Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the emergence of novel B3-derived lineages, ENPEN multicentre study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

Role of Polyphenols from the Aqueous Extract of Aloysia citrodora in the Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a mycotoxin considered a potent carcinogen for humans that contaminates a wide range of crops. Various strategies have been established to reduce or block the synthesis of AFB1 in food and feed. The use of aqueous extracts derived from plants with high antioxidant activity has been a subject of study in recent years due to their efficacy in inhibiting AFB1. In this study, we assessed the effect of Aloysia citrodora aqueous extract on Aspergillus flavus growth and on AFB1 production. A bio-guided fractionation followed by High Performance Liquid Chromatography (HPLC) and Mass spectrometry analysis of the active fraction were applied to identify the candidate molecules responsible for the dose-effect inhibition of AFB1 synthesis. Our results revealed that polyphenols are the molecules implicated in AFB1 inhibition, achieving almost a total inhibition of the toxin production (99%). We identified luteolin-7-diglucuronide as one of the main constituents in A. citrodora extract, and demonstrated that it is able to inhibit, by itself, AFB1 production by 57%. This is the first study demonstrating the anti-Aflatoxin B1 effect of this molecule, while other polyphenols surely intervene in A. citrodora anti-AFB1 activity.

Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus. Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus by Mate (Ilex paraguariensis), Rosemary (Rosmarinus officinalis) and Green Tea (Camellia sinensis) Extracts: Relation with Extract Antioxidant Capacity and Fungal Oxidative Stress Response Modulation.

Plant extracts may represent an ecofriendly alternative to chemical fungicides to limit aflatoxin B1 (AFB1) contamination of foods and feeds. Mate (Ilex paraguariensis), rosemary (Romarinus officinalis) and green tea (Camellia sinensis) are well known for their beneficial properties, which are mainly related to their richness in bioactive phenolic compounds. AFB1 production is inhibited, with varying efficiency, by acetone/water extracts from these three plants. At 0.45 µg dry matter (DM)/mL of culture medium, mate and green tea extracts were able to completely inhibit AFB1 production in Aspergillus flavus, and rosemary extract completely blocked AFB1 biosynthesis at 3.6 µg DM/mL of culture medium. The anti-AFB1 capacity of the extracts correlated strongly with their phenolic content, but, surprisingly, no such correlation was evident with their antioxidative ability, which is consistent with the ineffectiveness of these extracts against fungal catalase activity. Anti-AFB1 activity correlated more strongly with the radical scavenging capacity of the extracts. This is consistent with the modulation of SOD induced by mate and green tea in Aspergillus flavus. Finally, rutin, a phenolic compound present in the three plants tested in this work, was shown to inhibit AFB1 synthesis and may be responsible for the anti-mycotoxin effect reported herein.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Antifungal and anti-aflatoxigenic properties of organs of Cannabis sativa L.: relation to phenolic content and antioxidant capacities.

Aflatoxin B1 is a carcinogenic mycotoxin that frequently contaminates crops worldwide. Current research indicates that the use of natural extracts to combat mycotoxin contamination may represent an eco-friendly, sustainable strategy to ensure food safety. Although Cannabis sativa L. has long been known for its psychoactive cannabinoids, it is also rich in many other bioactive molecules. This study examines extracts from various organs of Cannabis sativa L. to determine their ability to limit aflatoxin production and growth of Aspergillus flavus. The results indicate that flower extract is most effective for limiting the synthesis of aflatoxin B1, leading to an almost-complete inhibition of toxin production at a concentration of 0.225 mg dry matter per gram of culture medium. Since flower extract is rich in phenolic compounds, its total antioxidant ability and radical-scavenging capacity are determined. Compared with other anti-aflatoxigenic extracts, the anti-oxidative potential of Cannabis sativa L. flower extract appears moderate, suggesting that its anti-mycotoxin effect may be related to other bioactive compounds.

A large-scale outbreak of hand, foot and mouth disease, France, as at 28 September 2021.

We report a large-scale outbreak of hand, foot and mouth disease (HFMD) in France. As at 28 September 2021, 3,403 cases have been reported (47% higher than in 2018-19). We prospectively analysed 210 clinical samples; 190 (90.5%) were enterovirus-positive. Most children presented with atypical HFMD. Coxsackievirus (CV)A6 (49.5%; 94/190) was predominant; no enterovirus A71 was detected. Dermatological and neurological complications of HFMD justify prospective syndromic and virological surveillance for early detection of HFMD outbreaks and identification of associated types.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

High Permissiveness for Genetic Exchanges between Enteroviruses of Species A, including Enterovirus 71, Favors Evolution through Intertypic Recombination in Madagascar.

Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains.

A New Hydrocellular Wound Dressing Used After Dermatologic Surgery: Dermatologic Surgeons' and Patients' Perceptions.

BACKGROUND: Optimum postsurgical healing requires appropriate dressing use. OBJECTIVE: This study assessed effectiveness and tolerance of a novel, hydrocellular dressing in dermatologic surgery using validated tools, describing its use in clinical practice, and comparing surgeons' and patients' perceptions of scar evolution. METHODS: This study examined direct suture closures of surgical excisions of small- to medium-sized skin lesions on the extremities or trunk. Dressings were changed 3 times/week. The Vancouver Scar Scale (VSS) and the Patient and Observer Scar Assessment Scales (POSAS) were used to assess outcomes. Data were collected at Day 0 (D0, FLC application), Day 15 to 21 (D15-21, suture removal), and Day 45 (D45) postprocedure by the surgeon and the patient. RESULTS: There were 128 patients (mean age: 55.1 years, 56.1% women). Mean length and width of the excisions were 3.5 × 1.65 cm and the most common FLC applied was 8 × 8 cm (67.7%). Most scars had normal pigmentation, pliability, and height at D15 to 21 and D45, as reported by patients and surgeons using VSS. Patient scores on visual analog scale (VAS) were high (>8/10) and global satisfaction measured by POSAS was generally high (>7/10 at D15-21; >8/10 at D45). CONCLUSION: These dressings were effective in managing surgical excisions, as assessed by VSS, VAS, and POSAS. Further controlled studies investigating various dressings in wound repair are needed.

Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20.

Enterovirus D68 (EV-D68) was detected in 93 patients from five European countries between 1 January 2019 and 15 January 2020, a season with expected low circulation. Patients were primarily children (n = 67, median age: 4 years), 59 patients required hospitalisation and five had severe neurologic manifestations. Phylogenetic analysis revealed two clusters in the B3 subclade and subclade A2/D. This circulation of EV-D68 associated with neurological manifestations stresses the importance of surveillance and diagnostics beyond expected peak years.

Multirecombinant Enterovirus A71 Subgenogroup C1 Isolates Associated with Neurologic Disease, France, 2016-2017.

In 2016, an upsurge of neurologic disease associated with infection with multirecombinant enterovirus A71 subgenogroup C1 lineage viruses was reported in France. These viruses emerged in the 2000s; 1 recombinant is widespread. This virus lineage has the potential to be associated with a long-term risk for severe disease among children.

Phylogeography of Coxsackievirus A16 Reveals Global Transmission Pathways and Recent Emergence and Spread of a Recombinant Genogroup.

Coxsackievirus A16 (CV-A16; Picornaviridae) is an enterovirus (EV) type associated with hand, foot, and mouth disease (HFMD) in children. To investigate the spatial spread of CV-A16, we used viral sequence data sampled during a prospective sentinel surveillance of HFMD in France (2010 to 2014) and phylogenetic reconstruction. A data set of 168 VP1 sequences was assembled with 416 publicly available sequences of various geographic origins. The CV-A16 sequences reported were assigned to two clades, genogroup B and a previously uncharacterized clade D. The time origins of clades B and D were assessed in 1978 (1973 to 1981) and 2004 (2001 to 2007), respectively. The shape of the global CV-A16 phylogeny indicated worldwide cocirculation of genetically distinct virus lineages over time and across geographic regions. Phylogenetic tree topologies and Bayes factor analysis indicated virus migration. Virus transportation events in clade B within Europe and Asia and between countries of the two geographic regions were assessed. The sustained transmission of clade D viruses over 4 years was analyzed at the township level in France and traced back to Peru in South America. Comparative genomics provided evidence of recombination between CV-A16 clades B and D and suggested an intertype recombinant origin for clade D. Time-resolved phylogenies and HFMD surveillance data indicated that CV-A16 persistence is sustained by continuing virus migration at different geographic scales, from community transmission to virus transportation between distant countries. The results showed a significant impact of virus movements on the epidemiological dynamics of HFMD that could have implications for disease prevention.IMPORTANCE Coxsackievirus A16 is one of the most prevalent enterovirus types in hand, foot, and mouth disease outbreaks reported in Southeast Asia. This study is based on epidemiological and viral data on HFMD caused by CV-A16 in a European country. The phylogeographic data complemented the syndromic surveillance with virus migration patterns between geographic regions in France. The results show how viral evolutionary dynamics and global virus spread interact to shape the worldwide pattern of an EV disease. CV-A16 transmission is driven by movements of infected individuals at different geographic levels: within a country (local dynamics), between neighboring countries (regional dynamics), and between distant countries (transcontinental dynamics). The results are consistent with our earlier data on EV-A71 and confirm the epidemiological interconnection of Asia and Europe with regard to EV infections.

Monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central France, 2014 to 2015.

BackgroundHuman enteric viruses are resistant in the environment and transmitted via the faecal-oral route. Viral shedding in wastewater gives the opportunity to track emerging pathogens and study the epidemiology of enteric infectious diseases in the community. Aim: The aim of this study was to monitor the circulation of enteric viruses in the population of the Clermont-Ferrand area (France) by analysis of urban wastewaters. Methods: Raw and treated wastewaters were collected between October 2014 and October 2015 and concentrated by a two-step protocol using tangential flow ultrafiltration and polyethylene glycol precipitation. Processed samples were analysed for molecular detection of adenovirus, norovirus, rotavirus, parechovirus, enterovirus (EV), hepatitis A (HAV) and E (HEV) viruses. Results: All wastewater samples (n = 54) contained viruses. On average, six and four virus species were detected in, respectively, raw and treated wastewater samples. EV-positive samples were tested for EV-D68 to assess its circulation in the community. EV-D68 was detected in seven of 27 raw samples. We collected data from clinical cases of EV-D68 (n = 17), HAV (n = 4) and HEV infection (n = 16) and compared wastewater-derived sequences with clinical sequences. We showed the silent circulation of EV-D68 in September 2015, the wide circulation of HAV despite few notifications of acute disease and the presence in wastewater of the major HEV subtypes involved in clinical local cases. Conclusion: The environmental surveillance overcomes the sampling bias intrinsic to the study of infections associated with hospitalisation and allows the detection in real time of viral sequences genetically close to those reported in clinical specimens.

[Enterovirus D68, a highly pathogenic and epidemic virus]. / Entérovirus D68 : un virus à haut potentiel pathogène et épidémique.

Since its discovery in 1962, enterovirus D68 (EV-D68) was one of the less frequently detected enteroviruses by the surveillance networks worldwide. In 2014, US pediatric hospitals reported increases in the number of children with severe respiratory illness. Following the alerts from the Center for Disease Control and Prevention, numerous cases of EV-D68 were reported in many countries. EV-D68 is associated with severe respiratory infections in children and adults, mostly in patients with underlying respiratory diseases. Like with poliovirus and EV-A71, rare but severe neurological complications may occur: acute flaccid myelitis is characterized by rapid onset of weakness and distinct abnormalities of the spinal cord gray matter on magnetic resonance imaging. Molecular epidemiology of strains isolated worldwide since the 90s shows a rapid evolution of the virus, reflecting its wide circulation in the general population. The recent emergence of EV-D68 underlines the unpredictable epidemic properties and the neurotropism of enteroviruses.

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response.

Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural anti-aflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated asan AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine's molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well asa targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin's biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase's families. Furthermore, this gene response was accompanied by a significant enhancement of catalase enzymatic activity. In conclusion, these data demonstrated that piperine inhibits AFB1 production while positively modulating fungal antioxidant status in A. flavus.

Aerosolization of Mycotoxins after Growth of Toxinogenic Fungi on Wallpaper.

Many fungi can develop on building material in indoor environments if the moisture level is high enough. Among species that are frequently observed, some are known to be potent mycotoxin producers. This presence of toxinogenic fungi in indoor environments raises the question of the possible exposure of occupants to these toxic compounds by inhalation after aerosolization. This study investigated mycotoxin production by Penicillium brevicompactum, Aspergillus versicolor, and Stachybotrys chartarum during their growth on wallpaper and the possible subsequent aerosolization of produced mycotoxins from contaminated substrates. We demonstrated that mycophenolic acid, sterigmatocystin, and macrocyclic trichothecenes (sum of 4 major compounds) could be produced at levels of 1.8, 112.1, and 27.8 mg/m2, respectively, on wallpaper. Moreover, part of the produced toxins could be aerosolized from the substrate. The propensity for aerosolization differed according to the fungal species. Thus, particles were aerosolized from wallpaper contaminated with P. brevicompactum when an air velocity of just 0.3 m/s was applied, whereas S. chartarum required an air velocity of 5.9 m/s. A. versicolor was intermediate, since aerosolization occurred under an air velocity of 2 m/s. Quantification of the toxic content revealed that toxic load was mostly associated with particles of size ≥3 µm, which may correspond to spores. However, some macrocyclic trichothecenes (especially satratoxin H and verrucarin J) can also be found on smaller particles that can deeply penetrate the respiratory tract upon inhalation. These elements are important for risk assessment related to moldy environments.IMPORTANCE The possible colonization of building material by toxinogenic fungi in cases of moistening raises the question of the subsequent exposure of occupants to aerosolized mycotoxins. In this study, we demonstrated that three different toxinogenic species produce mycotoxins during their development on wallpaper. These toxins can subsequently be aerosolized, at least partly, from moldy material. This transfer to air requires air velocities that can be encountered under real-life conditions in buildings. Most of the aerosolized toxic load is found in particles whose size corresponds to spores or mycelium fragments. However, some toxins were also found on particles smaller than spores that are easily respirable and can deeply penetrate the human respiratory tract. All of these data are important for risk assessment related to fungal contamination of indoor environments.

Ambulatory Pediatric Surveillance of Hand, Foot and Mouth Disease as Signal of an Outbreak of Coxsackievirus A6 Infections, France, 2014-2015.

The clinical impact of enteroviruses associated with hand, foot and mouth disease (HFMD) is unknown outside Asia, and the prevalence of enterovirus A71 (EV-A71) in particular might be underestimated. To investigate the prevalence of enterovirus serotypes and the clinical presentations associated with HFMD in France, we conducted prospective ambulatory clinic-based surveillance of children during April 2014-March 2015. Throat or buccal swabs were collected from children with HFMD and tested for the enterovirus genome. Physical examinations were recorded on a standardized form. An enterovirus infection was detected in 523 (79.3%) of 659 children tested. Two epidemic waves occurred, dominated by coxsackievirus (CV) A6, which was detected in 53.9% of enterovirus-infected children. CV-A6 was more frequently related to atypical HFMD manifestations (eruptions extended to limbs and face). Early awareness and documentation of HFMD outbreaks can be achieved by syndromic surveillance of HFMD by ambulatory pediatricians and rapid enterovirus testing and genotyping.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.