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Seroprevalence of human papillomaviruses and Chlamydia trachomatis and cervical cancer risk: nested case–control study

¹ Lund University, University Hospital at Malmö, SE-20502 Malmö, Sweden
² National Taiwan University, Taipei, Taiwan
³ National Defence Medical Center, Taipei, Taiwan

Correspondence
Joakim Dillner
joakim.dillner{at}med.lu.se

	ABSTRACT

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A nested case–control study of invasive and in situ cervical cancer was performed within a community-based cohort of 13 595 Taiwanese women assembled in 1991, with a follow-up period of 9 years. Baseline serum or plasma samples were analysed for antibodies against human papillomavirus (HPV) types 6, 16 and 18 and Chlamydia trachomatis. In total, 114 cases (42 incident cases identified during follow-up and 72 prevalent cases identified at baseline) and 519 matched controls were included in the study. HPV-16 seropositivity was strongly associated with cervical cancer (OR=6.33; 95 % CI 3.45–11.62). Overall, C. trachomatis was not associated with cervical cancer, but was associated with cervical cancer in analyses restricted to incident cases of cancer (OR=2.94; 95 % CI 1.17–7.42) or to cases in which serum samples were analysed (OR=3.13; 95 % CI 1.16–8.47). An antagonistic interaction between HPV-6 and -16 was found in a multiplicative model. These results suggest that different HPV types might interfere in cervical carcinogenesis and that C. trachomatis is associated with cervical cancer in prospective studies, and support the notion that HPV-16 seropositivity is strongly associated with cervical cancer.

	INTRODUCTION

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Cervical cancer is the most common cancer among women in Taiwan, with an age-standardized incidence rate of 22.1 per 100 000 person-years compared with a mean of 11.2 in developed countries (Parkin et al., 1993; Wang & Lin, 1996a). It is well established that the major cause of cervical cancer is persistent infection with oncogenic genital human papillomavirus (HPV) (Walboomers et al., 1999) and that both cervical cancer and HPV infection are associated with high-risk sexual behaviour (Dillner et al., 1996; Silins et al., 2002). The HPV type distribution varies among geographical regions but, worldwide, HPV-16 is the most common HPV type in cervical cancer (Clifford et al., 2003; Nauclér et al., 2004).

As the majority of HPV infections are transient (Evander et al., 1995; Hildesheim et al., 1994; Ho et al., 1998), viral genome detection is an imperfect marker of past HPV infection (Olsen et al., 1997). However, HPV antibody levels have been shown to be stable over time, even after more than a decade of follow-up (af Geijersstam et al., 1998; Shah et al., 1997), and correlate with life-time cumulative HPV exposure (Shah et al., 1997; Wang et al., 1997). Prospective seroepidemiological studies have found that the presence of IgG antibodies against HPV-16 is associated with an increased risk of cervical cancer (Shah et al., 1997; Wang et al., 1997), in particular in low-sexual-risk-taking populations (Dillner et al., 1997; Lehtinen et al., 1996).

In spite of the fact that exposure to oncogenic HPV increases with the number of sexual partners, there is no excess risk of developing cervical cancer in patients with a history of condylomata acuminata, which is caused by sexually transmissible infections with the benign HPV types 6 and 11 (Sigurgeirsson et al., 1991). A possible explanation is that benign HPV types might interfere with the oncogenicity of high-risk HPV types. Evidence of an antagonistic effect between benign and oncongenic HPV types has been found in seroepidemiological studies, where women seropositive for both HPV-6 and -16 showed a significantly decreased risk of developing cervical cancer compared with women seropositive for HPV-16 only (Luostarinen et al., 1999, 2004; Silins et al., 1999).

As only a small proportion of HPV-infected women actually develop cervical cancer (Herrero et al., 2000), many epidemiological studies have been conducted to investigate additional risk factors that may be involved in development of the disease. Several studies have reported an association between past Chlamydia trachomatis infection and cervical cancer (Anttila et al., 2001; Koskela et al., 2000; Smith et al., 2004). C. trachomatis is the most common sexually transmitted bacterial infection (Paavonen & Eggert-Kruse, 1999) and has been suggested to have a carcinogenic effect through modification of the ability to clear an HPV infection (Silins et al., 2005; Zenilman, 2001).

Two previous case–control studies have assessed the association between HPV infection and cervical cancer in Taiwan, although only on a limited number of invasive cancer cases (Liaw et al., 1995; Sun et al., 2002). However, these studies did not investigate HPV serology and therefore were not informative regarding the possible role of previous HPV infections. As the peak prevalence of HPV infection occurs when women initiate sexual relationships and cervical cancer develops 10–15 years after infection, most women who have been exposed to the virus and not developed disease will have cleared their infection when they are tested for the presence of the viral genome (Schiffman, 1992). Although only about 50–65 % of HPV-infected women seroconvert (Kirnbauer et al., 1993; Kjellberg et al., 1999), measurement of antibodies is still likely to reduce misclassification of past HPV exposure when investigating co-factors additional to HPV in the development of cervical cancer.

To investigate the cervical cancer risk of past HPV and C. trachomatis exposures, we therefore conducted a case–control study nested within a community-based cohort in Taiwan, where we measured the antibody response to HPV-6, -16 and -18 and C. trachomatis at baseline.

	METHODS

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Population.
The study was nested in a community-based cervical neoplasia screening project in Taiwan, described in detail elsewhere (Liaw et al., 1995). Briefly, an age-restricted population of 41 280 women aged 30–64 registered in local household registration offices from seven townships were invited by three consecutive mails. Two health examinations were performed in the periods from 1991 to 1993 and from 1993 to 1995 where 13 595 women received at least one health examination. Among these, 11 430 had a cervical Pap smear taken at least once. The Pap smear attendance rate ranged from 22.7 to 39.5 % according to area and from 22.3 to 30.6 % according to 5-year-interval age group. Baseline serum or plasma was collected from all of the study subjects. In addition, a questionnaire was carried out to record demographic characteristics, lifestyle habits, Pap smear history and reproductive and sexual history. Women who had never been married, who had a history of cervical neoplasia or who had had a hysterectomy were not eligible. Through linkage between the data file of the study and the national cancer registry, the national death certification system and the catastrophic illness registry, there were 117 cases who developed invasive cervical cancer or carcinoma in situ prior to 31 December 2000. The computerized national cancer registry in Taiwan was implemented in 1979 and has been collecting all cancer cases that have been diagnosed and reported from regional hospitals and medical centres. The universal health insurance was inaugurated in 1995 and almost all residents are included. As cancer is classified as a catastrophic disease, all cancer patients receive free diagnosis and treatment in regional hospitals and medical centres covered by the cancer registry. Therefore, the national cancer registry is considered complete. Both carcinoma in situ and invasive cervical cancers are reported to the registry. The universal health insurance also reimburses Pap smears annually for women aged 3 years or older. After reviewing the cases, three were excluded: two cases had been diagnosed before enrolment, and the security ID in the cancer registry did not correspond with the name for one case. Cases were grouped further into prevalent cases, incident cases and unscreened cases. Prevalent cases were defined as women who developed cancer within 1 year of the date of enrolment, or who had a biopsy with invasive cancer or carcinoma in situ at enrolment, or who had a Pap smear indicating invasive cancer or carcinoma in situ at enrolment. Incident cases were defined as women who developed cancer or carcinoma in situ more than 1 year after enrolment. A total of 114 cases had histologically confirmed squamous cell carcinoma comprising 60 with invasive cervical cancer and 54 with cervical carcinoma in situ. Women who were diagnosed with cervical cancer during follow-up, but who did not receive a Pap smear at enrolment, could not be classified with certainty as incident or prevalent cases and were referred to as ‘unscreened’ cases. Of the 114 cases, there were 72 prevalent cases (40 with carcinoma in situ and 32 with invasive cancer), 35 incident cases (12 with carcinoma in situ and 23 with invasive cancer) and seven unscreened cases (two with carcinoma in situ and five with invasive cancer).

Controls were matched individually to the cases and selected at random from eligible controls. The eligibility criteria were availability of baseline specimens (Pap smear and serum/plasma), no cervical neoplasia in any one of the three registries at the time when the matched case was diagnosed, and same gender, age, area of residence, type of sample (serum or plasma) and date of enrolment (±2 months). Up to six controls per case were selected, but only six matched controls were obtained for each of 50 cases. Twenty-six cases had five controls each, 11 cases had four controls each, nine cases had three controls each, six cases had two controls each, six cases had one control each and six cases had no eligible controls. Overall, we included 519 eligible controls matched to 108 cases in the analysis.

ELISA.
ELISA was performed in Sweden to detect IgG antibodies against HPV-6, -16 and -18 as described previously (Dillner et al., 1995). Virus-like particles (VLPs) comprising self-assembled L1 major capsid proteins generated in insect cells by recombinant baculovirus were coated onto ELISA plates (Kirnbauer et al., 1993). Human antibodies against VLPs were detected using a two-step ELISA with monoclonal antibodies against human IgG and a goat anti-mouse IgG horseradish peroxidase conjugate. For each serum or plasma sample, the difference in absorbance value obtained from plates coated with intact HPV VLPs and plates coated with control antigen (disrupted bovine papillomavirus VLPs) was calculated. A seropositive reference serum, obtained from patients with cervical intraepithelial neoplasia, was used on each plate as a positive control and reference serum. All samples were screened at a 1/30 dilution and samples above the pre-assigned cut-off levels for screening of 0.136 absorbance units for HPV-6 and 0.090 absorbance units for HPV-16 (Andersson-Ellström et al., 1996) were selected for confirmatory analysis. The cut-off value for confirmatory analysis for HPV-18 was set as half of the absorbance value of the reference serum. Confirmation and determination of antibody levels were performed using a sample titration series (1/10, 1/31.6, 1/100), where antibody levels were calculated using the parallel line model (PLL), which expresses antibody levels as units relative to a reference serum tested on each plate (Grabowska et al., 2002). The model is based on the assumption of parallelism between the lines obtained from the logarithm of the absorbance values on the y-axis and the dilution steps on the x-axis of the reference serum and the sample serums on each plate. A mean value of the slope of the reference and the sample is obtained. Hence, the PLL model will give the expected value for the slope, taking into account the slope of both the sample and the reference. The PLL units are then calculated as a function of the distance between the reference and the sample line. Before the start of the study, cut-off levels were set at 0.2445 PLL units for HPV-6, 0.3568 PLL units for HPV-16 and 1.3678 PLL units for HPV-18. The cut-off levels for HPV-16 and -18 were set by treating cervical cancer as a receiver-operated characteristic using an independent population-based case–control study (Silins et al., 2004) and for HPV-6 by treating HPV-6 infection as the characteristic.

Microimmunofluorescence.
Chlamydia-specific IgG antibodies were detected using microimmunofluorescence, as described previously (Koskela et al., 2000; Wang & Grayston, 1970). For C. trachomatis, serovars D–K were used. C. pneumoniae serovar IOL 207 served as a control antigen. All samples that were positive for titres 16 were analysed further in a titration series (16, 32, 64, 128 and 256). Titres of 64 were considered to be positive for both C. trachomatis and Chlamydia pneumoniae.

Data analysis.
SPSS and STATA software were employed for statistical analysis. Odds ratios (ORs) and corresponding 95 % confidence intervals (CIs) were estimated in univariate and multivariate conditional logistic regression models. All study variables were initially included in a multivariate logistic regression model. Variables that were not significantly associated with cervical cancer were then excluded in the final multivariate logistic regression model, except for the matched variables and variables previously described to be associated with cervical cancer, which were retained. The analysis of interaction between different HPV infections was estimated using both an additive and a multiplicative model. The additive model was performed by conditional logistic regression, calculating ORs with 95 % CIs for cervical carcinoma among subjects exposed to one or two HPV infections using the jointly unexposed subjects as reference (Silins et al., 1999). The observed risk was compared with the expected risk where the relative excess risk due to interaction (RERI) was defined as: RERI=RR(AB)–RR(A*)–RR(*B)+1, where RR(AB) is the relative risk (RR) when exposed to both causes A and B, RR(A*) is RR when exposed to only the first cause and RR(*B) is RR when exposed to only the second cause (Rothman & Greenland, 1998). In the absence of interaction, RERI equals zero, whilst there is evidence of interaction if the 95 % CI excludes zero (Hosmer & Lemeshow, 1992). In the multiplicative model, an interaction term was introduced into the conditional logistic regression model and a significance test was performed using a likelihood ratio test.

	RESULTS

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Socio-demographic characteristics of cases and controls are presented in Table 1. The mean age at baseline was 48.3 years for cases and 47.8 years for controls. Women with invasive cervical cancer tended to be older than women with carcinoma in situ (mean ages at baseline of 50.4 and 45.9, respectively). Only ten study subjects reported a history of smoking. Thirty-seven per cent of the subjects were illiterate, 43 % had only been to primary school and fewer than 1 % had been to university. Of the 633 study subjects, 603 reported having had only one lifetime sexual partner. The median age at first intercourse was 21 for cases and 22 for controls. Thirty-three per cent of cases and 37 % of controls reported having had a Pap smear taken earlier in life prior to enrolment. Former use of oral contraceptives was reported by 33 % of cases and 27 % of controls (Table 1).

	DISCUSSION

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In contrast to the low HPV-16 prevalence, we found that HPV-6 seroprevalence was 31 % among controls, which was higher than among controls in many other case–control studies (Silins et al., 1999; Matsumoto et al., 2003). The frequency of controls infected by HPV-18 was essentially similar to other case–control studies (Combita et al., 2003; Silins et al., 1999). The high prevalence of HPV-6 and -18 among the controls, even though only 2.7 % of the women reported having had more than one lifetime sexual partner, may have been due to the sexual behaviour of the husbands. A previous population-based case–control study from Taiwan reported that 71.9 and 58.3 % of husbands of women with cervical intraepithelial neoplasia and healthy controls, respectively, had visited a prostitute during the last year (Wang & Lin, 1996b). Another plausible explanation is the presence of alternative transmission routes in this population. Liaw et al. found 39 % of HPV DNA-positives cases with high-grade squamous intraepithelial lesions to be positive for HPV-52 and/or -58, which is a higher prevalence than reported from other geographical areas (Bosch et al., 1995; Liaw et al., 1995). Unfortunately, we were not able to test for antibodies to HPV-52 and -58. HPV-18 was not found to be associated with cervical carcinoma in our study, which was rather surprising as other studies have reported an association between antibodies against HPV-18 and risk of cervical carcinoma (Dillner et al., 1997; Wang et al., 1997). The assay was validated using known HPV-18-seropositive and -seronegative samples and the results were similar to previous studies, suggesting that the difference was not due to differences in assay performance. The fact that previous studies contained a substantial proportion of adenocarcinoma but the present study contained only squamous carcinoma may explain this difference, as HPV-18 in particular is associated with adenocarcinoma (Dillner et al., 1997).

Incident cases tended to be invasive rather than in situ cancers (66 % invasive cancers), whereas the opposite was true for prevalent cases (44 % invasive cancers). This was most likely due to the fact that the baseline examination included a Pap smear for all subjects, whereas the registry-based follow-up would have detected both symptomatic and screening-detected disease.

We performed analyses on incident cases and prevalent cases separately, as prospective data are more informative about the possible aetiological role of associations. HPV-16 was the only infection associated with cervical carcinoma among prevalent cases, whereas among incident cases C. trachomatis, HPV-6 and HPV-16 were associated with disease. An explanation is not immediately obvious, but it is possible that the more reliable prospective analyses had an increased ability to detect a co-factor role for high-risk-taking sexual behaviour.

There is ongoing debate among epidemiologists as to whether additive or multiplicative models should be used to detect interaction (Rothman & Greenland, 1998; Woodward, 1999). Hence, we evaluated interaction using both models. The antagonistic effect was significant between HPV-6 and -16 in the multiplicative model (P value=0.025) and the non-significant tendencies observed in the additive model were in concordance with previous studies that have demonstrated not only an antagonistic effect between HPV-6 and -16, but also a tendency for interaction between HPV-16 and -18 (Luostarinen et al., 1999, 2004; Silins et al., 1999). The point estimates of interaction in an adjusted analysis including history of smoking, C. trachomatis infection and other HPV infection were consistent with the estimates obtained in the univariate analysis. HPV serology has a high specificity but a limited sensitivity, where 50–65 % of women with past HPV infection seroconvert (Kirnbauer et al., 1993; Kjellberg et al., 1999). Hence, a significant proportion of women are misclassified as being seronegative and the ORs obtained might therefore be an underestimate. However, when Luostarinen et al. (1999) simulated different degrees of sensitivity, specificity and cross-reactivity in a similar HPV serological study to assess the effect of non-differential misclassification, they found corrected antagonism between different HPV types to be more pronounced under all combinations of sensitivity, specificity and cross-reactivity tested. However, it is possible that HPV-6 could mark some other exposure that is interacting with the HPV-16-associated cervical cancer risk. In this study, we tested only for antibodies against three HPV types, and as other HPVs are associated with both risk of various HPV infections and cervical cancer, other HPVs are potential confounders. Also, when interpreting statistical associations, the possibility that ‘significant’ findings may be due to chance must be considered.

Future studies that investigate interactions between HPV types would require larger case groups, as statistical power when studying interactions requires much larger studies than when studying the primary effect of a risk factor. Our study only included serological analyses of three HPV types. Taking into account the differences in geographical distribution of HPV types and that 15 types are considered to be high risk (Muñoz et al., 2003), it would also be desirable to conduct studies where the antibody response to more HPV types are investigated to obtain a more complete picture of interactions between HPV types and possibly reduce the risk of confounding. Probably, the most informative study design to elucidate whether one HPV infection protects against persistence of another HPV infection would be to test a cohort of HPV DNA-positive subjects at baseline for HPV seropositivities to other types and assess whether these seropositivities predict clearance of HPV infections.

Interassay variability can be a problem in serological studies, as ELISAs are sensitive to reaction conditions. When absorbance values are used as a cut-off level, strictly standardized reaction conditions are required, which may be difficult to achieve. We have demonstrated previously that measurement of antibody levels in PLL units relative to an internal standard significantly reduces the interassay variability and allows comparison of results from different studies.

C. trachomatis was not significantly associated with cervical cancer in our study. This was surprising, as many other studies have found an increased risk of cervical cancer for women with antibodies against C. trachomatis, both in analyses that have adjusted for HPV infection and in stratified analyses where only HPV DNA-positive cases and controls have been included (Koskela et al., 2000; Smith et al., 2004). However, whereas there was no increased risk among prevalent cases, we did find an increased risk among incident cases, which was not explained by the higher proportion of invasive cancer among incident cases (crude OR for incident invasive cancers=2.97; 95 % CI 1.36–6.51; crude OR for incident carcinoma in situ=3.15; 95 % CI 0.83–11.97). Whereas 17 % of the prevalent cases and corresponding matched controls had serum samples taken, up to 62 % of the incident cases and corresponding matched controls had serum samples taken. Existence of cross-reactivity between C. trachomatis and C. pneumoniae in plasma samples was suggested by significant clustering of seropositivities for these two agents, which was not detected in serum samples. Similar analysis comparing HPV prevalence in serum and plasma did not find any clustering among cases or controls. Microimmunofluorescence uses the entire C. trachomatis organism and is therefore a method distinct from ELISA, and a conceivable explanation for our findings is that there might be a non-specific reactivity with C. trachomatis antigen when plasma is analysed that decreases the specificity of the test. An alternative explanation is the fact that C. trachomatis antibodies decline over time (Puolakkainen et al., 1986), which would make earlier pre-diagnostic measurements more accurate. Finally, it is also possible that C. trachomatis only has an effect many years before the cancer starts and that non-causative C. trachomatis exposures occurring close to diagnosis (too short a lag time for an effect) will dilute associations in studies of prevalent cases.

In conclusion, women with past infections of both HPV-6 and -16 or HPV-16 and -18 appeared to have a decreased risk of developing cervical cancer compared with women with past infection with HPV-16 only. Whether the interactions seen were merely statistical associations or reflect biologically meaningful interferences remains to be shown and additional, larger studies will be necessary to understand fully the interplay between HPV types in cervical carcinogenesis. In particular, it would be interesting to know whether the order of infection with different viruses is of importance, an issue that could not be addressed in the current study. We confirmed that C. trachomatis is associated with cervical cancer in prospective studies. Our lack of association in the ordinary case–control study of prevalent cases has highlighted several possible methodological problems that need to be considered in further studies of C. trachomatis and cancer. Finally, the fact that HPV-16 seropositivity was associated so strongly with cervical cancer supports the view that, in this low-sexual-risk population, HPV-16 exposure per se is a major limiting factor determining cervical cancer incidence.

	ACKNOWLEDGEMENTS

 
We thank Carina Eklund for her help with the serological analysis. This study was supported by the Swedish Cancer Society.

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Received 28 August 2006; accepted 24 November 2006.

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