CDC Real-Time RT-PCR Support Strategies and Updates
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1 CDC Real-Time RT-PCR Support Strategies and Updates Fourth Annual SARInet Meeting May 23-26, 2017 Punta Cana, Dominican Republic Stephen Lindstrom, Ph.D. Diagnostics Development Team Virus Surveillance and Diagnosis Branch Influenza Division Centers for Disease Control and Prevention The findings and conclusions in this presentation are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention National Center for Immunization and Respiratory Diseases Influenza Division
2 Overview Testing strategies & algorithms for surveillance and diagnostic testing CDC rrt-pcr assays for detection and characterization of influenza Support strategies for rrt-pcr testing CLSIS protocols/procedures IRR - Reagent kits, control materials A(H1N1)pdm09 reactivity and assay update
3 H1 Influenza Viruses in Humans Type A H2 H3 H1 Avian H5 Avian H6 Avian H7 Avian H9 Avian H10 H3v H1v 1998/ B/Yam Potential Pandemic strains Seasonal Strains Type B B/Vic
4 Routine Influenza Testing Algorithm Routine Surveillance No Known risk for avian influenza Perform RT-PCR testing for seasonal human influenza A and influenza B viruses Negative InfA (+) InfB (+) Report Results Test for H3, pdm H1, pdminfa Test for YAM, VIC InfA unsubtypable possible novel influenza Based on risk assessment, test for H5, H7 and/or other novel strains w/rt-pcr InfA (+) H3 or pdm H1 (+) InfB (+) YAM or VIC (+) InfA inconclusive samples with possible SOIV Inconclusive samples with possible coinfection/laiv Report Results Report Results
5 Influenza Detection Algorithm and Sample Flow
6 CDC Influenza Virus Real-time RT-PCR Reagent Kits Influenza A/B Typing Panel (InfA, Inf B, RP) Influenza A Subtyping Panel (InfA, H3, pdmh1, pdminfa) Influenza B genotyping Panel (InfB, YAM, VIC, RP) Influenza A H5 (Asian lineage) Subtyping Panel (InfA, H5a, H5b, RP) Influenza A EuH7 Assay (EuH7)
7 Update: CDC Influenza A/Subtyping Kit (Ver2) CDC Human Influenza Real-Time RT-PCR Diagnostic Panel Kit Contents Application Influenza A Subtyping Kit (Ver2) FluIVD03-6 Primer and Probe Sets: InfA, H3, pdminfa, pdmh1, RP Controls: SIPC Specifically detects and differentiates seasonal influenza subtypes H3 and H1pdm09 Detects classical swine influenza viruses and swine triple reassortant viruses Update pdmh1 assay to ensure reactivity (Clade 6B.1) Remove H1 assay Update positive control SIPC Qualify ZEN quenched probes
8 Reactivity of Updated pdmh1 assay - Inclusivity Strain designation Conc. InfA pdminfa pdmh1 ver2 A/California/04/ (3/3) (3/3) (3/3) A/California/07/ (3/3) (3/3) (3/3) A/Colorado/14/ (3/3) (3/3) (3/3) A/Florida/27/ (3/3) (3/3) (3/3) A/Florida/62/ (3/3) (3/3) (3/3) A/Maryland/13/ (3/3) (3/3) (3/3) A/Minnesota/03/ (3/3) (3/3) (3/3) A/North Carolina/4/ (3/3) (3/3) (3/3) A/Utah/13/ (3/3) (3/3) (3/3) A/Washington/24/ (3/3) (3/3) (3/3)
9 Interpretation of Real-time RT-PCR Results InfA H3 pdm InfA pdmh1 B RP Result /- A/H /- A/H1pdm /- B Negative Inconclusive ± Influenza A - Unsubtypable Potential novel influenza Influenza A detected; Presumptive positive for Influenza A(H3N2) variant virus
10 Reactivity of CDC EuH7 Assay Invitrogen Quanta Strain Designation sub-type Lineage InfA EuH7 InfA EuH7 A/ANHUI/1/2013 H7N9 Eu A/DUCK/VIETNAM/NCVD-197/2009 H7N3 Eu A/TURKEY/ITALY/5425/07 H7N3 Eu A/Shoveler/Egypt/ NAMRU3/2007 H7N3 Eu A/Mallard/Netherlands/12/2000 H7N3 Eu A/Chicken/Italy/5074/1999 H7N1 Eu A/MEXICO/7218/2012 H7N3 N.Am. 2 + Undet + Undet 1 Eu = Eurasian lineage; 2 N.Am. = North American lineage.
11 Support for Public Health and Research Laboratories
12 Reagent ordering - IRR
13 CDC rrt-pcr Influenza Kit Distribution Globally CDC Influenza Virus Real-time RT-PCR Panel (RUO) Catalogue # Shipped 2014 Shipped 2015 Shipped 2016 Shipped 2017 Influenza A/B Typing Panel FluRUO Influenza A (H1/H3/H1 pdm09) Subtyping Panel FluRUO Influenza A (H3/H1pdm09) SubtypingPanel (VER 2) FluRUO Influenza B Lineage Genotyping Panel FluRUO Influenza A(H5) (Asian Lineage) Subtyping Panel (VER 3) FluRUO-06, FluRUO Influenza A/H7 (Eurasian Lineage) Assay FluRUO Total as of 4 th April 2017 in 117 countries
14 Countries receiving CDC rrt-pcr reagents 117 countries
15 CDC rrt-pcr Influenza Kit Distribution to PAHO Countries CDC Influenza Virus Real-time RT-PCR Panel (RUO) Catalogue # Shipped 2014 Shipped 2015 Shipped 2016 Shipped 2017 Influenza A/B Typing Panel FluRUO Influenza A (H1/H3/H1 pdm09) Subtyping Panel FluRUO Influenza A (H3/H1pdm09) Subtyping Panel (VER 2) FluRUO Influenza B Lineage Genotyping Panel FluRUO Influenza A(H5) (Asian Lineage) Subtyping Panel (VER 3) FluRUO-06, FluRUO Influenza A/H7 (Eurasian Lineage) Assay FluRUO Total as of 17 May laboratories in 22 countries
16 PAHO countries receiving reagents from IRR laboratories in 22 countries
17 CDC Sharepoint Site for Laboratory Support for Influenza Surveillance >320 users Provide public health and research laboratories access to multiple assays, procedures and methods depending on need (equipment, chemistry, etc.) Allow for coordinated communication with registered laboratories Provide timely notification of assay updates
18 Countries registered on CLSIS 90 countries
19 PAHO countries registered on CLSIS registrants 19 countries
20 CDC Sharepoint Site for Laboratory Support for Influenza Surveillance Real-time rrt-pcr laboratory protocols for influenza English, Spanish, Portuguese RNA extraction procedures (manual & automated) Package inserts for kits and controls Enzyme options Real-time PCR equipment options Primer/probe sequence information Manufacturing options for probe chemistry (ZEN, BHQ)
21 CDC Sharepoint Site for Laboratory Support for Influenza Surveillance UPDATES: Updated rrt-pcr procedures Assay specific protocols for FluA subtyping and FluB genotyping. Package insert for updated FluA subtyping kit (Ver2) Assay sequence updates to pdmh1 primers/probe FluB YAM/VIC genotyping primer/probe sequences
22 CDC Sharepoint Site for Laboratory Support for Influenza Surveillance PENDING UPDATES: Real-time PCR equipment procedures Qiagen Rotorgene Assay Manager protocol Automated Nucleic Acid isolation procedures Qiagen EZ1 Advanced XL Roche MP96 FluB YAM/VIC genotyping primer/probe sequences
23 CDC Sharepoint Site for Laboratory Support for Influenza Surveillance ONGOING ASSAY DEVELOPMENT: rrtpcr assays N. Am. H7 N. Am H5 FluB genotyping possible update Diagnostic sequencing Targeted Sanger sequencing for genotype identification H1pdm09 vs H1v genotypes Human H3 vs H3v genotypes B VIC deletion mutation variant
24 PAHO countries receiving support from CDC Support provided Countries Received IRR reagents 22 Registered on CLSIS (protocols) Countries that submitted samples to CDC since (46 users) 38 Total Countries in Region 46
25 Qualification of Real-time PCR Dx Instruments and Nucleic Acid Extraction Platforms
26 Real-time PCR Platforms LifeTechnologies/ABI 7300~7500Fast LifeTechnologies/ABI Step One LifeTechnologies ViiA7/QuantStudio Dx Qiagen Corbett Rotor-Gene Stratagene/Agilent Mx3000p/3005p Roche LC 2.0 Roche LC 96 BioRad CFX BioRad MiniOpticon BioRad icycler iq4/ iq5 3M Integrated Cycler Roche LC 480 Roche LC Nano
27 Real-time PCR Platforms? Thermo 7500Fast/FastDx Step One QuantStudio 3/5 QuantStudio Qiagen Rotor-Gene BioRad CFX Agilent 3M Roche Mx3005p Aria Integrated Cycler LC 480 LC 96 LC Nano
28 FDA cleared Real-time PCR Platforms Under Evaluation for Dx Qualification Thermo ABI QuantStudio Dx (QSDX) Qiagen RotorGene Dx (QMDX) 96 well Assay set up locked Results analysis and reporting locked 72 well Assay set up locked Results analysis and reporting locked
29 Automated Nucleic Acid Extraction Roche Kingfisher Magnapure Compact Magnapure LC Magnapure 96 Qiagen BioMerieux Qiagen Qiacube EZ1 Advanced XL Qiacube HT Qiasymphony EasyMag
30 Qiagen Extraction Platforms Qiacube: Purification of NA from up to 12 samples; spin-column format QIAsymphony: processing 1-96 samples - in batches of 24 EZ1 Advanced XL: Purification of nucleic acids from 1-6 or 1-14 samples QIAextractor: 8-96 samples per run.
31 kingfisher Magnetic bead extraction system Flexible format allows for multiple chemistry options Chemistry setup necessary Pre/post sample processing necessary Various platform throughputs 1-15; 24, 96 Multiple vendors Fisher Scientific, Ambion MagMax, Promega, etc.
32 Diagnostic Extraction Platform Under Evaluation Qiagen EZ1 Advanced XL Roche Magnapure samples minutes Reagents pre-packaged no set up Tube-to-tube extraction no post extraction processing GMP manufacturing for Dx testing 8-96 samples Reagents pre-packaged no set up Plate-to-plate extraction pre/post extraction processing GMP manufacturing for Dx testing
33 Extraction Platforms Under Consideration Qiagen Qiasymphony KingFisher
34 Diagnostics Development Team Lakshmi Malapati LaShondra Berman Bo Shu Christine Warnes Janna Murray Ji Liu Shannon Emery Marisela Rodriguez Brian Lynch Kai-Hui Wu
35 EPB: Lenee Blanton Desiree Mustaquim OD: Becky Garten Battelle Team: Stephen Burke (Project Manager) Janna Murray (IVD Product support) Chantelle Runnels (Quality Control) Kellie White (Quality Assurance) Acknowledgements OD/IRR Team: Erica Guthrie Sarah Schildecker No Man is an island DSR: Nicky Sulaiman Kanwar Bedi Dennis Bagarozzi
36 Thank You
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