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INTRODUCTION: A significant proportion of patients with Sexually Transmitted Infections (STIs) are coinfected with multiple pathogens. We report here development of a multiplex PCR for simultaneous detection of Chlamydia trachomatis (C.trachomatis), Neisseria gonorrhoeae (N.gonorrhoeae) and Trichomonas vaginalis (T.vaginalis) in genital specimens from women. METHODOLOGY: After detection of the organisms by routine techniques including PCR, culture and direct smear, multiplex-PCR was optimized to detect ompI gene of CT, parC of NG, ITS ribosomal RNA of TV as target genes. The limit of detection (LOD) was determined using serially diluted genomic DNA from known number of each pathogen. RESULTS: Totally 300 volunteers with mean age of 36.5 ±7.03 years were included and 266 (88.7%) had genitourinary clinical manifestations. Of 300 women, 150 (50.0%) were infected. Of them, 17 (5.7%) had N. gonorrhoeae, 98 (32.7%) T. vaginalis and 35 (11.7%) C. trachomatis. Multiplex-PCR revealed a total of 10 coinfections (3.3%) including 2 specimens of C. trachomatis/ N. gonorrhoeae, 3 specimens of C .trachomatis/ T. vaginalis and 5 specimens of N. gonorrhoeae/T. vaginalis coinfections. The sensitivity and specificity of multiplex-PCR for detecting N. gonorrhoeae were 100% and 98.59% (279 of 283) respectively and, for C. trachomatis and T. vaginalis were 100%. The LOD was 0.1 pg of DNA for C. trachomatis and N. gonorrhoeae, and 1.5 pg for T. vaginalis. CONCLUSIONS: The performance of this multiplex-PCR makes it a sensitive, rapid and affordable technique in clinical laboratory for simultaneous detection of STIs.
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BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. OBJECTIVE: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. MATERIALS AND METHODS: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. RESULTS: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). CONCLUSION: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.
