In an effort to decrease transmission during the first years of the COVID-19 pandemic, public health officials encouraged masking, social distancing, and working from home, and restricted travel. However, many studies of the effectiveness of these measures had significant methodologic limitations. In this analysis, we used data from the TrackCOVID study, a longitudinal cohort study of a population-based sample of 3,846 adults in the San Francisco Bay Area, to evaluate the association between self-reported protective behaviors including masking, physical distancing, travel and working outside the home, and incidence of SARS-CoV-2 infection. Participants without SARS-CoV2 infection were enrolled from August-December 2020 and followed monthly with testing and surveys (median of 4 visits). A total of 118 incident infections occurred (3.0% of participants). At baseline, 80.0% reported always wearing a mask; 56.0% avoided contact with non-household members some/most of the time; 9.6% traveled outside the state; and 16.0% worked 20 or more hours per week outside the home. These behaviors did not change markedly over time. Factors associated with incident infection included being Black or Latinx, having less than a college education, and having more household residents. The only behavioral factor associated with incident infection was working outside the home (aHR 1.62, 95% CI 1.02-2.59). Focusing on protecting people who cannot work from home could help prevent infections during future waves of COVID-19, or future pandemics from respiratory viruses. This focus must be balanced with the known importance of directing resources toward those at risk of severe infections.

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.

OBJECTIVE:Rapid-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.METHODS:Immunoglobulin 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.RESULTS:Autoantibodies 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.INTERPRETATION:Our 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.

Abstract Background Preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2_ infections in healthcare workers (HCWs) is critical for healthcare delivery. We aimed to estimate and characterize the prevalence and incidence of coronavirus disease 2019 (COVID-19) in a US HCW cohort and to identify risk factors associated with infection. Methods We conducted a longitudinal cohort study of HCWs at 3 Bay Area medical centers using serial surveys and SARS-CoV-2 viral and orthogonal serological testing, including measurement of neutralizing antibodies. We estimated baseline prevalence and cumulative incidence of COVID-19. We performed multivariable Cox proportional hazards models to estimate associations of baseline factors with incident infections and evaluated the impact of time-varying exposures on time to COVID-19 using marginal structural models. Results A total of 2435 HCWs contributed 768 person-years of follow-up time. We identified 21 of 2435 individuals with prevalent infection, resulting in a baseline prevalence of 0.86% (95% confidence interval [CI], .53%–1.32%). We identified 70 of 2414 incident infections (2.9%), yielding a cumulative incidence rate of 9.11 cases per 100 person-years (95% CI, 7.11–11.52). Community contact with a known COVID-19 case was most strongly correlated with increased hazard for infection (hazard ratio, 8.1 [95% CI, 3.8–17.5]). High-risk work-related exposures (ie, breach in protective measures) drove an association between work exposure and infection (hazard ratio, 2.5 [95% CI, 1.3–4.8). More cases were identified in HCWs when community case rates were high. Conclusions We observed modest COVID-19 incidence despite consistent exposure at work. Community contact was strongly associated with infections, but contact at work was not unless accompanied by high-risk exposure.

Objective: The study was designed to compare intentions to receive COVID-19 vaccination by race–ethnicity, to identify beliefs that may mediate the association between race–ethnicity and intention to receive the vaccine and to identify the demographic factors and beliefs most strongly predictive of intention to receive a vaccine. Design: Cross-sectional survey conducted from November 2020 to January 2021, nested within a longitudinal cohort study of the prevalence and incidence of SARS-CoV-2 among a general population-based sample of adults in six San Francisco Bay Area counties (called TrackCOVID). Study Cohort: In total, 3161 participants among the 3935 in the TrackCOVID parent cohort responded. Results: Rates of high vaccine willingness were significantly lower among Black (41%), Latinx (55%), Asian (58%), Multi-racial (59%), and Other race (58%) respondents than among White respondents (72%). Black, Latinx, and Asian respondents were significantly more likely than White respondents to endorse lack of trust of government and health agencies as a reason not to get vaccinated. Participants’ motivations and concerns about COVID-19 vaccination only partially explained racial–ethnic differences in vaccination willingness. Concerns about a rushed government vaccine approval process and potential bad reactions to the vaccine were the two most important factors predicting vaccination intention. Conclusions: Vaccine outreach campaigns must ensure that the disproportionate toll of COVID-19 on historically marginalized racial–ethnic communities is not compounded by inequities in vaccination. Efforts must emphasize messages that speak to the motivations and concerns of groups suffering most from health inequities to earn their trust to support informed decision making.

Division of HIV, Infectious Diseases & Global Medicine, San Francisco General Hospital, University of California, San Francisco (UCSF), CA, USA; Division of Infectious Diseases, UCSF, CA, USA; Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA Department of Family and Community Medicine, San Francisco General Hospital, University of California, San Francisco (UCSF), CA, USA; Department of Biochemistry and Biophysics, University of California, San Francisco (UCSF), CA, USA; Quantitative Sciences Unit, Stanford University School of Medicine, Stanford, CA, USA Division of Pediatric Infectious Diseases, Stanford University School of Medicine, Stanford, CA, USA; Division of Infectious Disease and Global Epidemiology, Department of Epidemiology and Biostatistics, UCSF, CA, USA

AbstractBackgroundHealthcare 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(+)Itpr3(tf)/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.

ROHHAD ( R apid-onset O besity with H ypothalamic Dysfunction, H ypoventilation and A utonomic D ysregulation) is a rare, yet severe pediatric disorder resulting in hypothalamic dysfunction and frequent sudden death. Genetic and other investigations have failed to identify an etiology or diagnostic test. Frequent co-occurrence of neuroblastic tumors (NTs) and cerebrospinal fluid inflammation point to an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Here, we screened antibodies from a curated cohort of ROHHAD patients (n=9) and controls (n=150) using a programmable phage display of the human peptidome (PhIP-Seq). Our ROHHAD cohort exhibited frequent association with NTs (8/9) and features consistent with autoimmune etiology. Autoantibodies to Zinc finger and SCAN domain-containing protein 1 (ZSCAN1) were discovered and orthogonally validated in 7 of 9 ROHHAD patients, all of whom had NTs, and shown to be absent in non-ROHHAD pediatric patients with NTs. Notably, human ZSCAN1 expression was confirmed in ROHHAD tumor and healthy human hypothalamus. Our results support the notion that tumor-associated ROHHAD is a pediatric PNS, potentially initiated by an immune response to peripheral NT. ZSCAN1 autoantibodies may aid in an accurate diagnosis of ROHHAD, thus providing a means toward early detection and treatment. Lastly, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches in addition to the classical rodent-based approaches for detecting novel PNS subtypes.

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.

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.

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 <1 year ($35,371 and $52,812), or had a hospital length of stay >2 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.

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.

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.).

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. Antibodies in CSF specific for viral peptides implicate enteroviruses in acute flaccid myelitis.

Abstract Background Since 2014 there have been global biennial outbreaks of acute flaccid myelitis (AFM), a rare but severe “polio-like” illness of as yet-unknown etiology primarily affecting children. Enteroviruses (EVs),, especially EV-D68 and EV-A71, have been implicated in association with AFM cases, but proving causality has been difficult as EVs are rarely isolated from cerebrospinal fluid. In addition, early identification of EV-associated AFM is challenging given that the diagnosis is reliant on potentially subjective clinical and radiological criteria with no specific biomarkers described to date. Methods We leveraged existing and newly generated data from a clinical CSF metagenomic assay for pathogen identification at University of California, San Francisco (UCSF) to interrogate the host response at the transcriptome level by RNA sequencing (RNA-Seq). These transcriptome RNA-Seq data were used to create statistical classification models to discriminate among viral infections that have been linked to AFM, including EV-D68, EV-A71, West Nile virus, and Powassan virus. The dynamic range of CSF cellularity (0 to >106 cells/mL), resulting in varying trancriptome coverage, as well as technical variation across samples required the development and validation of novel normalization techniques. In total, we analyzed ~50 CSF samples split into independent training and test sets. Results We were able to demonstrate a distinct signature of AFM that was able to predict the virus associated with AFM in blinded test samples with >80% accuracy. The key transcriptional features that best discriminated EV-A71 from EV-D68-associated AFM involved protein targeting, viral transcription, viral gene expression, and translation initiation pathways. Conclusion Here we demonstrate a novel approach to diagnosis of AFM that relies on host transcriptional biomarkers from cerebrospinal fluid. In the future, this method might allow earlier diagnosis of AFM to drive appropriate therapies and vaccines and predict patient outcomes, as well as guide research studies on the pathophysiology of EV-associated AFM. Disclosures All authors: No reported disclosures.

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.).

Background Since 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. Methods We 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. Results The most significantly enriched viral family by VirScan of CSF in AFM versus OND controls was Picornaviridae (mean rpK 11,266 versus mean rpK 950, p-adjusted < 0.001, Wilcoxon signed-rank test with Bonferroni adjustment). Enriched Picornaviridae peptides belonged almost entirely to the genus Enterovirus. 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. Conclusion Despite 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.

A 41-year-old woman was seen at the National Institutes of Health (NIH) Neuroimmunology Clinic in 2017 for recurrent episodes of fever, neck stiffness, and back and leg pain. In 2002, at age 26 years, she had several episodes of back and neck pain, malaise, and fever, each lasting 1 to 3 days (Fig 1). A chest x-ray and complete blood count were normal. Cerebrospinal fluid (CSF) during one of these episodes showed pleocytosis (60 white blood cells [WBC]/μl; 60% monocytes, 25% lymphocytes, 15% neutrophils), with elevated protein (96 mg/dl) and low glucose (26 mg/dl Table Supplementary Table 1). Magnetic resonance imaging (MRI) of the brain showed subtle fluid-attenuated inversion recovery (FLAIR) hyperintensity in the sulcal CSF. MRI of the spine was normal. Extensive investigations including CSF Mycobacterium tuberculosis (TB) complex polymerase chain reaction (PCR) and culture, Coccidioides antibodies, histoplasma antigen, cryptococcal antigen, and herpes simplex virus and varicella zoster virus PCRs were negative (see Supplementary Table 1). Nonetheless, she was treated empirically with valacyclovir for 2 weeks. She had had a recent exposure to TB and had converted from a negative purified protein derivative (PPD) skin test in 2001 to a positive result at the time of her presentation in 2002. Thus, she was also treated empirically for TB meningitis (TBM) with rifampin, pyrazinamide, and ethambutol for 1 year. Isoniazid (INH) was started but was discontinued after several weeks due to transaminitis and nausea. She did not receive adjunctive steroids. Her symptoms resolved until 2006 when, immediately following spinal epidural anesthesia during childbirth, she developed a fever with headache, neck stiffness, back pain, and night sweats. She was treated for endometritis but continued to have similar but less severe symptoms for several months. In early 2007, she acutely developed bilateral gluteal pain and left leg dysesthesias. CSF again showed pleocytosis (130 WBC/μl; 83% lymphocytes, 13% monocytes, 2% neutrophils, 2% other) with elevated protein (132 mg/dl) and low glucose (10 mg/dl). CSF TB PCR and culture, cryptococcal antigen, bacterial and fungal cultures, and viral PCRs, as well as CSF cytology and flow cytometry for malignant cells, were negative (see Supplementary Table 1). MRI of the lumbar spine now showed abnormal enhancement and nerve-root thickening in the caudal thecal sac, indicating arachnoiditis (see Fig 2A, B); brain, cervical, and thoracic spine MRI was normal. Repeat lumbar spine MRI 2 months later showed more extensive enhancement and clumping of the cauda equina. Computed tomography (CT) scans of the chest, abdomen, and pelvis, and a gallium scan, were unremarkable. In April 2007, she had a laminectomy and biopsy at L5/S1. The dura was thick, and there were adhesions within the thecal sac and scar tissue surrounding the nerve roots. Histology showed lymphohistiocytic inflammation and a poorly formed non-necrotizing granuloma. Mycobacterial cultures were negative. There was concern that premature discontinuation of INH in 2002 may have led to incomplete treatment of TBM, so she was again treated empirically for TB with rifampin, INH, pyrazinamide, ethambutol, and moxifloxacin for 1 year, as well as 3 weeks of prednisone 60 mg daily followed by a 3-week prednisone taper. This time, pyrazinamide was stopped early due to transaminitis and nausea, and ethambutol was stopped after several months when her CSF profile improved. Repeat lumbar punctures (LPs) showed normalization of protein and glucose with mild residual CSF pleocytosis (5–10 WBC/μl). Her symptoms improved significantly, and she resumed her daily activities. However, she continued to have intermittent mild low back pain, sometimes accompanied by chills. In 2015, at age 39 years, several days after a partial thyroidectomy for an incidentally discovered thyroid nodule, the patient again developed low back and leg pain, chills, headache, fever, and neck stiffness. She was treated with valacyclovir for possible herpes meningitis, as well as prednisone 60 mg daily for 5 weeks for arachnoiditis, with immediate improvement in symptoms. However, her pain recurred when prednisone was tapered gradually over the next several months, and she also developed pain with eye movement, urinary frequency and hesitancy, and subjective sensory changes in the left leg distally from the knee. MRI showed evidence of worsening lumbosacral arachnoiditis with thickening and enhancement of the cauda equina. There was also displacement of the cauda equina posteriorly and laterally by a loculated cystlike structure (see Fig 2A, B). She had another LP; however, only a small amount of fluid was obtained, possibly due to the loculation. The CSF was bloody (1,075 red blood cells [RBC]/μl) with 9 WBC/μl (78% lymphocytes, 22% neutrophils), elevated glucose (179 mg/dl), and low protein (13 mg/dl). Cryptococcal antigen and fungal cultures were negative. She had a whole body fluorodeoxyglucose positron emission tomography (FDG-PET) CT that was normal. The etiology of the arachnoiditis was thought to be postinfectious or autoimmune. She was treated with a 3-day course of intravenous methylprednisolone, followed by oral prednisone, with dramatic improvement; however, pain, neck stiffness, and fever again recurred when steroids were tapered several weeks later. She was subsequently maintained on prednisone, and any attempt to decrease to <35 mg/day resulted in worsening of her back pain, fatigue, and intermittent low-grade fevers and night sweats. Due to concern for neurosarcoidosis or another autoimmune disorder, she was placed on concomitant mycophenolate mofetil (up to 3,000 mg/day) for several months in 2016, without any improvement. While continuing the prednisone, she was transitioned from mycophenolate mofetil to methotrexate up to 15 mg/wk in early 2017, also with no improvement or ability to taper steroids. She developed bilateral cataracts attributed to chronic corticosteroid use. At the time of her presentation to the NIH Neuroimmunology Clinic in 2017, she had constant dull, aching pain in her back and buttocks that worsened with prolonged activity or stress. Every 1 to 3 months, she had several days of malaise and fever up to 38.3°C accompanied by more severe back and buttock pain. She complained of a sensation of urinary retention, although postvoid residuals were normal. She denied constipation or bowel incontinence, weakness or numbness, or neurological symptoms in her arms. Treatment of pain with pregabalin was minimally effective and caused drowsiness. Her pain responded to nonsteroidal anti-inflammatory drugs. She was born in Mumbai, India, immigrated to Arizona at age 22 years, and later moved to New York and then Maryland. She returned to India once, in 2009, and had no other foreign travel. Her medical history was significant for thrombocytopenia during early childhood, hepatitis B virus infection at age 12 years that subsequently resolved, left facial palsy in her teens associated with a herpetic rash in her left ear canal, and fever, headache, and malaise in 1998 at age 22 years, for which she was treated for malaria despite a negative blood smear. She had a sister with breast cancer and several distant family members with cancer, including leukemia, neuroblastoma, lung cancer, and a hepatoma. She had no family history of autoimmune or neurological disease, and no family members with frequent or severe infections. Her general physical and neurological evaluations were normal with the exception of mild atrophy in both legs without fasciculations and with preserved strength. Lumbar spine MRI again showed arachnoiditis with some extension superiorly compared to 2015 (see Fig 2C, D). Brain MRI showed a few small foci of leptomeningeal enhancement on postgadolinium FLAIR images. Blood was negative for rheumatologic testing, human immunodeficiency virus type 1 antibody, human T-cell lymphotropic virus type 1 and 2 antibodies, and Lyme serology. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were normal (see Supplementary Table 1). Given the patient's previously negative infectious workups as well as her response to steroids, her symptoms and MRI findings were thought to be secondary to an autoimmune process, perhaps triggered initially by an infection. As she had no response to therapies directed at lymphocytes (methotrexate, mycophenolate), she was given a 1-month trial of the IL-1 receptor antagonist anakinra (up to 200 mg/day), with no improvement in her chronic symptoms. However, about 1 week after discontinuation of anakinra, she developed her typical flare symptoms of malaise and worsening back pain. Her neurologic examination was stable. She had an LP at that time, which showed 156 RBC/μl, 30 WBC/μl (92% lymphocytes, 4% monocytes, 4% neutrophils), total protein 41 mg/dl, and glucose 47 mg/dl. Infectious studies, including mycobacterial culture, histoplasma antigen, and viral PCRs, were negative. She had an elevated IgG index of 2.41 (normal range = 0.26–0.62) and partially identical oligoclonal bands in CSF and serum (pattern 3). She had an elevated blood WBC count of 15,680 cells/μl (94% neutrophils) and normal ESR and CRP. Anakinra was restarted, and her flare symptoms improved over the next several weeks, although her chronic symptoms continued. Several months later, a surveillance lumbar spine MRI showed enlargement and new contrast enhancement of a subarachnoid nodule (see Fig 2E–H). An additional test was performed, and a diagnosis was made. This is a 41-year-old woman with a 15-year history of relapsing meningitis that progressed to chronic lumbar arachnoiditis, with several notable relapses following episodes of physical stress. Despite multiple investigations, no definitive etiology was identified, and the disease recurred despite multiple empirical treatment regimens for TB, herpesvirus infections, and inflammatory conditions. Several possible diagnoses should be considered at this stage. We focus our discussion around the findings of chronic arachnoiditis and granulomatous disease. The patient's initial presentation at age 26 years was of a subacute prodrome followed by acute meningism. An infectious cause was appropriately at the top of the differential. Given the subacute history and the monocyte-predominant CSF with WBC < 100 cells/μl, bacterial meningitis secondary to typical pathogens, although possible, was less likely.1, 2 Subacute causes of meningitis commonly occur in the setting of viral, parasitic, fungal, or atypical bacterial infections, such as TB.2 Multiple investigations for these types of pathogens were negative, and there was no evidence of systemic infection by CT or FDG-PET CT. However, TB PCR and culture are insensitive, and up to half of people with TBM have no evidence of systemic disease.3, 4 Given her recent exposure to TB and subsequent PPD conversion, CSF pleocytosis, hypoglycorrhachia, and the poor sensitivity of diagnostic tests for TBM, empiric treatment was appropriate given the disease's high morbidity and mortality.5-7 Her prolonged remission after empiric TB therapy was also reassuring. Her second attack 4 years later occurred acutely after an epidural anesthetic in the setting of pregnancy, when she was potentially more prone to infection. MRI showed new lumbar arachnoiditis. Efforts to culture or detect a pathogenic organism from the CSF were again unsuccessful, despite CSF pleocytosis and low glucose. Surgical biopsy revealed a poorly formed non-necrotizing granuloma. Necrotizing granulomas are a hallmark of TB; however, non-necrotizing granulomas can be found in TB-positive patients, and this finding should not dissuade the clinician from the diagnosis if clinical suspicion is high.8, 9 INH is a cornerstone medication in the therapy of TBM, and patients with INH resistance have significantly worse outcomes.10-13 The patient's initial TBM treatment did not include an adequate course of INH, and there was concern for recurrence. TB affects the spine predominantly in the form of extramedullary disease.14 Spinal arachnoiditis can occur as a complication of TBM and can infrequently be an asymptomatic finding.14-16 Cases of delayed lumbar arachnoiditis, occurring up to 15 years after effective treatment of the initial TBM, have been reported.17, 18 There are a multitude of infectious, autoimmune, and neoplastic causes of granulomatous disease in the central nervous system (CNS) (Table 1).19 Most notably in this patient, other infectious etiologies to consider would include fungal infections, neurosyphilis, and a variety of parasitic infections. Many of these conditions can cause chronic meningitis with a relapsing component, and broad diagnostic tests, such as cultures, may detect some (but not all) of these pathogens.20 Arachnoiditis is a rare condition characterized by chronic inflammation of the arachnoid and pia mater with increased production of collagen deposition between the two layers, leading to adhesions. Anatomically related cranial and radicular nerve roots become edematous and hyperemic before being entrapped and clumped together in the adhesive leptomeninges. Over time, the nerves atrophy due to diminished blood supply.21 Intracranial arachnoiditis can lead to cranial nerve abnormalities, with blindness possible in cases of optochiasmatic arachnoiditis.22 Lumbar arachnoiditis can cause back and lower limb pain, variable neurological deficits, and partial cauda equina syndrome. However, the clinical manifestations depend on the severity and location of disease.23 Any irritant or pathogen that causes chronic inflammation of the arachnoid mater can lead to adhesive arachnoiditis, and therefore, despite the rarity of the syndrome, the potential etiologies are broad. Older contrast agents used in CT myelograms (particularly ethyliodophentylate, which is no longer used), blood breakdown following subarachnoid hemorrhage, older anesthetic preservatives, and lumbar surgery have all been implicated.23 Other causes include infections, autoimmune conditions, and malignancy, as described below. Despite an extensive workup, a causative organism may not be found, due to poor sensitivities of diagnostic assays or unintentional omission of appropriate pathogen-specific investigations. Fungal infections such as Cryptococcus and Candida species can cause chronic meningitis and arachnoiditis.15, 22, 24, 25 However, this patient was not known to be immunocompromised or to have engaged in intravenous drug use, making fungal meningitis less likely. In addition, her clinical course and CSF profile were not consistent with coccidioidomycosis, which was suspected because she had lived in Arizona. Neurocysticercosis (NCC) can cause spinal arachnoid disease, usually in patients with basal arachnoid disease, and can be asymptomatic, although this was not considered in this patient (although she grew up in a country where NCC is endemic) because no parenchymal or subarachnoid cysts were detected on at least 8 brain MRIs over 15 years.26 She did have a cyst adjacent to the lumbar cord seen on MRI in 2015; however, in light of her overall presentation and lack of brain cysts, NCC was not investigated as a possible cause. Syphilis can rarely cause subacute meningitis with optochiasmal arachnoiditis and has also been implicated in lumbar arachnoiditis.27, 28 Schistosomiasis can manifest with spinal cord disease, either with transverse myelitis commonly involving the conus medullaris or with lumbar arachnoiditis.29 Neuroschistosomiasis occurs in endemic regions, such as Egypt, but neither India nor the USA is considered a high-risk area.30 Meningitis can also be seen in angiostrongyliasis, gnathostomiasis, and sparganosis parasitic infections, but usually with a much more acute course.31, 32 Case reports of vertebral disease and lumbar arachnoiditis secondary to Echinococcus granulosus, a zoonotic parasitic infection transmitted from dogs, have also been described.33, 34 Amoebic infections such as Balamuthia mandrillaris can cause granulomatous meningitis, but the course is usually much more rapid.35 An occult or indolent infection in her lumbar canal may have incited a larger-than-expected immune response after the introduction of an epidural needle, which would otherwise cause only a mild local inflammatory response. On a single LP in 2002, our patient had CSF eosinophilia, with eosinophils making up 1% of 147 WBC/μl. Eosinophilic meningitis, which is defined as the presence of at least 10% eosinophils of CSF WBC or at least 10 eosinophils/μl, is most often caused by a helminthic infection, most commonly Angiostrongylus catonensis. However, a wide variety of other infections as well as malignancy and autoimmune disease, including sarcoidosis, can also cause CSF eosinophilia, with variable blood eosinophilia.31 Postinfectious autoimmune neurological conditions are common. Paradoxical worsening of TBM is an immune-mediated response that can present up to a year after effective treatment, with 4% of patients developing spinal arachnoiditis.36 Postinfectious inflammatory lumbar arachnoiditis can also be seen following cryptococcal meningitis secondary to a postinfectious inflammatory response syndrome. Most cases have negative cryptococcal cultures in CSF.37 Neurosarcoidosis is an autoimmune non-necrotizing granulomatous condition that can present with both systemic and neurological disease.38 Neurosarcoidosis commonly causes a relapsing chronic meningitis, and chronic lumbar arachnoiditis has been described.39, 40 Neurological symptoms can be the initial presentation of neurosarcoidosis in 50% of patients, with 85% ultimately developing systemic disease but with a significant percentage continuing to have isolated CNS involvement.41 Features that prompt the consideration of neurosarcoidosis in our patient were her presentation with chronic meningitis and arachnoiditis, CSF pleocytosis, low CSF glucose (which can occur in 14% of patients), non-necrotizing granulomas on biopsy, and the relapsing nature of the disease in the same region of the CNS.41-43 Despite extensive investigation including CT of the chest, abdomen, and pelvis as well as FDG-PET/CT, there was no evidence of systemic sarcoidosis. Her later relapses responded extremely well to steroid therapy, and she became dependent on steroids, further implicating an autoimmune etiology. Primary CNS vasculitis can also present with granulomatous inflammation, but it rarely involves the spinal cord,44, 45 and blood vessel wall inflammation was not identified on biopsy. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis can also have CNS involvement, but repeat serum ANCA testing was negative. Invasive malignancy of the subarachnoid space is another important diagnostic consideration. Despite the patient's strong family history of cancer, there was no evidence of malignancy in repeated CSF cytology and flow cytometry examinations, on lumbar meningeal biopsy, or by whole body imaging. In addition, the sheer length of the 15-year disease course with no manifestations of systemic malignancy make the diagnosis less likely. However, this diagnostic possibility should always still be considered, especially given that systemic glucocorticoids can temporarily improve hematologic malignancies, and CSF cytology is notoriously insensitive. Despite the presumption of an autoimmune etiology, lingering concerns about an occult infection prompted enrollment of the patient in a research study at University of California, San Francisco to investigate her CSF with metagenomic next generation sequencing (mNGS), an unbiased approach to the identification of neuroinfectious diseases. The use of mNGS has gained momentum over the past several years, with several notable case reports and case series showcasing mNGS's ability to capture a broad range of infections with a single assay.24, 46-52 Total RNA is extracted from a patient's CSF, and complementary DNA (cDNA) is generated by reverse transcription with random hexamer primers. The cDNA is then converted into a library of random fragments, which is then sequenced on a massively parallel scale.53 The resulting genomic data are then processed through a bioinformatics pipeline, which removes human, low-complexity, redundant, and poor-quality sequences. The remaining sequences are searched against all known organisms in the National Center for Biotechnology Information's GenBank database to identify the source of the high-quality, nonredundant, high-complexity, nonhuman sequences.54-56 As a result, it is possible to identify the vast majority of known organisms, including viral, fungal, bacterial, and parasitic infections, whether or not they are being considered as part of the treating physician's differential diagnosis. In this case, mNGS of total RNA extracted from 500 μl of the patient's CSF generated 5,750,572 pairs of 135 nucleotide sequences. After computational filtering, there were 67,334 pairs of high-quality, nonhuman, and nonredundant sequences. Of these, 2,725 sequence pairs unambiguously aligned to the genus Taenia with 99 to 100% similarity to Taenia solium. Based on a z score–based statistical model comparing the abundance of organisms in the sample to "no template" water controls and uninfected CSF samples, T. solium was the highest ranking organism, with all other identified microbes consistent with frequent environmental contaminants.24 The diagnosis of NCC was confirmed with a clinical CSF cestode antigen assay and serology, which previously had not been performed.57 Retrospective review of the patient's earlier meningeal biopsy did not reveal evidence of cysticerci. NCC is caused by infection with T. solium, the pork tapeworm. The condition leads to single or multiple intraparenchymal, ventricular, and subarachnoid cysts. NCC is endemic to Central America, South America, Sub-Saharan Africa, and Asia.58 The hallmark of parenchymal NCC is the formation of a vesicular cyst that degenerates from a viable to a calcified form. During the viable stage, the parasite is thought to evade host defenses, and there is minimal to no immune response.59 Cyst degeneration occurs when the immune system detects the parasite. The pathogen can no longer evade the immune system, and a robust granulomatous inflammatory response occurs, which can lead to significant neurological morbidity.60, 61 The final, calcified stage contains a dead parasite and creates minimal inflammatory response.58, 62 In subarachnoid NCC, the parasite can lack typical cystic structures, making it more challenging to identify on imaging. Subarachnoid NCC can also cause pronounced inflammation, which can be difficult to control and treat.63, 64 Spinal NCC is a rarer manifestation of NCC, with extramedullary arachnoid disease constituting the majority of cases.26 Intracranial disease is also evident in most cases, but our patient only had subtle leptomeningeal enhancement on brain MRI and no intraparenchymal or intraventricular lesions.26 In retrospect, the cyst seen on the patient's lumbar spine MRI in 2015, and the enhancing intradural extramedullary lumbar nodule seen in 2017, likely represented degenerating cysts, although even after the diagnosis was made there was debate among neuroradiologists about whether the findings on MRI in 2015 represented a Taenia cyst versus arachnoid scarring. At the time, these were not identified as parasitic, given the lack of more typical brain cysts, as well as the larger clinical context of recurrent fever and constitutional symptoms, which are unusual features of NCC.65, 66 Nevertheless, NCC should be considered in any patient with chronic meningitis who has spent time in an endemic area, even without typical MRI findings. After the NCC diagnosis, the patient was started on dual antihelminthic therapy with praziquantel and albendazole. She was also started on the tumor necrosis factor α inhibitor etanercept to protect against an inflammatory reaction to degenerating cysts.67 On this therapy, she tolerated a steroid taper for the first time in 2 years, and after a year was able to discontinue steroids completely. Following 3 months of treatment, her MRI remained stable, CSF demonstrated reduced leukocytosis (10 WBC/μl; 91% lymphocytes, 7% monocytes, 2% neutrophils) with normal protein and glucose, and the CSF cestode antigen was no longer detectable. CSF cestode antigen was again undetectable after a year of treatment, and so antihelminthic treatment and etanercept were stopped, with the intention of stopping anakinra in the coming months. She continues to have fatigue and low back and buttock pain but has been able to increase her daily activities and has not had a severe symptom flare since starting specific therapy. This case thus highlights the utility of mNGS for the diagnosis of atypical presentations of common infections.24, 68, 69 The case also vividly illustrates that either improvement or lack of clinical deterioration in the setting of immunosuppression does not rule out an underlying infectious etiology, even after years of treatment. We thank the NIH neuroimmunology staff for their assistance in care of the patient and collection of samples, and the patient for her participation in the research study. E.S.B., A.V., A.N., J.L.D., and M.R.W. contributed to the conception and design of the study. E.S.B., E.M.O., T.N., D.S.R., A.V., A.N., L.M.K., H.A.S., K.C.Z., J.L.D., and M.R.W. contributed to the acquisition and analysis of data. E.S.B., P.S.R., and M.R.W. contributed to drafting the text and preparing the figures. Nothing to report. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.