A multiplex assay of Trichomonas vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae infections in genital specimens

Abstract

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.

Coinfections with multiple sexually transmitted pathogens in Republic of Korea, 2018–2020

“…A study of pregnant women who visited a hospital in Ghana showed that Candida (53%) coinfection was common in women with T. vaginalis infection 17 . In addition, in another study from Iran in which coinfection with sexually transmitted pathogens was confirmed using mPCR, 10/300 patients (3.3%) tested had coinfections confirmed, including 2 cases of C. trachomatis / N. gonorrhoeae , 3 cases of C. trachomatis / T. vaginalis , and 5 cases of N. gonorrhoeae / T. vaginalis coinfections 7 . According to a study conducted in Beijing, China, among the patients with coinfections, 60.6% of men and 71.4% of women were coinfected with U. urealyticum and C. trachomatis 2 …”

“…vaginalis coinfections. 7 According to a study conducted in Beijing, China, among the patients with coinfections, 60.6% of men and 71.4% of women were coinfected with U. urealyticum and C. trachomatis. 2 This study has several limitations.…”

“…Various methods are used to diagnose STIs, including culture or serological tests for each specific pathogen, but these methods are generally time‐consuming and lack sensitivity for identifying STIs in asymptomatic patients 7,8 . Considering the frequency of coinfection with STIs, the high number of asymptomatic cases, and the difficulties associated with culture methods, nucleic acid amplification testing is an appropriate method for diagnosing STIs 4 .…”

“…6 Various methods are used to diagnose STIs, including culture or serological tests for each specific pathogen, but these methods are generally time-consuming and lack sensitivity for identifying STIs in asymptomatic patients. 7,8 Considering the frequency of coinfection with STIs, the high number of asymptomatic cases, and the difficulties associated with culture methods, nucleic acid amplification testing is an appropriate method for diagnosing STIs. 4 Multiplex polymerase chain reaction (mPCR) is a sensitive and rapid method for simultaneous detection of STIs caused by multiple pathogens in clinical laboratories and has recently attracted attention as a method for detecting sexually transmitted pathogens using molecular diagnostics.…”

“…4,8 A significant proportion of patients with STIs are coinfected with multiple pathogens. 7 In this study, patients were tested for coinfections with 12 different sexually transmitted pathogens: Candida albicans, Chlamydia trachomatis, Gardnerella vaginalis, HSV1, HSV2, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Ureaplasma parvum, and Ureaplasma urealyticum. A symbiotic relationship between T. vaginalis and M. hominis has been reported in a Chinese study.…”