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Abstract Background Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.

Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures.

Aim: This cohort study aimed to describe functional mobility in Dravet syndrome, a developmental and epileptic encephalopathy., Method: Functional mobility was assessed in individuals (aged 3-25 years), diagnosed with Dravet syndrome, using the Functional Mobility Scale (FMS), Mobility Questionnaire 28 (MobQues28), and estimated walking distance. Secondary outcome variables were Gait Profile Score (GPS), walking velocity, age at independent walking, intellectual disability, seizure frequency, genetic variant type, and body mass index (BMI)., Results: Forty participants aged 3 years to 24 years 2 months (mean = 12 years 2 months) had a median MobQues28 of 79%, median scores of 5, 5, and 4 for the FMS 5 m, 50 m, and 500 m and a median estimated walking distance of 1 km to 3 km. Most difficulties were seen in walking up and down the stairs, walking over obstacles, kicking a ball, and running. MobQues28 scores showed a significant decrease (-6.6%, p = 0.016) in the age category of young adults (≥18 years). After correcting for age, MobQues28 was correlated to age at independent walking (-0.485, p = 0.002), GPS (-0.460, p = 0.003), and walking velocity (0.334, p = 0.038). Analysis of variance showed a significant effect of intellectual disability and BMI on MobQues28 (p = 0.029, p = 0.049). No effect of seizure frequency or genetic variant was found (p = 0.579, p = 0.337)., Interpretation: Functional mobility limitations were observed mainly in dual tasks and activities requiring stability, with limitations increasing from the age of 18 years. Age at independent walking, gait impairments, intellectual disability, and BMI can impact functional mobility in Dravet syndrome., What This Paper Adds: Most limitations were seen in dual task activities and activities that required more stability. Deterioration in functional mobility occurred in young adults. The more gait impairments, the more functional mobility limitations. Age at independent walking, intellectual disability, and body mass index can impact functional mobility., (© 2023 Mac Keith Press.)

Zoonotic hepatitis E virus (HEV) genotype 3 infections are the predominant cause of acute viral hepatitis in Europe, mostly associated with the consumption of HEV contaminated pork meat. In this study we looked at the HEV RNA positivity rate of pork meat products readily available from Belgian supermarkets and evaluated the overall HEV consumer exposure in a Belgian context. Two basic assessments were performed in a 'worst-case' scenario setting: one solely focusing on the contamination level of the product itself (ingredients and processing parameters) and another estimating the overall consumer exposure, taking into account consumption habits in Belgium. Non-thermal-processed ready-to-eat (i.e. ready for consumption without additional cooking step by consumer) pork meat products (e.g. raw dried sausages), had a high estimated HEV contamination level, while thermal-processed ready-to-eat pork meat products (e.g. pork liver pâté) had the highest overall consumer exposure estimates. Following these assessments, pork liver pâtés, raw dried hams and raw dried sausages (n = 54) were purchased from Belgian supermarkets (n = 3) and analyzed for HEV RNA by RT-PCR. In total, 31 % (n = 17) products tested positive. HEV RNA was found in 65 % of the pork liver pâtés, 15 % of raw dried hams and 0 % of raw dried sausages. Phylogenetic analysis of four isolates (all gt3c) from pork liver pâté samples showed similarities with human clinical cases from Germany and Belgium., Competing Interests: Declaration of competing interest All authors disclose no commercial associations that might create a conflict of interest in connection with this study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of this study was metagenomics analyses of acquired antibiotic-resistance genes (ARGs) in the intestinal microbiome of two important food-animal species in Hungary from a One Health perspective. Intestinal content samples were collected from 12 domestic pigs ( Sus scrofa ) and from a common carp ( Cyprinus carpio ). Shotgun metagenomic sequencing of DNA purified from the intestinal samples was performed on the Illumina platform. The ResFinder database was applied for detecting acquired ARGs in the assembled metagenomic contigs. Altogether, 59 acquired ARG types were identified, 51 genes from domestic pig and 12 genes from the carp intestinal microbiome. ARG types belonged to the antibiotic classes aminoglycosides (27.1%), tetracyclines (25.4%), β-lactams (16.9%), and others. Of the identified ARGs, tet (E), a bla OXA - 48-like β-lactamase gene, as well as cphA4 , ampS , aadA2 , qnrS2 , and sul1 , were identified only in carp but not in swine samples. Several of the detected acquired ARGs have not yet been described from food animals in Hungary. The tet (Q), tet (W), tet (O), and mef (A) genes detected in the intestinal microbiome of domestic pigs had also been identified from free-living wild boars in Hungary, suggesting a possible relationship between the occurrence of acquired ARGs in domestic and wild animal populations.

Along with (in) direct contact with animals and a contaminated environment, humans are exposed to antibiotic-resistant bacteria by consumption of food. The implications of ingesting antibiotic-resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal E. coli strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial E. coli plasmid donor concentration (10 5 and 10 7 CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 10 4 to 7.9 × 10 6 CFU cefotaxime resistant /Ml colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.

Food-producing animals are indicated as a reservoir of antibiotic resistance genes and a potential vector for transmission of plasmid-encoded antibiotic resistance genes by conjugation to the human intestinal microbiota. In this study, transfer of an antibiotic resistance plasmid from a commensal E. coli originating from a broiler chicken towards the human intestinal microbiota was assessed by using a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). This in vitro model mimics the human intestinal ecosystem and received a single dose of 10 9 E. coli MB6212, which harbors a plasmid known to confer resistance towards several antibiotics including tetracycline, sulfamethoxazole and cefotaxime. Since the degree of stress imposed by stomach pH and bile acids vary with the consumed meal size, the effect of meal size on E. coli donor survival and on plasmid transfer towards lumen and mucosal coliforms and anaerobes was determined. The administered commensal E. coli strain survived stomach acid and bile salt stress and was able to grow in the colon environment during the timeframe of the experiment (72 h). Transfer of antibiotic resistance was observed rapidly since cultivable transconjugant coliforms and anaerobes were already detected in the lumen and mucosa after 2 h in the simulated proximal colon. The presence of the resistance plasmid in the transconjugants was confirmed by PCR. Differences in meal size and adapted digestion had neither a detectable impact on antibiotic resistance transfer, nor on the survival of the E. coli donor strain, nor on short chain fatty acid profiles. The median number of resistant indigenous coliforms in the lumen of the inoculated colon vessels was 5.00 × 10 5 cfu/ml [min - max: 3.47 × 10 4 -3.70 × 10 8 cfu/ml], and on the mucosa 1.44 × 10 7 cfu/g [min-max: 4.00 × 10 3 -4.00 × 10 8 cfu/g]. Exact quantification of the anaerobic transconjugants was difficult, as (intrinsic) resistant anaerobic background microbiota were present. QPCR data supported the observation of plasmid transfer in the simulated colon. Moreover, inoculation of E. coli MB6212 had no significant impact on the microbial diversity in the lumen as determined by 16 S ribosomal gene based next generation sequencing on lumen samples. This study demonstrates that a commensal, antibiotic resistant E. coli strain present in food can transfer its antibiotic resistance plasmid relatively quickly to intestinal microbiota in the M-SHIME. The spread and persistence of antibiotic resistance genes and resistant bacteria in our intestinal system is an alarming scenario which might present clinical challenges, since it implies a potential reservoir for dissemination to pathogenic bacteria., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Food-producing animals represent one of the sources of antibiotic resistant commensal bacteria. There is an increasing awareness that these bacteria might have the potential to transfer their resistance genes to other (pathogenic) bacteria. In this study, 50 commensal Escherichia coli strains originating from food-producing animals and resistant to the "highest priority, critically important antibiotics" cefotaxime and/or ciprofloxacin, were selected for further characterization. For each strain (i) an antibiogram, (ii) the phylogenetic group, (iii) plasmid replicon type, (iv) presence and identification of integrons, and (v) antibiotic resistance transfer ratios were determined. Forty-five of these strains were resistant to 5 or more antibiotics, and 6 strains were resistant to 10 or more antibiotics. Resistance was most common to ampicillin (100%), sulfamethoxazole, ciprofloxacin (82%), trimethoprim, tetracycline (74%), cefotaxime, (70%) and ceftazidime (62%). Phylogenetic groups A (62%) and B1 (26%) were most common, followed by C (8%) and E (4%). In 43 strains, more than 1 replicon type was detected, with FII (88%), FIB (70%), and I1 (48%) being the most encountered types. Forty strains, positive for integrons, all harbored a class I integron and seven of them contained an additional class II integron. No class III integrons were detected. The antibiotic resistance transfer was assessed by liquid mating experiments. The transfer ratio, expressed as the number of transconjugants per recipient, was between 10 -5 and 10 0 for cefotaxime resistance and between 10 -7 and 10 -1 for ciprofloxacin resistance. The results of the current study prove that commensal E. coli in food-production animals can be a source of multiple resistance genes and that these bacteria can easily spread their ciprofloxacin and cefotaxime resistance.

The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H 2 O 2 , benzalkonium chloride, 55°C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i) Acanthamoeba castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34- to 4.72-log 10 cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H 2 O 2 (>92% decrease in median log 10 CFU/ml reduction) and 70% ethanol (>99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of Yersinia enterocolitica (74% decrease in median log 10 reduction), Escherichia coli (58%), and Salmonella enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%), and Listeria monocytogenes (99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria. IMPORTANCE The results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens., (Copyright © 2017 American Society for Microbiology.)

Broiler chicks are transported to production sites within one to 2 d post-hatch. Possible effects of this transportation are poorly understood and could vary among chicks from breeder flocks of different ages. The aim of the present study was to investigate the effects of transportation duration and parental flock age on chick welfare, productivity, and quality. After hatch in a commercial hatchery, 1,620 mixed-sex chicks from 29-wk old (young) and 1,620 chicks from 60-wk old (old) breeders were subjected to transportation of 1.5 h or 11 h duration. After transportation, 2,800 chicks were divided among 100 pens, with each pen containing 28 chicks from one transportation crate (2 or 3 pens per crate). From the remaining chicks, on average 6 chicks (min 4, max 8) per crate (n = 228) were randomly selected and assessed for chick quality, weighed, and culled for yolk sac weighing (one d). Chicks that had not been assigned to pens or were not used for post-transportation measurements, were removed from the experiment (n = 212). Mortality, ADG, BW, and feed conversion ( FC: ) of the experimental chicks were recorded until 41 d. Meat quality was measured for breast fillets (n = 47). No interaction effect of parental age and transportation duration was found for any variables. BW and yolk sac weight at one d were lower for chicks transported 11 h than 1.5 h and for chicks from young versus old breeders. The effect of parental flock age on BW persisted until slaughter. Additionally, parental age positively affected ADG until slaughter. Chick quality was lower in chicks from old versus young breeders. Chick quality and productivity were not affected by transportation duration. Mortality and meat quality were not affected by either parental age or transportation duration. To conclude, no long-term detrimental effects were found from long post-hatch transportation in chicks from young or old parent flocks. Based on these results, we suggest that 11 h post-hatch transportations under similar conditions do not impose long-term welfare or productivity risks., (© The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.)

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.

Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients., Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach., Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background., Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.

Objective: To assess long-term metabolic outcomes in children with diabetes mellitus that was diagnosed when they were <6 years old., Study Design: A cohort of 66 children with diabetes mellitus that had a duration of at least 5 years and was diagnosed before they were 6 years old. Thirty-four children were treated at diagnosis with multiple daily subcutaneous insulin injections (MDI), and all these children, except 3, were switched to continuous subcutaneous insulin infusion (CSII; group A). Thirty-two children received CSII as initial treatment (group B)., Results: Hemoglobin A1c values were significantly lower in patients receiving CSII than MDI during all the 8 years of follow-up except one (year 1: 6.9%+/-0.9% versus 7.6%+/-1%, P=.011 ; year 4: 7.4%+/-0.8% versus 8.1%+/-0.9%, P=.006; year 7: 7.6%+/-0.5% versus 8.3%+/-0.8%, P=.001). The incidence of severe hypoglycemia was greatly decreased for the CSII group (9.8 versus 22.3 episodes/100 patient-years, P=.016). In group A, hemoglobin A1c values increased during the study period, and in group B, they increased only during the first 2 years and remained constant thereafter. Only 9.1% of patients did not use or abandoned CSII., Conclusion: CSII in children<6 years of age enables better long-term metabolic control and lowers the risk of severe hypoglycemia better than MDI, especially when initiated at diagnosis., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

We report the case of a young adolescent with ataxia telangiectasia (AT) and life-threatening haemorrhage from the bladder due to a combination of bladder wall telangiectasis, immunosuppressive therapy and an infection with polyomavirus JC. BK and JC are both members of the polyomavirus family. BK virus is a known cause of haemorrhagic cystitis in bone-marrow and nephropathy in kidney transplant patients, whereas JC virus is mainly associated with progressive multifocal leukoencephalopathy and only rarely found in haemorrhagic cystitis. Although opportunistic infections are uncommon in AT and virus replication was described as being down-regulated in ATM (AT mutated protein)-deficient cells, clinicians should be aware that severe haematuria in a patient with AT and undergoing immunosuppressive therapy is suggestive for polyomavirus JC-induced haemorrhagic cystitis.

The seldom occurring androgen insensitivity syndrome is characterised by male karyotype in a phenotypical women. We report on a complete androgen insensitivity syndrome with the rare coincidence of bilateral Fallopian tubes and a testicular intraepithelial neoplasia. Additionally we discuss aspects of incidence, molecular background, characteristics, diagnostic pitfalls and therapy against the background of the current literature.

We report on a 10-year-old girl with severe aplastic anaemia who developed rhinocerebral infection caused by Absidia corymbifera and a possible co-infection caused by Alternaria alternata. Despite prolonged neutropenia, therapy with liposomal amphotericin B and posaconazole improved the clinical condition. Subsequently, the girl underwent allogeneic haematopoietic stem cell transplantation (HSCT) for the underlying disease, but the fungal infection remained under control with the antifungal treatment. No severe side effect of the antifungal drugs was noted. Unfortunately, the girl died 5 months after HSCT due to disseminated adenovirus infection.

A 45-year-old patient developed shortness of breath, intensive cough, hemoptysis, chest pain and acute bilateral pulmonary infiltrates following the inhalation of crack-cocaine. The bronchoalveolar lavage and transbronchial biopsy revealed infiltrations of polymorphonuclear neutrophils and the formation of foreign body granulomas. The diagnosis of a crack-syndrome was made and the patient rapidly improved under temporary discontinuation of cocaine inhalation and symptomatic therapy. Crack-cocaine is the free-base of cocaine-hydrochloride and its chemical properties allows it to be inhaled tobacco-like. Therefore the lungs become the principal organs exposed and affected. In addition to our findings, diffuse damage of the alveolar wall and capillary injury due to vasoconstriction and toxic action were reported, in some cases rapidly progressing into pulmonary oedema and ARDS. As the consumption of crack cocaine in Germany has markedly increased over the past decade, a higher prevalence of the reported syndrome has to be assumed.

Giant cell arteritis (GCA) is a disease chiefly found in elderly patients. Intracranial vessels are rarely involved in GCA. Here we report the case of a 19-year-old woman with GCA in the basilar and vertebral arteries. Two weeks after the first symptoms, she developed an aneurysmatical dilatation of the right vertebral artery which ruptured leading to subarachnoid hemorrhage. Although the ruptured right vertebral artery was clipped neurosurgically, she died two days later. Autopsy revealed GCA with focal medial necrosis and intimal thickening of the vertebral arteries and the basilar artery. No other arteries were affected. In the involved vessels, the media exhibited C1q immunoreactivity. At the intimal site of the internal elastic lamina there were increased levels of elastase. Other arterial diseases showing the pattern of GCA were excluded. This case demonstrates that GCA is not necessarily restricted to elderly people. Moreover, this case shows that a GCA-induced aneurysm is a very rare reason for subarachnoid hemorrhage even in young adults.