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 Neuroleptospirosis Neuroleptospirosis • Unanticipated bacterial infection • Unanticipated bacterial infection • 48-hour turnaround • 48-hour turnaround • Dramatic clinical improvement • Dramatic clinical improvement 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 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 • Wilson MR, Suan D, et al. Ann of Neurol 2017 • Wilson MR, Suan D, et al. Ann of Neurol 2017 23 24 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 (%) r e v o c m. r no 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 (%) r e v o c m. r no 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 (%) r e v o c m. r no 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,