IMPLEMENTATION OF GHOST FOR HAV OUTBREAK DETECTION
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1 National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention IMPLEMENTATION OF GHOST FOR HAV OUTBREAK DETECTION Sumathi Ramachandran,MS,MPH,PHD Molecular Epidemiology and Bioinformatics Team Division of Viral Hepatitis APHL Annual Meeting, June 2018
2 Background Hepatitis A virus (HAV) is primarily a human pathogen, transmitted by person-to person contact or ingestion of contaminated food or water Hepatitis A vaccine given within 2 weeks of exposure can prevent the disease In the USA, hepatitis A vaccination has been recommended for all children aged months since 2006, and consequently the number of acute cases has greatly fallen In recent years, large hepatitis A outbreaks have been observed in several states
3 Objectives Determine the genetic characteristics of HAV circulating in the United States Provide phylogenetic background of HAV for outbreaks investigation and molecular tracking of hepatitis A in the United States Transition from traditional Sanger sequencing to NGS & GHOST system for hepatitis A outbreaks investigations
4 CDC DVH HAV Sequence Datasets HAV Specimen Source ( ) # Positive / Tested Specimens # Unique Sequences Sentinel Counties ( ) 1234/ EIP Sites ( ) 472/ Border Infectious Disease Sites ( ) 378/ Food Safety A13FBM ( ) 105/ Various Outbreaks ( ) 2495/ Total 4684/
5 Genotype distribution of HAV circulating in USA HAV Specimens HAV Genotype (%) IA (%) IB (%) IIIA (%) Total # (97.6) 34 (2.1) 5 (0.3) 1612 Surveillance (82.3) 92 (15.9)** 5 (0.9) 577 Sub-total 2053 (93.8) 126 (5.8) 10 (0.5) (99.7) 1 (0.2) 1 (0.2) 602 Outbreaks ** 439 (23.2) 1453 (76.8)** 1 (0.1) 1893 Sub-total 1039 (41.6) 1454 (58.3)** 2 (0.1) 2495
6 HAV Genotype and Phylogenetic Sub-cluster International travel IDU MSM
7 Rising HAV Genotype IB in USA A13NM A16VA2 HAV Outbreaks Year Genotype IA (%) IB (%) Total A16VA (56.5) 30 (43.5) 69 A17CA, UT & KY (96.3) 1 (3.7) (46.2) 127 (53.4) (16.7) 1082 (83.3) (12.2) 417 (87.8) 475 Recent Outbreaks with Genotype IB Previous Cases with Genotype IB IA Middle East References Total 450 (21.4) 1657 (78.6) 2107 IB A16MI IA
8 Schematic Representation of the HAV Genome & Region Used for Molecular Analysis CDC SOP for HAV Molecular Analysis Traditional Sanger Sequencing (Up to April 2018) Next Generation Sequencing/ GHOST (Since May 2018)
9 Hepatitis A Outbreaks ( ) (VP1/P2B region, 315 bp in length) A13FBM435 A17CO Cluster A17USA Cluster3 A13FBM418 A17USA Cluster2 June 5 th 2018 update A16HI Cluster Samples processed at CDC 1924 Samples were PCR positive IA A13FBM424 A17USA Cluster1 A16HI Cluster1 Lab Technical Assistance: San Diego CA MI FL Outbreak Cluster (Case #) References IB CA 2017 Outbreak nt difference: 0.32%-0.64% A13FBM419 A17CA Cluster A A17CA480 A17CA300 A17CA232 A17CA Cluster D A17CA Cluster C A17CA Cluster B 2A17CA71 A16VA Cluster1 A13NM Cluster CA Clusters (A-D) A16VA Cluster2 A16MI Cluster2 A13FBM343 A16MI Cluster1 MI Clusters (1 & 2)
10 HAV Transmission Clusters ( ) MI Cluster 1 MI Cluster 2 CA Clusters B, C, D CA Cluster A (multiple states, n=12)
11 Next-Generation Sequencing Two Strategies for Viral Pathogens Whole-Genome Sequencing Single-Amplicon Sequencing 5 Co re E 1 E 2 NS 2 HAV Genome NS 3 NS 4 NS 5a NS 5b 3 - NGS reads cover the entire genome - Variable coverage of different genomic regions - Reads stagger - Consensus sequence obtained - Heterogeneity at individual positions NGS of VP1-2A gene variants Dominant major variant - NGS reads cover a single region - Sequences of individual variants - Deep coverage - Minority variants - Reads strictly aligned - Heterogeneity of the region - Intra-host population structure - Strong genetic strain identity - Increased multiplexing - Reduction in cost per specimen
12 National Center for HIV/AIDS, Global Health, Viral Outbreak Hepatitis, & Surveillance STD, Technology and TB Prevention (GHOST) Cyber-Molecular Detection of Hepatitis Viruses (HCV and HAV) Clinical samples GHOST Portal ( Amplicon Deep Sequencing: Miseq (Illumina) Not linked FASTQ/FASTA Upload Web-based Models Transmission cluster Source? Uploaded NGS files are automatically analyzed and the results are visualized in the form of transmission networks GHOST web-site contains bioinformatics tools for outbreak investigation and molecular surveillance Outbreak Surveillance
13 GHOST module for HAV Outbreak Investigation A17KY clusters (N=314 tested): A17KY Cluster Case # (%) A17CA Cluster A CA IB Cluster A 227 (85.7) CA IB Cluster B 1 MI IB Cluster 2 4 others IB 33 IA/IIIA 3 KY HAV cases GHOST Networks A17CA cluster A
14 HAV Intra-host variants - Kstep networks A17CA Cluster A A17KY recent cases A17KY early cases A17CA Cluster A
15 HAV Intra-host variants - Kstep networks MI Local ID MI_Sequences Genotype CDC_ID CDC_Sequences HAV MI Cluster 2 IB A16MI526 MI Cluster 2 HAV MI Cluster 2 IB A16MI527 MI Cluster 2 HAV MI Cluster 2 IB A16MI528 MI Cluster 2 HAV Unique IB A16MI529 MI Cluster 2 HAV MI Cluster 2 IB A16MI530 MI Cluster 2 HAV MI Cluster 2 IB A16MI531 MI Cluster 2 HAV MI Cluster 2 IB A16MI532 MI Cluster 2 HAV MI Cluster 2 IB A16MI533 MI Cluster 2 HAV Negative IB A16MI534 MI Cluster 2 HAV Negative IB A16MI535 Unique HAV MI Cluster 1 IB A16MI536 MI Cluster 1 HAV MI Cluster 2 IB A16MI537 MI Cluster 2 HAV MI Cluster 2 IB A16MI538 MI Cluster 2 HAV MI Cluster 2 IB A16MI539 NGS in progress HAV MI Cluster 1 IB A16MI541 MI Cluster 1 HAV MI Cluster 2 IB A16MI542 MI Cluster 2 HAV MI Cluster 1 IB A16MI543 MI Cluster 1 HAV MI Cluster 2 IB A16MI544 MI Cluster 2 HAV MI Cluster 2 IB A16MI545 MI Cluster 2 HAV MI Cluster 2 IB A16MI546 MI Cluster 2 HAV Unique IB A16MI547 HAV HAV MI Cluster 2 IB A16MI548 MI Cluster 2
16 GHOST Outreach Activities ( ongoing) CDC-NIH-NIDA-SAMHSA GHOST Center MA (Harvard University, 2018) Rural Opioid FOA (10 states) National Institute on Drug Abuse GHOST Trained States (22 states) & 2017 Workshops Phase 1 ( ongoing) GHOST HCV Pilot States TN NY NH MI AK CDC-APHL Phase 2 (2017- ongoing) GHOST HCV Implementation IA IN CO GA MT (2018) GHOST HAV Testing sites CA & SD County IN MI NY
17 Conclusions In the HAV outbreaks, genotype 1B was widely occurring among drug users/homeless, and genotype 1A among MSM NGS sequences represent complex intra-host HAV populations NGS sequencing coupled with GHOST eliminates the effects of population complexity and many technological artifacts on assessment of transmission links during outbreak investigations GHOST module for a more accurate identification of HAV strains in surveillance is in development Use by state labs is expected to help develop an improved GHOST module applicable to identification of HAV strains in both outbreak and surveillance settings
18 CDC Division of Viral Hepatitis Jeff Efird Monique Foster Megan Hofmeister Eyasu Teshale Noele Nelson Cynthia Jorgensen Brigette Ulin Blythe Ryerson Cecily Campbell Philip Spardling Alaya Koneru Tonya Hayden Saleem Kamili John Ward Paul Weidle Acknowledgments County of San Diego Health and Human Services Agency California Department of Public Health Utah Department of Health Kentucky Department of Health Michigan Department of Health and Human Services All other State Departments of Health Viral Hepatitis Laboratory (GHOST) Yulin Lin Lili Punkova Guo-Liang Xia Garrett Longmire Seth Sims Walker Gussler Amanda Poe Yury Khudyakov APHL 18
19 For more information, contact CDC CDC-INFO ( ) TTY: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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