American Journal of

EPIDEMIOLOGY
Volume 156 Copyright © 2002 by The Johns Hopkins
Bloomberg School of Public Health
Number 8
Sponsored by the Society for Epidemiologic Research
October 15, 2002 Published by Oxford University Press

ORIGINAL CONTRIBUTIONS

Herpes Simplex Virus and Risk of Cervical Cancer: A Longitudinal, Nested Case-
Control Study in the Nordic Countries

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Matti Lehtinen1, Pentti Koskela2, Egil Jellum3, Aini Bloigu2, Tarja Anttila4, Göran Hallmans5,
Tiina Luukkaala1, Steinar Thoresen6, Linda Youngman7, Joakim Dillner8, and Matti Hakama9
1 School of Public Health, University of Tampere, Tampere, Umeå, Sweden.
Finland. 6 The Cancer Registry of Norway, Oslo, Norway.
2 National Public Health Institute, Oulu, Finland. 7 Clinical Trial Service Unit, University of Oxford, Oxford,
3 Institute of Clinical Biochemistry, University of Oslo, Oslo, United Kingdom.
Norway. 8 Department of Clinical Microbiology, University of Lund,
4 Department of Microbiology, University of Oulu, Finland. Malmö, Sweden.
5 Department of Nutrition Research, University of Umeå, 9 Finnish Cancer Registry, Helsinki, Finland.

Received for publication November 19, 2001; accepted for publication May 28, 2002.

Human papillomaviruses (HPVs) play the major role in cervical carcinogenesis. The authors reevaluated the role of
herpes simplex virus type 2 (HSV-2) in this multistage process by conducting a longitudinal, nested case-control study
using 1974–1993 data and comparing the results with those from a meta-analysis of studies. A Nordic cohort of 550,000
women was followed up for an average of 5 years, after which 178 cervical carcinoma cases and 527 controls were
identified. HSV-2; HPV-16, HPV-18, and HPV-33; and Chlamydia trachomatis antibodies were determined at baseline
by HSV-2 glycoprotein gG-2 and HPV virus-like-particle enzyme immunoassays and by using the
microimmunofluorescence method. The relative risk of cervical carcinoma was calculated by conditional logistic
regression. Longitudinal studies on HSV-2 and cervical neoplasia were identified through MEDLINE (National Library
of Medicine, Bethesda, Maryland), and weighted mean relative risks were calculated. Smoking (relative risk = 1.6, 95%
confidence interval (CI): 1.1, 2.3) and HPV-16/HPV-18/HPV-33 (relative risk = 2.9, 95% CI: 1.9, 4.3) were both
associated with cervical carcinoma. The smoking- and HPV-16/HPV-18/HPV-33–adjusted relative risks for HSV-2 were
1.0 (95% CI: 0.6, 1.7) and 0.7 (95% CI: 0.3, 1.6), respectively, for HPV seropositives. In the meta-analysis, the relative
risk for HSV-2 was 0.9 (95% CI: 0.6, 1.3). In both sets of data, HSV-2 did not play a role in cervical carcinogenesis.

cervix neoplasms; herpes simplex; longitudinal studies; meta-analysis; retrospective studies

Abbreviations: CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; HPV, human papillomavirus; HSV-2,
herpes simplex virus type 2; RR, relative risk.

Since the end of the 1960s, herpes simplex virus type 2 study (1), together with inconsistent detection of HSV-2
(HSV-2) had been considered the major cause of invasive DNA and consistent identification of human papillomavirus
cervical carcinoma, but a longitudinal seroepidemiologic (HPV) DNA in cervical carcinoma (2), revised this paradigm

Reprint requests to Dr. Matti Lehtinen, University of Tampere, School of Public Health, POB 607, FI-33101 Tampere, Finland (e-mail: llmale@uta.fi).

687 Am J Epidemiol 2002;156:687–692

688 Lehtinen et al.

in the early 1980s. However, assessing an infectious etiology
of chronic disorders is difficult, and cohort studies that
include complete follow-up are less vulnerable to different
biases (3). Hence, most reliable results are provided by a
combination of longitudinal design, a population-based
setting, and state-of-the-science exposure assessment.
During the 1990s, considerable improvements took place
in the serologic diagnosis of herpes simplex and HPV infec-
tions. HSV-2 antibodies can now be determined by a glyco-
protein gG-2 enzyme-linked immunosorbent assay (ELISA)
that suffers only minimally from cross-reactivity between
herpes simplex virus type 1 and HSV-2 because a majority of
glycoprotein gG-2 is coded by a unique segment of the HSV-
2 genome (4–6). The HPV virus-like particle ELISA is a
highly type-specific and reproducible, albeit not very sensi-
samples to screen for congenital infections (11). The blood
samples are drawn at all maternity clinics, and about 98
percent of all pregnant women donate samples to the Finnish
Maternity Cohort bank. In 1993, this cohort had collected
710,000 samples from 390,000 women; the samples were
stored at –25°C.
The Janus Project was established in Norway in 1973 (12).
In 1991, the Janus Serum Bank had collected 424,000 serum
samples from 293,000 donors; again, the samples were
stored at –25°C. About 145,000 women were recruited
during routine health examinations. During phase I (1974–
1978) and phase II (1986–1991), the participation rates were
85 percent and 75 percent, respectively.
The Västerbotten Project was established in northern
Sweden in 1986 (13). Each year, all residents of Väster-

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tive, marker of cumulative HPV exposure (7–9). These assay
developments have substantially improved the potential of
serum sample banks to identify causes of anogenital cancers.
Natural history studies have been conducted with only
cervical intraepithelial neoplasia as the endpoint (10).
Linkage of population-based cancer registries with popula-
tion-based serum banks overcomes ethical problems related
to invasive cervical carcinoma being the endpoint. Applica-
tion of incidence density sampling for matched controls
increases the power to detect effects without bias. The lag
between serum withdrawal and diagnosis accounts for the
long latency period of invasive cervical carcinoma. By using
state-of-the-science laboratory assays and a nested case-
control design, we analyzed by far the largest known quan-
tity of serum sample material collected since the 1970s in the
Nordic countries (1974–1993) and compared the results
systematically with those from other longitudinal studies on
HSV-2 and cervical carcinoma.
MATERIALS AND METHODS
Serum banks
The study cohort consisted of 550,000 women who
donated blood samples to population-based serum banks in
Finland, Norway, and Sweden (table 1). In 1983, the Finnish
Maternity Cohort started to collect first-trimester blood
botten County aged 30, 40, 50, 60, and 69 years are invited
to participate in the health-promotion project, which
includes donating biologic samples for future medical
research. From 1986 to 1993, the participation rate was
about 65 percent. In 1993, the Västerbotten serum bank had
collected samples from 15,000 women; the samples were
stored at –80°C.
Cancer registries
The Finnish Cancer Registry, the Cancer Registry of
Norway, and the cancer registry at the Oncological Centre in
Umeå, which covers northern Sweden, are population based
and country- or countywide (14, 15). Notifications are
received from hospitals, laboratories, and physicians, and
reporting coverage is practically 100 percent.
Identification of cases and controls
Women with invasive cervical carcinoma as the primary
cancer diagnosis were identified by linkage of the serum
banks and cancer registries. The linkages were performed in
1994 by using personal identification numbers and resulted
in identification of 220 cases. Forty-two cases were
excluded: 24 did not have enough sera, two could not be
located, four had donated sera less than 15 days before the
diagnosis, and, for 12, the disease was not invasive.

TABLE 1. Characteristics of the Nordic cohort for a nested case-control study of the cervical carcinoma
risk associated with previous exposure to sexually transmitted microorganisms, 1974–1993

Period of Female serum Age at

Cases Controls
Serum bank Geographic location serum sample diagnosis
(no.) (no.)
sampling donors (no.) (years)

Janus Project Three counties in Norway 1974–1978 29,000 79 237 26–64

Several counties in 1986–1991 116,000 47 141 35–63
Norway
Västerbotten Project Västerbotten County, 1986–present 15,000 4 12 40–61
northern Sweden
Finnish Maternity Finland 1983–present 390,000 48 137 23–49
Cohort

Total Nordic countries 1974–present 550,000 178 527 23–64

Am J Epidemiol 2002;156:687–692

For each case, three female controls who were cancer free
at the time of the case’s diagnosis were selected randomly
and were matched for age at serum sampling (±2 years),
length of time that the serum sample was stored (±2 months),
and cohort area (Finland, Sweden, and subcohorts of the
Janus Serum Bank in Norway). The earliest prediagnostic
sample was chosen. If three controls could not be found, the
matching criteria were widened: the age of six controls
differed by more than 4 years (maximum, 4.4 years) from
that of the case; the maximum difference in storage time was
5 months. Samples from seven (1.3 percent) of 527 controls
could not be located.
Laboratory methods
Immunoglobulin G antibodies to HSV-2 were determined
by using a commercially available HSV-2 glycoprotein gG-
2 ELISA (Biokit SA, Barcelona, Spain) according to the
manufacturer’s recommendations. Immunoglobulin G anti-
bodies to HPV-16, HPV-18, and HPV-33 were determined
by a standard virus-like particle ELISA using predetermined
cutoff levels as an indication of exposure to the HPV types
(8, 9). Chlamydia trachomatis antibodies were determined
by using a standard microimmunofluorescence test (11).
Current smoking was defined by serum cotinine levels
using a quantitative ELISA modification (STC Technolo-
gies, Bethlehem, Pennsylvania). Because smoking is a risk
factor for passive smoking, the cutoff level of 20 µg/liter was
used to distinguish active smokers from nonsmokers.
Criteria for selecting studies for the meta-analysis
We followed the BMJ recommendations (16) for selecting
studies for review. All longitudinal studies on HSV-2 and
cervical neoplasia available until June 2001 were eligible for
inclusion. The studies were required to fulfill the following
two criteria: 1) follow-up with use of a cohort, nested case-
control approach among initially healthy women; and 2)
definition of cumulative exposure (previous or present infec-
tion measured by serology for HSV-2 among either the total
cohort or cases and controls) with a report of a relative risk
and its variance (confidence interval), corresponding odds
ratio, or pertinent frequency data (use of endpoints that could
be categorized into the following three groups: 1) invasive
cervical carcinoma, 2) inseparable invasive cervical carci-
noma/carcinoma in situ (alone or included in cervical
intraepithelial neoplasia), or 3) squamous cell carcinoma).
Unless otherwise indicated, we used smoking-adjusted and/
or matched (age, length of time of sample storage, country)
point estimates based on individual data.
In addition to manual searches, we searched through
MEDLINE (National Library of Medicine, Bethesda, Mary-
land) from 1966 to June 2001 by using the following search
terms from Medical Subject Headings (MeSH) of Index
Medicus and their combinations: herpes simplex virus and
cervical neoplasia, and case-cohort or cohort or follow-up or
longitudinal or prospective or retrospective study.
Am J Epidemiol 2002;156:687–692
Herpes Simplex Virus and Cervical Cancer Risk 689
Statistical analysis
Relative risks and their 95 percent confidence intervals
were estimated for invasive cervical carcinoma and squa-
mous cell carcinoma by using conditional logistic regression
(17) with Stata computer software (version 5.0; Stata Corpo-
ration, College Station, Texas). Unconditional logistic
regression analysis was applied for HPV-seropositive cases
and controls, including the matching variables in the model.
The weighted mean relative risk was calculated by taking
a weighted average of the log relative risk (RR) from eligible
studies, the weight assigned to each log RR being propor-
tional to the inverse of its variance (18). The variances were
derived from the published confidence intervals or from the
pertinent frequency data by means of standard formulae. The
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homogeneity of log(RRi) across the studies was tested by
using χ2 statistics.
RESULTS
We identified 178 cases with invasive cervical carcinoma,
150 of whom had cervical squamous cell carcinoma. Mean
lag time between serum sampling and diagnosis was 5 years
(range, 0.1–18) (table 1).
In univariate analysis, C. trachomatis, smoking, and HPV-
16/HPV-18/HPV-33 were associated with an increased risk
of invasive cervical carcinoma (RR = 2.5, 95 percent CI: 1.7,
3.9; RR = 1.6, 95 percent CI: 1.1, 2.4; and RR = 2.8, 95
percent CI: 1.9, 4.3, respectively). HSV-2 was associated
with a moderately increased risk (RR = 1.3, 95 percent CI:
0.8, 2.1). The crude risk of squamous cell carcinoma was
also low (RR = 1.4, 95 percent CI: 0.9, 2.4).
Stepwise adjustment for C. trachomatis, smoking, HPV-
16/HPV-18/HPV-33, and a combination of the latter two
factors removed the excess risk of both invasive cervical
carcinoma and squamous cell carcinoma (RR = 1.0, 95
percent CI: 0.6, 1.7 and RR = 1.1, 95 percent CI: 0.6, 1.9,
respectively; table 2). Among HPV-16/HPV-18/HPV-33
seropositives, HSV-2 was not associated with any excess
risk of invasive cervical carcinoma or squamous cell carci-
noma (RR = 0.7, 95 percent CI: 0.3, 1.6 and RR = 0.7, 95
percent CI: 0.3, 1.7, respectively).
Finally, with the exception of an increased risk (RR = 2.2,
95 percent CI: 0.5, 9.4) just prior to diagnosis, the lag did not
affect the point estimates for invasive cervical carcinoma
when the data were divided into four lag categories: 1–12,
13–59, 60–119, and ≥120 months (figure 1). On the other
hand, the point estimates for squamous cell carcinoma
tended to increase with increasing lag.
Six longitudinal seroepidemiologic studies (1, 19, 20–22,
the present study) on the association of HSV-2 with cervical
neoplasia (invasive cervical carcinoma and/or carcinoma in
situ) were identified. Altogether, more than 3 million person-
years of follow-up resulted in about 200 matched case-
control pairs, triplets, or quadruplets (table 3). Three studies
had applied glycoprotein gG-2 ELISA, and four studies had
applied the older serologic methods by using a ratio of HSV-
2 and herpes simplex virus type 1 antibody levels with a
predefined cutoff level of 0.85 (also known as the II/I ratio
(23)). Weighted mean relative risks of 0.7 (95 percent CI:
690 Lehtinen et al.

TABLE 2. Relative risk of cervical carcinoma associated with previous exposure to herpes simplex virus type 2, overall and among
human papillomavirus* seropositives, the Nordic countries, 1974–1993

Adjustment for Adjustment for

Adjustment for Adjustment for
Cases Controls Crude Chlamydia smoking and
smoking HPV*
Category trachomatis HPV*

% %
No. No. RR† 95% CI† RR 95% CI RR 95% CI RR 95% CI RR 95% CI
positive positive

Invasive cervical
carcinoma 178 15 525‡ 12 1.3 0.8, 2.1 1.2 0.7, 2.0 1.3 0.8, 2.1 1.0 0.6, 1.7 1.0 0.6, 1.7
Squamous cell
carcinoma 148 16 437 12 1.4 0.9, 2.4 1.3 0.8, 2.3 1.4 0.8, 2.4 1.1 0.6, 2.0 1.1 0.6, 1.9
Invasive cervical
carcinoma* 64 17 95 24 0.7 0.3, 1.6 0.7 0.3, 1.6 0.7 0.3, 1.6
Squamous cell
carcinoma* 59 17 85 24 0.7 0.3, 1.7 0.7 0.3, 1.6 0.7 0.3, 1.7

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* Human papillomavirus (HPV)-16, HPV-18, and HPV-33 seropositives combined.
† RR, relative risk; CI, confidence interval.
‡ Insufficient serum sample volume for two controls.

0.4, 1.3) and 0.9 (95 percent CI: 0.5, 1.9), respectively, were
obtained, indicating lack of any increased risk of cervical
neoplasia, irrespective of the method used to determine
HSV-2 antibodies (table 3). When the glycoprotein gG-2
ELISA was used, the grand mean relative risk for all longitu-
dinal studies was 0.9 (95 percent CI: 0.6, 1.3).
FIGURE 1. Human papillomavirus types 16, 18, and 33 and smok-
ing-adjusted relative risks of invasive cervical carcinoma (ICC, n =
178) and squamous cell carcinoma of the uterine cervix (SCC, n =
150) associated with herpes simplex virus type 2 infection, by lag
between serum sampling and cancer diagnosis in a Nordic cohort of
550,000 women followed up for an average of 5 years between 1974
and 1993. Relative risks for lag categories 1–12, 13–59, 60–119, and
≥120 months: ICC (downward triangles)—2.2 (95% confidence inter-
val (CI): 0.5, 9.4), 0.5 (95% CI: 0.2, 1.1), 1.6 (95% CI: 0.5, 5.7), and
1.7 (95% CI: 0.4, 6.5), respectively; SCC (upward triangles)—2.2
(95% CI: 0.5, 9.5), 0.5 (95% CI: 0.2, 1.2), 1.6 (95% CI: 0.4, 5.5), and
3.1 (95% CI: 0.7, 15), respectively.
DISCUSSION
In the present study, previous HSV-2 infection was not
associated with any excess risk of subsequent development
of cervical carcinoma. Similarly, in a meta-analysis of
comparable longitudinal seroepidemiologic studies that
yielded a remarkably narrow confidence interval, HSV-2
was not associated with any excess risk of cervical carci-
noma. Since the study by Choi et al. (19) was conducted, not
one of the longitudinal seroepidemiologic studies on HSV-2
and cervical neoplasia (1, 20–22) has found a significantly
increased relative risk, but all were underpowered to detect a
small excess risk. However, the upper 95 percent confidence
limit of our final meta-analysis, 1.3, indicates that if an
excess risk of cervical neoplasia were associated with HSV-
2, it would be very small.
Our results also indicate that the previous estimates of the
effect of HSV-2 on cervical neoplasia found in many cross-
sectional case-control studies (23–26) were biased. Possible
sources of bias include the cross-sectional design, inade-
quate power, misclassification, and confounding due to
uncontrolled risk factors, for example, HPV. Adjustment for
smoking and C. trachomatis, known risk factors for cervical
neoplasia/surrogates of risk-taking behavior (11, 27),
reduced our point estimates; further adjustment for HPV-16/
HPV-18/HPV-33 removed the excess risk from the point
estimate for women who tested positive for HSV-2 anti-
bodies. Moreover, HSV-2 antibodies were associated with
no excess risk for HPV-16/HPV-18/HPV-33–seropositive
women, also indicating that HSV-2 is not a cause of invasive
cervical carcinoma.
With few exceptions, HSV glycoprotein gG-2 ELISAs are
now considered highly sensitive (>95 percent) and reproduc-
ible (coefficient of variation, ≤5 percent) (6, 28). The test we
used should not have biased the relative risk toward unit risk.
Although our final relative risk estimates were consistent
with all of the previous longitudinal study results based on
the early nonstandardized tests, our increased crude relative
risk estimates for invasive cervical carcinoma and squamous
cell carcinoma suggest that the previous results were indeed
affected by a lack of test validity. Together with lower spec-

Am J Epidemiol 2002;156:687–692
 Herpes Simplex Virus and Cervical Cancer Risk 691

TABLE 3. Meta-analysis of longitudinal seroepidemiologic studies of herpes simplex virus type 2 and
cervical neoplasia* using the II/I ratio† or glycoprotein gG-2 enzyme-linked immunosorbent assay to
determine previous herpes simplex virus type 2 infection with serum antibodies

Mean follow-
Method used and Person-years of Cases
up time OR‡ 95% CI‡ Study and year (reference no.)
country follow-up (no.) (no.)
(years)

II/I
Canada 88,000 56 1.9 2.1 0.3, 15 Choi et al., 1977 (19)
Czech Republic 33,000 33 1.6 0.7 0.2, 3.0 Vonka et al., 1984 (1)
United States 7,000 23 3.5 0.8 0.2, 3.3 Adam et al., 1985 (21)
Finland 181,000 32 5.0 1.0 0.3, 2.8 Lehtinen et al., 1992 (20)

Total 309,000 144 2.8 0.9 0.5, 1.9

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gG-2
Czech Republic 33,000 33 1.6 2.0 0.5, 7.6 Vonka et al., 1984 (1)
United States 7,000 23 3.5 0.8 0.2, 2.8 Adam et al., 1985§ (21)
Finland 400,000 72 10.1 0.5 0.2, 1.0 Lehtinen et al., 1996 (22)

Total 440,000 138 6.6 0.7 0.4, 1.3

Present study 2,500,000 178 5.0 1.0 0.6, 1.8

Grand total¶ 2,940,000 316 5.3 0.9 0.6, 1.3

* Carcinoma in situ (alone or included in cervical intraepithelial neoplasia) and invasive cervical carcinoma.
† A ratio of herpes simplex virus type 2 and herpes simplex virus type 1 antibody levels with a predefined cutoff
level of 0.85 (23).
‡ OR, odds ratio; CI, confidence interval.
§ The gC-2 assay was used.
¶ Combines all studies in which glycoprotein gG-2 enzyme-linked immunosorbent assay was used.

ificity and sensitivity of the II/I assays and the early glyco-
protein gG-2 ELISAs, regression toward the mean probably
biased the previous results toward unit risk. In our longitu-
dinal study, we were able to assess a more accurate crude
relative risk; however, it proved to be confounded by HPV
exposure, as indicated by both adjusting for HPV-16/HPV-
18/HPV-33 antibodies and restricting the analysis to HPV-
seropositive cases and controls only. Thus, the meta-analysis
of the previous longitudinal studies yielded a probably
correct result for the wrong reasons or as a result of chance.
Why, then, did the cross-sectional studies find an associa-
tion? Invasive cervical carcinoma patients produce an
autoantibody response to an HSV-2–inducible tumor-
specific tissue polypeptide that is recognized by the II/I
assay (29). This finding also applies to the antibodies deter-
mined by HSV-2 nonstructural antigens (25, 26, 30, 31)
suggested to be found in invasive cervical carcinoma (32).
Cross-reactivity between viral infected cell protein (ICP) 8
and a homologous/functionally identical cellular protein,
proliferating cell nuclear antigen, which is abundant in inva-
sive cervical carcinoma, is one possible explanation (33–37).
It is plausible that ICP8 antibodies and proliferating cell
nuclear antigen autoantibodies, as well as the HSV-2 anti-
bodies and autoantibodies measured by the II/I assay, were
both indistinguishable and readily detectable in the incident
invasive cervical carcinoma cases.
Although our study did not associate a short time period
from serum sampling to cancer diagnosis with a significantly
increased risk, the possibility that cervical neoplasia predis-
poses to HSV-2 infection, that is, reverse causality, should
not be neglected as an explanation for the association found
in the previous cross-sectional case-control studies. Longitu-
dinal design removes much of the possible bias due to anti-
body response to the cross-reactive tumor-specific antigens,
or amplification of the virus by the occult neoplasia, irre-
spective of the serologic test used (1, 20, 30). Although the
nonsignificant increase of HSV-2–associated risk of squa-
mous cell carcinoma by increasing lag may deserve consid-
eration in even larger studies, we conclude that HSV-2 is not
likely to be causally associated with invasive cervical carci-
noma or squamous cell carcinoma.
ACKNOWLEDGMENTS
This study was supported by the Nordic Cancer Union and
the Academy of Finland. The Janus Serum Bank is owned by
the Norwegian Cancer Society. The serum samples were
provided following approval by the institutional review boards.

Am J Epidemiol 2002;156:687–692
692 Lehtinen et al.
The authors thank Prof. Vladimir Vonka for stimulating
discussions and Dr. Vera Abeler for histologic classification.
This is publication number 20 of the Nordic Biological
Specimen Banks study group on Cancer Causes and Control
(NBSBCCC).
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