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Herpes

Simplex

Virus

in Childhood

Erythema

Multiforme

32

PEDIATRICS

Vol. 89 No.

1 January 1992

William

L. Weston,

MD*;

Sylvia

L. Brice,

MD*;

Joy D. Jester,

MD*;

Alfred

T. Lane,

MDX;

Scott

Stockert*;

and

J. Clark

Huff,

MD*

ABSTRACT.

Although

an

association

between

herpes

simplex

virus

(HSV)

infection

and

erythema

multiforme

(EM)

minor

has

been

documented

in adults,

this

has

not

been

reported

in

the

pediatric

population.

This

study

assessed

the

potential

role

of HSV

infection

in the

path-ogenesis

of EM minor

in children.

Erythema

multiforme

skin

lesions

from

20 children,

aged

1 to 16 years,

were

examined

for the

presence

of HSV

by

using

the

polym-erase

chain

reaction.

The

children

included

all

fit strict

clinical

criteria

for EM minor.

Ten

had

a clinical

history

of

an

antecedent

herpes

infection

(“herpes-associated

EM”),

and

10

did

not

(“idiopathic

EM”).

Herpes

simplex

virus

DNA

was

detected

in skin

lesions

of 8 of 10 children

with

herpes-associated

EM and

in 8 of 10 with

idiopathic

EM.

Control

skin

biopsies

from

children

with

other

bullous

inflammatory

diseases

were

negative.

In

addi-tion,

no

HSV

could

be

detected

in

a biopsy

of normal

uninvolved

skin

of a child

in whom

HSV

was

present

in

lesional

skin.

In situ

hybridization

on

selected

biopsies

by

means

of an HSV-specific

riboprobe

confirmed

the

presence

of HSV

and

localized

it to the

epidermis.

It is

concluded

that

HSV

is a significant

precipitating

factor

for

EM

minor

in

children,

as it is in

adults,

and

that

clinicians

should

maintain

a high

index

of suspicion

of

HSV

even

in the

absence

of a known

history

of herpes

infection.

Pediatrics

1992;89:32-34;

erythema

multiforme,

herpes

simplex

virus,

polymerase

chain

reaction,

molecular

probes.

ABBREVIATIONS. EM, erythema multiforme; HSV, herpes sim-plex virus; PCR, polymerase chain reaction,

Erythema

multiforme

(EM)

of

the

minor

type

was

first

described

by

the

19th

century

dermatologist,

Ferdinand

von

Hebra.’

It is an

acute,

self-limited,

cutaneous

or

mucocutaneous

illness

with

character-istic

skin

lesions

consisting

of fixed

red

papules.

Many

lesions

may

evolve

to show

concentric

zones

of color

change

which

form

characteristic

taret

or iris lesions,

the

hallmark

of EM

minor

(Fig.

1).

Mucous

mem-brane

involvement

is mild

and

usually

restricted

to a

few

oral

erosions.

Erythema

multiforme

is most

com-monly

a

disease

of

young

adults.

It

is

frequently

recurrent

and

is often

associated

with

a preceding

herpes

simplex

virus

(HSV)

infection,

either

type

1 or

type

2. However,

the

role

HSV

plays

in the

pathogen-esis

of

EM

minor

has

not

been

fully

clarified.

Viral

From the 0Depariment of Dermatology, University of Colorado School of

Medicine, Denver, and the Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY.

Received for publication Oct 1 1, 1990; accepted Jan 15, 1991.

Reprint requests to (W.L.W.) B-153, 4200 E Ninth Ave. Denver, CO 80262. PEDIATRICS (ISSN 0031 4005). Copyright © 1992 by the American Acad-emy of Pediatrics.

cultures of EM skin lesions are routinely negative, yet

we

recently

demonstrated

HSV-specific

antigen

and

HSV

DNA

within

the

skin

lesions

of adults

with

EM

minor.’

Erythema

multiforme

minor

is less

common

in children, and unlike in adults, HSV has not been

reported

as

a significant

precipitating

factor.

To

in-vestigate

this

possible

association

in the

pediatric

age

group,

we examined

skin

biopsies

of EM lesions

from

20 children

for

the

presence

of HSV

DNA

by using

the polymerase chain reaction (PCR) and in situ

hy-bridization.

We

detected

HSV

DNA

in 8 of 10 cases

with

histories

of a preceding

HSV

infection

and

in 8

of 10 cases

without

obvious

preceding

HSV

infection.

Patient

Selection

METHODS

After informed consent was obtained, 20 children, aged I to 16, were entered into the study. This study was approved by the Human Subjects Committee at the University of Colorado Health Sciences Center. Each had a careful history and physical exami-nation, photography of skin lesions, and a 4-mm skin biopsy of an early papule or the outer border of a target lesion. Some children had a herpes labialis skin lesion still present at the time of onset of the EM minor, and these lesions were cultured for HSV. Some children had a subsequent herpes labialis lesion cultured for HSV. All children entered into the study satisfied the case definition of

erythema multiforme-with an acute cutaneous or mucocutaneous illness lasting less than 4 weeks characterized by symmetrically distributed, discrete, fixed, red skin papules, some of which evolved into characteristic target lesions.2 In addition, their skin biopsies demonstrated necrotic epidermal cells, vacuolar degeneration of the epidermis, a mononuclear cell inflammatory infiltrate, and absence of vasculitis. Biopsies were fixed in formalin and embedded in paraffin or fresh frozen. In one case, we obtained consent to biopsy normal uninvolved skin at the same time we biopsied an EM skin lesion.

If the child had a visible skin lesion compatible with a herpes lesion at the time of examination or a definite history of an HSV infection preceding an episode of EM or a positive HSV culture, the case was considered to be ‘herpes-associated erythema multi-forme’; if no definite history of HSV could be obtained the case was designated “idiopathic erythema multiforme.’ There were 10 cases in each category. In addition, eight paraffin-embedded biop-sies from children of similar ages with other bullous skin diseases such as linear IgA dermatosis, bullous lichen planus, and bullous

insect bite reactions were included as negative controls. Cultured human keratinocytes infected with HSV-1 served as a positive control.

Extraction

of DNA

for PCR

For formalin-fixed tissue, twenty-five 5-im sections were ob-tamed from each block, de-paraffini.zed in xylene, and rinsed with 95% ethanol.3 After drying, the tissues were resuspended in lysis buffer (10 mmol/L Tris-HC1, 1 mmol/L EDTA, pH 7.4, 0.1% sodium dodecyl sulfate) and incubated with proteinase K overnight at 37#{176}C.Processing of frozen tissues began with the proteinase K

step. All samples

were

phenol

extracted

twice,

and the

DNA was

ethanol precipitated, dried, then resuspended in 100 gL of

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(2)

‘F

ARTICLES 33

Fig. 1. Eight-year-old boy with herpes-associated erythema multi-forrne (EM) minor. Typical target lesions of EM minor are present on cheeks with crusted lesion on lower lip of healing antecedent

herpes labialis.

ED’rA buffer (10 mmol/L Tns-HC1, 1 mmol/L EDTA, pH 7.8) and

used as a source of tissue DNA for the PCR.

Amplification

with

the PCR

The PCR was performed as previously reported.3 Briefly,

oligo-nucleotide primers which flank a 92-base pair portion of the HSV genome coding for HSV DNA polymerase were used for amplifi-cation of the HSV DNA. These primers were previously demon-strated to be specific for HSV-1 and HSV-2, with no cross-reaction

with other herpesviruses (varicella-zoster virus, cytomegalovirus,

Epstein-Barr virus).34 To verify the quality of the extracted DNA, we also amplified all DNA samples with control primers which flank a I 10-base pair segment of the human genome coding for

i-globin. Additional control samples containing PCR reagents and

primers but no experimental DNA were included as controls for

potential contamination. Thirty cycles were performed using the DNA thermal cycler (Perkin-Elmer Cetus Instruments, Norwalk,

CT) at temperatures of 94#{176}Cfor denaturation, 58#{176}Cfor annealing,

and 72#{176}Cfor elongation.

Detection

of PCR-Amplified

DNA

The amplified DNA samples were separated on an 8%

poly-acrylamide gel. Both positive and negative control samples were

included with each gel. Samples were electrophoretically trans-ferred to ZetaProbe filters (BioRad, Richmond, CA). Internal oh-gonucleotides complementary to the amplified segments of DNA (both HSV and 13-globin) were end labeled with y 35S and hybrid-ized with the filters as previously reported.3 The filters were washed

at high stringency then exposed to Kodak XAR film overnight. Results were verified by repeating the PCR and Southern analysis

for all samples a second time.

In Situ

Hybridization

We used a 35S-labeled riboprobe complementary to the HSV DNA which codes for the viral envelope glycoprotein B as

previ-ously reported.3 Following standard prehybridization, the probe

was overlaid on frozen tissue sections; then the sections were

denatured at 95#{176}Cfor 15 minutes. Hybridization was accomplished overnight at 42#{176}C.Tissue sections were then dipped in Kodak

NTB-2 emulsion and examined microscopically after 3 to 5 days. For a positive control, rabbit skin cells infected with HSV-1 were

exam-med. Uninfected rabbit skin cells were negative controls. These

cells were pelleted, embedded in OCT, and 4 gmoh/L frozen sections were obtained. In addition, all specimens were hybridized with a control nboprobe specific for Epstein-Barr virus. This probe

was of similar length and guanine cytosine content as the HSV

probe.

HSV

NEG

HAEM

IPEM

,

p

BG11Obp

#{149}4

HSV92bpr

o

Fig. 2. Representative Southern analysis of DNA amplified by polymerase chain reaction PCR. DNA in the first lane of each pair was amplified by primers to 11-globin (BC) and in the second lane

by primers to herpes simplex virus (HSV). 35S-Iabeled internal

nucleotides produce dark bands with specific binding. BC is

dem-onstrated in all specimens. HSV is demonstrated in the HSV-positive control (HSV), herpes-associated erythema multiforme

(EM) minor (HAEM), and idiopathic EM minor (IPEM), but not in

negative control of linear IgA dermatosis (NEC). bp, base pair.

Statistical

Analysis

We used x2 analysis to compare the likelihood of detecting HSV

in skin lesions of children with EM with that of detecting HSV in

the skin lesions of children with other bullous skin diseases.

RESULTS

Ages

of patients

in the

herpes-associated

EM

group

ranged

from

8 to 16 years,

and

those

in the idiopathic

EM group from 1 to 15 years. The mean ages of the

two groups (herpes-associated = 1 1 .2 years,

idi-opathic

=

9.3

years)

were

not

significantly

different

by

x2

analysis

(P

=

.56).

A band

corresponding

to

HSV

DNA

was

detected

in 8 of 10 skin

biopsies

from

children

with

herpes-associated

EM;

from

8 of

10

biopsies

of idiopathic

EM;

in 0 of 8 biopsies

of other

bullous

skin

diseases;

and

in

none

of

the

“no-DNA”

samples

(Fig.

2).

Compared

with

the

other

bullous

skin

diseases,

the

frequency

of

positive

results

in

herpes-associated

EM

and

idiopathic

EM

was

highly

significant

by

x2

analysis

(P

<

.001).

In

all

DNA-containing

samples

a band

corresponding

to /3-globin

was

detected

(Fig.

2). One

child

in whom

HSV

DNA

was

detected

in EM

skin

did

not

have

demonstrable

HSV

DNA

in normal

uninvolved

skin

obtained

si-multaneously.

One

child

1 year

of

age

in

the

idi-opathic

EM

group

had

HSV

in his

EM

skin

biopsy,

and

the

two

whose

biopsies

were

negative

were

both

15 years

old.

Repetition

of experiments

confirmed

the

initial

results.

By in situ

hybridization

with

the

HSV

riboprobe,

grains

were

seen

predominantly

over

ker-atinocytes

as previously

reported

in adults

with

her-pes-associated

EM.3

Grains

were

observed

over

HSV-1-infected rabbit skin cells but not over uninfected

cells.

No

specific

binding

was

observed

with

the

Epstein-Barr

virus

control

riboprobe.

DISCUSSION

Erythema

multiforme

minor

has

been

considered

a

hypersensitivity

phenomenon,

potentially

induced

by

multiple factors such as drugs or infectious agents. In

the adult

population,

the most

prevalent

precipitating

factor

is an antecedent

infection

with

HSV.

Although

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(3)

34

HSV

IN

ERYTHEMA

MULTIFORME

EM

minor

is predominantly

a disease

of adults,

it is

estimated

that

up

to 20%

of cases

occur

in the

pedi-atric

age

group.2

Despite

this,

the

potential

etiologic

factors

for

children

with

EM

minor

have

not

been

established.

In the

few

clinical

series

describing

EM

minor

in children,

the

absence

of a role

for HSV

has

been

notable.2’57To

investigate

further

the

potential

significance

of herpes

infection

in the

pathogenesis

of EM minor

in children,

we examined

the

cutaneous

EM

lesions

of 20 subjects

1 6 years

old

and

younger

for the

presence

of HSV

by using

the

PCR

and

in situ

hybridization.

By

PCR,

we

detected

HSV

DNA

in

80%

of these

children.

In situ hybridization

confirmed

the

presence

of

HSV

DNA

within

skin

lesions

and

localized

it

predominantly

to

the

epidermis.

These

results

parallel

those

we

previously

reported

in

adults.3

The

PCR

is a highly

sensitive

and

specific

technique

which

has

proved

extremely

useful

in

the

study

of viral

and

other

infectious

diseases.

However,

because

of

the

capacity

of this

procedure

to detect

minute

quantities

of any

given

DNA

species,

signifi-cant

problems

with

potential

contamination

and

with

interpretation

of positive

results

exist.

In this

study,

we

handled

all

DNA

specimens

with

sterile

or

dis-posable

reagents

and

equipment

to reduce

the

risk

of

cross-contamination,

and

we included

many

negative

controls

(including

specimens

with

no

DNA)

with

each

experiment,

to quickly

detect

false-positive

re-sults.

In

addition,

each

specimen

was

examined

at

least

twice

to confirm

positive

or negative

findings.

As previously

noted,

the

herpes

primers

were

specific

for

HSV-1

and

HSV-2

and

did

not

amplify

DNA

of

other

herpesviruses.

In situ

hybridization

is a less

sensitive

technique

but

offers

the

advantage

of

localizing

the

DNA

of

interest

within

the

tissue

specimen,

in this

case

the

epidermis

of

EM

skin

lesions.

Thus,

in

addition

to

confirming

the

results

of

PCR,

this

technique

also

provided

evidence

that

HSV

was

present

within

ker-atinocytes

in the skin

and

not

limited

to HSV-infected

blood

cells

that

might

have

been

included

in

the

dermal

portion

of biopsy

specimens.

Inasmuch

as we did

not

detect

HSV

in any

biopsies

of other

bullous

inflammatory

dermatoses,

nor

in the

uninvolved

skin

of the child

with

active

EM (in whom

HSV

was

detected

in lesional

skin),

we

suggest

that

HSV

has

a role

in the

formation

of EM

skin

lesions.

Our

hypothesis

is that

the inflammatory

skin

response

in

EM

minor

is herpes-specific

and

represents

the

expected

host

immune

response

to the

expression

of

HSV

antigens

on

the

keratinocyte

surface.8

This

is

consistent

with

the finding

that keratinocyte

injury

is

the earliest

histopathologic

sign

of EM minor.2’8’9

How

the virus

gets

to keratinocytes

after

a preceding

herpes

labialis

lesion

is

not

yet

explained.

We

favor

the

possibility

that

there

is

a

transient

herpes

viremia

during

early

phases

of

the

preceding

herpes

labialis

infection

and

the

virus

disseminates

to distant

sites

in

the

skin.

We

cannot

yet

exclude

the

possibility

that

HSV

may

be latent

within

focal

areas

of skin,

and

the

EM

minor

skin

lesion

is an

immunologic

response

at

a site

where

latent

virus

has

expressed

viral

proteins.

Why

some

children

get

EM

skin

lesions

following

herpes

labialis

while

the

vast

majority

do

not

is not

known.

It was

of

particular

interest

that

so

many

of

the

children

in

this

study

with

“idiopathic

EM’

also

had

detectable

HSV

in

their

skin

lesions.

We

had

also

found

this

to be

the

case

in adults,

which

suggested

the

possibility

of

subclinical

herpes

infection.

In

adults

with

recurrent

EM,

we

have

on

occasion

ob-served

subtle

lesions

which

preceded

the onset

of EM

minor.

While

these

lesions

did

not

progress

to typical

herpetic

vesicles,

they

did

recur

at the

same

site

and

were

HSV

positive

by

viral

culture.

Based

on

the

results

of this

study,

we

encourage

clinicians

to

consider

HSV

as

a major

precipitating

factor

of

EM

minor

in

infants

and

children

even

in

the

absence

of herpes

infection.

In addition,

we

advise

against the routine treatment of children with EM

minor

with

systemic

steroids

in

view

of

the

high

likelihood

that

HSV

is present.2’102

ACKNOWLEDGMENTS

This work was supported by grant A! 16637 from the National Institute of Allergy and Infectious Diseases and grant AM 07411 from the National Institute of Arthritis and Musculoskeletal and

Skin

Diseases, National Institutes of Health.

REFERENCES

1. von Hebra F; Fagge CH, trans. On Diseases of the Skin Including the Exanthemata. London, England: New Sydenham Society; 1866;1:285-289

2. Brice SL, Huff JC, Weston WL. Erythema multiforme. Curr Probl

Der-matol. 1990;2:3-25

3. Brice SL, Krzemeien D, Weston WL, HuffJC. Detection of herpes simplex virus DNA in cutaneous lesions of erythema multiforme. IInvest

Der-matol. 1989;193:183-187

4. Cao M, Xaio X, Egbert B, Darragh TM, Yen TSB. Rapid detection of

cutaneous herpes simplex virus infection with the polymerase chain

reaction. IInvest Dermatol. 1989;92:391-392

5. Dikland WJ, Oranje Al’, Solz E, van Joost TH. Erythema multiforme in

childhood and early infancy. Pediatr Dermatol. 1986;3:135-139 6. Hellgren L, Hersle K. Erythema multiforme. Acta Allergol.

1965;21:45-51

7. Brown ZA, Ashley R, Douglas J. Neonatal herpes simplex infection: relapse after initial therapy and transmission from a mother with an

asymptomatic genital herpes infection and erythema multiforme. Pediatr InfectDis1,1987;6:1057-1061

8. Orton PW, Huff JC, Tonnesen MG, Weston WL. Detection of a herpes

simplex viral antigen in skin lesions of erythema multiforme. Ann Intern Med. 1984;101:48-50

9. Howland WW, Golitz LE, Huff JC, Weston WL. Erythema multiforme: clinical, histopathologic, and immunologic study. IAm Acad Dermatol. 1984;10:438-446

10. Huff JC, Weston WL. Recurrent erythema multiforme. Medicine

(Balti-more) 1989;68:133-140

1 1

,

Huff JC. Acyclovir for recurrent erythema multiforme caused by herpes simplex.

I

Am Acad Dermatol. 1988;18:197-199

12. Renfro L, Grant-Kels JM, Feder HM Jr, et al. Controversy: are systemic

steroids indicated in the treatment of erythema multiforme? Pediatr Dermatol. 1989;6:43-50

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1992;89;32

Pediatrics

T. Lane

William L. Weston, Sylvia L. Brice, Joy D. Jester, Scott Stockert, J. Clark Huff and Alfred

Herpes Simplex Virus in Childhood Erythema Multiforme

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1992;89;32

Pediatrics

T. Lane

William L. Weston, Sylvia L. Brice, Joy D. Jester, Scott Stockert, J. Clark Huff and Alfred

Herpes Simplex Virus in Childhood Erythema Multiforme

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