PCR-Detected Chlamydia trachomatis Infections From the Uterine Cervix of Young Women From the General Population

Abstract

DIAGNOSIS OF GENITAL Chlamydia trachomatis infections is important, because untreated infections in women may have great impact on public health, both on an individual level and on the community level. A variety of methods for detecting chlamydial infection exist.1 Previously, diagnosis by culture was “the gold standard.” Within the last few years, methods that use amplification of nucleic acids have been introduced. Polymerase chain reaction (PCR) and ligase chain reaction using primers specific for the endogenous plasmid are very sensitive methods.2–4 In female sexually transmitted disease (STD) populations, the prevalence of C. trachomatis infection has been reported to be as much as 27.1%,5 whereas in women from the general population, it has ranged from 4.5% to 9.2%.2,3,6,7 Within the last decade, the prevalence has shown a decreasing trend, most likely because of increased screening and treatment.8 Until recently, risk factors for C. trachomatis have mostly been examined using nonamplification detection methods. The objective of this study is to investigate the prevalence of and the risk factors for genital C. trachomatis in cervical scrapes as detected by PCR in a random sample of younger Danish women from the general population. From May 1991 to January 1993, a population-based cohort of 11,088 women (aged 20-29 years) was established with the purpose of studying risk factors for cervical neoplasia with emphasis on human papillomavirus (HPV) infection. Enrollment and data collection procedures are described elsewhere.9 Briefly, all participants were personally interviewed and had a gynecologic examination in which material for HPV and C. trachomatis testing were collected. A sample of 525 women was randomly chosen from the cohort. They were all tested for genital infection with C. trachomatis. However, the samples from 3 women were not adequate for testing; therefore, the remaining 522 women formed the study population for this article. Cervical swabs from the ectocervix and endocervix were taken using cotton-tipped swabs, which were placed in a tube with 2 ml of Tris HCl-EDTA buffer and kept frozen at −80°C until they were used. After thawing and vigorous vortexing, the swabs were removed. Ten μl of crude cell suspensions of these cervical scrapes were boiled for 10 minutes, chilled on ice, and subsequently used for PCR analyses. To examine whether the samples were suitable for PCR amplification, they were screened by a PCR specific for the human beta-globin gene.3 Subsequently, C. trachomatis PCR was performed as described previously.4 Polymerase chain reaction products were analyzed by a nonradioactive enzyme immunoassay (EIA).10 Dilutions of C. trachomatis serovar L2 DNA were used as positive controls. Cut-off values used in PCR-EIA were OD values corresponding to 0.01 to 0.1 inclusion forming unit (IFU) L2 DNA.4 Our PCR test is slightly more sensitive than the commercially available C. trachomatis detection systems (e.g., Amplicor [Roche Diagnostic Systems, Basel, Switzerland] 1 IFU), and the specificity is similar to those reported for the commercially available tests (>99.5%). Initial evaluation of data included univariate analysis, whereby the association between each variable and outcome was measured by the prevalence odds ratio (POR) and 95% confidence interval (CI). Variables related to having genital C. trachomatis were subsequently evaluated by means of a multiple logistic regression analysis to adjust for potential confounding. The prevalence of C. trachomatis was 6.7% (35/522). Age was a strong determinant for having genital chlamydial infection (Table 1). Women 20 to 22 years of age and 23 to 24 years of age had, respectively, a 5.1 times and 2.6 times higher risk of chlamydial infection than 25- to 29-year-old women (95% CI: 2.0-12.8 and 0.9-7.2, respectively).TABLE 1: Determinants for PCR-Detected Genital Chlamydia trachomatis Among 522 Young Women From CopenhagenLikewise, lifetime number of sex partners was a predictor for genital C. trachomatis. After adjustment, women with five to nine partners were 4.8 times more likely to have chlamydial infection than women with zero to four partners (95% CI: 1.8-12.7). Having had 10 or more sex partners increased the risk 2.8 times, although it was not a significant increase (95% CI: 0.9-8.8). Women who had used a barrier contraceptive method (condom or diaphragm) for ≥5 years had a 60% decreased risk of chlamydial infection compared with women who had never used this method or who had only used it for ≤1 year, although the association was not significant. However, the trend was significant (p < 0.02). No association was observed with the use of oral contraceptives (OC), neither with ever use of OC nor with duration of OC use (data not shown). In the crude analysis, ever pregnant women had a significantly lower risk of C. trachomatis infection than women who had never been pregnant (POR = 0.3; 95% CI: 0.1-0.9). However, after adjustment, the association was no longer significant. We also observed a tendency toward women who reported a history of genital chlamydial infection having a decreased risk of current C. trachomatis detection (POR = 0.4). However, the association did not reach statistical significance. Finally, no significant association with detection of C. trachomatis was observed for smoking, education, and “sexual” variables other than from lifetime number of sex partners (e.g., age at first intercourse and partners before the age of 20 years) (data not shown). The prevalence of genital chlamydial infection in our randomly sampled study group of women from the general population resembles that found by other investigators in population-based studies using PCR and ligase chain reaction.2,3 The determinants for having genital C. trachomatis in our plasmid-PCR-based study are in accordance both with studies using other diagnostic methods5–7 and with the newer PCR-based method.11–14 Younger age, increasing number of sex partners, and increasing years without use of barrier contraception are the major risk factors. The use of OCs as a risk factor for C. trachomatis has been a matter of debate. It has been suggested that OC increases the accessible transitional zone (ectopy), thereby making the cervix more susceptible to infection with C. trachomatis.15 However, another suggested explanation is that the ectopy increases the likelihood of detecting the infection because of more effective sampling in OC users.16 We have used a very sensitive method that might detect C. trachomatis independently of the size of the transitional zone and, therefore, may have overcome the problem of detection-bias. However, to address the effect of OC use more accurately, an analysis including the timing of OC use in relation to the occurrence of C. trachomatis infection is needed. Studies have indicated that pregnant women have an increased risk of genital C. trachomatis.17 This has been attributed to increased hormonal levels and lowered immunity during pregnancy. In the current study, ever pregnant women had a decreased risk (although not significant) of current C. trachomatis infection compared with never pregnant women. A similar association has been shown previously regarding another STD (HPV).18 Number of recent partners has been shown to be a good predictor of genital chlamydial infection, and it could be hypothesized that young, ever pregnant women have had a recent period of monogamy in connection with the pregnancy, and likewise, their partners have probably also been monogamous. Therefore, the risk of currently being infected with genital chlamydial infection is decreased. Being a current smoker has been shown to be correlated positively with sexual habits.19 Therefore, it is a variable that has to be taken into account when risk factors for an STD are evaluated to adjust for the possible confounding effect. However, in line with Willmott,19 we found no effect of smoking per se on the risk of currently being infected with C. trachomatis. Immunity against genital C. trachomatis has been much discussed. In the current study, the risk of C. trachomatis infection increased with lifetime number of sexual partners until a certain point, whereafter it leveled off. This could indicate that women with multiple sex partners acquire immunity against chlamydial infection. Like C. trachomatis, HPV is sexually transmitted, and both infections frequently occur in younger populations. Therefore, the risk of repeated infections with HPV or C. trachomatis is much higher than for STDs with a low prevalence. For HPV, it has been shown that repeated exposure can lead to acquired immunity.18,20 The women in the study group were also tested for genital HPV DNA, which was detected by PCR in 15.5% of the women (81/522). In Figure 1, the prevalence of C. trachomatis and HPV according to number of partners is shown. The profiles of the prevalence curves are very similar. For C. trachomatis, we also stratified by age and the profile of the prevalence curve in each age stratum was identical to the one shown in Figure 1. Accordingly, as in the case of HPV, it can be hypothesized that repeated exposure leads to acquired immunity against C. trachomatis. However, for C. trachomatis, several different outer membrane protein genotypes have been identified. Therefore, immunity may be strain specific and/or shortlived. Finally, we found a tendency toward a “protective” effect of former genital chlamydial infection (self-reported). This may also support that immunity against C. trachomatis exists.FIG. 1: Prevalence of genital HPV and C. trachomatis according to lifetime number of sex partners.However, it should be emphasized that our results are based on a small number of infected individuals, and it can not be excluded that the associations are confounded by behavioral changes over time that we could not adjust for. Further investigation of the immune response to evaluate the extent of spontaneous regression of C. trachomatis is needed-ideally in a prospective design.