INTRODUCTION

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The diagnosis of primary or recurrent genital herpes simplex virus (HSV) infections, which are mainly caused by HSV type 2 (HSV-2), is based on clinical symptoms, culture of clinical specimens, viral detection by nucleic acid amplification, and HSV antigen detection assays (4, 30, 34). HSV-1 and HSV-2 are closely related (13), and for the study of humoral responses to HSV infection, complement fixation assays, enzyme-linked immunoassays (EIA) with crude antigens, immunofluorescence assays, and neutralization assays all lack specificity due to the cross-reactivity of antibodies against HSV-1 and HSV-2 (3, 4, 5). Assays using type-specific HSV antigens which can be used to differentiate between HSV-1- or HSV-2-specific antibodies have been described (2, 7, 8, 18, 21, 24, 28; D. Alexander et al., Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. C-101, p. 18, 1996), with the immunoblot assay (Western blotting [WB]) considered the "gold standard" because it has been most extensively validated (1, 4). An alternative to WB which does not require affinity-purified antigen is detection of type-specific antibody by blocking monoclonal antibody (MAb) (28). Serological assays and especially type-specific assays can be used in seroepidemiological surveys and other studies of the transmission of genital herpes (10, 26, 29).

The objective of the present study was to compare an MAb-blocking EIA for HSV-2 antibody detection with a strip immunoblot assay (SIA) for HSV-1- or HSV-2-specific antibodies using serum samples from a sexually transmitted disease (STD) clinic population.

	MATERIALS AND METHODS

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The study population consisted of 1,683 STD clinic attendees (582 women and 1,101 men) who originally participated in a prevalence study of Chlamydia trachomatis infection during the period from 1986 to 1988. This cohort has been described previously (31, 32).

Serum specimens. A total of 1,612 serum specimens from the STD clinic population were available for this study, out of 1,683 specimens collected between 1986 and 1988. All specimens were stored at 20°C prior to testing.

MAb-blocking assay. Type-specific antibodies to HSV-2 were detected using an MAb-blocking EIA (with the MAb AP-1, against HSV-2 glycoprotein G-2 [gG-2]) at the Virus Reference Division, Central Public Health Laboratory, London, United Kingdom (17). This assay is a direct modification of the validated MAb-blocking radioimmunoassay (RIA) (28). Briefly, wells of Greiner microtiter plates were coated with an HSV-2-infected-cell lysate at a 1:25 dilution in phosphate-buffered saline (PBS) overnight at 4°C, followed by detergent (1.5% Triton X-100 and 0.5% Nonidet P-40 in PBS) for 30 min at room temperature. After incubation for 2 h at 37°C with PBS containing 10% fetal calf serum, wells of coated plates were incubated successfully for 1 h at 37°C with the following in PBS containing 10% fetal calf serum and 0.2% Tween 20: a 1:4 dilution of test serum, a 1:16,000 dilution of HSV-2-specific MAb, and a 1:1,000 dilution of horseradish peroxidase-conjugated anti-mouse MAb. Plates were washed three times between each stage using 0.05% Tween 20 in PBS. Colorimetric detection using tetramethyl benzidine substrate at room temperature in the dark followed, with the reaction stopped after 20 min by the addition of 2 M H₂SO₄. The optical density of each well was then measured immediately at 450 nm (and at 620 nm for reference) (OD_450/620), and the percent blocking of each serum was calculated by comparison with diluent controls and with the mean OD_450/620 of four wells containing a positive control serum (as described in reference 22, but substituting OD_450/620 measurements for measurements of counts per minute). Sera were considered positive for HSV-2 antibody if they gave a blocking value of 50%. This cutoff was set using the mean result plus three standard deviations for sera from a population of children (n = 213) considered negative for antibody to HSV-2. An in-house WB assay was used to confirm the sensitivity and specificity of the testing strategy (17).

SIA. The Chiron RIBA HSV-1 and -2 SIA (Chiron Diagnostics, Emeryville, Calif.) utilizes antigens immobilized on nitrocellulose strips as described by Alexander and colleagues (Alexander et al., Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996). gB-1 is an HSV-1-specific peptide and gG-1 is a recombinant protein specific for HSV-1. gG-2 is a recombinant protein from HSV-2, and HSV-2 gD-2 is cross-reactive for HSV-1 and HSV-2 antibodies due to 85% conservation. The gG antigens were expressed in yeast as N-terminal-fusion proteins with superoxide dismutase; a C-terminal truncation of gD-2 was expressed in Chinese hamster ovary cells. The assay was carried out according to the instructions of the manufacturer at the Municipal Health Service of Amsterdam. Control bands for two levels of human immunoglobulin G (IgG) were also spotted on the immunostrip. A test was valid only if the human IgG control band was present. The intensity of the reaction was scored relative to that of the IgG control bands as follows: , absent; 1+, equal to the intensity of the level I IgG control band; 2+, greater than level I and less than the level II IgG band; 3+, equal to the intensity of the level II IgG control band; 4+, greater than the level II IgG band. Antibody status was based on the banding pattern. The cross-reactive HSV-2 gD-2 band indicated only the presence of HSV antibody. The HSV-1 gG-1 and/or gB-1 band and the HSV-2 gG-2 band indicated the presence of HSV-1- and HSV-2-specific antibodies, respectively. Single-band reactivity to HSV-2 gG-2, HSV-1 gG-1, or HSV-1 gB-1 in the absence of the HSV-2 gD-2 band was interpreted as HSV antibody negative. Reactivity to only HSV-2 gD-2 was interpreted as the presence of HSV antibody, not typed.

Discrepancy analysis and gold standard. Specimens with discrepant results were further analyzed by HSV-2 WB assay using HSV-2 proteins as the antigen. This was carried out at the Institute of Virology in Rotterdam, The Netherlands, as previously described (1). Immunoblots without visible bands were treated with a colloidal gold stain (Aurodye; Jansen Life Sciences), and in some cases bands became visible after prolonged incubation, indicating the presence of HSV-2 antibody at a low concentration. Sera were considered to contain HSV-2-specific antibody if results were positive by both EIA and SIA; if the result by EIA and SIA were discrepant, the HSV-2 WB result was considered decisive. If WB retesting gave an equivocal result, then the serum was assumed to contain HSV-2-specific antibody.

Statistical analysis. Data were analyzed with confidence interval analysis (16) and Epi Info, version 6.3 (12).

	RESULTS

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HSV-2-specific antibody results were obtained by MAb-blocking EIA and SIA for 1,612 sera (Table 1). In the EIA, 541 (33.6%) sera were positive and 1,068 sera were negative for HSV-2 antibody, with 3 sera giving equivocal results. SIA detected HSV-2-specific antibody in 555 (34.4%) serum specimens; 144 (26%) of these 555 sera were positive for HSV-2 antibody only, and 411 (74%) sera contained HSV-1 and HSV-2 antibody. Another 16 sera were positive for HSV antibody but could not be typed. HSV-1 antibody was detected by SIA in 1,155 (71.6%) sera, with 744 (64%) of these sera containing HSV-1 antibody alone and 411 (36%) sera containing antibody to both HSV types. Comparison of the EIA and SIA results revealed that 512 of the 541 EIA HSV-2-positive sera were also positive for HSV-2 antibody by SIA, resulting in a concordance of 94.6% for the SIA relative to the EIA. Of 555 SIA HSV-2-positive sera, 512 were EIA positive, giving a concordance of 92.3% for the EIA relative to the SIA (Table 1).

Pattern of SIA bands in sera with discrepant results. Eight serum specimens with discrepant results were HSV-2 antibody positive by EIA and HSV-1 antibody positive by SIA. Twenty-one sera were HSV-2 antibody positive by EIA and negative by SIA for HSV-1 and HSV-2 antibodies. Eighteen of these 21 sera were reactive by SIA for the gG-2 band. However, these 21 samples were interpreted as SIA negative due to the absence of reactivity to the HSV-2 gD-2 band.

Discrepancy analysis by HSV-2 WB. Confirmation by WB of discrepant SIA and EIA HSV-2 antibody results is reported in Table 2. Five of the eight EIA HSV-2-positive and SIA HSV-1-positive sera were available in sufficient quantity to be tested by HSV-2 WB. Four of these five were HSV-2 antibody negative, and one serum specimen tested positive by HSV-2 WB. Nineteen of the 21 EIA HSV-2 antibody-positive and SIA HSV-1- and HSV-2-negative sera could be tested by HSV-2 WB. Of these, one serum specimen was HSV-2 WB negative, three reacted equivocally, and two were positive. For the remaining 13 serum specimens, bands were seen on the immunoblots only after Aurodye colloidal gold stain was added, indicating the presence of HSV-2 antibody at a low concentration.

Sensitivity and specificity of EIA and SIA relative to the gold standard (HSV-2 WB). Sensitivity and specificity characteristics of both assays (presented in Table 3) are based on the findings reported in Table 2. Eighty of the 87 sera with discrepant EIA and SIA results could be tested by WB (n = 52) or by COBAS HSV-2 (n = 28). The calculated assay characteristics (Table 3) are based on the assumption that the WB assay used to resolve discrepant EIA and SIA results was the gold standard. Seven sera with equivocal WB results were assumed to contain HSV-2-specific antibody.

	DISCUSSION

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This study demonstrates a high degree of concordance between the HSV-2-antibody-specific EIA and the HSV-1 and -2 SIA. Analysis of the EIA HSV-2 antibody-positive and SIA HSV antibody-negative serum specimens by HSV-2 WB indicated the presence of HSV-2 antibody in 15 of 19 specimens, albeit at a low concentration. Similarly, 9 of 13 of the EIA HSV-2 antibody-negative and SIA HSV-2 antibody-positive specimens could be confirmed by HSV-2 WB. Of the EIA HSV-2-negative and SIA nontypeable serum specimens, most (12 of 15) could not be confirmed by HSV-2 WB, whereas all 28 EIA HSV-2-negative and SIA HSV-1- and HSV-2-positive sera were confirmed as HSV-2 antibody positive by the COBAS assay.

Discrepancies between the assays may be due to differences in the presented antigens or in their ability to detect a type-specific humoral response following primary infection. In the blocking assay, an MAb (AP1) raised against gG-2 was used. Humoral responses to all HSV-2 infections may not include reactivity to this epitope. Differences between the sensitivities of both assays were magnified by the group of 28 EIA HSV-2-negative and SIA HSV-1- and HSV-2-positive sera which had been tested by COBAS EIA using gG-2 as the antigen. In this group, 20 sera gave a weak reaction against the HSV-2-specific gD-2 antigen spotted on the SIA. A detailed analysis of differences between various gG-based serologic assays was reported recently (27). No single explanation was found, although inconsistent test results were partly associated with weakly positive specimens.

The correlation between the presence of HSV-2-specific antibody detected by EIA and primary episodes of HSV-2 genital herpes has previously been studied in this population by Van de Laar et al. (31). In that study, medical history and clinical presentation were used to determine whether a symptomatic patient had primary genital herpes, and swabs from genital lesions were obtained for viral culture. A low rate of detection of HSV-2-specific antibody in current primary episodes of genital herpes was obtained. Only 19 of 34 (56%) sera from primary HSV-2 episodes contained HSV-2 antibody (31). The findings of that study were confirmed by the present study, as the SIA detected exactly the same sera containing HSV-2 antibody in the subgroup of individuals with primary HSV-2 infection. According to the literature, HSV type-specific antibody may not be detected reliably within 8 weeks of onset (2, 11, 20, 28). In the present study, the interval between onset of primary infection and the development of specific antibodies could not be determined, as only one serum specimen was available from cases of primary HSV infection. As discussed earlier, for the population in the present study, sensitivity differences between the EIA and SIA were not accounted for by differences in the ability to detect type-specific antibody during a primary episode.

In the development of the original MAb-blocking RIA, WB results using HSV-1 and HSV-2 antigens were used separately to validate the assay in a panel of sera collected from 64 individuals with culture-typed genital herpes (28). Twenty-one sera were obtained from patients with HSV-2 first episodes, and 25 sera were from patients with HSV-2 recurrences. In that study, WB analysis demonstrated greater sensitivity than the MAb-blocking RIA for detecting HSV-2 antibody following HSV-2 first episodes, with sensitivities of 19 of 21 (91%) and 16 of 21 (76%) true positives, respectively.

Other comparative studies of recently developed commercial HSV type-specific antibody assays have been described (14, 18, 19; A. J. Vyse, M. J. Slomka, D. W. G. Brown, D. Lewis, and J. A. Corney, EUROGIN Int. Conf. Herpes Viruses Genital Pathol., 1996). The commercial availability of those assays offers possible diagnostic tools for the clinical management of patients with genital herpes. However, the role of serological testing in the care of HSV-infected individuals remains to be established. A retrospective study suggested that serology would be more useful in diagnosing and managing recurrent disease than for primary HSV infection (25). Recurrent genital herpes is often asymptomatic or atypical (23, 35). The assays evaluated in the present study may be useful for the diagnosis of recurrent genital herpes. The case has been presented for screening to identify asymptomatic HSV-2 infections among STD clinic attendees. Here it is argued that this screening can provide a means of controlling the spread of genital herpes (9). Several issues require clarification before such an approach can be introduced, although according to some authors the patients are prepared to accept the findings of type-specific serology (6, 22; R. Brugha, D. Brown, A. Meheus, and A. Renton, Editorial, Sex. Transm. Infect. 75:142-144, 1999). Similarly, the benefit of using these tests to diagnose genital herpes just before term must be determined and will be dependent on the incidence of neonatal herpes. For example, in The Netherlands there is a very low incidence of neonatal herpes, with approximately half of the cases due to transmission of HSV-1 by health care workers (33).

We conclude that both the SIA and the blocking EIA are suitable assays for the detection of antibody against HSV-2, showing a high level of agreement in detecting HSV-2 antibody in the population studied. With regard to the applicability of either assay, the blocking EIA is a useful tool for large epidemiological studies, whereas the SIA proved to be a slightly more sensitive method.