SYLVAN, STAFFAN P. E. MD, PhD*; VON KROGH, GEO MD, PhD†; TIVELJUNG, A. PhD‡; SIWERTH, BRITT-MARIE*; HENRIKSSON, LISBETH*; NORÉN, LENA MD‡; ASP, ANNA-KARIN MA§; GRILLNER, LENA MD, PhD‡
WOMEN INFECTED WITH genital Chlamydia trachomatis (CT) can develop reproductive sequelae, including infertility and ectopic pregnancy. 1–5 The proposition that CT is a potential cofactor in the progression of oncogenic papillomavirus infection to cervical cancer is currently under debate. 6,7 In men, the infection accounts for 30% to 50% of visits to physicians for nongonococcal urethritis. 8,9 However, in both sexes, clinical signs and symptoms of CT infection are mild or nonspecific in up to 70% of infected cases, 10 and the perpetuation of infection might be explained by describing the identified cases metaphorically as the tip of the iceberg.
Genital CT infection has been notifiable by law in Sweden since 1988. 11,12 As part of the case-finding strategy, the current legislation also requires physicians treating index cases to perform partner notification, in which all identifiable partners are contacted and informed that they have the duty to consult a physician and provide a specimen in order to establish whether they are infected. 13
In the period 1989 to 1994, the reported number of cases in Sweden, as diagnosed by CT culture and/or enzyme immunoassay, declined from 29,319 to 13,785. It was believed that legislative changes were the major factor in this decline. Since 1995, however, the prevalence of CT in Sweden has been on a constant increase again: the number of reported cases was 19,284 in the year 2000. The proportion of males in Sweden in whom CT was identified increased from 35% to 43% during the period 1989 to 2000. The increased detection rate may in part be due to the routine introduction of more sensitive DNA polymerase chain reaction (PCR) and ligase chain reaction amplification tests 14–18 but also may possibly be explained by behavioral changes in the population.
The current study was conducted in the context of the current rise in CT cases, the development of new diagnostic strategies, and an outreach to community-based youth centers for screening sites. Two community-based youth-health clinics in Stockholm were enrolled, and the potential of recruiting more men for screening was specifically investigated. The CT isolates detected were differentiated into genovars by genotyping with restriction fragment length polymorphism analysis. 19
In Sweden there have been youth clinics in each county since the late 1970s. 20 These clinics serve subjects aged 13 to 23 years, are free, offer voluntary and personal choices for counseling without parents’ consent, and focus on a broad range of youth-related issues. The staff includes a midwife with the authority to prescribe birth control pills, a psychologist and/or a social assistant, and a consultant physician in the field of gynecology or venereology. The services are related to individual social and psychological issues of significance for teenagers and adolescents, contraception counseling, birth control pill prescription, physical examination, and screening for sexually transmitted diseases (STDs) when indicated. Historically, 90% of visitors to the youth clinics have been female. However, in recent years, males have accounted for 20% to 25% of new visitors in some of the youth clinics that have actively focused on attracting young men by designating special boys-only visiting hours.
In order to provide advisory service to young people early in life, most youth clinics offer routine visits to 8- to 9-year-old Swedish elementary school pupils for lectures about the basics of sexual development and health. At this stage the target group generally comprises pupils 14 to 16 years old, an age at which most girls have reached a relatively higher level of physical and psychological maturity and (more commonly than among boys that age) have had their sexual debut. 21 No standard curriculum has been developed for the sex education classes. The youth clinic staff also aims to provide information about the type, purpose, and accessibility of the services offered to young people.
The current pilot study on CT screening was undertaken between September 1999 and February 2000 at two county-based youth-health clinics in the outer metropolitan area of Stockholm. Each clinic serves approximately 4000 youths in the age group of 13 to 23 years, which represents 11% to 12% of the total population in the two respective communities. The focus was on providing broad information to sexually active individuals about CT as a cause of frequent and commonly nonsymptomatic infection associated with reproductive health risks.
The planning phase comprised the preparation of elaborate, uniform teaching material for use in high schools to educate students about the symptoms, prevalence, and complications of CT, as well as the protective benefit of condoms against common STDs such as CT infection, gonorrhea, genital herpes, and condyloma acuminatum. Before test enrollment, one of the investigators (G. K.) lectured to 20 to 30 male pupils in each secondary grade class (average age, 17 years) at the two high schools in the county, while midwives from the youth clinics educated the female pupils. The protection against STDs offered by condoms was underscored, and the students were encouraged to attend the youth clinics for free CT urine testing and STD counseling. The potential personal advantages to sexually active adolescents of both sexes of attending the clinic for regular CT screening were particularly emphasized.
In order to recruit participants who could not be reached through this educational effort, information about the study also appeared in local newspapers and on placards at the schools, local shopping centers, and libraries. These notices focused on the asymptomatic nature of genital chlamydial infection and gave information about the study sites and study procedures. No economic incentive was offered to respondents for completing the interview or providing a urine specimen. No control group was evaluated.
At the time of CT testing enrollment, youth clinic visitors who attended because of routine partner notification procedures were excluded, but all other attendees who wished to participate were included. Study participants were asked to fill in an anonymous questionnaire that included sexual and social behavior, country of ethnic origin, and any genital symptoms. Individual questionnaires and urine samples were labeled with a unique code guaranteeing anonymity; only coded test results and questionnaires were subsequently compiled and sent to the county medical officer for unidentifiable statistical analysis. Patients who were infected with CT were treated for free at the youth-health clinic according to established local guidelines (usually with oral doxycycline, administered for 9 days) and subsequently participated in the partner notification service protocol in accordance with the Communicable Disease Act of 1988.
Polymerase chain reaction.
The urine samples were refrigerated at 2 °C to 8 °C during storage and transported to the microbiology laboratory at the Karolinska Hospital within 24 hours. Urine samples were evaluated by means of urine-based PCR with use of the Cobas Amplicor C trachomatis test (Roche Diagnostics).
C trachomatis– positive urine specimens were subsequently used for genotyping. 22 As a reference for the restriction enzyme cleavage, a set of C trachomatis strains was used (kindly provided by Dr. Kenneth Persson, Malmö Allmänna, Sjukhus, Sweden). C trachomatis serotypes C 252, D 36, E 1,449, F 373, G 349, H 1,230, and K 173, kept in formalin, underwent DNA extraction with use of a QIAamp DNA mini kit (Qiagen) after repeated prewashing in phosphate buffered saline.
DNA was recovered following a slightly modified protocol with use of the QIAamp kit. Before DNA extraction, 0.5-ml urine specimens were washed in 0.5 ml C trachomatis/ Neisseria gonorrhoeae urine wash buffer (Amplicor; Roche), incubated at 37 °C for 15 minutes, and subsequently centrifuged at 12,500 g for 5 minutes. The pellet was dissolved in 180 μl ATL buffer, and DNA extraction proceeded according to the manufacturer's protocol (Qiagen). Finally, DNA was eluted in 100 μ ultrapure water.
Two sets of PCR primers to the C trachomatis major outer membrane protein gene were used for a nested PCR approach. The first step of the PCR was carried out with primers CT1 and CT2, 22 5′-GCCGCTTTGAGTTCTGCTTCCTC-3′ and 5′-ATTTACGTGAGCAGCTCTCTCAT-3′, respectively. The nested set of PCR primers, CT1-N and CT5-N, was located proximal to the outer primer pair: CT1-N = 5′-CCTTGCAAGCTCTGCCTGTG-3′: CT5-N = 5′-TCGATCAAGCGAGTCTCAACTG-3′.
Amplifications were carried out in a final volume of 50 μ containing 20 μM primer, 1.5 mmol/l MgCl2, 0.2 mmol/l deoxyribonucleoside triphosphates, and 1 U Taq polymerase (PerkinElmer Biosystems). In the first-step PCR, the primer combination CT1/CT5 was used with 1 μ DNA extracted from reference serotypes or 10 μ DNA extracted from urine specimens. The PCR was performed in a thermocycler (Eppendorf) as follows: denaturation at 94 °C for 4 minutes, 30 cycles with denaturation at 94 °C for 30 seconds, annealing at 50 °C for 30 seconds, extension at 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes. The expected PCR product fragment size was approximately 1130 base pairs (bp).
In the nested PCR, conditions were as mentioned above, except that the primer combination CT1-N/CT5-N was used with 1 μ of undiluted PCR product from the first-step PCR. Amplification was carried out as follows: denaturation at 94 °C for 4 minutes, 30 cycles with denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes. Nested PCR products were analyzed by agarose gel electrophoresis, yielding a visible product of approximately 1090 bp.
Amplified DNA fragments were digested with Eco RI, Hinf I, and Hpa II, respectively. Digestion was performed with 5 U of restriction enzyme on 10 μ of PCR product, with use of the recommended assay buffer (Roche), overnight at 37 °C. Analysis of digested DNA was carried out by agarose gel electrophoresis (3% agarose) and staining with ethidium bromide.
The statistical analysis consisted of univariate analysis followed by logistic regression to determine independent factors associated with PCR positivity.
The study sample comprised 108 male and 341 female sexually active adolescents who all consented to be interviewed as well as to provide a urine specimen. Demographic and reproductive health characteristics are summarized in Table 1. The majority of participants were Swedish (84.2%) and were free of genitourinary symptoms at the time of screening (77.4% of males, 83.9% of females). A very early sexual debut (13 years of age or younger) was reported by 10.6% of the boys and 6.8% of the girls. A history of STD was reported by 21.9% of boys and 15.0% of girls, and chlamydial infection and condyloma were the most commonly reported prior infections (data not shown).
All participants had been sexually active during the past 12 months; 14.4% of the males and 35.0% of the females reported a single partner, whereas 59.6% and 56.6%, respectively, reported two to four sex partners. Most participants were single, whereas 13.9% and 15.6% reported being married or living with their current partner. Fewer than 40% of the attendees were smokers. During their most recent intercourse, only one in five had consumed an alcoholic beverage and more than 75% had not used a condom. Approximately one quarter of the participants reported regular use of condoms.
Chlamydial Infection Detection
Table 2 shows the prevalence of CT infection: 5.9% (20/341) among women and 9.3% (10/108) among men (NS). Two factors were statistically independently associated with CT infection. First, males with genitourinary symptoms, such as dysuria and penile discharge, were positive for infection significantly more often (30.4%) than were nonsymptomatic males (3.4%;P < 0.001; odds ratio [OR] = 10.84; 95% CI = 2.47–47.59). A similar difference was not observed for females: 5.6% with symptoms versus 6.0% without symptoms were infected (OR = 0.92; 95% CI = 0.25–3.34). Second, a history of STD was associated with a higher rate of CT infection (P < 0.02; OR = 2.75; 95% CI = 1.21–6.24). Traditional markers of high-risk behavior, such as use of contraceptive pills, smoking, alcohol intake before intercourse, and not having a regular partner, were not associated with a higher infection rate.
The nested PCR approach yielded visible products with 7 reference strains and 30 clinical urine samples (selected positive samples). Digestion of the expected PCR product from different serotypes was performed in silico with use of the NetWare Webcutter 2.0 (http://www.firstmarket.com/cutter/cut2.html; Max Heiman, Yale University, New Haven, CT). DNA sequences from all known serotypes of the CT-MOMP gene were cut, and the restriction enzyme cleavage patterns are summarized in Table 3.
The Eco RI, Hinf I and Hpa II digestions allowed discrimination between serotypes C, D, E, F, G, H, and K on the basis of the expected PCR product. The restriction enzyme cleavage patterns were those expected by in silico digestion.
The 30 selected CT–positive PCR products from urine specimens were typed by restriction fragment length polymorphism analysis. The serovar distribution at the two youth-health centers is shown in Figure 1. Serovar E was the most prevalent serovar for both men (60%) and women (60%). Genotypes F and K were second most prevalent for men (20%), and genotype D was second most prevalent for women (15%). Genotypes K and F were found in lower percentages of cases. Serovar E was most prevalent at both youth-health clinics. The second most prevalent serovar was F in one of the two youth-health centers and serovar K in the other. One strain was impossible to type.
Most reports on STD prevalence and incidence are based primarily on patient-initiated self-referral to medical providers and tend to underreport asymptomatic cases and to underrepresent persons who have limited access to medical care. 23 A further constraint of official STD reporting systems is the lack of detailed information about the persons infected, because generally only minimal demographic and behavioral traits are available. Selective CT screening has been a common strategy because universal screening is too costly in most settings. 24 When applied at youth clinics and STD clinics, selective screening may provide richer clinical and behavioral data. In Sweden, presumably healthy sexually active females undergo routine CT testing in youth clinics when they seek a prescription for birth control pills. This is a feasible means of case-finding based on convenience sampling and may potentially be more effective than random or universal sampling strategies. 19,25
A similarly systematic strategy for convenience sampling among sexually active adolescent males has not yet arisen within the Swedish national tradition, although some youth clinics in recent years have actively focused on attracting young men by designating special boys-only visiting hours. The availability of noninvasive screening techniques, such as urine-based DNA amplification methods, may potentially increase the success of CT screening services, while decreasing the anxiety of clients who fear invasive procedures. This is obviously of particular importance for asymptomatic young men who fear the indisputably unpleasant invasive technique previously used for urethral sampling.
The current study presents the results of convenience sampling among well-informed youths with presumed high-risk behavior. Thus, the sampling frame described here has its own set of selection biases, and, therefore, the study is not truly population-based. In addition, because of the limited sample, caution is warranted in extrapolating this figure to 13- to 23-year-old females in the Stockholm metropolitan area or on a national level.
All of the young women and men who attended were willing to complete an inquiry form and to provide a urine specimen for CT testing. Among the women without any genitourinary symptoms, the prevalence of CT infection was 6.0%, which did not differ from the 5.9% prevalence rate among symptomatic women. Our figures are slightly higher than the 3.9% prevalence rate observed among young Parisian females 26 but are in concordance with the 5.6% incidence detected among Finnish women attending a family planning clinic, 27 as well as the 5.4% prevalence among abortion-seeking Norwegian women. 28
Altogether, 9.3% of males tested positive for CT. Males who reported dysuria and/or some discharge on the questionnaire generally described these symptoms as mild and/or transient when their history was subsequently obtained during clinical follow-up measurements. Among the CT–positive men, 30% consistently reported a complete absence of symptoms. The detection rate among truly nonsymptomatic men was 3.7%, which is somewhat lower than the 5.4% prevalence observed among male teenagers and young adults in community-based urine screening programs in the United States. 24
Nonsymptomatic or mildly symptomatic CT–infected people may remain unidentified and therefore continue to spread the infection to their partners. In the current study the prevalence tended to be correlated to partner change during the previous year, yet there was no statistical difference between patients who had only 1 partner (3.9%), those who had 2 to 4 partners (7.8%), and those who had 5 or more partners (12%). Although no statistically significant correlation was detected, we do find it notable that the CT prevalence tended to be higher among patients who were either married or reported a cohabitation relationship (10.9%) than among those who were single (5.1%). Apparently, adolescents living in a “steady relationship” seem to have at least the same risk of being CT–infected as those having changed partners within the previous year. Therefore, we believe that all adolescents in a steady sexual relationship need information and encouragement about undergoing testing for CT because of the possibility that previously contracted infection might be transmitted by either one in the couple.
The data collected on self-reported sexual and contraceptive behaviors in our population are associated with high validity and reliability, because such sensitive topics were approached confidentially through self-administered questionnaires that the investigators were not permitted to view. A major concern for future individualized postintervention educational and behavioral efforts is the low self-reported use of condoms among the study population. Whereas 70% of the females reported use of contraceptive pills, >75% of the study population, regardless of sex, had not used a condom during their most recent intercourse, and only 27% used condoms regularly.
We believe that partner notification alone may not be sufficient for an optimal reduction in the spread of CT but that targeted screening programs among sexually active people should also be implemented as part of case-finding strategies. With the exception of our finding that an increase in risk of CT was associated with prior infection, no additional selective screening criteria that could guide local programs emerged from the current pilot study. It has been suggested that prevention programs based on a case-finding strategy should also focus on repeated testing for individuals of both sexes who have had an STD during the preceding year. 29–34 This recommendation is further substantiated by our finding that a significant increase (P < 0.01) in the risk of infection was associated with a history of STD.
The need for additional follow-up for these youths is elucidated by the finding that 15% to 22% of the study group had been evaluated for STD in the past. In addition to intensified contact-tracing for patients with repeated CT infections, such an extended follow-up might contribute to a reduction in the pool of infectious individuals. We did not detect any correlation between increased risk and behavioral factors such as inconsistent use of condoms by the males, current use of contraceptive pills by the females, smoking habits, or alcohol consumption during the previous intercourse.
In the current study it was shown that CT genotyping was successful for 97% (29) of the 30 CT–positive urine samples. Similar to reports from other European centers, the most prevalent omp1 genotype in women was E, 35,36 followed by F. Serovars F and E are known to be associated with increased and decreased risk of severe upper genital tract infections, respectively. 36 It would be of interest to determine if infection by certain genotypes carries increased risk of tubal scarring or is associated with the development of cervical squamous cell carcinoma. 6,7 Moreover, the typing of CT genotypes in routinely collected urine specimens provides an opportunity to study the epidemiology of CT genotypes and to assess whether genotype-specific properties confer a transmission advantage in an asymptomatically infected youth population. 37
Estimations of prevention effectiveness have mostly concerned chronic diseases and tend not to take into account the dependence of one individual's health outcome on the health outcome of other individuals. The prevalence and incidence of CT may increase or decline as a reflection of the natural progression of epidemics or as a result of changing behaviors, although behavioral changes may or may not be related to the intervention. In the United States universal screening has been shown to be more cost-effective than selective screening at a CT prevalence >3%, 24 whereas in Sweden general screening has been estimated to be cost-effective at a prevalence of ≥5%. 38
The current study showed no difference in CT prevalence between women lacking any symptoms (5.5%) and women with some type of genitourinary symptom (6.0%), and the symptoms in the men were vague and often transient. Therefore, we find it prudent to recommend that all sexually active male and female clients of youth-health clinics in Sweden be offered convenience screening for CT, regardless of any symptoms or presumed risk behavior. In order to optimize this strategy, sexual health services for young men should become widely accessible, and the services should be advertised in an informal and engaging way. 39
By their nature, noninvasive amplification methods for CT detection also offer the opportunity for cost-effective screening of youths outside traditional facilities, including schools and homes. 40–48 Thus, although self-sampling for CT may become of great future use, the challenge will be to integrate these innovations into a comprehensive yet cost-effective youth-oriented system of STD prevention 49,50 that includes active population- based information as well as individual counseling when test results are positive.
We believe that a comprehensive approach to adolescent health should include the development of aggressive youth-centered and -targeted campaigns to help initiate and maintain healthy choices, instead of simply addressing the unintended consequences of high-risk activity. 51–53 Interventions of variable content may lead to desired changes in knowledge, attitudes, perceptions, self-efficacy, skills, and behavior, such as positive changes in male condom use, number of sex partners, and practice of unprotected sex. We support the view that any intervention providing active health information to young people at risk of STD may yield such changes. A body of literature has clearly shown that theory-driven sexual risk–reduction interventions that address abstinence rather than emphasizing social skills such as sexual communication and condom use are less effective in enhancing STD-preventive behaviors. 54–60
We envision a comprehensive STD-prevention strategy that includes community interventions to enhance access to health care and STD services as well as community-based screening programs. Interventions could be developed that address the issue of routine STD evaluation as part of periodic health examinations. In this area, educating clients and assisting health care providers with public health STD programs may have a significant impact on the demand and delivery of STD screening services. Interventional opportunities will be missed unless the importance of discussing sexual behavior on a comprehensive educational level is appreciated. Although many youths are out of school, our study emphasizes the strategic possibility of enhancing sexual health education in schools. This study has yielded locally relevant data that might prove to be useful in the development of interventions targeted toward Swedish adolescents.
In conclusion, the integration of school- and community-based educational programs with the use of behavioral surveys and collection of urine specimens for CT testing in representative samples is feasible for case-finding purposes. CT serovar typing is a simple and sensitive method of CT classification that may facilitate epidemiologic studies. If more widely adopted, these strategies could have an impact on the spread of genital CT and thus improve the reproductive health of young women and men, as well as reduce the risk of cervical cancer.
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