Elisa for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis

Journal

of Virological

7 ( 1983) 1 I l- I25

Meihods,

117

Elsevier

ELISA

FOR THE DETECTION

THE CEREBROSPINAL

R.M. COLEMAN’. ‘Deparrmenr

(Accepted

Emory

University

OF PATIENTS

R.J. WHITLEY*,

University

of Alabama,

SIMPLEX WITH

AL,

ANTIGENS

IN

ENCEPHALITIS

H. KEYSERLING’

School of Medicine, Birmingham,

VIRUS

Atlanta,

and A.J. NAHMIAS’

GA. and ‘Department

ofpediatrics

U.S.A.

27 May 1983)

An inhibition antigens

FLUID

P.D. BAILEY’,

of Pediarrics,

and Microbiology,

OF HERPES

enzyme-linked

in cerebrospinal

type 1 IHSV-I)

immunosorbent

assay (ELISA)

fluid (CSF) has been developed.

for the detection

A Triton

X-100 extract

infected HEp-2 cells was used to coat wells of polyvinyl

globulin

served as the reference

Positive

results were obtained

(65%) older individuals

antibody

and the CSF specimens

in CSF specimens

with HSV culture

positive

negative

brain biopsies.

The assay was negative

patients

with bacterial

meningitis

patients

within

herpetic

infection.

herpes simplex

10 days of onset, The overall

virus

ELISA

and

from

chloride

of herpes simplex virus of herpes simplex virus

plates. Rabbit anti-HSV-1 of 1 : 4.

were tested at a tinal dilution

I l/18 (61%) neonates with HSV infection,

brain biopsies,

and in 4/29 (14%) patients

with CSF from 14 infants without

10 with cryptococcal

meningitis.

15/23

with culture

HSV infections,

The test was positive

from 30 in IO/21

1l/14 within I I-20 days, and in 5/6 more than 20days after onset of the sensitivity

CSF

of the assay was 63% and the specificity

HSV antigen

was 95%.

encephalitis

INTRODUCTION

Herpes morbidity

simplex encephalitis (HSE) is a devastating disease of high mortality and (Whitley et al., 1980; Nahmias and Whitley, 1981). However, it has been

recently demonstrated in newborns and older individuals that mortality and morbidity can be reduced by appropriate antiviral therapy (Whitley et al., 1977; Whitley et al., 1981). To date, in some newborns and in almost all older patients, diagnosis has to be based on brain biopsy since herpes simplex virus (HSV) is most often not isolated from the cerebrospinal fluid (CSF) of patients with HSE (Whitley et al., 1980a, b; Nahmias et al., 1981). We have recently reported that determination of HSV antibodies in serum and CSF is not usually helpful for the early diagnosis ofthis entity (Nahmias et al., 1982). The present report presents our experience with an enzyme-linked immunosorbent inhibition assay for the detection of HSV antigens in CSF specimens.

0166-0934/83/$03.00

0 1983 Elsevier Science Publishers

B.V.

118

MATERIALS

AND METHODS

Patient specimens Cerebrospinal

fluids were obtained

from patients

with brain biopsy confirmed

or

suspected herpes encephalitis, bacterial meningitis, or cryptococcal meningitis. Many of the CSF specimens from the patients with or without HSV positive brain biopsies were obtained from the Collaborative Antiviral Study Group and most were assayed under code. CSF specimens from neonates without HSV infection were also used as controls. Most of the CSF specimens had been frozen at -70°C some for as longas 3yr. Brain biopsies, CSF, and other clinical specimens were inoculated on primary rabbit kidney cells for virus isolation. HSV isolates were serotyped by direct immunofluorescence tests (Nahmias antibodies by either indirect

et al., 1971a). Serum specimens were tested for HSV immunofluorescence (Nahmias et al., 1971b), passive

hemagglutination assay (ELISA).

and Nahmias,

(Schneweis

1971), or enzyme-linked

immunosorbent

Viruses HSV-1 (F strain) and HSV-2 (MS strain) were propagated in HEp-2 cells grown in minimal essential medium containing 0.16% sodium bicarbonate, 0.3% HEPES buffer, 5% fetal calf serum, 5% newborn calf serum, and 10 &ml of gentamicin. After HSV infection, the cells were maintained in the same medium with 2% newborn calf serum. Antigen preparation HSV-1 and HSV-2 infected HEp-2 cells were allowed to progress to a 4’-cytopathic effect (24-48 h). The cells were harvested, sonicated in pH 8.6 Tris-glycine buffer containing

2% Triton

X-100 (Vestergaard

et al., 1977), clarified

by centrifugation

for

10 min (250 Xg), aliquoted, and stored at -70°C. Control antigen of uninfected HEp-2 cells was prepared by the same method. Supernatants of HSV infected cells and uninfected HEp-2 monolayers were also stored at -70°C. Other control antigens included supernatants from varicella-infected human foreskin fibroblasts (HFF) 5 days after infection, supernatants from AD- 169 cytomegalovirus (CMV)-infected HFF 5 days after infection, and supernatants from uninfected HFF. Antibody

preparation

Antiserum prepared in rabbits against HSV-I was kindly supplied by Dr. B. Norrild (University of Copenhagen, Denmark). The virus was grown in rabbit cornea1 cells to

119

maximum

cytopathic

effect and were then sonicated

(PBS) with 0.1% Triton

in phosphate

X- 100. Each rabbit was injected subcutaneously

buffered

saline

with 0.5 ml of

vaccine (equal volumes infected rabbit cornea1 cells and Freund’s incomplete adjuvant). The rabbits were given three biweekly injections, a booster injection after 1 mth, and bled 7 days later. precipitation al., 1980).

The globulin

and adjusted

fraction

was prepared

to 35-40 mg protein/ml

as previously

by ammonium described

sulfate

(Norrild

et

Enzyme-linked immunosorbent assays Optimal

dilutions

of the antigens

for coating

the wells, the antiserum,

and the

peroxidase-labeled conjugates were determined by block titrations. Cooke 96-well polyvinyl chloride flat-bottom plates (Dynatech Laboratories, Inc., Alexandria, VA) were cleated with 100 ul of antigen overnight at 4°C. Buffers were 0.05 M sodium carbonate, pH 9.6, with 0.02% sodium azide for dilution of coating antigen; PBS, pH 7.4, with 1% bovine serum albumin and 10% inactivated fetal calf serum for dilution of CSF specimens, antisera, and conjugates; and PBS with 0.05% Tween 20 for washing the microplates. For the inhibition assay for antigen, 50 pl of CSF was incubated with 100 pl of the appropriate dilution of the anti-HSV-1 globulin and 50 ul of buffer in an uncoated polystyrene microtiter plate for 2 h at 37°C. Negativecontrols,consistingofbufferonly and supernatants from uninfected HEp-2 cells, were included in each assay. The positive control was the supernatant from HSV-1 infected HEp-2 cells. Fifty ul of the antige,n-antibody mixture was added to each of four wells of an HSV-1 antigen-coated plate (previously washed 3 X for 3 min each). After incubation for 2 h at 37”C, the plate was washed three times before adding 100 ul of peroxidase-corrjugated goat anti-rabbit IgG (Miles Laboratories,

Elkhart,

IN). After a further incubation

of 45 min at 37”C,

the plate was washed three times and 200 ul of pH 6.0 substrate solution containing 40 mg of O-phenylenediamine-2HCl (Eastman Kodak Co., Rochester, NY) and 0.005% H,O, in 100 ml of substrate buffer (7.74 g of citric acid and 17.93 g of Na,HPO,/I of water) was added. The reaction

was terminated

after 15 min at room temperature

with

50 ul of 4 N H,SO,. The absorbance was measured at 450 nm. The arithmetic mean and standard deviation were calculated for each specimen tested. A CSF specimen was considered positive if the mean absorbance was at least three standard deviations less than the mean of the negative controls. Variation coefficients (C,) were calculated as the SD expressed as a percentage of the mean. The antibody assay was performed in HSV-1, HSV-2 and uninfected HEp-2control antigen-coated wells. For screening 100 ul of a 1 : 100 dilution of the CSF specimens was incubated in antigen-coated wells for 2 h at 37°C. The plates were washed three times, 100 ul of peroxidase-conjugated goat anti-human IgG (Miles Laboratories, Elkhart, IN) was added. After incubation at 37°C for 45 min, the plates were washed, substrate added, and the absorbance measured as described for the antigen assay. If

120

the screening dilution was positive, the specimens were further diluted and the titer was determined as the highest dilution which demonstrated greater absorbance in the HSV-1 and/or

HSV-2 antigen

wells compared

to the HEp-2

control

wells.

RESULTS

Standardization

of the assays

For the antibody assay, the optimal concentration of the HSV-1 antigen was 1 : 3,200 and of the HSV-2 antigen was 1 : 1,600. Box titrations demonstrated that for the inhibition assay the optimal dilution of the HSV-1 antigen for coating the plates was 1 : 5,000 and that for the reference HSV- 1 globulin was a dilution of 1 : 12,800 (final = 1 : 25,600). Evidence for the specificity of the inhibition assay for HSV antigen(s) was obtained by demonstrating that HSV-1 and HSV-2 supernatants were inhibitory, whereas CMV, varicella, or control non-infected cell supernatants were not. Representative results obtained in the inhibition assay for detection of HSV antigens are shown in Table 1. Maximal enzyme activity for the negative control using non-infected cell supernatants was A,,, = 0.28-0.35. Coefficient of variation was generally <20. The mean of the negative control minus 3 SD was 0.24 for this assay which corresponded to a 20% reduction. The A,,, nm of most positive CSF specimens was at least 20% lower than the negative control.

TABLE ELISA

1 inhibition

assay for detection

+’ HSV antigens*

c,**

Reduction*** (%I

Positive control

0.14 k 0.01

7

Negative

0_30 * 0.02

1

I

0.21 i 0.01

30

2

0.24 i 0.01

5 4

3

0.19 * 0.02

II

37

4

0.25f

0.01

4

I7

5

0.29 + 0.01

3

3

6

0.30 + 0.01

3

0

0.30 * 0.03

IO

0

x

0.28 It 0.004

I

CSF

*

control

Underlined

values are at least 3 so less than the mean ofthe negatlvrcontrol.

mean adsorbance ** CoelTicient ***q’ Reduction

of quadruplicate

values + SD.

of variation. compared

to negative

control.

53 0 20

Results areexpressed

as the

121

HSV antigens and antibodies The antigen

ofne~~borns

in the CSF

assay was positive

in at least one CSF specimen

obtained

from 11 of the

18 newborns with HSV infections, including 9 of 12 with central nervous system (CNS) manifestations and 2 of 6 with no apparent acute CNS disease (Table 2). HSV antigen(s) were detected in the CSF within 10 days after onset in 6 of 12 patients, within 11 to 20 days in 4 of 5 infants, and in more than 20days in 1 neonate. The assay was negative with CSF from 14 infants without HSV infections. HSV was cultured from the CSF of only three ofthe infants with CNS involvement. Viruses were isolated from other clinical specimens of 13 of the HSV infected infants. Two of the isolates were HSV-1, 12 were HSV-2, and two were not serotyped. HSV antigens und anfibodies

in rhe CSF of older pafients

The results of the inhibition ELISA with CSF of patients with brain biopsy positive HSV encephalitis are shown in Table 3. The assay was positive in the CSF from 15 of TABLE: 2 Detection

of HSV antigens

(Ag) and antibodies

Type of

Days after

disease

onset

(Ab) in CSF of newborns

No. with ELlSA

No. Ag’ -

No. Ag+ -

Total no.

Total no.

patients

specimens

4/l

4/10

(5)

4/4

8114

(3)

l/l

6/9

reactions

Ag’

Ag-

Ag-

-Ap’ Ab-

Ab’

2


2(2)**

22)

0

II-20

3(7)

I(l) 1(5)

0

0

l(1)

2(2)

215

2/5

;

HSV infection with CNS*

3(b)

involvement >20

(I)

(I)

HSV infection without apparent


20

CNS

involvement

1I-20

0

0

0

l(2)

O/l

o/2

>20

0

0

0

(1)

o/o

O/l

N.A.***

0

0

4(4)

lO(11)

O/l4

O/15

numbers

with repeat specimens

No HSV infection

*

CNS = central

** Number

nervous

of patients

only be bracketed. ***Not

applicable.

system.

(number

ofspecimens);

on the same patient would

122

TABLE

3

DetectIon

of antigens (Ag) and antibodies

Days

No. with ELISA

(Ab) in CSF t’rom patients with HSV-posltlw

reactions

No. Ag’

braln hiops)

No. Ag’

after onset

AC’ --z-

Aem 2

Ag’

AC_ &

Ali_

Ab’

Ah’

Ab-

Total

90

l(l)*

3(3)

0

5(9)

4/9

I I-20

l(I)

h(7)

2(3)

(1)

7/9

>20

0

4(ll)

(4)

l(3)

4/5

*

Number

o( patients (number

no.

patients

Total no. specimens

4/13 WI2 II/IX

01 specimens).

23 (65%) patients tested and 23 of 43 (53%) total specimens from these patients. CSF from 4 of 9 patients were antigen positive within 10 days after onset, 7 of 9 within 1 l-20 days, and 4 of 5 more than 20 days after onset. When CSF specimens were positive for both antigen and antibody the range of antibody titers was 1 : 100 to 1 : 12,800, with a mean of 1 : 800. In those cases with negative antigen reactions, the range of antibody titers was 1 : 800-l : 6,400, with a mean of 1 : 1.600. CSF from 4 of the 29patients (14%) with HSV-negative brain biopsies were positive in the antigen assay (Table 4). Two of these 4 patients had demonstrated a four-fold increase in serum antibody titers and negative CSF antibody by passive hemagglutination and/or indirect immunofluorescence. Thirty CSF specimens from patients with bacterial meningitis and ten from patients with cryptococcal meningitis were found to be negative in the antigen assay. Yet, the CSF titer A HSV

from one of the cryptococcal meningitis patients demonstrated an HSV antibody of i : 100. summary of our total experience to date with the inhibition assay for detecting antigen in CSF specimens is presented in Table 5. The antigen assay was positive

TABLE Dctcction

4 of antigens (Ag) and antibodies

Day

No. with ELISA

(Ab) in CSF from patients uith

reactions

HSV-negative

No. Ag’

brain biopsy No. As+

after onxt

A&!’ -

< IO I I-20 >2O *

AC c

Total

no.

Total no.

patients

specimens

3/1x

Ab’

Ab’

Ab-

3(j)*

0

0

14( 15)

3/17

0

l(I)

l(2)

f)(8)

l/b’

l/l1

0

0

4(4)

o/4

2/6

(2) Number

AC i

of patlcnts (number

of specimens)

123

TABL.E 5 Summary Patient

of detection group

of HSV antigens No. patients

(Ag) in CSF Age/No.

tested (%)

Total (‘7r)

Days after onset

Infants

I I-20

>20

6/12* (50%)

415 (80%)

l/l

(100%)

1l/l8 (61%)

4/ 9

(44%)

719 (77%)

415

( 80%)

15/23 (65qx)

3/17

(18%)

l/8 (12.5%)

O/4(

with HSV

infection HSV-brain

biopsy

positive HSV-lbrain

biopsy

negative Infants

210

4/29 (14’7i’)

0%)

without

HSV infection*

o/14 (0%)

Bactel-ial meningitis*

o/30 (0%‘)

Cryptococcal meningitis* *

O/IO (0%)

D,lys after onset, data not available

on these groups

in at least one CSF specimen from 26 of the 41 (634)0 neonates or older patients with proven herpes encephalitis (two other HSV-infected neonates without acute CNS manifestations were also antigen positive). Herpes simplex virus was isolated from the CSF of only three of these newborns. Only 4 of the 83 (5%) HSE-negative demonstrated a positive reaction in their CSF.

patients

DISCIJSSION

Detection

of HSV antigen(s)

radioimmunoassay

in CSF by ELISA has not previously

for the detection

of HSV antigens

been reported.

A

in the CSF has been reported

(Chen et al., 1978). Because of the advantages of ELISA in permitting even small laboratories to perform such assays, we have concentrated our efforts in using this method. Earlier efforts at coating the plate directly with CSF specimens failed in preliminary tests. Although there have been other methods reported by other investigators for detecting HSV antigens (Miranda et al., 1977; Chen et al., 1978; Vestergaard and Jensen, 1981; Pronovost et al., 1981; Cleveland et al., l982), we have concentrated on the inhibition assay. The inhibition assay is relatively easy to perform since it requires only one specific antibody, the anti-species conjugates are readily available, and the test can be completed in about 6 h. Concern regarding the inhibition assay was the possible effect of antigen-antibody complexes (Yolken and Stopa, 1980). Our current results do not appear to support a relationship between the finding of antigen with or without antibody in the CSF.

124

Thus, patients) antigens

antigens

were detected

in 20 CSF

specimens

(from

brain

biopsy

positive

which had antibody titers ranging up to 1 : 12,80O(mean 1 : 800). Conversely, were nor detected in seven CSF specimens (from this same patient group)

with antibody

titers ranging

from

1 : 800-l

: 6,400 (mean

1 : 1,600).

Further investigation is needed to determine which antigen(s) are being detected in this assay. The positive reactions in CSF from infants known to be infected with HSV-2 (12/18) and in CSF from adults with HSV-1 from brain biopsies suggest that cross-reacting antigens to the two HSV types are being detected. If the specific antigens could be identified, monoclonal antibodies might be used in this test (Pereira et al., 1980). Particularly, there is need for improving results of this assay with specimens taken early after the onset of the disease (< 10 days). In this critical early period after onset, only half of the CSF specimens from neonates or brain biopsy positive adults were positive in the assay. In contrast, 75% of the specimens taken 1 l-20 days after onset and slightly more of those taken more than 20 days after onset were positive. Current efforts are therefore devoted toward increasing the sensitivity ofthe assay by the use of monoclonal antibodies and methods such as the biotin-avidin system (Guesdon et al., 1979). The ability to detect HSV antigens in CSF from patients with HSE would be a useful diagnostic tool as HSV is infrequently isolated from the CSF, particularly in non-neonate patients (Whitley et al., 1980a, b; Nahmias et al., 1981). In addition, early detection of HSE is critical since appropriate antiviral therapy can reduce both mortality and morbidity (Whitley et al., 1980; Nahmias and Whitley, 1981). At the present time, the false positive reactions (5%) and the sensitivity (63%) of the inhibition ELISA indicate that isolation of the virus from a brain biopsy will still be necessary for the early and definitive diagnosis of HSE in patients, particularly those beyond

the newborn

age. In addition,

other causes of the encephalitis,

HSV-negative

as indicated

brain biopsy may help establish

by Whitley

et al. (1981).

ACKNOWLEDGEMENTS

We thank Dr. B. Norrild, Univ. of Copenhagen, Denmark, for supplying the reference serum, Dr. W. Feldman, Emory Univ., for CSF specimens from patients with bacterial meningitis, and S. Blumer, CDC, Atlanta, GA, for CSF specimens from patients with cryptococcal meningitis. We also wish to thank N. Barton and G. Kerrick for assistance with patients and clinical records. These studies were supported by NIH-NIDR Training Grant DE-07074-05, Easter Seals Research Foundation Grant N-8229, and NIH-NIAID sub-contract NO l-Al12667 and NIH-NIAID Program Project Grant AI-19554-01. REFERENCES

Chen, A.B.. T. Ben-Porat.

R.J. Whitlq

and A.S. Kaplan.

1978. Virology

91. 324

125

Cleveland,

P.H., D.D. Richman,

Clin. Microbial.

D.C. Redfield,

D.R. Disharoon,

P.S. Binder and M.N. Oxman,

1982. J.

16, 676.

Guesdon,

J., T. Ternynck

Miranda,

Q.R., G.D.

and S. Avrameas,

Nahmias.

A.J. and R.J. Whitley,

Bailey, A.S. Fraser

Nahmias,

A.J., I. Del Buono,

Nahmias,

A.J., I. Del Buono,

1979, J. Histochem.

and H.F. Tenoso,

1981, Pediatr.

J. Pipkin,

27, 1131. Dis. 136 (Suppl.).

S 304.

Rev. 2, 659.

R. Hutton

K. Schneweis,

Cytochem.

1977, J. Infect.

and C. Wickliffe,

D.S. Gordon

197la,

Appl.

Microbial.

22, 455.

and D. Thies, 197lb, Proc. Sot. Exp. Biol. Med.

138, 21. Nahmias,

A.J.,

R.J. Whitley,

A. Visintine,

Norrild,

B., S.L. Shore, T.L. Cromeans

Pereira,

L.. T. Klassen

and J.R. Baringer,

Pronovost,

A.D.,

Schneweis,

K.E. and A.J. Nahmias.

A. Baumgarten

Vestergaard,

B.F. and 0. Jensen,

and R.F. Schinazi Vestergaard, Whitley,

(Elsevier

Y. Takei and C.A. Alford,

and A.J. Nahmias, 1980, Infect.

and G.D.

Immun.

1981, J. Clin. Microbial.

1981, in: The Human B. Norrild

Immun.

28. 38.

29, 724.

1971, 2. Immunitaetsforsch.

North-Holland,

B.F.. O.J. Bjerrum.

Hsiung,

Jr., 1982, J. Infect. Dis. 145, 829.

1980. Infect.

Allerg.

Herpesviruses,

13, 97.

Klin. Immunol.

eds. A.J. Nahmias,

141, 471. W.R. Dowdle

Inc., New York) p. 391. and P.C. Grauballe,

R.J.. S.J. Soon& R. Dolin, G.J. Galasso.

1977, J. Virol. 24. 82.

L.T. Chien and C.A. Alford,

1977, N. Engl. J. Med. 297,

289. Whitley.

R.J., A.J. Nahmias,

Whitley, R.J., A.J. Nahmias, trics 66, 495. Whitley, Alfcnd, Yolken,

R.J., S. Soong,

A.M. Visintine,

C.L. Fleming

S.J. Soong. G.G. Galasso.

M.S. Hirsch,

A.W.

Karchmer,

and C.A. Alford,

R. Dolin,

1981, N. Engl. J. Med. 304, 313. R.H. and P.J. Stopa,

1980, J. Clin. Microbial.

1980a, Pediatrics

C.L. Fleming and C.A. Alford,

II, 546.

G. Galasso,

66. 489.

Jr., 1980b, Pedia-

J.K. Dunnick

and C.A.