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Department of Pathology, Vrije Universiteit Medical Center, Amsterdam
Laboratory for Pathology and Medical Microbiology, PAMM Institute, Eindhoven, The Netherlands
Unit of Field and Intervention Studies, International Agency for Research on Cancer, Lyon, France
División de Investigación, Instituto Nacional de Cancerología, Bogota, Colombia
The natural course of Chlamydia trachomatis infection and its risk factors were studied in Colombian women with normal cytological results, during a 5-year period. Eighty-two women who were found to be positive for C. trachomatis at the start of the study were studied at 6-month intervals. At each visit, a cervical scrape sample was obtained for detection of C. trachomatis by use of C. trachomatis endogenous-plasmid polymerase chain reaction (PCR)enzyme immunoassay and VD2-PCRreverse line blot assay. Of the women studied, 67% had a single-serovar infection, 10% had a mixed-serovar infection, and 23% had an infection with an unidentified type. An inversed rate of clearance of C. trachomatis infection was observed with oral contraceptive use (hazard ratio [HR], 1.7 [95% confidence interval {CI}, 1.12.7]) and first sexual intercourse at 20 years of age (HR, 4.3 [95% CI, 2.38.0]). Serovars of group B (B, D, and E) and C (H, I, J, and K) had a decreased rate of clearance (rate ratio, 0.4 [95% CI, 0.10.9]), compared with that for serovars of the intermediate group (F and G). At 4 years of follow-up, 94% of the women had cleared their infections.
Chlamydia trachomatis is an obligate, intracellular, nonmotile, gram-negative bacterium recognized as one of the most common sexually transmitted agents in the world. Approximately 70% of infections with C. trachomatis run an asymptomatic course that remains undetected and can result in severe complications, such as pelvic inflammatory disease, tubal infertility (6%21% of Chlamydia-infected women), pelvic pain (18%24% of Chlamydia-infected women), and ectopic pregnancy (7%9% of those who become pregnant) [1, 2]. In addition, an epidemiological association between the presence of antibodies to C. trachomatis and cervical intraepithelial neoplasia (CIN) lesions has been observed [36], but its direct role in the development of cervical lesions is still unclear.
More than 19 different serovars and variants have been identified and classified according to their pathogenic potential: serovars A, B, Ba, and C have been commonly associated with trachoma, DK with urogenital infections, and L1, L2, and L3 with lymphogranuloma venereum [7]. In addition, on the basis of amino acid similarity, these serovars have been grouped into the following serogroups or classes: group B (B, Ba, D, Da, E, L1, L2, and L2a), group C (A, C, H, I, Ia, J, Ja, K, and L3), and the intermediate group (F and G) [8].
Epidemiological studies have shown that the highest prevalence of C. trachomatis infection is in women <25 years of age. However, little is known about the natural history of C. trachomatis infection, mainly because the infection can easily be treated with antibiotics.
Therefore, only a few prospective and retrospective studies of C. trachomatis infection have been performed, some of them with low numbers of participants and short follow-up [912]. More prospective studies of the natural course of C. trachomatis infection are needed to advance our knowledge of C. trachomatis infection in terms of transmission, clearance rates, late complications, and associated risk factors [6].
We studied C. trachomatis infection, as a risk factor for CIN, in a population-based cohort (a group of low-income women from Bogota, Colombia) study of the natural history of human papillomavirus (HPV) infection and the role that other risk factors play in CIN. Baseline data on C. trachomatis infection in this population show that the prevalence of C. trachomatis infection is 5.3% [13].
In the present study, we performed a 5-year follow-up analysis of asymptomatic C. trachomatispositive women with normal cytological results at baseline. Furthermore, the relationships between C. trachomatis serovars, various characteristics of the women, and other risk factors in the clearance of C. trachomatis infection were analyzed.
PATIENTS, MATERIALS, AND METHODS
Patients and study design.
The design of the study has been described in more detail elsewhere [14] and was made to reveal the natural course of HPV infection, with detection of CIN grade III (CIN III) as the clinical end point. A total of 1995 women (90.7%) were finally included in the study cohort. Informed consent was obtained from all participants included in the study, and the ethical committee of the International Agency for Research on Cancer approved the study protocol for HPV and cofactors. Clinical specimens and questionnaire data were retrospectively used for study of the natural history of C. trachomatis infection, which has been suggested to be one of the cofactors in HPV-related cervical carcinogenesis. In brief, between November 1993 and November 1995, the Colombian National Cervical Cancer Institute conducted a census at 4 different health centers in Bogota. Two thousand women 1885 years of age were randomly identified in the census and invited to participate in the study. Additionally, to obtain more information on sexually active adolescents, 200 females 1317 years of age attending an adolescent clinic that gives advice on contraceptives were also invited to participate. At recruitment, all participants answered a structured questionnaire, filled in by a medical doctor, on sociodemographic characteristics, lifelong sexual behavior, reproductive history, smoking, antibiotic use, and dietary habits. After the interview, all women were asked to undergo a gynecological examination and to provide a cervical scrape sample for cytological examination and future testing (HPV and cofactors). Women with cervical dysplasia or with any relevant clinical symptoms (e.g., cervical discharge) were subsequently examined and treated according to standard medical protocols.
Follow-up consisted of 1 visit every 69 months, up to December 2000. At each visit, a short questionnaire and clinical specimens, as described for the first visit, were obtained. Women's C. trachomatis infection status was not known during follow-up and did not influence clinical management, except when women displayed symptoms.
The analysis described here was performed on a subset of the study cohort. We selected 84 women with normal cytological results who were positive for C. trachomatis infection and attended 1 follow-up visit. For analysis, the following criteria were applied: women were excluded from the analysis at the first occurrence of C. trachomatissensitive antibiotic use or presence of CIN III in the biopsy specimen. Two women reported antibiotic use during the study and were excluded. Nine women used antibiotics after clearance of C. trachomatis infection and were included in the study. In total, 82 women were analyzed.
Collection and processing of samples.
After performance of a Pap smear, the remaining material from the endocervical brush and spatula were used for C. trachomatis testing. It was placed in a tube containing 5 mL of PBS 1× plus 0.05% thiomersal. Cells were detached from the spatula and endocervical brush by vortexing and were centrifuged at 3000 g for 10 min. The cell pellet was resuspended in 1 mL of buffer (10 mmol/L Tris-HCl [pH 8.3]) and stored at -70°C until use. For analysis, a 100-L aliquot was boiled for 10 min at 100°C, cooled on ice, and centrifuged for 1 min at 3000 g; 10 L of this pretreated crude-cell suspension was used for polymerase chain reaction (PCR) analysis [15]. To assess the quality of the target DNA, a 209 bpamplifying -globin PCR was performed by use of the primer combination BGPCO3 and BGPCO5, as described elsewhere [16].
Detection of C. trachomatis by use of plasmid PCR.
The detection of C. trachomatis was performed as described elsewhere [17]. Plasmid endogenous-specific primers Bio PL6.1 and PL6.2 were used for PCR amplification. The PCR amplification consisted of DNA denaturation for 4 min at 95°C followed by 40 cycles of amplification by use of a 9600 thermocycler (Perkin-Elmer). Each cycle included a denaturation step of 1 min at 95°C, an annealing step of 1 min at 55°C, and a chain-elongation step of 1.5 min at 72°C. The final elongation step was extended for another 4 min.
The biotinylated PL6.1/PL6.2 PCR products were detected by use of an EIA, as described elsewhere [18, 19]. In brief, in this assay, 5 L of the biotinylated PCR products was captured in streptavidin-coated wells of a microtiter plate (Roche). The captured DNA was denaturated by alkaline treatment, and the denaturated PCR product was hybridized to a digoxigenin-labeled Chlamydia-specific probe. The unbound probe was removed, and the hybrids were detected by use of anti-dig Fab fragments labeled with alkaline phosphatase (Roche). Paranitrophenyl phosphate (Sigma) was used as substrate. Finally, the optical density (OD) was measured at 405 nm by use of a Labsystem Multiscan reader. In our assay, a cutoff point was defined as 3 times the mean OD of the negative controls. As a positive control, a 10-fold dilution series of C. trachomatis L2 DNA was used, as described elsewhere [20], resulting in a detection sensitivity corresponding to 0.010.1 inclusion forming units.
C. trachomatis typing by use of a nested VD2-PCRreverse line blot (RLB) assay.
A nested VD2-PCR assay was used to amplify the VD2 region of the omp1 gene of C. trachomatis, as described elsewhere [21]. In brief, 2 sets of primers (general and nested) were used to amplify segments of 220 bp and 166 bp, respectively. The following primers were used for the primary VD2-PCR of C. trachomatis: Bio-MCTV2S (Bio-5-GTATTYTGTA CAYTRGGAGCM-3) and MCTV2AS (5-CCYCARTCCCASAYAGCTGC-3) and, for the nested PCR, Bio-MCTV2N (Bio-5-AGGAAAYTCNGCWTCYTTCAA-3) and MCTV2AN (5-CTGCNCGAGCNCCNACYCT-3). The amplification conditions for both PCRs, described elsewhere [21], resulted in biotinylated VD2-PCR products.
RLB analyses were performed as described elsewhere [2123]. In brief, C. trachomatisspecific oligonucleotide probes containing a 5-amino group were covalently attached to a Biodyne C membrane (Pall BioSupport), in parallel lines, by use of a miniblotter. Nine different oligonucleotide probes (Isogen) directed against the VD2 region of the MOMP gene were used to identify 9 different serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K). After binding of the oligonucleotide probes, the membrane was removed from the miniblotter and rotated 90°. The slots of the miniblotter that were perpendicular to the oligonucleotide-probe lines were filled with 10 L of each biotinylated VD2-PCR product generated. Hybridization was performed in the miniblotter and was visualized by use of peroxidase-labeled streptavidine, which interacted with the biotin of the probe, followed by enhanced chemiluminescense detection, as described in detail elsewhere [2123].
Statistical methods.
The time to clearance of C. trachomatis infection was modeled by use of methods for interval-censored survival time data. Thus, if clearance was achieved, it occurred between 2 visits. The date of clearance was defined as the middle of the interval between a positive and a negative C. trachomatis test by use of cervical scrape samples. The survival function, which describes the probability that a C. trachomatis infection has cleared (as a function of time since enrollment), was estimated by use of Kaplan-Meier analysis. Different factors potentially associated with persistence/clearance of C. trachomatis infection were considered: serovar analysis, HPV infection, age, education, number of regular sex partners, age at first sexual intercourse, oral contraceptive use, intrauterine contraceptive device (IUD) use, parity, and smoking.
We performed Cox regression analysis to estimate the age-adjusted rate ratios (RRs) and 95% confidence intervals (CIs) for clearance of C. trachomatis infection, according to the different risk factors. Finally, variables significantly related to the rate of clearance were included in a multivariate Cox model.
We made 2 different analyses of clearance: (1) at the level of C. trachomatispositive women, independent of serotype; and (2) at the level of specific C. trachomatis serovar infection. -globin PCR-negative samples and inadequate cytological results (at any point in time) were considered to be invalid.
RESULTS
Characteristics of the cohort.
Eighty-two women who had normal cytological results and attended 1 follow-up visit were included in the analysis. In total, we tested 544 cervical scrape samples obtained at enrollment and follow-up visits. The median follow-up time was 5.7 years, the median interval between visits was 8 months, and the median number of visits was 6.5.
Table 1 shows the characteristics of the study population divided into 2 age groups. Information about race/ethnicity was not included, since these data are extremely hard to access in epidemiological studies in South America, where almost everyone is of mixed ethnicity.
Most of the women (57.3%) were 30 years of age (70.7% were >25 years of age), had an intermediate educational level (39%), reported a single lifelong sex partner (82.9%), and had had 1 or 2 full-term pregnancies (56.1%). Oral contraceptives were used by 47.6% of the women, and an IUD was used by 66.7% of the women. Only 28.1% of the women reported ever smoking, 39.0% had their first sexual intercourse between 17 and 19 years of age, and 21.9% were infected with HPV. Special attention was given to information about antibiotic use during follow-up.
Analysis of C. trachomatispositive women.
At the beginning of the study, 82 women had C. trachomatis infection, detected by use of C. trachomatis plasmid PCR as the primary method. Approximately 46% of the infections were persistent at 1 year, 18% at 2 years, and 6% at 4 years of follow-up, as determined by use of plasmid PCR without any consideration for serotypes. In 94% of the women, C. trachomatis infections had cleared at 4 years of follow-up (figure 1). Although the mean time of follow-up was >5 years, the numbers at 5 years were too small for determination of percentages.
In addition, risk determinants were analyzed for clearance of C. trachomatis infection. Rates of clearance were higher for women who had ever used oral contraceptives and for women who had their first sexual intercourse at 20 years of age (figure 2). No significant associations were found between C. trachomatis clearance and age, education, number of sex partners, parity, IUD use, smoking, or HPV infection.
Table 2 shows crude RRs of clearance of C. trachomatis infection, by several characteristics and multivariate analysis. Factors significantly and independently associated with a higher rate of clearance were oral contraceptive use (RR, 1.7 [95% CI, 1.12.7]) and first sexual intercourse at 20 years of age (RR, 4.3 [95% CI, 2.38.0]). The other factors studiedage, number of sex partners, education, parity, IUD use, smoking, and HPV infectionwere not significantly associated with the probability of clearance of C. trachomatis infection.
C. trachomatis serovarspecific analysis.
The 82 C. trachomatispositive women were subjected to omp1-PCR-RLB analysis, for determination of serotype, and this was successfully done for 63 women. Table 1 shows the serogroups of C. trachomatis infections at entry into the study: 55 (67%) were single-serovar infections, 8 (10%) were mixed-serovar infections, and 19 (23%) were of an unknown type. Of the serovars identified in single-serovar infections, 36.4% belonged to the intermediate group (F and G), 34.6% to group B (B, D, and E), and 29.1% to group C (H, I, J, and K). The most common serovars identified were D (23.6%), F (21.8%), G (14.5%), and E (10.9%). Of the C. trachomatis mixed-serovar infections, the most common infections detected were D-E (50%).
The natural course of C. trachomatis infection, by serovar, was determined only for single-serovar infections (n = 55). Infection was classified as persistent if the same serotype was found at the following visit. The majority of the C. trachomatispositive women had an identical serotype in the following cervical scrape sample, with the exception of 2 women (1 had subsequent infections with serotypes G, D, G, and K-G, and 1 had subsequent infections with serotypes G and D and then was found to be negative).
Analysis of C. trachomatis serovars showed differences in the rates of clearance. The rate of clearance was lower for serovars of groups B and C than for serovars of the intermediate group (figure 3).
Table 3 shows crude RRs of clearance of serotype infection, by several characteristics and multivariate analysis. Serovars of group B (Ba, D, and E) were significantly associated with a lower rate of clearance (RR, 0.4 [95% CI, 0.10.9]). As in the general analysis of C. trachomatis infection, the factors significantly and independently associated with a higher rate of clearance were oral contraceptive use (RR, 2.6 [95% CI, 1.45.1]) and first sexual intercourse at 20 years of age (RR, 3.4 [95% CI, 1.29.3]).
DISCUSSION
We have shown in the present long-term follow-up study that, in 94% of women with normal cytological results, C. trachomatis DNA could not be detected in cervical scrape samples by use of the sensitive plasmid PCR-EIA [17, 19], strongly suggesting that C. trachomatis infection was cleared from the cervix epithelium. Approximately 54% of the infections were cleared at 1 year and 82% were cleared at 2 years of follow-up. These results are in line with those reported by others, who have shown rates of clearance of C. trachomatis infection near 50% at 1 year of follow-up [9, 12, 19]. Although persistence of C. trachomatis infection has been observed in humans, animal models, and in vitro systems [11, 19, 2428], the mechanisms used by C. trachomatis to persist are largely unknown.
The rate of clearance of C. trachomatis infection with serovars of group B (B, D, and E) was lower than that of serovars of the intermediate group (F and G). We found, however, a trend in which group C (H, I, J, and K) had a lower rate of clearance than did the intermediate group. Although grouped serovars were analyzed, to reach sufficient statistical power, additional analyses restricted to the most frequently found individual serovars of group B and the intermediate group showed identical results for the individual serovars D and E and F and G (data not shown). These results are in line with those reported by Dean et al. [11], who suggested a significant association between serovars of group C and persistence of C. trachomatis infection, determined by use of PCR and sequencing, and with those reported in another study of women, by Morré et al. [12]. In the latter study, serotype E infections were found 2 times more frequently in women with persistent C. trachomatis infection and suggested a possible association between specific C. trachomatis variants and persistence of C. trachomatis infection. An explanation for the higher rate of persistence of serovars of groups B and C might be that they are more able to escape the host immune response. Moreover, in a mouse model, genital serovar D showed more invasiveness than did other serovars [29], which might support its higher rate of persistence. Alternatively, this might lead to quicker clearance, by eliciting a stronger host response. However, serovar D possesses more omp1 gene diversity than do other prevalent serovars [3032], which might help to circumvent the proper host immune response. Fortunately, at baseline and during follow-up, serum samples were obtained from all participating women for future immunological studies; it would be very interesting to correlate host humoral responses to C. trachomatis with clearance of C. trachomatis infection.
C. trachomatis is sensitive to treatment with tetracycline, macrolides, and fluoroquinolones, which could interfere with the rates of clearance of C. trachomatis infection. In the present study, 11 women used antibiotics during follow-up, but 9 used them after C. trachomatis infection had been cleared. The 2 women who used antibiotics while infected with C. trachomatis were excluded from the study. Even when all 11 women were excluded from the study, the rates of clearance were similar (data not shown).
Of the women found to be infected with C. trachomatis at baseline, 23% could, unfortunately, not be typed by use of the VD2-PCR-RLB assay [21]. Additional analysis showed that the initial plasmid PCR was weakly positive in these cases and that these women were all found to be negative for C. trachomatis at the following visit. This indicates that the infections of these women involved low copy numbers and were on their way to being cleared and, therefore, could not be detected by the typing assay, which is known to be slightly less sensitive than the initial plasmid PCR.
In the present study, oral contraceptive use was associated with a higher rate of clearance of C. trachomatis infection and was principally observed in women <30 years old (data not shown). Oral contraceptive use has been associated with an increased risk of C. trachomatis infection in some studies of prevalence [33]. Serological studies have shown a relationship between hormonal contraception use and modification of immune response or susceptibility to C. trachomatis infection [34, 35]. In a rodent model, Kaushic et al. [36] showed that giving estradiol to ovariectomized rats enhanced the immune response and led to complete protection from C. trachomatis infection, without any sign of inflammation. In contrast, when Kaushic et al. gave the rats progesterone, the rats became heavily infected, and infection was accompanied by acute inflammatory response, showing that, depending on the hormonal environment, the immune system can be altered. Differences in hormonal composition of the contraceptives might explain these results.
Women 20 years of age at first sexual intercourse had a higher rate of clearance of C. trachomatis infection than did females <16 years of age at first sexual intercourse. The hormonal changes associated with puberty that occur in females <16 years of age and probably affect the C. trachomatis immune response might explain this effect. The association observed between onset of C. trachomatis infection and stage of menstrual cycle in humans [35, 37] also seems to confirm that hormonal status influences the rate of clearance of C. trachomatis infection. In the present study, a group of females 1317 years of age who attended an adolescent clinic that gives advice on contraceptives could have biased the results obtained. In contrast to oral contraceptive use and age at first sexual intercourse, age, parity, number of sex partners, IUD use, smoking, and HPV infection were not associated with rate of clearance of C. trachomatis infection in the present follow-up study.
Unfortunately, the infection status of the male partners was not known and could not be evaluated in the present study. Since all study women had 12 lifetime sex partners, their infections were most likely related to the behavior of their male partners. Repeated infection from an untreated male sex partner might have biased the present study in such a way that the duration of infection was increased.
In conclusion, in the present study, it has been shown that 94% of the women cleared their C. trachomatis infections, as determined by use of plasmid PCR with cervical scrape samples. Clearance of C. trachomatis infection is associated with oral contraceptive use and first sexual intercourse at 20 years of age. Furthermore, C. trachomatis serovars of groups B and C are associated with decreased rates of clearance.
Acknowledgments
We thank all the study participants, gynecologists, nurses, and social workers who collaborated on the fieldwork (human papillomavirus study group: Mauricio González, Joaquín Luna, Gilberto Martínez, Edmundo Mora, Gonzalo Pérez, José Maria Fuentes, Constanza Gómez, Eva Klaus, Constanza Camargo, Cecilia Tobón, Teodolinda Palacio, Carolina Suárez, Claudia Molina, and Alex Torres); Martyn Plummer, for useful comments on the manuscript; S. Morré, for helpful discussions; and R. van Andel, R. Pol, D. Kramer, and M. Lettink, for technical support.
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