Norovirus (L McIntyre) 6 May 2003

Transcription

BC Epidemiology and Laboratory Investigation
of Norovirus
(Norwalk-like viruses)
Lorraine McIntyre
BC Food Protection Symposium
May 6, 2003
A research and teaching centre affiliated with UBC
.
.
Early studies on infectious
gastroenteritis
Zahorsky J, Arch Pediatr 1929;46:391
-Winter vomiting disease
Reimann HA et al., Proc Soc Exp Biol Med
1945;59:8
-Transmitted gastro by oral and aerosol
exposure to stool filtrates.
Comparison of Gastro
Outbreaks in
2 Schools
Quesnel –2002
Norwalk, OH –1968
838 students
232 students
355 symptomatic
116 symptomatic
Vomiting, Diarrhea
Secondary attack
32%
Vomiting, nausea,
diarrhea
Norwalk Virus Discovery
Kapikian et al., J. Virol 1972;10:1075
Norwalk outbreak stool passaged in
volunteers
Performed Immune electron microscopy
with stool and acute/convalescent sera
Visualized clumped 30 nm virus
particles
Positive Strand RNA Viruses
Families:
Picornaviridae
Caliciviridae
Astroviridae
Flaviviridae
Togaviridae
Coronaviridae
Enterovirus, Poliovirus, Rhinovirus
Norovirus
Human Astro virus
West Nile Virus
Rubella virus
Human Corona virus
Atmar & Estes, 2001
Copyright © 2001 by Lippincott Williams & Wilkins
Norwalk-like Virus
(Norovirus)
Method of Detection RT-PCR
Validation Studies
Role of GI Outbreak Co-ordinator
Activity in British Columbia
Reverse Transcription
Polymerase Chain Reaction
Reverse Transcriptase PCR
Positive strand RNA (about 7.6 kb)
Step 1 - make a cDNA template
Step 2 - amplify using PCR
One tube reaction system
Molecular Method Extraction
Sample + Lysis buffer
prepare stool
suspension in DEPCwater, centrifuge
add 100 µl to GITC
containing lysis buffer
from NucliSens
(Organon Teknika)
Purified Nucleic acid
Nucleic acid
Silica
Washes Proteins
Lipids
etc.
Elution buffer
Centrifugation
Diagram from Organon Teknika
Lysis Buffer
Ethanol
Acetone
Silica
Viral RNA
Viral RNA
Protein
Silica
Viral RNA
Viral RNA
Viral RNA
Silica
Viral RNA
Series of wash
steps purify the
silica bound nucleic
acids
Nucleic acids are
eluted with elution
buffer
Diagram from Organon Teknika
manual or automated
extraction using
NucliSens Extractor
with the NucliSens
commercial kit - “Boom
silica gel extraction
method”
extractor run time is
only 41 min
Non-Structural Polyprotein
5
3
Amplification Region
4754-4773 5093-5305
2C: helicase
3C: protease
3D: RNA-dependent RNA polymerase
Region B within ORF1 (3’ end)
100 b.p. ladder ( Marker )
RT-PCR Amplicon ( 213 bp )
1. PCR + Control
2. PCR Sensitive Control
3. PCR - Control
PCR Control
1. + Extraction Control
2. - Extraction Control
3. Extraction Reagent Control
Extraction Control
PCR Inhibition Control
1 Sample + 7 Controls
If we have only
one sample …
Overall Performance of
RT - PCR vs. EM
6X better than EM
7
6
5
Finds Norwalk-
4
like virus in 50%
of stools
3
2
1
0
RTPCR
EM
Finds Norwalk-
like virus in 24%
of vomitus
Temperature / pH Studies
Temperature
13 weeks at 4oC and -20oC
Up to 6 months at -80, -20, 4oC and 2
months at room temp
pH
Solutions pH 2 to 7, overnight, then over
week-end
Laboratory Benefits
Improvement in rapid detection of
Norovirus outbreaks - fewer samples
need to be submitted
TAT 2.4 days
75% reduction in samples split to
Enterics and Virus Isolation
Role of Outbreak Co-ordinator in
Laboratory Investigation
Initial Notification
Consult with PHI about outbreak
Set-up outbreak code name
Inform internal outbreak lab group by email notification
Advise PHI on collection, delivery and
possible etiology
Role of Outbreak Co-ordinator in
Receipt of Samples
Arrange for specimen delivery to other
sections: Virus Isolation, Enterics,
Parasitology, Environmental Services
(Food, Molecular Testing for NLV)
Phone results to PHI
Update outbreak group on results
Investigation of Large GI
Outbreaks
“A guide to the
of GI Disease Outbreaks”
– for large GI outbreaks
“Outbreak Kits” – to
facilitate collection of
stool specimens and
testing of viruses,
bacteria and parasites in
laboratory
Gastrointestinal Outbreaks over the last 10 Years
200
Total #
Confirmed Norwalk
180
160
120 outbreaks
were predicted
Tota l Num be rs
140
120
100
June 2002
80
60
40
20
0
1993
1994
1995
1996
1997
1998
Year
1999
2000
2001
2002
2003
-2
F e 001
br
ua
ry
M
ar
ch
Ap
ril
M
ay
Ju
ne
Ju
A u ly
S e gu
pt s t
em
b
O er
ct
No obe
ve r
m
De be
ce r
m
Ja ber
n2
F e 002
br
ua
ry
M
ar
ch
Ap
ril
M
ay
Ju
ne
Ju
A u ly
S e gu
pt s t
em
b
O er
ct
No obe
ve r
m
De be
ce r
m
Ja ber
n2
F e 003
b2
M 003
ar
-2
A p 003
r-2
00
3
Ja
n
2001/2002 Total Reported Outbreaks
& Norovirus Confirmed Outbreaks
60
50
Total #
Confirmed Norwalk
40
30
20
10
0
Reported Outbreaks by Health Authority in 2002
Provincial Lab
1%
Health Canada
5%
Fraser
23%
Vancouver Island
15%
4.00
2.44
3.86
Vancouver
Coastal
20%
7.04
3.77
Northern
9%
Interior
27%
2002 Final Results
189 GI Outbreak Notifications
172 were laboratory investigated
(no specimens for 17 OB’s)
Norovirus
112.5
Torovirus
0.5
Listeria moncytogenes
2
Salmonella ssp.
2
Clostridium perfringens
2
Rotavirus
1
60%
Confirmed Norovirus Outbreaks by Health Authority in 2002
Health Canada
6%
Provincial Lab
0%
Vancouver Island
13%
Fraser
28%
2.14
2.44
Vancouver Coastal
15%
1.68
3.77
4.27
Northern
10%
Interior
28%
Seasonal Distribution of Confirmed Norovirus Outbreaks in
Institutions from Nov 2000 to Feb 2003
70
60
50
40
30
20
10
LTCFs
Hospitals
0
Schools&Daycares
N
00
ov
-
b
Fe
01
M
ar0
1
01
n
u
-J
10
l
Ju
Catering&Restaurants
Oc
t01
N
01
ov
-
b
Fe
02
M
a
r02
02
n
u
-J
Camps&Cruiseships&Other
02
l
u
J
t0
Oc
2
N
02
ov
-
b
Fe
03
Percentage of Confirmed Norovirus outbreaks by
Establishment from Nov 2000 to Feb 2003
Camps &
Cruiseships & Other
7%
Catering &
Restaurants
7%
Schools & Daycares
6%
Hospitals
11%
LTCFs
69%
Table 1 – Environmental Microbiology (Food)
Confirmed Food Poisoning Incidents
January 1, 2002 to December 31, 2002
Etiology
Bacillus cereus
B. cereus and S. aureus
Campylobacter ssp.
Clostridium perfringens enterotoxin
Clostridium perfringens enterotoxin
Clostridium perfringens
L. monocytogenes (serotype 1-2a)
L. monocytogenes (serotype 4b)
L. monocytogenes (serotype 4b)
Salmonella Heidelberg Group B
S. aureus enterotoxin type A
Yersinia enterocolitica
Specimen/Food Vehicle
Chinese Food
Chinese Food
Fondue (chicken)
Stool / Banquet food
Stool / Catered Christmas lunch
BBQ Chicken
Smoked salmon cream cheese
Cheese
Cheese
Chicken
Sponge Cake
Cheese
No. Ill
2
2
6+
35
19
14
0
28
82
8
1
2
Table 2 – Environmental Microbiology (Food)
Confirmed Norovirus Food Poisoning Incidents
January 1, 2002 to December 31, 2002
Suspect Food Vehicle
Borscht and perogies
Hot-dog or Church pot-luck
Chinese Food
Cheese Whiz and Beef Jerky
Hamburger
Eggs (breakfast meal)
Crab Meat
Smoked Turkey Meat
Eggs (breakfast meal)
Custard
Pizza
Bakery sweets
Caesar salad
Fried Chicken
Milk
Hamburger
Pizza
No. Ill
1
2 (or 36)
3
2
3
1
1
1
1
1
3
5
2
2
3
1
2
Onset (hrs)
8.0
3.0
8.0
22.0
1.5
2.5
0.5
2.0
2.5
24.0
2.0
4.0
2.0
24.0
?
6.0
12.0
BC Food Poisonings 2002
12 + 17 solved
168 investigations
17.3% solved (7% bacterial)
Bacterial = 41%
Viral = 59%
ABI Prism 310/3100
Genetic Analyzer
Molecular
Molecular Epidemiology
Epidemiology of
of Norwalk-like
Norwalk-like
viruses
from
British
Columbia
viruses from British Columbia
2 positives per outbreak
RT-PCR amplicon sequenced using Munroe primers
Sequence edited
Compare to prototypic virus sequence
Compare to all outbreak strains
“sequevar” BCCDC YY-NNN
Molecular Epidemiology of Norwalk-like viruses
from British Columbia
Molecular
Epidemiology of
of Norwalk-like
Norwalk-like viruses
viruses
from
from British
British Columbia
Columbia
Norovirus (Valid and protovirus groups)
Genogroup I
-
Norwalk
Southampton
Desert Shield
Chiba
Musgrove
Hesse
Winchester
Genogroup II
- Hawaii
- Melksham
- Toronto/Mexico
- Bristol/Lordsdale
- Hillingdon
- Seacroft
- Leeds
- Amsterdam?
GII-5 Hillingdon
GII-5 White River
GII-n Ernfurt
GII-2 Melksham
GII-1 Hawaii
GII-1b Wortley
GII-3 Toronto
GII-4 Bristol
GII-7 Gwynedd
GII-7 Leeds
GII-8 Amsterdam
GII-8 Idaho Falls
GII-6 Florida
G11-6 Seacroft
GIV-1 Fort Lauderdale
GIV-1 Alphatron
GI-4 Chiba
GI-4 New Orleans
GI-5 Appalachicola Bay
GI-5 Musgrove
GI-1 Norwalk
GI-2 Southampton
GI-6 Hesse
GI-3 Desert Shield
GIII-1 Jena
60.8
60
50
40
30
20
Nucleotide Substitutions (x100)
10
0
BCCDC 01-003
BCCDC 97-004
BCCDC 97-006
BCCDC 94-004
BCCDC 96-003
BCCDC 02-002
BCCDC 94-001
BCCDC 01-004
BCCDC 02-003
BCCDC 01-024
BCCDC 02-005
BCCDC 01-005
BCCDC 96-004
BCCDC 01-002
BCCDC 01-018
BCCDC 94-005
BCCDC 96-007
BCCDC 01-025
BCCDC 97-003
BCCDC 01-026
BCCDC 02-004
BCCDC 02-014
BCCDC 02-017
BCCDC 02-018
BCCDC 02-010
BCCDC 02-009
BCCDC 97-002
BCCDC 93-001
BCCDC 01-013
BCCDC 01-014
Bristol Virus
BCCDC 94-002
BCCDC 94-003
BCCDC 00-001
BCCDC 97-001
BCCDC 01-008
BCCDC 95-001
BCCDC 96-002
BCCDC 96-006
BCCDC 01-015
BCCDC 95-002
BCCDC 01-006
BCCDC 00-002
BCCDC 01-017
BCCDC 97-005
BCCDC 01-020
BCCDC 00-003
BCCDC 01-009
BCCDC 01-021
BCCDC 01-007
BCCDC 96-001
BCCDC 01-023
BCCDC 02-021
BCCDC 02-006
BCCDC 01-011
BCCDC 01-010
BCCDC 01-022
BCCDC 02-001
BCCDC 01-012
BCCDC 01-001
BCCDC 01-019
BCCDC 02-008
BCCDC 02-007
BCCDC 02-012
BCCDC 02-013
BCCDC 02-011
BCCDC 02-015
BCCDC 02-019
BCCDC 02-020
BCCDC 96-005
53.3
50
40
30
Nucleotide Substitutions (x100)
20
10
0
Specimen # FV02-755 Gateby 2002, Vernon
DNA sequencing of a 172nt fragment obtained by RT-PCR of ORF 1
at the 3’ end of the RNA dependent RNA polymerase gene has
determined it to be a member of the protovirus group
Bristol/Lordsdale.
Phylogenetic analysis of this isolate with other representative
members of this group has determined that the isolate belongs to
sequevar
BCCDC 02-007 (The term sequevar is a termed
coined at BC CDC to represent a unique or distinct lineage of the
virus). Please note that the tested sequence accounts for only a
fraction of the 8000 nt genome, which has been demonstrated to
denote specific virus genotype and genogroups.
Molecular Epidemiology of Norwalklike viruses from British Columbia

1994 Î 3 strains
1995 Î 1 strain
1996 Î 7 strains
1997 Î 6 strains
1998 Î --1999 Î --2000 Î 3 strains
2001 Î 26 strains
2002 Î 30 strains
2003 Î 15 strains (Apr 14)
Molecular Epidemiology of Norwalklike viruses from British Columbia
2000 – 3 strains
2001 – 43 outbreaks (26 strains)
14 outbreaks (32.6%) BCCDC 00-001
2002 – 106 outbreaks (30 strains)
2003 – 56 outbreaks (13 strains - end of Feb)
Molecular Epidemiology of Norwalk-like
viruses in LTCF
2000
75% (00-001)
No secondary sources
2001
33% (01-001)
restaurant/non-outbreak
2002
44% (02-007)
All sources/province wide
2003
82% (02-007)
All sources/province wide
Two Catering Outbreaks
FO#139 - 25 Nov 02
FO#141 - 26 Nov 02
Catering Company “A” - Victoria
owner - sandwiches & wraps
4 groups, at risk 101, ill 59
BCCDC 02-019
Catering Company “B” - Vancouver
staff (at home with sick child)
sponge cake
church, 178 at risk, 49
BCCDC 02-029
viruses in Hospitals 2002-2003
2002 º 9 outbreaks
–
–
–
–
–
–
–
–
–
Burnaby Hospital
Chilliwack General
Prince George Hospital
Trail Hospital
Saanich PH (ECU2)
Royal Jubilee
Saanich PH (south)
Victoria General
Lady Minto Hospital
BC CDC 02-022
BC CDC 02-019
BC CDC 02-007
BC CDC 02-027
BC CDC 02-007
BC CDC 02-019
BC CDC 02-019
BC CDC 02-019
BC CDC 02-019
viruses in Hospitals 2002-2003
2003 º 10 outbreaks
–
–
–
–
–
–
–
–
–
–
Cranbrook Hospital
Chilliwack General
Fernie Hospital
Burnaby General
Burnaby General (2B)
St Paul’s Hospital
Powell River Hospital
VGH Psych
VGH Surg
Royal Inland
BC CDC 02-007
BC CDC 02-007
BC CDC 02-026
BC CDC 02-007
BC CDC 02-007/02-024
BC CDC 02-007
BC CDC 02-024
BC CDC 02-015
BC CDC 02-007
BC CDC 02-007
viruses in Cruise ships
MS Statendam
BCCDC 01-010
LTCF/non-outbreak
Denali National Park, Alaska
BCCDC 02-006
No other sources
BCCDC 02-007
Multiple sources
Princess Line
–
–
–
–
Ocean Princess
Star Princess
Sun Princess
Sea Princess
MS Ryndam
MS Radiance of the Seas
BCCDC 02-019
Chilliwack General Hospital
“A tale of a fateful trip”
Just sit right back and you'll hear a
tale,
A tale of a fateful trip,
That started from this tropic port,
Aboard this tiny ship.
July 24, 2002
BCCDC 02-007
Molecular
Epidemiology of
of Norwalk-like
viruses
from
British
Columbia
BCCDC 02-007
“Ring of Fire”
Molecular
Epidemiology of
of Norwalk-like
viruses
from
from British
British Columbia
Columbia
August 8, 2002
Molecular
Epidemiology of
of Norwalk-like
viruses
from
British
Columbia
“A tale of a fateful trip” (July 24, 2002)
41 st Norwalk-like out break since January 2001
Virus emerged in Chilliwack/Vancouver June 5th
Detected Hyatt Regency Hotel June 12th
Detected in LTCF -> Vancouver/Richmond/Kelowna/Penticton
Detected -> Quesnel /Prince George /Kelowna /Summerland /
Surrey /Kitimat /Vernon /Preston
November 7th Prince George
100 mile house/Agassiz/Burnaby/Vancouver/Vernon/Victoria
Molecular
Epidemiology of
of Norwalk-like
viruses
from
British
Columbia
Norwalk Virus: What you need to know (www.cruisecritic.com)
Carnival's Fascination has joined Holland America's Amsterdam and
Statendam, and Disney's Magic, to become the latest in a series of
cruise ships to "come down with a virus." The Norwalk Virus, that is.
The highly contagious Norwalk strain until recently was largely limited
in its plague to ships sailing Alaska in summer (in 2002 alone,
passengers on Alaska-Northwest Canada-bound ships including
Princess' Ocean Princess, Royal Caribbean's Radiance of the Seas
and Holland America's Ryndam, were afflicted in significant numbers).
But what seems like a rampant outbreak spread among a variety of
cruise ships from different cruise lines in Florida has raised awareness
to almost hysterical levels.
Synopsis Â
No longer seasonal in British Columbia
Predominate strain each year
Predominate strain usually appears early in the year ->00-001/01-001/ 02-007
Persists, timeframe variable
No predilection for particular setting?
Once gone – stays gone?
Rocky Mountains not a barrier - Cochrane, Alberta
Thanks
Alan McNabb, Supervisor, Molecular Diagnostics
Dr. Martin Petric, Clinical Virologist
Min Kuang-Lee, Technologist
Joe Fung, Supervisor, Environmental Microbiology