Opportunistic

Pathogens

ELSEVIER Opportunistic Pathogens 9 (1997) 3Y ~~41

Evaluation of new reagents for typing IgG to HSV-1 and HSV-2

Fernando de Ory *, Maria Eulalia Guisasola, Inmaculada Casas, Jo& Manuel Echevarria

Scrckio c/e Miuvhiolop’u Dicrpzdstiu, Centro Nucionui c/c Microbiolo~irr. Irrsrituro de S&d Curios III, .~fujcrduhonrkr. 28220 hltrdrid. Spain

Received 25 April 1997; accepted 26 June 1997

Abstract

Until recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-I and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases). normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-I. Only one sample was reactive in the HSV-I assay, and negative by the indirect ELISA. Type specific HSV-I and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection. 8 1997 Elsevier Science B.V.

Kc~~~or-ds: Herpes simplex virus type 1: Herpes simplex virus type 2; Glycoprotein G; ELISA: Sexually transmitted diseases

1. introduction

Serological diagnosis of herpes simplex virus (HSV) infections and identification of individuals previously infected by HSV-2, have been hampered until recently by the lack of suitable type specific assays. Bearing in mind the strong crossreactivity between HSV-1 and HSV-2, the correct characterization of type-specific an- tibody response was difficult to reach. Because the HSV-1 and HSV-2 glycoprotein G (gG1 and gG2. respectively) only contains type-specific antigenic deter- minants [I], several serological methods, including ELISA [2], enzyme immunodot [3], and Western blot [4,5] have been developed to detect type-specific anti- HSV using these proteins.

*Corresponding author. Tel.: + 34 1 5097901: fax: + 34 1 5097966: e-mail: fory(‘qisciii.es

1386-2618~97~%17.00 61 1997 Elsevier Science B.V. All rights reserved. PII S 1386.2618(97)00006-S

Recently, a Spanish company has marketed commer- cial kits for HSV-1 and HSV-2 specific IgG using recombinant (for HSV-1 assay) or purified (for HSV-2 assay) gG. The aim of this paper is to present an evaluation of these reagents.

2. Material and methods

Eighty two serum samples were used in the study. They were grouped as follows. (a) Thirty samples from 13 cases of HSV infections (Table 1). Cases 1 and 2 were from congenital infections; and the remaining were from postnatal infections, including meningitis (case 3) and encephalitis (cases 4-13). The etiological diagnosis was based on serology, and/or on detection of HSV DNA in CSF samples paired with the correspond- ing serum, as shown in Table 1. (b) Twenty four

40

Table I

Cases from HSV infections

case Age Days after onset CFT” EIA G’ EIA M” gGl’ gG7’ CSF’

I c

2C

3M

4E

5E

6E

IE

8E

9E

IO E

II E

12 E

I3 E

0 days

23 days

25 years

15 years

73 years

58 years

67 years

2 years

64 years

45 years

33 years

I8 years

30 41 23 60

4

20 8

23

50 2

I7 80

30 II

30

16 256

32 512

32

32 <64 256 256

<64 I28 I28

164 I28

164 256

<IO0 450

<IO0 450

< 100

0.3 I .o

0.8 0.7

2; n

n+6

27

120 IO 30

67 years

14 24

2 15

512 8

64

i64 <64

512 ~64 128

<R 32 32

~64 I28

x.7 7.3 1.0 9.2

3000

6500 12000

125 000 I20 000

IO 000 50 000

8000 3000

30 000 20.9 23.0

12.0 22.3 13.0 22.1

18000 40 000

20 000 550

2000

100 IO 000 IO 000

6500 16000

Pos Pos Ncg Pos Pos

Pos Neg Pos

Neg

Pos

% Pos

Neg

NC.g

W Pos

Neg Neg

200 < 100

0.1 0. I

< 100 <IO0

700 3000

4000 IO00

2200

1400 900

6500 950

I200

< 100 1000

850 IO 000

900

4000 <IO0 <IO0

<IO0 na

<IO0 900 950

0.6 0.4 0.8 0.3 0.7

< 100 < 100 < 100 < 100 < 100 < 100

<IO0 0.6 2.8

< IO0 na <IO0

0.2 0.5

n a na

na na HSV-2

na HSV-I Ncg

na HSV-I na na na Neg HSV nil

HSV na HSV

na HSV-I

Neg na Neg na HSV-I na

Neg HSV-I HSV-I

.* C. congenital: M, meningitis; E. encephalitis. ’ Reciprocal of titer.

’ HSVI +2 IgG: reciprocal of H titer or sample absorbance/cut off ratio. ” HSVI +2 IgM. ’ HSVI or HSV2 glycoprotein G 1gG: reciprocal of titer or sample absorbancc/cuf off ratio.

’ PCR result in CSF taken at the same time than the serum; na, not available.

samples from 20 patients attended in clinics for sexually transmitted diseases (STD). (c) Twenty eight samples for normal children aged 2-5 years.

The serological characterization of the cases was done by indirect ELISA and complement fixation test (CFT). The ELISA used was from a commercial source (Enzygnost HSV IgG, Behring. Germany). Quantitative or qualitative determinations were carried out. In quan- titative determinations. seroconversion was defined as a two fold titer increase between acute and convalescent samples. CFT was done by a standard procedure [6] using a commercial antigen (Virion, Switzerland) and 4 U of complement. CFT seroconversion was defined as a four fold variation in titer between acute and convales- cent samples. None of these two methods can differenti- ate type-specific anti-HSV antibodies. DNA detection in CSF samples was made by polymerase chain reaction assays (PCR) [7,8].

Indirect ELISA kits for detecting IgG antibody

against HSV-I (ELISA gl) and HSV-2 (ELISA 82) (BioElisa HSV-I y BioElisa HSV-2, Biokit SA, Barcelona, Spain) were evaluated. Briefly, samples di- luted (1 / 100) were inoculated into microplate wells con- taining the corresponding HSV-1 or HSV-2 glycoprotein G and incubated for 1 h at 37°C. The plates were washed five times and an anti-human IgG conjugated with peroxidase was added. After incuba- tion for 30 min at 37°C. the plates were washed as previously, and the substrate of the enzyme (3-3’,5-5’ tetrametyl benzidine) was added. The enzymatic reac- tion was stopped by adding H$O,lN and the ab- sorbance was read at 450 nm. A sample was recorded positive if the absorbance value was higher than the mean value of two determinations of the cut off control included in the kit. For the quantitative determinations, ten fold dilutions were assayed and the titer was cal- culated as the dilution showing the same absorbance value than the cut off control.

3. Results and discussion

The methods evaluated gave a type specific identifica- tion in 9/13 of the HSV recent infections studied (Table 1). In both congenital cases, seroconversions to HSV-2 were found. No serological response to HSV-1 or HSV- 2 was detected in the patient with meningitis caused by HSV-2 (case 3). Among cases with encephalitis, a sero- conversion or a two fold titer increase was seen in six cases to HSV-1 (cases 4, 5, 6, 8, 9 and 10). and in one case to HSV-2 (case 11). In cases with HSV-1 serocon- version having a CSF sample available. PCR for HSV was ever positive. In the single case showing HSV-2 seroconversion, the CSF sample was taken 10 days after the onset of neurological symptoms and was negative for HSV by PCR. In the three remaining cases (7, 12 and 13) no significant rise in antibody titer between acute and convalescent samples was seen.

Among samples from STD clinics (group b), a posi- tive result for HSV-2 IgG was obtained in four cases (20%). Nineteen cases (95%) were positive in the ELISA gl assay. Full agreement with indirect ELISA and CFT results was found. No cases positive for HSV-2 IgG and negative for HSV-1 IgG were detected in this group of patients.

Finally, among samples from normal children no positive results for HSV-2 IgG were obtained, whereas seven samples were positive for HSV-1 I&G. Six of these were positive in the Enzygnost assay.

In relation to the sensitivity of the assays, some aspects should be considered. In two cases (one menin- gitis and one encephalitis, cases 3 and 12, Table 1) with follow up samples taken at 20 and 24 days, respectively, no reactivity to HSV-1 or -2 was detected. This lack of reactivity could respond to a delayed antibody response to the gG in comparison with other proteins included in the ELISA and CFT. In a further case, a significant variation between acute and convalescent samples was, however. detected as early as 17 days (case 5). The anti-gG assays were less sensitive than the standard ELISA in terms of antibody titers, which were from three to more than 100 times lower. depending on the case. In the two cases of congenital infection studied, the anti-HSV-2 assay showed similar titers than the CFT.

The samples from normal children were included in the study in order to evaluate the specificity of the ELISA g2 assay. No positive results for anti-HSV-2 were obtained among children positive for anti-HSV- 1, thus suggesting that the method is highly specific.

Typing of HSV-specific antibody is rarely required for management of patients with acute HSV infections, but may be relevant for other purposes, such as epi- demiological studies or prevention of vertical virus transmission. Screening of pregnant women for anti- HSV-2 could be useful for identifying those at risk of transmission of HSV-2 to the newborn, and such se- lected women could be then monitored for virus shed- ding in cervix during the weeks prior to delivery. Since the anti-HSV-2 assay evaluated here seems to be highly specific, it seems likely that it may be useful for pre- venting neonatal HSV-2 infections, which may have serious consequences for the newborn. This possibility in under current investigation.

Acknowledgements

We thank Dr Angels Diez, from Biokit SA, Barcelona, Spain, for providing the kits for HSV-1 and HSV-2 antibodies.

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