Sexually Transmitted Diseases, October 2005, Vol. 32, No. 10, p.599 – 604 DOI: 10.1097/01.olq.0000179887.01141.c3 Copyright © 2005, American Sexually Transmitted Diseases Association All rights reserved.

Reinfections, Persistent Infections, and New Infections After General Population Screening for Chlamydia trachomatis Infection in The Netherlands IRENE K. VELDHUIJZEN, MSC,* JAN E. A. M. VAN BERGEN, MD, MPH,†‡ HANNELORE M. GÖTZ, MD, MPH,* CHRISTIAN J. P. A. HOEBE, MD, PHD,§ SERVAAS A. MORRE´, PHD,储 AND JAN HENDRIK RICHARDUS, MD, PHD,*¶ FOR THE PILOT CT STUDY GROUP

From the *Municipal Public Health Service, Rotterdam, The Netherlands; †STI AIDS Netherlands, The Netherlands; the ‡Department of General Practice, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands; the §Municipal Public Health Service, Eastern South Limburg, The Netherlands; the 储Laboratory of Immunogenetics, Section Immunogenetics of Infection Diseases, VU University Medical Center, Amsterdam, The Netherlands; and the ¶Department of Public Health, Erasmus MC, University Medical Center Rotterdam, The Netherlands

Objectives: The objectives of this study were to determine the rate of new infections and reinfections or persistent infections with Chlamydia trachomatis to define appropriate screening intervals and to identify risk factors for reinfection. Design: This was a cross-sectional study among a subsample of participants in a population-based screening. Setting: This study was conducted in urban and rural areas in The Netherlands. Participants: A total of 21,000 15- to 29-year-old women and men were invited for home-based urine testing. One year after the study, a subsample of 299 participants were offered retesting. Main Outcome Measures: The authors studied the rate of infection with C. trachomatis. Serovar determination was used to potentially discriminate between new infections and reinfections or persistent infections. Results: Nine C. trachomatis infections were found among 187 responders (4.8% confidence interval, 1.7–7.9). The prevalence was 10.4% (5 of 48) in previous positives and 2.9% (4 of 139) in negatives. Three of 5 repeatedly positive participants were infected with a different C. trachomatis serovar. Conclusions: Our study indicates that infected persons found in a systematic, population-based screening should be rescreened within 1 year. Optimal screening intervals still need to be determined.

CHLAMYDIA TRACHOMATIS IS THE most common bacterial sexually transmitted infection worldwide.1 It is often asymptomatic and can be persistent of nature for prolonged periods.2,3 Chlamydial infections are an important preventable cause of reproductive morbidity and can cause pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, reduced fertility, and infertility.4,5 Repeated infections with C. trachomatis increase the risk of such complications.6 Screening for chlamydial infections has been shown to reduce the incidence of PID and ectopic pregnancy.7–10 The availability of sensitive detection methods on urine and highly effective single-dose treatment has made screening for infections with C. trachomatis feasible. Whether to implement a national screening program and in which modality is currently under debate in The Netherlands, like in several other countries. Because reinfection with C. trachomatis is possible after treatment, and previously negative individuals can acquire a new infection, it is obvious that screening should be offered regularly. To determine the frequency with which screening should take place (screening interval), knowledge of the rate of reinfection or persistent infection and new infection is required. C. trachomatis can be subdivided into different serovars based on variation in the major outer membrane protein. Serovar determination can be used as a tool to track transmission because it can help to distinguish between new infections and persistent infections or reinfections. The follow up and rescreening of individuals with a positive test result needs extra attention. Persons who have been infected with C. trachomatis are at risk of becoming reinfected or having a persistent infection. A reinfection can be acquired from current or previous partners who have not been treated or from a new partner. A persistent infection can occur as a result of treatment failure.

PILOT CT Study Group: J. E. A. M. van Bergen, J. Broer, PhD (Municipal Public Health Service Groningen, The Netherlands), A. J. J. Coenen, MSc (STI AIDS Netherlands, The Netherlands), H. Go¨tz, F. de Groot, BA (Municipal Public Health Service Groningen, The Netherlands), C. J. P. A. Hoebe, S. A. Morre´, J. H. Richardus, D. T. van Schaik, MSc (STI AIDS Netherlands, The Netherlands), I. K. Veldhuijzen, and M. Verhooren, BA (Municipal Health Service “Hart voor Brabant,” The Netherlands). JvB was the project leader and has initiated the CT pilot. JvB, HG, JB, IV, CH, JR, AC, FdG, DvS, MV, and SM contributed to the study design and protocol and collected and interpreted data. IV performed the statistical analysis. The manuscript was written by IV and JvB and critically reviewed by SM, HG, JB, JR, DvS, CH, AC, FdG. Prof. P. J. E. Bindels, A. J. P. Boeke, MD, PhD, Prof. J. D. F. Habbema, J. A. R. van den Hoek, MD, PhD, L. Jacobi, MSc, and S. A. Morre´, PhD, were scientific advisors (scientific advisory board). The authors thank J. Spaargaren (Municipal Health Service, Amsterdam) for typing coordination of the C. trachomatis strains. This study has been financed by ZonMw (The Netherlands Organisation for Health Research and Development) who has no commercial interests and had no role in study design, organization of the study, and/or writing of the manuscript. S. A. Morre´ is supported by Tramedico BV, The Netherlands, the Falk Foundation, Germany, the Department of Internal Medicine of the VU University Medical Centre, and Foundation of Immunogenetics, Amsterdam, The Netherlands. Correspondence: Jan van Bergen, MD, MPH, SOA AIDS Nederland, Keizersgracht 390-392, 1016 GB Amsterdam, The Netherlands. E-mail: [email protected]. Received for publication November 5, 2004, and accepted February 15, 2005.

599

600

VELDHUIJZEN ET AL

The need for rescreening after a positive test and subsequent treatment has been argued in a number of studies that were mainly conducted in selected clinic populations and where reinfection rates between 7% and 34%11–14 were found. However, in a systematic home-based screening program among registered patients of 15 general practices in Amsterdam, none of the 56 patients who had received treatment were found to have reinfections at the second screening 1 year after the initial screening.15 Although one expects lower reinfection rates in the general population, some studies in more population-based settings (e.g., general practice, population sexually transmitted disease surveillance register) also reported high rates of reinfection between 13% and 29%.16 –18 In prospective follow-up studies, participants were mostly tested several times in the first 6 months after treatment. Kjaer et al found a cumulative reinfection rate of 29% in 6 months follow up and recommended retesting 3 to 6 months after treatment.18 In a study by Whittington et al, 7% of women were reinfected at 4 months after treatment, Schillinger et al found 11% after 6 months.11,17 The Centers for Disease Control and Prevention in the United States recommends annual screening of all sexually active women ⬍25 years and rescreening of all women with chlamydial infection 3 to 4 months after treatment.19 Based on high prevalence and reinfections rates in the population under study, Burstein et al recommend chlamydia screening for all sexually active females ⬍25 years at least twice yearly.20 The purpose of this study was to determine the rates of infection 1 year after screening in initial C. trachomatis-negative and -positive persons and to identify risk factors for reinfection. Moreover, because it is difficult to distinguish between persistent infection or reinfection and newly acquired infections, we wanted to discriminate between type of reinfection using C. trachomatis serovar determination. Methods Study Population A large population-based study, the CT PILOT project, was conducted from September 2002 through March 2003 and was designed to investigate prevalence of C. trachomatis infections in rural and urban areas in The Netherlands. A total of 21,000 women and men aged 15 to 29 years received a package by mail with a urine sampling kit and a questionnaire concerning demographic data (sex, age, self-reported ethnicity, education), symptoms, history of sexually transmitted infection, and sexual behavior. Urine analysis was done by a nucleic amplification test (PCR; Roche, Basel, Switzerland). The method of sampling and screening as well as response rates, nonresponse, prevalence, and risk factors are described elsewhere.21,22 The study was approved by the appropriate medical ethics committee. The acceptability of the screening method was studied in all 156 C. trachomatis (CT)-positives and a random sample of 600 CTnegatives from the CT PILOT. The results of the acceptability study, including response analysis, are described in this journal.23 The reinfection study described here was conducted among 299 respondents in the acceptability study who agreed to be screened again after a year. The testing procedure was similar to the procedure used in the first screening. Also, concerning the laboratory testing, specimens were pooled by 5 and retested individually to confirm positive samples. Participants were asked to complete a questionnaire with questions on symptoms in the previous 4 weeks and sexual behavior to identify risk factors that were associated with new infection or reinfection. All participants received the test result by mail and were asked to contact the Municipal Health Service for treatment in case of a positive result. The data collected

Sexually Transmitted Diseases

●

October 2005

in the reinfection study were linked to the data from the initial study on the basis of an identification number. Chlamydia trachomatis Serovar Determination Typing was performed by either amplification of the total omp1 gene followed by restriction fragment length polymorphisms (RFLP) or by sequence analysis of the variable segment 2 (VS-2) of the omp1 gene. For details on the polymerase chain reaction (PCR)-based RFLP and VS-2 sequencing, see the Appendix. Statistical Analysis Statistical analysis was done with SPSS statistical software version 10.0 (SPSS, Inc., Chicago, IL). Prevalences were calculated with 95% confidence intervals. Differences in characteristics between participants and nonresponders and between prevalence and several characteristics were analyzed using the chi-square (for trend) or Fisher exact test. A P value ⬍0.05 was considered statistically significant. Results Response A new test kit was sent to 299 participants in the acceptability study who had agreed to receive an invitation for rescreening 1 year after the first screening offer. The respondents of the acceptability study were comparable to respondents in the original screening except for sex; relatively more women participated in the acceptability study. The response to the rescreening offer was 63%; 187 participants returned a urine sample (Table 1). The response did not differ by sex or by the initial test result. Participation in the youngest age group of 15 to 19 years was lower than participation in the 20- to 29-year age group, 53% and 67%, respectively (P ⫽ 0.022). Participants who had never been sexually active at the time of the initial screening participated less in the rescreening (42%) compared with sexually active participants (65%, P ⫽ 0.024). Four participants (2%) had never been sexually active at the time of the second screening. Follow up of CTpositive participants after the first screening showed that all participants in the reinfection study who initially tested positive test had been treated for their CT infection.

TABLE 1.

Response and Characteristics of Respondents

Overall Sex Men Women Age group 15–19 20–29 Sexually active‡ Yes No Result initial test Positive Negative

n*

N†

Percent

187

299

63%

59 129

96 203

61% 64%

49 139

92 207

53% 67%

174 11

269 26

65% 42%

48 139

71 228

68% 61%

P Value 0.727 0.022 0.024 0.345

*Number of respondents. †Number invited for rescreening. ‡At the time of the initial screening.

time points, identical VS-2 sequences were identified, which were also identical between the 2 persons. From the 5 repeatedly positive participants, additional information on the probable reason for reinfection and treatment could be retrieved. Their physician had treated all of the 5 participants after the initial screening. Of the 3 participants who were infected with a new serovar, 2 had mentioned one partner in the 6 months preceding the first screening round and both partners were treated. Both participants did not report new partners in the 2 months before the rescreening, but did report one partner in the preceding 6 months, and it is not known if these were the same partners as mentioned after the first screening. The other participant mentioned 2 partners in the previous 6 months before the initial screening. The current partner indicated to go to his own physician for treatment, but at the second screening, the participant said this never happened. Besides, she had 2 other partners since the previous screening. Serovar determination showed infection with another serovar after the second screening. A new partner most likely reinfected this participant. The 2 participants with identical serovars at both screenings both mentioned 2 partners in the 6 months before the initial screening, of whom only one was treated. One of the participants mentioned a new partner in the 2 months preceding the rescreening. These participants were possibly reinfected with the same strain through an untreated partner or by a new partner with the same type of serovar.

Prevalence, Incidence, and Risk Factors We found 9 C. trachomatis infections among 187 participants (4.8%; 95% confidence interval [CI], 1.7–7.9). Five of 48 previously positive participants tested positive (10.4%; 95% CI, 1.8 – 19.1) and 4 of 139 previously CT-negative persons, (2.9%; 95% CI, 0.1–5.7) (Fisher exact test P ⫽ 0.05). The average time of follow up was 1.1 year (range, 0.9 –1.3 years). Although demographic characteristics like sex, age, education, ethnicity, and urbanization were not statistically significant associated with reinfection, some associations were present. Women were more often infected than men (6.2% [8 of 129] and 1.7% [one of 58], respectively, P ⫽ 0.28). Prevalence was highest in the youngest age group: 8.2% (4 of 49) in 15 to 19 year olds, 4.5% (3 of 67) in 20 to 24 year olds, and 2.8% (2 of 71) in 25 to 29 year olds (P ⫽ 0.19). With regard to risk of infection for sexual behavioral risk factors, having a new sexual partner in the 2 months before screening was, although not statistically significant, associated with C. trachomatis infection; 12.5% (3 of 24) of participants who had a new sex partner in the previous 2 months tested positive compared with 4.2% (6 of 143) of participants who did not (Fisher exact test P ⫽ 0.122) (Table 2). Serovar Determination To investigate whether persons were reinfected with the same serovar, PCR-based RFLP analysis on the omp1 gene was performed. Three persons were clearly infected with another serovar as compared with their initial identified C. trachomatis serovar (Table 3). Two persons had the most prevalent serovar, serovar E, at both time points. To investigate in detail whether these serovar E isolates were identical for both time points, variable segment 2 (VS-2) of the omp1 gene was sequenced. For both persons at both

TABLE 2.

601

RESCREENING FOR CHLAMYDIAL INFECTION

Vol. 32 ● No. 10

Discussion We found a substantial number of reinfections when we offered rescreening a year after the initial screening. Reinfection differed by initial infection status and was 10.4% (5 of 48) in previous

Prevalence According to Initial Test Result Positive First Test

Overall Sex Men Women Age group 15–19 20–24 25–29 AAD* 1 2 3 4 5 Ethnicity Dutch Not Dutch Education Low Intermediate High New partner in last 2 mo Yes No

Negative First Test

Total

Positive

N

Percent

Positive

N

Percent

Positive

N

%

P Value

5

48

10.4%

4

139

2.9%

9

187

4.8%

0.050 0.278

0 5

9 39

0.0% 12.8%

1 3

49 90

2.0% 3.3%

1 8

58 129

1.7% 6.2%

1 2 2

9 16 23

11.1% 12.5% 8.7%

3 1 0

40 51 48

7.5% 2.0% 0.0%

4 3 2

49 67 71

8.2% 4.5% 2.8%

2 2 1 0 0

14 10 11 9 4

14.3% 20.0% 9.1% 0.0% 0.0%

1 1 1 0 1

39 30 26 17 27

2.6% 3.3% 3.8% 0.0% 3.7%

3 3 2 0 1

53 40 37 26 31

5.7% 7.5% 5.4% 0.0% 3.2%

4 0

41 6

9.8% 0.0%

3 1

128 10

2.3% 10.0%

7 1

169 16

4.1% 6.3%

1 2 1

17 21 9

5.9% 9.5% 11.1%

1 1 2

27 58 53

3.7% 1.7% 3.8%

2 3 3

44 79 62

4.5% 3.8% 4.8%

2 3

8 38

25.0% 7.9%

1 3

16 105

6.3% 2.9%

3 6

24 143

12.5% 4.2%

0.188

0.694

0.522 0.952

0.122

*AAD indicates area address density: AAD1 ⫽ very high urban (⬎2500 addresses/km2); AAD2 ⫽ high urban (1500 –2500 addresses/km2); AAD3 ⫽ moderate urban (1000 –1500 addresses/km2); AAD4 ⫽ low urban (500 –1000 addresses/km2); and AAD5 ⫽ rural (⬍500 addresses/km2).

602 TABLE 3.

VELDHUIJZEN ET AL

Sexually Transmitted Diseases

●

October 2005

Chlamydia trachomatis Serovar Analysis t⫽0 No.

CT⫹

Serovar

CT⫹

Serovar

Gender

1 2 3 4 5

⫹ ⫹ ⫹ ⫹ ⫹

H E Ga E E

⫹ ⫹ ⫹ ⫹ ⫹

E E* G E* D

F F F F F

Dutch Dutch Dutch Dutch Dutch

6 7 8 9

— — — —

— — — —

⫹ ⫹ ⫹ ⫹

F —† E F

M F F F

Dutch Dutch Dutch Cameroon

Infected t ⫽ 0 and t ⫽ 1

Infected only at t ⫽ 1

t⫽1 Ethnicity

*Sequencing of variable segment 2 (VS-2) of the omp1 gene revealed that the strains were identical both on serovar level and sequence level for both patients on both time points. †This isolate was positive twice in the commercial C. trachomatis polymerase chain reaction (Roche) but could not be typed most likely resulting from DNA degradation or low titer infection.

positives and 2.9% (4 of 139) in negatives (P ⫽ 0.05). The prevalence of infection in previous negatives was comparable with the prevalence in the sexual active participants in the initial screening (2.3%).21 Although not statistically significant, the highest rate of infection in previous negatives was among the youngest age group and in participants who had a new sex partner in the 2 months before screening. In previous positives, the rate of reinfection was similar by age group but higher in those with a recent new sex partner. Having a new partner is associated with age; 33% of participants aged 15 to 19 years had a new partner in the previous 2 months compared with 11% of participants older than 19. Among the 5 previous positives, 3 were infected with a different serovar. They were possibly infected by new partners but could also have acquired the new serovar through old partners who dated outside the relationship. Failing partner treatment was identified as a potential reason for reinfection in 2 other cases, although a reinfection with the same strain (serovar E) by a different partner cannot be excluded, especially because serovar E is the most prevalent serovar (40%) in The Netherlands.24 Both persons who had serovar E at both time points were from completely different geographic location in The Netherlands, which makes it likely that in The Netherlands, another serovar E strain is present as compared with the reference serovar E strain. This emphasizes the epidemiologic value of sequencing to assess serovars, because additional information is obtained as compared with the PCRbased RFLP analysis. A limitation of our study is the small number of subjects, which affects the generalization of the results and the extent to which statistical inferences can be made. Power was just sufficient to detect the difference in infection rate by initial infection status but not enough to identify other risk factors for reinfection. A further limitation of the study is the fact that selection bias cannot be excluded. The C. trachomatis-negative participants were randomly selected, but less than half participated in this study. However, when we compared the C. trachomatis-negative participants in the CT PILOT study with the previously negative responders in the reinfection study, there were no differences in sex, age group, ethnicity, or number of lifetime partners. Non-Dutch participants were underrepresented in the CT PILOT study, and with a nonresponse rate of 37% among those who accepted rescreening after 1 year, some selection bias cannot be excluded. The use of detailed serovar determination seems useful to distinguish between new infections and persistent infections or rein-

fections. However, if the same serovar is detected, we cannot distinguish among a newly acquired infection with the same strain, a persistent infection, and the same infection being transmitted through an untreated partner. Another limitation of the PCR-based RFLP analysis to determine serovars is the fact that only one serovar will be identified. In the case of double C. trachomatis serovar infections, although unlikely, it cannot exclude the fact that identification of different serovars at different time points is still the result of persistent infections. The higher rate of C. trachomatis infection in previously positive individuals points at the need for rescreening of these individuals and implies that rescreening should take place after less than a year. Besides, the higher risk of reinfection in persons with a previous positive C. trachomatis test stresses the need for improved partner treatment strategies and upgrading of counseling performance in primary care. Home-based rescreening through the mail has been found to be feasible and acceptable.18,25,26 New methods such as patient-delivered partner treatment,17,27,28 homebased screening of partners,29 and screening through the Internet30 appear to have much better yield than conventional partner notification and opens new avenues for better outcome in the future. Our study has a striking difference compared with a previous population-based rescreening study in Amsterdam, where no reinfections were found.15 In terms of (partner) treatment, our study represented current practice in The Netherlands. The patient provided the general practitioner with a study leaflet containing the test results as well as the recommended treatment and partner treatment. A routine test of cure is currently not included in the recommendations on management of C. trachomatis infections. In the Valkengoed study, the participating general practitioners had received some training before the screening, although afterward, they still indicated that more training on this issue was needed.15 This underscores the need for additional training in counseling in primary care when embarking on a screening program. Another point of difference with this study is the fact that the Valkengoed study protocol consisted of a test of cure after the first screening. They found 5 patients (8% [5 of 62]) were still positive 4 weeks after the treatment, although they reported to have taken treatment, as did their partners. These persons were treated again with a different regimen, but in our study design, these cases could become classified as “persistent infections.”

603

RESCREENING FOR CHLAMYDIAL INFECTION

Vol. 32 ● No. 10

Conclusion We found high reinfection rates among persons tested positive in a first screening round. Our study indicates that infected persons found in a systematic, population-based screening should be rescreened within 1 year. Optimal screening intervals still need to be determined. References 1. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. Geneva: World Health Organization, 2001. 2. Golden MR, Schillinger JA, Markowitz L, et al. Duration of untreated genital infections with Chlamydia trachomatis. Sex Transm Dis 2000; 27:329 –337. 3. Dean D, Suchland RJ, Stamm WE. Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping. J Infect Dis 2000; 182:909 –916. 4. Westro¨m L, Joesoef R, Reynolds G, et al. Pelvic inflammatory disease and fertility: A cohort of 1,844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex Transm Dis 1992; 19:185–192. 5. Stamm WE. Chlamydia trachomatis infections of the adult. In: Holmes KK, Sparling PF, Mardh PA, eds. Sexually Transmitted Diseases, 3rd ed. New York: McGraw-Hill, 1999. 6. Hillis SD, Nakashima A, Amsterdam L, et al. The impact of a comprehensive chlamydia prevention program in Wisconsin. Fam Plann Perspect 1995; 27:108 –111. 7. Kamwendo F, Forslin L, Bodin L, et al. Decreasing incidences of gonorrhea- and chlamydia-associated acute pelvic inflammatory disease. A 25-year study from an urban area of central Sweden. Sex Transm Dis 1996; 23:384 –391. 8. Egger M, Low N, Smith GD, et al. Screening for chlamydial infections and the risk of ectopic pregnancy in a county in Sweden: Ecological analysis. BMJ 1998; 316:1776 –1780. 9. Scholes D, Stergachis A, Heidrich FE, et al. Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection. N Engl J Med 1996; 334:1362–1366. 10. Ostergaard L, Andersen B, Moller JK, Olesen F. Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: A cluster-randomized 1-year follow-up study. Clin Infect Dis 2000; 31:951–957. 11. Whittington WL, Kent C, Kissinger P, et al. Determinants of persistent and recurrent Chlamydia trachomatis infection in young women: Results of a multicenter cohort study. Sex Transm Dis 2001; 28: 117–123. 12. Rietmeijer CA, Van Bemmelen R, Judson FN, Douglas JM Jr. Incidence and repeat infection rates of Chlamydia trachomatis among male and female patients in an STD clinic: Implications for screening and rescreening. Sex Transm Dis 2002; 29:65–72. 13. Richey CM, Macaluso M, Hook EW. Determinants of reinfection with Chlamydia trachomatis. Sex Transm Dis 1999; 26:4 –11. 14. Barnett SD, Brundage JF. Incidence of recurrent diagnoses of Chlamydia trachomatis genital infections among male and female soldiers of the US Army. Sex Transm Infect 2001; 77:33–36. 15. van Valkengoed IG, Morre SA, van den Brule AJ, et al. Follow-up, treatment, and reinfection rates among asymptomatic Chlamydia trachomatis cases in general practice. Br J Gen Pract 2002; 52:623– 627. 16. Xu F, Schillinger JA, Markowitz LE, et al. Repeat Chlamydia trachomatis infection in women: analysis through a surveillance case registry in Washington State, 1993–1998. Am J Epidemiol 2000; 152:1164 –1170. 17. Schillinger JA, Kissinger P, Calvet H, et al. Patient-delivered partner treatment with azithromycin to prevent repeated Chlamydia trachomatis infection among women: A randomized, controlled trial. Sex Transm Dis 2003; 30:49 –56. 18. Kjaer HO, Dimcevski G, Hoff G, et al. Recurrence of urogenital Chlamydia trachomatis infection evaluated by mailed samples obtained at home: 24 weeks’ prospective follow up study. Sex Transm Infect 2000; 76:169 –172.

19. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines 2002. MMWR 2002; 51:32–35. 20. Burstein GR, Zenilman JM, Gaydos CA, et al. Predictors of repeat Chlamydia trachomatis infections diagnosed by DNA amplification testing among inner city females. Sex Transm Infect 2001; 77:26 –32. 21. van Bergen JEAM, Götz HM, Richardus JH, Hoebe CJPA, Broer J, Coenen ATT, for the PILOT CT Study Group. Prevalence of urogenital Chlamydia trachomatis increases significantly with level of urbanisation and suggests targeted screening approaches: Results from the first national population-based study in The Netherlands. Sex Transm Infect 2005; 81:17–23. 22. Götz HM, van Bergen JEAM, Veldhuijzen IK, Broer J, Hoebe CJPA, Richardus JH, for the PILOT CT Study Group. A prediction rule for selective screening of Chlamydia trachomatis infection. Sex Transm Infect 2005; 81:24 –30. 23. Götz HM, Veldhuijzen IK, van Bergen JEAM, Hoebe CJPA, de Zwart O, Richardus JH, for the PILOT CT Study Group. Acceptability and consequences of screening for Chlamydia trachomatis by home based urine testing. Sex Transm Dis. 32:557–562. 24. Spaargaren J, Smit C, Verhaest I, et al. Analysis of Chlamydia trachomatis serovar distribution changes in The Netherlands (1986 – 2002). Sex Transm Infect 2004; 80:151–152. 25. Bloomfield PJ, Steiner KC, Kent CK, Klausner JD. Repeat chlamydia screening by mail, San Francisco. Sex Transm Infect 2003; 79:28 – 30. 26. Sparks R, Helmers JR, Handsfield HH, et al. Rescreening for gonorrhea and chlamydial infection through the mail: A randomized trial. Sex Transm Dis 2004; 31:113–116. 27. Kissinger P, Clayton JL, O’Brien ME, et al. Older partners not associated with recurrence among female teenagers infected with Chlamydia trachomatis. Sex Transm Dis 2002; 29:144 –149. 28. Klausner JD, Chaw JK. Patient-delivered therapy for chlamydia: Putting research into practice. Sex Transm Dis 2003; 30:509 –511. 29. Ostergaard L, Anderson B, Moller JK, et al. Managing partners of people diagnosed with Chlamydia trachomatis: A comparison of two partner testing methods. Sex Transm Infect 2003; 79:358 – 361. 30. Novak DP, Edman AC, Jonsson M, et al. The internet, a simple and convenient tool in Chlamydia trachomatis screening of young people. Eurosurveillance 2003; 8:171–176. 31. Lan J, Ossewaarde JM, Walboomers JM, et al. Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR. J Clin Microbiol 1994; 32:528 –530. 32. Morre SA, Ossewaarde JM, Lan J, et al. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as confirmed by omp1 nucleotide sequence analysis. J Clin Microbiol 1998; 36:345–351.

Appendix Chlamydia trachomatis Serovar Determination Typing was performed by either amplification of the total omp1 gene followed by restriction fragment length polymorphisms (RFLP) or by sequence analysis of the variable segment 2 (VS-2) of the omp1 gene. For RFLP-based serovar typing analysis, the omp1 gene was amplified using the following primers: sero1A (initial sense) ATG AAA AAA CTC TTG AAA TCG G; sero2A-T (initial antisense 1) TTT CTA GAT TTC ATT TTG TT; sero2A-C (initial antisense 2) TTT CTA GAT TTC ATC TTG TT; CTOMPN1 (nested sense) CTT TGA GTT CTG CTT CCT; and CTOMPN2 (nested antisense) AGG AAA CGA TTT GCA TTG T. The PCR mixture, templated by 2 ␮L of total DNA (final volume 25 ␮L) contained 50 mmol/l NaCl, 2 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH8.0), 200 ␮mol/L (each) deoxynucleotide triphosphate (dNTP), 800 pg of each primer, and 0.5 U Taq polymerase (Supertaq, Eurogentec). The PCR reaction was carried out in a thermocycler (PTC-200 Peltier Thermal Cycler; MJ Research Inc., MA) starting with 3 minutes of denaturation at 94°C and continuing for 30 cycles of amplification.

604

VELDHUIJZEN ET AL

Each cycle consisted of denaturation at 93°C for 30 seconds, annealing at 45°C for 30 seconds, and chain elongation at 72°C for 1.30 minutes. The PCR product was checked on agarose gel for its length. Subsequently, 5 ␮L was digested using different restriction enzymes as described previously.31 Serovars and variants were identified by their RFLP patterns after polyacrylamide gel electrophoresis.32 The VS-2 sequencing was used for samples with lowquality DNA of which the 1.1 kB of omp1 could not be amplified

Sexually Transmitted Diseases

●

October 2005

efficiently. VS-2 was amplified in a seminested PCR using the following primers: prime 1 (PCR sequencing) TGT ATA TTY TGT ACA YTR GGA GC, primer 2 (PCR) CRT TYA AYT CTT CRA YTT TAG G, primer 3 (PCR sequencing) TGA GCG TAT TGG AAA GAA GC. Amplification was started with 3 minutes of denaturation at 94°C and continued for 50 cycles (5 seconds at 93°C, 20 seconds at 55°C, 20 seconds at 72°C). Ten microliters of PCR product was used as input for the sequence reaction.