Norovirus Testing: What Happens at “The Lab” Dr. Amy Woron Amy.Woron@TN.gov 615-262-6462 Molecular Biologist TN Dept. of Health Laboratory Services
Norovirus GI and GII Assay Differentiates between GI and GII Can have results in as little as 5 hours 2009 data below TN Grand Region County GI pos GII pos GI and GII pos East Hamilton 5 9 Henderson 2 Knox 1 Middle Davidson Robertson Sumner Williamson 3 Wilson West Gibson Out of State N/A GI GII
CaliciNet Maybe. There are multiple patients all with norovirus GII. Are they all related? Maybe.
National Norovirus Outbreak Network CaliciNet USA Best tool is the epi investigation ….. CaliciNet can help National Norovirus Outbreak Network
Current Pitfalls in Norovirus Surveillance No national laboratory surveillance system Tracking multi-state (international) outbreaks with a potential common source is a challenge Only a fraction of the states report to the CDC No standard protocol among laboratories for sequence analysis and genotyping 5
Objectives of CaliciNet Improve surveillance of norovirus (sapovirus, other GI viruses) Allow for real-time data exchange at the state and national level Rapid genotyping and compare norovirus sequences Link geographically different clusters of illness to a common (food) source Facilitate the identification of new emerging norovirus strains
States Participating in CaliciNet WA WA ME MT MT ND ND ME ME OR OR MN MN ID VT NH SD SD WI WI NY NY MA WY WY MI MI RI CT IA IA PA PA NE NE NJ NV NV OH OH MD MD DE UT UT IN IN CO CO CA CA IL IL WV WV VA VA DC KS KS MO MO KY KY NC NC TN AZ AZ OK OK AR AR SC SC NM NM MS MS AL AL HI GA GA TX TX LA LA AK AK FL FL States with CaliciNet certified participants March 2010 (n = 16) 7
Confirmed and Suspected Etiology of 1270 Foodborne Outbreaks Reported to CDC, 2006 Other/Multiple 2% Unknown 29% Norovirus 40% Chemical 5% Parasites 1% Bacteria 23% CDC 2009 MMWR
From Stool Cup to Sequence Norovirus testing: From Stool Cup to Sequence Stool specimen RNA Extraction RT-PCR testing Real-time RT-PCR Conventional RT-PCR positive Compare norovirus sequence with database Cycle sequencing 9
Norovirus regions targeted by detection and typing RT-PCR assays Hel Pro Pol VP1 VP2 7654 1 5' 3' ORF 3 ORF 1 ORF 2 RT* C D 85/98 bp 330/344 bp 177/253 bp * RT = TaqMan realtime RT-PCR Norovirus detection Norovirus typing
Required fields for CN upload Epi Lab Page 8; CN Manual v.3
Multiple Alignment
Dendrogram
Nucleotide Polymorphisms
GI Reference Strains
GII Reference Strains
Similarity to PulseNet BioNumerics Encourage state level participation Increase real-time information flow Ability to view/compare outbreaks at the national level Another tool in the epidemiology tool belt
CaliciNet Summary A novel national surveillance system for linking norovirus outbreak clusters potentially to a common source Network for standardized norovirus genotyping and classification Potential to identify new emerging norovirus strains 19
Leslie Barclay lbarclay@cdc.gov (CaliciNet scripts) CDC CaliciNet Team Contact information: Leslie Barclay lbarclay@cdc.gov (CaliciNet scripts) Nicole Gregoricus ngregoricus@cdc.gov (Laboratory protocols) Jan Vinjé jvinje@cdc.gov (Supervisor CaliciNet) 20 20
Future Study Human Astrovirus testing … Human Sapovirus testing … Food testing for norovirus … Slides with the CDC CaliciNet logo courtesy of Dr. Jan Vinjé, CaliciNet Team Leader, CDC
Questions?