Case report: Novel treatment regimen for enterovirus encephalitis in SCID

Most non-polio enterovirus infections in immunocompetent individuals are acute and self-limiting in nature; however, infection can be severe, chronic and have devastating outcomes in immunocompromised hosts. Therapeutic strategies have predominantly involved supportive care, with the lack of approved antiviral treatments proving challenging for management. We report a case of an 8-month-old child who presented with severe enterovirus encephalitis following gene therapy for X-linked severe combined immunodeficiency (X-SCID) and who demonstrated clinical and microbiological improvement after a novel regimen of favipiravir, fluoxetine, and high-dose intravenous immunoglobulin (IVIg). The patient presented 6 weeks post–gene therapy with rapid neurological deterioration in the context of incomplete immune reconstitution, with microbiological and radiological evidence confirming enterovirus encephalitis. His neurologic examination stabilised 8 weeks after treatment, and he subsequently demonstrated excellent immune recovery. This is the first case report of combined therapy with favipiravir, fluoxetine, and high-dose IVIg in the context of severe enterovirus encephalitis in an immunocompromised host. This case highlights the importance of considering enterovirus encephalitis in immunocompromised patients presenting with both acute and chronic neurological signs, as well as developmental regression. The demonstrated treatment success and the associated low risk of toxicity warrant further investigation of this therapeutic regimen

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Background: Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017. Methods: Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS. Results: Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees. Conclusions: Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans

Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples

Changing composition of SARS-CoV-2 lineages and rise of Delta variant in England.

BACKGROUND: Since its emergence in Autumn 2020, the SARS-CoV-2 Variant of Concern (VOC) B.1.1.7 (WHO label Alpha) rapidly became the dominant lineage across much of Europe. Simultaneously, several other VOCs were identified globally. Unlike B.1.1.7, some of these VOCs possess mutations thought to confer partial immune escape. Understanding when and how these additional VOCs pose a threat in settings where B.1.1.7 is currently dominant is vital. METHODS: We examine trends in the prevalence of non-B.1.1.7 lineages in London and other English regions using passive-case detection PCR data, cross-sectional community infection surveys, genomic surveillance, and wastewater monitoring. The study period spans from 31st January 2021 to 15th May 2021. FINDINGS: Across data sources, the percentage of non-B.1.1.7 variants has been increasing since late March 2021. This increase was initially driven by a variety of lineages with immune escape. From mid-April, B.1.617.2 (WHO label Delta) spread rapidly, becoming the dominant variant in England by late May. INTERPRETATION: The outcome of competition between variants depends on a wide range of factors such as intrinsic transmissibility, evasion of prior immunity, demographic specificities and interactions with non-pharmaceutical interventions. The presence and rise of non-B.1.1.7 variants in March likely was driven by importations and some community transmission. There was competition between non-B.1.17 variants which resulted in B.1.617.2 becoming dominant in April and May with considerable community transmission. Our results underscore that early detection of new variants requires a diverse array of data sources in community surveillance. Continued real-time information on the highly dynamic composition and trajectory of different SARS-CoV-2 lineages is essential to future control efforts. FUNDING: National Institute for Health Research, Medicines and Healthcare products Regulatory Agency, DeepMind, EPSRC, EA Funds programme, Open Philanthropy, Academy of Medical Sciences Bill,Melinda Gates Foundation, Imperial College Healthcare NHS Trust, The Novo Nordisk Foundation, MRC Centre for Global Infectious Disease Analysis, Community Jameel, Cancer Research UK, Imperial College COVID-19 Research Fund, Medical Research Council, Wellcome Sanger Institute.National Institute for Health Research, Medicines and Healthcare products Regulatory Agency, DeepMind, EPSRC, EA Funds programme, Open Philanthropy, Academy of Medical Sciences Bill,Melinda Gates Foundation, Imperial College Healthcare NHS Trust, The Novo Nordisk Foundation, MRC Centre for Global Infectious Disease Analysis, Community Jameel, Cancer Research UK, Imperial College COVID-19 Research Fund, Medical Research Council, Wellcome Sanger Institute

Comparison of sensitivity and specificity of molecular and conventional methods in the diagnosis of bacterial infections

The aim of this project was to compare the specificity and sensitivity of molecular and culture methods in diagnosis of bacterial infections. During 2006-2009 we analyzed: 326 blood samples, 136 Cerebrospinal fludis, 80 pleuric fluids, 40 articular fluids, 52 tissues, 295 gastric-sputum-bronchial egests και 270 cervical specimens from patients admitted to the University Hospital of Larissa. Broad range 16S rRNA PCR was applied to sterile clinic specimens, and it was found to be more sensitive than cultures of: CSF (100% against 30%), pleuritic fluids (100% against 33%), articular fluids (100% against 0%) και orthopaedic infection tissues (100% against 52,4%). In contrast broad range 16S rRNA PCR method in blood studies was less sensitive (34,6%) than cultures. PCR with primers specific for pathogenic bacteria was applied to specimens with natural floral. Molecular methods are substandard especially in the ability of detecting mycobacteria having a percentage of sensitivity only 19,4%. However, they have an important advantage in identification of mycobacteria using direct culture, and in determination of mutations encoding resistance to rifampicin and isoniazid. In addition the PCR method is useful for diagnosis of Chlamydia infections in comparison with immunofluorescence. In conclusion, molecular techniques provide important assistance in successful diagnosis of bacterial infections.Στόχος της παρούσας εργασίας ήταν η σύγκριση της ειδικότητας και της ευαισθησία των μοριακών τεχνικών σε σχέση με τις κλασσικές συμβατικές μεθόδους στη διάγνωση των βακτηριακών λοιμώξεων. Στο χρονικό διάστημα 2006-2009 αναλύθηκαν με τις δύο μεθόδους: 326 αίματα, 136 ΕΝΥ, 80 πλευριτικά υγρά, 40 αρθρικά υγρά, 52 ιστοί, 295 γαστρικά-πτύελα-βρογχικές εκκρίσεις και 270 τραχηλικά δείγματα ασθενών από το Πανεπιστημιακό Νοσοκομείο Λάρισας. Στα κλινικά δείγματα χωρίς μικροβιακή χλωρίδα εφαρμόσαμε την ευρέως φάσματος 16S rRNA PCR, και στα αποτελέσματα προέκυψε μεγαλύτερο ποσοστό ευαισθησίας συγκριτικά με την καλλιέργεια : ΕΝΥ (100% έναντι 30%), πλευριτικά υγρά (100% έναντι 33%), αρθρικά υγρά (100% έναντι 0%) και ιστοί ορθοπεδικών λοιμώξεων (100% έναντι 52,4%). Aντίθετα, η εφαρμογή της ευρέως φάσματος PCR στο αίμα είχε μικρότερη ευαισθησία (34,6%) από την κλασική καλλιέργεια. Στα κλινικά δείγματα με φυσιολογική χλωρίδα εφαρμόσαμε PCR με εκκινητές ειδικούς για παθογόνα βακτήρια. Συγκεκριμένα στην ανίχνευση μυκοβακτηριδίων η μοριακή μέθοδος υστερεί των κλασσικών μεθόδων με ποσοστό ευαισθησίας μόνο 19,4%. Συνεισφέρει όμως σημαντικά στην ταυτοποίηση μυκοβακτηριδίων από άμεσο καλλιέργημα και στον προσδιορισμό των μεταλλάξεων που κωδικοποιούν αντοχές σε δύο κύρια αντιφυματικά αντιβιοτικά, τη ριφαμπικίνη και την ισονιαζίδη. Επίσης ουσιαστική βοήθεια φαίνεται να προσφέρει η PCR στη διάγνωση χλαμυδιακών λοιμώξεων σε σχέση με τον ανοσοφθορισμό. Συμπερασματικά, οι μοριακές τεχνικές βοηθούν σημαντικά στη διάγνωση των βακτηριακών λοιμώξεων

Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative.

There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era

Toll-Like Receptor 4 Gene (TLR4), but Not TLR2, Polymorphisms Modify the Risk of Tonsillar Disease Due to Streptococcus pyogenes and Haemophilus influenzae▿ †

Tonsillar disease (recurrent tonsillitis and/or tonsillar hypertrophy) is one of the most common human disorders, with Streptococcus pyogenes (group A beta-hemolytic streptococcus [GAS]) and Haemophilus influenzae representing the most common pathogens. Until now, no study has investigated why some individuals are more susceptible to tonsillar infections caused by specific bacteria than others. The aim of this study was to uncover possible associations between common Toll-like receptor gene (TLR) polymorphisms and tonsillar disease. The TLR2-R753Q, TLR4-D299G, and TLR4-T399I polymorphisms were determined in a cohort of 327 patients subjected to tonsillectomy due to recurrent tonsillitis (n = 245) and tonsillar hypertrophy (n = 82) and 245 healthy bone marrow donors. Associations of the aforementioned polymorphisms with the isolated bacterial strains after tonsillectomy were also investigated. Interestingly, carriers of the TLR4 polymorphisms displayed an approximately 3-fold increased risk for GAS infections (for TLR4-D299G, odds ratio [OR] = 2.81, 95% confidence interval [CI] = 1.16 to 6.79, P = 0.038; for TLR4-T399I, OR = 3.01, 95% CI = 1.29 to 7.02, P = 0.023), and this association was more profound in patients with recurrent tonsillitis. On the contrary, the presence of the TLR4-T399I polymorphism was associated with a 2-fold decreased risk of Haemophilus influenzae carriage (OR = 0.38, 95% CI = 0.15 to 0.96, P = 0.038). In the end, no significant differences were observed, considering the genotype and allele frequencies of the above-mentioned polymorphisms, between patients and controls. Our findings indicate that, regarding tonsillar infections, TLR4 polymorphisms predispose individuals to GAS infection, while they are protective against Haemophilus influenzae infection. This result further elucidates the role that host immune genetic variations might play in the susceptibility to common infections and tonsillar disease

Detection of Enterovirus D68 in Wastewater Samples from the UK between July and November 2021

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68′s known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed

Sequence analysis of iVDPV strains.

<p>Neighbour-joining tree representing phylogenetic relationships through the entire capsid coding sequence (2637 nt) between iVDPV isolates from the case study (shown as a number that corresponds to the date of isolation in the format <i>ddmmyy</i>), Sabin 2 vaccine strain and other type 2 VDPV and wild polioviruses. EMBL Data Library accession numbers for published capsid sequences are shown in the tree. Numbers at nodes indicate the percentage of 1000 bootstrap pseudoreplicates supporting the cluster. The sequence of PV1-Mahoney reference strain was introduced as an outgroup for the correct rooting of the tree. Isolates from the patient are labelled with blue circles on the tree, other iVDPV isolates are indicated in yellow, cVDPVs in red, VDPVs found in sewage samples in green and wild polioviruses in purple.</p

Neutralization of poliovirus strains by sera from fully immunised humans.

<p>The graphs represent comparison of neutralization titres in 40 sera from UK adults against iVDPV isolate 160198 versus MEF-1 (A) or versus Sabin 2 (B) vaccine strains in cell culture assays. The values are expressed as reciprocals (Log2) of the highest dilution of serum that protected 50% of the cell cultures determined by the Karber formula.</p