Enterovirus and Parechovirus meningitis in children: a review of the epidemiology, diagnostic challenges, and significance of on-site CSF virology tests in tropical paediatric patients’ care

Enteroviruses and Parechoviruses are increasingly recognized as the cause of aseptic meningitis, especially in the paediatric age group. However, because of indistinguishable clinical features with bacterial meningitis, many clinicians cannot make a clear distinction in disease presentation, and a large number of cases go undiagnosed. Although polymerase chain reaction is the current standard diagnostic approach, it takes many hours or days to get a result and these tests are not available at primary and secondary levels of care in many resource-poor countries. Furthermore, diagnosis is often difficult in children due to nonspecific cellular and biochemical cerebrospinal fluid findings. Some affected children may develop neurologic or/and systemic complications, resulting in prolonged hospital admission, increasing the risk of avoidable deaths, and healthcare expenditures. This review focuses on epidemiology, presentation, and diagnosis of Enterovirus and Parechovirus meningitis, highlighting the challenges in diagnosis and the potential roles of on-site CSF virology tests in improving the quality of paediatric patient’s care. The information provided should help early case detection, thereby ensuring avoidance of unnecessary antibiotics, minimal complications, a short period of hospital stays, and a reduction in healthcare-associated costs. Keywords: Aseptic meningitis; Enterovirus; Parechovirus; Diagnostic challenge; On-site virology test; Children   FrenchTitle: Méningite à Entérovirus et Parechovirus chez les enfants: un examen de l’épidémiologie, des défis diagnostiques et de l’importance du tests virologique sur site du LCR dans les soins aux patients pédiatriques tropicaux   Les entérovirus et les parechovirus sont de plus en plus reconnus comme la cause de la méningite aseptique, en particulier dans le groupe d'âge pédiatrique. Cependant, en raison des caractéristiques cliniques indiscernables de la méningite bactérienne, de nombreux cliniciens ne peuvent pas faire une distinction claire dans la présentation de la maladie, et un grand nombre de cas ne sont pas diagnostiqués. Bien que la réaction en chaîne par polymérase soit l'approche diagnostique standard actuelle, il faut plusieurs heures ou jours pour obtenir un résultat et ces tests ne sont pas disponibles aux niveaux de soins primaires et secondaires dans de nombreux pays pauvres en ressources. En outre, le diagnostic est souvent difficile chez les enfants en raison de découvertes non spécifiques du liquide céphalo-rachidien cellulaire et biochimique. Certains enfants atteints peuvent développer des complications neurologiques ou systémiques, entraînant une hospitalisation  prolongée, augmentant le risque de décès évitables et les dépenses de santé. Cette revue se concentre sur l'épidémiologie, la présentation et le diagnostic de la méningite à entérovirus et parechovirus, mettant en évidence les défis du diagnostic et les rôles potentiels des tests virologiques sur place dans le LCR dans  l'amélioration de la qualité des soins aux patients pédiatriques. Les informations fournies devraient contribuer à la détection précoce des cas, garantissant ainsi d'éviter les antibiotiques inutiles, des complications minimales, une courte période d'hospitalisation et une réduction des coûts associés aux soins de santé. Mots clés: méningite aseptique; Entérovirus; Parechovirus; Défi diagnostique; Test de virologie sur place;  Enfant

Viral Etiology of Central Nervous System Infections and Community-Acquired Sepsis in Southeast Asia: Unravelling the Unknown

My PhD research consists of a series of studies on the application of metagenomic next-generation sequencing (mNGS) to search for viral etiology in patients presenting with community-acquired sepsis or central nervous system (CNS) infections. I first developed a mNGS workflow for the sensitive detection of a broad range of viruses in clinical samples (Chapter 2). I then used this optimized method to search for viruses in 665 patients presenting with community-acquired sepsis of unknown origin enrolled in an observational study across Thailand and Vietnam in 2013-2015. While the mNGS analysis revealed significant insights into the epidemiology of sepsis in both countries, the analysis also led to the first detection of a recently discovered flavivirus - human pegivirus 2 (HPgV-2) - in a serum sample of a Vietnamese patient co-infected with HIV and HCV. This represents the first detection of HPgV-2 in Vietnam. Therefore, I conducted further research to unravel its epidemiology in Vietnam (Chapter 4). In Chapter 5, I used mNGS to analyze 204 cerebrospinal fluid (CSF) from patients with CNS infections of unknown origin enrolled from hospitals across central and southern Vietnam in 2012-2016. Enteroviruses were the most common viruses detected, especially in children and young adults. To inform future research directions, I conducted a pilot of 66 consecutive CSF samples collected from patients presenting with CNS infections admitted to the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam (Chapter 6). mNGS accurately detected a wide range of pathogens that were also detected by routine diagnostic methods, but also increased the diagnostic yield from 22.7% (15/66) to 34.8% (23/66) (Chapter 6). Finally, in Chapter 7, I provide an overview about my research findings, and propose some future directions based on the main findings obtained during my PhD research

Molecular epidemiology of acute respiratory virus infections

Acute respiratory virus infections are very common but can also cause severe disease. In my thesis, I have analysed the molecular epidemiology of acute respiratory virus infections caused by enterovirus D68 and coronaviruses. In Paper I, we used real-time PCR and Sanger sequencing to analyse the outbreak of enterovirus D68 in Stockholm in 2016. We found that the outbreak was caused by the subclade B3, and we also described three patients with neurological manifestations. The virus sequences were closely related to concurrent sequences from North America. In Paper II, we developed an assay for whole-genome sequencing of enterovirus D68 a next-generation platform. By using the assay on the samples from the 2016 outbreak, we found that the outbreak was caused by multiple independent introductions of the virus. We also estimated the time to the most common recent ancestor for the subclades B1 and B3 to 2009. In Paper III, we used the whole-genome sequencing assay in a European multicentre study of enterovirus D68 circulation in the 2018 season. We also included sequences in public repositories. We found that the viruses in 2018 belonged to subclades A2 and B3 and that sequences in subclade B3 originated from the circulation in 2016. We also found that enterovirus D68 had a rapid geographic mixing and that residues on the surface of the virus particle had an elevated substitution rate of amino acids. Hence, we proposed asymptomatic reinfections of adults to explain both rapid geographical dispersal and selective pressure on the surface residues. In Paper IV, we analysed stored results from routine clinical diagnostics for the four common cold coronaviruses. The data contained the results from September 2009 to April 2020. At the species level, we found a pattern of alternating biennial circulation, and we also found the circulation of Betacoronaviruses to peak earlier than that of Alphacoronaviruses. In Paper V, we investigated Sweden’s first SARS-CoV-2 pandemic wave in 2020. We analysed stored respiratory samples with real-time PCR for SARS-CoV-2 and found that community transmissions started earlier than previously appreciated. We also se-quenced stored SARS-CoV-2-positive samples. To these sequences, we added infor-mation from contact tracing records and combined them with data from public reposi-tories. Among cases exposed abroad, we mainly found clades 20B and 20A, whereas clade 20C dominated domestic infections. Furthermore, we found the proportion of clade 20C to be correlated with the cumulative number of deaths due to COVID-19. We interpreted this as early undetected introductions of clade 20C having had a significant impact on the further course of the pandemic in Sweden

The Behavioral Ecology of the Tibetan Macaque

This open access book summarizes the multi-disciplinary results of one of China’s main primatological research projects on the endemic Tibetan macaque (Macaca thibetana), which had continued for over 30 years, but which had never been reported on systematically. Dedicated to this exceptional Old World monkey, this book makes the work of Chinese primatologists on the social behavior, cooperation, culture, cognition, group dynamics, and emerging technologies in primate research accessible to the international scientific community

The Behavioral Ecology of the Tibetan Macaque

This open access book summarizes the multi-disciplinary results of one of China’s main primatological research projects on the endemic Tibetan macaque (Macaca thibetana), which had continued for over 30 years, but which had never been reported on systematically. Dedicated to this exceptional Old World monkey, this book makes the work of Chinese primatologists on the social behavior, cooperation, culture, cognition, group dynamics, and emerging technologies in primate research accessible to the international scientific community. One of the most impressive Asian monkeys, and the largest member of its genus, the Tibetan macaque deserves to be better known. This volume goes a long way towards bringing this species into the spotlight with many excellent behavioral analyses from the field. - Frans de Waal, Professor of Psychology, Emory University, USA. Macaques matter. To understand primate patterns and trends, and to gain important insight into humanity, we need to augment and expand our engagement with the most successful and widespread primate genus aside from Homo. This volume focuses on the Tibetan macaque, a fascinating species with much to tell us about social behavior, physiology, complexity and the macaque knack for interfacing with humans. This book is doubly important for primatology in that beyond containing core information on this macaque species, it also reflects an effective integrated collaboration between Chinese scholars and a range of international colleagues—exactly the type of collaborative engagement primatology needs. This volume is a critical contribution to a global primatology. - Agustín Fuentes, Professor of Anthropology, University of Notre Dame, USA. I have many fond memories of my association with Mt. Huangshan research beginning in 1983, when together with Professor Qishan Wang we established this site. It is such a beautiful place and I miss it. It is gratifying to see how far research has progressed since we began work there, becoming more internationalized and very much a collaborative endeavor under the long-term direction of Professor Jin-Hua Li and colleagues. This book highlights the increased interest in this species, representing a variety of disciplines ranging from macro aspects of behavior, cognition and sociality, to micro aspects of microbes, parasites and disease, authored by a group of renowned Chinese and international primatologists. I applaud their efforts and expect more interesting work to come from this site in the years ahead. - Kazuo Wada, Professor Emeritus, Kyoto University, Japan

Blueberry polyphenols as natural antimicrobial agents against foodborne viruses: Towards understanding their mechanism and applications in food systems

Foodborne viruses are recognized as public health concerns worldwide and therefore effective strategies to control their spread are being researched. Blueberries are known for their health benefits and antimicrobial properties. This study aimed to (1) determine the antiviral effects of blueberry juice (BJ) and blueberry proanthocyanidins (B-PAC) against the infectivity of hepatitis A virus (HAV), Aichi virus (AiV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) at 37oC over 24-h using standard plaque assays; (2) evaluate antiviral effects in model foods (apple juice (AJ) and 2% milk) and simulated gastrointestinal conditions at 37oC; and (3) determine the mechanism of action of B-PAC by comparing activity of monomeric catechins, procyanidin B2, B-type PAC from blueberries (B-PAC) and A-type PAC from cranberries (C-PAC), effects on viral structure using transmission electron microscopy, and on adsorption and replication. FCV-F9, MNV-1, HAV and AiV titers were reduced to undetectable levels after 5 min, 3-h, 30 min and 3-h with 1, 1, 2, and 5 mg/ml B-PAC, respectively. BJ reduced FCV-F9, MNV-1, AiV to undetectable levels and HAV by ~2 log PFU/ml after 3, 6, 24 and 24-h, respectively. All tested viruses were reduced to undetectable levels within 15 min with B-PAC (1, 2 and 5 mg/ml) in AJ (pH 3.6). B-PAC effects decreased in milk, where FCV-F9, MNV-1, HAV and AiV were reduced by ~1 log PFU/ml with 5 mg/ml after 24-h. B-PAC at 5 mg/ml in simulated intestinal fluid reduced all tested viruses to undetectable levels within 30 min. Monomeric catechins and procyanidin B2 were less effective than the polymeric B-PAC. Time of addition assays indicated that B-PAC had modest effects in preventing viral adsorption, with no significant effect on viral replication. TEM observations revealed moderate effects on virus structure with either damaged viral capsid or virus binding to B-PAC, possibly preventing virus attachment to host cells. Overall, this study demonstrated the ability of BJ and B-PAC to reduce viral titers in model food systems and under simulated gastric conditions, suggesting their potential as preventive and therapeutic options against foodborne viral illnesses

Emerg Infect Dis

PMC4550154611

Molecular signals of arms race evolution between RNA viruses and their hosts

Viruses are intracellular parasites that hijack their hosts’ cellular machinery to replicate themselves. This creates an evolutionary “arms race” between hosts and viruses, where the former develop mechanisms to restrict viral infection and the latter evolve ways to circumvent these molecular barriers. In this thesis, I explore examples of this virus-host molecular interplay, focusing on events in the evolutionary histories of both viruses and hosts. The thesis begins by examining how recombination, the exchange of genetic material between related viruses, expands the genomic diversity of the Sarbecovirus subgenus, which includes SARS-CoV responsible for the 2002 SARS epidemic and SARS-CoV-2 responsible for the COVID-19 pandemic. On the host side, I examine the evolutionary interaction between RNA viruses and two interferon-stimulated genes expressed in hosts. First, I show how the 2′-5′-oligoadenylate synthetase 1 (OAS1) gene of horseshoe bats (Rhinolophoidea), the reservoir host of sarbecoviruses, lost its anti-coronaviral activity at the base of this bat superfamily. By reconstructing the Rhinolophoidea common ancestor OAS1 protein, I first validate the loss of antiviral function and highlight the implications of this event in the virus-host association between sarbecoviruses and horseshoe bat hosts. Second, I focus on the evolution of the human butyrophilin subfamily 3 member A3 (BTN3A3) gene which restricts infection by avian influenza A viruses (IAV). The evolutionary analysis reveals that BTN3A3’s anti-IAV function was gained within the primates and that specific amino acid substitutions need to be acquired in IAVs’ NP protein to evade the human BTN3A3 activity. Gain of BTN3A3-evasion-conferring substitutions correlate with all major human IAV pandemics and epidemics, making these NP residues key markers for IAV transmissibility potential to humans. In the final part of the thesis, I present a novel approach for evaluating dinucleotide compositional biases in virus genomes. An application of my metric on the Flaviviridae virus family uncovers how ancestral host shifts of these viruses correlate with adaptive shifts in their genomes’ dinucleotide representation. Collectively, the contents of this thesis extend our understanding of how viruses interact with their hosts along their intertangled evolution and provide insights into virus host switching and pandemic preparedness

Aérosols viraux en milieux de soins : contrôle, mesure et caractérisation

Les éclosions virales constituent des menaces persistantes pour les établissements de soins. En plus de compromettre la santé des usagers, du personnel et des visiteurs, ces éclosions représentent d'énormes défis de gestion des ressources humaines, matérielles et financières. La pandémie de SRAS-CoV-2 sévissant depuis plus d'une année a mis en lumière la méconnaissance du rôle de l'air dans la transmission des virus. Des technologies de traitement de l'air pourraient contribuer au contrôle des virus aérosolisés et éventuellement à la protection des occupants des milieux de soins. Dans le cadre de ce doctorat, une stratégie de traitement de l'air utilisant l'ozone a été testée pour inactiver des bioaérosols viraux. Afin d'obtenir un portrait de la contamination aérienne en milieu hospitalier, une campagne d'échantillonnage a été menée lors de trois éclosions d'influenza. Enfin, la production d'aérosols pendant le traitement d'échantillons en laboratoire clinique a été examinée. Dans la première étude, le norovirus murin ainsi que les bactériophages PhiX174, Phi6, PR772 et MS2 ont été nébulisés dans une chambre d'aérosols rotative et exposés à un traitement de l'air utilisant l'ozone à différents niveaux d'humidité relative. Le norovirus murin a été exposé à 0,23 ppm d'ozone et à 20% et 85% d'humidité relative alors que les bactériophages ont été exposés à 1,13 ppm d'ozone et à trois humidités relatives, soit 20%, 55% et 85%. Pour tous les virus, des temps d'exposition de 10, 40 et 70 minutes ont été évalués. Ce traitement a été comparé à une condition de référence, qui consistait en une exposition à l'air. Les aérosols ont été récupérés à l'aide d'un échantillonneur d'air et les virus ont été quantifiés en culture et par biologie moléculaire. Des ratios infectieux ont été calculés afin de déterminer la réduction de l'infectiosité virale attribuable au traitement à l'ozone. Une inactivation d'au moins deux ordres de grandeur a été observée après 40 minutes d'exposition à l'ozone à 85% d'humidité relative pour PhiX174, MS2 et MNV-1. Une exposition à la condition de référence à 20% d'humidité relative pendant 10 minutes a été suffisante pour une inactivation similaire des bactériophages PR772 et Phi6. Ce même traitement de l'air a ensuite été évalué pour l'inactivation d'aérosols d'influenza et du virus respiratoire syncytial. Toutefois, dans le cas de ce second virus, la perte d'infectiosité lors des procédés d'aérosolisation et d'échantillonnage était trop importante pour pouvoir l'exposer à l'ozone. Concernant l'influenza, des concentrations d'ozone de 0,23 et 1,70 ppm ont été testées à des niveaux faibles et élevés d'humidité relative. Deux suppléments, l'un de nature lipidique et l'autre de nature protéique, ont été ajoutés au lysat viral afin de quantifier l'effet protecteur qu'ils pourraient procurer aux virus aérosolisés. Une condition sans supplément a aussi été testée à des fins de comparaison. Une exposition pendant 80 minutes à une concentration d'ozone de 1,70 ppm combinée à une humidité relative élevée a engendré la meilleure inactivation, soit une réduction de quatre ordres de grandeur, pour les aérosols sans supplément ou additionnés de supplément protéique Lors de la troisième étude, l'air d'un milieu hospitalier en contexte d'éclosion grippale a été échantillonné à trois reprises. L'efficacité de récupération de trois appareils, dont deux fonctionnant à haut débit et un à bas débit, a été évaluée. Cette campagne a révélé une variabilité des concentrations aériennes d'influenza A et B entre les éclosions. Bien que des concentrations maximales de l'ordre de 10⁵ copies d'ARN/m³ aient été détectées, aucun virus infectieux n'a été quantifié. Finalement, la génération d'aérosols pendant le traitement d'échantillons sanguins et urinaires dans un laboratoire clinique de biochimie a été examinée. Les employés redoutaient de produire des aérosols contenant du SRAS-CoV-2 infectieux à partir d'échantillons récoltés chez des patients infectés par la COVID-19. Pour ce projet, une culture liquide d'une bactérie modèle a été employée en remplacement des échantillons cliniques. Trois méthodes de collecte ont été utilisées pour évaluer la production d'aérosols, soit le prélèvement d'air par un appareil standard, l'emploi de boîtes indicatrices et l'écouvillonnage de surfaces. Aucune bactérie n'a été récupérée par ces trois méthodes d'échantillonnage, ce qui indique que les procédures de traitement étudiées n'ont produit qu'une faible quantité d'aérosols.Viral outbreaks are recurring threats to healthcare facilities. While putting the health of users, staff and visitors at risk, these outbreaks represent enormous challenges in the management of human, material and financial resources. The SARS-CoV-2 pandemic. The SARS-CoV-2 pandemic, which has been raging for more than a year, has highlighted the misunderstanding of the role of air in the transmission of viruses. Air treatment technologies could contribute to the control of airborne viruses and eventually to the protection of healthcare occupants. During this doctoral program, an air treatment strategy using ozone was assessed for the inactivation of viral bioaerosols. To obtain a global portrait of airborne contamination in a hospital environment, an air sampling campaign was conducted during three influenza outbreaks. At last, the aerosol production during sample treatment in a clinical laboratory was examined. In the first study, murine norovirus and bacteriophages PhiX174, Phi6, PR772 and MS2 were nebulized in a rotative aerosol chamber and exposed to an air treatment using ozone at different relative humidity levels. The murine norovirus was exposed to 0.23 ppm of ozone and 20% and 85% of relative humidity while the bacteriophages were exposed to 1.13 ppm of ozone and three relative humidity: 20%, 55% and 85%. For all viruses, exposure times of 10, 40 and 70 minutes were evaluated. This treatment was compared to a reference condition, which was air exposure. The aerosols were collected with an air sampler and viruses were quantified using both culture and molecular biology. Infectious ratios were calculated to determine the viral infectivity reduction that was attributable to ozone. An inactivation of at least two orders of magnitude was obtained for an ozone exposure of 40 minutes at 85% of relative humidity for PhiX174, MS2 and MNV-1. Exposure to the reference condition at 20% of relative humidity for 10 minutes was sufficient for a similar inactivation of bacteriophages PR772 and Phi6. The same air treatment was then evaluated for the inactivation of influenza or respiratory syncytial virus aerosols. However, the infectivity loss of the respiratory syncytial virus during aerosolization and sampling processes was too elevated for ozone exposure. For influenza, ozone concentrations of 0.23 and 1.70 ppm were tested at low and high relative humidity levels. Two supplements, one lipid-based and the other protein-based were added to the viral lysate to quantify their protective effect for airborne viruses. A condition without a supplement was also tested for comparison purposes. Exposure to 1.70 ppm of ozone at high relative humidity for 80 minutes yielded the greatest inactivation, which was a reduction of four orders of magnitude for aerosols without supplement or with the protein-based supplement. In the third study, the air in a hospital environment during influenza outbreaks was sampled three times. The collection efficiency of three air samplers, two high flowrate and one low flowrate, was evaluated. This campaign revealed a variability between outbreaks in regards to airborne influenza A and B concentrations. While concentrations of up to 10⁵ RNA copies/m³ were detected, no infectious virus could be quantified. Lastly, aerosol generation during blood and urine sample treatment in a clinical biochemistry laboratory was examined. Employees feared producing infectious SARS-CoV-2 containing aerosols from samples collected from COVID-19 infected patients. For this project, a liquid culture of a model bacteria was employed to replace clinical samples. The sampling methods were used to evaluate aerosol production: air sampling using a standard device, the use of settling plates and surface swabbing. No bacteria were recovered by these three sampling methods, which indicates that the studied sample treatment procedures produce low quantities of aerosols