lmmunocytochemical Confirmation of Oral and Respiratory Herpesvirus Infection by the Immunoperoxidase Method Raj K. Gupta, M.D., F.I.A.C., Andrew J. Buchanan, B.Sc.(Hons.), A.N.Z.I.M.L.T., and Robert Fauck, C.T. (I.A.C.)

Recognition of diagnostic viral changes in general, and due to herpesvirus infection (HVI) in particular, from a variety of materials examined cytologically has added a new dimension to diagnostic cytology. Such recognition provides valuable information to the clinician when no infection is suspected. Previously, the diagnosis of HVI of the respiratory tract was only possible at postmortem. tissue culture, or diagnostic rise in antibody titer. This study describes our experience in the diagnosis of HVI of the respiratory tract initially suggested by sputum cytology and later confirmed by the immunoperoxi- dase method. The importance of sputum cytology in the diagnosis of WVI is emphasized. Diagn Cytopathol

Key Words: Herpesvirus infection (HVI); Immunoperoxidase; Cytology; Herpes simplex virus type 1 (HSV-1); Herpes simplex virus type 2 (HSV-2)

1981;3:287-90.

Cell changes due to herpesvirus infection (HVI) in smears of the female genital tract are well known.'-' A few studies have also described HVI cell changes in material from the neonate,* esophageal washing^,^ cerebrospinal fluid," and body cavity fluids." Cytologic examination of sputum is now an established procedure in the diagnosis of malignancies of lung. However, it has also been shown that the diagnosis of nonmalignant conditions of the respiratory tract is also possible by this method." One of these conditions is due to HVI,'2+'3 which is characterized by the presence of multinucleate and/or single cells with

Received October 26,1986. Accepted January 15,1987. From the Cytology Unit, Department of Pathology, Wellington

Hospital and Wellington Clinical School of Medicine, Wellington, New Zealand.

Address reprint requests to Raj K. Gupta, MD., F.I.A.C., Head, Cytology Unit, Wellington Hospital, Private Bag, Wellington, New Zealand.

enlarged, molded, opaque basophilic nuclei of ground- glass appearance. A careful scrutiny of the material usually discloses the presence of homogenous intranuclear inclusions, and experimental data have shown that the inclusions are a late event in the course of HVI in vitro.14 Previously, the diagnosis of HVI of the respiratory tract was only possible at postmortem or by the time-consum- ing method of tissue culture and/or diagnostic rise of antibody titer. In recent years, the application of immu- noperoxidase techniques in cases of HVI has provided a useful, rapid, and relatively simple method of diagnosis in cytologic materiaI.15-22

This study relates our experience in the detection of unsuspected HVI of the respiratory tract in deep-cough samples of sputum by the Papanicolaou smear method and confirmation by the immunoperoxidase technique. The sputum samples had been submitted for various respiratory illnesses during an 8-yr period ending October 1986.

Materials and Methods From 18,373 cases in which sputum cytology had been performed, Papanicolaou stained smears were reviewed in 12 cases in which a diagnosis of HVI infection had been made. In all cases, sputum samples had been received in a fresh unfixed state, and direct cellular spreads were made on at least four slides from grossly significant areas in specimen (i.e., bloody, mucoid, nonpurulent, tissue flecks; nonsaliva) and immediately fixed in 95% alcohol. All slides were screened, and findings of HVI cells were reported with a recommendation for examination of oral cavity for ulcers and cytology of oral scrapings. In all 12 cases, clinical notes were reviewed for patient details.

Q 1987 IGAKU-SHOIN MEDICAL PUBLISHERS, INC. Dlagnostk Cytopathology, Vol3, No 4, December 1987 287

N

m

W

c

Y)

0

v

0

C

w

4

P m

Tabl

e I.

Clin

ical

, Cyt

olog

ic, a

nd Im

rnun

oper

oxid

ase D

ata

in T

wel

ve P

atie

nts

With

Her

pes V

irus

Infe

ctio

n

Sput

um

Ora

l scr

apes

M

ulti-

M

ulii-

Im

mun

oper

oxid

ase

4

Sing

le

nucl

eate

Si

ngle

nu

clea

te

Sput

um

Ora

l scr

apes

> !+

__

_

Sing

le

Mul

tinuc

leat

e Si

ngle

M

ultin

ucle

ate

celIs

ce

ils

cells

C

ase

number

Age

Se

x A

ssoc

iate

dclin

ical

dise

ase

ON

H

I ON

HI

ON

H

I ON

HI

cells

ce

lls

cells

ce

lls

Cou

rse

1 69

M

CO

RD

,cer

ebro

vasc

ular

acci

dent

; F

R F

F N

ot d

one

HSV

- 1

HSV

-1

Not

don

e D

ied;

T

/1 y

es; O

CN

U

C+

+ C

+ +

no au

tops

y N+

N+

C+

+ C

+ +

N-

N*

2 79

M

Pneumonia;CORDT/Iyes;OCNU F

R F

F N

ot d

one

HSV

-I

HSV

-1

Not

don

e R

ecov

ered

3 79

M CORD, he

mop

tysi

s,w

eigh

t loss,?

F F

F F

Not

don

e H

SV- 1

H

SV-1

N

ot d

one

Rec

oVed

C

A lu

ng; T

/I y

es; O

CN

U

C+

C

+ +

lung

s; P

neum

onia

on

chem

othe

ra-

Cf

+

C+

+

py; T

/I no; O

CN

U

N+

N

+

N-

N*

4 22

M

Syn

ovio

maw

ithm

etas

tasi

sbon

e;?

R R

F F

Not

don

e H

SV-2

H

SV-2

N

ot d

one

Recovered

5 65

M

CORD,adenocarcinomalung;T/I F

R

F

F N

ot d

one

HSV

- 1

HSV

-1

Not

don

e D

ied;

no; O

CN

U

C+

+

C+

+

no a

utop

sy

N*

N*

6 61

M

CO

RD

, hem

opty

sis;

T/I

yes

; F

FF

F

Non

e H

SV-1

H

SV-1

N

one

Rec

over

ed

OCNU

C+

C

++

N

- N%

C+

+

Ct +

N-

N-

OC

NU

C

+

C+

N

%

N&

7 72

M

CO

RD

; T/I

yes;

OC

NU

F

FM

M

Non

e H

SV-1

H

SV-1

N

one

Rec

over

ed

8 58

F

Lu

ng a

bces

s; C

OR

D; T/1 ye

s;

FF

FF

N

one

HSV

-2

HSV

-2

None

Rec

over

ed

9 52

M Alwholic;cirrhosis;CORD;T/I

F

R F

F F

R

M

M

HSV-1

HSV

-1

HSV

- 1

HSV

-I

Rec

over

ed

yes;

OC

UP

C+

+ C

+ +

C+

+

C+

+

N+

N*

N%

N?

C+

C

+

C+

Ct

no a

utop

sy

N-

N-

N-

N-

10

82

M

CO

RD

; T/I

yes

; OC

UP

F F

M

M

R R

F F

HSV

-1

RSV-

1 H

SV- 1

H

SV-I

D

ied;

11

59

M L

eukemiainremissiononchemo-

F F

M

M

F R

F F

HSV

-2

HSV

-2

HSV

-2

HSV-2

Rec

over

ed

ther

apy;

T/I

no;

OC

UP

C+

C+

C+

C+

no; O

CU

P C

+ Jr

c+

t C

+ +

C+

+ N

+_

Nt

N+

N?

12

74

F Pneumonia;pleuraleffusion;T/I

F F

M

M

F F

M

M

HSV

-1

HSV

- 1

HSV- 1

HSV

-I

Rec

over

ed

N+

N

+

N-

N?

Key

: OC

NU

, ora

l cav

ity-n

o ul

cers

; OC

UP,

ora

l cav

ity-u

lcer

s pr

esen

t; T

/I, t

rach

eost

omy

and/

or in

tuba

tion;

ON

, opa

que n

ucle

i; H

I, he

rpes

incl

usio

ns; C

OR

D, c

hron

ic ob

stru

ctiv

e res

pira

tory

di

seas

e; R, r

are:

l-l

O/s

mea

r; F

, few

: lO

-SO

/smea

r; M, m

any:

abo

ve 5

0/sm

ear;

HSV

-1, p

ositi

ve b

y im

mun

oper

oxid

ase

stai

n; H

SV-2

, po

sitiv

e by

im

mun

oper

orid

ase stain; C

, cyt

opia

sm o

f he

rpes

-infe

cted

cel

ls; N, n

ucle

us of

her

pes-

infe

cted

cells

; + +,

inte

nse s

tain

ing;

+, m

oder

atel

y in

tens

e sta

inin

g; f, tr

ace

stai

ning

; -, n

o st

aini

ng.

ORAL AND RESPIRATORY HERPES

Immunoperoxidase Study HVI cells displaying ground-glass appearance or inclu- sions in two of the four Papanicolaou-stained smears of sputum in each of the 12 cases and two slides of oral scrapes (cases 9-12; Table I) were first signaled by ink circles. These circles were then reproduced on the under surface of the slide by means of a diamond-tipped etcher for immunoperoxidase staining. The coverslips were removed by immersion of the smears overnight in xylene, and all the smears were destained in 1% acid alcohol. One of the smears of sputum in each case plus one smear of oral scrape from each of cases 9-12 were stained using the HSV-2, rabbit anti-HSV-2 serum (Dako, Copenhagen, Denmark) diluted 1:400 in pH 7.6 Tris-buffered saline (Sigma Chemicals, St. Louis, MO), while second smears of sputum and oral scrapes from cases 9-12 were stained using the primary antibody rabbit anti-HSV- 1 (Dako, McIntyre, Copenhagen, Denmark).

HSV-2 and HSV-1 (supplied by Dako) are supposed to be fairly specific, with minimal cross-reactivity . This has also been shown in the studies of Anderson et al.” and Vernon.lg Also, cross reactivity was not convincingly seen in any of our cases. The exact steps of immunoperoxidase procedure for HSV-2 were the same as used by Anderson et al.”; for HSV-1, the steps applied were the same as used by Vernon.” Histologic sections of esophagus known to contain virocytes served as positive controls while smears from cases of cytomegalovirus (CMV), human papilloma virus (HPV), and tissue sections of lungs with squamous-cell carcinoma and adenocarcinoma acted as negative controls in all cases. Additionally, specific con- trols included substitution of rabbit antisera of different specificity for the primary antiserum and substitution of buffer alone for primary antiserum.”

A positive result of immunoperoxidase staining was read when golden-brown to black precipitate in the cyto- plasm and/or the nucleus of the cells affected by HVI was present.

Results Table I summarizes the clinical, cytologic, and immuno- peroxidase findings in the 12 cases in whom the diagnosis of HVI was made. As shown in Table I, five cases, despite sputum findings of HVI, showed no oral lesions and oral scrapings were not submitted. In three cases (6,7, and 8) with HVI cells in sputum, no ulcers in the oral cavity were found and oral scrapings were also negative for HVI cells. In the remaining four cases, sputum and oral scrapings from ulcers in Papanicolaou-stained smears showed cells diagnostic of HVI; this finding was further confirmed by immunoperoxidase method (Figs. 1 and 2). The number of cells infected due to HVI varied from a few to many. These were easily recognizable in Papanicolaou-stained preparations. Nine of the 12 cases had an uneventful

Fig. 1. Sputum smear showing immunostaining in HVI cell (shown by arrows) using immunoperoxidase method ( ~ 9 5 0 ) .

recovery, while three patients later died. No autopsy was done in these three cases to ascertain whether HVI was in any way related to death.

Discussion A finding in the cases analyzed in this study was that the majority of patients (eight of 12) had had a tracheostomy and/or intubation due to chronic respiratory disease. The presence of HVI cells was also found to be more common in males. The number of cells infected did not correlate with the severity of the disease process or ultimate course. Similar observations have been made by Frable et al.’* Nine of the 12 cases fully recovered. Whether such a recovery was attributed to an early diagnosis of HVI by sputum cytology and treatment, or to a specific therapy for the medical condition for which the patient was being treated, remained somewhat uncertain. None of the cases were investigated by antibody titers, viral cultures, or electron microscopy. The cytologic and immunoperoxi- dase findings in sputum were considered quite reliable for the diagnosis of HVI.

Most studies have indicated that the HVI diagnosed from cytologic material is due to the herpes simplex virus.l-3.W 1 It is our feeling that in Papanicolaou-stained smears alone, a differentiation between herpes simplex virus types 1 and 2 is not possible unless the technique of immunoperoxidase is applied. This seems also to have been suggested by other studies using the immunoperoxi- dase techniques in cytologic material.’”* The mode of

Fig. 2. Oral scrape in above case with similar immunopositivity in HVI cells (shown by arrows) ( ~ 9 5 0 ) .

Diagnostic Cytopathology, Yo1 3. No 4, December 1987 289

GUPTA ET AL.

spread of HVI remained somewhat puzzling. While in four cases the finding of oral lesions and HVI in oral scrapes could account for aspiration as being the probable cause of respiratory infection, it was not possible to adequately explain the reason in eight cases. In five of these, oral scrapes had not been examined; in three cases, cytological examination or oral scrapes was negative. It was therefore considered reasonable to assume that at least in some of these cases, HVI was possibly due to associated severe medical problems such as pneumonia, an immunosuppressed state, and chronic respiratory disease.

Based on this study, an interesting finding was the specific staining for HSV-2 in the cytaplasm of HVI cells by immunoperoxidase in three out of 12 smears of sputum. Also, in one of these, the cytoplasm of HVI cells in oral scrapes (case 11) specifically stained for HSV-2. In the past, it has mostly been believed that in extrageni- tal sites the HVI is mainly due to HSV-1. While this appeared to be true in nine of our 12 cases in the smears of sputum using immunoperoxidase stains, it was not so in the remaining three cases. The exact reasons for the finding of HSV-2 in these cases remained unclear. An editorial in the L ~ n c e t ~ ~ discussed a changing pattern of HVI in humans, possibly ascribed to the practice of orogenital contact. No such history was present in these three cases.

The incidence of HVI in genitals and extragenital sites is increasing. Our study further indicates a need for its recognition. In a site like the respiratory tract, a simple method of diagnosis is by an initial sputum cytology, followed by immunoperoxidase staining, which seems eminently suited for the purpose of identification of HVI. It is also a noninvasive method of investigation, which is particularly important when dealing with the immuno- suppressed patient.

Acknowledgment The authors gratefully acknowledge the secretarial assistance of Jillian Lamboo in the typing of this manuscript. The technical assistance of Heather Baitd is also acknowledged.

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290 Diagnostic Cytopathology. Yo1 3, No 4, December 1987