Rise

in

seroprevalence

of

herpes

simplex

virus

type

among

highly

sexual

active

homosexual

men

and

an

increasing

association

between

herpes

simplex

virus

type

and

HIV

over

time

-LRB-

-RRB-

Objectives

Herpes

simplex

virus

type

and

type

-LRB-

and

-RRB-

are

both

highly

prevalent

The

rate

of

genital

transmission

is

reportedly

increasing

over

time

is

considered

to

be

an

important

risk

factor

for

HIV

transmission

We

therefore

studied

changes

in

the

and

prevalence

in

a

large

cohort

of

men

who

have

sex

with

men

-LRB-

MSM

-RRB-

over

a

time

period

Methods

Among

HIV-infected

and

HIV-uninfected

MSM

participating

in

the

Amsterdam

Cohort

Studies

seroprevalence

of

and

was

determined

and

prevalence

rate

ratios

-LRB-

PRR

-RRB-

and

confidence

intervals

were

calculated

Results

Between

and

the

and

prevalence

decreased

among

HIV-uninfected

MSM

-LRB-

P

-RRB-

but

remained

stable

among

HIV-infected

MSM

prevalence

increased

among

men

with

at

least

sexual

partners

over

lifetime

-LRB-

PRR

P

-RRB-

The

association

between

HIV

infection

and

became

stronger

over

time

-LRB-

PRR

P

-RRB-

Conclusions

Seroprevalence

of

and

remained

high

among

HIV

infected

MSM

from

to

The

association

of

HIV

and

increased

during

the

HIV

epidemic

Since

the

proportion

of

sexual

transmission

of

is

rising

it

is

important

to

study

the

potential

role

of

as

risk

factor

for

HIV

acquisition

Introduction

Herpes

simplex

virus

type

-LRB-

-RRB-

is

widespread

in

the

general

population

while

herpes

simplex

virus

type

-LRB-

-RRB-

is

more

restricted

to

risk

groups

such

as

men

who

have

sex

with

men

-LRB-

MSM

-RRB-

prevalence

is

around

in

the

general

population

-LSB-

-RSB-

Transmission

usually

occurs

during

childhood

through

oral

contact

and

normally

causes

oropharyngeal

infection

Childhood

transmission

has

declined

in

industrialised

countries

resulting

in

a

lower

prevalence

of

and

leaving

a

larger

population

of

adolescents

at

risk

for

sexual

transmission

of

Earlier

studies

have

reported

sexually

related

risk

factors

for

infection

in

women

-LSB-

-RSB-

Among

those

persons

attending

the

STD

clinic

and

blood

donors

infection

is

associated

with

younger

age

of

first

intercourse

-LSB-

-RSB-

However

less

is

known

about

sexually

related

risk

factors

for

infection

among

MSM

-LSB-

-RSB-

infection

is

usually

transmitted

sexually

and

is

considered

as

a

marker

for

sexual

risk

behaviour

in

populations

-LSB-

-RSB-

In

HIV-infected

MSM

the

prevalence

of

is

as

high

as

-LSB-

-RSB-

while

being

in

the

general

population

-LSB-

-RSB-

is

a

risk

factor

for

HIV

acquisition

especially

in

the

African

setting

-LSB-

-RSB-

and

in

MSM

-LSB-

-RSB-

infected

persons

are

more

susceptible

for

HIV

-LSB-

-RSB-

Moreover

HIV-infected

persons

are

more

likely

to

have

subclinical

reactivation

of

and

are

therefore

more

likely

to

transmit

the

virus

-LSB-

-RSB-

We

previously

demonstrated

a

decline

in

the

prevalence

of

among

MSM

in

Amsterdam

between

which

could

be

explained

by

a

decrease

in

sexual

risk

behaviour

-LSB-

-RSB-

However

in

the

second

half

of

the

their

sexual

risk

behaviour

increased

after

effective

HIV

therapy

became

generally

available

This

may

have

caused

an

increase

in

the

prevalence

of

and

possibly

also

in

since

We

here

studied

the

trend

in

and

prevalence

among

homosexual

men

over

a

time

period

-LRB-

-RRB-

and

whether

risk

factors

for

infection

changed

during

this

period

Methods

Study

population

In

an

open

and

prospective

cohort

study

on

HIV

seroconversion

and

AIDS

among

sexually

active

HIV-negative

and

positive

homosexual

men

was

started

The

Amsterdam

Cohort

Study

-LRB-

ACS

-RRB-

is

still

ongoing

although

entry

criteria

with

respect

to

HIV

status

and

age

have

changed

over

time

From

until

May

both

HIV-positive

and

HIV-negative

men

were

included

From

May

until

February

only

HIV-negative

men

were

allowed

in

the

study

From

February

through

HIV-positive

and

HIV-negative

men

could

enter

the

study

but

since

they

must

be

year

of

age

At

an

ACS

visit

a

standardised

questionnaire

is

administered

regarding

demographics

sexual

behaviour

and

medical

history

for

sexually

transmitted

infections

-LRB-

STI

-RRB-

Blood

samples

are

collected

for

immunologic

and

virologic

testing

and

for

storage

For

this

study

stored

sera

-LRB-

collected

at

the

first

cohort

visit

-RRB-

taken

from

ACS

participants

with

at

least

two

cohort

visits

-LRB-

-LRB-

-RRB-

-RRB-

were

tested

for

and

Laboratory

methods

Sensitive

and

specific

FDA

approved

serological

assay

for

was

used

-LRB-

HerpeSelect

by

FOCUS

technologies

USA

-RRB-

Its

manufacturer

recommends

an

index

value

as

positive

However

there

is

evidence

that

using

this

cut-off

value

in

studies

yields

a

high

rate

of

false

positive

results

in

populations

with

multiple

infections

such

as

those

in

Africa

-LSB-

-RSB-

Raising

the

positive

cut-off

will

increase

the

specificity

-LSB-

-RSB-

Since

the

optimal

cut-off

for

our

target

population

has

not

been

established

samples

with

results

in

the

range

of

and

were

re-tested

with

a

highly

specific

Western

blot

ELISA

and

Western

blot

results

were

concordant

for

samples

The

proportion

of

samples

that

were

positive

with

both

ELISA

and

Western

blot

increased

with

increasing

index

value

-LRB-

Fig.

-RRB-

Based

on

these

results

we

consider

a

cut-off

value

of

as

being

positive

and

an

index

value

is

classified

as

negative

The

index

value

had

concordance

with

Western

blot

results

while

the

index

value

of

showed

concordance

There

were

no

differences

in

Western

blot

outcomes

between

HIV-infected

and

HIV

uninfected

MSM

Fig.

of

serology

by

ELISA

and

Western

blot

samples

were

tested

with

ELISA

and

re-tested

with

the

Western

blot

to

identify

discrepancies

between

the

two

assays

Blood

samples

were

tested

for

and

at

the

Public

Health

Laboratory

of

the

Health

Service

of

Amsterdam

The

Western

blot

was

conducted

at

the

Institute

for

Pathology

and

Medical

Research

-LRB-

ICPMR

-RRB-

in

Sydney

Australia

Blood

samples

are

also

tested

for

HIV

antibodies

by

enzyme

linked

immunosorbent

assay

-LRB-

ELISA

-RRB-

-LRB-

Abbot

Laboratories

North

Chicago

Illinois

USA

Vironostika

Organon

Teknika

Boxtel

the

Netherlands

-RRB-

and

when

positive

are

confirmed

by

Western

blot

Variables

and

statistical

analyses

The

statistical

analyses

were

based

on

the

data

collected

at

entry

of

the

cohort

Variables

used

in

this

study

were

calendar

year

of

ACS

entry

HIV-status

age

nationality

education

age

of

first

homosexual

contact

lifetime

sexual

partners

and

self-reported

history

of

syphilis

and

gonorrhoea

in

the

past

years

Variables

concerning

sexual

practices

included

orogenital

anogenital

and

oroanal

contact

in

the

prior

months

Some

changes

in

the

questions

were

made

in

the

questionnaires

between

and

regarding

the

sexual

practices

Oroanal

contact

was

not

asked

for

from

to

resulting

in

approximately

missing

values

on

this

variable

From

onward

the

most

important

difference

was

that

orogenital

contact

with

ejaculation

was

asked

while

in

the

previous

years

orogenital

contact

in

general

was

asked

The

percentage

of

participants

not

having

orogenital

contact

was

somewhat

higher

for

the

years

and

The

prevalence

of

and

at

the

ACS

entry

was

determined

and

risk

factors

for

and

were

assessed

by

calculating

prevalence

rate

ratios

-LRB-

PRR

-RRB-

with

their

confidence

interval

-LRB-

CI

-RRB-

Odds

ratios

could

not

be

interpret

as

relative

risks

since

the

rare

event

assumption

was

not

reached

Therefore

PRR

's

were

directly

estimated

using

a

modified

Poisson

regression

approach

-LSB-

-RSB-

This

approach

provided

a

correctly

estimated

standard

error

for

the

estimated

relative

risk

Since

inclusion

criteria

with

respect

to

age

and

HIV-status

changed

over

time

all

risk

factor

analyses

were

adjusted

for

age

and

HIV-status

Variables

that

were

statistically

significant

were

included

in

the

multivariate

model

using

a

stepwise

forward

approach

forcing

age

and

HIV-status

in

the

model

We

tested

whether

risk

factors

changed

over

time

by

testing

for

interaction

between

variables

under

investigation

and

calendar

time

in

the

multivariate

model

Calendar

time

therefore

was

categorised

as

Confounding

was

defined

to

be

present

when

the

included

variable

caused

a

change

of

the

prevalence

ratio

by

more

than

Interaction

was

defined

to

be

present

when

the

addition

of

an

interaction

term

improved

the

original

model

and

the

P-value

was

Statistical

significance

was

defined

as

a

P-value

To

reduce

residual

confounding

when

measuring

the

association

between

HSV

and

sexual

practices

three

variables

measuring

sexual

practices

over

the

prior

months

were

included

in

the

model

at

the

same

time

together

with

the

lifetime

sexual

partners

We

modelled

time

trends

in

the

and

prevalence

with

calendar

time

as

a

continuous

variable

using

restricted

cubic

splines

with

four

knots

resulting

in

a

smoothly

varying

curve

For

MSM

the

index

value

was

missing

and

MSM

had

a

missing

index

value

Participants

with

a

missing

index

value

for

both

HSV-infections

were

not

included

in

the

analyses

MSM

with

a

missing

index

value

for

one

HSV-infection

but

with

a

known

HSV

status

of

the

other

HSV-infection

were

included

in

the

analyses

for

the

known

HSV

serostatus

Finally

sensitivity

analyses

for

were

conducted

by

using

the

cut-off

value

of

as

recommended

by

the

manufacturer

and

by

using

the

cut-off

value

of

excluding

those

with

an

index

value

in

the

grey

area

-LRB-

between

and

-RRB-

However

time

trends

in

prevalence

and

risk

factors

found

were

comparable

to

when

was

the

cut-off

value

-LRB-

data

not

shown

-RRB-

Results

General

characteristics

Between

and

a

total

of

MSM

had

at

least

two

visits

General

characteristics

of

the

total

study

group

are

presented

in

Table

Of

the

-LRB-

-RRB-

MSM

were

antibody

positive

while

-LRB-

-RRB-

of

the

men

were

antibody

positive

Of

the

total

group

-LRB-

-RRB-

were

positive

for

both

Participants

were

predominantly

of

Dutch

nationality

-LRB-

-RRB-

and

had

a

median

age

of

years

-LRB-

interquartile

range

-RRB-

Table

and

sexual

characteristics

of

homosexual

men

for

the

total

study

group

and

for

participants

and

participants

separately

between

and

with

the

prevalence

ratios

for

and

with

their

confidence

-LRB-

CI

-RRB-

Overall

-LRB-

CI

-RRB-

Overall

-LRB-

-RRB-

-LRB-

-RRB-

Year

of

study

entry

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Index

value

-LRB-

-RRB-

-LRB-

-RRB-

Nationality

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Sexual

partners

in

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Age

of

first

homosexual

contact

-LRB-

median

IQR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

HSV

-LRB-

-RRB-

-LRB-

-RRB-

HIV

infection

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

of

gonorrhoea

in

the

past

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

of

syphilis

in

the

past

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Orogenital

contact

in

the

past

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Anogenital

contact

in

the

past

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Oroanal

in

the

past

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aSince

ACS

inclusion

criteria

have

changed

over

times

all

analyses

were

adjusted

for

age

-LRB-

per

increase

-RRB-

and

HIV

status

at

cohort

entrybAnalyses

are

also

adjusted

for

the

sexual

techniques

and

number

of

partners

to

exclude

residual

confoundingcPer

year

of

increase

Prevalence

of

and

over

time

There

was

an

overall

decline

in

the

prevalence

of

both

and

between

and

-LRB-

Tables

-RRB-

To

investigate

time

trends

in

HSV

seroprevalence

we

included

an

interaction

term

between

time

and

HIV-status

-LRB-

Fig.

b

-RRB-

Among

HIV-negative

MSM

the

prevalence

decreased

significantly

over

time

P

-LRB-

Fig.

-RRB-

Among

HIV-positive

MSM

the

prevalence

remained

stable

over

time

P

-LRB-

Fig.

-RRB-

Table

model

of

risk

factors

associated

with

P-value

-LRB-

a

-RRB-

infectionYear

of

study

entry

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

HIV

-LRB-

-RRB-

Northern

or

Central

-LRB-

-RRB-

Non

-LRB-

-RRB-

Education

-LRB-

-RRB-

-LRB-

-RRB-

Sexual

partners

in

-LRB-

-RRB-

-LRB-

-RRB-

History

of

Gonorrhoea

in

the

past

-LRB-

-RRB-

History

of

Syphilis

in

the

past

yearsOrogenital

contact

in

the

past

-LRB-

-RRB-

Anogenital

contact

in

the

past

-LRB-

-RRB-

Oroanal

contact

in

the

past

-LRB-

-RRB-

P-value

-LRB-

b

-RRB-

infectionYear

of

study

entry

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

HIV

-LRB-

-RRB-

HSV

-LRB-

-RRB-

History

of

Syphilis

in

the

past

-LRB-

-RRB-

Orogenital

contact

in

the

past

-LRB-

-RRB-

Anogenital

contact

in

the

past

-LRB-

-RRB-

Oroanal

contact

in

the

past

-LRB-

-RRB-

Fig.

-LRB-

a

-RRB-

prevalence

in

the

Amsterdam

Cohort

Study

among

MSM

according

to

the

HIV

status

-LRB-

HIV

positive

HIV

negative

-RRB-

and

the

confidence

interval

-LRB-

b

-RRB-

prevalence

in

the

Amsterdam

Cohort

Study

among

MSM

according

to

the

HIV

status

-LRB-

HIV

positive

HIV

negative

-RRB-

and

the

confidence

interval

The

prevalence

significantly

decreased

among

HIV-negative

men

P

-LRB-

Fig.

-RRB-

Results

from

the

regression

models

showed

that

after

adjustment

for

changes

in

age

nationality

education

and

changes

in

sexual

risk

behaviour

the

decline

in

prevalence

among

HIV

negative

MSM

remained

significant

-LRB-

PRR

adjusted

P

-RRB-

In

contrast

the

prevalence

remained

stable

over

time

for

men

infected

with

HIV

-LRB-

P

-RRB-

Again

this

result

was

observed

after

controlling

for

age

demographic

characteristics

and

sexual

behaviour

Risk

factors

Risk

factors

for

and

infection

adjusted

for

age

and

HIV-status

are

presented

in

Table

In

the

final

model

calendar

year

HIV-status

nationality

and

number

of

lifetime

sexual

partners

remained

independent

predictors

for

infection

-LRB-

Table

-RRB-

For

infection

earlier

year

of

study

entry

positive

HIV-status

co-infection

a

history

of

syphilis

and

sexual

behaviour

remained

independent

predictors

-LRB-

Table

-RRB-

Changing

risk

factors

over

time

Different

interaction

terms

were

included

in

the

model

It

appeared

that

the

effect

of

calendar

year

differed

between

HIV-infected

and

HIV-uninfected

MSM

for

both

and

As

shown

in

Fig.

the

association

between

HIV

and

became

stronger

over

time

This

was

due

to

the

decline

in

prevalence

over

time

among

HIV-negative

MSM

but

not

among

HIV-positive

MSM

-LRB-

Fig.

-RRB-

For

the

association

with

HIV

infection

increased

with

calendar

year

and

was

highest

after

-LRB-

Fig.

-RRB-

For

also

the

effect

of

calendar

time

differed

with

respect

to

nationality

number

of

lifetime

sexual

partners

and

with

co-infection

As

shown

by

the

regression

model

a

decrease

in

infection

over

time

was

observed

only

among

MSM

with

Dutch

or

Northern/Central-European

nationality

The

association

between

and

having

non-European

origin

became

stronger

over

time

The

adjusted

PRR

was

-LRB-

P

-RRB-

before

and

the

adjusted

PRR

became

-LRB-

P

-RRB-

for

the

time

period

after

Also

the

association

between

and

a

higher

number

of

lifetime

sexual

partners

became

stronger

after

Figure

shows

the

prevalence

of

infection

over

time

according

to

the

number

of

sexual

lifetime

partners

A

decrease

in

the

prevalence

was

seen

among

MSM

with

fewer

than

partners

-LRB-

P

-RRB-

while

among

MSM

with

more

than

partners

the

prevalence

increased

between

and

-LRB-

P

-RRB-

Fig.

-LRB-

a

-RRB-

Prevalence

of

and

the

confidence

interval

over

time

among

HIV

negative

MSM

according

to

number

of

lifetime

sexual

partners

partners

-LRB-

b

-RRB-

Prevalence

of

and

the

confidence

interval

over

time

among

HIV

negative

MSM

according

to

number

of

lifetime

sexual

partners

partners

For

the

effect

of

calendar

time

differed

with

respect

to

the

number

of

lifetime

sexual

partners

and

with

co-infection

A

large

number

of

lifetime

partners

also

was

strongly

associated

with

However

a

decrease

in

the

prevalence

was

seen

among

all

categories

of

lifetime

sexual

partners

-LRB-

Fig.

-RRB-

but

this

decrease

was

stronger

for

MSM

with

fewer

than

partners

and

for

those

MSM

with

partners

Discussion

In

the

present

study

we

demonstrated

an

overall

decrease

in

and

prevalence

among

HIV-negative

MSM

but

not

among

HIV-positive

MSM

In

the

period

the

association

between

and

HIV

among

MSM

became

stronger

over

time

and

prevalence

increased

in

highly

sexually

active

HIV-negative

MSM

To

our

knowledge

this

is

the

first

study

based

on

almost

years

of

and

prevalence

data

among

MSM

The

decrease

seen

in

the

seroprevalence

of

and

over

time

could

not

be

explained

by

changes

in

demographic

characteristics

or

sexual

behaviour

The

decrease

in

is

likely

to

reflect

a

decrease

in

childhood

transmission

by

the

oropharyngeal

contact

of

Since

fewer

individuals

are

infected

in

childhood

with

there

is

a

growing

population

of

persons

at

risk

at

the

time

they

become

sexually

active

resulting

in

a

larger

proportion

of

sexual

transmission

of

Two

risk

factors

for

infection

that

could

be

important

in

its

sexual

transmission

were

identified

by

this

study

First

HIV

infection

is

associated

with

seropositivity

Second

the

prevalence

of

was

higher

among

highly

sexual

active

MSM

-LRB-

at

least

lifetime

sexual

partners

-RRB-

An

association

also

shown

earlier

by

others

-LSB-

-RSB-

HIV

infection

in

this

respect

may

reflect

an

epidemiological

marker

for

sexual

risk

behaviour

for

transmission

prevalence

did

not

decrease

in

those

infected

with

HIV

and

we

consider

that

genital

infection

has

a

growing

role

in

the

acquisition

of

HIV

Likewise

prevalence

did

not

decline

over

time

among

those

infected

with

HIV

whereas

a

decline

was

noted

among

HIV

uninfected

MSM

Although

is

sexually

transmitted

we

did

not

find

an

association

between

a

higher

number

of

lifetime

partners

and

infection

was

also

highly

prevalent

among

MSM

with

lifetime

partners

Mainly

between

and

there

were

no

major

differences

in

the

proportion

of

MSM

infected

with

This

suggests

that

is

highly

sexual

transmissible

and

when

having

a

low

number

of

life

time

partner

the

risk

of

receiving

a

infection

is

still

very

high

The

results

of

this

study

show

a

protective

effect

of

orogenital

contact

for

infection

which

might

be

explained

by

the

fact

that

anogenital

contact

is

a

stronger

predictor

for

infection

All

variables

measuring

sexual

practices

are

included

in

the

analyses

at

the

same

time

Since

most

MSM

practised

all

the

techniques

during

the

same

time

period

these

practices

could

not

be

analysed

as

independent

risk

factors

The

stronger

effect

of

anogenital

contact

might

have

overruled

the

effect

of

orogenital

contact

resulting

in

a

protective

effect

of

orogenital

contact

The

overall

prevalence

of

in

this

study

is

similar

to

that

among

MSM

in

San

Francisco

in

but

higher

than

the

prevalence

found

in

more

recent

studies

in

the

US

-LSB-

-RSB-

The

lower

prevalence

in

those

more

recent

studies

probably

reflects

the

decline

in

prevalence

over

time

as

found

in

our

study

Russell

et

al.

found

high

prevalence

rates

of

among

HIV-infected

MSM

in

Australia

-LSB-

-RSB-

The

seroprevalence

was

more

than

twice

as

high

as

among

HIV-uninfected

MSM

there

was

no

significant

difference

in

prevalence

between

HIV-infected

and

HIV-uninfected

MSM

Several

epidemiological

studies

have

described

an

association

between

HIV

and

-LSB-

-RSB-

is

recognised

as

a

risk

factor

for

HIV

acquisition

in

MSM

In

addition

may

up-regulate

HIV

and

increase

local

HIV

replication

on

mucosal

surfaces

leading

to

an

increased

risk

of

HIV

transmission

Our

study

is

the

first

to

show

an

increase

in

the

association

between

and

HIV

since

suggesting

that

may

play

a

growing

role

in

driving

the

HIV

epidemic

in

MSM

If

this

is

the

case

prevention

of

may

well

contribute

to

the

prevention

of

HIV

among

highly

sexually

active

MSM

Although

serological

screening

for

among

MSM

is

still

under

debate

the

increasing

association

between

seropositivity

and

HIV

is

an

argument

in

its

favour

Several

reasons

against

screening

have

been

raised

such

as

the

lack

of

a

reliable

serological

test

We

are

aware

of

the

low

specificity

of

the

various

serological

assays

However

these

serological

assays

might

be

useful

as

a

screening

tool

when

used

with

an

increased

cut-off

value

less

individuals

will

be

classified

as

false

positive

A

second

argument

against

serological

screening

is

that

infection

is

largely

asymptomatic

and

condom

use

appears

only

partially

protective

against

HSV

transmission

These

factors

complicate

the

prevention

of

and

genital

infection

However

it

has

been

shown

that

half

of

the

patients

initially

unaware

of

their

infection

are

able

to

recognise

symptoms

after

being

educated

to

do

so

-LSB-

-RSB-

Also

knowledge

of

the

status

of

a

sexual

partner

has

been

associated

with

a

reduced

risk

of

transmission

-LSB-

-RSB-

Antiviral

drugs

used

as

suppressive

therapy

will

lower

the

frequency

of

recurrences

by

-LSB-

-RSB-

A

combined

approach

of

offering

serological

screening

to

highly

sexual

active

MSM

together

with

encouraging

condom

use

to

reduce

the

risk

of

HSV

transmission

and

using

suppressive

therapy

among

those

with

recurrent

lesions

might

eventually

play

an

effective

part

in

controlling

the

HIV

epidemic

among

MSM

One

limitation

of

our

study

is

its

cross-sectional

design

As

a

consequence

we

can

not

reveal

the

relation

between

HIV

and

HSV

infection

being

unable

to

determine

which

occurred

first

As

both

HIV

and

HSV

are

sexually

transmitted

diseases

their

association

may

well

reflect

shared

sexual

behavioural

practices

leading

to

transmission

as

well

as

a

biological

relation

Longitudinal

studies

in

which

incident

HIV

and

HSV

cases

are

captured

are

therefore

needed

to

give

more

insight

into

the

relationship

between

HIV

and

HSV

as

affected

by

changes

in

sexual

risk

behaviour

The

results

of

this

study

have

two

implications

for

HIV

and

HSV

research

among

highly

sexually

active

MSM

First

it

appears

that

and

HIV

are

now

more

strongly

related

than

in

the

early

days

of

the

HIV

epidemic

As

a

vaccine

against

for

MSM

is

not

yet

available

a

determination

of

the

extent

to

which

the

prevention

of

specially

aimed

for

MSM

at

high

risk

for

HIV

can

contribute

to

controlling

the

HIV

epidemic

is

needed

Second

since

the

extent

of

sexual

transmission

of

is

rising

we

need

to

clarify

its

potential

role

as

a

risk

factor

for

HIV

acquisition

in

longitudinal

studies

O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O B O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O B O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O O B O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O


The Rotterdam Study : objectives and design update The Rotterdam Study is a prospective cohort study ongoing since 1990 in the city of Rotterdam in the Netherlands . The study targets cardiovascular , neurological , ophthalmological and endocrine diseases . As of 2008 about 15,000 subjects aged 45 years or over comprise the Rotterdam Study cohort . The findings of the Rotterdam Study have been presented in some 600 research articles and reports -LRB- see http://www.epib.nl/rotterdamstudy -RRB- . This article gives the reasons for the study and its design . It also presents a summary of the major findings and an update of the objectives and methods . Introduction The Rotterdam Study was designed in the mid-1980s as a response to the demographic changes that were leading to an increase of the proportion of elderly people in most populations -LSB- 1 -RSB- . It was clear that this would produce a strong rise in elderly people living with diseases , as most diseases cluster at the end of life , and that to discover the causes of diseases in the elderly one would have to study risk factors of those diseases -LSB- 2 -RSB- . A major approach to finding causes is the prospective follow-up study , which has proven quite effective in finding causes of heart disease and cancer . Remarkably , in the 1980s there were hardly any follow-up studies focussing on the elderly . The design of the Rotterdam Study The basic design of the study was straight-forward : a prospective cohort study among 7,983 persons living in the well-defined Ommoord district in the city of Rotterdam in the Netherlands -LRB- 78 % of 10,215 invitees -RRB- . They were all 55 years of age or over and the oldest participant at the start was 106 years -LSB- 3 -RSB- . The study started with a pilot phase in the second half of 1989 . From January 1990 onwards participants were recruited for the Rotterdam Study . Figure 1 gives a diagram of the various cycles of the study . Fig. 1Diagram of examination cycles of the Rotterdam Study -LRB- RS -RRB- . RS1 refers to the baseline examination of the original cohort -LRB- pilot phase 07/1989 - 12/1989 ; cohort recruitment 01/1990 - 09/1993 -RRB- . RS2 , RS3 and RS4 refer to re-examination of the original cohort members . RSPlus1 refers to the extension of the cohort with persons in the study district that became 55 years since the start of the study or those of 55 years or over that migrated into the study district . RSPlus2 refers to the re-examination of the extension cohort . RSYoung1 refers to the baseline examination of all persons aged 45 and over living in the study district that had not been examined -LRB- i.e. mainly comprising those aged 45 -- 55 years -RRB- In 1999 , 3,011 participants -LRB- out of 4,472 invitees -RRB- who had become 55 years of age or moved into the study district since the start of the study were added to the cohort . In 2006 , a further extension of the cohort was initiated in which about 6,000 subjects aged 45 -- 54 years , living in the Ommoord district , were invited -LRB- expected number of participants about 4,000 -RRB- . By the end of 2008 , the Rotterdam Study is therefore expected to comprise about 15,000 subjects aged 45 years or over . The participants were all examined in some detail at baseline . They were interviewed at home -LRB- 2 h -RRB- and then had an extensive set of examinations -LRB- a total of 5 h -RRB- in a specially built research facility in the centre of their district . These examinations focussed on possible causes of invalidating diseases in the elderly in a clinically state-of-the-art manner , as far as the circumstances allowed . The emphasis was put on imaging -LRB- of heart , blood vessels , eyes , skeleton and later brain -RRB- and on collecting bodily fluids that enabled further in-depth molecular and genetic analyses . These examinations were repeated every 3 -- 4 years in characteristics that could change over time . And so we had examination cycles from 1990 to 1993 , from 1993 to 1995 , from 1997 to 1999 , from 2000 to 2001 , from 2002 to 2004 , from 2004 to 2005 and from 2006 to 2008 -LRB- Fig. 1 -RRB- . The participants in the Rotterdam Study are followed for a variety of diseases that are frequent in the elderly -LRB- and many are also in the not so elderly -RRB- : coronary heart disease , heart failure and stroke , Parkinson disease , Alzheimer disease and other dementias , depression and anxiety disorders , macular degeneration and glaucoma , diabetes mellitus and osteoporosis . The Rotterdam Study has been approved by the institutional review board -LRB- Medical Ethics Committee -RRB- of the Erasmus Medical Center and by the review board of the Netherlands Ministry of Health , Welfare and Sports . The approval has been renewed every 5 years . Separate approval has been obtained for the introduction of major new elements in the study -LRB- e.g. MRI investigations -RRB- . In the remainder of this article the objectives and major findings will be presented with an update of the methods for cardiovascular diseases , neurological diseases , ophthalmologic diseases , psychiatric diseases , endocrine diseases , as well as for genetic and for pharmaco-epidemiologic studies . Cardiovascular diseases Objectives Research on the epidemiology of cardiovascular diseases focuses on three primary areas of interest : studies on risk factors for atherosclerosis and coronary heart disease , studies on the detection of subjects at high risk of coronary heart disease , and studies on cardiovascular conditions in older age . Two groups of putative risk factors for atherosclerosis and coronary heart disease are included . The first are endocrine factors , including estrogens and androgens , insulin and insulin-like growth hormone I , and thyroid gland and adrenal gland hormones . The second group contains factors involved in haemostasis , inflammation and endothelial function . Research also focuses on genetic factors in these areas in relation to risk of coronary heart disease . The ability of classical cardiovascular risk factors to identify subjects at high risk of coronary heart disease is limited . Risk stratification may be improved when based on the presence of atherosclerosis . To this end , repeated measurements of non-coronary atherosclerosis and measurements of coronary , carotid and aortic arch calcification have been included in the study . Another line of research focuses on cardiovascular diseases in the elderly that are in large part the consequence of ischemic heart disease , like heart failure and atrial fibrillation . An important topic in this area is the early diagnosis of heart failure using echocardiographic assessment of asymptomatic systolic and diastolic dysfunction of the left ventricle . Atrial fibrillation is another major chronic condition frequent at older age . Examination of the determinants and prognosis of atrial fibrillation is part of this research line . Major findings Findings on determinants of atherosclerosis and coronary heart disease include the relation between hypothyroidism and myocardial infarction -LSB- 4 -RSB- and the association of genetic variation in the estrogen receptor alpha gene with risk of myocardial infarction in women -LSB- 5 -RSB- . The study demonstrated that high levels of CRP -LSB- 6 -RSB- and lipoprotein-associated phospholipase A2 activity -LSB- 7 -RSB- were associated with risk of coronary heart disease and stroke . Genetic findings in this area include a positive association of coronary heart disease with genetic variation in the complement factor H gene -LSB- 8 -RSB- but not with variation in the CRP gene -LSB- 6 -RSB- . The study showed that carotid intima-media thickness and plaques measured by ultrasound , the ankle-arm index , and aortic calcification detected by X-ray are independent predictors of risk of coronary heart disease -LSB- 9 -RSB- . The study also showed that vessel wall stiffness , as measured by aortic pulse wave velocity -LRB- PWV -RRB- , improved prediction of cardiovascular disease beyond the classical risk factors -LSB- 10 -RSB- . The Rotterdam Study was the first to publish results from population-based research on the predictive value for coronary heart disease of coronary calcification assessed by electron-beam CT -LSB- 11 -RSB- . The study enabled accurate assessment of the incidence and lifetime risk of heart failure and atrial fibrillation in an elderly population -LSB- 12 , 13 -RSB- . It was shown that atherosclerosis and inflammation are also involved in these conditions -LSB- 14 , 15 -RSB- . Methods update Repeated measures of non-coronary atherosclerosis included carotid intima-media thickness and plaques by ultrasound , the ankle-arm index and aortic calcification by X-ray -LSB- 9 -RSB- . Electron-beam CT and multi-detector CT were used to accurately quantify calcification in the coronary , aortic arch and carotid arteries -LSB- 11 -RSB- . In the second additional cohort of the Rotterdam Study -LRB- RS Young1 , see Fig. 1 -RRB- , the measurement of plaque vulnerability with high-resolution MRI of the carotid arteries will be added . Other outcome measures include electrocardiography , echocardiography and measures of vessel wall stiffness -LRB- PWV and carotid distensibility -RRB- -LSB- 10 -RSB- . Determinants were assessed by physical examinations , collection of blood samples , and by questionnaires and interview . The role of genetic factors is studied using the candidate gene approach and more recently the genome wide association study . Clinical cardiovascular outcomes are collected during our continuous follow-up and include non-fatal myocardial infarction and cardiac death , revascularizations , heart failure and atrial fibrillation . The methods of diagnosis of these outcomes have been described in previous papers -LSB- 6 , 12 , 13 -RSB- . Neurological diseases Objectives Neuroepidemiologic research in the Rotterdam Study focuses on the frequency , etiology and early recognition of the most frequent neurologic diseases in the elderly , including dementia -LRB- in particular Alzheimer disease -RRB- , Parkinson disease and stroke . In neurodegenerative and cerebrovascular disorders clinical symptoms typically become manifest late in the disease course , the occurrence of clinical disease does not reflect the underlying spectrum of disease-related pathology , and most of the clinical syndromes are etiologically heterogeneous . Therefore , an additional research focus is on the causes and consequences of pre-symptomatic brain pathology that can be assessed with non-invasive imaging modalities . Major findings Neurodegenerative and cerebrovascular diseases are highly frequent in the elderly . The prevalence increases from age 55 to 65 years to age 90 years and above from less than 1 % to over 40 % for dementia -LSB- 16 -RSB- , from less than 0.5 % to more than 4 % for Parkinson disease -LSB- 17 -RSB- , and from approximately 1 % to nearly 10 % for stroke . The incidence figures follow this pattern of a strong increase with age over the entire age range , with the age-specific incidence of dementia being identical for men and women at least until the age of 85 -LSB- 18 -RSB- but with men having a higher age-specific incidence of both stroke and Parkinson disease than women throughout the age range -LSB- 19 , 20 -RSB- . Vascular pathology and vascular risk factors are associated with worse cognitive performance -LSB- 21 -RSB- , which also translates in people with vascular pathology or risk factors for vascular disease having an increased risk of dementia , including Alzheimer disease -LSB- 22 -RSB- . Moreover , several life style factors are associated with the risk of dementia and Alzheimer disease -LSB- 23 -- 25 -RSB- , suggesting that onset of dementia may at least partly be delayed or prevented . Commonly used drugs may have a role in this -LSB- 26 -RSB- . The classical risk factors for stroke also predict risk of stroke in the Rotterdam Study -LSB- 27 -RSB- . More recently identified risk factors , including inflammatory markers , may be etiologically relevant but thus far add little to the identification of people at risk -LSB- 28 -RSB- . Possibly underlying this is that a large amount of stroke goes clinically undetected -LSB- 29 -RSB- . Nearly 20 % of elderly people have at least one silent brain infarct , and thereby a nearly fourfold increased risk of clinical stroke , a more than doubled risk of dementia including Alzheimer disease , and an increased risk of depression -LSB- 29 -RSB- . Neuroimaging reveals that brain pathology is widespread -LSB- 30 -RSB- and can go clinically undetected for a long time . In addition to the silent infarcts , many apparently healthy elderly have ischemic changes in their cerebral white matter that are associated with an increased risk of dementia , stroke and depression -LSB- 31 -RSB- . Also brain atrophy , especially of the hippocampus , is already present years before onset of even the earliest sign of cognitive impairment or subjective complaints -LSB- 32 -RSB- . This emphasizes the need to shift the attention in etiologic research of neurodegenerative and cerebrovascular disease to the causes of pre-symptomatic and underlying brain changes . Methods update Assessment of dementia and Alzheimer disease In the baseline and follow-up examinations participants undergo an initial screen for dementia with the Mini Mental State Examination -LRB- MMSE -RRB- and the Geriatric Mental Schedule -LRB- GMS -RRB- , followed by an examination and informant interview with the Cambridge Examination for Mental Disorders of the Elderly -LRB- CAMDEX -RRB- in screenpositives -LRB- MMSE < 26 or GMS > 0 -RRB- , and subsequent neurological , neuropsychological and neuroimaging examinations -LSB- 16 , 18 -RSB- . Of subjects who can not be reexamined in person , information is obtained from the GPs and the regional institute for outpatient mental health care . A consensus panel makes the final diagnoses in accordance with standard criteria -LRB- DSM-III-R criteria ; NINCDS-ADRDA ; NINDS-AIREN -RRB- . Assessment of parkinsonism and Parkinson disease Participants are screened in the baseline and follow-up examinations for cardinal signs of parkinsonism -LRB- resting tremor , rigidity , bradykinesia or impaired postural reflexes -RRB- . Persons with at least one sign present are examined with the Unified Parkinson Disease Rating Scale and a further neurologic exam . Parkinson disease is diagnosed if two or more cardinal signs are present in a subject not taking antiparkinsonian drugs , or if at least one sign has improved through medication , and when all causes of secondary parkinsonism -LRB- dementia , use of neuroleptics , cerebrovascular disease , multiple system atrophy or progressive supranuclear palsy -RRB- can be excluded -LSB- 17 , 19 -RSB- . Assessment of stroke and stroke subtypes History of stroke at baseline was assessed through interview and verified in medical records . Putative incident strokes get identified through the linkage of the study database with files from general practitioners , the municipality , and nursing home physicians ' files , after which additional information -LRB- including brain imaging -RRB- is collected from hospital records . A panel discusses all potential strokes and subclassifies strokes into ischemic , hemorrhagic or unspecified -LSB- 20 , 28 -RSB- . Assessment of cognitive function Global cognitive function is measured through the Mini Mental State Examination -LRB- MMSE -RRB- in all surveys . From the third survey onwards we added a 30 min test battery that was designed to assess executive function and memory function , and which includes a Stroop test , a Letter Digit Substitution Task , a Word Fluency Test , and a 15 words Word List Learning test . Rotterdam Scan Study : brain imaging within the Rotterdam Study In 1991 , a random sample of 111 participants underwent axial T2-weighted magnetic resonance -LRB- MR -RRB- imaging to assess presence and severity of white matter lesions -LSB- 33 -RSB- . In 1995 , a random sample of 563 non-demented participants underwent brain MR imaging in the context of the Rotterdam Scan Study . The scanning protocol included series of axial proton-density , T2-weighted and T1-weighted images , as well as a high-resolution 3D-HASTE sequence -LSB- 31 -RSB- . From August 2005 onwards , a dedicated 1.5 Tesla scanner is operational in the research centre of the Rotterdam Study , and brain imaging is performed in all study participants without contra-indications . The scanning protocol includes 4 high-resolution axial sequences -LRB- 3D T1-weighted ; 2D PD-weighted ; 2D FLAIR ; and 3D T2 * GRE -RRB- , 2D phase-contrast imaging , and diffusion tensor imaging -LRB- DTI -RRB- . Psychiatric diseases Objectives The aim of the psychiatric research in the Rotterdam Study is to investigate the determinants , correlates and consequences of common psychiatric problems in the elderly . The focus has been on depressive disorders but anxiety disorders , sleep disturbances and complicated grief are also being studied . Major findings The one-month prevalence of both major and minor depression at baseline was 1.5 % , the prevalence of anxiety disorder was 8.2 % . We found evidence for the vascular depression hypothesis . More severe coronary and extra-coronary atherosclerosis were associated with a higher prevalence of depression , as were cerebral haemodynamic changes -LSB- 34 , 35 -RSB- . However , we could not rule out that earlier depressive episodes may have contributed to the development of atherosclerosis . Moreover , our data did not support a specific symptom profile of vascular depression as previously defined -LSB- 36 -RSB- . Most of our studies of nutritional and metabolic factors provided no support for an etiological role in depression . Folate deficiency , homocysteine , fatty acid ratio -LRB- omega-6 / omega-3 -RRB- and vitamin E had little or no effect on the risk of depression after control for health and health related behaviour -LSB- 37 -- 39 -RSB- . In contrast , low levels of vitamin B12 and high levels of inflammatory protein IL-6 convey a strong risk for depression , although the latter effect may be confined to high-risk groups -LSB- 40 -RSB- . Several SNPs have been investigated , mostly with negative results . However , we found that an ER-alpha polymorphism had a substantial effect on anxiety but not on depression in women -LSB- 41 -RSB- . Methods update In the first years of the Rotterdam Study psychiatric data collection was very limited . However , from the third examination onwards , which began in 1997 -LRB- see Fig. 1 -RRB- , depressive symptoms and disorders are have been ascertained in all participants . The 4,603 participants with valid depression scores in this examination thus constitute the baseline population for longitudinal studies of depression . Because assessment of anxiety disorders , sleeping disturbances and complicated grief were added in the fourth examination , they can currently be studied only cross-sectionally . Assessment of determinants Psychiatric research in the Rotterdam Study focuses on biological risk factors . The vascular depression hypothesis was tested with different measures of atherosclerosis , arterial stiffness and cerebral blood flow -LSB- 34 , 35 -RSB- . We also examined whether blood levels of vitamins and fatty acids , immune parameters , and markers of folate metabolism increased the likelihood of depression -LSB- 37 -- 40 -RSB- . In one ongoing project , diurnal patterns of cortisol secretion are related to psychiatric and other disorders . Studies of genetic polymorphisms and brain morphology are underway . Current data collection includes a dexamethasone suppression test to measure hypothalamic-pituitary-adrenal axis activity in all participants , which is unique in a population-based study . Assessment of outcomes Depression is assessed using a two step procedure . First , participants completed the Center for Epidemiologic Studies Depression scale -LRB- CES-D -RRB- during the home interview to measure self-reported depressive symptoms -LSB- 42 -RSB- . As a second step , screen positive subjects , who scored above a well-established CES-D cut-off of 20 points , have a psychiatric interview using the Present State Examination -LRB- PSE/SCAN -RRB- -LSB- 43 -RSB- . This semi-structured interview is conducted by a clinician and yields DSM-IV diagnoses including major and minor depression and dysthymia . The following anxiety disorders are assessed with a slightly adapted Munich version of the Composite International Diagnostic Interview : generalized anxiety disorder , specific and social phobia , agoraphobia without panic disorder , and panic disorder -LSB- 44 -RSB- . Sleep quality and disturbance is measured with the Pittsburgh Sleep Quality Index . In addition , sleep duration and fragmentation are assessed with actigraphy , a method that infers wakefulness and sleep from the presence or absence of limb movement -LSB- 45 -RSB- . The Inventory of Complicated Grief is used to identify traumatic grief -LSB- 46 -RSB- . This is a condition distinct from normal grief and bereavement-related depression , characterized by symptoms like disbelief about the death and searching for the deceased . Ophthalmologic diseases Objectives The primary objectives of the ophthalmological part of the Rotterdam Study are to study frequency and risk factors of common and invalidating eye diseases with emphasis on age-related macular degeneration -LRB- AMD -RRB- and primary open angle glaucoma -LRB- POAG -RRB- . Another line of research focuses on the associations between retinal vessel abnormalities and cardiovascular and neurologic diseases . Major findings AMD was the main cause of blindness in the high age-group -LSB- 47 -RSB- . Major risk factors that were found for AMD are smoking , atherosclerosis , hyperopia -LSB- 48 -- 50 -RSB- . The APO-E ε-4 allele showed an inverse association with AMD -LSB- 51 -RSB- . First-degree relatives of patients with late ARM developed ARM at an increased rate at a relatively young age -LSB- 52 -RSB- . The heterogeneity of genetic risk among AMD families is considerable , and the proportion of high-risk families is relatively small -LSB- 53 -RSB- . The anticipated protective effect of cholesterol-lowering drugs on AMD could not be substantiated -LSB- 54 -RSB- . A protective effect on AMD was demonstrated in participants using a diet rich of antioxidants and zinc intake in our study population . Support for a multifactorial origin of AMD was found in the cumulative effects of aberrant CFH and CRP genes , inflammations and smoking as genetic and environmental stimulators of the complement cascade -LSB- 55 -RSB- . The prevalence estimates of POAG in Rotterdam was highly dependent on the applied criteria -LSB- 56 -RSB- . That was a major reason for proposing a classification system without final , subjective adjudication -LSB- 57 -RSB- . Systemic blood pressure and hypertension were associated with elevated intraocular pressure but not with prevalent POAG -LSB- 58 -RSB- . Relatives of patients with POAG have a strongly increased risk of glaucoma . Enlarged cup-disc ratio was the earliest and most prominent feature of familial aggregation -LSB- 59 -RSB- . The incidence of OAG rises significantly with age . Most of the patients with incident OAG were unaware of having OAG -LSB- 60 -RSB- . The incidence of visual field loss rises fivefold between 55 and 80 years to 20/1 ,000 person years in the general population -LSB- 61 -RSB- . Glaucoma is the main cause for visual field loss , followed by stroke -LSB- 62 -RSB- . Larger retinal venular diameters are associated with generalized atherosclerosis , inflammation and cholesterol levels . Retinal venular diameters are variable and may play their own independent role in predicting cardiovascular disorders -LSB- 62 -RSB- . Dilated retinal venules at baseline were predictive for stroke , cerebral infarction , dementia , white brain matter lesions , impaired glucose tolerance , diabetes mellitus and mortality -LSB- 63 , 64 -RSB- . In systemic hypertension , the arteriolar diameters are narrow and arteriolar and venular narrowing precedes the onset of hypertension -LSB- 65 -RSB- . Methods update Repeated ophthalmic measurements include best corrected ETDRS visual acuity , refractive error , Goldmann applanation tonometry , keratometry , slitlamp examination of the anterior segment and visual field testing . In pharmacological mydriasis we make colour photographs of the lens , 35 degree photography of the macular area , and simultaneous stereoscopic imaging of the optic disc and macular area . Digital photography of the macular area and optic disc are added since the fourth follow-up examination . Scanning laser ophthalmoscopy -LRB- HRT II -RRB- measurements of the optic disc , macular pigment density measurements and optical coherence tomography of the macular area and optic disc were added during follow-up . For the assessment of AMD we use stereoscopic 35 ° macular images centred on the fovea . The images are graded according to the International Classification and Grading System for AMD -LSB- 66 -RSB- . The procedure for assessing POAG includes Goldmann applanation tonometry , visual field screening , ophthalmoscopy and stereoscopic fundus photography -LSB- 57 -RSB- . Assessment of retinal vascular diameters is done by analyses of digitized fundus transparencies with a semi automated system -LSB- 58 , 67 -RSB- . Genetic studies Objectives The first objective of the laboratory team is to collect , store and manage the biological tissues sampled in the Rotterdam Study . The second objective of the group concerns genotyping and assessment of biomarkers . Major findings Both from biomarker studies as well as molecular genetic studies important findings have emerged . For example , among the biomarker analyses the study documenting the relationship between homocysteine and osteoporosis has been novel -LSB- 68 -RSB- and since widely replicated . Several candidate gene studies have also yielded major new insights coming from both exploratory studies as well as from collaborative replication efforts across all disease-oriented research lines in the Rotterdam Study . A unique feature of the Rotterdam Study is exploited by studying the relationship between pleiotropic gene variants and not one but multiple diseases and disease-related endpoints . For example , the studies on the promoter region of the IGF-1 gene revealed a series of consistent associations ranging from birth weight to diabetes -LSB- 69 -RSB- , while other consistent associations involve the estrogen receptor alpha -LRB- ESR1 -RRB- gene in relation to osteoporosis -LSB- 70 -RSB- , osteoarthritis , height , myocardial infarction -LSB- 5 -RSB- , age-at-menopause , and depression . The Rotterdam Study is involved as a major collaborative centre for studies for novel genes coming from genome-wide analysis -LRB- GWA -RRB- studies , including CFH in age-related macula degeneration -LSB- 71 -RSB- , NOS1AP in QT interval -LSB- 72 -RSB- , and several SNPs involved in height , type 2 diabetes , and breast cancer . Finally , Rotterdam Study investigators play a leading role in the emerging large global consortia focussing on the contribution of complex disease gene variants by prospective meta-analyses across many epidemiological cohorts , such as for osteoporosis in the GENOMOS Study and EUROSPAN . Methods update Data collection , storage and management At each examination , blood , serum , plasma -LRB- citrate , heparine and EDTA based -RRB- , sputum , and urine are collected . Fasting blood samples are collected along with challenged samples as part of a glucose tolerance test . Sputum is collected before and after a dexamethasone-suppression test . Sputum is frozen at − 196 °C before and after the challenge and stored at − 80 °C . To obtain serum and plasma , tubes are centrifuged according to a protocol standardising time and conditions from the drawing of blood to centrifugation . All samples are snap frozen at − 196 °C using liquid nitrogen and stored at − 80 °C . RNA is isolated from blood within 5 h after sampling and stored at − 20 °C . DNA is isolated from blood and extraction has been recently automated using a Hamilton STAR pipetting platform and AGOWA magnetic bead technology . DNA sample storage is in Matrix 2D-barcode tubes in 96-well format . Overnight urine samples are collected , frozen at − 196 °C and stored at − 80 °C . For data management , an in-house customized laboratory management system has been developed . Sample retrieval will be automated with an in-house customized laboratory track and trace system . Blood assessments For all participants , serum cholesterol , HDL , LDL , triglycerides , glucose and glucose levels are assessed . In urine , micro albumin and creatinine are determined in all participants . There have been a large number of specific blood/serum/plasma-based biomarker assessments including steroids -LRB- e.g. estrogens , androgens , vitamin D , cortisol -RRB- , interleukins , CRP , IGF1 , insulin , iron-parameters -LRB- iron , ferritin and transferrin saturation -RRB- , fibrinogen , homocysteine , folic acid , riboflavine , pyridoxine , SAM/SAH ratio , cobalamine , Lp-PLA2 , Fas/Fas-L , vitamins , a-beta42 / 40 and thyroid hormones -LRB- TSH -RRB- . Genotyping facilities Affiliated laboratory facilities include a medium/high-throughput platform for candidate gene studies and genome wide association -LRB- GWA -RRB- analyses . The facilities use high-end automated machinery including a Caliper/Zymark ALH 3000 pipetting robot -LRB- including a TwisterII , and integrated plate sealer , plate reader -LRB- OD 260/280 -RRB- , a Tecan EVO 150 Freedom pipetting robot , a Deerac Equator NS808 nanoliter liquid dispenser , 15 electronic PCR machines -LRB- ABI 9700 , 2 × 384 -RRB- , an ABI7900HT Taqman machine -LRB- running 1 ng gDNA in 2 μl reactions -RRB- , a WAVE 3500HT dHPLC , and two ABI3100 sequencing machines . DNA sample handling is centred on 384-well plates . Candidate gene studies are done mostly using Taqman genotyping with throughputs at 30,000 genotypes per day . Continuous efforts are focussed on reducing the required amount of genomic DNA , which is now down to 1 ng per genotype . Genome-wide genotyping studies are based on 500/1 ,000 K Affymetrix arrays and 317 , 550 and 1,000 K Illumina arrays with throughputs at 100 -- 300 arrays per week . The genotyping facility has been partly sponsored by NWO investment grants -LRB- 911-03-012 ; 175.010.2005.011 -RRB- , is part of the ErasmusMC Biomics core facility , and serves as knowledge centre for polymorphism analysis attracting national and international interested parties , both academic and industrial . Candidate gene studies We have genotyped > 200 polymorphisms across the complete cohort and conducted a large number of candidate gene studies in the Rotterdam Study . These mostly concern individual potentially functional single nucleotide polymorphisms -LRB- SNPs -RRB- per gene , but sometimes also haplotype tagging SNPs -LRB- e.g. ESR1 , ESR2 , HSD11B1 , fibrinogen -RRB- , and also high-density SNP screening -LRB- e.g. the vitamin D receptor gene -LSB- 73 -RSB- -RRB- . The candidate genes studied include the apolipoprotein E gene -LRB- APOE -RRB- , the angiotensin-converting enzyme -LRB- ACE -RRB- , the gene encoding angiotensinogen -LRB- AGT -RRB- , angiotensin II type 1 receptor -LRB- AT1R -RRB- gene , G protein beta3 -LRB- GNB3 -RRB- , adducine gene , Cholesteryl Ester Transfer Protein -LRB- CETP -RRB- , Hepatic Lipase , Phosphodiesterase 4D -LRB- PDE4D -RRB- , ALOX5AP encoding 5-lipoxygenase activating protein , a polymorphism in the regulatory region of the Insulin-like Growth Factor 1 -LRB- IGF-1 -RRB- gene , the hemochromatosis -LRB- HFE -RRB- gene , Complement factor H gene -LRB- CFH -RRB- , and several polymorphisms in genes from the estrogen - , thyroid - , cortisol - , vitamin D - , IGF - , and Wnt-signalling pathways , the homocysteine pathway , and several matrix molecules . Genome wide association -LRB- GWA -RRB- studies A recently developed approach to identify robust genetic factors for complex disease is the Genome Wide Association -LRB- GWA -RRB- analysis . This is based on genotyping epidemiological cohorts with ultra-high density SNP arrays with up to 1 million SNPs . The method has already been shown to successfully identify genetic factors for several traits and diseases including age-related macula degeneration , inflammatory bowel disease , body mass index , type 2 diabetes and breast cancer . Through a large grant from the Dutch research organization NWO one of the world 's largest GWA studies is conducted involving 10,000 DNA samples from the Rotterdam Study . This GWA is based on the Illumina 550 K arrays and finished by the end of 2007 . The GWA data will be useful for all research lines within the Rotterdam Study , and will also serve as a control GWA dataset for other research centres in and outside the Netherlands for both SNP frequencies as well as copy number variations -LRB- CNVs -RRB- . In addition to this , a pilot study has been performed on 500 women from the Rotterdam Study using the Affymetrix 500 K platform allowing direct platform comparisons and merging of data to further increase genome coverage . In addition , our group has been active in developing new software for GWA analyses -LSB- 74 -RSB- . Pharmaco-epidemiologic studies Objectives A major objective of the pharmaco-epidemiologic studies is to investigate the role of drugs as determinants of disease in the Rotterdam Study . This includes studying efficacy and effectiveness of drugs , as well as adverse reactions to drugs . Major findings Important findings have been published on pharmaco-epidemiological topics concerning the main outcomes in the Rotterdam Study . Studies about the association between dementia and antihypertensive drugs -LSB- 75 -RSB- and NSAIDs -LSB- 26 -RSB- have strongly suggested a protective effect of both groups of drugs . Several studies have been performed on cardiovascular topics -LSB- 76 -- 78 -RSB- . In one of these studies , NSAIDs were associated with an increased risk of heart failure -LSB- 79 -RSB- . In line with the suspicion that QTc-prolonging drugs may cause sudden cardiac death , it was demonstrated in the Rotterdam Study that a prolonged QTc is indeed an important risk factor -LSB- 77 -RSB- . Furthermore , in one study it was demonstrated that high-dose corticosteroids increase the risk of atrial fibrillation -LSB- 78 -RSB- . In the important area of locomotor diseases , studies have demonstrated that thiazide diuretics protect against hip fracture -LSB- 79 -RSB- and that statins reduce the risk of vertebral fracture -LSB- 80 -RSB- . On the other hand , the risk that long-term use of certain NSAIDs may aggravate signs of osteoarthritis has been emphasized -LSB- 81 -RSB- . In the area of ophthalmological diseases , a protective effect of cholesterol-lowering agents on macular degeneration has been suggested -LSB- 82 -RSB- . In other areas , such as pharmacogenetics and other causes of interactions between drugs , several important findings have been published -LSB- 83 -- 91 -RSB- . Methods update For several reasons , a drug is a highly attractive determinant in clinical epidemiology . First , drugs are probably the most important therapeutic intervention in health care . Despite rigorous clinical research before registration , many important effects of drugs are discovered after marketing . Second , all marketed drugs have proven biological activity , meaning that it concerns a determinant which really matters . Third , and as a consequence of the availability of complete medication histories in Dutch health care , the role of drug exposure can be assessed in a detailed way . In the Rotterdam Study , there is an almost complete coverage of the population as of 1 January , 1991 thanks to the fact that all pharmacies which serve the Ommoord region are on one computer network . To date , almost 3 million prescriptions have been delivered to the population of the Rotterdam Study and of each prescription , details are available about the product name and contents , ATC-code , dosage and duration of drug therapy . Drugs are a group of determinants , which can be studied in association with a large variety of diseases . In the Rotterdam Study , there is a strong interest in the association between drugs and the cardiovascular , neurological , endocrinological and ophthalmological diseases , which have been the main topics since starting . However , there is also important information about the association with psychiatric diseases , cancer , and chronic obstructive pulmonary disease . Moreover , important information about secondary outcomes , such as drug blood levels , other laboratory information , and information about hospital discharge diagnoses , is gathered on a continuous basis to facilitate pharmaco-epidemiological studies . Management of the Rotterdam Study The Rotterdam Study is directed by a Management Team comprising Jan Heeringa , MD , study coordinator , Eric Neeleman , head IT , Frank van Rooij , head data-management , and the scientific principal investigators Albert Hofman -LRB- PI Rotterdam Study , chairman -RRB- , Monique Breteler -LRB- PI Neurological diseases -RRB- , Cornelia van Duijn -LRB- PI Genetic studies -RRB- , Gabriel Krestin -LRB- PI Radiology -RRB- , Huibert Pols -LRB- PI Endocrinology -RRB- , Bruno Stricker -LRB- PI Pharmaco-epidemiology -RRB- , Henning Tiemeier -LRB- PI Psychiatric diseases -RRB- , André Uitterlinden -LRB- PI Genome wide analysis -RRB- , Johannes Vingerling -LRB- PI Ophthalmologic diseases -RRB- and Jacqueline Witteman -LRB- PI Cardiovascular diseases -RRB- .

O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O B I O B I O O O O O O O O O B I O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O

alzheimer disease
cohort study
dementia
diabetes
genome wide analysis
glaucoma
heart failure
macular degeneration
myocardial infarction
osteoporosis
parkinson disease
stroke