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.
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.
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
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
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.
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.
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.
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.
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.
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).
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).
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.
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