chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , and trichomonas vaginalis are known pathogens in sexually transmitted infection ( sti ) . however , most of those species present atypical symptoms and some patients have no symptoms [ 1 - 3 ] .
therefore , physicians sometimes have difficulty in making a differential diagnosis . moreover , except for chlamydia trachomatis and mycoplasma genitalium , the treatment regimens between the species differ .
in addition , routine bacterial culture may give negatives result for commercial sexual workers or asymptomatic people who recently experienced unprotected sexual contact and have acquired an sti [ 1 - 3 ] .
thus , there are neglected asymptomatic patients in the community who can serve as a reservoir of the sti [ 1 - 4 ] .
generally , ureaplasma urealyticum is not known as a clinical pathogen but it can be a cause of urethritis , especially in patients resistant to routine treatment .
mycoplasma hominis also does not play a great role in the pathogenesis of urethritis , but it can be a heavy pathogen in immunocompromised patients .
recently , many reports have revealed that microorganisms such as ureaplasma urealyticum and mycoplasma hominis can contribute not only to lower genitourinary infection but also to infertility . therefore ,
identification of the incidence of asymptomatic sti such as urethritis or cervicitis and determination of the prevalence of these 6 species in asymptomatic people is very important .
accordingly , we conducted a screening test for sti by using multiplex polymerase chain reaction ( pcr ) in asymptomatic people .
the institutional review board of the catholic university of korea , college of medicine , approved the study protocol , and all patients provided written informed consent to participate in the study ( irb no : sc10snsi0091 ) . from july 2010 to december 2010 , 802 persons who came 
 to the hospital for a general checkup participated voluntarily and a brief questionnaire was obtained . sexually active , asymptomatic people aged between 20 and 60 years were enrolled .
we defined ' sexually active ' as having had sexual intercourse in the preceding 3 months .
people with genitourinary symptoms such as urethral or cervical discharge , dysuria , or itching in the genital area were also excluded .
first - voided urine specimens were collected in sterile 50 ml screw - cap plastic bottles . in women ,
the specimens from the 709 men and women were equilibrated to room temperature and centrifuged at 5000xg for 15 minutes .
the supernatant was discarded , and the pellet was resuspended in 1 ml 1xpbs before dna extraction .
genomic dna was extracted from the pretreated specimens ( swab or urine ) by using the qiaamp dna mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
pcr amplification was performed with the seeplex std6 ace detection kit ( seegene , seoul , korea ) according to the 
 manufacturer 's instructions .
the kit contains sets of primers that were specifically designed from highly conserved regions of genetic sequences for the 6 organisms ( chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis ) by use of seegene dpo technology .
an internal control was included in the pcr mix to detect the presence of pcr inhibitors .
for the negative control , sterile deionized water was used as the pcr template instead of nucleic acid . the positive control contained in the kit
amplified fragments were separated by use of the labchip dx seeplex assay system(caliper , hopkinton , ma , usa ) .
amplified pcr products were separated and detected by automated gel electrophoresis with the labchip dx seeplex assay system ( caliper , hopkinton , ma , usa ) .
the pcr products from each sample ( 20 l ) were transferred to 96-well plates and placed in the labchip dx instrument .
the samples were loaded automatically on the seeplex chip and detected sequentially according to the sample order .
analysis was performed with designated software ( seegene viewer ) that presents each of the samples and identifies the fragments that yield a positive readout for the bands of interest in the presented results . a tabulated matching matrix provides a simple readout , identifying matching bands to the type of sti pathogens .
from july 2010 to december 2010 , 802 persons who came 
 to the hospital for a general checkup participated voluntarily and a brief questionnaire was obtained . sexually active , asymptomatic people aged between 20 and 60 years were enrolled .
we defined ' sexually active ' as having had sexual intercourse in the preceding 3 months .
people with genitourinary symptoms such as urethral or cervical discharge , dysuria , or itching in the genital area were also excluded .
first - voided urine specimens were collected in sterile 50 ml screw - cap plastic bottles . in women
the specimens from the 709 men and women were equilibrated to room temperature and centrifuged at 5000xg for 15 minutes . the supernatant was discarded , and the pellet was resuspended in 1 ml 1xpbs before dna extraction .
genomic dna was extracted from the pretreated specimens ( swab or urine ) by using the qiaamp dna mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
pcr amplification was performed with the seeplex std6 ace detection kit ( seegene , seoul , korea ) according to the 
 manufacturer 's instructions .
the kit contains sets of primers that were specifically designed from highly conserved regions of genetic sequences for the 6 organisms ( chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis ) by use of seegene dpo technology .
an internal control was included in the pcr mix to detect the presence of pcr inhibitors .
for the negative control , sterile deionized water was used as the pcr template instead of nucleic acid . the positive control contained in the kit
amplified fragments were separated by use of the labchip dx seeplex assay system(caliper , hopkinton , ma , usa ) .
amplified pcr products were separated and detected by automated gel electrophoresis with the labchip dx seeplex assay system ( caliper , hopkinton , ma , usa ) .
the pcr products from each sample ( 20 l ) were transferred to 96-well plates and placed in the labchip dx instrument .
the samples were loaded automatically on the seeplex chip and detected sequentially according to the sample order .
analysis was performed with designated software ( seegene viewer ) that presents each of the samples and identifies the fragments that yield a positive readout for the bands of interest in the presented results . a tabulated matching matrix provides a simple readout , identifying matching bands to the type of sti pathogens .
the mean age in this study was 45.48.1 years . among the 709 persons , 229 ( 32.3% ) had at least one microorganism in his or her genitourinary tract .
the prevalences of chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis in asymptomatic people were 5.6% ( 40/709 ) , 0.4% ( 3/709 ) , 0.3% ( 2/709 ) , 22.1% ( 157/709 ) , 11.6% ( 82/709 ) , and 1.1% ( 8/709 ) respectively(table 1 ) . among all persons ,
50 ( 7.1% ) had pathogens of sti in their urinary tract ( table 2 ) .
of these patients , the number of patients with a single pathogen was 47 ( 6.6% ) , for which the number with isolated chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , or trichomonas vaginalis was 37 , 1 , 1 , and 8 , respectively .
three double - infections , i.e. , samples containing 2 or more pathogens , were identified , and each sample had chlamydia trachomatis . when trichomonas vaginalis was identified , it was either isolated alone or co - infected with mycoplasma hominis only and no other species .
the total number of people who had at least two microorganisms in their genitourinary tract was 55 ( 7.5% ) . among these 55 persons ,
48 ( 6.8% ) had two different species whether they were pathogens or not , 6 ( 0.8% ) persons had three different species , and 1 patient had four different species .
the prevalences of ureaplasma urealyticum and mycoplasma hominis were 22.1% and 11.6% , respectively . in people who were infected with chlamydia trachomatis , the prevalences of ureaplasma urealyticum ( 35.0% ) and mycoplasma hominis ( 22.5% ) were higher than in those not infected with chlamydia trachomatis ( table 1 , 2 ) .
chlamydia trachomatis infection is the most prevalent sexually transmitted bacterial infection among women and men worldwide .
chlamydia trachomatis is usually described as an obligate intracellular pathogen and accounts for 30% to 40% of the etiopathogenesis of urethritis . however , about half of infected subjects are asymptomatic . thus , there are many silent infections in the community . mason et al showed that the prevalence of chlamydia trachomatis in asymptomatic men was 4% ( 14/349 ) .
takahashi et al reported that chlamydia trachomatis was detected in 6% of samples from healthy people .
similarly , in our study , chlamydia trachomatis was observed in 40 persons ( 5.6% ) .
thus , most asymptomatic patients are neglected unless they visit the clinic to be evaluated for sti by chance or face complications such as pelvic inflammatory disease .
neisseria are fastidious gram - negative cocci that require nutrient supplementation to grow in laboratory cultures . although gonorrhea can be diagnosed by inspection of the yellowish discharge from the urethra , many patients infected with neisseria gonorrhoeae have no discharge .
furthermore , it is known that 10% of infected males and 50% of infected females are asymptomatic .
hein et al studied 2672 sexually active adolescents and reported that prevalence rates of asymptomatic gonorrhea were 1.9% among boys and 7.0% among female adolescents .
regardless of age group , a surveillance study reported prevalence rates of gonorrhea in healthy people of 0.06% to 0.18% . in our study , the prevalence in asymptomatic people was 0.4% , and the result was easily obtained by pcr test with a urine or endocervical swab sample .
mycoplasma genitalium was first isolated in 1981 from two men with nongonococcal urethritis ( ngu ) .
results from a meta - analysis of 19 studies on patients with ngu showed that mycoplasma genitalium was found in 21% compared with 7% of patients without ngu .
ross et al reported a detection rate of 0.6% for mycoplasma genitalium in asymptomatic british persons .
currently , nucleic acid amplification tests ( naats ) are usually recommended to detect this pathogen .
wikstrm and jensen reported that mycoplasma genitalium is a common cause of persistent or recurrent urethritis among men treated with doxycycline , and erythromycin appears to be less efficient than azithromycin in eradicating the infection .
therefore , when physicians encounter patients who complain of continuous symptoms of urethritis , they should consider drug - refractory urethritis such as that caused by mycoplasma genitalium .
it is another pathogen of sti of the urogenital tract , and men with this infection rarely exhibit symptoms . in women , greenish - yellow frothy vaginal discharge and itching
trichomoniasis is treated and cured with metronidazole or tinidazole , which should be prescribed to any sexual partners as well because they may be asymptomatic carriers .
sutton et al reported that the prevalence of trichomonas vaginalis in reproductive - aged women was 3.1% . in our study , the total prevalence rate was 1.1% in asymptomatic people , and prevalence rates in asymptomatic men and women were 0.2% and 2.5% , respectively . screening for these pathogens is important not only to identify infected symptomatic individuals for the diagnosis and management of their infections but also to identify asymptomatic individuals who serve as reservoirs for infection [ 1 - 4 ] . in our study , there were 50 ( 7.1% ) infected people among 709 asymptomatic people ; thus , this proportion should not be neglected . among the 50 infected patients , 37 with chlamydia trachomatis , 1 with neisseria gonorrhoeae , 1 with mycoplasma genitalium , 8 with trichomonas vaginalis , 2 with chlamydia trachomatis with neisseria gonorrhoeae , and 1 with chlamydia trachomatis with mycoplasma genitalium were confirmed .
thus , regardless of chlamydia trachomatis , 13 patients should be considered as candidates for antimicrobial treatment .
these facts suggest the possibility of prescribing the wrong medicines if practitioners give symptomatic patients some regimens empirically . therefore , the need to detect multiple species at once should be raised . in this sense , use of the multiplex pcr assay to screen asymptomatic people may be important because the treatment regimen could be optimized for people with positive results , especially for those with multiple organisms .
however , ureaplasma urealyticum is frequently isolated from the urethra of healthy men , and some studies have reported that there is no significant difference in its prevalence between men with ngu and men without ngu .
nevertheless , there are some reports that ureaplasma urealyticum serves as a cause of persistent ngu . for that reason , ureaplasma urealyticum
therefore , ureaplasma infection must be considered in patients with treatment failure for ngu or patients with multiple sexual partners .
if no species except ureaplasma are detected , symptoms and leukocyte numbers in first voided urine samples are helpful for clarifying the diagnosis of ureaplasma infection .
mycoplasma hominis is frequently identified from the genitourinary tract . in general , it is known as a commensal species but it can work as a pathogen in special conditions such as in an immunocompromised state . generally , the rate of colonization of mycoplasma hominis in the urogenital tract was reported to be between 4% and 13% in men and between 21% an 54% in women .
our study showed that the incidence of this species was 9.1% in men and 15.4% in women . in our study , the incidences of ureaplasma urealyticum and mycoplasma hominis were 22.1% and 11.6% .
nevertheless , those incidences were increased up to 35.0% and 22.5% when the samples had chlamydia trachomatis .
the reason for this increase with chlamydial infection is not clear , and a limitation of our study is that we did not include people who had symptoms .
therefore , comparison of the prevalence of these species in symptomatic urethritis patients is necessary . as described previously , most pathogens causing sti as well as commensal microorganisms are difficult to cultivate by routine microbiological diagnosis .
however , naats , such as pcr , are useful for the identification of microorganisms that are difficult to cultivate and for those that grow slowly . in another recent study in korea , the multiplex pcr kit ( seegene inc . , seoul , korea )
was used to detect causative microorganisms of sti in patients with chronic prostatitis and vaginitis . in multiplex pcr , more than one target sequence can be amplified by including more than one pair of primers in the reaction .
multiplex pcr has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility .
furthermore , when the clinical sample amount is limited , multiplexing allows more targets to be analyzed by using a single aliquot of sample material .
although there are some worries that multiplex pcr in the clinical setting may have difficulties such as poor sensitivity or specificity , false - negative results , and nonspecific interactions , horii et al showed that the multiplex pcr assay had an overall sensitivity of 96% and specificity of 100% compared with uniplex pcr assays . furthermore , there was no cross - reaction with other microorganisms .
the multiplex pcr assay has recently made it convenient for clinicians to test for causative organisms simultaneously from many clinical fields .
similarly , we succeeded in easily generating results by use of multiplex pcr with only a single sample per person .
asymptomatic individuals play a role as reservoirs for sti . in our study , there were 50 ( 7.1% ) infected people among 709 asymptomatic people ; therefore , this proportion should not be neglected .
an sti screening test should be considered as a part of a general checkup , especially in high - risk groups , e.g. , commercial sexual workers , people who have multiple sexual partners , and immunocompromised persons [ 1 - 3,6,25 ] . by use of multiplex pcr
, physicians can easily identify many microorganisms at once , and treating asymptomatic persons contributes to the prevention of sti in the community .
the prevalences of chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis in asymptomatic people were 5.6% , 0.4% , 0.3% , 22.1% , 11.6% , and 1.1% , respectively .
we hope that these results will be used as baseline data for future studies . in our study , about 32.3% of asymptomatic people had at least one microorganism , and excluding ureaplasma urealyticum and mycoplasma hominis from the count , the prevalence rate of silent sti was 7.1% with multiplex pcr
therefore , screening for sti should be considered as a part of a general checkup , especially in high - risk groups .
 purposeour purpose was to conduct a screening test for urethritis or cervicitis as a sexually transmitted disease ( std ) by using multiplex polymerase chain reaction(pcr ) and to determine the prevalence of chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis in asymptomatic people.materials and methodsfrom july 2010 to december 2010 , 709 persons who came to the hospital for a general checkup were tested . 
 multiplex pcr assays were done with first voided urine samples or endocervical swabs by use of the seeplex std6 ace detection kit.resultsthe mean age in this study was 45.48.1 years . among the 709 persons , 229 ( 32.3% ) 
 had a positive result for at least one microorganism , 48 ( 6.8% ) had two different species , 6 ( 0.8% ) had three different species , and 1 person had four different species . 
 the overall prevalence of asymptomatic stds such as urethritis or cervicitis was 7.1% ( 50/709 ) . 
 the prevalence rates of chlamydia trachomatis , neisseria gonorrhoeae , mycoplasma genitalium , ureaplasma urealyticum , mycoplasma hominis , and trichomonas vaginalis infection in asymptomatic persons were 5.6% ( 40/709 ) , 0.4% ( 3/709 ) , 0.3% ( 2/709 ) , 22.1% ( 157/709 ) , 11.6% ( 82/709 ) , and 1.1% ( 8/709 ) , respectively.conclusionswith only a single sample , we could identify the prevalence rates of six microorganisms and the overall proportion of urethritis or cervicitis in asymptomatic people . 
 this proportion can not be neglected ; therefore , screening tests for sexually transmitted diseases such as urethritis or   ervicitis should be recommended to asymptomatic people . 