norovirus testing: what happens at the lab dr. amy woron [email protected] 615-262-6462 molecular...
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Norovirus Testing:What Happens at “The Lab”
Dr. Amy [email protected]
Molecular BiologistTN Dept. of Health
Laboratory Services
Norovirus GI and GII Assay• Differentiates between GI and GII• Can have results in as little as 5 hours
GII
GI
TN Grand Region County
GI pos
GII pos
GI and GII pos
East Hamilton 5 9
East Henderson 2
East Knox 1
Middle Davidson 1 5
Middle Robertson 1 1
Middle Sumner 1 5
Middle Williamson 3 1
Middle Wilson 2
West Gibson 1
Out of State N/A 2
2009 data below
CaliciNet
• There are multiple patients all with norovirus GII.
• Are they all related?
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
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
States with CaliciNet certified participants March 2010 (n = 16)
AZAR
CACO
DE
FL
GA
ILIN
IA
KS
KY
ME
MD
MN
MO
MT
NENV
NM
NY
NC
ND
OH
OK
OR
PA
RI
SC
SD
TX
UT
VA
WA
WV
WIWY
AK
MI
ME
LA
MS AL
AZAR
CACO
CT
FL
GA
ID
ILIN
IA
KS
KY
ME
MD
MA
MN
MO
MT
NENV
NH
NM
NY
NC
ND
OH
OK
OR
PA
SC
SD
TN
TX
UT
VT
VA
WA
WV
WIWY
AK
HI
DC
MI
NJ
LA
MS AL
Confirmed and Suspected Etiology of 1270 Foodborne Outbreaks Reported to CDC, 2006
CDC 2009 MMWR
Other/Multiple2%
Unknown29%
Chemical5%
Parasites1%
Bacteria23%
Norovirus40%
Real-time RT-PCR
RNA Extraction
Norovirus testing: From Stool Cup to Sequence
positive
Cycle sequencing
Conventional RT-PCR
Compare norovirus sequence with database
Stool specimen
RT-PCR testing
Norovirus regions targeted by detection and typing RT-PCR assays
3'
ORF 2
ORF 3
C D177/253 bp
ORF 1
RT*85/98 bp
5'
Hel Pro Pol1
7654VP1
330/344 bp
VP2
Norovirus detection Norovirus typing* RT = TaqMan realtime
RT-PCR
Required fields for CN upload
Page 8; CN Manual v.3
Epi Lab
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
CDC CaliciNet Team Contact information:Leslie Barclay [email protected] (CaliciNet scripts)Nicole Gregoricus [email protected] (Laboratory protocols)Jan Vinjé [email protected] (Supervisor CaliciNet)
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
