Repurposing rapid diagnostic tests to detect falsified vaccines in supply chains.

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose but have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global 'Prevent, Detect and Respond' strategy.

Innovative method for rapid detection of falsified COVID-19 vaccines through unopened vials using handheld Spatially Offset Raman Spectroscopy (SORS).

Preventing, detecting, and responding to substandard and falsified vaccines is of critical importance for ensuring the safety, efficacy, and public trust in vaccines. This is of heightened importance in context of public health crisis, such as the COVID-19 pandemic, in which extreme world-wide shortages of vaccines provided a fertile ground for exploitation by falsifiers. Here, a proof-of-concept study explored the feasibility of using a handheld Spatially Offset Raman Spectroscopy (SORS) device to authenticate COVID-19 vaccines through rapid analysis of unopened vaccine vials. The results show that SORS can verify the chemical identity of dominant excipients non-invasively through vaccine vial walls. The ability of SORS to identify potentially falsified COVID-19 vaccines was demonstrated by measurement of surrogates for falsified vaccines contained in vaccine vials. In all cases studied, the SORS technique was able to differentiate between surrogate samples from the genuine COVISHIELD™ vaccine. The genuine vaccines tested included samples from six batches across two manufacturing sites to account for any potential variations between batches or manufacturing sites. Batch and manufacturing site variations were insignificant. In conjunction with existing security features, for example on labels and packaging, SORS provided an intrinsic molecular fingerprint of the dominant excipients of the vaccines. The technique could be extended to other COVID-19 and non-COVID-19 vaccines, as well as other liquid medicines. As handheld and portable SORS devices are commercially available and widely used for other purposes, such as airport security, they are rapidly deployable non-invasive screening tools for vaccine authentication.

The Queen Square Encephalitis Multidisciplinary Team Meeting - experience over three years, pre and post the COVID-19 pandemic.

BACKGROUND: Patients with suspected encephalitis continue to represent a diagnostic and therapeutic challenge, even in highly resourced centres. In February 2018, we set up a monthly in-person multidisciplinary team meeting (MDT). We describe the experience and outcomes of the MDT over three years. METHODS: A retrospective analysis was performed to summarise patient demographics, MDT outcomes and final diagnoses. RESULTS: Over the three-year period, 324 discussions of 238 patients took place. Cases were diverse; approximately 40% related to COVID-19 or brain infection, 40% autoimmune or other inflammatory disorders and 20% encephalitis mimics or uncertain aetiologies. Feedback from an online survey sent to referring teams and attendees highlighted the value of the MDT; 94% reported the discussion was useful and 69% reported resulting change in patient management. CONCLUSIONS: Multidisciplinary input is crucial in this challenging area, ensuring that all diagnostic avenues are explored and opening doors to novel diagnostics and therapeutics. It also supports clinicians dealing with unwell patients, including in centres where less specialist input is available, and when decisions have to be made where there is little or no evidence base.

Pathophysiology, diagnosis, and management of neuroinflammation in covid-19.

Although neurological complications of SARS-CoV-2 infection are relatively rare, their potential long term morbidity and mortality have a significant impact, given the large numbers of infected patients. Covid-19 is now in the differential diagnosis of a number of common neurological syndromes including encephalopathy, encephalitis, acute demyelinating encephalomyelitis, stroke, and Guillain-Barré syndrome. Physicians should be aware of the pathophysiology underlying these presentations to diagnose and treat patients rapidly and appropriately. Although good evidence has been found for neurovirulence, the neuroinvasive and neurotropic potential of SARS-CoV-2 is limited. The pathophysiology of most complications is immune mediated and vascular, or both. A significant proportion of patients have developed long covid, which can include neuropsychiatric presentations. The mechanisms of long covid remain unclear. The longer term consequences of infection with covid-19 on the brain, particularly in terms of neurodegeneration, will only become apparent with time and long term follow-up.

Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

Exploring the Potential of Iminosugars as Antivirals for Crimean-Congo Haemorrhagic Fever Virus, Using the Surrogate Hazara Virus: Liquid-Chromatography-Based Mapping of Viral N-Glycosylation and In Vitro Antiviral Assays.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a pathogen of increasing public health concern, being a widely distributed arbovirus and the causative agent of the potentially fatal Crimean-Congo haemorrhagic fever. Hazara virus (HAZV) is a genetically and serologically related virus that has been proposed as a surrogate for antiviral and vaccine testing for CCHFV. Glycosylation analysis of HAZV has been limited; first, we confirmed for the first time the occupation of two N-glycosylation sites in the HAZV glycoprotein. Despite this, there was no apparent antiviral efficacy of a panel of iminosugars against HAZV, as determined by quantification of the total secretion and infectious virus titres produced following infection of SW13 and Vero cells. This lack of efficacy was not due to an inability of deoxynojirimycin (DNJ)-derivative iminosugars to access and inhibit endoplasmic reticulum α-glucosidases, as demonstrated by free oligosaccharide analysis in uninfected and infected SW13 and uninfected Vero cells. Even so, iminosugars may yet have potential as antivirals for CCHFV since the positions and importance of N-linked glycans may differ between the viruses, a hypothesis requiring further evaluation.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

A systematic review of brain imaging findings in neurological infection with Japanese encephalitis virus compared with Dengue virus.

OBJECTIVES: Japanese encephalitis virus (JEV) and dengue virus (DENV) represent important causes of encephalitis in Asia. Brain imaging may provide diagnostic clues about the etiology of infectious encephalitis. We performed a systematic review of brain imaging findings in Japanese encephalitis (JE) and DENV neurological infection (dengue) to identify characteristic lesions. METHODOLOGY: Five databases were searched. We included all study types and imaging techniques. Laboratory methods were categorized using diagnostic confidence levels. Imaging data were synthesized, and focal findings are presented as proportions for JE and dengue and for subgroups based on diagnostic confidence. PRINCIPAL FINDINGS: Thalamic lesions were the most reported magnetic resonance imaging finding in both diseases but appeared to occur more often in JE (74% in 23 studies) than dengue (29.4% in 58 studies). In cases diagnosed with antigen or nucleic acid tests, thalamic lesions were reported frequently in both JE (76.5% in 17 studies) and dengue (65.2% in 23 studies). SIGNIFICANCE: The results suggest that thalamic lesions frequently occur in both JE and dengue encephalitis. No radiological findings were found to be pathognomonic of either disease. Although brain imaging may support a diagnosis, laboratory confirmation with highly specific tests remains crucial.

Mouse models of Japanese encephalitis virus infection: A systematic review and meta-analysis using a meta-regression approach.

BACKGROUND: Japanese encephalitis (JE) virus (JEV) remains a leading cause of neurological infection across Asia. The high lethality of disease and absence of effective therapies mean that standardised animal models will be crucial in developing therapeutics. However, published mouse models are heterogeneous. We performed a systematic review, meta-analysis and meta-regression of published JEV mouse experiments to investigate the variation in model parameters, assess homogeneity and test the relationship of key variables against mortality. METHODOLOGY/ PRINCIPAL FINDINGS: A PubMed search was performed up to August 2020. 1991 publications were identified, of which 127 met inclusion criteria, with data for 5026 individual mice across 487 experimental groups. Quality assessment was performed using a modified CAMARADES criteria and demonstrated incomplete reporting with a median quality score of 10/17. The pooled estimate of mortality in mice after JEV challenge was 64.7% (95% confidence interval 60.9 to 68.3) with substantial heterogeneity between experimental groups (I^2 70.1%, df 486). Using meta-regression to identify key moderators, a refined dataset was used to model outcome dependent on five variables: mouse age, mouse strain, virus strain, virus dose (in log10PFU) and route of inoculation. The final model reduced the heterogeneity substantially (I^2 38.9, df 265), explaining 54% of the variability. CONCLUSION/ SIGNIFICANCE: This is the first systematic review of mouse models of JEV infection. Better adherence to CAMARADES guidelines may reduce bias and variability of reporting. In particular, sample size calculations were notably absent. We report that mouse age, mouse strain, virus strain, virus dose and route of inoculation account for much, though not all, of the variation in mortality. This dataset is available for researchers to access and use as a guideline for JEV mouse experiments.

The role of viral genomics in understanding COVID-19 outbreaks in long-term care facilities.

We reviewed all genomic epidemiology studies on COVID-19 in long-term care facilities (LTCFs) that had been published to date. We found that staff and residents were usually infected with identical, or near identical, SARS-CoV-2 genomes. Outbreaks usually involved one predominant cluster, and the same lineages persisted in LTCFs despite infection control measures. Outbreaks were most commonly due to single or few introductions followed by a spread rather than a series of seeding events from the community into LTCFs. The sequencing of samples taken consecutively from the same individuals at the same facilities showed the persistence of the same genome sequence, indicating that the sequencing technique was robust over time. When combined with local epidemiology, genomics allowed probable transmission sources to be better characterised. The transmission between LTCFs was detected in multiple studies. The mortality rate among residents was high in all facilities, regardless of the lineage. Bioinformatics methods were inadequate in a third of the studies reviewed, and reproducing the analyses was difficult because sequencing data were not available in many facilities.

STROBE-metagenomics: a STROBE extension statement to guide the reporting of metagenomics studies.

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.

The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings.

Preliminary clinical data indicate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with neurological and neuropsychiatric illness. Responding to this, a weekly virtual coronavirus disease 19 (COVID-19) neurology multi-disciplinary meeting was established at the National Hospital, Queen Square, in early March 2020 in order to discuss and begin to understand neurological presentations in patients with suspected COVID-19-related neurological disorders. Detailed clinical and paraclinical data were collected from cases where the diagnosis of COVID-19 was confirmed through RNA PCR, or where the diagnosis was probable/possible according to World Health Organization criteria. Of 43 patients, 29 were SARS-CoV-2 PCR positive and definite, eight probable and six possible. Five major categories emerged: (i) encephalopathies (n = 10) with delirium/psychosis and no distinct MRI or CSF abnormalities, and with 9/10 making a full or partial recovery with supportive care only; (ii) inflammatory CNS syndromes (n = 12) including encephalitis (n = 2, para- or post-infectious), acute disseminated encephalomyelitis (n = 9), with haemorrhage in five, necrosis in one, and myelitis in two, and isolated myelitis (n = 1). Of these, 10 were treated with corticosteroids, and three of these patients also received intravenous immunoglobulin; one made a full recovery, 10 of 12 made a partial recovery, and one patient died; (iii) ischaemic strokes (n = 8) associated with a pro-thrombotic state (four with pulmonary thromboembolism), one of whom died; (iv) peripheral neurological disorders (n = 8), seven with Guillain-Barré syndrome, one with brachial plexopathy, six of eight making a partial and ongoing recovery; and (v) five patients with miscellaneous central disorders who did not fit these categories. SARS-CoV-2 infection is associated with a wide spectrum of neurological syndromes affecting the whole neuraxis, including the cerebral vasculature and, in some cases, responding to immunotherapies. The high incidence of acute disseminated encephalomyelitis, particularly with haemorrhagic change, is striking. This complication was not related to the severity of the respiratory COVID-19 disease. Early recognition, investigation and management of COVID-19-related neurological disease is challenging. Further clinical, neuroradiological, biomarker and neuropathological studies are essential to determine the underlying pathobiological mechanisms that will guide treatment. Longitudinal follow-up studies will be necessary to ascertain the long-term neurological and neuropsychological consequences of this pandemic.

Correction to: Relevance of biomarkers across different neurodegenerative diseases.

An amendment to this paper has been published and can be accessed via the original article.

Relevance of biomarkers across different neurodegenerative diseases.

BACKGROUND: The panel of fluid- and imaging-based biomarkers available for neurodegenerative disease research is growing and has the potential to close important gaps in research and the clinic. With this growth and increasing use, appropriate implementation and interpretation are paramount. Various biomarkers feature nuanced differences in strengths, limitations, and biases that must be considered when investigating disease etiology and clinical utility. For example, neuropathological investigations of Alzheimer's disease pathogenesis can fall in disagreement with conclusions reached by biomarker-based investigations. Considering the varied strengths, limitations, and biases of different research methodologies and approaches may help harmonize disciplines within the neurodegenerative disease field. PURPOSE OF REVIEW: Along with separate review articles covering fluid and imaging biomarkers in this issue of Alzheimer's Research and Therapy, we present the result of a discussion from the 2019 Biomarkers in Neurodegenerative Diseases course at the University College London. Here, we discuss themes of biomarker use in neurodegenerative disease research, commenting on appropriate use, interpretation, and considerations for implementation across different neurodegenerative diseases. We also draw attention to areas where biomarker use can be combined with other disciplines to understand issues of pathophysiology and etiology underlying dementia. Lastly, we highlight novel modalities that have been proposed in the landscape of neurodegenerative disease research and care.

Harnessing Dengue Rapid Diagnostic Tests for the Combined Surveillance of Dengue, Zika, and Chikungunya Viruses in Laos.

Recent expansions of vector-borne diseases highlight the need for improved surveillance, especially in resource-poor settings. Dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) share the same vectors as well as similar clinical presentations, suggesting that combined surveillance would be useful. We hypothesized that blood spotted on dengue rapid diagnostic tests (RDTs) could be harnessed for sample collection in remote areas for subsequent detection of DENV, CHIKV, and ZIKV by reverse transcription real-time polymerase chain reaction (RT-qPCR). CHIKV and ZIKV dilutions were spotted on dengue RDTs (SD BIOLINE Dengue DUO, Standard Diagnostics, Gyeonggi-do, Republic of Korea), dried, and extracted. As reference, aliquots of each viral dilution were directly extracted. Using specific RT-qPCR tests, both viruses were successfully detected from RDT extracts. However, the limit of detection was slightly lower in comparison to direct extracts, two logfold for CHIKV and one logfold for ZIKV. For analysis of temperature stability, DENV dilutions were spotted on RDTs and stored for up to 2 months at -80°C, 4°C, or 35°C before testing. Storage of RDTs for 2 months at 35°C did not compromise detection of RNA by RT-qPCR; only minimal degradation was observed. This proof-of-principle study demonstrates the potential of using dengue RDTs for DENV/CHIKV/ZIKV combined surveillance in areas without access to laboratory facilities. Further investigations are needed for evaluation of tri-viral surveillance under field conditions using patient samples. Large-scale implementation of surveillance for these viruses is of crucial public health importance for the early detection of epidemics. This method also has important implications for improving understanding of the molecular epidemiology of the three viruses.

A need to raise the bar - A systematic review of temporal trends in diagnostics for Japanese encephalitis virus infection, and perspectives for future research.

OBJECTIVE: Japanese encephalitis virus infection (JE) remains a leading cause of neurological disease in Asia, mainly involving individuals living in remote areas with limited access to treatment centers and diagnostic facilities. Laboratory confirmation is fundamental for the justification and implementation of vaccination programs. We reviewed the literature on historical developments and current diagnostic capability worldwide, to identify knowledge gaps and instill urgency to address them. METHODS: Searches were performed in Web of Science and PubMed using the term 'Japanese encephalitis' up to 13th October 2019. Studies reporting laboratory-confirmed symptomatic JE cases in humans were included, and data on details of diagnostic tests were extracted. A JE case was classified according to confirmatory levels (Fischer et al., 2008; Campbell et al., 2011; Pearce et al., 2018; Heffelfinger et al., 2017), where level 1 represented the highest level of confidence. FINDINGS: 20,212 published JE cases were identified from 205 studies. 15,167 (75%) of these positive cases were confirmed with the lowest-confidence diagnostic tests (level 3 or 4, or level 4). Only 109 (53%) of the studies reported contemporaneous testing for dengue-specific antibodies. CONCLUSION: A fundamental pre-requisite for the control of JEV is lacking - that of a simple and specific diagnostic procedure that can be adapted for point-of-care tests and readily used throughout JE-endemic regions of the world.

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

A 9-month retrospective evaluation of the aetiology and management of patients presenting with encephalitis/meningoencephalitis at a South London hospital.

Encephalitis causes high morbidity and mortality. An incidence of 4.3 cases of encephalitis/100 000 population has been reported in the UK. We performed a retrospective evaluation of the diagnosis and management of adults admitted to hospital with a clinical diagnosis of encephalitis/meningoencephalitis. Clinical, laboratory and radiological data were collated from electronic records. Thirty-six patients, median age 55 years and 24 (67%) male were included. The aetiology was confirmed over nine months in 25 (69%) of whom 16 were infections (six viral, seven bacterial, two parasitic and one viral and parasitic co-infection); 7 autoimmune; 1 metabolic and 1 neoplastic. Of 24 patients with fever, 15 (63%) had an infection. The median time to computed topography, magnetic resonance imaging and electroencephalography (EEG) was 1, 8 and 3 days respectively. Neuroimaging was abnormal in 25 (69%) and 17 (89%) had abnormal EEGs. Only 19 (53%) received aciclovir treatment. Six (17%) made good recoveries, 16 (44%) had moderate disability, 8 (22%) severe disability and 6 (17%) died. Outcomes were worse for those with an infectious cause. In summary, a diagnosis was made in 69.4% of patients admitted with encephalitis/meningoencephalitis. Autoimmune causes are important to consider at an early stage due to a successful response to treatment. Only 53% of patients received aciclovir on admission. Neuroimaging and EEG studies were delayed. The results of this work resulted in further developing the clinical algorithm for managing these patients.