HUMAN PAPILLOMAVIRUS TYPE 16 (HPV-16), herpes simplex virus type 2 (HSV-2), HIV, Chlamydia trachomatis and Treponema pallidum cause sexually transmitted infections (STIs).1–4 The proportion of asymptomatic infections varies but is considerable for all of these microorganisms,5–8 which makes it difficult to clinically monitor their spread, especially in rapidly changing socioeconomic conditions.9 However, recent developments in laboratory methods with high sensitivity, specificity, and reproducibility now enable determination of type-specific immunoglobulin G antibodies also to HPV-16 and HSV-2.10,11
There is a dose-response association between number of lifetime sexual partners and HPV-16, HSV-2, and C trachomatis seroprevalence.12–14 Therefore, antibodies to STIs should refer to a population with risk-taking sexual behavior and multiple infections that predispose to concomitant seropositivity for STIs in women. We assessed the seroprevalences of STIs and the excesses of concomitant seropositivity, assuming no concomitant seropositivity above that which would be expected at random for the different STIs in a population-based cohort of pregnant women attending women's consultation clinics in in Tallinn, Estonia from 1996 to 1997.
Estonia (area, 45,216 km2) is a country with approximately 1.5 million inhabitants, 834,270 of whom are women.15 Approximately one third of the population is located at the capital of Estonia, Tallinn.15 The Estonian fertile-aged female population by age and reproductive status is described in Table 1. Women's consultation clinics are the main public healthcare institutions responsible for antenatal care in Estonia. The proportion of private clinics and gynecologists providing antenatal care is low. In 1997, 70% of all pregnant women in Estonia started their antenatal visits before week 12 of gestation, and the average number of antenatal visits was 11.16 Venous blood samples are always drawn for screening of congenital infections before week 12 of gestation.
From 1996 to 1997 all five consultation clinics and one major private clinic in Tallinn participated in a study about HPV-16 seroprevalence among young pregnant women. After obtaining informed consent, 3055 samples of venous blood were collected simultaneously with routine blood samples during the first trimester of pregnancy (and in some cases were followed by an elective abortion). The blood samples were collected from the clinics daily and the sera were stored at −20 °C at the Institute of Experimental and Clinical Medicine, Tallinn. The unique personal identification numbers used in Estonia made it possible to obtain additional information about reproductive history of the women by linkage to the Estonian Medical Birth Registry and Estonian Medical Abortion Registry. We identified 1904 sera from women who had no previous full-time pregnancies. A random sample of sera from 1163 women was chosen for the HPV-16 seroprevalence study, and sera from 1110 women were available for antibody analyses to the STIs (Table 2). In addition, hepatitis B virus (HBV), hepatitis C virus (HCV), and Chlamydia pneumoniae antibodies were determined.
Serum antibodies to HBV, HCV, HIV, HPV-16, HSV-2, C trachomatis, C pneumoniae, and T pallidum were determined as follows:
Enzyme immunoassay was used for the determination of antibodies to HBV surface antigen (Enzygnost Anti-HBs II; Behring Diagnostics, Marburg, Germany). Recombinant HCV antigens were used for the determination of antibodies to HCV by enzyme immunoassay (Innotest HCV Ab III; Innogenetics, Zwijndrecht, Belgium). Purified immunodominant antigens of the core and envelope proteins of HIV type 1 (Weiss isolate) and an immunodominant epitope of HIV type 2 were used for the determination of antibodies to HIV types 1 and 2 by enzyme immunoassay (Murex HIV1+2, Murex Biotech Limited, Dartford, UK). HPV-16 capsids expressed in baculovirus and comprising both L1 and L2 capsid proteins were used as antigens in enzyme-linked immunosorbent assay (ELISA).10 Each ELISA plate contained three positive control sera (serum pools from women with cervical cancer or cervical intraepithelial neoplasia). A total of 40 sera from virginal women were used as negative controls. For each serum, the difference in optical densities obtained with plates coated with intact HPV-16 capsids and plates coated with a control antigen (disrupted bovine papillomavirus capsids) was calculated.17 The assays for all positive results were repeated and samples with discrepant results were reanalyzed. The preassigned cut-off level of optical density 0.100 was used.
Purified HSV-2 glycoprotein G was used for the determination of type-specific immunoglobulin G antibodies by ELISA (Bioelisa HSV-2 IgG; Biokit SA, Barcelona, Spain). Pooled C trachomatis serovars GFK, CHJI, BED (Washington Research Foundation, Seattle, WA) and C pneumoniae strain K6 were used for C trachomatis and C pneumoniae immunoglobulin G antibodies by the microimmunofluorescence method.18 A cut-off level of 1/16 was used. Mouse monoclonal antigens (Chlamyset FA; Orion Diagnostica, Espoo, Finland) were used for the quality control of each antigen patch. C trachomatis-positive/C pneumoniae-negative sera, C trachomatis-negative/C pneumoniae-positive sera, and low-titred and high-titred C trachomatis/C pneumoniae-positive human control sera were used.
An in vitro agglutination test with gelatin particle carriers sensitized with purified pathogenic T pallidum was used for the determination of T pallidum antibodies (Serodia-TP·PA, Fujirebio Inc., Tokyo, Japan). Positive results were confirmed by demonstrating specific immunoglobulin antibodies with an assay based on the labeled and unlabeled recombinant proteins TpN15, TpN17, and TpN47, which carry the immunodominant epitopes of T pallidum (ICE*Syphilis Detection Pack; Murex Biotech Limited, Dartford, UK).
Coinfection with STIs was defined as concomitant seropositivity for a minimum of two different STIs in a woman. Seroprevalence is defined as proportion of women seropositive for an infection among all women. The expected prevalences of concomitant seropositivity for two three, and four STIs were estimated assuming pairwise, threewise, and fourwise independence of the infections as follows: EQUATION 1 where P(X) and P(Y) are denoted for the seroprevalences of infections X and Y, P(X,Y) for the seroprevalences of both infections X and Y, and Pe refers to the expected prevalences. Expected number Ne of women with simultaneous seropositivity was estimated as follows: EQUATION 2 Chi-square statistics were used to estimate the statistical significance between observed and expected numbers of women with coinfections.
During February 1996 and November 1997, 3055 sera were collected from 2943 young pregnant women in Tallinn. A total of 1904 sera were donated by 1823 women who had not had a full-term pregnancy. Among these women, 1466 gave birth, 345 underwent an abortion, and for 12 women the relevant pregnancy outcome remained unknown. According to the Estonian Medical Birth Registry, from 1996 to 1997 3236 primiparous mothers 15 years to 30 years were registered in Tallinn. Sera from 152, 1484, and 161 primiparous mothers in consecutive age groups (17 years or younger, 18 years to 25 years, and 26 years to 30 years) were stored (Table 3). Mothers 25 years and 26 years of age mostly contributed to the last age group.
Overall, 581 of 1110 women (52%) were seropositive for one or more of the microorganisms. Prevalences of specific antibodies to HPV-16, HSV-2, C trachomatis, and T pallidum were 36.1%, 12.7%, 20.2%, and 2.4%, respectively (Table 4). No HIV-seropositive persons were found. Moderate trends of increasing seroprevalence by age were observed for HPV-16 and HSV-2 antibodies but not for the bacterial microorganisms (Table 4). The relative proportions of seronegative results were comparable for the first four age groups.
A total of 134 women (23.1%) were seropositive for two STIs, 33 women (5.6%) were seropositive for three STIs, and 4 women (0.7%) were seropositive for four different STIs. Overall, 410 women (70.6%) had antibodies to only one STI (Table 5). The latter comprised mainly HPV-16-seropositive women, whereas 54% to 81% of women who were HSV-2, C trachomatis, or T pallidum seropositive were concomitantly seropositive for at least one of the other STIs (Table 5).
The observed number of women with concomitant seropositivity for at least two STIs exceeded the expected number, which assumed the observed seroprevalences and no coinfections above the random (i.e., independent of spread of the STIs) (Tables 4 and 6). Statistically significant differences between observed and expected numbers of the combinations were found for all the combinations (P range, 0.000-0.015) (Table 6). Antibodies to HBV, HCV, and C pneumoniae were determined as controls. For empirical verification of our method, we replaced C trachomatis seropositive results with C pneumoniae seropositive results and did not find excess of concomitant seropositivity to HPV-16, HSV-2, and T pallidum (Table 7). This was also the case when T pallidum seropositive results were replaced with HBV, HCV, or HBV and HCV seropositive results (data not shown).
Among the 1110 women, serologic evidence of 793 STIs were found in 581 women. Two hundred fifty-seven, 49, and 4 women were seropositive for a minimum of two, three, and four STIs, respectively, whereas 178.8, 14.1, and 0.25 women were expected to be infected by chance, respectively. Finally, we compared the estimated expected numbers of women with concomitant seropositivity with observed numbers. The proportions of excess concomitant seropositivity for STIs among women with concomitant seropositivity for two, three, and four STIs were 30% (257-178.8/257) (Table 6), 71% (49-14.1/49), and 94% (4-0.25/4), respectively. For the four youngest age groups the corresponding figures were 32%, 63%, and 95%.
Our results indicate that a majority of pregnant Estonian women younger than 30 years have been exposed to one or more STIs. The relative proportions and age distributions of the seroprevalences were different between the three most common agents: HPV-16, HSV-2, and C trachomatis. The figures for HSV-2 seroprevalence are comparable with those in Finland and Sweden, where the seroprevalences in the similar age groups ranged from 13.9% to 17.4% and 17% to 32% in the late 1980s and early 1990s.19,20 Seroprevalence of C trachomatis among a comparable Swedish population was 24.7%,21 which also comes close to our figures. Seroprevalence of HPV-16 was 1.5- to 2-fold higher among pregnant women in Tallinn, Estonia than in Stockholm (20%) or Helsinki (24%).22,23 Differences in time, study materials, and primiparous women versus all pregnant women may not totally explain the difference, and other factors such as rapidly changing behavioral and socioeconomic factors may be involved.9
We expected to identify subjects with coinfections of multiple STIs if seropositive persons with different STIs were markers of sexual behavior. Therefore, we assessed the observed numbers of women concomitantly seropositive for two, three, or four STIs and compared the observed numbers with expected numbers on basis of the prevalences of any STIs occurring alone and assuming total independence of spread. Given the seroprevalences, 193 subjects concomitantly seropositive for the different STIs were expected by chance whereas 310 were observed, yielding 38% additional STIs among those with concomitant seropositivity. This is a much smaller figure than 71% and 94% of additional that STIs we found among seropositive persons for three and four STIs, respectively. However, only 5%37 of all the women were seropositive to three or more STIs. In a population with a prevalence of 20% for C trachomatis and 36% for HPV-16, the presence (i.e., prevalence of women with risk-taking sexual behavior) is probably much higher than the 5%. When only the most prevalent STIs-HPV-16, HSV-2, and C trachomatis-were considered, 32% additional infections (250-170,5/250) were observed. If seropositivity to T pallidum (2.4%) was accepted as a proxy of an STI core group (i.e., group with high risk-taking sexual behavior24,25), the Tallinn core group had a relatively small effect on concomitant seropositivity to multiple STIs.
Various large-scale studies have evaluated by means of relative risks or odds ratios the possibility of using STI antibodies as a surrogate of sexual behavior. Seropositivity for C trachomatis was independently associated with early sexual experience and number of lifetime partners.12,21 Also, seropositivity for HPV-16 capsids, measured by odds ratios, was a strong marker of number of lifetime partners.1,13 Seropositivity for HSV-2 has been launched as an objective marker of sexual behavior.14,26 We used chi-square statistics as an indicator of the strength of the association between two STIs. P values were highly significant, indicating an association. By replacing C trachomatis with C pneumoniae or infections with low rates of sexual transmission,27 we observed no such associations. However, antibodies to a minimum of two STIs did not identify the same women. Therefore, measures of the strength of the association, relative risks or odds ratios28 and chi-square statistics may not be considered as conclusive evidence that seropositivity can be regarded as a surrogate for risk-taking behavior. In fact, traditionally recognized behavioral correlates of sexual behavior (e.g., number of life-time partners) or serology have been reported not to identify all cases in communities with high prevalences of HSV-2 or HPV infections.12,29 We arrived at the same conclusions. Most likely, the interplay between sexual behavior and coinfection of different STIs at the individual or population level is more complex (e.g., because of different transmission probabilities of the STIs or different levels of assortative spread30–32) than number of lifetime partners, which is well reflected by STI seropositivity. This cannot be studied in detail without a questionnaire about sexual behavior.
Transmission of an STI within a population is influenced by behavioral and sociocultural factors and changing environmental reservation.30–32 Sexual behavior is without doubt the most important single factor; however, the reverse seems not to be true. The observed relatively low proportion of additional infections among women seropositive for two STIs (30%) suggests that seropositivity to these STIs is not an accurate measure of sexual behavior. The use of three or more STI antibodies has a low sensitivity (i.e., underestimates the size of female population with risk-taking behavior).
The current study was first attempt to estimate the seroprevalences of several STIs in Estonia at the population level. We found that seropositivity to multiple STIs was not common, and low rates of nonsexual transmission4,5,17,33 make alternative modes of transmission an unlikely explanation. Within the sexual transmission there are determinants increasing or decreasing the probability of infection. Furthermore, multiple exposures to closely related STIs might cause perturbation of the measurable specific immune responses. As a practical application of our findings, it is questionable whether any of the STI antibodies, which are commonly referred as surrogates of sexual behavior, can alone adequately reflect such behavior in women.
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