CO

MP

AR

ISO

N O

F C

ULT

UR

E F

OR

CYT

OP

ATH

IC E

FFE

CT

TO S

HE

LL

VIA

L A

ND

FLU

OR

ES

CE

NT

AN

TIB

OD

Y S

TAIN

FO

R V

IRA

L D

IAG

NO

SIS

K. W

ilkin

s,H

. McI

ntire

, J.

Col

eman

, Dep

artm

ent o

f Pat

holo

gy, U

nive

rsity

of M

issi

ssip

pi M

edic

al C

ente

r, Ja

ckso

n, M

S.

Intr

oduc

tion:

Our

cla

ssic

vira

l cul

ture

for d

iagn

osis

has

con

sist

ed o

f ino

cula

ting

seve

ral m

onol

ayer

s of

ac

tivel

y gr

owin

g hu

man

or a

nim

al c

ells

, inc

ubat

ing

and

then

obs

ervi

ng th

e ce

lls fo

r the

cy

topa

thic

effe

ct (C

PE).

Cul

ture

s ha

d to

be

mai

ntai

ned,

typi

cally

, for

one

to tw

o w

eeks

an

d so

met

imes

as

long

as

twen

ty-e

ight

day

s de

pend

ing

upon

the

viru

s. C

PE to

ok m

any

form

s an

d w

as a

t tim

es d

iffic

ult t

o ap

prec

iate

. Cul

ture

s ha

d to

be

mai

ntai

ned

over

man

y da

ys w

ith m

ultip

le m

edia

cha

nges

whi

le c

aref

ully

pre

vent

ing

cell

subs

trate

co

ntam

inat

ion.

W

e pr

esen

t a c

ompa

rison

of c

ultu

re fo

r viru

ses

usin

g lo

ng tu

bes

and

CPE

read

ing

vers

us s

hell

vial

and

flor

esce

nt m

onoc

lona

l ant

ibod

y bl

ind

stai

ning

usi

ng th

e D

iagn

ostic

H

ybrid

s (D

HI)

Met

hod.

Sum

mar

y of

Res

ults

Obs

erva

tions

and

Com

paris

on C

hart

s

Test

ing

for V

ario

us V

iruse

sH

erpe

s Si

mpl

ex (H

SV):

Thirt

y un

know

n H

SV s

ampl

es w

ere

inoc

ulat

ed o

nto

MR

C-5

ce

lls. C

oncu

rrent

ly, s

hell

vial

s co

ntai

ning

gen

etic

ally

eng

inee

red

baby

ham

ster

kid

ney

cells

, EL

VIS™

(DH

I) ce

lls, w

ere

inoc

ulat

ed. E

LVIS

cel

ls a

re e

ngin

eere

d w

ith a

HSV

pro

mot

er

gene

link

ed to

an

Esc

heric

hia

coli

LacZ

repo

rter g

ene.

In th

e pr

esen

ce o

f X-G

al, i

nfec

ted

cells

turn

blu

e (S

tabe

ll et

al.,

199

3, S

tabe

ll an

d O

livo,

199

2).T

he M

RC

-5 c

ells

wer

e ex

amin

ed d

aily

for u

p to

7 d

ays

for C

PE. T

he E

LVIS

cel

ls w

ere

fixed

and

sta

ined

at 2

4 ho

urs

and

exam

ined

for t

he p

rese

nce

of b

lue

cells

. Pos

itive

MR

C-5

cel

l cul

ture

s w

ere

stai

ned

with

a F

luor

esce

in Is

othi

ocya

nate

(FIT

C) l

abel

ed m

onoc

lona

l ant

ibod

ies

(Che

mic

on)

to H

SV-1

and

HSV

-2. T

he E

LVIS

cul

ture

s w

ere

stai

ned

with

a (F

ITC

) lab

eled

mon

oclo

nal

antib

ody

(DH

I) to

HSV

-2. T

he p

rese

nce

of a

ny c

ells

fluo

resc

ing

was

con

side

red

posi

tive

for

HSV

-2. I

f no

fluor

esce

nt c

ells

wer

e de

tect

ed in

the

ELVI

S sa

mpl

es, t

hey

wer

e rin

sed

and

rest

aine

d w

ith a

FIT

C la

bele

d m

onoc

lona

l ant

ibod

y to

HSV

-1 (D

HI).

The

pre

senc

e of

flu

ores

cenc

e w

as c

onsi

dere

d po

sitiv

e fo

r HSV

-1.

Res

pira

tory

Viru

ses:

Twen

ty-fo

ur u

nkno

wn

resp

irato

ry v

irus

sam

ples

wer

e in

ocul

ated

on

to R

MK,

HEP

2 an

d M

RC

-5 c

ell l

ines

. Con

curre

ntly

, she

ll vi

als

cont

aini

ng R

-Mix

(DH

I) w

ere

also

inoc

ulat

ed. R

-Mix

, a m

ixtu

re o

f a m

ink

lung

cel

l lin

e (M

v1Lu

) and

A-5

49, h

as b

een

show

n to

hav

e ex

celle

nt s

ensi

tivity

for r

espi

rato

ry v

iruse

s (S

t.G

eorg

e et

al.,

200

2;

Bare

nfan

ger e

t al.,

200

1). C

onve

ntio

nal c

ultu

res

wer

e ev

alua

ted

twic

e a

wee

k fo

r CPE

and

te

sted

for h

emag

glut

inat

ion

on d

ay 1

0. R

-Mix

cul

ture

s w

ere

fixed

and

sta

ined

at 2

4 ho

urs,

48

hou

rs a

nd 5

day

s us

ing

a FI

TC la

bele

d pa

n an

ti-re

spira

tory

viru

s an

tibod

y so

lutio

n (D

HI).

Po

sitiv

e cu

lture

s of

eac

h w

ere

furth

er e

valu

ated

by

stai

ning

with

spe

cies

spe

cific

FIT

C

labe

led

antib

odie

s (D

HI)

for I

nflu

enza

A a

nd B

, Par

ainf

luen

za 1

,2 a

nd 3

, and

Res

pira

tory

S

yncy

tial V

irus.

Ade

novi

rus

was

incu

bate

d w

ith a

n an

ti-ad

enov

irus

mon

oclo

nal a

ntib

ody

(Bar

tels

) fol

low

ed b

y a

FITC

labe

led

anti-

mou

se a

ntib

ody

(Bar

tels

). En

tero

viru

s:Tw

enty

-four

unk

now

n en

tero

viru

s sa

mpl

es w

ere

inoc

ulat

ed o

nto

RM

K, A

-54

9 an

d M

RC

-5 c

ells

. Sup

er E

-Mix

(DH

I) sh

ell v

ials

wer

e al

so in

ocul

ated

. Sup

er E

-Mix

is a

m

ixtu

re o

f buf

falo

gre

en m

onke

y ki

dney

cel

ls (B

GM

K) g

enet

ical

ly e

ngin

eere

d to

con

tain

hu

man

dec

ay a

ccel

erat

ion

fact

or (h

DAF

) and

A-5

49 c

ells

(Hua

ng e

t al.,

200

2; B

uck

et a

l.,

2002

). C

onve

ntio

nal c

ultu

re tu

bes

wer

e ev

alua

ted

for C

PE tw

ice

wee

kly.

Cul

ture

s po

sitiv

e fo

r CP

E a

nd c

ultu

res

still

neg

ativ

e af

ter 1

0 da

ys w

ere

stai

ned

with

a p

an F

ITC

labe

led

anti-

ente

rovi

rus

antib

ody

solu

tion

(Che

mic

on).

E-M

ix c

ultu

res

wer

e st

aine

d w

ith th

e pa

n en

tero

viru

s an

tibod

y so

lutio

n on

day

s 2

and

5. C

ultu

res

that

sho

wed

fluo

resc

ence

, bot

h co

nven

tiona

l as

wel

l as

E-M

ix c

ultu

res,

wer

e fu

rther

eva

luat

ed b

y st

aini

ng w

ith s

peci

es

spec

ific

FITC

labe

led

antib

odie

s (C

hem

icon

) for

Cox

sack

ie A

and

B a

nd E

chov

iruse

s.

Ente

rovi

ruse

s w

ere

incu

bate

d w

ith a

n an

ti-en

tero

viru

s m

onoc

lona

l ant

ibod

y (C

hem

icon

) fo

llow

ed b

y a

FITC

labe

led

anti-

mou

se a

ntib

ody

(Bar

tels

)C

ytom

egal

oviru

s (C

MV)

and

Var

icel

la Z

oste

r Viru

s (V

ZV):

Thirt

y-fo

ur u

nkno

wn

CM

V an

d VZ

V sa

mpl

es w

ere

sim

ulta

neou

sly

inoc

ulat

ed o

nto

MC

R-5

cel

ls a

nd H

&V-M

ix

(DH

I). H

&V-M

ix is

a m

ixtu

re o

f MR

C-5

cel

l and

CV-

1 ce

lls (A

frica

n gr

een

mon

key

kidn

ey

cells

) (H

uang

et a

l. 20

02).

CM

V co

nven

tiona

l cul

ture

tube

s w

ere

obse

rved

dai

ly fo

r CPE

w

hile

VZV

cul

ture

s w

ere

eval

uate

d 3

times

a w

eek.

Pos

itive

CM

V co

nven

tiona

l cul

ture

s an

d th

ose

still

not

pos

itive

on

day

28 w

ere

stai

ned

with

FIT

C la

bele

d m

onoc

lona

l ant

ibod

ies

to

CM

V (C

hem

icon

). Po

sitiv

e VZ

V cu

lture

s an

d th

ose

still

nega

tive

on d

ay 1

4 w

ere

stai

ned

with

a F

ITC

labe

led

mon

oclo

nal a

ntib

ody

to V

ZV(M

erifl

uor).

H&V

-Mix

cul

ture

s fo

r CM

V w

ere

stai

ned

on d

ays

2 an

d 3

for C

MV

with

a F

ITC

labe

led

mon

oclo

nal a

ntib

ody

(DH

I). V

ZV

cultu

res

wer

e st

aine

d on

day

s 2

and

5 fo

r VZV

with

a F

ITC

labe

led

mon

oclo

nal a

ntib

ody

(Mer

ifluo

r

Cel

l Lin

es:C

ultu

res

of M

RC

-5 (h

uman

em

bryo

nic

lung

), R

MK

(rhes

us m

onke

y ki

dney

), H

EP2

(hum

an la

ryng

eal c

arci

nom

a), A

-549

(hum

an lu

ng c

arci

nom

a) w

ere

purc

hase

d fro

m D

HI f

or u

se in

con

vent

iona

l viru

s cu

lture

s.B

asic

Des

ign:

Kno

wn

viru

s sa

mpl

es w

ere

purc

hase

d (D

HI),

sim

ulta

neou

sly

inoc

ulat

ed

onto

bot

h co

nven

tiona

l lon

g tu

bes

and

shel

l via

ls a

nd in

cuba

ted

at 3

7°C

. Pos

itive

co

nven

tiona

l cul

ture

s w

ere

dete

rmin

ed b

y C

PE, h

emag

glut

inat

ion

or b

lind

stai

ning

. Sh

ell v

ials

wer

e de

term

ined

to b

e po

sitiv

e us

ing

Dia

gnos

tic H

ybrid

s In

c. c

riter

ia (s

ee

belo

w).

The

sam

ples

wer

e tre

ated

as

if th

ey w

ere

patie

nt s

ampl

es. T

he te

chno

logi

st

eval

uatin

g th

e re

sults

was

una

war

e of

whi

ch s

ampl

es w

ere

posi

tive

and

for w

hich

vi

rus.

For

all

cultu

res

the

trans

port

med

ia w

as re

mov

ed a

t the

tim

e of

inoc

ulat

ion

and

repl

aced

by

refe

ed a

nd re

fed

twic

e w

eekl

y.

Her

pes

Sim

plex

Viru

s C

ultu

res:

Both

the

shel

l via

l cul

ture

s an

d th

e co

nven

tiona

l cul

ture

s co

rrect

ly d

etec

ted

all o

f the

pos

itive

sam

ples

(10

of

10),

and

neith

er in

corre

ctly

iden

tifie

d a

nega

tive

cultu

re a

s po

sitiv

e (2

0 of

20)

(Tab

le 1

). Al

l the

po

sitiv

e EL

VIS

cell

cultu

res

wer

e de

tect

ed w

ithin

24

hour

s. P

ositi

ve c

onve

ntio

nal c

ultu

res

requ

ired

48

hour

s. A

dditi

onal

ly, o

ur p

roce

dure

for c

onve

ntio

nal

cultu

res

requ

ired

eval

uatio

n un

til d

ay 7

.

Num

ber

Exp

ecte

d R

esult

Result

Day

Result

Day

HS

V 1

Neg

Neg

1N

eg

7H

SV

2N

eg

Neg

1N

eg

7H

SV

3H

SV

-1H

SV

-11

HS

V-1

2H

SV

4N

eg

Neg

1N

eg

7H

SV

5H

SV

-1H

SV

-11

HS

V-1

2H

SV

6H

SV

-2H

SV

-21

HS

V-2

2H

SV

7N

eg

Neg

1N

eg

7H

SV

8N

eg

Neg

1N

eg

7H

SV

9H

SV

-1H

SV

-11

HS

V-1

2H

SV

10

Neg

Neg

1N

eg

7H

SV

11

Neg

Neg

1N

eg

7H

SV

12

HS

V-2

HS

V-2

1H

SV

-22

HS

V 1

3N

eg

Neg

1N

eg

7H

SV

14

Neg

Neg

1N

eg

7H

SV

15

Neg

Neg

1N

eg

7H

SV

16

HS

V-2

HS

V-2

1H

SV

-22

HS

V 1

7H

SV

-1H

SV

-11

HS

V-1

2H

SV

18

Neg

Neg

1N

eg

7H

SV

19

Neg

Neg

1N

eg

7H

SV

20

Neg

Neg

1N

eg

7H

SV

21

Neg

Neg

1N

eg

7H

SV

22

HS

V-2

HS

V-2

1H

SV

-22

HS

V 2

3N

eg

Neg

1N

eg

7H

SV

24

Neg

Neg

1N

eg

7H

SV

25

HS

V-1

HS

V-1

1H

SV

-12

HS

V 2

6N

eg

Neg

1N

eg

7H

SV

27

Neg

Neg

1N

eg

7H

SV

28

HS

V-2

HS

V-2

1H

SV

-22

HS

V 2

9N

eg

Neg

1N

eg

7H

SV

30

Neg

Neg

1N

eg

7

Tab

le 1

: H

erpes s

imple

x C

om

pari

so

n

Shell

Via

lL

ong T

ub

e

Mat

eria

l and

Met

hods

:N

umbe

rE

xpec

ted

Res

ult

Res

ult

Day

Res

ult

Day

Res

p 1

Neg

Neg

5N

eg10

Res

p 2

Neg

Neg

5N

eg10

Res

p 3

Flu

BF

lu B

1F

lu B

3R

esp

4R

SV

RS

V1

RS

V3

Res

p 5

Par

aFlu

2P

araF

lu 2

1P

araF

lu 2

3R

esp

6N

egN

eg5

Neg

10R

esp

7R

SV

RS

V1

RS

V3

Res

p 8

Flu

AF

lu A

1F

lu A

3R

esp

9A

deno

Ade

no1

Ade

no3

Res

p 10

Neg

Neg

5N

eg10

Res

p 11

RS

VR

SV

1R

SV

3R

esp

12F

lu A

Flu

A1

Flu

A3

Res

p 13

Flu

AF

lu A

1F

lu A

3R

esp

14N

egN

eg5

Neg

10R

esp

15P

araF

lu 1

Par

aFlu

11

Par

aFlu

13

Res

p 16

Par

aFlu

3P

araF

lu 3

1P

araF

lu 3

3R

esp

17N

egN

eg5

Neg

10R

esp

18N

egN

eg5

Neg

10R

esp

19A

deno

Ade

no1

Ade

no3

Res

p 20

Flu

BF

lu B

1F

lu B

3R

esp

21N

egN

eg5

Neg

10R

esp

22N

egN

eg5

Neg

10R

esp

23N

egN

eg5

Neg

10R

esp

24F

lu A

Flu

A1

Flu

A3

Tab

le 2

: R

espi

rato

ry V

irus

es c

ompa

riso

n

She

ll V

ial

Lon

g T

ube

Ente

rovi

rus

Cul

ture

s:Th

e Su

per E

-Mix

cul

ture

s co

rrect

ly id

entif

ied

all e

nter

oviru

s po

sitiv

e cu

lture

s (1

3 of

13)

with

in 4

8 ho

urs

with

out i

ncor

rect

ly

iden

tifyi

ng a

neg

ativ

e sa

mpl

e as

pos

itive

(11

of 1

1)

(Tab

le 3

). C

onve

ntio

nal c

ultu

res

corre

ctly

iden

tifie

d al

l pos

itive

cul

ture

s w

ithin

48

hour

s. N

egat

ive

cultu

res

wer

e he

ld a

nd e

valu

ated

by

our p

roce

dure

un

til d

ay 1

0.

Num

ber

Exp

ecte

d R

esul

tR

esul

tD

ayR

esul

tD

ay

Ent

ero

1N

egN

eg5

Neg

10E

nter

o 2

Neg

Neg

5N

eg10

Ent

ero

3E

cho

viru

sE

cho

viru

s2

Ech

ovir

us2

Ent

ero

4C

oxs

acki

e BCo

xsac

kie

B2

Cox

sack

ie

B2

Ent

ero

5E

cho

viru

sE

cho

viru

s2

Ech

ovir

us2

Ent

ero

6N

egN

eg5

Neg

10E

nter

o 7

Neg

Neg

5N

eg10

Ent

ero

8E

cho

viru

sE

cho

viru

s2

Ech

ovir

us2

Ent

ero

9C

oxs

acki

e BCo

xsac

kie

B2

Cox

sack

ie

B2

Ent

ero

10

Neg

Neg

5N

eg10

Ent

ero

11C

oxs

acki

e BCo

xsac

kie

B2

Cox

sack

ie

B2

Ent

ero

12

Ech

ovi

rus

Ech

ovi

rus

2E

chov

irus

2E

nter

o 1

3E

cho

viru

sE

cho

viru

s2

Ech

ovir

us2

Ent

ero

14

Neg

Neg

5N

eg10

Ent

ero

15C

oxs

acki

e BCo

xsac

kie

B2

Cox

sack

ie

B2

Ent

ero

16C

oxs

acki

e ACox

sack

ie A

2C

oxs

acki

e A

2E

nter

o 1

7N

egN

eg5

Neg

10E

nter

o 1

8N

egN

eg5

Neg

10E

nter

o 1

9Ent

ero

viru

sEnt

ero

viru

s2

Ent

ero

viru

s2

Ent

ero

20

Ech

ovi

rus

Ech

ovi

rus

2E

chov

irus

2E

nter

o 2

1N

egN

eg5

Neg

10E

nter

o 2

2N

egN

eg5

Neg

10E

nter

o 2

3N

egN

eg5

Neg

10E

nter

o 2

4E

cho

viru

sE

cho

viru

s2

Ech

ovir

us2

Tab

le 3

: E

nter

ovi

rus

Co

mpa

riso

n

She

ll V

ial

Lo

ng T

ube

Res

pira

tory

Cul

ture

s:R

-Mix

cul

ture

s co

rrect

ly

iden

tifie

d al

l pos

itive

resp

irato

ry s

ampl

es (1

4 of

14)

w

ithin

24

hour

s w

ith n

o fa

lse

nega

tives

(10

of 1

0)

(Tab

le 2

). C

onve

ntio

nal c

ultu

res

also

iden

tifie

d al

l po

sitiv

e sa

mpl

es; h

owev

er, s

ome

conv

entio

nal

cultu

res

requ

ired

as lo

ng a

s 10

day

s to

bec

ome

posi

tive.

Cyt

omeg

alov

irus

and

Varic

ella

Zos

ter V

irus

Cul

ture

s:H

&V-M

ix c

ultu

res

dete

cted

all

posi

tive

CM

V sa

mpl

es (9

of 9

) with

no

fals

e po

sitiv

es (1

5 of

15

) with

in 4

8 ho

urs

(Tab

le 4

). Th

e sh

ell v

ial c

ultu

res

corre

ctly

iden

tifie

d al

l pos

itive

VZV

sam

ples

(4 o

f 4)

with

out f

alse

pos

itive

s (6

of 6

) with

in 4

8 ho

urs

(Tab

le

5). C

onve

ntio

nal c

ultu

res

also

cor

rect

ly id

entif

ied

all

posi

tive

CM

V sa

mpl

es b

ut s

ome

cultu

res

took

as

long

as

8 d

ays

to b

ecom

e po

sitiv

e an

d by

pro

cedu

re w

ere

held

for 2

8 da

ys. C

onve

ntio

nal c

ultu

res

faile

d to

id

entif

y 2

of 4

VZV

pos

itive

sam

ples

and

requ

ired

14

days

for t

he tw

o sa

mpl

es th

at w

ere

iden

tifie

d.

Expe

cted

Re

sult

Day

Neg

28CM

V8

CMV

3Ne

g28

Neg

28CM

V8

Neg

28CM

V3

Neg

28CM

V3

Neg

28Ne

g28

Neg

28CM

V8

Neg

28Ne

g28

CMV

6Ne

g28

CMV

6Ne

g28

Neg

28Ne

g28

CMV

6Ne

g28

Num

ber

Resu

ltDa

y

VZV

1Ne

g14

VZV

2Ne

g14

VZV

3Ne

g14

VZV

4Ne

g14

VZV

5V

ZV14

VZV

6Ne

g14

VZV

7Ne

g14

VZV

8Ne

g14

VZV

9Ne

g14

VZV

10

VZV

14Ne

gN

eg3

VZV

VZV

2

Neg

Neg

3VZ

VVZ

V2

VZV

VZV

2Ne

gN

eg3

VZV

VZV

2Ne

gN

eg3

Neg

Neg

3Ne

gN

eg3

Resu

ltD

ay

Tabl

e 5: V

aric

ella

zoste

r Co

mpa

rison

Expe

cted

Res

ult

Shell

Vial

Long

Tub

e

CMV

24Ne

g3

Neg

CMV

23CM

V2

CMV

CMV

22Ne

g3

Neg

CMV

21Ne

g3

Neg

CMV

20Ne

g3

Neg

CMV

19CM

V2

CMV

CMV

18Ne

g3

Neg

CMV

17CM

V2

CMV

CMV

16Ne

g3

Neg

CMV

15Ne

g3

Neg

CMV

14CM

V2

CMV

CMV

13Ne

g3

Neg

CMV

12Ne

g3

Neg

CMV

11Ne

g3

Neg

CMV

10CM

V2

CMV

CMV

9Ne

g3

Neg

CMV

8CM

V2

CMV

CMV

7Ne

g3

Neg

CMV

6CM

V2

CMV

CMV

5Ne

g3

Neg

CMV

4Ne

g3

Neg

CMV

3CM

V2

CMV

CMV

2CM

V2

CMV

CMV

1Ne

g3

Neg

Resu

ltRe

sult

Day

Tabl

e 4: C

ytom

egalo

virus

Com

paris

on

Num

ber

Shell

Vial

Long

Tub

e

Dis

cuss

ion:

The

shel

l via

l tec

hniq

ues

usin

g th

e D

iagn

ostic

Hyb

rid’s

ELV

IS c

ell

(HSV

), R

-Mix

cel

ls (r

espi

rato

ry v

iruse

s),

Supe

r E-M

ix c

ells

(ent

erov

iruse

s) a

nd

H&

V-M

ix c

ells

(CM

V an

d VZ

V) w

ere

able

to

cor

rect

ly id

entif

y 10

0% o

f sam

ple

viru

ses

test

ed.C

onve

ntio

nal v

iral c

ultu

res

corre

ctly

iden

tifie

d 10

0% o

f the

HSV

, re

spira

tory

viru

ses,

ent

erov

iruse

s an

d C

MV

but m

isse

d 2

VZV

pos

itive

sam

ples

. Nei

ther

te

chni

que,

in th

is s

tudy

, fal

sely

iden

tifie

d a

nega

tive

sam

ple

as p

ositi

ve.

The

maj

or a

dvan

tage

of t

he D

iagn

ostic

H

ybrid

s sh

ell v

ial t

echn

ique

is d

ecre

ased

tu

rn a

roun

d tim

e.H

SV c

ultu

res

decr

ease

d tu

rn a

roun

d tim

e to

24

hour

s co

mpa

red

to 2

-7

days

usi

ng th

e co

nven

tiona

l cul

ture

m

etho

d. R

espi

rato

ry c

ultu

res

had

a de

crea

sed

turn

aro

und

time

to 2

4 ho

ur

com

pare

d to

3-1

0 da

ys. E

nter

oviru

ses

had

a de

crea

se to

48

hour

s co

mpa

red

to 2

-10

days

. CM

V c

ultu

res

wer

e po

sitiv

e w

ithin

48

hour

s w

ith th

e D

iagn

ostic

Hyb

rids

tech

niqu

e co

mpa

red

to 3

-8 d

ays

for t

he c

onve

ntio

nal

tech

niqu

e. N

egat

ive

cultu

res

wer

e he

ld fo

r ob

serv

atio

n fo

r 28

days

. VZV

turn

aro

und

time

was

redu

ced

to 4

8-72

hou

rs c

ompa

red

to 1

4 da

ys fo

r con

vent

iona

l cul

ture

s.

Respi

rato

ry Lo

ng

Tube

Respi

rato

ry Sh

ell

Vial

Herpe

s*** L

ong

Tube

Herpe

s Sh

ell V

ial

Enter

ovir

us Lo

ng

Tube

Enter

ovir

us Sh

ell

Vial

CMV*

*** L

ong

Tube

CMV

Shell

Vial

VZV

Long

Tu

beVZ

V Sh

ell V

ial

Mater

ialNu

mber

of Tu

bes/V

ials

63

41

63

42

42

Numb

er of

Refee

ds3

13

13

18

15

1Da

ys rea

d for

CPA

20

70

20

120

60

Cost

of Ce

lls$9

.44$7

.50$6

.30$3

.60$9

.44$7

.50$5

.20$5

.00$6

.30$5

.00Co

st of

Refee

d Med

ia$1

.32$0

.89$1

.10$0

.31$2

.21$0

.53$1

.10$0

.59$1

.10$0

.59Co

st of

Stain*

$9.60

$11.0

0$9

.80$4

.91$9

.60$1

1.00

$9.80

$12.6

2$9

.80$1

0.35

Cost

of He

madso

rption

$2.00

Acces

sionin

g3 m

in3 m

in3 m

in3 m

in3 m

in3 m

in3 m

in3 m

in3 m

in3 m

inSe

t Up P

er Tu

be/V

ial1.7

min

1.7 m

in1.7

min

1.7 m

in1.7

min

1.7 m

in1.7

min

1.7 m

in1.7

min

1.7 m

inRe

ad C

PA Pe

r Tub

e/Vial

0.2 m

in0.2

min

0.2 m

in0.2

min

0.2 m

in0.2

min

0.2 m

in0.2

min

0.2 m

in0.2

min

Refee

d Per

Tube

/Vial

0.1 m

in0.1

min

0.1 m

in0.1

min

0.1 m

in0.1

min

0.1 m

in0.1

min

0.1 m

in0.1

min

Stain

10 m

in10

min

6 min

6 min

10 m

in10

min

10 m

in10

min

10 m

in10

min

Total

Tech

Time

26.4

min

18.4

min

18.4

min

10.8

min

26.4

min

18.4

min

32.6

min

16.5

min

26.6

min

16.5

min

Cost

Tech

Time

**$9

.77$6

.81$6

.80$4

.00$9

.77$6

.81$1

2.62

$6.11

$9.84

$6.11

Cost

Mater

ials

$22.3

6$1

9.39

$17.2

0$8

.82$2

2.36

$19.3

9$1

6.10

$18.2

1$1

7.20

$15.9

4To

tal C

ost$3

2.13

$26.2

0$2

4.00

$12.8

2$3

2.13

$26.2

0$2

8.72

$24.3

2$2

7.04

$22.0

5

** A

ssume

s $0.3

7 per

min

Table

6: C

ost A

nalys

is She

ll Vial

Com

pared

to C

onve

ntion

al Vi

rus C

ulture

s

Tech

nolog

ist Ti

me

*Assu

mes P

ositiv

e Cult

ures w

ith M

axim

um St

ai n**

*With

typin

g**

**As

sumes

28 da

ys

A co

mpa

rison

of t

he c

ost (

Tabl

e 6)

sho

ws

that

per

sa

mpl

e, in

our

labo

rato

ry, t

he D

iagn

ostic

Hyb

rids

tech

niqu

es a

re s

light

ly le

ss e

xpen

sive

than

our

pr

evio

us m

etho

ds u

sing

CPE

as

an e

nd p

oint

. Ba

renf

ange

r et a

l (20

01) f

ound

that

usi

ng R

-Mix

was

sl

ight

ly m

ore

expe

nsiv

e th

an c

onve

ntio

nal c

ultu

res

for

resp

irato

ry c

ultu

res.

The

she

ll vi

al te

chni

que

is e

asie

r to

lear

n, ta

kes

muc

h le

ss tr

aini

ng ti

me

and

end

poin

ts

are

less

sub

ject

ive

than

CPE

.

Con

clus

ion:

Dia

gnos

tic H

ybrid

s sh

ell v

ial

tech

niqu

e re

duce

s tu

rn a

roun

d tim

e w

ithou

t sac

rific

ing

sens

itivi

ty o

r sp

ecifi

city

, has

a c

ost t

hat i

s ro

ughl

y eq

uiva

lent

if n

ot le

ss e

xpen

sive

, and

is

easi

er to

lear

n w

ith d

ecre

ased

trai

ning

tim

e an

d a

less

sub

ject

ive

end

poin

t tha

n co

nven

tiona

l cul

ture

s us

ing

CPE

.

Cos

t Ana

lysi

s

Con

clus

ion

Ref

eren

ces:

Bare

nfan

ger,

J., D

rake

, C.,

Mue

ller,

T., T

rout

t, T.

, O’B

rien,

J. a

nd G

uttm

an, K

., 20

01. R

-Mix

cel

ls a

re fa

ster

, at

leas

t as

sens

itive

and

mar

gina

lly m

ore

cost

ly th

an c

onve

ntio

nal c

ell l

ines

for t

he d

etec

tion

of re

spira

tory

vi

ruse

s. J

Clin

Viro

l 22:

101

-110

. Bu

ck, G

.E.,

Wie

sem

ann,

M. a

nd S

tew

art,

L., 2

002.

Com

paris

on o

f mix

ed c

ell c

ultu

re c

onta

inin

g ge

netic

ally

en

gine

ered

BG

MK

and

CaC

o-2

cells

with

RT-

PCR

and

con

vent

iona

l cel

l cul

ture

for t

he d

iagn

osis

of

ente

rovi

ral m

enin

gitis

. J C

lin V

irol;

25: S

13-S

18.

Hua

ng, Y

.T.,

Hite

, S.,

Dua

ne, V

. and

Yan

, H.,

2002

. CV-

1 an

d M

RC

-5 m

ixed

cel

ls fo

r sim

ulta

neou

s de

tect

ion

of h

erpe

s vi

ruse

s an

d va

ricel

la z

oste

r in

skin

lesi

ons.

J C

lin V

irol;

24: 3

7-44

.H

uang

, Y.T

., Ya

m, P

., Ya

n, H

. and

Sun

, Y.,

2002

. Eng

inee

red

BGM

Kce

lls fo

r sen

sitiv

ity a

nd ra

pid

dete

ctio

n of

ent

erov

iruse

s. J

Clin

Mic

robi

ol; 4

0(2)

: 366

-371

.St

abel

l, E.

C.,

and

Oliv

o, P

.D. 1

992.

Isol

atio

n of

a c

ell l

ine

for r

apid

and

sen

sitiv

e hi

stoc

hem

ical

ass

ay fo

r the

de

tect

ion

of h

erpe

s si

mpl

ex v

irus.

J V

irol M

etho

ds38

: 195

-204

.St

abel

l, E.

C.,

O’R

ourk

e, S

.R.,

Sto

rch,

G.A

. and

Oliv

o, P

.D.,

1993

. Eva

luat

ion

of a

gen

etic

ally

eng

inee

red

cell

line

and

a hi

stoc

hem

ical

β-g

alac

tosi

dase

ass

ay to

det

ect h

erpe

s si

mpl

ex v

irus

in c

linic

al s

peci

men

s. J

Clin

M

icro

biol

31:

279

6-27

98.

St. G

eorg

e, K

., Pa

tel,

N.M

., H

artw

ig, R

.A.,

Scho

ll, D

.R.,

Jolli

ck, J

.A.,

Kauf

fman

n, L

.M.,

Evan

s, M

.R. a

nd

Rin

aldo

, C.R

., 20

02. R

apid

and

sen

sitiv

e de

tect

ion

of re

spira

tory

viru

s in

fect

ions

for d

irect

ed a

ntiv

iral

treat

men

t usi

ng R

-Mix

cul

ture

s. J

Clin

Viro

l24:

107

-115

.