Share this article on:

Molecular Epidemiology of Genital Chlamydial Infection Among Male Patients Attending an STD Clinic in Taipei, Taiwan

Yu, Mei-ching RN, MBA*; Li, Lan-hui MS†; Li, Shu-ying PhD‡; Tang, Li-hui RN‡; Tai, Yu MPH§; Chen, Kow-tong MD, PhD§

Sexually Transmitted Diseases: August 2007 - Volume 34 - Issue 8 - pp 570-573
doi: 10.1097/01.olq.0000253375.59976.24
Article

Objective: To assess the epidemiology of genital Chlamydia trachomatis infection among men attending an STD clinic in Taipei, Taiwan.

Study: Between July 2004 and June 2005, a total of 426 first-void urine specimens from male patients were tested for C. trachomatis by using a urine-based PCR DNA amplification assay.

Results: The overall prevalence of genital C. trachomatis infection was 16.4%. Youth, current symptoms, the presence of N. gonorrhoeae infection, and inconsistent use of condoms were positively associated with C. trachomatis infection. The most common chlamydia strain present was genotype E, followed by D and Da, F, K, J, G, and H.

Conclusions: C. trachomatis genital infection was prevalent among male patients attending a STD clinic in Taipei. Young Taiwanese men attending STD clinics should be counselled on condom use.

C. trachomatis genital infection was prevalent among male patients attending an STD clinic in Taipei. The most common chlamydia strain present was genotype E, followed by D and Da.

From the *Department of Chemical Engineering, Tatung University, Taiwan; the †Taipei City STD Control Center, Taipei, Taiwan; the ‡Mycotic Diseases Laboratory, Center of Laboratory Research and Diagnostics, Center for Disease Control, Taiwan; and the §Department of Public Health, College of Medicine, National Cheng-Kung University, Tainan, Taiwan

The authors are grateful to the staff of the Taipei City STD Control Center, Taiwan, for their assistance with the interviewing of study participants. The authors also thank the Taipei City government and Center for Disease Control, Department of Health, Taiwan, for providing financial support.

Correspondence: Kow-Tong Chen, MD, PhD, Department of Public Health, College of Medicine, National Cheng- Kung University, Tainan, Taiwan. No. 1, University Road, Tainan, Taiwan. E-mail: ktchen@mail.ncku.edu.tw.

Received for publication July 10, 2006, and accepted November 8, 2006.

CHLAMYDIA TRACHOMATIS REMAINS THE most commonly sexually transmitted disease (STD) in the world, with an estimated 89 million individuals affected each year.1 Typing of C. trachomatis strains is an important goal in the field of epidemiology as well as for clinical and basic research on chlamydial infections. In recent years epidemiologic studies of C. trachomatis have been based on the classification of surface antigens.2 One of these is the major outer membrane protein (MOMP), which is the predominant surface protein and the antigen that defines individual C. trachomatis serotypes.3

Characterisation of C. trachomatis strains can provide valuable information regarding the variants circulating in the community. With increased knowledge of the epidemiology of chlamydial infection, efforts to prevent the spread of the disease are likely to be more effective. There is little information on male chlamydial infection in Taiwan. Thus, this study was undertaken with the aim of determining the prevalence and genotypes of C. trachomatis infection in male patients attending a national STD clinic in Taipei, Taiwan.

Back to Top | Article Outline

Materials and Methods

Study Population and Data Collection

Between July 2004 and June 2005, all male attendees at the STD Clinic of the Taipei City STD Control Center were invited to participate in this study, regardless of the reason for their visit. The protocol for this study was approved by the Ethics Committee of the Taipei STD Control Center, Taipei, Taiwan. Participants were asked to complete a questionnaire, which included demographic information, sexual history, and current symptoms (burning urination or urethral discharge or genital skin rash/ulceration). Each participant provided a first void urine specimen that was tested for C. trachomatis and Neisseria gonorrhoeae.

Back to Top | Article Outline

STD Diagnosis

Initial identification of C. trachomatis/N. gonorrhoeae was by the Amplicor PCR method (PCR Roche Diagnostic Systems, Branchburg, NJ), with urine specimens processed according to the manufacturer's instructions.4 An internal control was run for detecting inhibitors.5

Back to Top | Article Outline

DNA Extraction

DNA was extracted from urine samples that were positive after the Amplicor PCR test by use of the QIAamp viral RNA minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and as previously reported.6

Back to Top | Article Outline

PCR

Amplification of an approximately 1130 bp fragment encompassing VS1-4 of ompA was performed using the outer primer pair NLO-NRO,7 and reamplification of a 584 bp fragment spanning VS1-2 was conducted based on the inner primer pair MOMP87-C214 (Table 1).8 The first PCR step was carried out in a final reaction volume of 25 μL, containing 0.4 μmol/L each NLO/NRO outer primer pair, 5μL (50 ng) extracted DNA and 12.5 μL 2 × PCR master mix (MBI, Fermentas). The amplification conditions consisted of initial polymerase activation at 95°C for 5 minutes; 35 cycles of 94°C for 60 seconds, 54°C for 60 seconds, and 72°C for 80 seconds; and a final elongation step at 72°C for 10 minutes. A negative control (water) was included in each run. In the second PCR step, 3 μL product from the first PCR step and 0.4 μmol/L each MOMP87-C214 inner primer pair was added to a final volume of 25 μL. The PCR conditions were: 95°C for 5 minutes; 35 cycles of 94°C for 50 seconds, 56°C for 50 seconds and 72°C for 50 seconds; and a final elongation step at 72°C for 10 minutes. The amplified products were electrophoresed in 2% agarose gel and visualized by staining with ethidium bromide.

Back to Top | Article Outline

DNA Sequencing

The amplified DNA was purified by using the QIAquick PCR Purification Kit (Qiagen), and strands of 584 and 1130 bp fragments of the ompA segment were sequenced using the BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems). The reaction mixture was loaded onto a 3730 Avant genetic analyzer (Applied Biosystems). The primer sets used for sequencing were MOMP87-C214 and NLO-NRO. All the PCR products were sequenced bidirectionally.

Back to Top | Article Outline

Blast Analysis and Alignments

The consensus sequences were compared to sequences of known C. trachomatis strains by using the BLAST search tool at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The sequences were assembled into alignments by using the reference strains A/Sa1 (M58938), B/IU-1226 (AF063208), B16 (AY950630), B/TW-5 (M17342), C/TW3 (M17343), D/B-120 (X62918), Da/TW-448 (X62921), E/Bour (X52557), F/ICCal3 (X52080), G/UW57 (AF063199), H/Wash (H/UW4) (X16007), H/IS1075 (AF414959), J/UW36 (AF063202), J/isolate 6,858 (AY950622), K/UW31 (AF063204), K/isolate 4,011 (AY950621), L1/440 (M36533), L2/434 (M14738), L3/404 (M55700). Multiple alignments were performed with the BioEdit version 7.0 software.

Back to Top | Article Outline

Statistical Analysis

Statistical analysis was conducted using SPSS software for Windows (version 10.0; SPSS). All variables were analyzed using the χ2 test or Fisher exact test for categorical variables, and the Student t test for continuous variables. All variables that revealed significant results in the context of univariate analysis were subsequently entered stepwise into a multiple logistic regression analysis procedure, for which, all measured effects were controlled for by means of controlling the influence of all other variables at time of testing. Significance was defined by an α level of 0.05 or less.

Back to Top | Article Outline

Results

Characteristics of Study Population

Of 498 eligible male patients, 47 (9%) were homosexual, and 473 (95%) had conclusive results. The 426 patients who were both heterosexual and had conclusive results were included in the analyses. The characteristics of the study population are indicated in Table 2. The median age of study patients was 28 years (range from 17 to 50 years); 54% were ≤30 years old; 75% were single; 76% had finished senior high school (>12 years). Condoms were used consistently during sexual contacts by 55%. Forty-eight percent reported experiencing symptoms.

Of the 426 samples, the prevalence of C. trachomatis and N. gonorrhoeae infection were 16.4% (70/426) and 10.3% (44/426), respectively.

Back to Top | Article Outline

Risk Factors for C. trachomatis Infection

In the univariate analysis, youth (P <0.001), occurrence of N. gonorrhoeae infection (P <0.001), inconsistent condom use (P = 0.0004), and experiencing symptoms (P = 0.01) were positively correlated with chlamydial infection. There was no significant difference in the frequency of chlamydial infection when looking at marital status and education level (Table 2).

The multivariate model showed that youth (≤30 years), the occurrence of gonococcal infection, inconsistent condom use, and experiencing symptoms were independently associated with chlamydial infection (Table 3).

Back to Top | Article Outline

Genotyping Analysis

The most common Chlamydia strain present was genotype E (25%), followed by D and Da (22%), F (17%), K (12%), J (11%), G (11%), H (1%), and Ba (0%) (Table 4). In addition, 9% of strains were nontypable.

Of the 70 patients identified as C. trachomatis positive, 41 (59%) had urogenital symptoms at the time of visit. Eighty-one percent (13/16) infected with genotype E had no symptoms, while 26% (14/54) of those with the non-E genotype had no symptoms (P <0.001, Fisher exact test).

Back to Top | Article Outline

Discussion

This study identified chlamydia as the most important cause of STDs in urban Taiwan. The pathogen was detected in urine samples obtained from 70 (16.4%) of the 426 patients enrolled. The most common genotype in descending order of prevalence, were E, D, and Da, F, K, J, G, and H.

Other studies have shown that D, E, and F are the most common genotypes in different parts of the world, accounting for 55%–84% of patients with urogenital infections.9,10 The high prevalence of serotype J was noted in the United States.11 The prevalence of serotypes in Taipei seems more correlated with serotype prevalence in Japan.10

Several studies indicate that identical genotypes or serovars have been associated with asymptomatic or symptomatic infection in different studies.12,13 In the current study, overall, 41% of the C. trachomatis infections seemed to be asymptomatic. However, serotype E was more frequently (81%) associated with asymptomatic infection. These results may suggest that genotype E is less pathogenic.12

The previous study reports that the onset of STD is late in some Asian countries due to the sexual activity beginning after adolescence, with the earliest first sexual intercourse with one's spouse or fiancé, and slow acquisition of income for use in commercial sex.14 However, the results of this study show that the frequency of chlamydial infection has a strong association with youth (less than 30 years old), and is more consistent in developed countries.15

In this study, patients reporting inconsistent condom use had a higher risk of chlamydial infection than those reporting consistent condom use. These results emphasize that consistent condom use could have a significant positive impact in Taiwan, much as it did in other countries.16

Out of the 70 C. trachomatis-positive tests from routine analysis, DNA was not amplified by the ompA PCR in 9%. This is consistent with the previous study that reported failure rates of 1% to 24%.17

However, we acknowledge that our study had several limitations. Firstly, the study population was collected from patients who visited a National STD Clinic, and as such, the study population may not be truly representative of all people aged 17 to 50 years of age in Taipei. Secondly, while our STD clinic probably accurately represents the experiences of other STD clinics in the country, the data derived from this clinic may not appropriately reflect the subpopulation of C. trachomatis-infected Taiwanese patients presenting at other urology hospitals (clinics).

Despite these limitations, the results of this study clearly showed a high prevalence of C. trachomatis genital infection among male patients attending a STD clinic in Taipei. Young Taiwanese men attending STD clinics should be counseled on condom use during intercourse.

Back to Top | Article Outline

References

1. Gerbase AC, Rowley JT, Heymann DH, Berkley SF, Piot P. Global prevalence and incidence estimates of selected curable STDs. Sex Transm Infect 1998;74:s12–s16.
2. Brunham R, Yang C, Maclean I, Kimani J, Maitha G, Plummer F. Chlamydia trachomatis from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the major outer membrane protein (omp1) gene. J Clin Invest 1994;94:458–463.
3. Baehr W, Zhang YX, Joseph T, et al. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc Natl Acad Sci USA 1988;85:4000–4004.
4. Bunnell RE, Dahlberg L, Rolfs R, et al. High prevalence and incidence of sexually transmitted diseases in urban adolescent females despite moderate risk behaviors. J Infect Dis 1999;180:1624–1631.
5. Chernesky MA, Jang D, Sellors J, et al. Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women. Mol Cell Probes 1997;11:243–249.
6. Lysen M, Osterlund A, Rubin CJ, Persson T, Persson I, Herrmann B. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections during 1 year of contact tracing in a Swedish County. J Clin Microbiol 2004;42:1641–1647.
7. Ngandjio A, Clerc M, Fonkoua MC, et al. Restriction endonuclease patterns of the omp1 gene of reference Chlamydia trachomatis strains and characterization of isolates from Cameroonian students. J Med Microbiol 2004;53:47–50.
8. Yuan Y, Zhang YX, Watkins NG, Caldwell HD. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. Infect Immun 1989;57:1040–1049.
9. Fredlund H, Falk L, Jurstrand M, Unemo M. Molecular genetic methods for diagnosis and characterization of Chlamydia trachomatis and Neisseria gonorrhoeae: Impact on epidemiological surveillance and interventions. APMIS 2004;112:771–784.
10. Yamazaki T, Hagiwara T, Kishimoto T, et al. Distribution of Chlamydia trachomatis serovars among female prostitutes and non-prostitutes in Thailand, and non-prostitutes in Japan during the mid-90s. Jpn J Infect Dis 2005;58:211–213.
11. Gaydos CA, Bobo L, Welsh L, Hook EW III, Viscidi R, Quinn TC. Gene typing of Chlamydia trachomatis by polymerase chain reaction and restriction endonuclease digestion. Sex Transm Dis 1992;19:303–308.
12. van de Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med 1996;72:261–265.
13. Geisler WM, Suchland RJ, Whittington WL, Stamm WE. The relationship of serovar to clinical manifestations of urogenital Chlamydia trachomatis infection. Sex Transm Dis 2003;30:160–165.
14. Parish WL, Laumann EO, Cohen MS, et al. Population-based study of chlamydial infection in China—A hidden epidemic. JAMA 2003;289:1265–1273.
15. Groseclose SL, Zaidi AA, Delisle SJ, Levine WC, St Louis ME. Estimated incidence and prevalence of genital Chlamydia trachomatis infections in the United States, 1996. Sex Transm Dis 1999;26:339–344.
16. Warner L, Newman DR, Austin HD, et al. Condom effectiveness for reducing transmission of gonorrhea and Chlamydia: The importance of assessing partner infection status. Am J Epidemiol 2004;159:242–251.
17. Jonsdottir K, Kristjansson M, Hjaltalin OJ, Steingrimsson O. The molecular epidemiology of genital Chlamydia trachomatis in the greater Reykjavik area, Icelancd. Sex Transm Dis 2003;30:249–256.
© Copyright 2007 American Sexually Transmitted Diseases Association