IHRC, Inc. Supporting public health worldwide

Bala Swaminathan, Ph.D.Vice-President, IHRC, Inc.

Atlanta, GA, USA

EpidemiologyEpidemiology: 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 BiologyMolecular 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”SubtypingMolecular subtyping

Synergy between two seemingly disparate scientific disciplines

Example of Molecular Subtyping

1135 Kb

76.8 Kb

33.3 Kb

216.9 Kb

452.7 Kb

Fragment

Size

PulseNet Universal Reference Standard

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

13

13

33

Jan Feb Mar Apr May Jun Jul Aug Sep

No.

of

cases

Month

2

4

6

8

10

12

14

16

18

20

22

Oct Nov Dec

E. Coli O157 Outbreak – Minnesota, 2000

Courtesy: John Besser, MN State Health Dept

Statistical Association with Consumption of Hamburger from Cub Foods; 2000

Nov DecN

o.

of

case

s

2

4

6

8

10

12

14

16

18

20

22

With PFGEOR = 17.1; p =

0.005-SIGNIFICANT-

No.

of

case

s

2

4

6

8

10

12

14

16

18

20

22

Nov Dec

Without PFGEOR = 1.93; p = 0.31-NOT significant-

Courtesy: John Besser, MN State Health Dept

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

With PFGEOR = 17.1; p = 0.005

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

22

No

. of

ca

se

s

2

4

6

8

10

12

14

16

18

20

With PFGEOR = 17.1; p = 0.005OR = 17.1; p = 0.005

Criminal investigation:

Outbreak investigation:

1 in 200

Odds of exposure given illness

11Courtesy: John Besser, Minnesota Dept. of Health

What are the Standards of Evidence for Molecular Epidemiology?

Strong epidemiologic association between illness in outbreak-related cases and implicated food

Pathogen isolated from implicated foodPathogen isolates subtyped validated methodsPathogen 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

EXHX01.0047

EXHX01.0124

Extra band at approx. 145Kb

No. entries in The PulseNet database before8/15/2006N= 22,532

157 (0.7%)

594 (2.6%)

E. coli E. coli O157 OutbreakO157 Outbreak 0609mlEXH-2

For outbreak detection, must use stringent criteria to define subtype of outbreak strain unless epidemiologic findings indicate the need more inclusive criteria

0123456789

10

'86 '89 '90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02

Year

Cas

es p

er m

illio

n po

pula

tion

Impact of Molecular Epidemiology on Food Regulation

Incidence of reported cases and outbreaks of listeriosis in the

United States, 1986-2002*

*Data from active surveillance systems, Some data are preliminary

PulseNet beginssubtyping Listeria

Multistate outbreak

Single state outbreak

Impact of Molecular Epidemiology on Food Regulatory Policy – Recent Example

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

0

10

20

30

40

50

60

70

1 8 15 22 29 36 43 50 57 64 71

Day of Outbreak

Nu

mb

er o

f C

ases

outbreak detected 1993

Meat recall

1993 Western States E. coli O157 Outbreak

726 cases4 deaths

39 d

2002 Colorado E. coli O157 Outbreak

0

10

20

30

40

50

60

70

1 8 15 22 29 36 43 50 57 64 71

Day of Outbreak

Nu

mb

er o

f C

as

es

outbreak detected 2002

18 d

CL PHL: 0-7 dPHL: 4-7 d

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

2005 - 2007 Salmonella Typhiumrium

Stuffed chicken products, pot pies Millions

2007 Salmonella Wandsworth

“Veggie bootie” snacks unknown

2000 - 2006 E. coli O157:H7 Sprouts, bagged lettuce, fresh spinach unknown

Total = 513,950,000 lbsother recent notable outbreaks:

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 EpidemiologyMLVA typing

Already in use for E. coli O157:H7 subtyping in PulseNet

SNP (single nucleotide polymorphism) analysisUnder development and evaluation

Whole genome sequencingOn the horizon

Multilocus VNTR Analysis(MLVA)

Variable Number Tandem Repeats (VNTRs) in non-coding sequences Conserved repeat motif found in the genome

Example: TAACCG Variable numbers of repeat units among isolates of the same

speciesMLVA examines the number of repeats at multiple loci to

determine genetic relationships

TAACCG

TAACCGTAACCG

TAACCGTAACCGTAACCGTAACCGTAACCG

TAACCGTAACCGTAACCGTAACCG

Isolate AIsolate BIsolate CIsolate D

1245

Number of repeats

InsertionInsertion

DeletionDeletion

VNTR_vals

MLVA_composite

100

80604020

F5733

H6436

G5308

F6141

H2306

01-577

F7382

F8751

F8768

F7383

F7384

C9523

C9581

C9815

G5244

A7793

F7349

F7350

F7351

F7353

F7354

F6749

F6750

A8184

EDL933

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0047

EXHX01.0047

EXHX01.1264

EXHX01.1264

EXHX01.0047

EXHX01.0047

EXHX01.0001

EXHX01.0001

EXHX01.0001

EXHX01.0001

EXHX01.0004

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.1514

EXHX01.0283

EXHX01.0029

EXHX01.0028

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0015

EXHA26.0548

EXHA26.0015

EXHA26.0015

EXHA26.0250

EXHA26.0250

EXHA26.0001

EXHA26.0001

EXHA26.0001

EXHA26.0001

EXHA26.0585

EXHA26.0014

EXHA26.0536

EXHA26.0014

EXHA26.0014

EXHA26.0598

EXHA26.0014

EXHA26.0014

EXHA26.0715

EXHA26.0711

GA / Stool

GA / Stool

ME / Environmental

GA / Meat

CT / Stool

VA / Stool

NJ / Stool

CO / Stool

CO / Ground beef

NJ / Hamburger

NJ / Fatal case

WA / Sporadic

CA / Outbreak

AZ / Sporadic

WA / Sporadic

OR / Stool

WI / Stool

WI / Stool

WI / Taco meat

WI / Stool

WI / Stool

NY / Fatal case

NY / Sibling

MI / Stool

MI / Hamburger

1998

1998

1992

1998

1996

2001

2000

2002

2002

2000

2000

1993

1993

1993

1993

03-1982

2000

2000

2000

2000

2000

1999

1999

06-1982

05-1982

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.

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.

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.

.

.

.

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.

.

.

.

.

.

.

.

GA water park outbreak

CT apple cider outbreak

CO outbreak

Western States outbreak

WI restaurant outbreak

NY County Fair

MI outbreak

NJ outbreak

Clustering of outbreak isolates and some selected sporadic isolates by MLVA

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.

Strain Year SG Clade Outbreak

No.

cases

No. (%)

hosp.

No. (%)

HUS

RIMD-

0509952*1996 1 1

Radish sprouts,

Sakai, Japan8,355 398 (5) 0 (0)

93-111 1993 9 2Hamburger,

Northwest U.S.583 171 (29) 41 (7)

EDL-933* 1982 12 3Hamburger,

MI and OR47 33 (70) 0 (0)

TW14359 2006 30 8Spinach,

Western U.S.204 104 (51) 31 (15)

TW1458a 2006 30 8Lettuce,

Eastern U.S.71 53 (75) 8 (11)

350 EHEC O157 outbreaks in the USA (1982-2002) 8,598 1,493 (17) 354 (4)

Manning, et al. (2008)