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

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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,
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    INVESTIGATION INTO THE LINK BETWEEN WATER QUALITY AND MICROBIOLOGICAL SAFETY OF SELECTED FRUIT AND VEGETABLES FROM FARMING TO PROCESSING STAGES (VOLUME 1) Report to the WATER RESEARCH COMMISSION and DEPARTMENT OF AGRICULTURE, FORESTRY AND FISHERIES Edited by EM du Plessis1 and L Korsten1 Research team L Korsten1, EM Buys2, B Pillay3, M Taylor4 1Department of Microbiology and Plant Pathology, University of Pretoria 2Department of Food Science, University of Pretoria 3Department of Microbiology, University of KwaZulu-Natal 4Department of Medical Virology, University of Pretoria/National Health Laboratory Service WRC Report No. 1875/1/15 ISBN 978-1-4312-0693-3 August 2015 Obtainable from Water Research Commission Private Bag X03 GEZINA, 0031 [email protected] or download from www.wrc.org.za DISCLAIMER This report has been reviewed by the Water Research Commission (WRC) and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the WRC nor does mention of trade names or commercial products constitute endorsement or recommendation for use. © Water Research Commission ii EXECUTIVE SUMMARY Background and motivation An increase in the consumption of fresh and minimally processed fruits and vegetables globally (Ayers and Westcot, 1 985; Beuchat, 2002) has resulted in one of the most important health challenges in terms of foodborne diseases. In the United States of America (USA) these dietary changes resulted in doubling of produce-associated disease outbreaks per year from 1973-1992 (USA Food and Drug Administration, 1998). However, over the past 10-15 years the number of foodborne disease outbreaks has declined. The number of CDC outbreak reports varied between 1243 to 1417 during 2000-2002 in comparison to only 800 to 850 outbreaks reported from 2009-2013 (http://www.foodsafetynews.com/2015/06/the-prevalence-of-foodborne-illness; accessed 12 July 2015).
  • Icosahedral Viruses Defined by Their Positively Charged Domains: a Signature for Viral Identity and Capsid Assembly Strategy

    Icosahedral Viruses Defined by Their Positively Charged Domains: a Signature for Viral Identity and Capsid Assembly Strategy

    Support Information for: Icosahedral viruses defined by their positively charged domains: a signature for viral identity and capsid assembly strategy Rodrigo D. Requião1, Rodolfo L. Carneiro 1, Mariana Hoyer Moreira1, Marcelo Ribeiro- Alves2, Silvana Rossetto3, Fernando L. Palhano*1 and Tatiana Domitrovic*4 1 Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil. 2 Laboratório de Pesquisa Clínica em DST/Aids, Instituto Nacional de Infectologia Evandro Chagas, FIOCRUZ, Rio de Janeiro, RJ, 21040-900, Brazil 3 Programa de Pós-Graduação em Informática, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil. 4 Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil. *Corresponding author: [email protected] or [email protected] MATERIALS AND METHODS Software and Source Identifier Algorithms Calculation of net charge (1) Calculation of R/K ratio This paper https://github.com/mhoyerm/Total_ratio Identify proteins of This paper https://github.com/mhoyerm/Modulate_RK determined net charge and R/K ratio Identify proteins of This paper https://github.com/mhoyerm/Modulate_KR determined net charge and K/R ratio Data sources For all viral proteins, we used UniRef with the advanced search options (uniprot:(proteome:(taxonomy:"Viruses [10239]") reviewed:yes) AND identity:1.0). For viral capsid proteins, we used the advanced search options (proteome:(taxonomy:"Viruses [10239]") goa:("viral capsid [19028]") AND reviewed:yes) followed by a manual selection of major capsid proteins. Advanced search options for H.