, Brocothrix thermosphacta, Lactobacillus curvatus, and Lactobacillus brevis. These were determined to arise from food contact surfaces such as cutting blades, leg hooks, Ertalon and polyurethane conveyor belts, working tables, and the hands of the operators. Importantly, it was determined that cleaning and disinfection was largely inadequate. Air was also determined to be an important vector of psychrotrophic bacteria in the processing environment, potentially contaminating the products directly or indirectly.

Altered CD4+ T cell immunity in nurses occupationally exposed to viral pathogens.

Pathogen exposure, including but not limited to herpesviruses, moulds the shape of the immune system, both at a basal state and in response to immune challenge. However, little is known about the impact of high exposure to other viruses on baseline immune signatures and how the immune system copes with repetitive exposures to maintain a balanced functionality. Here we investigated baseline immune signatures, including detailed T cell phenotyping, antigen-specific CD4+ and CD8+ T cell responses and cytokine profile in paediatric (PED) nurses, who have high occupational exposure to viral pathogens including varicella zoster virus (VZV) and respiratory viruses, and in neonatal intensive care unit (NICU) nurses, as a control group with infrequent occupational exposure. Our results show a lower CD4+ T cell response to two VZV proteins (IE62 and gE) and to tetanus toxoid (TT) in PED nurses who are cytomegalovirus (CMV)-seronegative, compared to CMV-seronegative NICU nurses, and that the decline might be more pronounced the more sustained the exposure. This decline might be due to an attrition of VZV- and TT-specific T cells as a result of the continuous pressure on the CD4+ T cell compartment. Moreover, our data suggest that the distinct T cell phenotypes known to be associated with CMV-seropositivity might be less prominent in PED nurses compared to NICU nurses, implying a plausible attenuating effect of occupational exposure on CMV-associated immunosenescence. Overall, this pilot study reveals an impact of occupational exposure to viral pathogens on CD4+ T cell immunity and supports further investigation in a larger cohort.

Hepatitis C: The beginning of the end-key elements for successful European and national strategies to eliminate HCV in Europe.

Hepatitis C virus (HCV) infection is a major public health problem in the European Union (EU). An estimated 5.6 million Europeans are chronically infected with a wide range of variation in prevalence across European Union countries. Although HCV continues to spread as a largely "silent pandemic," its elimination is made possible through the availability of the new antiviral drugs and the implementation of prevention practices. On 17 February 2016, the Hepatitis B & C Public Policy Association held the first EU HCV Policy Summit in Brussels. This summit was an historic event as it was the first high-level conference focusing on the elimination of HCV at the European Union level. The meeting brought together the main stakeholders in the field of HCV: clinicians, patient advocacy groups, representatives of key institutions and regional bodies from across European Union; it served as a platform for one of the most significant disease elimination campaigns in Europe and culminated in the presentation of the HCV Elimination Manifesto, calling for the elimination of HCV in Europe by 2030. The launch of the Elimination Manifesto provides a starting point for action in order to make HCV and its elimination in Europe an explicit public health priority, to ensure that patients, civil society groups and other relevant stakeholders will be directly involved in developing and implementing HCV elimination strategies, to pay particular attention to the links between hepatitis C and social marginalization and to introduce a European Hepatitis Awareness Week.

Can Flanders resist the measles outbreak? Assessing vaccination coverage in different age groups among Flemish residents.

The Belgian strategic plan to eliminate measles contains several vaccination strategies including routine immunisation programmes and catch-up campaigns. A new expanded programme on immunisation-based survey (2016) assessed the uptake of the recommended measles-mumps-rubella (MMR) vaccine in three different cohorts: toddlers, adolescents and parents of toddlers. A two-stage cluster sampling technique was used to select 875 toddlers (age 18-24 months) and 1250 adolescents (born in 2000) from 107 municipalities in Flanders. After consent of the parent(s), 746 (85.2%) families of toddlers and 1012 (81.0%) families of adolescents were interviewed at home. Measles vaccination coverage was high at 18-24 months (96.2%) and 81.5% were vaccinated at recommended age. Toddlers who had two siblings or a non-working mother or changed vaccinator were more at risk for not being vaccinated. Coverage of the teenager dose reached 93.5% and was lower in adolescents with educational underachievement or whose mother was part-time working or with a non-Belgian background. Only 56.0% of mothers and 48.3% of fathers remembered having received at least one measles-containing vaccine. Although measles vaccination coverage in toddlers meets the required standards for elimination, administration of the teenager dose of MMR vaccine and parent compliance to the recent measles catch-up campaign in Flanders leave room for improvement.

Assessing the risk of measles resurgence in a highly vaccinated population: Belgium anno 2013.

Despite long-standing two-dose measles-mumps-rubella (MMR) vaccination, measles outbreaks still occur in highly vaccinated European populations. For instance, large measles outbreaks occurred in France (2008­13), the United Kingdom (2012­13) and the Netherlands (2012). Based on a multicohort model approach, using spatial serological survey data, MMR vaccination coverage data and data on social contacts, we found effective reproduction numbers significantly higher than 1 for measles in Belgium. This indicates that at one of the expected re-introductions, a measles outbreak is likely to spread, especially when it occurs during school term. The predicted average effective reproduction number increased over a 30-year time span from 1.3 to 2.2 and from 1.9 to 3.2 for basic reproduction numbers of 12 and 18, respectively. The expected relative measles incidence was highest in infants under one year of age, in adolescents and young adults. In conclusion, gradually increasing proportions of susceptible adolescents and young adults provide through their highly active social life an avenue for measles to resurge in large outbreaks upon re-introduction in Belgium, especially during school terms. Infants form an important vulnerable group during future measles outbreaks.

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

Universal hepatitis B vaccination in Belgium: impact on serological markers 3 and 7 years after implementation.

Hepatitis B virus (HBV) can be eliminated by effective universal vaccination. In Belgium, a free-of-charge HBV vaccination programme in infants with catch-up in adolescents was introduced in 1999. To evaluate the effects in <20-year-olds, seroprotection (anti-HBs >11 mIU/ml, according to the assay) and markers of infection (anti-HBc, HBsAg) were assessed in 2443 residual sera collected 7-8 years after implementation of the programme. The maximal prevalence of a solely anti-HBs seroprotective ('vaccinated') serostatus was 82·9% at age 1 year and 60·5% at age 13 years. A clear increase was found in age cohorts targeted by the campaign after a similar serosurvey conducted 4 years earlier. The prevalence of HBV infection remained unchanged at a low level (1·8% in 2006) similar to pre-vaccination data (1993-1994). We conclude that universal HBV vaccination has achieved overall high levels of vaccine-induced immunity, despite regional variations, which may give rise to pockets of susceptible young adults in the future.

Rotavirus vaccination coverage and adherence to recommended age among infants in Flanders (Belgium) in 2012.

In Belgium, rotavirus vaccination has been recommended and partially reimbursed since October 2006. Through a retrospective survey in 2012, we estimated the coverage rate of the rotavirus vaccination in Flanders among infants born in 2010. Using a standardised questionnaire, 874 families were interviewed at home, collecting information on demographic characteristics, socio-economic background and documented vaccination history (updated from medical files and vaccination database, if needed). Adherence to the recommended age for vaccination (8, 12 and 16 weeks) was also assessed. The coverage rate for two doses of rotavirus vaccination was 92.2% (95% confidence interval: 90.2-93.8). Respectively 31.7% and 10.1% of the children received their first and second dose at the recommended age. Incomplete vaccination was often a deliberate choice of the parents. Only eight children (1%) were vaccinated after the maximum age of 26 weeks. Factors identified by multiple logistic regression as related to incomplete vaccination were: living in the province of Antwerp, unemployed mother, and three or more older siblings in the household. Four years after introduction, the coverage rates were surprisingly high for a vaccine that is not fully reimbursed and not readily available in the vaccinator's fridge, which is the case for the other recommended infant vaccines.

Epidemiology and outcome of invasive pneumococcal disease among adults in Belgium, 2009-2011.

This epidemiological study examined morbidity and case fatality of invasive pneumococcal disease (IPD) in adults in Belgium as well as distribution and antibiotic susceptibility of Streptococcus pneumoniae serotypes.Adults hospitalised with microbiologically proven IPD were prospectively enrolled. The study started in 2009 with patients aged ≥50 years, whereas in 2010 and 2011, patients aged ≥18 years were included. The clinical presentation, patient profile, treatment, outcome, and mortality were recorded during hospitalisation.Outcome was also assessed one month afterdischarge. Of the 1,875 patients with IPD identified, 1,332 were included in the analysis. Bacteraemic pneumonia, affecting 1,049 of the patients, was the most frequent IPD type (79%), and chronic obstructive pulmonary disease and cancer were the main comorbidities.One-third of patients required admission to intensive care unit. A total of 208 (16%) patients died during hospitalisation and an additional 21 (2%) within one month after discharge. Case fatality rates of ≥20%were observed in patients with chronic heart failure, hepatic disease, and renal insufficiency. Serotypes 7F, 1, 19A, and 3 were the most prevalent and together accounted for 47% (569/1,214) of all IPD cases and 42% (80/189) of mortality. Of the patient isolates, 21% (255/1,204) were resistant to erythromycin and 22% (264/1,204) to tetracycline. Penicillin non-susceptibility was mostly found in serotype 19A isolates. These baseline data are essential when assessing the impact of pneumococcal conjugate vaccination in adults in the future.

Antimicrobial activity of traditional medicinal plants from Ankober District, North Shewa Zone, Amhara Region, Ethiopia.

CONTEXT: Traditional medicinal plants have long been used in Ethiopia to treat human and livestock ailments. Despite a well-documented rich tradition of medicinal plant use in the country, their direct antimicrobial effects are still poorly known. OBJECTIVE: To investigate the antimicrobial activity of 19 medicinal plant species that were selected based on the ethnobotanical information on their traditional use to treat infectious diseases in Ankober District. METHODS: About 23 different ethanol extracts of plants obtained by maceration of various parts of 19 medicinal plant species were studied for potential antimicrobial activity using a broth microdilution method against Bacillus cereus, Bacteroides fragilis, Candida albicans, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. RESULTS: Plant extracts from Embelia schimperi Vatke (Myrsinaceae) showed the strongest antibacterial activity with a minimum inhibitory concentration (MIC) value of 64 µg/ml against B. cereus, L. monocytogenes, and S. pyogenes. Growth inhibitory activities were also observed for extracts of Ocimum lamiifolium Hochst. (Lamiaceae) against S. pyogenes, and those of Rubus steudneri Schweinf. (Rosaceae) against S. epidermidis at an MIC value of 128 µg/ml. Generally, 74% of ethanol extracts (17 extracts) showed antimicrobial activity against one or more of the microbial strains tested at an MIC value of 512 µg/ml or below. DISCUSSION AND CONCLUSIONS: Results confirm the antimicrobial role of traditional medicinal plants of Ankober and warrant further investigations on promising medicinal plant species so as to isolate and characterise chemicals responsible for the observed strong antimicrobial activities.

The state of hepatitis B and C in the Mediterranean and Balkan countries: report from a summit conference.

The burden of disease due to chronic viral hepatitis constitutes a global threat. In many Balkan and Mediterranean countries, the disease burden due to viral hepatitis remains largely unrecognized, including in high-risk groups and migrants, because of a lack of reliable epidemiological data, suggesting the need for better and targeted surveillance for public health gains. In many countries, the burden of chronic liver disease due to hepatitis B and C is increasing due to ageing of unvaccinated populations and migration, and a probable increase in drug injecting. Targeted vaccination strategies for hepatitis B virus (HBV) among risk groups and harm reduction interventions at adequate scale and coverage for injecting drug users are needed. Transmission of HBV and hepatitis C virus (HCV) in healthcare settings and a higher prevalence of HBV and HCV among recipients of blood and blood products in the Balkan and North African countries highlight the need to implement and monitor universal precautions in these settings and use voluntary, nonremunerated, repeat donors. Progress in drug discovery has improved outcomes of treatment for both HBV and HCV, although access is limited by the high costs of these drugs and resources available for health care. Egypt, with the highest burden of hepatitis C in the world, provides treatment through its National Control Strategy. Addressing the burden of viral hepatitis in the Balkan and Mediterranean regions will require national commitments in the form of strategic plans, financial and human resources, normative guidance and technical support from regional agencies and research.

Antibody persistence and immune memory in adults, 15 years after a three-dose schedule of a combined hepatitis A and B vaccine.

A combined hepatitis A and B vaccine is available since 1996. Two separate open-label primary studies evaluated the immunogenicity and safety of this hepatitis A and B vaccine (720 EI.U of HAV and 20 µg of HBsAg) in 306 healthy subjects aged 17-43 years who received three doses of the vaccine following a 0, 1, and 6 months schedule. These subjects were followed up annually for the next 15 years to evaluate long-term persistence of anti-HAV and anti-HBs antibodies. The subjects whose antibody concentrations fell below the cut-offs between Year 11 and Year 15 (anti-HAV: <15 mIU/ml; anti-HBs: <10 mIU/ml) were offered an additional dose of the appropriate monovalent hepatitis A and/or B vaccine. In subjects who received the additional vaccine dose, a blood sample was collected 1 month after vaccination. At the Year 15 time point, all subjects in Study A and Study B were seropositive for anti-HAV antibodies and 89.3% and 92.9% of subjects in the respective studies had anti-HBs antibody concentrations ≥10 mIU/ml. Four subjects (two in each study) received an additional dose of monovalent hepatitis B vaccine and mounted anamnestic responses to vaccination. No vaccine-related serious adverse events were reported. This study confirms the long-term immunogenicity of the three-dose regimen of the combined hepatitis A and B vaccine, as eliciting long-term persistence of antibodies and immune memory against hepatitis A and B for up to at least 15 years after a primary vaccination.

Detection of human papillomavirus DNA in urine. A review of the literature.

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

The health and economic burden of chickenpox and herpes zoster in Belgium.

Varicella-zoster virus causes chickenpox (CP) and after reactivation herpes zoster (HZ). Vaccines are available against both diseases warranting an assessment of the pre-vaccination burden of disease. We collected data from relevant Belgian databases and performed five surveys of CP and HZ patients. The rates at which a general practitioner is visited at least once for CP and HZ are 346 and 378/100 000 person-years, respectively. The average CP and HZ hospitalization rates are 5·3 and 14·2/100 000 person-years respectively. The direct medical cost for HZ is about twice as large as the direct medical cost for CP. The quality-adjusted life years lost for ambulatory CP patients consulting a physician is more than double that of those not consulting a physician (0·010 vs. 0·004). In conclusion, both diseases cause a substantial burden in Belgium.