MKGS Vol 45 No 2 April-Juni 2012.indd
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Volume 45 Number 2 June 2012
Research Report
Seroprevalence of Herpes Simplex virus types 1 and 2 and their
association with CD4 count among HIV-positive patients
Irna Sufiawati1, Sunardhi Widyaputra2, and Tony S. Djajakusumah3
1Department of Oral Medicine, Faculty of Dentistry, University of Padjadjaran
2Department of Oral Pathology, Faculty of Dentistry, University of Padjadjaran
3Department of Dermatology & Venereology, Faculty of Medicine, University of Padjadjaran
Bandung - Indonesia
ABSTRACT
Background: Herpes simplex virus (HSV) is a common cause of viral opportunistic infections among HIV-positive patients.
Frequent, more severe and prolonged episodes of recurrent HSV infection can be a source of significant morbidity and mortality
among HIV-positive patients with advanced immunosuppression, reflected by low CD4 count. However, conflicting results have also
been reported. Purpose: The aim of this study was to investigate the seroprevalence of HSV type 1 (HSV-1) and type 2 (HSV-2) in
HIV-positive patients compare with the rate in HIV-negative patients, and to evaluate their association with CD4 count. Methods:
A cross sectional study was conducted among 145 subjects consisting of 80 HIV-positive and 65 HIV-negative patients attending the
top referral hospital in Bandung, West Java, Indonesia. The serum obtained was assayed for the presence of HSV-1 and HSV-2 IgG
antibodies using ELISA kits. Data were analyzed using a Chi-square test, t-tests and analysis of variance (ANOVA). Results: There
were no significant differences in HSV-1 seroprevalence between HIV-positive patients (71%) and HIV-negative patients (66%). HSV-
2 seroprevalence was significantly higher in HIV-positive patients (30%) than HIV-negative patients (5%). The titers of HSV-1 IgG
antibodies in HIV-positive patients (mean 24.63 ± 19.06 IDU) were significantly lower than those of HIV-negative patients (mean 44.62
± 33.22 IDU). In contrast, HSV-2 IgG antibody titers in HIV-positive patients (mean 13.31 ± 20.28 IDU) were significantly higher than
HIV-negative patients (mean 4.42 ± 10.99 IDU). There was no significant correlation between HSV-1 and HSV-2 seropositivity and
CD4 count among HIV-positive patients. However, most of HSV-2 seropositive patients had CD4 count < 200 cells/mm3. Conclusion:
Seroprevalence of HSV-1 and HSV-2 among HIV-positive patients was high with no correlation with CD4 count.
Key words: HSV, IgG, HIV, CD4
ABSTRAK
Latar belakang: Herpes simplex virus (HSV) adalah penyebab infeksi virus oportunistik yang paling umum pada pasien HIV-
positif. Infeksi HSV rekuren yang sering terjadi, lebih berat, dan episode yang berkepanjangan dapat menjadi penyebab morbiditas
dan mortalitas yang signifikan pada pasien HIV-positif dengan imunosupresi lanjut, ditandai dengan jumlah CD4 yang rendah.
Namun, hasil yang bertentangan juga telah dilaporkan. Tujuan: Penelitian ini bertujuan untuk mengetahui seroprevalensi HSV tipe 1
(HSV-1) dan tipe 2 (HSV-2) pada pasien HIV-positif dibandingkan dengan pasien HIV-negatif, dan untuk mengevaluasi hubungannya
dengan jumlah CD4. Metode: Penelitian potong lintang ini dilakukan pada 145 subjek yang terdiri dari 80 pasien HIV-positif dan 65
pasien HIV-negatif yang berkunjung ke rumah sakit pusat rujukan di Bandung, Jawa Barat, Indonesia. Antibodi IgG HSV-1 dan HSV-
2 di dalam serum diperiksa dengan menggunakan ELISA. Data dianalisis dengan uji Chi-square, t-test dan ANOVA, nilai p < 0.05
dianggap signifikan secara statistik. Hasil: Seroprevalensi antibodi IgG HSV-1 pada pasien HIV-positif (71%) tidak berbeda secara
signifikan dengan pasien HIV-negatif (66%). Namun, seroprevalensi HSV-2 secara signifikan lebih tinggi pada pasien HIV-positif
(30%) dibandingkan dengan pasien HIV-negatif (5%). Titer antibodi IgG HSV-1 pada pasien HIV-positif (mean 24.63 ± 19.06 IDU)
secara signifikan lebih rendah dibandingkan pasien HV-negatif (mean 44.62 ± 33.22 IDU). Sedangkan, titer antibodi IgG HSV-2 pada
pasien HIV-positif (mean 13.31 ± 20.28 IDU) secara signifikan lebih tinggi dibandingkan pasien HIV-negatif (mean 4.42 ± 10.99
IDU). Tidak ada hubungan yang signifikan antara seropositivitas HSV-1 dan -2 dengan jumlah CD4. Namun, sebagian besar pasien
115Sufiawati, et al.: Seroprevalence of Herpes Simplex virus types 1 and 2 and their association
INTRODUCTION
The increase in the immunocompromised populations
due to HIV infection is a factor that can contribute to the
change in epidemiology of herpesviruses-associated disease.
The hallmark of herpesviruses infection is the ability to
establish latent, life-lasting, and periodically reactivating
infections in the host. The immunosuppressive state induced
by HIV-1 may facilitate herpes viruses’ reactivation or
re-infection.1 On the other hand, it is growing evidence
that herpevirus infection may increase an pasienal’s
susceptibility to HIV infection,2 and could interact with
HIV to accelerate disease progression.3,4 To date, there are
eight known human herpes viruses, they are herpes simplex
virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV),
and cytomegalovirus (CMV), Epstein-Barr virus (EBV) and
human herpesvirus-6, human herpesvirus-7, and HHV-8
(Kaposi’s sarcoma herpes virus).5 Among herpesviruses
family members, HSV is the most common co-infection
and pathogenic in HIV-1-positive patients.6
Herpes simplex viruses (HSV), an alpha-herpesvirus,
are categorized into two types, herpes type 1 (HSV-1)
and herpes type 2 (HSV-2). For each virus, the primary
mode of transmission is different and there is a tendency
to infect different anatomical sites, causing a wide variety
of mucocutaneous infections. HSV-1 is mainly localized
around the oral region and HSV-2 around the genital region,
however it is quite possible to transmit the virus to either
region. The prevalence of HSV-1 infection in general
populations is high in most geographic areas worldwide
ranges from roughly 65% to 90% and has been found to be
higher than HSV-2 infection.7,8 Studies from different parts
of the world demonstrated that rates of HSV-1 infection
were much higher in HIV-positive patients or with a high
risk of HIV than in the general population. The prevalence
rates of HSV type 1 (HSV-1) among HIV-1 infected people
ranging from 90% to 100%.9,10
HSV-2 prevalence is highly variable and depends on
many factors, including country and region of residence,
population subgroup, sex, and age. Prevalence of HSV-2
infection in the general population in developing Asian
countries appears to be lower (10–30%) than developed
regions.11 In the United States, HSV-2 seroprevalence
was 16.2%.12 HSV-2 seroprevalence in Central and South
America are estimated at 20% to 60%.7,11 In Europe,
HSV-2 seropositivity varies by region ranging from
4.2% to 23.9%.13 Sub-Saharan Africa has the highest
HSV-2 seroprevalence in the world, reaching 80% in
adult population.7 HSV type 2 affects 50–90% of HIV-
1infected people higher than in the general population.9,11,14
HSV-2 infection reported as a major risk factor for HIV
acquisition.15 A meta-analysis of the association between
HSV-2 infection and risk of HIV-1 acquisition reviewed
31 studies have demonstrated that prevalent HSV-2 is
associated with a 2- to 4-fold increased risk of HIV-1
acquisition.16,17 These epidemiological evidence indicated
a strong relationship exist between HSV-2 and HIV.
Previous studies have demonstrated that HSV infections
are associated with a compromised immune system in
HIV-positive patients. Hoots et al.5 reported that there
was a statistically significant association between HSV
seropositivity and the degree of immunosuppression, as
reflected by cluster difference 4 (CD4) count. Other studies
showed that in HIV-positive patients, asymptomatic HSV
shedding increases with lower CD4 count.36,37 Another
study also confirmed that risk factors for increased HSV
shedding among HIV-positive men were low CD4 cell
count.38 However other studies have shown conflicting
results, Santos et al.39 reported a weak and statistically non-
significant association of HSV and CD4 count. Patients with
HSV infection can present with severe manifestations even
after their CD4 count increases to > 500 cells/mm3.40 It has
been suggested that immune reconstitution inflammatory
syndrome (IRIS), usually occurs in individuals with a
rapidly rising CD4 count, associated with severe HSV
lesions after HAART initiation.
Due to the apparent evolving epidemiological trends of
herpesviruses infection in HIV-positive people, this study
was conducted to assess the seroprevalence of HSV-1
and HSV-2, and their correlation with CD4 count among
HIV-positive patients in Bandung, West Java, Indonesia.
Since more herpesviruses infections are asymptomatic, the
seroepidemiological studies are critical in understanding
the pattern and distribution of infection, which have not
been previously investigated among HIV-positive patients
in West Java, Indonesia.
MATERIALS AND METHODS
Data were collected in a cross-sectional study from
January until March 2012 in a referral hospital in Bandung,
West Java. We recruited 80 patients who were diagnosed
as HIV-positive patients. We also enrolled 65 sex and age-
matched healthy volunteers as controls. Ethical clearance
was obtained from the Institutional Review Board of the
seropositif HSV-2 memiliki jumlah CD4 < 200 sel/mm3. Kesimpulan: Seroprevalensi HSV-1 dan HSV-2 pada pasien HIV-positif
adalah tinggi, tetapi tidak berkorelasi dengan jumlah CD4.
Kata kunci: HSV, IgG, HIV, CD4
Correspondence: Irna Sufiawati, c/o: Departemen Penyakit Mulut, Fakultas Kedokteran Gigi Universitas Padjadjaran. Jl. Sekeloa
Selatan No.1 Bandung 40132. E-mail: irnasufiawati@yahoo.com. Fax: +62222532805.
116 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 2 June 2012: 114–120
Ethical Committee Hasan Sadikin Hospital and patients
gave written informed consent for participation.
Samples of venous blood (5 ml) was drawn from all
the enrolled subjects into EDTA blood collection tubes
and immediately kept at +4° C. HIV status was confirmed
by Enzyme-linked immunosorbent assay (ELISA). CD4
testing was done using a BD FACSCount™ cytometer.
The sera were obtained on the same day and stored at -
20° C freezer in aliquots until tested. The presence of IgG
antibodies against HSV-1 and HSV-2 were examined using
ELISA kits (Indec Diagnostic, Indonesia), in accordance
with the manufacturer’s instructions. Positive and negative
standard sera, accompanying the kit were included in each
assay.
Data were entered and analyzed in SPSS 11.0 for
windows. Differences in HSV-1 and -2 seropositivity rates
among different groups were evaluated using the Chi-square
test. Mean titer levels were compared between HIV-positive
patients and HIV-negative controls using two sample t-tests.
The statistical significance of correlation between HSV-1
and HSV-2 IgG titers with CD4 count was obtained using
ANOVA. P-values < 0.05 were considered statistically
significant. The mean, median, mode and standard deviation
has also been done by using the same software.
RESULTS
Of all 145 subjects included in the analysis, the HIV
group comprised 80 HIV-positive patients (42 male and 38
female), the mean age was 30.8 + 8.3 years (median 31.5
years, range 1-55). The control group consisted of 65 HIV-
negative patients (34 male and 31 female), the mean age
was 29.1 + 12.1 years (median 28 years, range 1-56 years).
There was no statistically significant different between
HIV-positive patients and HIV-negative control in age
(p > 0.05) and gender distributions (p > 0.05). The majority
of HIV-positive patients were adequately controlled as
determined by CD4 count ranging from (mean 393.4 +
210.8 cells/mm3) (Table 1).
The results showed that seroprevalence of HSV-1
IgG was found slightly higher in HIV-positive patients
(71%) than in HIV-negative patients (63%), however the
different was no statistically significant (p > 0.05). While,
we found that HSV-2 seroprevalence was significantly
higher in HIV-positive than HIV-negative patients (30%
vs. 5%, respectively; p < 0.05). HSV-1 and-2 IgG antibodies
were not found in 34% HIV-negative patients and in 20%
HIV-positive patients. Further, we identified that of all
subjects there were a number of subjects gave a positive
test for both HSV-1 and-2 IgG antibodies. Out of 80 HIV-
positive patients, 21% of them have both HSV-1 and-2
IgG antibodies, significantly higher (p < 0.05) than those
of HIV-negative patients (3%) (Figure 1).
Immunoglobulin G antibodies against to HSV-1 are
more frequently found in HIV-positive patients with CD4
count > 500 cells/mm3. In contrast, many HIV-positive
patients who were HSV-2 seropositive had CD4 count
< 200 cells/mm3. In detail, HSV-1 IgG antibodies were
found in 13% of HIV-positive patients with CD4 count
< 200 cells/mm3, 22% of them have CD4 count ranging
from 200 to 349 cells/mm3, 28% of them have CD4 count
350-499 cells/mm3, and many of them (37%) have CD4
count more than 500 cells/mm3. In contrast, only 17% of
HIV-positive patients with CD4 count > 500 cells/mm3
had HSV-2 IgG antibodies, 28% of them have CD4 count
Table 1. Characteristics of study participants
Characteristics
HIV-positive patients
(n = 80)
HIV-negative patients
(n = 65)
p
Age (yeard old)
Mean ± SD (range) 29 ± 13 (1-58) 31 ± 8 (1-55) 0.24
Median 28 32
Gender (%)
Female 38 31 0.37
Male 42 34 0.42
CD4 Counts (cell/mm3)
Mean ± SD (range) 394 ± 209 ND –
Median 393
Note: n: number of subjects; ND: not determined; *p < 0.05, statistically significant
Figure 1. Seroprevalence of IgG antibody against HSV-1
and HSV-2 among HIV-positive and HIV-negative
patients.
117Sufiawati, et al.: Seroprevalence of Herpes Simplex virus types 1 and 2 and their association
< 200 cells/mm3, 22% of them have CD4 count 200-349
cells/mm3, and 33% of them have CD4 count 350-499
cells/mm3 (Figure 2). There were no significant correlation
between HSV-1 and HSV-2 seropositivity and CD4 count
(p > 0.05).
The mean titer of IgG antibodies against both two herpes
simplex viruses were statistically significantly different
compared to HIV-negative control group. The titer of
HSV-2 IgG antibodies were detected significantly higher
(p < 0.05) in HIV-positive patients compared with HIV-
negative patients. In contrast, the titers of HSV-1 IgG
antibodies in HIV-positive patients were significantly lower
(p < 0.05) than in HIV-negative patients. In addition, Table
2 showed that there were no significant correlation between
the titer of both HSV-1 and HSV-2 IgG antibodies and
CD4 count (p > 0.05). However, when we used post hoc
analysis (2-tail p-values for pairwise independent groups
t-tests), we found a significant different in the titer of HSV-
2 between HIV-positive patients with CD4 T-cell count
< 200 cells/mm3 and those patients with CD4 count > 500
cells/mm3 (p = 0.0233).
DISCUSSION
To our knowledge, this is the first study to compare
the seroprevalence of HSV type 1 and 2 antibodies in
HIV-positive and HIV-negative patients in a large public
referral hospital serving the urban and surrounding rural
area in Bandung, West-Java, the province with the highest
burden of HIV in Indonesia. We were particularly interested
in determining the seroprevalence of these herpes viruses in
HIV-positive patients and their associations with immune
status as measured by CD4 count, since there is little
known about this. Our study demonstrated that overall IgG
antibodies againts HSV-1 and HSV-2 were more prevalent
in both HIV-positive than HIV-negative patients (Tabel 1).
However, result from statistical analysis showed that only
HSV-2 seroprevalence rates were significantly higher in
HIV-positive than in HIV negative patients.
In this study, seroprevalence of HSV-1 IgG was found
slightly higher in HIV-positive than in HIV negative
patients. This results were not much different with other
studies that showed the prevalence of HSV-1 infection
in general populations worldwide is high ranges from
roughly 65% to 90%.7,8 The high prevalence of HSV-1
clearly shows that most people are infected with some
type of infection at least once in their lifetime. Most of
them are asymptomatic during the initial stage, they remain
undiagnosed for long periods of time or even throughout
the life. However, the seroprevalence rates of HSV-1 in
HIV-positive patients in our study lower than in the other
Figure 2. Distribution of HSV-1 and HSV-2 seropositive
patients by the rate of CD4 count.
Table 2. The titers of IgG antibody against HSV-1 and HSV-2 and their correlation with CD4 count
Virus Type
Virus Titers (IDU)
p
HIV-Positive Patients
HIV-Negative
Patients
HSV-1
Mean ± SD 24.63 ± 19.06 44.62 ± 33.22 0.0000118
Median 21.8 52.6
HSV-2
Mean ± SD 13.31 ± 20.28 4.42 ± 10.99 0.0019
Median 2.1 2
CD4 T-cell counts (cells/mm3)
p
< 200 200 – 349 350 – 499 ≥ 500
HSV-1
Mean ± SD 23.14 ± 22.789 20.38 ± 17.734 21.09 ± 17.734 25.69 ± 17.774 0.8013
Median 13.85 17.45 22 24.1
HSV-2
Mean ± SD 22.76 ± 27.392 11.18 ± 18.647 13.43 ± 21.351 6.12 ± 11.675 0.1665
Median 7.25 1.5 2 1.8
Note: SD: standard deviation; ID U (Indec Units); *p < 0.05, statistically significant
118 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 2 June 2012: 114–120
epidemiological studies carried out in various countries
within the range of 90–100%.9,10,18 The high prevalence
of HSV-1 IgG antibody in HIV-positive patients than in
general population is possibly because many of them were
acquired HSV-1 through sexual activities. It is well known
that HSV-1 is usually acquired orally during childhood via
non-sexual contacts, however the virus can be transmitted
also through sexual contacts. HSV-2 is the main causative
agent of genital herpes worldwide.19 However, recent data
reported that HSV-1 has also emerged as a major causative
agent of genital herpes in some developed countries,20,21
and the increase in the frequency of genital herpes caused
by HSV-1 compared with genital HSV-2 infection.22,23
The results of this study also showed that HSV-1 IgG
seroprevalence was higher than HSV-2 in both groups
which is in agreement with the reported results from
different regions of the world where the prevalence of
HSV-1 prevalence is almost always greater than HSV-
2 prevalence.7 A recent study also reported a higher
prevalence rate of IgG antibody against HSV-1 than HSV-2
in both groups.24 HSV-1 infections usually occur earlier
in life. It has been suggested that a high percentage of
HSV-2 antibodies because of prior HSV-1 protected from
subsequent HSV-2 infection. In developed countries, while
childhood acquisition of HSV-1 has decreased, HSV-2
seroprevalence has increased, suggesting the possible
protective effect of HSV-1 against HSV-2 infection.24
A prior infection with HSV-1 has an acquired immune
response that confers moderate protection against getting
HSV-2, and reduces its severity.25 A study reported that
previous HSV type 1 infection appeared to reduce the
risk for acquisition of HSV type 2 infection by 40%.26 It
is not known whether previous genital HSV-1 infection
modifies the risk of HSV-2 acquisition more substantially
than previous oral HSV-1 infection.27 However, HSV-1
infections are still at risk of HSV-2 acquisition. There
are conflicting results from studies on the risk of HSV-1
positive patients of acquiring HSV-2 that are reported that
previous HSV-1 infection does not reduce susceptibility to
subsequent genital HSV-2.28
Furthermore, our results indicate statistically significant
a higher prevalence of HSV-2 IgG in HIV-positive patients
when compared to 5% HIV-negative patients. This finding
is consistent with most previous studies in other countries.
HSV-2 infection is present in 30 to 70% of those in Europe
and 50 to 90% of those in Africa among patients with HIV
infection.16 A study reported that the HIV-positive men shed
HSV-2 orally more frequently than did the HIV-negative
men.29 However, other studies have identified that genital
shedding of HSV-2 is higher in HIV-positive patients.30
The higher rate of HSV-2 seroprevalence of HSV-2 among
HIV-AIDS patients because HSV-2 is transmitted via
sexual contact with an HIV-positiveperson. Several studies
demonstrated that behavioral and sexually transmitted
infection (STIs) as predictors of HSV-2 acquisition.16,17,31
Primary genital HSV-2 occurring in an HIV-1-infected
person is a marker for unsafe sexual practices. Genital ulcer
caused by HSV-2 provides a site for HIV entry on HIV
negative patients and the associated inflammation increases
the number of activated cells that can be targeted by HIV. In
contrast, many HIV-1-infected patients are already infected
with HSV-2 at the time of HIV-1 acquisition and having
herpes doubled the risk of subsequently catching HIV.
Epidemiological studies found that at least a 2- to 4-fold
increased risk of acquiring HIV among patients infected
with HSV-2,16,32 and may account for 40–60% of new HIV
infections in high HSV-2 prevalence populations.2,31-33
Susceptibility to HIV is higher among patients who have
recently acquired HSV-2. Nonetheless, it also found an
increased risk of HIV infection even when herpes infection
appeared dormant or was causing no symptoms.34 The
high prevalence and incidence of HSV-2 infection among
HIV-positive patients compared with the general population
suggests a critical need for screening and preventive
programs among this targeted group. This would be of help
in prevention of HIV infection.
It has been stated that more frequently virus infections
are associated with a compromised immune system in
HIV-positive patients. Prior study confirmed that immune-
suppressed patients are more vulnerable to common virus
infections.4 Our findings showed many HIV-positive
patients who were HSV-1 seropositive had CD4 count
more than 500 cells/mm3. In contrast to HSV-1, many
HIV-positive patients who were HSV-2 seropositive had
CD4 count < 200 cells/mm3. Some studies also reported that
there were an association between HSV-2 seropositivity and
CD4 count,35 but others have shown conflicting results.39
Interestingly, when we analysis of a comparison of IgG
antibody titers, the results showed significantly higher
titers of IgG antibody against both HSV-1 and HSV-2 in
HIV-positive patients compared to HIV-negative patients.
We also found a significant different in the titer of HSV-
2 between HIV-positive patients with CD4 cell count
< 200 cells/mm3 and those patients with CD4 count > 500
cells/mm3. However, we did not find significant correlation
between the titer of both herpes viruses and CD4 count.
There are some possibilities could explain this finding.
First, HSV infection did not modulate the relationship
of HIV-1 to CD4+ T cell count suggests that the effect of
HSV-2 infection on CD4+ T cell count manifests prior to
acquisition of HIV-1.41 Second, it is suggested that CD8+
T-cells are a critical component of the response to HSV
infection but not CD4+ T-cells. The level of anti-HSV
antibody did not have any impact on the percentage or
absolute number of late-differentiated CD4+ T-cells.42 In
contrast, HSV-1 infection reduced the number of infiltrating
CD8+ T cells.43 A study also confirmed that among HIV-
positive patients, the frequency of HSV-2 -specific CD-8
T cells is inversely related to HSV-2 severity.44
In conclusion, seroprevalence of HSV-1 and HSV-2
among HIV-positive patients was high with no correlation
with CD4 count. This study may increase the understanding
about the spread of herpes simplex virus and may be
valuable for guiding prevention efforts of recurrent herpes
119Sufiawati, et al.: Seroprevalence of Herpes Simplex virus types 1 and 2 and their association
simplex virus disease among HIV-positive patients. In
addition, the detection of IgG antibodies against herpes
simplex virus may help seropositive people identify
symptoms and protect their partners from acquiring HIV,
or vice versa, protect HIV-positive patients from acquiring
the most common viral opportunistic infection.
ACKNOWLEDGEMENT
We are grateful to Sharaf M. Tugizof, Ph.D., DSc., at
the university of California, San Francisco (UCSF), USA,
for his kind support and great expertise, and valuable
suggestion in this study. We would like also to thank the
staff of the clinical pathology laboratory, Hasan Sadikin
Hospital, Bandung, Indonesia, for their laboratory help.
This work was supported by the Directorate General
of Higher Education, Ministry of National Education
Indonesia.
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