Challenges in the Clinical Recognition of Acute Flaccid Myelitis and its Implications.

OBJECTIVES: To explore the challenges in diagnosing acute flaccid myelitis (AFM) and evaluate clinical features and treatment paradigms associated with under recognition. STUDY DESIGN: This was a retrospective multicenter study of pediatric patients (≤18 years) who were diagnosed with AFM from 2014 to 2018 using the Centers for Disease Control and Prevention's case definition. RESULTS: In 72% of the cases (126 of 175), AFM was not considered in the initial differential diagnosis (n = 108; 61.7%) and/or the patient was not referred for acute care (n = 90; 51.4%) at the initial clinical encounter, and this did not improve over time. Although many features of the presentation were similar in those initially diagnosed with AFM and those who were not; preceding illness, constipation, and reflexes differed significantly between the 2 groups. Patients with a non-AFM initial diagnosis more often required ventilatory support (26.2% vs 12.2%; OR, 0.4; 95% CI, 0.2-1.0; P = .05). These patients received immunomodulatory treatment later (3 days vs 2 days after neurologic symptom onset; 95% CI, -2 to 0; P = .05), particularly intravenous immunoglobulin (5 days vs 2 days; 95% CI, -4 to -2; P < .001). CONCLUSIONS: Delayed recognition of AFM is concerning because of the risk for respiratory decompensation and need for intensive care monitoring. A non-AFM initial diagnosis was associated with delayed treatment that could have a clinical impact, particularly as new treatment options emerge.

Risk Factors for Infection and Health Impacts of the Coronavirus Disease 2019 (COVID-19) Pandemic in People With Autoimmune Diseases.

BACKGROUND: People with autoimmune or inflammatory conditions taking immunomodulatory/suppressive medications may have higher risk of novel coronavirus disease 2019 (COVID-19). Chronic disease care has also changed for many patients, with uncertain downstream consequences. METHODS: We included participants with autoimmune or inflammatory conditions followed by specialists at Johns Hopkins. Participants completed periodic surveys querying comorbidities, disease-modifying medications, exposures, COVID-19 testing and outcomes, social behaviors, and disruptions to healthcare. We assessed whether COVID-19 risk is higher among those on immunomodulating or suppressive agents and characterized pandemic-associated changes to care and mental health. RESULTS: In total, 265 (5.6%) developed COVID-19 over 9 months of follow-up (April-December 2020). Patient characteristics (age, race, comorbidity, medications) were associated with differences in social distancing behaviors during the pandemic. Glucocorticoid exposure was associated with higher odds of COVID-19 in models incorporating behavior and other potential confounders (odds ratio [OR]: 1.43; 95% confidence interval [CI]: 1.08, 1.89). Other medication classes were not associated with COVID-19 risk. Diabetes (OR: 1.72; 95% CI: 1.08, 2.73), cardiovascular disease (OR: 1.68; 95% CI: 1.24, 2.28), and kidney disease (OR: 1.76; 95% CI: 1.04, 2.97) were associated with higher odds of COVID-19. Of the 2156 reporting pre-pandemic utilization of infusion, mental health or rehabilitative services, 975 (45.2%) reported disruptions therein, which disproportionately affected individuals experiencing changes to employment or income. CONCLUSIONS: Glucocorticoid exposure may increase risk of COVID-19 in people with autoimmune or inflammatory conditions. Disruption to healthcare and related services was common. Those with pandemic-related reduced income may be most vulnerable to care disruptions.

Acute flaccid myelitis: cause, diagnosis, and management.

Acute flaccid myelitis (AFM) is a disabling, polio-like illness mainly affecting children. Outbreaks of AFM have occurred across multiple global regions since 2012, and the disease appears to be caused by non-polio enterovirus infection, posing a major public health challenge. The clinical presentation of flaccid and often profound muscle weakness (which can invoke respiratory failure and other critical complications) can mimic several other acute neurological illnesses. There is no single sensitive and specific test for AFM, and the diagnosis relies on identification of several important clinical, neuroimaging, and cerebrospinal fluid characteristics. Following the acute phase of AFM, patients typically have substantial residual disability and unique long-term rehabilitation needs. In this Review we describe the epidemiology, clinical features, course, and outcomes of AFM to help to guide diagnosis, management, and rehabilitation. Future research directions include further studies evaluating host and pathogen factors, including investigations into genetic, viral, and immunological features of affected patients, host-virus interactions, and investigations of targeted therapeutic approaches to improve the long-term outcomes in this population.

A human-derived 3D brain organoid model to study JC virus infection.

Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches.

Improvement in depressive symptoms after antiretroviral therapy initiation in people with HIV in Rakai, Uganda.

Depression is common following HIV infection and often improves after ART initiation. We aimed to identify distinct dimensions of depression that change following ART initiation in persons with HIV (PWH) with minimal comorbidities (e.g., illicit substance use) and no psychiatric medication use. We expected that dimensional changes in improvements in depression would differ across PWH. In an observational cohort in Rakai, Uganda, 312 PWH (51% male; mean age = 35.6 years) completed the Center for Epidemiologic Studies-Depression (CES-D) scale before and up to 2 years after ART initiation. Twenty-two percent were depressed (CES-D scores ≥ 16) pre-ART that decreased to 8% after ART. All CES-D items were used in a latent class analysis to identify subgroups with similar change phenotypes. Two improvement phenotypes were identified: affective-symptom improvement (n = 58, 19%) and mixed-symptom improvement (effort, appetite, irritability; n = 41, 13%). The affect-improvement subgroup improved on the greatest proportion of symptoms (76%). A third subgroup was classified as no-symptom changes (n = 213, 68%) as they showed no difference is symptom manifestation from baseline (93% did not meet depression criteria) to post-ART. Factors associated with subgroup membership in the adjusted regression analysis included pre-ART self-reported functional capacity, CD4 count, underweight BMI, hypertension, female sex(P's < 0.05). In a subset of PWH with CSF, subgroup differences were seen on Aß-42, IL-13, and IL-12. Findings support that depression generally improves following ART initiation; however, when improvement is seen the patterns of symptom improvement differ across PWH. Further exploration of this heterogeneity and its biological underpinning is needed to evaluate potential therapeutic implications of these differences.

Complement component 3 from astrocytes mediates retinal ganglion cell loss during neuroinflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterized by varying degrees of secondary neurodegeneration. Retinal ganglion cells (RGC) are lost in MS in association with optic neuritis but the mechanisms of neuronal injury remain unclear. Complement component C3 has been implicated in retinal and cerebral synaptic pathology that may precede neurodegeneration. Herein, we examined post-mortem MS retinas, and then used a mouse model, experimental autoimmune encephalomyelitis (EAE), to examine the role of C3 in the pathogenesis of RGC loss associated with optic neuritis. First, we show extensive C3 expression in astrocytes (C3+/GFAP+ cells) and significant RGC loss (RBPMS+ cells) in post-mortem retinas from people with MS compared to retinas from non-MS individuals. A patient with progressive MS with a remote history of optic neuritis showed marked reactive astrogliosis with C3 expression in the inner retina extending into deeper layers in the affected eye more than the unaffected eye. To study whether C3 mediates retinal degeneration, we utilized global C3-/- EAE mice and found that they had less RGC loss and partially preserved neurites in the retina compared with C3+/+ EAE mice. C3-/- EAE mice had fewer axonal swellings in the optic nerve, reflecting reduced axonal injury, but had no changes in demyelination or T cell infiltration into the CNS. Using a C3-tdTomato reporter mouse line, we show definitive evidence of C3 expression in astrocytes in the retina and optic nerves of EAE mice. Conditional deletion of C3 in astrocytes showed RGC protection replicating the effects seen in the global knockouts. These data implicate astrocyte C3 expression as a critical mediator of retinal neuronal pathology in EAE and MS, and are consistent with recent studies showing C3 gene variants are associated with faster rates of retinal neurodegeneration in human disease.

Early factors associated with later conversion to multiple sclerosis in patients presenting with isolated myelitis.

OBJECTIVE: To identify early clinical and paraclinical factors that may help predict later conversion to multiple sclerosis (MS) in patients presenting with isolated myelitis (ie, 'transverse myelitis' without clinical or radiological evidence of inflammation/demyelination elsewhere in the central nervous system). METHODS: In this retrospective cohort study, we included patients with isolated myelitis who were followed clinically and radiologically at our specialised myelopathy clinic. We excluded patients with MS at the onset, aquaporin-4-IgG seropositivity, myelin oligodendrocyte glycoprotein-IgG seropositivity or other identified aetiology. Logistic regression was used to identify factors predictive of conversion to MS (defined by the 2017 McDonald criteria). RESULTS: We included 100 patients, followed for a median of 4.3 years. Conversion to MS occurred in 25 of 77 patients (32%) with short-segment myelitis (longest lesion spanning <3 vertebral segments on MRI) as compared with 0 of 23 patients (0%) with longitudinally extensive myelitis (p=0.002). Among patients with short-segment myelitis, factors identified as highly predictive of conversion to MS using multivariate logistic regression included cerebrospinal fluid (CSF)-restricted oligoclonal bands (OCB) (OR (OR) 9.2, 95% CI 2.1 to 41.0, p=0.004), younger age (OR 1.1 for each year younger, 95% CI 1.0 to 1.1, p=0.04) and longer follow-up (OR 1.3 for each year longer, 95% CI 1.0 to 1.6, p=0.04). Conversion to MS occurred at a median of 2.8 years after myelitis onset. CONCLUSIONS: Short-segment MRI cord lesion(s), CSF-restricted OCB, younger age and longer follow-up are all factors predictive of conversion to MS in patients presenting with isolated myelitis.

Spinal fistulas documented by contrast enhanced computed tomography during myelopathy workup: a lost opportunity.

PURPOSE: Low-flow spinal arteriovenous fistulas (SAVFs) with intradural venous drainage typically manifest with a progressive venous hypertensive myelopathy (VHM) in older patients. VHM is difficult to identify. MRI is often nonspecific, and many cases are initially misdiagnosed, most often as transverse myelitis. The workup of myelopathic patients frequently includes thoracic and/or abdominal contrast-enhanced CT (CECT) that are generally not reviewed by neuroradiologists. The purpose of this work was to investigate how often abnormal enhancing intracanalar structures corresponding to the draining veins of a low-flow SAVF were documented by CECT. MATERIALS AND METHODS: We evaluated 92 consecutive patients with low-flow SAVFs and VHM treated at our institution between 2009 and 2018. The study group included 22 of these patients with at least one thoracoabdominal CECT available for review. The control group consisted of 20 consecutive myelopathy patients with negative angiography and at least one thoracoabdominal CECT. Intracanalar enhancing structures were classified either as (i) conspicuous or (ii) equivocal or absent. RESULTS: One CECT in the study group was technically inadequate. Conspicuous intracanalar enhancing structures were observed in 20 of the remaining 21 patients with SAVFs (95.2%) and in 2 of 20 control patients (10%). None of the enhancing intracanalar structures was mentioned in official study reports. CONCLUSIONS: The presence of enhancing vascular structures within the spinal canal on thoracoabdominal CECT obtained during the workup of myelopathies appears to represent a powerful but currently underappreciated tool for the detection of low-flow SAVFs.

Prenatal cytomegalovirus, rubella, and Zika virus infections associated with developmental disabilities: past, present, and future.

Prenatal infections have long been recognized as important, preventable causes of developmental disabilities. The list of pathogens that are recognized to have deleterious effects on fetal brain development continues to grow, most recently with the association between Zika virus (ZIKV) and microcephaly. To answer clinical questions in real time about the impact of a novel infection on developmental disabilities, an historical framework is key. The lessons learned from three historically important pathogens: rubella, cytomegalovirus, and ZIKV, and how these lessons are useful to approach emerging congenital infections are discussed in this review. Congenital infections are preventable causes of developmental disabilities and several public health approaches may be used to prevent prenatal infection. When they cannot be prevented, the sequelae of prenatal infection may be treatable. WHAT THIS PAPER ADDS: The list of prenatal infections associated with developmental disabilities continues to increase. Lessons learned from rubella, cytomegalovirus, and Zika virus have implications for new pathogens. Severity of illness in the mother does not correlate with severity of sequelae in the infant.

Beta-amyloid (Aß) uptake by PET imaging in older HIV+ and HIV- individuals.

The causes of cognitive impairment among older HIV+ individuals may overlap with causes among elderly HIV seronegative (HIV-) individuals. The objective of this study was to determine if beta-amyloid (Aß) deposition measured by [18F] AV-45 (florbetapir) positron emission tomography (PET) is increased in older HIV+ individuals compared to HIV- individuals. Forty-eight HIV+ and 25 HIV- individuals underwent [18F] AV-45 PET imaging. [18F] AV-45 binding to Aß was measured by standardized uptake value ratios (SUVR) relative to the cerebellum in 16 cortical and subcortical regions of interest. Global and regional cortical SUVRs were compared by (1) serostatus, (2) HAND stage, and (3) age decade, comparing individuals in their 50s and > 60s. There were no differences in median global cortical SUVR stratified by HIV serostatus or HAND stage. The proportion of HIV+ participants in their 50s with elevated global amyloid uptake (SUVR > 1.40) was significantly higher than the proportion in HIV- participants (67% versus 25%, p = 0.04), and selected regional SUVR values were also higher (p < 0.05) in HIV+ compared to HIV- participants in their 50s. However, these group differences were not seen in participants in their 60s. In conclusion, PET imaging found no differences in overall global Aß deposition stratified by HIV serostatus or HAND stage. Although there was some evidence of increased Aß deposition in HIV+ individuals in their 50s compared to HIV- individuals which might indicate premature aging, the most parsimonious explanation for this is the relatively small sample size in this cross-sectional cohort study.

Chronic Dengue Virus Panencephalitis in a Patient with Progressive Dementia with Extrapyramidal Features.

OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.

Acute Flaccid Myelitis: A Clinical Review.

Acute flaccid myelitis (AFM) is an emerging disorder primarily affecting children that is characterized by acute flaccid paralysis accompanied by abnormalities of the spinal cord gray matter on magnetic resonance imaging. In most cases, prodromal fever or respiratory symptoms occur, followed by acute-onset flaccid limb weakness. Respiratory, axial, bulbar, facial, and extraocular muscles may also be affected. The clinical manifestations have been described as "polio-like," due to striking similarities to cases of poliomyelitis. The primary site of injury in AFM is the anterior horn cells of the spinal cord, resulting in a motor neuronopathy. Seasonal peaks of cases have occurred in the United States every 2 years since 2012. However, AFM remains a rare disease, which can make it challenging for physicians to recognize and differentiate from other causes of acute flaccid paralysis such as Guillain-Barre syndrome, spinal cord stroke, and transverse myelitis. Epidemiological evidence suggests that AFM is linked to a viral etiology, with nonpolio enteroviruses (in particular enterovirus D68) demonstrating a plausible association. The epidemiology, possible etiological factors, clinical features, differential diagnosis, treatment, and outcomes of AFM are discussed in this review.

Guillain-Barré Syndrome Associated with Zika Virus Infection in Colombia.

BACKGROUND: Zika virus (ZIKV) infection has been linked to the Guillain-Barré syndrome. From November 2015 through March 2016, clusters of cases of the Guillain-Barré syndrome were observed during the outbreak of ZIKV infection in Colombia. We characterized the clinical features of cases of Guillain-Barré syndrome in the context of this ZIKV infection outbreak and investigated their relationship with ZIKV infection. METHODS: A total of 68 patients with the Guillain-Barré syndrome at six Colombian hospitals were evaluated clinically, and virologic studies were completed for 42 of the patients. We performed reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays for ZIKV in blood, cerebrospinal fluid, and urine, as well as antiflavivirus antibody assays. RESULTS: A total of 66 patients (97%) had symptoms compatible with ZIKV infection before the onset of the Guillain-Barré syndrome. The median period between the onset of symptoms of ZIKV infection and symptoms of the Guillain-Barré syndrome was 7 days (interquartile range, 3 to 10). Among the 68 patients with the Guillain-Barré syndrome, 50% were found to have bilateral facial paralysis on examination. Among 46 patients in whom nerve-conduction studies and electromyography were performed, the results in 36 patients (78%) were consistent with the acute inflammatory demyelinating polyneuropathy subtype of the Guillain-Barré syndrome. Among the 42 patients who had samples tested for ZIKV by RT-PCR, the results were positive in 17 patients (40%). Most of the positive RT-PCR results were in urine samples (in 16 of the 17 patients with positive RT-PCR results), although 3 samples of cerebrospinal fluid were also positive. In 18 of 42 patients (43%) with the Guillain-Barré syndrome who underwent laboratory testing, the presence of ZIKV infection was supported by clinical and immunologic findings. In 20 of these 42 patients (48%), the Guillain-Barré syndrome had a parainfectious onset. All patients tested were negative for dengue virus infection as assessed by RT-PCR. CONCLUSIONS: The evidence of ZIKV infection documented by RT-PCR among patients with the Guillain-Barré syndrome during the outbreak of ZIKV infection in Colombia lends support to the role of the infection in the development of the Guillain-Barré syndrome. (Funded by the Bart McLean Fund for Neuroimmunology Research and others.).

Vitamin D is not associated with HIV-associated neurocognitive disorder in Rakai, Uganda.

We investigated whether vitamin D is associated with HIV-associated neurocognitive disorder (HAND). HIV-infected (HIV+) antiretroviral therapy (ART)-naïve adults in rural Uganda underwent a neurocognitive battery for determination of HAND stage at baseline and after 2 years. Baseline serum 25-hydroxyvitamin D (25OH-D) and serum and cerebrospinal fluids (CSF) vitamin D-binding protein (VDBP) were obtained. Of the 399 participants, 4% (n = 16) were vitamin D deficient (25OH-D < 20 ng/mL). There was no association between 25OH-D, serum or CSF VDBP, and HAND stage at baseline or follow-up. Future studies in a population with higher levels of vitamin D deficiency may be warranted.

Interleukin-6 is associated with mortality and neuropsychiatric outcomes in antiretroviral-naïve adults in Rakai, Uganda.

Serum interleukin-6 (IL-6) and D-dimer have been associated with multiple adverse outcomes in HIV-infected (HIV+) individuals, but their association with neuropsychiatric outcomes, including HIV-associated neurocognitive disorder (HAND) and depression, headaches, and peripheral neuropathy have not been investigated. Three hundred ninety-nine HIV+ antiretroviral therapy (ART)-naïve adults in Rakai, Uganda, were enrolled in a longitudinal cohort study and completed a neurological evaluation, neurocognitive assessment, and venous blood draw. Half of the participants had advanced immunosuppression (CD4 count < 200 cells/µL), and half had moderate immunosuppression (CD4 count 350-500 cells/µL). All-cause mortality was determined by verbal autopsy within 2 years. HAND was determined using Frascati criteria, and depression was defined by the Center for Epidemiologic Studies-Depression (CES-D) scale. Neuropathy was defined as the presence of > 1 neuropathy symptom and > 1 neuropathy sign. Headaches were identified by self-report. Serum D-dimer levels were determined using ELISA and IL-6 levels using singleplex assays. Participants were 53% male, mean age 35 + 8 years, and mean education 5 + 3 years. Participants with advanced immunosuppression had significantly higher levels of IL-6 (p < 0.001) and a trend toward higher D-dimer levels (p = 0.06). IL-6 was higher among participants with HAND (p = 0.01), with depression (p = 0.03) and among those who died within 2 years (p = 0.001) but not those with neuropathy or headaches. D-dimer did not vary significantly by any outcome. Systemic inflammation as measured by serum IL-6 is associated with an increased risk of advanced immunosuppression, all-cause mortality, HAND, and depression but not neuropathy or headaches among ART-naïve HIV+ adults in rural Uganda.

Comparative quantitative clinical, neuroimaging, and functional profiles in children with acute flaccid myelitis at acute and convalescent stages of disease.

AIM: To quantify characteristics in acute flaccid myelitis (AFM) at acute and convalescent stages. METHOD: This was a retrospective case series of children with AFM evaluated at a single institution in the USA (2014-2017). Acute inflammatory/ischemic myelopathies were excluded. Neurological assessments and segmental quantitative analysis of signal abnormalities on magnetic resonance imaging (MRI) of the brain and spinal cord were performed. RESULTS: Sixteen patients (11 males, five females) were evaluated. Median age at onset was 4 years (interquartile range [IQR] 3-6y). All had parainfectious acute-onset limb weakness, lower motor neuron examination, and spinal fluid pleocytosis. On acute spinal cord MRI, longitudinally extensive T2 hyperintensities were identified throughout the spinal cord mostly within grey matter; five out of 12 patients had dorsal brainstem T2 hyperintensities. At a median of 2 months follow-up (IQR 2-3mo), spinal cord MRI improved in seven out of nine patients although focal T2 hyperintensities persisted in cervical and lumbar grey matter. At a median follow-up of 4 months (IQR 2-6mo), Medical Research Council sum score rose from a median of 29 to 32; distal muscle groups improved more than proximal ones; four out of 16 patients were ventilator-dependent; and two out of 16 patients were quadriplegic. INTERPRETATION: While patients may show marked improvement on neuroimaging from acute to convalescent stages, the majority of children with AFM have limited motor recovery and continued disability. Clinicians should consider the timing of clinical and neuroimaging exams when assessing diagnosis and prognosis. WHAT THIS PAPER ADDS: During the 2014 to 2017 acute flaccid myelitis outbreak in the USA, clinical recovery was better in distal than proximal muscle groups. Lumbar spinal cord showed more residual abnormalities at convalescence.

Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics.

The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

Rotenone exerts developmental neurotoxicity in a human brain spheroid model.

Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 µM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 µM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.

Emerging Viral Infections and Their Impact on the Global Burden of Neurological Disease.

Emerging viral infections of the nervous system represent a major global public health concern in the 21st century. They are caused primarily by RNA viruses and are mostly associated with acute or subacute encephalitis. The spectrum of associated central or peripheral nervous system disorders is broad, and results either from a direct viral effect or due to the host immune responses against the infection. Emerging viral infections impose substantial neurological morbidity and mortality, particularly in low- and middle-income regions. In the past five decades, vector-borne viruses primarily transmitted by arthropods, or arboviruses, have been responsible for epidemics with a high burden of neurological disease, like the 2015-2016 Zika virus epidemic in the Americas. Viruses that have become neurovirulent for humans after geographical expansion include West Nile, Dengue, and Zika viruses. Factors such as animal migration, disruption of ecological niches, and cross-species contact have caused old viruses to reappear and cause neurological disease, as is the case of Ebola virus. In addition to these biological challenges, current preventive strategies, vaccination, and diagnostic and therapeutic approaches remain limited. We review the clinical-virological features and global impact of the most relevant emerging viral infections of the nervous system as they are projected over the 21st century.

Neurological Implications of Zika Virus Infection in Adults.

The 2015-2016 epidemic of Zika virus (ZIKV) in the Americas and the Caribbean was associated with an unprecedented burden of neurological disease among adults. Clinically, Guillain-Barre syndrome (GBS) predominated among regions affected by the ZIKV epidemic, but the spectrum of neurological disease in the adults appears broader as cases of encephalopathy, encephalitis, meningitis, myelitis, and seizures have also been reported. A para-infectious temporal profile of ZIKV-associated GBS (ZIKV-GBS) has been described in clinical studies, which may suggest a direct viral neuropathic effect. However, ZIKV neuropathogenesis has not yet been fully understood. Mechanisms for ZIKV-GBS and other neurological syndromes have been hypothesized, such as adaptive viral genetic changes, immunological interactions with other circulating flaviviruses, and host and factors. This review summarizes the current evidence on ZIKV-associated neurological complications in the adults.