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We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S–internal transcribed ribosomal gene spacer (28S–ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids. A universal method enables high-specificity, unbiased pathogen detection from diverse body fluids using metagenomic sequencing and may accelerate clinical decisions.

ObjectiveRapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD.MethodsImmunoglobulin G (IgG) from pediatric ROHHAD patients (n=9), non-inflammatory individuals (n=100) and relevant pediatric controls (n=25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively.ResultsAutoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed.InterpretationOur results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

Purpose We describe the design of a longitudinal cohort study to determine SARS-CoV-2 incidence and prevalence among a population-based sample of adults living in six San Francisco Bay Area counties. Methods Using an address-based sample, we stratified households by county and by census-tract risk. Risk strata were determined by using regression models to predict infections by geographic area using census-level sociodemographic and health characteristics. We disproportionately sampled high and medium risk strata, which had smaller population sizes, to improve precision of estimates, and calculated a desired sample size of 3400. Participants were primarily recruited by mail and were followed monthly with PCR testing of nasopharyngeal swabs, testing of venous blood samples for antibodies to SARS-CoV-2 spike and nucleocapsid antigens, and testing of the presence of neutralizing antibodies, with completion of questionnaires about socio-demographics and behavior. Estimates of incidence and prevalence will be weighted by county, risk strata and sociodemographic characteristics of non-responders, and will take into account laboratory test performance. Results We enrolled 3842 adults from August to December, 2020, and completed follow-up March 31, 2021. We reached target sample sizes within most strata. Conclusions Our stratified random sampling design will allow us to recruit a robust general population cohort of adults to determine the incidence of SARS-CoV-2 infection. Identifying risk strata was unique to the design and will help ensure precise estimates, and high-performance testing for presence of virus and antibodies will enable accurate ascertainment of infections.

Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)1-6. Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)2. CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM.

Importance Cerebrospinal fluid (CSF) cytologic testing and flow cytometry are insensitive for diagnosing neoplasms of the central nervous system (CNS). Such clinical phenotypes can mimic infectious and autoimmune causes of meningoencephalitis. Objective To ascertain whether CSF metagenomic next-generation sequencing (mNGS) can identify aneuploidy, a hallmark of malignant neoplasms, in difficult-to-diagnose cases of CNS malignant neoplasm. Design, setting, and participants Two case-control studies were performed at the University of California, San Francisco (UCSF). The first study used CSF specimens collected at the UCSF Clinical Laboratories between July 1, 2017, and December 31, 2019, and evaluated test performance in specimens from patients with a CNS malignant neoplasm (positive controls) or without (negative controls). The results were compared with those from CSF cytologic testing and/or flow cytometry. The second study evaluated patients who were enrolled in an ongoing prospective study between April 1, 2014, and July 31, 2019, with presentations that were suggestive of neuroinflammatory disease but who were ultimately diagnosed with a CNS malignant neoplasm. Cases of individuals whose tumors could have been detected earlier without additional invasive testing are discussed. Main outcomes and measures The primary outcome measures were the sensitivity and specificity of aneuploidy detection by CSF mNGS. Secondary subset analyses included a comparison of CSF and tumor tissue chromosomal abnormalities and the identification of neuroimaging characteristics that were associated with test performance. Results Across both studies, 130 participants were included (median [interquartile range] age, 57.5 [43.3-68.0] years; 72 men [55.4%]). The test performance study used 125 residual laboratory CSF specimens from 47 patients with a CNS malignant neoplasm and 56 patients with other neurological diseases. The neuroinflammatory disease study enrolled 12 patients and 17 matched control participants. The sensitivity of the CSF mNGS assay was 75% (95% CI, 63%-85%), and the specificity was 100% (95% CI, 96%-100%). Aneuploidy was detected in 64% (95% CI, 41%-83%) of the patients in the test performance study with nondiagnostic cytologic testing and/or flow cytometry, and in 55% (95% CI, 23%-83%) of patients in the neuroinflammatory disease study who were ultimately diagnosed with a CNS malignant neoplasm. Of the patients in whom aneuploidy was detected, 38 (90.5%) had multiple copy number variations with tumor fractions ranging from 31% to 49%. Conclusions and relevance This case-control study showed that CSF mNGS, which has low specimen volume requirements, does not require the preservation of cell integrity, and was orginally developed to diagnose neurologic infections, can also detect genetic evidence of a CNS malignant neoplasm in patients in whom CSF cytologic testing and/or flow cytometry yielded negative results with a low risk of false-positive results.

ImportanceMalignant neoplasms of the central nervous system (CNS) are frequently not detected by cerebrospinal fluid (CSF) flow cytometry or cytology, and clinical phenotypes can overlap with inflammatory meningoencephalitis.ObjectiveTo determine whether an existing CSF metagenomic next-generation sequencing (mNGS) assay can identify a hallmark of malignant neoplasms — aneuploidy — in difficult-to-diagnose cases of CNS malignancy.DesignTwo retrospective, case-control studies included a total of 155 samples from patients with an eventual diagnosis of a CNS malignancy (n=59 patients) and controls with other CNS diseases (n=73 patients). The first study was used to evaluate test performance in positive and negative controls. The second study was used to assess the potential utility of aneuploidy detection in patients whose CSF was sent for mNGS because of suspected neuroinflammatory disease who were ultimately found to have a CNS malignancy.SettingThis is a single site study at a large tertiary care center, University of California San Francisco, that enrolled from 2014 to 2019.ParticipantsThe test performance case-control study enrolled positive control patients with a CNS malignancy (n=47 patients) and negative controls with other neurologic diseases (n=56 patients) who had had CSF flow cytometry and/or cytology performed. The second case-control study enrolled patients with suspected neuroinflammatory disease who were ultimately diagnosed with a CNS malignancy (n=12) and other neurologic disease controls (n=17).Main Outcome(s) and Measure(s)The primary outcome measures were the performance characteristics of detecting aneuploidy in CSF by a cell-free DNA mNGS assay compared to cytology and/or flow cytometry and the tumor fraction in CSF from patients with CNS malignancies.ResultsAcross the two case-control studies, the overall sensitivity of the CSF mNGS assay for detecting aneuploidy in patients ultimately diagnosed with a CNS malignancy was 75% (63-96%, 95% CI), and specificity was 100% (96-100%, 95% CI). Notably, CSF mNGS detected aneuploidy in 64% of the non-diagnostic cytology and flow cytometry cases in the test performance study and in 55% of the cases with suspected neuroinflammatory disease who were ultimately diagnosed with a CNS malignancy. Of the cases in whom aneuploidy was detected, 90% had multiple chromosomal copy number variants with tumor fractions ranging from 31% to 49%.Conclusions and RelevanceMetagenomic NGS of CSF, originally designed to diagnose neurologic infections, detects evidence of CNS malignancies (i.e., aneuploidy) in cases where CSF flow cytometry and/or cytology were negative with a low risk of false positive results.3 Key PointsQuestionCan CSF metagenomic NGS, a test designed to diagnose infections, also detect genetic signatures of cancer in patients with suspected neuroinflammatory disease?FindingsAcross two case-control studies of patients with negative CSF cytology and/or flow cytometry, CSF mNGS detected genetic evidence for a malignancy with a sensitivity of 75% (63-85%, 95% CI) and specificity of 100% (96-100%, 95% CI).MeaningCSF mNGS, an assay with low sample volume requirements that does not require the preservation of cell integrity, has the potential to complement cancer detection by CSF flow cytometry and cytology.

BackgroundHealthcare personnel (HCP) are prioritized for earliest SARS-CoV-2 vaccine administration, yet relatively few data exist on HCP’s knowledge, motivations, concerns, and intentions regarding COVID-19 vaccines.MethodsWe conducted a cross-sectional survey Nov.16-Dec.8, 2020 among HCP enrolled in a cohort study at three Northern California medical centers serving diverse roles including COVID-19 patient care. Eligible HCP were adult (age≥18) on-site employees of the University of California, San Francisco, San Francisco General Hospital, and Stanford Healthcare. A one-time electronically-administered survey was sent to cohort HCP on November 16, 2020 and responses analyzed.ResultsOverall, among 2,448 HCP invited, 2,135 completed the COVID-19 vaccine survey (87.2% response rate). HCPs had mean age 41 years, were 73% female, and had diverse jobs including COVID-19 patient contact. Enthusiasm for vaccination was overall strong, and more HCP (1,453, 69%) said they would definitely/likely receive vaccine if formally FDA-approved versus if approved via emergency use authorization only (785, 35%). While 541 (25%) respondents wanted to be among the earliest to receive vaccine, more desired vaccination after the first round (777, 36%) or >2 months after vaccinations began (389, 18%). Top factors increasing motivation for vaccination included perceiving risk from COVID-19 to self (1,382, 65%) or to family/friends (1355, 63%). Top concerns were vaccine side effects, cited by 596 (28%), and concerns about political involvement in FDA’s approval process (249, 12%).ConclusionsHCP were enthusiastic about COVID-19 vaccination for individual protection and protecting others, but harbored concerns about vaccine side effects. Our data may inform emerging vaccine education campaigns.Key PointsAmong 2,135 healthcare personnel surveyed, we found enthusiasm for COVID-19 vaccination both for individual benefit and protecting others. However, healthcare personnel rated their knowledge of COVID-19 vaccines as only moderate and harbored concerns about vaccine side effects. Education raising awareness of vaccine efficacy and side effects may help maximize vaccine uptake.

Introduction Agenesis of the corpus callosum (AgCC) is characterized by the congenital partial or complete absence of the corpus callosum. Several strains of mice have been reported to carry AgCC, with the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain consistently showing a complete absence of the corpus callosum, as well as a variable reduction in the size of the hippocampal commissure. While much research has focused on the social deficits of the BTBR strain, little research on its cognitive behavior has been conducted. The goal of our study was to compare two facets of executive functioning, spatial working memory, and sustained attention between the BTBR and C57BL/6J (B6) strains. Methods Spatial working memory was measured utilizing a delayed matching‐to‐position (DMTP) task and sustained attention was measured utilizing an operant task in which mice were trained to distinguish signal and nonsignal events. Results Both the BTBR and B6 mice demonstrated a predictable decline in performance on the DMTP task as the delay interval increased and predictable increase in performance on the sustained attention task as the duration of the signal event increased. Although no significant differences were found between strains on the performance of these tasks, there was a significant difference in learning the association between lever pressing and food reward. Histological investigation confirmed the complete absence of commissural fibers from the corpus callosum, but also the hippocampal commissure, counter to a previous study. Conclusion The results suggest spatial working memory and sustained attention are unaffected by the absence of these commissural fibers alone., The cognitive deficits associated with agenesis of the corpus callosum have been understudied in the BTBR mouse. We studied working memory and sustained attention in this mouse model of AgCC utilizing operant paradigms. Surprisingly, our results from mice with histologically confirmed absence of the corpus callosum and hippocampal commissure were similar to the results from B6 controls.

Author(s): Beck, Erin S; Ramachandran, Prashanth S; Khan, Lillian M; Sample, Hannah A; Zorn, Kelsey C; O'Connell, Elise M; Nash, Theodore; Reich, Daniel S; Venkatesan, Arun; DeRisi, Joseph L; Nath, Avindra; Wilson, Michael R

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF. Grants supporting this project include the National Multiple Sclerosis Society and the American Academy of Neurology award FAN-1608-25607 (R.D.S.), Clinical Research Training Scholarship P0534134 (P.S.R.), Sandler and William K. Bowes Jr Foundations (M.R.W., J.L.D., L.M.K., H.A.S., K.C.Z.), Rachleff Family Foundation (M.R.W.), and NINDS of the NIH under award K08NS096117 (M.R.W.) and F31NS113432 (K.E.L.). This study was partially supported by a grant from the Spanish National Health Institute [grant number PI15CIII-00020] and the European Regional Development Fund (FEDER funds). UCSF Biomedical Sciences Program (I.A.H., K.E.L.), UCSF Medical Scientist Training Program (K.E.L.), and the Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., J.L.D., E.D.C.) also supported this project. Sí

Author(s): Fulton, Brent D; Proudman, David G; Sample, Hannah A; Gelfand, Jeffrey M; Chiu, Charles Y; DeRisi, Joseph L; Wilson, Michael R | Abstract: OBJECTIVE:To estimate healthcare expenditures that could be impacted by advanced diagnostic testing for patients hospitalized with meningitis or encephalitis. METHODS:Patients hospitalized with meningitis (N = 23,933) or encephalitis (N = 7,858) in the U.S. were identified in the 2010-2014 Truven Health MarketScan Commercial Claims and Encounters Database using ICD-9-CM diagnostic codes. The database included an average of 40.8 million commercially insured enrollees under age 65 per year. Clinical, demographic and healthcare utilization criteria were used to identify patient subgroups early in their episode who were at risk to have high inpatient expenditures. Healthcare expenditures of patients within each subgroup were bifurcated: those expenditures that remained five days after the patient could be classified into the subgroup versus those that had occurred previously. RESULTS:The hospitalization episode rate per 100,000 enrollee-years for meningitis was 13.0 (95% CI: 12.9-13.2) and for encephalitis was 4.3 (95% CI: 4.2-4.4), with mean inpatient expenditures of $36,891 (SD = $92,636) and $60,181 (SD = $130,276), respectively. If advanced diagnostic testing had been administered on the day that a patient could be classified into a subgroup, then a test with a five-day turnaround time could impact the following mean inpatient expenditures that remained by subgroup for patients with meningitis or encephalitis, respectively: had a neurosurgical procedure ($83,337 and $56,020), had an ICU stay ($34,221 and $46,051), had HIV-1 infection or a previous organ transplant ($37,702 and $62,222), were age l1 year ($35,371 and $52,812), or had a hospital length of stay g2 days ($18,325 and $30,244). DISCUSSION:Inpatient expenditures for patients hospitalized with meningitis or encephalitis were substantial and varied widely. Patient subgroups who had high healthcare expenditures could be identified early in their stay, raising the potential for advanced diagnostic testing to lower these expenditures.

A 37-year-old man with a history of seminoma presented with vertigo, ataxia, and diplopia. An autoantibody specific for kelch-like protein 11 (KLHL11) was identified with the use of programmable phage display. Immunoassays were used to identify KLHL11 IgG in 12 other men with similar neurologic features and testicular disease. Immunostaining of the patient's IgG on mouse brain tissue showed sparse but distinctive points of staining in multiple brain regions, with enrichment in perivascular and perimeningeal tissues. The onset of the neurologic syndrome preceded the diagnosis of seminoma in 9 of the 13 patients. An age-adjusted estimate of the prevalence of autoimmune KLHL11 encephalitis in Olmsted County, Minnesota, was 2.79 cases per 100,000 men. (Funded by the Rochester Epidemiology Project and others.).

BackgroundMetagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test.MethodsIn a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review.ResultsWe enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment.ConclusionsRoutine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

BackgroundSince 2014, the United States has experienced a biennial spike in pediatric acute flaccid myelitis (AFM). Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF) and only inconsistently identified from the respiratory tract, serum, or stool.MethodsWe interrogated CSF from children with AFM (n=42) and pediatric controls with other neurologic diseases (OND) (n=58). Samples were incubated with T7 bacteriophage expressing 481,966 sixty-two amino acid peptides with a fourteen amino acid overlap tiled across all known vertebrate virus and arbovirus genomes, an adaption of the VirScan method. Antibody-bound phage were deep sequenced to quantify enriched peptides with normalized counts expressed as reads per hundred thousand (rpK). EV antibody findings were confirmed with ELISA using whole viral protein 1 (VP1) from contemporary enterovirus (EV) A71 and D68 strains. Separately, metagenomic next-generation sequencing (mNGS) of CSF RNA, both unbiased and with targeted enrichment for EVs, was performed.ResultsThe most significantly enriched viral family by VirScan of CSF in AFM versus OND controls wasPicornaviridae(mean rpK 11,266 versus mean rpK 950, p-adjusted < 0.001, Wilcoxon signed-rank test with Bonferroni adjustment). EnrichedPicornaviridaepeptides belonged almost entirely to the genusEnterovirus.The mean EV VP1 ELISA signal in AFM (mean OD 0.51) was significantly higher than OND controls (mean OD 0.08, p-value < 0.001, Mann-Whitney test). mNGS did not detect additional enterovirus RNA in CSF.ConclusionDespite the rare detection of EV RNA in the CNS of patients with AFM, a pan-viral serologic assay identified high levels of CSF EV antibodies in AFM CSF compared to CSF from OND controls. These results provide further evidence for a causal role of non-polio enteroviruses in AFM.

Solid organ transplant patients are vulnerable to suffering neurologic complications from a wide array of viral infections and can be sentinels in the population who are first to get serious complications from emerging infections like the recent waves of arboviruses, including West Nile virus, Chikungunya virus, Zika virus, and Dengue virus. The diverse and rapidly changing landscape of possible causes of viral encephalitis poses great challenges for traditional candidate‐based infectious disease diagnostics that already fail to identify a causative pathogen in approximately 50% of encephalitis cases. We present the case of a 14‐year‐old girl on immunosuppression for a renal transplant who presented with acute meningoencephalitis. Traditional diagnostics failed to identify an etiology. RNA extracted from her cerebrospinal fluid was subjected to unbiased metagenomic deep sequencing, enhanced with the use of a Cas9‐based technique for host depletion. This analysis identified West Nile virus (WNV). Convalescent serum serologies subsequently confirmed WNV seroconversion. These results support a clear clinical role for metagenomic deep sequencing in the setting of suspected viral encephalitis, especially in the context of the high‐risk transplant patient population., Unbiased metagenomic deep sequencing identifies West Nile virus in the cerebrospinal fluid of a child who had a renal transplant and presented with acute meningoencephalitis.

ObjectiveTo identify molecular correlates of primary angiitis of the CNS (PACNS) through proteomic analysis of CSF from a biopsy-proven patient cohort.MethodsUsing mass spectrometry, we quantitatively compared the CSF proteome of patients with biopsy-proven PACNS (n = 8) to CSF from individuals with noninflammatory conditions (n = 11). Significantly enriched molecular pathways were identified with a gene ontology workflow, and high confidence hits within enriched pathways (fold change >1.5 and concordant Benjamini-Hochberg–adjusted p < 0.05 on DeSeq and t test) were identified as differentially regulated proteins.ResultsCompared to noninflammatory controls, 283 proteins were differentially expressed in the CSF of patients with PACNS, with significant enrichment of the complement cascade pathway (C4-binding protein, CD55, CD59, properdin, complement C5, complement C8, and complement C9) and neural cell adhesion molecules. A subset of clinically relevant findings were validated by Western blot and commercial ELISA.ConclusionsIn this exploratory study, we found evidence of deregulation of the alternative complement cascade in CSF from biopsy-proven PACNS compared to noninflammatory controls. More specifically, several regulators of the C3 and C5 convertases and components of the terminal cascade were significantly altered. These preliminary findings shed light on a previously unappreciated similarity between PACNS and systemic vasculitides, especially anti-neutrophil cytoplasmic antibody–associated vasculitis. The therapeutic implications of this common biology and the diagnostic and therapeutic utility of individual proteomic findings warrant validation in larger cohorts.

The central nervous system (CNS) has a dedicated network of blood vessels to support the physiological activity of the brain, spinal cord and meninges. Consequently, inflammation of CNS vasculature can have devastating effects on neurological function. A lack of understanding regarding the molecular pathology of CNS vasculitis impedes the development of better diagnostics and effective therapies. Here, we analyze the proteome of cerebrospinal fluid from patients with biopsy-confirmed Primary Angiitis of the Central Nervous System (PACNS) relative to non-inflammatory control patients and patients with Reversible Cerebral Vasoconstrictive Syndrome (RCVS), a syndrome that clinically mimics PACNS in several aspects. In PACNS, we find significant elevation of apolipoproteins, immunoglobulins and complement cascade components. Notably, we find a bias towards activation of the alternative complement pathway with elevated levels of the terminal cascade component, complement C5. Given the recent treatment successes of Anti-Neutrophil Cytoplasmic Antibody (ANCA) vasculitis with the C5 receptor inhibitor, CCX168 (Avacopan), our results suggest that complement C5 inhibitors may also prove useful as therapeutic interventions for PACNS.

We used unbiased metagenomic next-generation sequencing to diagnose a fatal case of meningoencephalitis caused by St. Louis encephalitis virus in a patient from California in September 2016. This case is associated with the recent 2015–2016 reemergence of this virus in the southwestern United States.

Background As purported causal factors are identified for autism spectrum disorder (ASD), new assays are needed to better phenotype animal models designed to explore these factors. With recent evidence suggesting that deficits in social motivation are at the core of ASD behavior, the development of quantitative measures of social motivation is particularly important. The goal of our study was to develop and validate novel assays to quantitatively measure social motivation in mice. Methods In order to test the validity of our paradigms, we compared the BTBR strain, with documented social deficits, to the prosocial C57BL/6J strain. Two novel conditioning paradigms were developed that allowed the test mouse to control access to a social partner. In the social motivation task, the test mice lever pressed for a social reward. The reward contingency was set on a progressive ratio of reinforcement and the number of lever presses achieved in the final trial of a testing session (breakpoint) was used as an index of social motivation. In the valence comparison task, motivation for a food reward was compared to a social reward. We also explored activity, social affiliation, and preference for social novelty through a series of tasks using an ANY-Maze video-tracking system in an open-field arena. Results BTBR mice had significantly lower breakpoints in the social motivation paradigm than C57BL/6J mice. However, the valence comparison task revealed that BTBR mice also made significantly fewer lever presses for a food reward. Conclusions The results of the conditioning paradigms suggest that the BTBR strain has an overall deficit in motivated behavior. Furthermore, the results of the open-field observations may suggest that social differences in the BTBR strain are anxiety induced.

Objective: Immunodeficient patients are particularly vulnerable to neuroinvasive infections that can be challenging to diagnose. Metagenomic next-generation sequencing can identify unusual or novel microbes and is therefore well suited for investigating the etiology of chronic meningoencephalitis in immunodeficient patients. Methods: We present the case of a 34 year-old man with X-linked agammaglobulinemia from Australia suffering from three years of meningoencephalitis that defied an etiologic diagnosis despite extensive conventional testing, including a brain biopsy. Metagenomic next-generation sequencing of his cerebrospinal fluid and brain biopsy tissue was performed to identify a causative pathogen. Results: Sequences aligning to multiple Cache Valley virus genes were identified via metagenomic next-generation sequencing. Reverse transcription polymerase chain reaction and immunohistochemistry subsequently confirmed the presence of Cache Valley virus in the brain biopsy tissue. Interpretation: Cache Valley virus, a mosquito-borne orthobunyavirus, has only been identified in three immunocompetent North American patients with acute neuroinvasive disease. The reported severity ranges from a self-limiting meningitis to a rapidly fatal meningoencephalitis with multi-organ failure. The virus has never been known to cause a chronic systemic or neurologic infection in humans. Cache Valley virus has also never previously been detected on the Australian continent. Indeed, our research subject traveled to North and South Carolina and Michigan in the weeks prior to the onset of his illness. This report demonstrates that metagenomic next-generation sequencing allows for unbiased pathogen identification, the early detection of emerging viruses as they spread to new locales, and the discovery of novel disease phenotypes. This article is protected by copyright. All rights reserved.

Hepatitis E virus (HEV) infection uncommonly causes chronic hepatitis and neurologic disease. We describe a case of genotype 3a HEV meningoencephalitis diagnosed by metagenomic next-generation sequencing, illustrating the power of an unbiased molecular approach to microbial testing and the first reported case of HEV infection presumably acquired through lung transplantation.

Background Metagenomic next-generation sequencing (mNGS) of CSF can identify nearly all pathogens in a single test. We previously validated a CSF mNGS assay in a licensed clinical laboratory. To date, the utility of mNGS for infectious disease diagnosis has been described in case reports and small case series, but not in a large-scale clinical trial. Methods The PDAID (“Precision Diagnosis of Acute Infectious Diseases”) study was a 1-year nationwide prospective study across 8 tertiary care hospitals to evaluate the performance and utility of a clinical metagenomic sequencing assay for diagnosis of meningitis, encephalitis, or myelitis from cerebrospinal fluid (CSF) (ClinicalTrials.gov number NCT02910037). We recruited acutely ill hospitalized inpatients lacking a diagnosis at the time of enrollment. CSF samples were processed and analyzed by mNGS testing within 1 week of receipt in the clinical microbiology laboratory, with sequencing results reported in the patient medical record and used to make contemporaneous treatment decisions. Weekly clinical microbial sequencing boards were convened to discuss mNGS results with treating physicians, and clinical impact evaluated by surveys, chart review, and direct clinician feedback. Results A total of 204 patients were enrolled. Patients were severely ill (ICU 48%, average length of stay 26 days, overall 30-day mortality 7.4%). Fifty-nine neurologic infections were diagnosed in 57 patients (27.9%). mNGS identified 15 (25.4%) infections that were missed by all conventional microbiological tests, including emerging and/or uncommon pathogens such as St. Louis encephalitis virus, hepatitis E virus acquired by lung transplant, and Nocardia farcinica. Twelve of the 15 mNGS-only diagnoses (80%) had clinical impact, with 9 of 15 (60%) guiding appropriate treatment. For diagnosis of infections by direct detection CSF testing, mNGS had 79.1% sensitivity and 98.8% specificity, versus 65.1% sensitivity and 99.4% specificity by conventional testing. Conclusion A significant proportion of neurologic infections are missed despite extensive diagnostic testing performed in tertiary care hospitals. Clinical metagenomic CSF testing was found to be useful in increasing the number of diagnosed neurologic infections and providing actionable information for physicians. Disclosures All authors: No reported disclosures.

Importance Identifying infectious causes of subacute or chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed. Objective To present a case series of patients with diagnostically challenging subacute or chronic meningitis using metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) supported by a statistical framework generated from mNGS of control samples from the environment and from patients who were noninfectious. Design, Setting, and Participants In this case series, mNGS data obtained from the CSF of 94 patients with noninfectious neuroinflammatory disorders and from 24 water and reagent control samples were used to develop and implement a weighted scoring metric based onzscores at the species and genus levels for both nucleotide and protein alignments to prioritize and rank the mNGS results. Total RNA was extracted for mNGS from the CSF of 7 participants with subacute or chronic meningitis who were recruited between September 2013 and March 2017 as part of a multicenter study of mNGS pathogen discovery among patients with suspected neuroinflammatory conditions. The neurologic infections identified by mNGS in these 7 participants represented a diverse array of pathogens. The patients were referred from the University of California, San Francisco Medical Center (n = 2), Zuckerberg San Francisco General Hospital and Trauma Center (n = 2), Cleveland Clinic (n = 1), University of Washington (n = 1), and Kaiser Permanente (n = 1). A weightedzscore was used to filter out environmental contaminants and facilitate efficient data triage and analysis. Main Outcomes and Measures Pathogens identified by mNGS and the ability of a statistical model to prioritize, rank, and simplify mNGS results. Results The 7 participants ranged in age from 10 to 55 years, and 3 (43%) were female. A parasitic worm (Taenia solium, in 2 participants), a virus (HIV-1), and 4 fungi (Cryptococcus neoformans,Aspergillus oryzae,Histoplasma capsulatum, andCandida dubliniensis) were identified among the 7 participants by using mNGS. Evaluating mNGS data with a weightedzscore–based scoring algorithm reduced the reported microbial taxa by a mean of 87% (range, 41%-99%) when taxa with a combined score of 0 or less were removed, effectively separating bona fide pathogen sequences from spurious environmental sequences so that, in each case, the causative pathogen was found within the top 2 scoring microbes identified using the algorithm. Conclusions and Relevance Diverse microbial pathogens were identified by mNGS in the CSF of patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for 1 year, the first reported case of CNS vasculitis caused byAspergillus oryzae, and the fourth reported case ofC dubliniensismeningitis. Prioritizing metagenomic data with a scoring algorithm greatly clarified data interpretation and highlighted the problem of attributing biological significance to organisms present in control samples used for metagenomic sequencing studies.

Author(s): Wilson, Michael R; Shanbhag, Niraj M; Reid, Michael J; Singhal, Neel S; Gelfand, Jeffrey M; Sample, Hannah A; Benkli, Barlas; O'Donovan, Brian D; Ali, Ibne KM; Keating, M Kelly; Dunnebacke, Thelma H; Wood, Matthew D; Bollen, Andrew; DeRisi, Joseph L | Abstract: ObjectiveIdentification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis.MethodsWe present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent.ResultsSequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive.InterpretationThe diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.