Journal of Virological Methods 199 (2014) 29–34

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Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Evaluation of commercial herpes simplex virus IgG and IgM enzyme

immunoassays
Kristin Liermann, Anna Schäfler, Andreas Henke, Andreas Sauerbrei ∗
Institute of Virology and Antiviral Therapy, German Reference Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena,
Germany

a b s t r a c t

Article history:
Received 21 October 2013
Received in revised form
Serological methods are used widely for the determination of herpes simplex virus (HSV) IgG and IgM
25 November 2013
antibodies in virological laboratories. The present study evaluates the automated performance of the
Accepted 3 January 2014
Available online 10 January 2014
Virion\Serion (Würzburg, Germany) and Orgentec (Mainz, Germany) enzyme-linked immunosorbent
assays (ELISA) for the determination of HSV type-common and type-specific IgG and IgM antibodies. Two
hundred sixty-three sera from HSV-negative children, healthy blood donors as well as patients without
Keywords:
Laboratory diagnostic reagents and with acute HSV infections were included. The Serion ELISAs classic HSV 1 + 2, HSV 1 and HSV 2 IgG
HSV-1 showed sensitivities between 89.1% and 98.0% and specificities from 82.8% to 100%. Sensitivities of the
HSV-2 Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG were calculated as 91.0–96.0% and 88.5–95.4% accom-
Genital herpes panied by specificities between 93.1% and 100%. The HSV type-common Serion IgM ELISA revealed also
ELISA a high sensitivity and specificity. However, the single-type HSV-1 and HSV-2 IgM ELISAs from both com-
Glycoprotein G panies did not detect reliably HSV-1- and HSV-2-specific IgM antibodies. In conclusion, the automated
performance of Serion ELISAs classic HSV 1 + 2, HSV 1 and HSV 2 IgG as well Orgentec ELISAs Anti-HSV-1
and Anti-HSV-2 IgG provide highly dependable results for identifying HSV-1 and HSV-2 IgG-positive or
-negative individuals. While HSV type-common IgM ELISAs can be useful to confirm acute newly acquired
HSV infections, the use of single-type IgM ELISAs on the basis of whole-virus antigen is dispensable.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction infections are observed mainly in adolescents and adults. In devel-

Herpes simplex virus (HSV) has been described as one of the
most common pathogens affecting humans. Once acquired during
primary infections mostly in infancy, the virus remains latent life-
long in sensory local ganglia. Endogenous viral reactivations may
occur frequently resulting in recurrent infections such as labial or
genital herpes. Both, primary and recurrent HSV infections may
lead to substantial physical and psychological morbidity. There are
two types, HSV-1 and HSV-2, which show genetic differences and
differ also in the way of transmission, the body site affected pre-
dominantly and the seroprevalence. The viruses are transmitted
mainly by direct contact. The type 1 affects preferably the body
above the waist, and HSV-2 infections occur primarily below the
waist. While newly acquired HSV-1 infections occur mostly dur-
ing infancy and childhood, HSV-2 infections and recurrent HSV-1
∗ Corresponding author at: Institute of Virology and Antiviral Therapy, Jena
University Hospital, Friedrich Schiller University of Jena, Hans-Knoell-Strasse 2,
D-07745 Jena, Germany. Tel.: +49 3641 9395700; fax: +49 3641 9395702.
E-mail address: Andreas.Sauerbrei@med.uni-jena.de (A. Sauerbrei).
oped countries such as Germany, the seroprevalence of HSV-1
increases gradually in childhood and adolescence and reaches lev-
els up to about 90% in adults (Wutzler et al., 2000; Sauerbrei et al.,
2011). In contrast, a significant increase of HSV-2 IgG antibodies
is observed usually with the start of sexual activity (Smith and
Robinson, 2002; Sauerbrei et al., 2011). The seroprevalence of HSV-
2 varies as a function of age, sex, number of life-time sexual partners
and socio-economic status (Smith and Robinson, 2002) and ranges
from 10% to 60% (Wutzler et al., 2000; Gupta et al., 2007). Although
HSV-2 is the main cause of genital herpes, genital lesions may often
harbor HSV-1 (Gupta et al., 2007).
During the symptomatic phase, the direct detection of the virus
using polymerase chain reaction (PCR) and/or virus isolation in cell
cultures has been considered as the gold standard for the labora-
tory diagnosis of HSV infections (Sauerbrei et al., 2000; Finnström
et al., 2009). The domain of HSV serology is usually the determina-
tion of HSV type-specific IgG antibodies to identify virus carriers,
in particular of HSV-2, transferring potentially the virus to suscep-
tible persons (Sauerbrei and Wutzler, 2004; Bentley et al., 2012).
Herpes simplex virus type 2-specific assays offer the opportu-
nity to confirm the clinical diagnosis of previous HSV-2 infection

0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jviromet.2014.01.001
30 K. Liermann et al. / Journal of Virological Methods 199 (2014) 29–34
without using relatively expensive and labor-intensive methods
for viral detection (Bentley et al., 2012). In combination with the
PCR technique, the type-specific HSV serology can also be useful
to distinguish between the early phase of primary infections and
recurrent HSV-2 infections (Brown, 2002). Thus, HSV type-specific
serological assays may be of special value in view of the care of
pregnant women and their newborns. In contrast, the significance
of HSV-specific IgM antibodies for diagnosing acute HSV infections
is overestimated frequently since HSV IgM can only be detected
with a time delay of up to 10 days after exposure (Page et al., 2003)
and can cross-react with other herpes viruses, especially with the
varicella-zoster virus (VZV) (Sauerbrei, 2007). In addition, IgM anti-
bodies may be reactive not only in newly acquired HSV infections
but also in cases of asymptomatic HSV reactivation without clini-
cal relevance (Sauerbrei et al., 2000). The latter may result in false
conclusions for the further diagnostic and therapeutic approaches.
Additionally, the determination of HSV type-specific IgM antibod-
ies seems to make little sense because both virus types show a high
cross-reactivity and the reliability of diagnostic IgM assays based on
type-specific epitopes in a single viral protein suffers from limited
sensitivity.
The objective of the present study was to evaluate the HSV
type-common and type-specific IgG and IgM enzyme-linked
immunosorbent assays (ELISA) provided by the manufacturers
Institut Virion\Serion GmbH (Würzburg, Germany) and Orgentec
Diagnostika (Mainz, Germany) for the diagnosis of acute and latent
HSV infections. To this end, 263 sera from HSV-negative children,
healthy blood donors as well as patients without and with acute
HSV infections were included in this study.
2. Materials and methods
2.1. Serum panels
To compare the performance of different HSV IgG and IgM
ELISAs, a total of 263 sera were included. All sera were taken ran-
domly between 1997 and 2012 from healthy children between 5
months and 3 years of age, healthy voluntary blood donors aged
18 to 65 years or hospitalized patients between 14 and 70 years
of age, living in towns and their rural surroundings of the fed-
eral state of Thuringia/Germany. Concerning the blood donors,
informed consent for the use of their sera was obtained prior to
processing samples. For children aged up to 18 years, consent
was not sought. In accordance to recommendations of the Cen-
tral Ethical Committee of Germany (Zentrale Ethikkommission bei
der Bundesärztekammer, 2003), no patient consent is required for
studies on anonymised residual diagnostic samples. In this study,
the second use of sera was approved by the Ethical Committee of the
Jena University Hospital, Germany (process numbers: 3233-08/11,
3670-01/13). Sera were tested utilizing reference procedures (see
chapter 2.2.) for the determination of HSV-specific IgG and IgM
antibodies and stored in aliquots at −20 ◦ C without interruption.
Only samples with concordant results in all reference assays were
included.
According to the results of reference tests, all sera were classi-
fied into six panels which are summarized in Table 1. Serum panel
2 was used to exclude cross-reactions of HSV antigens to VZV-
specific antibodies. Panel 6 consisted of 25 anti-HSV IgM-positive
residual diagnostic samples from patients with symptomatic acute
HSV infections such as herpes genitalis, herpetic gingivostomati-
tis or herpes simplex pneumonia. These sera were sent to the
German Reference Laboratory of HSV and VZV for examining HSV-
specific IgM and IgG antibodies between 2000 and 2012. In 11
patients, the clinical diagnosis was confirmed by detection of viral
DNA using PCR or determination of viral antigens using indirect
immunofluorescence antibody test (IFAT) supporting the positive
IgM results. In further 8 sera, the Enzygnost® Anti-HSV IgM ELISA
(Siemens Healthcare Diagnostics, Marburg, Germany) was positive,
and in the remaining 6 patients with herpes genitalis, HSV-2-
specific IgG was detectable. Whereas all sera (Table 1) with the
exception of panel 5 were tested in the Serion ELISAs, sera of panels
1–3 were only tested partially in the Orgentec assays. The reason
was that several sera were not available for both. All sera were
allowed to attain room temperature immediately before ELISA test-
ing. Antibody testing was carried out blindly in groups of up to 75
serum samples. Serum samples were tested in duplicate on differ-
ent days.
2.2. Reference procedures
For the determination of HSV type-specific IgG class antibod-
ies, the HerpeSelect 1 ELISA IgG and the HerpeSelect 2 ELISA IgG
(Focus Diagnostics, Cypress, CA, United States) were used. These
ELISAs licensed by the United Food and Drug Administration use
recombinant glycoprotein gG-1 and gG-2 antigens, respectively.
They were available as one of the first tests to determine HSV type-
specific antibodies but distributed formerly by the companies Gull
and Meridian. Because of robust sensitivity and specificity, these
assays have been used as standard methods in many HSV sero-
prevalence studies (Ashley-Morrow et al., 2004; Delany-Moretlwe
et al., 2010; Sauerbrei et al., 2011). The sensitivity and specificity
of the HerpeSelect ELISAs for samples from sexually active adults
have been shown to be 91.2% (HSV-1) to 96.1% (HSV-2) and 92.3%
(HSV-1) to 97% (HSV-2) (Focus Diagnostics, 2011a,b). All tests were
carried out manually according to the instruction for use (IFU).
Samples were considered positive if the index value was greater
than 1.1. Negative samples had an index value of less than 0.9
and those with index values between 0.9 and 1.1 were considered
equivocal.
The immunoblot assay recomLine HSV-1 & HSV-2 IgG (Mikro-
gen, Neuried, Germany) was used for confirmatory examination
of HSV type-specific IgG antibodies. This test is based on nitro-
cellulose membranes blotted with purified recombinant gG-1 and
gG-2 as well as with proteins common to both types of HSV.
The assay was performed manually taking into account the IFU.
According to the manufacturer’s publications (Mikrogen, 2011),
the HSV-1 assay has been shown to have 99% (in routine diagnos-
tic samples) to 100% (in samples from blood donors) sensitivity
and 88.7% (routine diagnostic samples) to 94.6% (blood donors)
specificity. For the HSV-2 immunoblot, the sensitivity was given
as 75.0% (blood donors) to 93.8% (routine diagnostic samples) and
the specificity as 92.5% (routine diagnostic samples) to 100% (blood
donors).
For semi-quantitative detection of IgM class antibodies to HSV,
a modification of the IFAT was used as described previously
(Sauerbrei et al., 2000). In short, before measuring IgM, serum sam-
ples were pretreated with anti-human IgG (BAG Health Care, Lich,
Germany). Sera diluted initially 1:20 were incubated with HSV-2 US
strain-infected HEp-2 cells grown on microscopic glass slides. Non-
infected HEp-2 cells were used as controls to exclude anti-cellular
antibodies. Fluorescein-labelled anti-human IgG or IgM from rabbit
(Dako, Hamburg, Germany) served as conjugates. Sera with titers
of ≥1:20 were considered IgM positive. In addition, this modified
IFAT was used for the determination of VZV-specific IgG antibod-
ies. Varicella-zoster virus prototype Oka strain-infected A549 cells
grown on glass microscope slides served as antigens (Sauerbrei
et al., 1999), and titers of ≥1:5 were regarded as positive. This test
has been recommended as alternative to the fluorescent antibody
to membrane antigen assay for the determination of VZV immune
status (Sauerbrei et al., 2004).
 K. Liermann et al. / Journal of Virological Methods 199 (2014) 29–34 31

Table 1
Panels of sera.

Serum panel Characteristics Number of sera Years in which

sera were taken

1 HSV-1/2- and VZV-seronegative healthy children, between 5 months and 3 50 1997–2006

years of age
2 HSV-1/2-seronegative and VZV- seropositive healthy children, between 5 50 1997–2006
months and 3 years of age
3 HSV-1- and HSV-2-seropositive healthy voluntary blood donors aged 18–65 51 1997–2006
years
4 HSV-1-seropositive and HSV-2-seronegative voluntary blood donors aged 50 2003–2008
18–65 years
5 HSV-1-seronegative and HSV-2-seropositive voluntary blood donors or 37 1997–2012
patients without acute HSV infections, 14-65 years of age
6 Anti-HSV IgM-positive patients with acute HSV infections (IgM titers ≥1:20), 25 2000–2012
18–38 years of age
Total number of sera 263

2.3. Enzyme-linked immunosorbent assays to be tested
The Serion ELISAs classic HSV 1 + 2 IgG, HSV 1 IgG, HSV 2 IgG,
HSV 1 + 2 IgM, HSV 1 IgM and HSV 2 IgM (Institut Virion\Serion
GmbH, Germany) evaluated in this study are based on the indi-
rect ELISA technique. The tests allow qualitative and quantitative
immunoenzymatic determination of IgG class antibodies against
HSV. Microtiter plates for Serion ELISAs classic HSV 1 + 2 IgG and
IgM are coated with a mixture of purified whole-virus antigens of
both HSV-1 and HSV-2. In contrast, the Serion ELISA classic HSV 1
IgG uses recombinant gG-1 as antigen, and in Serion ELISA classic
HSV 2 IgG, recombinant gG-2 is bound on the surface of microtiter
wells. The use of the envelope proteins gG-1 in HSV-1 and gG-2
in HSV-2 IgG ELISA allows differentiation of type-specific IgG anti-
body response. The microtiter plates for Serion ELISAs classic HSV
1 IgM and HSV 2 IgM are coated with the corresponding whole-
virus antigen of HSV to ensure immediate and sensitive detection
of acute infections (Institut Virion\Serion GmbH, 2013). Higher
antibody concentrations to the corresponding antigen indicate
the HSV type of infection. All assays were performed automat-
ically by the use of the ImmunomatTM system (Virion\Serion)
following the IFU. The processing was performed analogous to the
manual use. Prior to testing IgM, samples were pretreated auto-
matically with Serion rheumatoid factor absorbens according to
the manufacturer’s instructions. Results were quantitatively calcu-
lated automatically by means of the ImmunomatTM system’s own
software. All samples were tested twice, and the mean values of
the individual quantitative results were calculated. On this basis,
the final qualitative results were determined. Results were given
in units per milliliter (U/ml). Positive results were equivalent to
>30 U/ml, borderline results to 20–30 U/ml, and results of <20 U/ml
were interpreted as negative.
The Orgentec ELISAs Anti-HSV-1 IgG, Anti-HSV-2 IgG, Anti-HSV-
1/2 IgM, Anti-HSV-1 IgM and Anti-HSV-2 IgM are also based on the
indirect enzyme-linked immune reaction. The ELISA Anti-HSV-1
IgG uses gG-1 and the ELISA Anti-HSV-2 IgG gG-2 coating antigen
for the determination of HSV type-specific IgG class antibodies.
In contrast, the microtiter plates of the ELISA Anti-HSV-1/2 IgM
are coated with whole-virus antigen from both, the HSV-1 and
the HSV-2, and the ELISAs Anti-HSV-1 IgM and Anti-HSV-2 IgM
use the whole-virus antigen of the corresponding virus type. All
ELISAs were performed using the Alegria® test strips in combina-
tion with the fully automated random access Alegria® analyzer
according to the IFU. By means of the Sensotronic Memorized
Calibration (SMC® ) technology, data encoded on the barcode are
transferred from the test strip to the instrument, and the assay
is processed and evaluated automatically (Orgentec Diagnostika,
2013). In the Alegria® test strips, rheumatoid factor was inte-
grated. Thus, absorption occurred during sample processing, and
no sample preparation or pre-dilution was required. All samples
were tested twice. In case of discrepancies between both qualita-
tive test results, re-tests were carried out, and the most frequent
result (including the original test result) was accepted. Results were
expressed in U/ml. Results equivalent to >25 U/ml were interpreted
as positive, 20–25 U/ml as borderline and <20 U/ml as negative. For
IgM ELISAs, a second cut-off value of 12.5 U/ml was recommended.
2.4. Statistical methods
Sensitivity was calculated as the proportion of positive sera
(HSV-specific IgG or IgM positive using the reference tests) that
were identified correctly as positive by the tested ELISAs for HSV
type-common IgG/IgM or HSV type-specific IgG. Specificity referred
to the proportion of negative sera (HSV-specific IgG or IgM negative
using the reference tests), which had a negative test result using
the tested ELISAs for HSV type-common or type-specific IgG/IgM
(Altman and Bland, 1994). A different approach was used to include
the borderline results into statistical calculation. Firstly, all border-
line results were classified as positive and secondly, all borderline
results were classified as negative. Thus, in case of borderline
results, a range for sensitivity and specificity was given. Differences
between sensitivity/specificity of the HSV type-specific IgG Serion
ELISAs and the corresponding Orgentec ELISAs were analyzed sta-
tistically by the Fisher’s exact test, and p values were subject to a
significance level of 5%. Kappa statistics was used to compare the
agreement between the ELISAs tested and the reference procedures
against that which might be expected by chance (Cohen, 1960). For
this calculation, all borderline results were excluded. When Kappa
indices are between 0.81 and 1.00, the strength of agreement has
been regarded as almost perfect (Landis and Koch, 1977). In general,
the 95% confidence intervals (CI) were calculated.
3. Results
The results for evaluation of Serion ELISAs classic HSV 1 + 2 IgG,
HSV 1 IgG, HSV 2 IgG as well as the ELISAs Anti-HSV-1 IgG and
Anti-HSV-2 IgG from Orgentec using serum panels 1–5 are sum-
marized in Table 2. Concerning serum panels 1 and 2, 50 sera each
were included in the evaluation of Serion ELISAs. In contrast, 25
sera each were tested using the Orgentec ELISAs. Furthermore, the
panels 3 and 5 consisted of 51 and 34 sera, respectively, for the
Serion ELISAs while 50 and 37 sera were tested using the Orgentec
ELISAs. There was an absolute concordance between the results of
the Serion ELISA HSV 1 + 2 IgG for panels 3 and 4, the Serion ELISA
HSV 2 IgG for serum panels 2, 4 and 5, and the Orgentec ELISA
Anti-HSV 2 IgG for serum panels 1 and 4 with the reference pro-
cedures. In another cases, a small number of discrepant results in
comparison to the reference tests were observed.
32 K. Liermann et al. / Journal of Virological Methods 199 (2014) 29–34

Table 2
Results of anti-HSV IgG in serum panels 1–5 using Serion ELISA classic HSV 1 + 2 IgG, Serion ELISA classic HSV 1 IgG, Serion ELISA classic HSV 2 IgG as well as the ELISAs
Anti-HSV-1 IgG and Anti-HSV-2 IgG IgG from Orgentec in comparison to reference tests.

Serum Number of Serion ELISA Serion ELISA Serion ELISA Number of sera Orgentec ELISA Orgentec ELISA
panel sera (Serion) classic HSV 1 + 2 classic HSV 1 IgG classic HSV 2 IgG (Orgen-tec) Anti-HSV-1 IgG Anti-HSV 2 IgG
IgG

Pos. Bord. Neg. Pos. Bord. Neg. Pos. Bord. Neg. Pos. Bord. Neg. Pos. Bord. Neg.

1 50 0 2 48 1 6 43 0 1 49 25 0 2 23 0 0 25
2 50 1 0 49 3 6 41 0 0 50 25 0 1 24 1 0 24
3 51 51 0 0 44 6 1 47 2 2 50 44 4 2 44 3 3
4 50 50 0 0 46 3 1 0 0 50 50 47 1 2 0 0 50
5 34 25 3 6 1 6 27 34 0 0 37 0 3 34 33 3 1

Based on these findings, the type-specific Serion HSV IgG ELISAs
reached sensitivity (Table 3) between 89.1–98.0% (HSV-1 IgG)
and 95.3-97.6% (HSV-2 IgG) and specificity between 82.8–97.0%
(HSV-1 IgG) and 99.3–100% (HSV-2 IgG). The sensitivity of the
Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG was 91.0–96.0%
and 88.5–95.4%, respectively, and the specificity was calculated
as 93.1–100.0% and 99.0%, respectively. There were no statistical
differences between sensitivity/specificity of the HSV type-specific
IgG Serion ELISAs and the corresponding Orgentec ELISAs, except
for the specificity of HSV-1 IgG ELISAs when borderline results were
classified as positive (p ≤ 0.05). After comparison of the results with
those of the reference assays by the Kappa statistics (Table 3), the
Kappa indices for the Serion HSV IgG ELISAs were computed at
0.93 (CI 95%: 0.88–0.98) to 0.98 (CI 95%: 0.96–1.00), and for the
Orgentec HSV IgG ELISAs, the Kappa indices were between 0.94 (CI
95%: 0.90–0.99) and 0.95 (CI 95%: 0.91–100). Finally, the results of
the HSV-1 IgG ELISAs from Virion\Serion and Orgentec showed a
Cohen’s Kappa of 0.98 (CI: 0.95–1.00), and the Kappa index was 0.94
(CI 95%: 0.88–1.00) when the HSV-2 IgG ELISAs were compared.
The results of anti-HSV IgM in sera of panel 6 using type-
specific and type-common Serion as well as Orgentec IgM ELISAs
are shown in Table 4. The type-common Serion HSV IgM ELISA
was positive in 22 out of 25 sera from patients with active HSV
infection. Only one serum revealed a negative result and 2 sera
a borderline result. In comparison, the Orgentec ELISA Anti-HSV-
1/2 IgM classified 13 sera (cut-off 12.5 U/ml:20) as positive and 12
(cut-off 12.5 U/ml:5) sera as negative. Out of 6 HSV-1 infections,
the type-specific Serion IgM ELISAs identified 5 and the Orgentec
ELISAs Anti-HSV-1 IgM and Anti-HSV-2 IgM identified 3 (cut-off
12.5 U/ml:4) sera as IgM-positive to HSV-1. Out of 10 HSV-2 infec-
tions, the single-type IgM Serion ELISAs identified in 3 sera and
the corresponding Orgentec ELISAs in 4 (cut-off 12.5 U/ml:4) sera
IgM antibodies against HSV-2. Using the type-specific Serion IgM
ELISAs, testing 9 sera with unclassified virus type of HSV infection
revealed HSV-1-specific IgM antibodies in 6 cases and HSV-2 IgM
in none case. By the use of the HSV type-specific Orgentec IgM
ELISAs, HSV-1 IgM was detected in 3 (cut-off 12.5 U/ml:3) sera and
HSV-2 IgM in 1 (cut-off 12.5 U/ml:2) serum. On the basis of these
findings, the type-common HSV IgM Serion ELISA had a sensitiv-
ity of 88.0–96.0% and specificity of 87.4–94.7%. The sensitivity of
the Orgentec Anti-HSV-1/2 IgM ELISA was calculated as 52.0% (cut-
off 25 U/ml) and 80.0% (cut-off 12.5 U/ml), respectively (specificity
not tested, Table 3). The sensitivity of the HSV type-specific ELISAs
was not calculated because of the small number of samples and the
lack of suitable reference assays. After testing sera of panels 1–3, all
single-type IgM tests showed a high specificity between 87.4–94.7%
and 99.3–100% (Table 3).
4. Discussion
Herpes simplex virus causes frequent infections which may be
associated with a variety of symptoms differing in severity of symp-
toms. Although the PCR technique has been accepted as method of
choice for diagnosis of acute infections, HSV-serological methods
are used widely in virological laboratories. Herpes simplex virus
type-specific IgG assays are potentially useful to identify HSV car-
riers, in particular of HSV-2 (Swiss herpes management forum,
2004; Centers for Disease Control and Prevention, 2010). These
serological tests have been available commercially for more than
one decade. Despite this fact, many laboratories do not offer HSV
type-specific serology, but provide only type-common HSV sero-
logic tests (Zahariadis and Severini, 2010; Bentley et al., 2012).
The present study demonstrates a comprehensive head-to-head
comparison of the HSV IgG and IgM ELISAs provided by the man-
ufacturers Virion\Serion and Orgentec. The tests were chosen
since they are CE-marked assays used widely in Europe, espe-
cially in Germany, and they are performed and evaluated fully
automatically.

Table 3
Range of sensitivity and specificity of Serion ELISAs classic HSV IgG and IgM as well as the Orgentec HSV IgG and IgM ELISAs and Kappa indices after comparison with reference
tests. 95% confidence intervals are given in brackets.

ELISA Sensitivity (%) Specificity (%) Kappa

Serion ELISA classic HSV 1 + 2 IgG 93.3 (87.4–96.7) to 95.6 (90.2–98.2) 97.0 (90.8–99.2) to 99.0 (93.8–99.9) 0.94 (CI 95%: 0.89–0.98)
Serion ELISA classic HSV 1 IgG 89.1 (81.0–94.2) to 98.0 (92.3–99.7) 82.8 (75.1–88.6) to 97.0 (92.0–99.0) 0.93 (CI 95%: 0.88–0.98)
Serion ELISA classic HSV 2 IgG 95.3 (87.7–98.5) to 97.6 (91.0–99.6) 99.3 (95.8–100) to 100.0 (96.9–100.0) 0.98 (CI: 95%: 0.96–1.00)

Orgentec ELISA Anti-HSV-1 IgG
Orgentec ELISA Anti-HSV-2 IgG IgG
Serion ELISA classic HSV 1 + 2 IgM
Serion ELISA classic HSV 1 IgM
Serion ELISA classic HSV 2 IgM
91.0 (83.2–95.5) to 96.0 (89.5–98.7) 93.1 (85.0–97.2) to 100.0 (94.7–100.0) 0.95 (CI 95%: 0.91–1.00)
88.5 (79.4–94.1) to 95.4 (88.0–98.5) 99.0 (93.8–99.9) 0.94 (CI 95%: 0.90–0.99)
88.0 (67.7–96.8) to 96.0 (77.7–100.0) 87.4 (80.8–92.1) to 94.7 (89.5–97.5) 0.96 (CI 95%: 0.88–1.00)
Not calculated 93.3 (87.8–96.6) to 96.0 (91.2–98.4) Not calculated
Not calculated 99.3 (95.8–100.0) to 100.0 (96.9–100.0) Not calculated

Orgentec ELISA Anti-HSV-1/2 IgM Cut-off 25 U/ml: 52.0 (CI 95%: Not tested Not calculated
31.8–71.7) Cut-off 12.5 U/ml: 80.0 (CI
95%: 58.7–92.4)

Orgentec ELISA Anti-HSV-1 IgM Not calculated 99.0 (CI 95%: 93.8–99.9) Not calculated
Orgentec ELISA Anti-HSV-2 IgM Not calculated 99.0 (CI 95%: 93.8–99.9) Not calculated
 K. Liermann et al. / Journal of Virological Methods 199 (2014) 29–34 33

Table 4
Results of anti-HSV IgM in serum panel 6 using Serion ELISAs classic HSV 1 + 2 IgM, HSV 1 IgM, HSV 2 IgM as well as the ELISAs Anti-HSV-1/2 IgM, Anti-HSV-1 IgM and
Anti-HSV-2 IgM from Orgentec (results in brackets are on the basis of the lower cut-off value).

Infection ELISA Number of sera

positive borderline negative

HSV-1 (n = 6) Serion ELISA classic HSV 1 + 2 IgM 6 0 0

Serion ELISA classic HSV 1 IgM 5a 0 1
Serion ELISA classic HSV 2 IgM 4a 0 2
Orgentec ELISA Anti-HSV-1/2 IgM 4 (5) 0 (0) 2 (1)
Orgentec ELISA Anti-HSV-1 IgM 4 (4)b 0 (0) 2 (2)
Orgentec ELISA Anti-HSV-2 IgM 3 (4)b 1 (0) 2 (2)
HSV-2 (n = 10) Serion ELISA classic HSV 1 + 2 IgM 9 1 0
Serion ELISA classic HSV 1 IgM 5c 4 1
Serion ELISA classic HSV 2 IgM 6c 0 4
Orgentec ELISA Anti-HSV-1/2 IgM 4 (9) 0 (0) 6 (1)
Orgentec ELISA Anti-HSV-1 IgM 2 (6)d 1 (0) 7 (4)
Orgentec ELISA Anti-HSV-2 IgM 5 (8)e 1 (0) 4 (2)
HSV type not classified (n = 9) Serion ELISA classic HSV 1 + 2 IgM 7 1 1
Serion ELISA classic HSV 1 IgM 6a 1 2
Serion ELISA classic HSV 2 IgM 4a 1 4
Orgentec ELISA Anti-HSV-1/2 IgM 5 (6) 0 (0) 4 (3)
Orgentec ELISA Anti-HSV-1 IgM 4 (5)f 0 (0) 5 (0)
Orgentec ELISA Anti-HSV-2 IgM 3 (4)f 1 (0) 5 (4)
a
In all sera, higher antibody concentrations in HSV-1 IgM ELISA than in HSV-2 IgM ELISA
b
In 3 (4) sera, higher antibody concentrations in HSV-1 IgM ELISA than in HSV-2 IgM ELISA
c
In 3 sera, higher antibody concentrations in HSV-2 IgM ELISA than in HSV-1 IgM ELISA
d
In 0 (4) sera, higher antibody concentrations in HSV-2 IgM ELISA than in HSV-1 IgM ELISA
e
In 4 (4) sera, higher antibody concentrations in HSV-2 IgM ELISA than in HSV-1 IgM ELISA
f
In 3 sera, higher antibody concentrations in HSV-1 IgM ELISA than in HSV-2 IgM ELISA, and in 1 (2) sera, higher antibody concentrations in HSV-2 IgM ELISA than in
HSV-1 IgM ELISA

The results of 235 sera from healthy children, healthy volun-
tary blood donors as well as patients without acute HSV infections
revealed high sensitivity between 89.1 and 98.0% as well as high
specificity between 82.8% and 100% for the HSV type-specific IgG
Serion ELISAs. Comparable results with sensitivities between 88.5%
and 96.0% and specificities between 93.1% and 100% were achieved
after testing the HSV type-specific IgG ELISAs distributed by Orgen-
tec on the basis of 187 sera. For the interpretation of findings, it has
to be considered that all borderline results were included into sta-
tistical calculation. However, it is stated in the IFU of the Serion
ELISAs classic that “in cases where the results are within the bor-
derline range a definitive interpretation of the result is not possible”
(Institut Virion\Serion GmbH, 2013). Using Kappa statistics, the
strength of agreement was almost perfect between the ELISAs and
the reference assays on the one hand and between both ELISAs on
the other hand. These findings confirm the results of numerous pre-
vious studies (Sauerbrei and Wutzler, 2004; Morrow and Friedrich,
2006; Sauerbrei and Wutzler, 2007; Zahariadis and Severini, 2010)
and demonstrate that many distributed HSV type-specific IgG
ELISAs, which are based on the recombinant viral envelope antigens
gG-1 and gG-2, are reliable assays for accurate identifying HSV-1 or
HSV-2 IgG-positive or -negative individuals. This means that these
persons had experienced a primary infection by HSV-1 usually dur-
ing childhood or by HSV-2 usually during adolescence or adulthood.
A negative result excludes most likely a recurrent infection caused
by the corresponding HSV type. However, a primary HSV infec-
tion can only be excluded in association with negative results in
PCR and/or viral culture. Although there is a strong consensus that
assays on the basis of gG-1 and gG-2 (Bergström and Trybala, 1996)
are most accurate for discriminating between latent HSV-1 and
HSV-2 infections, recent studies have shown that gC-1 could be
a useful alternative antigenic target to determine seroprevalence
of HSV-1 with comparable success as gG-1 (Scheper et al., 2010;
Sauerbrei et al., 2011). For the interpretation of HSV type-specific
ELISA results, it should be, however, considered that specificity can
be reduced in populations with a high prevalence of HIV-caused
diseases (Delany-Moretlwe et al., 2010). If no differentiation of HSV
type-specific immunity is required, the type-common Serion ELISA
classic HSV 1 + 2 IgG provides reliable information on the experi-
enced HSV infection regardless of the virus type. This ELISA revealed
also high sensitivity and specificity in this study.
In contrast to HSV IgG ELISAs, there is a general confusion
regarding the quality of the results obtained by ELISAs that are
commercially available for the detection of HSV IgM antibodies
(Ohana et al., 2000). As the present study shows, the type-common
HSV Serion IgM ELISA revealed also a high sensitivity of 88.0–96.0%
and high specificity of 87.4–94.7% for detection of HSV IgM in sam-
ples obtained from patients with acute HSV infection, from healthy
children and from blood donors. The sensitivity of the correspond-
ing Orgentec ELISA Anti-HSV-1/2 IgM was reduced on the basis of
the cut-off value of 25 U/ml recommended primarily and increased
to 80% when a lower detection limit of 12.5 U/ml was assumed.
According to the IFU from Orgentec, sera with test results ran-
ging from 12.5 to 25.0 U/ml can be classified as “reactive” taking
into account the respective local conditions, e.g. the patient collec-
tive investigated (Orgentec Diagnostika, 2013). Since it is assumed
that HSV reactivations are associated with lower IgM concentra-
tions than primary infections, the definition of two cut-off values
aims to differentiate between primary and recurrent infections.
However, there is no evidence to support this approach. A limi-
tation is that the specificity of the Orgentec ELISA Anti-HSV-1/2
IgM was not calculated. Furthermore, cases from acute VZV infec-
tions were not included in this study to exclude cross-reactions to
VZV-specific IgM antibodies. In contrast to the high specificity, the
single-type HSV IgM ELISAs from both Virion\Serion and Orgen-
tec had a limited detection rate of HSV IgM antibodies and did
not detect reliably type-specific IgM. These results suggest that
only type-common HSV IgM ELISAs with high sensitivity and speci-
ficity may be useful to confirm acute HSV infections. This can apply
to IgM as a marker for newly acquired HSV infections especially
caused by HSV-2 (Page et al., 2003). However, IgM results appear
to be ineffective at discriminating early from established infections
(Morrow and Friedrich, 2006) since IgM can also be positive in
recurrent infections regardless of the presence of clinical symp-
toms. This means that the positive predictive value of HSV IgM
assays for acute HSV infection is low. A further drawback is that the
34 K. Liermann et al. / Journal of Virological Methods 199 (2014) 29–34
determination of IgM antibodies has practically no significance for
timely diagnosis of acute primary and recurrent HSV infection. The
use of HSV type-specific IgM ELISAs, which are offered with increas-
ing frequency, is practically superfluous. This relates to HSV-1 and
HSV-2 IgM ELISAs coated with the corresponding whole-virus or
type-specific gp-1 or gp-2 antigens. While the first have main lim-
itations in HSV type specificity, the latter most likely produce test
sensitivity inferior to preparations containing many different viral
antigens. In conclusion, results of the HSV IgM serology should not
be relied upon to make decisions for antiviral treatment of HSV
infections.
Acknowledgements
This work was supported by the Institut Virion\Serion
GmbH (Würzburg, Germany) and Orgentec Diagnostika (Mainz,
Germany).
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