{"paper_id":"d48aabb2-ec6c-48a6-968d-b0e71daca497","body_text":"Chlamydia trachomatis  is a common bacterial sexually transmitted disease (STD) worldwide. Chlamydial urogenital infection has increased at a fast rate, with more than 2.8 million new cases diagnosed each year [ 1 ]. In Malaysia, a high prevalence of chlamydial antibody was detected among urban citizens [ 2 ], particularly young females and sex workers [ 3 ]. Long term asymptomatic persistency of the pathogen among 50–70 % of individuals results in wide spread of the disease [ 4 ], and leads to delayed treatment, which in turn causes prostatitis and epididymitis in males or infertility in females [ 5 ]. In females, the pathogen can ascend the reproductive tract to the endometrial epithelium and fallopian tubes, leading to pelvic inflammatory disease (PID) in approximately 20–40 % of the infected patients; among which 11 % develop tubal factor infertility while 9 % show ectopic pregnancy [ 6 ].  C. trachomatis -mediated inflammation in the genital tract can result in cervicitis or endometriosis, causing lesions, abnormal mucopurulent discharge and thickening of the uterus inner layer [ 6 ]. Inflammation can also contribute to hormonal disorders, i.e., polycystic ovarian syndrome (PCOS) and irregular menses [ 7 ]. Additionally, newborn infants can be infected at birth when delivered through an infected birth canal, causing severe conjunctivitis or pneumonia [ 8 ]. Fig. 1 Plasmid copy numbers among  C. trachomatis -infected patients with different clinical parameters. Dot plot graphs show the ratio of plasmid: Momp  as determined by quantitative real-time PCR assay. Each dot represents data from a single patient in each group ( n  = 92). The  P  values were measured with unpaired Student’s  t -test. Data was considered significant when * P  < 0.05.  n.s. : non-significant\nPlasmid copy numbers among  C. trachomatis -infected patients with different clinical parameters. Dot plot graphs show the ratio of plasmid: Momp  as determined by quantitative real-time PCR assay. Each dot represents data from a single patient in each group ( n  = 92). The  P  values were measured with unpaired Student’s  t -test. Data was considered significant when * P  < 0.05.  n.s. : non-significant\nIn recent years, several research groups suggest that the  C. trachomatis  without plasmid demonstrates a weaker degree of virulence. The plasmid-deficient strains show impaired ability to infect the female mouse genital tract [ 9 ,  10 ].  C. trachomatis  infection damages the reproductive tract by activating TLR2 and initiates a Th1-dependent inflammatory response. In a study using plasmid-cured  C. muridarum  mutant infection in a murine model, TLR2-dependent cytokine was not detected and no sign of oviduct damage can be observed, suggesting that the plasmid-cured mutant has lost virulence [ 9 ]. Also, intra-vaginal immunization of the plasmid-deficient strains provide protection in the murine model of genitourinary tract infection [ 11 ]; as well as in trachoma infected macaques [ 12 ] due to the failure to trigger a Toll-like receptor 2-dependent immune response [ 13 ]. These findings have led to research on use of plasmid-deficient strains as a potential human vaccine. In fact, an intravaginal infection of attenuated plasmidless  C. trachomatis  L2 has been shown to provide protection to the host because it is nonpathogenic and raises systemic antibody in the C3H/HeJ mouse model [ 11 ].\nDespite compelling evidence in animal models, there is no equivalent finding from human clinical study confirming the significance of plasmid in  C. trachomatis  infection. A recent study in human ocular  C. trachomatis  infection reveals that no direct association exists between plasmid copy numbers and disease severity [ 14 ]. Therefore, further work should be done to establish the plausible connection. In this study, we investigated the prevalence of plasmid-bearing (+) or plasmid-free (−)  C. trachomatis  infection and the plasmid copy numbers among obstetrics and gynecology patients in Kuala Lumpur, the capital city of Malaysia. By associating the data with the patients’ clinical presentations, we evaluated the prevalence and risk of plasmid (+) and plasmid (−) variants in affecting gynecological disorders.\n\nThe patient cohort from this study comprised 180 female patients of child-bearing age (mean: 30.95 years old; IQR: 27–35) who visited the Obstetrics and Gynecology clinic at the University of Malaya Medical Centerfrom 2010 to 2014 (Table  1 ). A total number of 124 patients were married, 13 were single or had divorced, and the remaining 43 patients did not reveal their marital status. Most of the patients were of Malay ethnicity (68.3 %), followed by Indians (13.3 %), Chinese (12.7 %), and others (5.5 %), reflecting the demographics of the multiethnic Malaysian population. A majority of the patients recruited to the study presented with gynecological complications or subfertility issues, and had a higher risk of STD infection. The main reason for patients’ visit to the clinic was due to infertility (37 %). Other reasons included abdominal pain, bleeding, menorrhagia, irregular menstrual cycles and others. Two patients had cystectomy whereas one had a history of resolved PID. Table 1 Patient demographics. Numbers (percentages) of patients with or without  C. trachomatis  infection; and patients infected with plasmid (−) or plasmid (+) variants.  n  = 180. The  P  values for all variables were measured with Fisher’s exact test. For age, a  t -test ( a ) was used.  n.s. : non-significant Parameters All patients ( n  = 180) No infection ( n  = 88) \n C. trachomatis  infection ( n  = 92) \n P  value OR (95 % CI) Plasmid (−) \n C. trachomatis \n ( n  = 6) Plasmid (+) \n C. trachomatis \n ( n  = 86) \n P  value OR (95 % CI) Age (years)  Mean [IQR] 30.9 [27–35] 30.7 [27–34.25] 31.29 [27–35] 0.60  n.s. \n a \n 30.2 [26.75–32.25] 33.3 [27–35] 0.59  n.s. \n a \n  Maximum 45 44 45 36 45  Minimum 20 20 20 26 20 Marital status  Married 124 (68.8 %) 55 (44.4 %) 69 (55.6 %) 0.078  n.s. \n 1.800 (0.94–3.41) 3 (4.3 %) 66 (95.6 %) 0.16  n.s. \n 0.303 (0.056–1.62)  Single 12 (6.7 %) 6 (50 %) 6 (50 %) 1.00  n.s. \n 0.953 (0.29–3.07) 0 (0 %) 6 (100 %) 1.00  n.s. \n 0.952 (0.048–18.87)  Divorced 1 (0.5 %) 0 (0 %) 1 (100 %) 1.00  n.s. \n 2.902 (0.11–72.24) 0 (0 %) 1 (100 %) 1.00  n.s. \n 4.385 (0.16–118.8)  unknown 43 (23.8 %) 27 (62.7 %) 16 (37.3 %) 0.053  n.s. \n 0.475 (0.23–0.96) 3 (18.7 %) 13 (81.2 %) 0.60  n.s. \n 5.615 (1.02–3.09) Ethnicity  Malay 123 (68.3 %) 64 (52.1 %) 59 (47.9 %) 0.26  n.s. \n 0.6705 (0.35–1.26) 4 (6.7 %) 55 (93.2 %) 1.00  n.s. \n 1.127 (0.19–6.51)  Indian 24 (13.3 %) 11 (45.9 %) 13 (54.1 %) 0.82  n.s. \n 1.152 (0.48–2.72) 1 (7.6 %) 12 (92.3 %) 1.00  n.s. \n 1.233 (0.13–11.50)  Chinese 23 (12.7 %) 10 (43.5 %) 13 (56.5 %) 0.65  n.s. \n 1.284 (0.53–3.10) 1 (7.6 %) 12 (92.3 %) 1.00  n.s. \n 1.233 (0.13–11.50)  Others 10 (5.5 %) 3 (30 %) 7 (70 %) 0.33  n.s. \n 2.333 (0.58 to 9.32) 0 (0.0 %) 7 (100.0 %) 1.00  n.s. \n 0.815 (0.13–11.50)\nPatient demographics. Numbers (percentages) of patients with or without  C. trachomatis  infection; and patients infected with plasmid (−) or plasmid (+) variants.  n  = 180. The  P  values for all variables were measured with Fisher’s exact test. For age, a  t -test ( a ) was used.  n.s. : non-significant\nGenital  C. trachomatis  infection in each patient was first diagnosed by detection of the  C. trachomatis Momp  ( OmpA ) gene. Total DNA was first isolated from the patients’ endocervical swabs and PCR amplified using a human β-globin control primer to ensure the successful extraction of DNA from the endocervical swabs (data not shown). The presence of  C. trachomatis  DNA was detected with  Momp  primer using a nested PCR amplification method (outer primers followed by inner primer amplification) [ 15 ]. The results were further confirmed using a quantitative real-time PCR method with inner primer alone. MOMP is expressed on the cell surface with a porin activity that is often used for diagnosis of  C. trachomatis  [ 16 ]. Over half of the patients, i.e., 51.1 % (92/180), were diagnosed with genital  C. trachomatis  infection, suggesting a high incidence of sexually transmitted chlamydial infection among the female patients recruited (Table  2 ). No significant difference was detected among patients by age, marital status or ethnic background. We then sequenced the  Momp  PCR product and performed a nucleotide BLAST to assess the diversity of  C. trachomatis  strains in the local population. Interestingly, our data revealed that all samples examined showed a D serotype (data not shown). Table 2 Chlamydial infection in patients with different symptoms. Numbers (percentages) of patients with or without  C. trachomatis  infection.  n  = 180. The  P  values for all variables were measured with Fisher’s exact test . n.s. : non-significant Parameters All patients ( n  = 180) Non-infected ( n  = 88) \n C. trachomatis \n -infected ( n  = 92) \n P  value Odd Ratio (95 % CI) Fertility  Fertile 112 76 (67.9 %) 36 (32.1 %)  Infertile 68 12 (17.6 %) 56 (82.4 %) <0.0001 *** \n 9.852 (4.70 to 20.63)   - 1° or 2° infertility 43 4 (9.3 %) 39 (90.7 %) < 0.0001 *** \n 20.580 (6.83 to 62.02)   - Miscarriage 25 8 (32.0 %) 17 (68.0 %) 0.0013 ** \n 4.486 (1.77 to 11.36) Reproductive tract  Normal lining 143 79 (55.2 %) 64 (44.8 %)  Inflammation 37 9 (24.3 %) 28 (75.7 %) 0.00088 *** \n 3.84 (1.69 to 8.72)   - Mucopurulent Cervicitis 25 9 (36.0 %) 16 (64.0 %) 0.0876  n.s. \n 2.167 (0.90 to 5.23)   - Endometriosis 12 0 (0.0 %) 12 (100.0 %) 0.0001 *** \n 30.43 (1.77 to 524.2) Menstrual Cycle  Regular 130 70 (53.8 %) 60 (46.2 %)  Irregular 50 18 (36.0 %) 32 (64.0 %) 0.0452 * \n 2.074 (1.05 to 4.06) Hormonal Disorder  Undiagnosed 170 87 (51.2 %) 83 (48.8 %)  PCOS 10 1 (10.0 %) 9 (90.0 %) 0.0184 * \n 9.434 (1.17 to 76.14) * P <0.05 ** P <0.01 *** P <0.001\nChlamydial infection in patients with different symptoms. Numbers (percentages) of patients with or without  C. trachomatis  infection.  n  = 180. The  P  values for all variables were measured with Fisher’s exact test . n.s. : non-significant\n* P <0.05 ** P <0.01 *** P <0.001\nOverall,  C. trachomatis  infection showed an association with infertility (OR:9.85, 95 % CI:4.70–20.63,  P  < 0.0001 *** ) (Table  2 ). Among patients who were fertile, only 32.1 % (36/112) were infected by  C. trachomatis , whereas among infertile patients, 82.4 % (56/68) had  C. trachomatis  infection. Notably, a high  C. trachomatis  prevalence of 90.7 % (39/43,) was detected among patients who were diagnosed with primary (1°) or secondary (2°) infertility (OR:20.580, 95 % CI:6.83–62.02,  P  < 0.0001 *** ). Meanwhile, 68 % (17/25) of patients who experienced miscarriage were infected by  C. trachomatis  (OR:4.486, 95 % CI:1.77–11.36,  P  = 0.0013 ** ).\nThe inflammation (cervicitis with mucopurulent discharge or endometriosis) of patients’ reproductive tract was recorded by clinicians during examination.  C. trachomatis  infection showed a close connection with the inflammation in the reproductive system (OR:3.84, 95 % CI:1.69–8.72,  P  = 0.0008 *** ) (Table  2 ). Our data showed that approximately 64 % (16/25) of patients who were diagnosed with mucopurulent cervicitis (OR:2.167, 95 % CI:0.90–5.23,  P  = 0.087) and 100 % (12/12) of patients with endometriosis (OR:30.43, 95 % CI:1.77–524.2,  P  < 0.0001 *** ) had  C. trachomatis  infection.\nIn addition, we showed that  C. trachomatis  infection exhibited significant association with irregular menstrual cycles (OR:2.07, 95 % CI:1.05–4.06,  P  = 0.045 * ) as well as PCOS (OR:9.43, 95 % CI:1.17–76.14,  P  = 0.018 * ), when compared to the uninfected controls.\nNext, we investigated if the patients enrolled were infected by plasmid (+) or (−)  C. trachomatis  variants. Total DNA extracted from the vaginal swabs of the  C. trachomatis -infected patients were amplified with two sets of cryptic plasmid primers (which target  Pgp1  and  Pgp8  respectively) using quantitative real-time PCR analysis. Of the 92  C. trachomatis- infected patients, we detected that 93.5 % (86/92) were caused by plasmid (+)  C. trachomatis  whereas only 6.5 % (6/92) were caused by plasmid (−)  C. trachomatis  (Table  1 ).\nWhen comparing the clinical parameters of the patients, we noted no significant correlation between the plasmid and the tendency for the patients to exhibit infertility (OR:0.76, 95 % CI:0.13–4.40,  P  = 0.562), inflammation (OR:5.26, 95 % CI:0.28–98.68,  P  = 0.155), irregular menses (OR:2.81, 95 % CI:0.31–25.24,  P  = 0.315) and PCOS (OR:1.59, 95 % CI:0.08–30.6,  P  = 0.529) (Table  3 ). Among the patients with infertility, 92.8 % (52/56) cases were diagnosed with plasmid (+)  C. trachomatis , compared to only 7.14 % (4/56) cases with plasmid (−)  C. trachomatis . Similarly, the majority of the patients who had inflammation in the reproductive tract (96.4 %, 27/28) and PCOS (100 %, 9/9) were caused by plasmid (+)  C. trachomatis . Among the 6 patients infected by plasmid (−)  C. trachomatis , 2 were fertile while 4 had infertility. However, 5 among the 6 showed no sign of inflammation and had regular menstrual cycles. Table 3 \n C. trachomatis  plasmid in patients with different symptoms. Numbers (percentages) of patients infected with plasmid (−) or plasmid (+)  C. trachomatis  variants.  n  = 92. The  P  values for all variables were measured with Fisher’s exact test.  n.s. : non-significant Parameters \n C. trachomatis- infected patients ( n  = 92) Plasmid (−)  C. trachomatis  ( n  = 6) Plasmid (+)  C. trachomatis  ( n  = 86) \n P  value Odd Ratio (95 % CI) Fertility  Fertile 36 2 (5.55 %) 34 (94.4 %)  Infertile 56 4 (7.14 %) 52 (92.8 %) 0.5629  n.s. \n 0.764 (0.13 to 4.40)   - 1° or 2° infertility 39 3 (7.6 %) 36 (92.3 %) 0.5295  n.s. \n 0.755 (0.14 to 3.95)   - Miscarriage 17 1 (5.8 %) 16 (88.2 %) 0.6939  n.s. \n 0.875 (0.09 to 8.01) Reproductive tract  Normal lining 64 5 (7.8 %) 59 (92.1 %)  Inflammation 28 1 (3.5 %) 27 (96.4 %) 0.4045  n.s. \n 2.288 (0.25 to 20.55)   - Mucopurulent Cervicitis 16 0 (0.0 %) 16 (100 %) 0.1550  n.s. \n 5.269 (0.28 to 98.68)   - Endometriosis 12 1 (8.3 %) 11 (91.6 %) 0.5786  n.s. \n 0.733 (0.078 to 6.88) Menstrual Cycle  Regular 60 5 (8.3 %) 55 (91.6 %)  Irregular 32 1 (3.1 %) 31 (96.8 %) 0.3153  n.s. \n 2.818 (0.31 to 25.24) Hormonal Disorder  Undiagnosed 83 6 (72.2 %) 77 (92.7 %)  PCOS 9 0 (0 %) 9 (100 %) 0.5293  n.s. \n 1.59 (0.083 to 30.6)\nC. trachomatis  plasmid in patients with different symptoms. Numbers (percentages) of patients infected with plasmid (−) or plasmid (+)  C. trachomatis  variants.  n  = 92. The  P  values for all variables were measured with Fisher’s exact test.  n.s. : non-significant\nTo examine if the abundance of plasmid in  C. trachomatis  is associated with the presence of the clinical symptoms, we quantified the plasmid copy numbers in each patient (Figure  1 ). The average C T  value from two cryptic plasmid primers ( Pgp1  and  Pgp8 ) for each patient was obtained. Relative plasmid copy numbers per bacterium (ratio for plasmid: Momp ) for all 92  C. trachomatis -infected patients were calculated. In general, we noticed no direct connection between fertility, the reproductive tract lining and menstrual cycle, consistent with the previous studies in human ocular infection and  in vitro  tissue tropism [ 14 ,  17 ]. Notably, PCOS patients showed a higher plasmid copy number (2.386 ± 0.284 versus 1.734 ± 0.096,  P  = 0.0472), relative to the non-PCOS patients. Although endometriosis patients also demonstrated a comparatively higher plasmid copy number, (2.193 ± 0.308 versus 1.714 ± 0.110,  P  = 0.114), no statistical significance was noted.\n\nIn this study, we reported a high prevalence (51.1 %) of  C. trachomatis  infection among female adults of child bearing age, with subfertility or gynecological problems who visited Obstetrics and Gynecology clinics in Malaysia. This indicates that there is a pressing need for wider population screening to increase awareness and prevent the spread of the disease among the community. Most of the patients who demonstrated symptoms were diagnosed with genital  C. trachomatis  infection, including infertility (82.4 %), reproductive system lesions (75.7 %), irregular menses (64 %) and PCOS (90 %), suggesting the  C. trachomatis  is a leading factor for female reproductive system disorders.\nAlthough a high rate of genital  C. trachomatis  prevalance was detected in our study, the patients recruited were suspected to be at risk of the bacterial infection based on the clinical examination. The rates of  C. trachomatis  infection vary in different studies depending on the group of patients recruited and the study region. For example, a study using 50 infertile female patients showed a 40 % infection rate by  C. trachomatis  [ 18 ] whereas other studies reported only a 8 % [ 19 ] or 15 %  C. trachomatis  infection rate. Among patients with tubal infertility, the prevalence of  C. trachomatis  was reported to be 38.3 % among 120 patients [ 20 ]. Consistent to the previous study, the presence of plasmid (+)  C. trachomatis  was high (93.5 %). However, the prevalence using real-time PCR amplification depends highly on the assay sensitivity. For data validation, a glycogen-positive test for bacterial isolates can be used to confirm the presence of plasmid in the bacteria [ 21 ].\nVarious research groups have established that the plasmid (+)  C. trachomatis  demonstrated a higher virulence in animal models of ocular or genital infections [ 9 – 14 ]. Consistent with these findings, our results showed that high percentages of those who had infertility (92.8 %), inflammation in the reproductive tract (96.4 %), irregular menses (96.8 %) and PCOS (100 %) were diagnosed with plasmid (+) but not plasmid (−) variants of  C. trachomatis . Although these observations provide support for previous work which suggests the usage of plasmidless  C. trachomatis  as a potential human vaccine [ 11 ], caution must be taken as some of the patients who showed symptoms were infected by the plasmid (−) strains. Also note that the genetic diversity of human populations may contribute to results contradicting experiments performed using inbred animals. Therefore, additional surveys which involve a larger human cohort should be conducted to ensure the over-all safety of the plasmidless strains among individuals from diverse backgrounds.\nPlasmid (+) or (−)  C. trachomatis  strains demonstrated no difference with regards to growth kinetics, plaquing efficiency and size but showed a defect in glycogen granule accumulation and intrainclusion movement [ 10 ]. A recent study showed that  C. trachomatis  is capable of inducing alteration to global host histone modifications and double strand break repair, thus generating an environment favorable for malignant transformation [ 22 ]. In fact,  C. trachomatis  infection poses risk for infected individuals to develop cervix intraepithelial neoplasia [ 23 ,  24 ], in a similar way to other tumor-inducing pathogens. Extra-chromosomal plasmid DNA may play a role in integrating with the host genome which leads to this pathological damage. The conserved 7.5 kb cryptic plasmid is a small, non-conjugative and non-integrative extrachromosomal DNA that contains genes encoding 8 proteins [ 25 ]. The plasmid  Pgp3  encodes for immunogenic trimers which trigger specific antibody production in infected individuals [ 26 ,  27 ]; and is secreted into the cytosol of infected cells during chlamydial infection [ 28 ,  29 ]. Meanwhile,  Pgp4  encodes for a protein which comprises a putative helix-loop-helix domain which functions as transcriptional regulator for virulence-associated genes [ 10 ,  30 ]. Therefore, expression of plasmid genes may be crucial in leading to clinical symptoms. To support this notion, a quantitative protein analysis could be carried out in the future to measure the amount of plasmid-derived proteins in the patient sample.\n\nIn conclusion, we suggest that plasmids maybe a potential risk factor for reproductive system disorders including infertility, inflammation in the reproductive tract, irregular menses and PCOS in genital chlamydial infection. However, continued work is required to substantiate plasmids as a virulence factor in future studies with a larger human cohort.\n\nA total number of 180 female patients of child-bearing age who visited the Obstetrics and Gynecology Outpatient Clinics at the University of Malaya Medical Centre voluntarily participated in the study from 2010 to 2014. Detailed clinical information on reasons for referral, gynecological history including menstruation, symptoms of genital and urinary tract infection, obstetric and medical histories were documented. The subjects’ vulva and cervix were examined for the presence of lesions, warts, ulcers, ectopy, erythma and discharge. Patients with a positive urine pregnancy test, recent antibiotic therapy, yeast infection and genital tuberculosis were excluded from the study. The participants were briefed that their blood and vaginal swab samples will be used for research purposes, and written consent was obtained. This study has been approved by the University of Malaya Medical Centre Medical Ethics Committee.\nEndocervical swabs were collected by using a Floqswab (Copan, Brescia, Italy) and transferred to a laboratory in a UTM-RT universal transport medium tube (Copan). The samples were vortex mixed and the cells were centrifuged at 400× g for 10 min. The cells recovered were lysed and separated with phenol chloroform. The DNA was precipitated using 1:10 volume of 3 M sodium acetate and isopropanol at −20 °C overnight incubation. The samples were washed and eluted with TE buffer.\nDiagnosis of  C. trachomatis  was performed as described [ 15 ]. Nested PCR was performed to amplify the genes that encode for Major Outer Membrane Protein (MOMP) and plasmid using outer and inner primers (Table  4 ). PCR samples were run in a Veriti Thermal Cycler (Applied Biosystems, Essex, UK) at the following conditions: 95 °C for 3 min, 30 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min using HelixAmp Taq DNA polymerase (Nanohelix, Daejeon, South Korea). The first round PCR was amplified using 50 ng of DNA isolated from endocervical swabs with outer primer pairs. Then, 1 μl of first round PCR product was used as the template in the second round PCR and amplified with inner primer pairs. The amplicons were run on gel electrophoresis using 1.5 % agarose gel and viewed under a UV transilluminator. Human β-globin gene was used as a positive control to confirm successful DNA retrieval from patient swabs. PCR amplification for human β-globin primer was run separately in PCR master mix containing a total volume 50 μl as follows: 1 μl template DNA, 200 mM of each of dNTP, 25 pmol of each primer, 4 mM Tune Up solution, 5 μl of PCR buffer and 2.5 units of HelixAmp Taq DNA polymerase recombinant (Nanohelix, Daejeon, South Korea). PCR samples were run at the following conditions: 95 °C for 3 min, 30 cycles amplification consisted of 95 °C for 30 s, 50 °C for 1 min, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 7 min. Table 4 PCR primers used in  C. trachomatis  conventional PCR and real-time PCR diagnosis. For nested PCR amplification of  Momp  and plasmid, outer primer pairs were used at first round PCR, while inner primer pairs were used at second round PCR amplification. For real-time PCR diagnosis,  Momp  inner primer pairs, plasmid  Pgp8  inner primer pairs and plasmid  Pgp1  primer pairs were used Target genes Primer Sequence (5′-3′) Amplicon size \n Momp \n Outer forward TTGTTTTCGACCGTGTTTTG 455 bp Outer reverse AGCRTATTGGAAAGAAGCBCCTAA Inner forward AAACWGATGTGAATAAAGARTT 395 bp Inner reverse TCCCASARAGCTGCDCGAGC Plasmid  Pgp8 \n Outer forward TTGGCYGCTAGAAAAGGCGATT 212 bp Outer reverse TCCGGAACAYATGATGCGAAGT Inner forward AACCAAGGTCGATGTGATAG 150 bp Inner reverse TCAGATAATTGGCGATTCTT Plasmid  Pgp1 \n Forward TTCTTTGATGGCTTCCCAAC 456 bp Reverse ACGATTTTCTCCAACCGATG \n β-globin \n Forward GAAGAGCCAAGGACAGGTAC 268 bp Reverse CAACTTCATCCACGTTCACC\nPCR primers used in  C. trachomatis  conventional PCR and real-time PCR diagnosis. For nested PCR amplification of  Momp  and plasmid, outer primer pairs were used at first round PCR, while inner primer pairs were used at second round PCR amplification. For real-time PCR diagnosis,  Momp  inner primer pairs, plasmid  Pgp8  inner primer pairs and plasmid  Pgp1  primer pairs were used\nA quantitative real-time PCR assay was used to confirm the diagnosis and to determine the plasmid copy numbers in patient samples. DNA samples isolated from the vaginal swabs of  C. trachomatis -infected patients were prepared. Mastermix containing 1 μl template DNA, 1× SsoAdvanced Universal SYBR Green Supermix (Biorad, Hercules, CA), 10 pmol primers were run using a Mx3000 Stratagene thermacycler (Agilent Technologies, Santa Clara, CA). Primers used were inner primers for  Momp , inner primer for plasmid  Pgp8 , and plasmid  Pgp1  primers. Plasmid copy number for each patient was calculated as below: \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ Plasmid\\; copy\\; number=\\frac{Average\\left({C}_TPgp1+{C}_TPgp8\\right)}{C_T MOMP} $$\\end{document} Plasmid copy number = Average C T P g p 1 + C T P g p 8 C T MOMP\nAmplification of an approximately 871 bp fragment of  OmpA  was performed by nested PCR. The first PCR step was carried out with outer primer pair (Table  1 ) using 10 μl of DNA extracted from swabs. Amplification was performed in a final reaction volume of 50 μl containing 0.3 μM of each primer, 0.2 mM of dNTPs, 3 μl of TuneUp solution (Nanohelix, Korea) and 5U of HelixAmp™ Taq  DNA polymerase (Nanohelix, Korea). The first amplification conditions consisted of initial polymerase activation at 94 °C for 2 min; 40 cycles of 94 °C for 45 s, 60 °C for 45 s and 72 °C for 90 s and a final elongation step at 72 °C for 5 min. In the second round PCR, 3 μl of product from the first PCR step was amplified using inner primer pairs (Table  1 ). Nested PCR conditions consisted of 95 °C for 5 min; 40 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min and a final elongation step at 72 °C for 10 min. The amplified products were visualized by electrophoresis on 1.5 % agarose gel stained with GelRed solution (Biotium).\nThe 871 bp  OmpA  fragments obtained were purified using a QIAquick Gel Extraction kit (Qiagen) and processed using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The reaction mixtures were loaded onto a 3730xL DNA Analyzers (Applied Biosystems). The primers used for sequencing were inner primer pairs. Nucleotide sequence data were assembled by Bioedit and residues corresponding to flanking primers were excluded from analysis. Sequences were submitted to the standard nucleotide BLAST search engine at the National Center for Biotechnology Information ( blast.ncbi.nlm.nih.gov/Blast.cgi ) to determine the genotype.\nStatistical analysis was done using GraphPad PRISM version 5. For normally distributed data, an unpaired  t -test was used. For categorical data (clinical symptoms), Fisher’s exact test was used, with a no-symptom group as control. Odd ratio (OR) and 95 % confidence interval (CI) were calculated. Statistical significance was determined at  P  < 0.05 * ,  P  < 0.01 **  and  P  < 0.001 *** .\n\nAll data and materials are available upon request.","source_license":"CC-BY-4.0","license_restricted":false}