The prevalence of Chlamydia trachomatis infection among Saudi women attending the infertility clinic in Central Saudi Arabia

Patients and selection criteria

This study was conducted between October 2012 to July 2013 at 2 tertiary hospitals, King Khalid University Hospital (KKUH) and King Abdulaziz University Hospital (KAUH) in Riyadh, Saudi Arabia. We recruited married female patients aged between 19 and 46 years old who were attending the infertility clinic of both hospitals. For comparison, we invited married female patients who were attending the gynecology clinic of both hospitals as the control group. The control group were of the same age group as the infertile group, provided that they never consulted the clinic for any infertility problem. Women who had a history of medical or surgical diseases, were pregnant at the time of the study, and who had taken antibiotics 30 days prior to the study were excluded from this study. Both groups fulfilled the inclusion criteria during the period of the study and agreed to participate by signing an informed and written consent. The Institutional Review Board of King Saud University granted the approval for the conduction of this study.

Demographic characteristics of the patients were noted using a preformed data collection sheet. Signs and symptoms including burning micturition, genital bleeding, irregular menstruation, vaginal discharge, lower abdominal pain, history of PID, abortion, ectopic pregnancy, premature delivery, low birth weight, and infertility duration were also obtained. The type and etiologic cause of infertility was also obtained from the infertility group of patients. Physical pelvic examination was conducted by a gynecologist.

Specimen collection

Two endocervical swabbing by speculum examination were carried out to all patients by the attending gynecologist. The first swab was used before sampling to clean off the excess mucus, while the second was rubbed and were rotated several times over the endocervical cells in the cervical canal to collect samples for assay. Swabs were withdrawn from the cervical canal without touching the vaginal surface, and were placed into the Copan universal transport medium (UTM-RT, Copan Medical Diagnostic Laboratories, Hamilton, New Jersey, USA). All swabs were stored at 4°C until transported to the laboratory. The DNA extraction was performed using automated DNA extraction by MagNA Pure Compact Nucleic Acid Isolation Kit and MagNA Pure Compact system by Roche Diagnostics, Indianapolis, Indiana, USA. A 136 bp fragment of the C. trachomatis genome gene was amplified with specific primers and detected with probes labelled with Light Cycler Red 640 (Roche Diagnostics, Indianapolis Indiana, USA). The polymerase chain reaction (PCR) was monitored by an additional PCR product of 278 bp. The master mix for real time PCR was prepared using LightCycler^® Fast Start DNA Master HybProbe (Roche Diagnostics, Indianapolis, Indiana, USA). To avoid contamination, mixing of the reagents (except of the DNA template) was performed in a separate room, away from the room where DNA purification were carried out. The reaction mixture was prepared in a cold reaction tube, 15 µl of the reaction mix was transferred to a light cycler 2.0 capillary. A 5 µl of template DNA was added to each capillary for a final reaction volume of 20 µl. One negative control was always included in each run by replacing the template DNA with water. A positive control was included in each run by replacing the template DNA with one of the control DNA from the standard row provided with the light mix kit (TIB Molbiol, Germany). The LightCycler PCR program was composed by: fast start Taq DNA polymerase activation carried out in 95°C for 10 minutes, followed by cycling: 95°C (20°C/second [s]) for 5 seconds, 55°C (20°C/s) for 5 s, and 72°C (20°C/s) for 15 s, repeated 50 times. Melting assay ended the analysis: samples were heated to 95°C (20°C/s) hold for 20 s, cooled to 40°C (20°C/s) hold for 20 s, and then heated slowly at 0.2°C/s up to 85°C, finally cooled to 40°C (20°C/s). The PCR results were obtained within 50 minutes (50 cycles and melting curve); data analysis was performed, as described in the LightCycler instrument operator’s manual.

Statistical analysis

All collected data were encoded and analyzed using the Statistical Package for Social Sciences version 19.0 (IBM Corp., Armonk, NY, USA). We present the frequencies and percentages for different items of nominal variables, and mean, standard deviation and range for numerical variables. We used chi-square test to compare between infertile group and the non-infertile group, and to compare between primary infertility and secondary infertility with respect to all nominal variables. For smaller number of cases, we used Fisher’s exact test to compare between variables. Correlation between variables was carried out using the Pearson correlation. We assumed there was a statistically significant when p<0.05.