Transcript Molecular Epidemiology: Impact on Food Regulation and

Bala Swaminathan, Ph.D.
Vice-President, IHRC, Inc.
Atlanta, GA, USA
IHRC, Inc.
Supporting public health worldwide
Epidemiology
 Epidemiology: the study of the distribution and
determinants of health-related states in specified
populations, and the application of this study to control
health problems.
 Epidemiologists
 collect data about an entire population through surveillance
systems or descriptive epidemiological studies.
 use these data to generate hypotheses about the relationships
between exposure and disease.
 test the hypotheses by conducting analytical studies such as
cohort or case-control studies.
 use the findings from these studies to develop, recommend
and/or implement some form of community intervention to
end the health problem and prevent its recurrence.
Molecular Biology
 Molecular biology involves the study of macromolecules
(DNA, RNA, proteins) and the macromolecular
mechanisms found in living things, such as the molecular
nature of the gene and its mechanisms of gene replication,
mutation, and expression.
 In the context of infectious disease epidemiology, the
molecular biologic approach involves molecular
characterization of disease –causing organisms and their
subdivision by their DNA, RNA and/or proteins.
 DNA “fingerprinting”
 Subtyping
 Molecular subtyping
Synergy between two seemingly disparate scientific disciplines
Example of Molecular Subtyping
PulseNet Universal Reference Standard
Fragment
Size
1135 Kb
452.7 Kb
216.9 Kb
76.8 Kb
33.3 Kb
A typical E. coli O157:H7 PFGE Gel
•National network of public health laboratories
–State and local public health departments
and Federal agencies (CDC, USDA-FSIS, FDA)
•Routinely perform standardized molecular
subtyping of foodborne disease-causing
bacteria
•Share DNA “fingerprints” electronically in
real-time via Internet
•Dynamic database of DNA “fingerprints” at
CDC
Participation in PulseNet International
33
13
13
E. Coli O157 Outbreak – Minnesota, 2000
22
20
18
No. of cases
16
14
12
10
8
6
4
2
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Courtesy: John Besser, MN State Health Dept
22
22
20
20
18
18
16
16
14
14
No. of cases
No. of cases
Statistical Association with Consumption of
Hamburger from Cub Foods; 2000
12
10
8
12
10
8
6
6
4
4
2
2
Nov
Dec
Without PFGE
OR = 1.93; p = 0.31
-NOT significant-
Nov
Dec
With PFGE
OR = 17.1; p = 0.005
-SIGNIFICANT-
Courtesy: John Besser, MN State Health Dept
Criminal investigation:
22
20
Outbreak investigation:
16
No. of cases
Odds of exposure
given illness
18
1 in 200
14
12
10
8
6
4
2
With PFGE
OR = 17.1; p = 0.005
Courtesy: John Besser, Minnesota Dept. of Health
11
What are the Standards of Evidence for
Molecular Epidemiology?
 Strong epidemiologic association between illness in
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
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outbreak-related cases and implicated food
Pathogen isolated from implicated food
Pathogen isolates subtyped validated methods
Pathogen subtyping data corroborate epidemiologic
findings (case patient isolates are
indistinguishable/nearly indistinguishable from
implicated food isolates)
If subtyping data do not corroborate epidemiologic
findings, appropriate and acceptable explanation of
discrepancy
E. coli O157 Outbreak
0609mlEXH-2
Extra band at
approx. 145Kb
No. entries in
The PulseNet
database before
8/15/2006
N= 22,532
EXHX01.0124
157 (0.7%)
EXHX01.0047
594 (2.6%)
For outbreak detection, must use stringent criteria to define subtype of
outbreak strain unless epidemiologic findings indicate the need more inclusive
criteria
Impact of Molecular Epidemiology on Food
Regulation
Incidence of reported cases and outbreaks of listeriosis in the
United States, 1986-2002
Cases per million population
*
Multistate outbreak
10
9
8
7
6
5
4
3
2
1
0
Single state outbreak
'86
PulseNet begins
subtyping Listeria
'89 '90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02
*Data from active surveillance systems,
Some data are preliminary
Year
Impact of Molecular Epidemiology on
Food Regulatory Policy – Recent Example
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Recent outbreaks involving frozen processed
foods that are not fully-cooked but require
microwave cooking or conventional cooking
before consumption.
Largest of these outbreaks spanned a period of
more than one year, and caused illness in more
than 400 people in 41 states.
Vehicle of transmission in this outbreak
frozen pot pies containing poultry meat
Pathogen was Salmonella serotype
Typhimurium or a monophasic variant of the
same serotype .
Two other salmonellosis outbreaks detected
and investigated in Minnesota between 2005
and 2006.
Frozen, pre-browned, single-serving,
microwavable stuffed-chicken entrees were
involved in both outbreaks.
Between 1998 and 2005, Minnesota had
detected two more outbreaks caused by
similar products

Common features of all outbreaks
 Molecular epidemiology enabled public
health authorities to recognize and
promptly investigate the outbreaks
 Posting of the outbreak pattern on the
national PulseNet database served as the
trigger for other states to look for cases in
their own states
 although the packages of the products
implicated in these outbreaks had
cooking instructions which, if strictly
followed, may have inactivated the
Salmonella, the presentation and
packaging of the product may have led
the consumer to assume that they were
fully cooked and, therefore, only needed
to be heated to an appropriate
temperature for consumption.
Remedies: Better labeling and Consumer Education
Public Health Impact of Molecular Epidemiology
1993 Western States E. coli O157 Outbreak
70
Number of Cases
outbreak
detected 1993
726 cases
4 deaths
39 d
60
50
40
30
20
Meat recall
10
0
1
8
15
22
29
36
43
50
57
64
71
Day of Outbreak
2002 Colorado E. coli O157 Outbreak
70
Number of Cases
60
50
outbreak detected 2002
40
CL PHL: 0-7 d
PHL: 4-7 d
30
20
10
0
1
8
18
d
15
22
29
36
43
50
57
64
71
Day of Outbreak
If only 5 cases of E. coli O157:H7 infections were averted by the recall of ground beef
in the Colorado outbreak, the PulseNet system would have recovered all costs for
start up and operation for 5 years. (Elbasha et al. Emerg. Infect. Dis. 6:293-297, 2000)
Largest U.S. Food Recalls (> 750,000 lbs) in which Molecular Epidemiology
Has Played a Prominent Role
Year
Pathogen
Food
Food recalled (lbs)
2007
E. coli O157:H7
Ground beef
21,700,000
2007
E. coli O157:H7
Ground beef
5,700,000
2007
E. coli O157:H7
Ground Beef
800,000
2006
Salmonella Tennessee
Peanut Butter
345,000,000
2005
Salmonella Enteritidis
Almonds
13,000,000
2003
E. coli O157:H7
Blade Tenderized Frozen Steak
750,000
2002
Listeria monocytogenes
Ready-to-eat poultry products
27,400,000
2002
E. coli O157:H7
Ground beef
18,600,000
2000
Listeria monocytogenes
Ready-to-eat poultry products
16,900,000
2000
E. coli O157:H7
Ground beef
1,100,000
1998
Listeria monocytogenes
Hot dogs, deli meats
35,000,000
1998
Salmonella Agona
Toasted oats cereal
3,000,000
1997
E. coli O157:H7
Frozen ground beef
25,000,000
Total = 513,950,000 lbs
other recent notable outbreaks:
2005 - 2007
Salmonella Typhiumrium
Stuffed chicken products, pot pies
Millions
2007
Salmonella Wandsworth
“Veggie bootie” snacks
unknown
E. coli O157:H7
Sprouts, bagged lettuce, fresh spinach
unknown
2000 - 2006
17
Molecular Epidemiology: Further
Improvements Needed
 Reduce delays in pathogen
subtyping and submission of
patterns to national databases
 Implement more discriminating
and epidemiologically relevant
subtyping methods to
complement or replace existing
methods; PFGE will continue to
be used for the next few years
 Reduce/eliminate disparities in
state/local capacities for
molecular epidemiology of
foodborne diseases
 Develop/implement innovative
strategies for timely and routine
gathering of epidemiologic data
independently and in parallel
with molecular subtyping
 “Team Diarrhea” concept works;
Can the “Team Diarrhea”
approach be replicated in other
states, regionally or nationally?
Next Generation Subtyping Methods
for Molecular Epidemiology
 MLVA typing
 Already in use for E. coli O157:H7 subtyping in PulseNet
 SNP (single nucleotide polymorphism) analysis
 Under development and evaluation
 Whole genome sequencing
 On the horizon
Multilocus VNTR Analysis
(MLVA)
 Variable Number Tandem Repeats (VNTRs) in non-coding
sequences
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
Conserved repeat motif found in the genome
 Example: TAACCG
Variable numbers of repeat units among isolates of the same species
 MLVA examines the number of repeats at multiple loci to
determine genetic relationships
Number of
repeats
Isolate A
TAACCG
Isolate B
TAACCGTAACCG
Isolate C
TAACCGTAACCGTAACCGTAACCG
Isolate D
TAACCGTAACCGTAACCGTAACCGTAACCG
1
2
4
5
Variable Number Tandem Repeats
VNTRs
Insertion
Deletion
Multiple Locus VNTR Analysis can be
developed from low-pass sequence data
80
60
40
20
MLVA_composite
100
Clustering of outbreak isolates and some
selected sporadic isolates by MLVA
VNTR_vals
F5733
EXHX01.0224
EXHA26.0536
GA / Stool
1998
H6436
EXHX01.0224
EXHA26.0536
GA / Stool
1998
G5308
EXHX01.0224
EXHA26.0536
ME / Environmental
1992
F6141
EXHX01.0224
EXHA26.0536
GA / Meat
1998
H2306
EXHX01.0224
EXHA26.0536
CT / Stool
1996
01-577
EXHX01.0047
EXHA26.0015
VA / Stool
2001
F7382
EXHX01.0047
EXHA26.0548
NJ / Stool
2000
F8751
EXHX01.1264
EXHA26.0015
CO / Stool
2002
F8768
EXHX01.1264
EXHA26.0015
CO / Ground beef
2002
F7383
EXHX01.0047
EXHA26.0250
NJ / Hamburger
2000
F7384
EXHX01.0047
EXHA26.0250
NJ / Fatal case
2000
C9523
EXHX01.0001
EXHA26.0001
WA / Sporadic
1993
C9581
EXHX01.0001
EXHA26.0001
CA / Outbreak
1993
C9815
EXHX01.0001
EXHA26.0001
AZ / Sporadic
1993
G5244
EXHX01.0001
EXHA26.0001
WA / Sporadic
1993
A7793
EXHX01.0004
EXHA26.0585
OR / Stool
03-1982
F7349
EXHX01.0011
EXHA26.0014
WI / Stool
2000
F7350
EXHX01.0011
EXHA26.0536
WI / Stool
2000
F7351
EXHX01.0011
EXHA26.0014
WI / Taco meat
2000
F7353
EXHX01.0011
EXHA26.0014
WI / Stool
2000
F7354
EXHX01.0011
EXHA26.0598
WI / Stool
2000
F6749
EXHX01.1514
EXHA26.0014
NY / Fatal case
1999
F6750
EXHX01.0283
EXHA26.0014
NY / Sibling
1999
A8184
EXHX01.0029
EXHA26.0715
MI / Stool
06-1982
EDL933
EXHX01.0028
EXHA26.0711
MI / Hamburger
05-1982
.
GA. water park outbreak
.
CT. apple cider outbreak
.
CO. outbreak
NJ .outbreak
.
.
Western
States outbreak
.
.
.
WI. restaurant
.
outbreak
.
.
.
NY
County Fair
.
.
MI. outbreak
Table 1. SNP genotype (SG) and clade for several Escherichia coli O157:H7 outbreak
strains along with hospitalization and hemolytic uremic syndrome (HUS) rates by
outbreak.
No.
Strain
Year
SG
Clade
RIMD-
Outbreak
No. (%)
cases
No. (%) hosp.
HUS
8,355
398 (5)
0 (0)
583
171 (29)
41 (7)
47
33 (70)
0 (0)
204
104 (51)
31 (15)
71
53 (75)
8 (11)
8,598
1,493 (17)
354 (4)
Radish sprouts, Sakai,
1996
1
1
0509952*
Japan
Hamburger, Northwest
93-111
1993
9
2
U.S.
Hamburger,
EDL-933*
1982
12
3
MI and OR
Spinach,
TW14359
2006
30
8
Western U.S.
Lettuce,
TW1458a
2006
30
8
Eastern U.S.
350 EHEC O157 outbreaks in the USA (1982-2002)
Manning, et al. (2008)