1 Disclosure 2 Defining Our Terms 3 Meningitis 4 Encephalitis 5 Myelitis 6

Home , Alphaflexiviridae, Alphatetraviridae, Avihepatovirus, Avisivirus, Carmotetraviridae, Cosavirus

5/5/20

Disclosure

Solving Neurologic Mysteries: Next-Generation Approaches to Diagnosis

Michael Wilson, MD, MAS Associate Professor UCSF Weill Institute for Neurosciences Department of Neurology Division of Neuroimmunology and Glial Biology

1 2

Defining Our Terms Meningitis

• Meningitis: inflammation of the meninges • Encephalitis: inflammation of the brain • Myelitis: inflammation of the spinal cord

3 4

Encephalitis Myelitis

5 6

1 5/5/20

Meningoencephalitis Encephalitis: Morbidity and Mortality

• ~20,000 cases/year in the United States • $2 billion in inpatient costs • ~10% mortality • Survivors are frequently disabled – speech, memory, mobility

7 8

Encephalitis of Unknown Etiology Diagnostic Challenges

• > 50% unknown cases in 26 of 41 studies in recent meta-analysis • > 100 pathogens cause encephalitis • Not explained by # of pathogens tested for • Wide variation • California Encephalitis Project • Geography • 7 years (1,570 cases): 63% of cases unknown • Season • Patient demographic • Epidemics

• Granerod J, et al. Neurology 2010;75:924-932 • Glaser CA, et al. CID 2006;43:1565-1577 9 10

Emerging Neurologic Infections Zika Virus

• West Nile virus • Phylogenetic analyses indicate Zika was introduced to Brazil in 2013 • Periodic measles, mumps outbreaks • Chikungunya virus • 18 months before it was detected • Zika virus • Powassan virus • Nearly 2 years before recognized as a cause of microcephaly, • Nipah and Hendra viruses meningoencephalitis and Guillain-Barré syndrome • Enterovirus A71 • Chandipura virus • Monkeypox virus • Ebola virus • PML in HIV and other immunosuppressed patients • Rate of severe neurological symptoms caused by emerging viruses • Arenavirus (LCMV-like) in solid organ transplant pts • 39% commonly do so • Dengue virus in the Florida Keys • 10% rarely or occasionally do so • ? SARS-Coronavirus-2 ?

• Lipkin WI. Nat Rev Micro 2013;11(2):133-41 • Tyler KL. Arch Neurol. 2009; parts 1 and 2 • Faria NR, et al. Science 2016 11 12

2 5/5/20

Candidate-Based Approach

Dubey, et. al. Annals of Neurology, 2018 13 14

Metagenomics

NEB Sequencing Library Prep

15 16

NEB Sequencing Library Prep Read-Pairs Retained Time (min) Component Target Four Niches to Fill Raw Sequence 19,574,979 100% 0.0 NA NA (.fastq files)

Hg38/PanTro4 Removal of 1,632,130 8.3% 5.2 STAR v2.4.2 RepBase Human Reads • Novel, highly divergent pathogens PriceSeqFilter 816,065 4.1% 5.8 read-pairs Quality Filter v1.1.2 • Pathogens not linked with a clinical phenotype cd-hit-dup Compression of 96,913 0.49% 5.9 read-pairs v4.6.4-2015 • Rare pathogen not on the differential Redundant Reads lzw-filter 92,074 0.47% 6.3 read-pairs • “Rule out” infection in suspected autoimmune cases LZW Complexity (script) Filter bowtie2 Paired-End 2,232 0.01% 6.9 Hg38/RepBase Human Removal v2.2.4 gmap/gsnapl NCBI nt Alignment to 1,683 <0.01% 7.4 v2015-12 2015 nt database rapsearch2 NCBI nr Alignment to 1,467 <0.01% 9.3 v2.23 2015 nr database Taxnomic MySQL NCBI 9.4 Stats/Reporting v5.5.53 Taxonomy

Abbreviations: NT, nucleotide; NR, non-redundant protein. 17 18

3 5/5/20

Early Success / Proof Of Concept Early Success / Proof Of Concept

Neuroleptospirosis Neuroleptospirosis • Unanticipated bacterial infection • Unanticipated bacterial infection • 48-hour turnaround • 48-hour turnaround • Dramatic clinical improvement • Dramatic clinical improvement

Balamuthia mandrillaris • Unanticipated amoebic infection • New cause of endophthalmitis • Wilson MR, et al. NEJM 2014 • Wilson MR, et al. NEJM 2014 • Wilson MR, et al. Ann Neurol 2015

19 20

Early Success / Proof Of Concept Early Success / Proof Of Concept

Balamuthia mandrillaris Balamuthia mandrillaris • Unanticipated amoebic infection • Unanticipated amoebic infection • New cause of endophthalmitis • New cause of endophthalmitis • Wilson MR, et al. NEJM 2014 • Wilson MR, et al. NEJM 2014 • Wilson MR, et al. Ann Neurol 2015 • Wilson MR, et al. Ann Neurol 2015

21 22

Early Success / Proof Of Concept Early Success / Proof Of Concept

Neuroleptospirosis Cache Valley virus Neuroleptospirosis Cache Valley virus • Unanticipated bacterial infection • New cause of chronic • Unanticipated bacterial infection • New cause of chronic • 48-hour turnaround encephalitis • 48-hour turnaround encephalitis • Dramatic clinical improvement • Undetected spread of an • Dramatic clinical improvement • Undetected spread of an arbovirus to a new continent arbovirus to a new continent

4 5/5/20

Early Success / Proof Of Concept Early Success / Proof Of Concept

Neuroleptospirosis Cache Valley virus Neuroleptospirosis Cache Valley virus • Unanticipated bacterial infection • New cause of chronic • Unanticipated bacterial infection • New cause of chronic • 48-hour turnaround encephalitis • 48 hour turnaround encephalitis • Dramatic clinical improvement • Undetected spread of an • Dramatic clinical improvement • Undetected spread of an arbovirus to a new continent arbovirus to a new continent

Balamuthia mandrillaris Balamuthia mandrillaris Candida dubliniensis • • Unanticipated amoebic infection • Unanticipated amoebic infection Unanticipated fungal • New cause of endophthalmitis • New cause of endophthalmitis infection • Wilson MR, et al. NEJM 2014 • Wilson MR, et al. NEJM 2014 • IV drug use not disclosed • Wilson MR, et al. Ann Neurol 2015 • Wilson MR, et al. Ann Neurol 2015 • Wilson MR, Suan D, et al. Ann of Neurol 2017 • Wilson MR, Suan D, et al. Ann of Neurol 2017 • Wilson MR, O’Donovan B, Gelfand J, et al. JAMA Neurol 2018 25 26

Pitfalls I Pitfalls I

• Low input nucleic acid from • Low input nucleic acid from • NGS library kit benchmarking cerebrospinal fluid (picogram cerebrospinal fluid (picogram • Depletion of Abundant Sequences quantities) quantities) by Hybridization (DASH) • Human background • Human background

Figure 3.

A Patient 1 Human ChrM (12S +16S) B. mandrillaris 16S 10 0.04 untreated untreated DASHed DASHed age (%) e r v o m. c no r

0 0 500 genomic position 3500 200 genomic position 1200 B Patient 2 Human ChrM (12S +16S) C. neoformans 18S 10 1.2 untreated untreated DASHed DASHed age (%) e r v o m. c no r

0 0 500 genomic position 3500 350 genomic position 550

C Patient 3 Human ChrM (12S +16S) T. solium 18S 18 12 untreated untreated DASHed DASHed age (%) e r v o m. c no r 0 0 500 genomic position 3500 500 genomic position 2500 • Gu W, Crawford ED, O’Donovan B, Wilson MR, et al. Genome Biol 2016 • Ramani V, Shendure J. Genome Biol 2016 27 28

Pitfalls II Pitfalls II

• Environmental contamination • Environmental contamination • Weighted z score-based scoring algorithm to separate bona fide pathogen sequences for spurious environmental sequences

• Wilson MR*, O’Donovan B*, Gelfand J*, et al. JAMA Neurol 2018 29 30

5 5/5/20

Case 1 Case 1

• 40 year-old physician with 15 years of relapsing myelitis and • 40 year-old physician with 15 years of relapsing myelitis and arachnoiditis arachnoiditis • Evaluated and treated at top academic medical centers • Evaluated and treated at top academic centers • Immigrated to US from India at age 22; lived in AZ, NY and MD • No history of intravenous drug use • No known animal, mosquito or tick exposures

• Beck E, et al. Ann Neurol 2019 • Beck E, et al. Ann Neurol 2019 31 32

Case 1 Case 1

• 2002: Subacute onset of back pain and malaise -> night sweats, fever, anorexia, severe headache and neck stiffness • CSF • WBC 454 (25% lymphocytes, 15% neutrophils, 60% monocytes) • Protein 96 • Glucose 23

33 34

Case 1 Case 1

• Treated for tuberculous meningitis for 3 months (had to stop due to • Treated for tuberculous meningitis for 3 months (had to stop due to medication side effects) medication side effects) • 2006: epidural anesthesia for childbirth -> Temp 102F, headache, neck stiffness, back pain, night sweats -> got antibiotics • …but low-grade symptoms for months before acute onset bilateral gluteal and left leg pain

35 36

6 5/5/20

Case 1 Case 1

• CSF • Significant improvement but intermittent low back pain and chills • WBC 130 (83% lymphocytes, 2% neutrophils, 13% monocytes) • Glucose 10 • Protein 132 • Laminectomy at L5-S1 for biopsy -> non-specific inflammation • 1 more year of anti-tuberculosis therapy

37 38

Case 1 Case 1

• Significant improvement but intermittent low back pain and chills • Since May 2015 has been on >35 mg of daily prednisone plus… • 2015: days after thyroid surgery developed low back and leg pain and • Mycophenolate mofetil (up to 3000mg daily) for 8 months numbness with chills, headache, fever and neck stiffness • Methotrexate (Jan 2017 to present) • Given valacyclovir + prednisone with dramatic improvement • Chronic, dull aching pain in back and buttocks that worsens with • Recurrent symptoms when steroids tapered sitting, bending or twisting or moving her head • Monthly gets general malaise, Temp 101F and more severe throbbing pain for 1-2 weeks

39 40

Case 1 Cysticercosis

• CSF • Taenia solium tapeworm • RBC 156 • Two intermediate hosts • WBC 30 (92% lymphocytes, 4% neutrophils, 4% monocytes) • Pig • Protein 41 • Human • Glucose 47 • Definitive host: human • IgG index 2.41

41 42

7 5/5/20

Precision Diagnosis of Acute Infectious CLIA Validation Diseases (PDAID) Study Design • Establishment of: • 8 academic medical centers around the U.S. • Quality metrics • Prospective enrollment of patients with acute meningitis +/- • Threshold values encephalitis or exacerbation of chronic meningitis • Limits of detection (0.16 to 313 genomic copies or colony forming • No diagnosis at the time of enrollment units per milliliter for each representative organism type) • Adults and children

• Wilson MR, Sample HA, et al. NEJM 2019 • Miller S, et al. Genome Research 2019 • ClinicalTrials.gov number, NCT02910037 43 44

Results Results

• 204 patients enrolled • 10/13 mNGS-only infections not entertained by treating physicians • 58 infections diagnosed in 57 patients (27.9%) (St. Louis encephalitis virus, hepatitis E virus, etc) or had been missed

mNGS alone Co-identified Standard alone N=13 (22.4%) N=19 (31%) N=26 (44%)

• Wilson MR, Sample HA, et al. NEJM 2019 • Wilson MR, Sample HA, et al. NEJM 2019 • Chiu CY, et al. Emerg Inf Dis 2017 • Chiu CY, et al. Emerg Inf Dis 2017 • Murkey JA, et al. Open Forum Inf Dis 2017 • Murkey JA, et al. Open Forum Inf Dis 2017 45 46

Results Results

• 10/13 mNGS-only infections not entertained by treating physicians • Clinical false negatives (n=26) (St. Louis encephalitis virus, hepatitis E virus, etc) or had been missed • Diagnosed by serology (n=11) • CSF mNGS 80% sensitive and 98.2% specific for infections identified by • Diagnosed in samples other than CSF (n=7) direct detection methods in CSF • Low pathogen titer in CSF (n=8) • Traditional direct detection CSF assays were 67.5% sensitive and 99.4% specific

8 5/5/20

Neuroinvasive viruses diagnosed by CSF Disadvantages of Direct Detection Assays serology • Short time windows for detection for some pathogens • West Nile virus • Compartmentalized / focal infections may shed little to no genetic • Powassan virus material into the cerebrospinal fluid • Japanese encephalitis virus • Eastern equine encephalitis virus • Measles virus • Poliovirus • Varicella zoster virus • Rabies virus

49 50

Approaches to Antibody Discovery VirScan Design What we would like: • • Next-generation sequencing-compatible Final number of representative virus genomes: 76,700 • Super cheap • All vertebrate, tick and mosquito viruses • Plate-based • Collapsed on 98% amino acid identity • Robot friendly • Final number of 62 amino acid peptides overlapping by 14 amino • High signal to noise ratio acids: 481,966 • Scalable

• Xu GJ, Kula T, Xu Q, Li MZ, Vernon SD, Ndung’u T, et al. Science. 2015 • Johnson TP, et al. Ann Neurol 2019 • Schubert RD, et al. Nat Med 2019 51 52

Utilize Phage to Display Fragments of All Vertebrate and Arboviruses

• Xu GJ, Kula T, Xu Q, Li MZ, Vernon SD, Ndung’u T, et al. Science. 2015 Magnetic Bead • Schubert RD, et al. Nat Med 2019 53 54

9 5/5/20

Magnetic Bead Magnetic Bead 55 56

Acute Flaccid Myelitis

Magnetic Bead

57 58

Acute Flaccid Myelitis AFM Controversies

• Upsurge in AFM cases in 2014 (n=120), 2016 • ~40% of AFM children have EVD68 in body compartments outside the (n=149) and 2018 (n=210) CNS • Baseline incidence of 22-35 cases/year • Almost none of these children have EV detected in the CSF • Median age of 6 years old • No alternate agent has been identified in 60% of patients • Slightly more boys than girls • 80% of cases preceded by a prodromal illness • Asymmetrical paralysis over hours to days • Cases now reported in 14 countries on 6 continents

• Messacar K, Tyler KL. JAMA 2019 59 60

10 5/5/20

AFM Etiology Study CSF VirScan Detects Enterovirus Ab in AFM

a dsDNA dsRNA (+)ssRNA • CSF samples from children with AFM (n=42) • AFM: mean Picornaviridae reads Birnaviridae Alphaflexiviridae Adenoviridae Picobirnaviridae Alphatetraviridae 11,082 rpK, IQR 16,850 Alloherpesviridae Reoviridae Arteriviridae • Ascoviridae Totiviridae Astroviridae Median age: 37.8 months, IQR 11 to 64 months Asfarviridae Caliciviridae Baculoviridae Carmotetraviridae • OND: mean Picornaviridae reads Herpesviridae (-)ssRNA Coronaviridae • 2018 (n=36), 2014 (n=4), 2016 (n=2) Hytrosaviridae Dicistroviridae Iridoviridae Arenaviridae Flaviviridae rpK 1121 IQR 974 Lavidaviridae Bornaviridae Hepeviridae • Malacoherpesviridae Filoviridae Iflaviridae CSF samples from children with other neurologic diseases (n=58) • -8 p-adjusted = 6.3 x 10 Wilcoxon signed-rank test with Marseilleviridae Fimoviridae Mesoniviridae Bonferroni adjustment Mimiviridae Hantaviridae Narnaviridae • Median age: 120 months, IQR, 66 to 174 months Nudiviridae Nairoviridae Nodaviridae Papillomaviridae Nyamiviridae Permutotetraviridae Polydnaviridae Orthomyxoviridae Picornaviridae Polyomaviridae Paramyxoviridae Polycipiviridae • Infectious (40%), Autoimmune (38%), Non-inflammatory (10%), Poxviridae Peribunyaviridae Roniviridae Phasmaviridae Secoviridae Malignancy (5%), Unknown (7%) Phenuiviridae Solinviviridae ssDNA Pneumoviridae Togaviridae Rhabdoviridae Tymoviridae Anelloviridae Sunviridae Bidnaviridae Tospoviridae Circoviridae ssRNA/DNA-RT Genomoviridae Hepadnaviridae Metaviridae Parvoviridae Unclassified Smacoviridae Retroviridae b c • Schubert RD, et al. Nat Med 2019 Picornaviridae Enterovirus

us p<.001

us 0.4 us vi r us us us us us vi r us us us us us us vi r us vi r vi r us us vi r us 0.3 vi r vi r 61 62 uvi r unsagivi r o b vihepat o visivi r 0.2 Cardi o Cos a Aalivi r Ampivi r Aphth o Crohivi r Dicipivi r Enter o Erb o Gallivi r Hepat o Hunnivi r K Aquam a A A K Limnipivi r

0.1 us us proportion of read s us us us us us

a us us us vi r us us us us us vi r vi r vi r ivi r us 0.0 uvi r vi r us vi r vi r vi r dsDNA dsRNA (+)ssRNA ivi r AFM OND r Birnaviridae Alphaflexiviridae o b arech o asse r otamipivi r rem o esch o orchivi r Unclassified Me g Mischivi r Mos a Oscivi r P P Ros a P Rab o Sa k T Salivi r Sapel o Senec a Adenoviridae Picobirnaviridae Alphatetraviridae Sicinivi r T T Alloherpesviridae Reoviridae Arteriviridae OND (xAFM - xOND) AFM Ascoviridae Totiviridae Astroviridae Asfarviridae Caliciviridae Baculoviridae Carmotetraviridae Herpesviridae (-)ssRNA Coronaviridae Hytrosaviridae Dicistroviridae Iridoviridae Arenaviridae Flaviviridae Lavidaviridae Bornaviridae Hepeviridae Malacoherpesviridae Filoviridae Iflaviridae Marseilleviridae Fimoviridae Mesoniviridae Mimiviridae Hantaviridae Narnaviridae Nudiviridae Nairoviridae Nodaviridae Papillomaviridae Nyamiviridae Permutotetraviridae Polydnaviridae Orthomyxoviridae Picornaviridae Polyomaviridae Paramyxoviridae Polycipiviridae Poxviridae Peribunyaviridae Roniviridae Phasmaviridae Secoviridae Phenuiviridae Solinviviridae ssDNA Pneumoviridae Togaviridae Rhabdoviridae Tymoviridae Anelloviridae Sunviridae Bidnaviridae Tospoviridae Circoviridae ssRNA/DNA-RT AFM CSF antibodies target multiple EV Genomoviridae Hepadnaviridae CSF VirScan Detects Enterovirus Ab in AFMMetaviridae Parvoviridae Unclassified Smacoviridae Retroviridae antigens b c a Picornaviridae Enterovirus • Enriched Picornaviridae peptides 1 500 1000 1500 2000 2193aa

us p<.001

us 0.4 us vi r us us us

us 5’ VP4 VP2 VP3 VP1 2A 2B 2C 3A 3C 3D 3’ us vi r belonged almost entirely to the genus us us us us us us vi r us vi r vi r us us vi r us 0.3 vi r )) vi r

Enterovirus uvi r 8 OND unsagivi r visivi r o b vihepat o 0.2 - x 6 Aalivi r Ampivi r Aphth o Cardi o Cos a A Crohivi r Dicipivi r Enter o Erb o Gallivi r Hepat o Hunnivi r K Aquam a A K Limnipivi r

AFM 4 • x 69% (29/42) of AFM cases versus 7% ( 2 0.1 2 us us proportion of read s log us us us us us us us us (4/58) of OND controls considered vi r us us us us us vi r vi r vi r ivi r us 0.0 uvi r vi r us b

vi r 600 610 620 620 1760 1770 1780 vi r vi r ivi r AFM OND r positive for EV antibodies by VirScan o b

arech o asse r EV-A71 otamipivi r rem o esch o orchivi r Unclassified Me g Mischivi r Mos a Oscivi r P P P Rab o Ros a Sa k Salivi r Sapel o Senec a T Sicinivi r T T EV-D68 (x - x ) EV-A EV-A OND AFM OND AFM EV-A EV-B EV-A EV-B EV EV-B EV-B EV-D EV-F EV-D EV-J EV-J EV-A EV EV-C EV-E Yak-EV EV-G EV-J EV-G RV-B EV-B EV-B RV-B EV EV-B EV-A RV-B EV-B EV-B EV-B RV-C RV-B EV-B Yak-EV EV-B EV-H RV-B EV-F EV EV-D EV-B RV-A EV-B • Schubert RD, et al. Nat Med 2019 • Schubert RD, et al. Nat Med 2019 • Mishra N, et al. mBio 2019 63 64

No Difference in EV Antibody Detection Autoimmune Encephalitis Based on Presence of Viral Nucleic Acid

a b • Natural experiments with autoantibodies targeting individual proteins EV by ELISA EV by VirScan important for CNS function 3 p<.001 0.5 p<.001

p=ns p< .001 p = ns p < .001 0.4 • Can result in dramatic clinical phenotypes that can mimic other 2 0.3 diseases

0.2

OD 450n m 1 0.1 proportion of read s

0 0.0

OND OND

EV Previously Detected EV Previously Detected EV Previously Undetected EV Previously Undetected

• Schubert RD, et al. Nat Med 2019 65 66

11 5/5/20

Neuromyelitis optica NMDA R Ab encephalitis Encephalitis in the 21st Century: Rise of Autoantibodies • Incidence in Olmsted County, MN 1995-2015:

• Infectious Encephalitis 0.8/100,000 • Autoimmune 1.0/100,000 https://www.youtube.com/watch?v=OyPgb6V08ok

LGI1 Ab encephalitis

• Autoimmune 1995-2005 0.4/100,000 • Autoimmune 2006-2015 1.2/100,000 https://www.youtube.com/watch?v=QlEHp8oYhsQ • Marignier R, et al. Brain 2010 • Dalmau J, et al. Ann Neurol 2007 Dubey, et. al. Annals of Neurology, 2018 • Fukata Y, et al. Neurosci Res 2017 67 68

PhIP-Seq: A Rapid Way to Discover Linear Autoimmune Targets Antibody Discovery Pipeline

• Rodent brain slice staining • Immunoprecipitation à Mass spectrometry

• O’Donovan BD, Mandel-Brehm C, et al. https://www.biorxiv.org/content/10.1101/502187v1 Under review • Larman HB, et al. Nat Biotechnol 2011 69 70

Case 2 Case 2

• 37 y.o. right-handed man with a history of testicular seminoma status post left • CSF orchiectomy in 2012 • WBC 13 (0-5) • Presented with 9 months of vertigo and that acutely worsened with constant • RBC 11 double vision for five months • Protein 51 (15-45) • Patient reports it is like constantly 'walking in a canoe' • Glucose 56 • Right hand tremor • 14 unique oligoclonal bands • Stopped driving and working because of vision and balance problems • IgG index 2.1 (<0.6) • Not able to pick up his small children

Mandel-Brehm C, Dubey D, et al. NEJM 2019 Mandel-Brehm C, Dubey D, et al. NEJM 2019 71 72

12 5/5/20

Case 2

• Dramatic improvement with pulse steroids • Tumor screen with whole body PET/CT negative • Started monthly maintenance IVIg…BUT

Mandel-Brehm C, Dubey D, et al. NEJM 2019 Mandel-Brehm C, Dubey D, et al. NEJM 2019 73 74

Epilogue Autoantibody Discovery

• Phage display identified a candidate autoantigen: Kelch-like protein 11 • Component of a cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex that mediates the ubiquitination of target proteins

Mandel-Brehm C, Dubey D, et al. NEJM 2019 75 76

Mayo Collaboration Mayo Collaboration

• Additional 12 patients identified • Additional 12 patients identified • All men with a history of testicular cancer • All men with a history of testicular cancer • 8 men developed their neurologic syndrome before the diagnosis • 8 men developed their neurologic syndrome before the diagnosis of testicular cancer of testicular cancer

Mandel-Brehm C, Dubey D, et al. NEJM 2019 Mandel-Brehm C, Dubey D, et al. NEJM 2019 77 78

13 5/5/20

KLH11 Encephalitis Conclusions

• Second known autoantigen associated with testicular cancer • CSF mNGS is now clinically available with a ~7 day turnaround time • Age-adjusted male-specific prevalence of autoimmune KLHL11 • CSF mNGS increases diagnostic yield in patients with meningitis and encephalitis in Olmsted County, MN: encephalitis • 2.79/100,000 person-yrs (95% CI 0, 6.65/100,000 person-years) • Additional results conveyed in the clinical microbial sequencing board • Viral genotyping • Accurate species identification • Resistance mutations • Pathogen reads below the reporting threshold

Mandel-Brehm C, Dubey D, et al. NEJM 2019 79 80

Conclusions Conclusions

• mNGS can be insensitive for detecting infections that are • Comprehensive viral antibody profiling can shed additional light on • Compartmentalized neuroviral infections, especially when direct detection methods are • Serologically diagnosed insensitive • Very low abundance in the CSF • Autoantibody profiling with phage display can identify paraneoplastic • CSF with very high pleocytosis (e.g., >1000 WBC) syndromes that defy detection by traditional methods • Limitations of phage display • Conformational epitopes not well represented • Lack of post-translational modifications

81 82

Acknowledgements

• Joseph DeRisi • Wilson Lab • CIAPM Demonstration Project • Charles Chiu • Stephen Hauser • Ryan Schubert • Sam Pleasure • Steve Miller • Akshaya Ramesh • UCSF Center for Advanced Technology • NID Cohort • Prashanth Ramachandran • Eric Chow • Many collaborators! • Sandler Foundation • Isobel Hawes • Hannah Sample • Bowes Foundation • Ravi Dandekar • Kelsey Zorn • UCSF CTSI KL2 • Lillian Khan • Edwina Tran • NINDS K08 • Jeff Gelfand • Antibody Team • NIAID R01 • Michael Geschwind • Caleigh Mandel-Brehm • CIAPM • Mayo Clinic • Brian O’Donovan • American Brain Foundation • Sean Pittock • Debbie and Andy Rachleff • CZ Biohub • Divyanshu Dubey • Marcus Foundation • CDC • Emily Crawford • Weill Innovation Fund • Steve Oberste • Chaz Langelier • CZ Biohub • Jennifer Anstadt • Cristina Tato • Fei Fan Ng • John Pak • Jim Sejvar • Wesley Wu