Development of Duplex Polymerase Chain Reactionin-house assay to detect of Chlamydia trachomatis and Neisseria gonorrhoeaefrom genital discharge in Patient with Sexual Transmitted Infections

Abstract

Abstract

The incidence of sexually transmitted infections (STIs) with discharge in Indonesia to high-risk groups provide dominant pattern of Chlamydia trachomatis(CT) and Neisseriagonorrheae(NG) infection. Pathogenesis change of CT and NGinfection suggeststhat routine syndromic approach diagnostic is no longer accurate. To develop detection system of CTand NG using duplex polymerase chain reaction (PCR) assay to genital discharge in patient with STIs. Three phases: firstly was PCR assay optimalization to annealing time and temperature, primer concentration, centrifugation time and elution volume, secondly, specificity test and thirdly duplex PCR assay application to clinical specimen. Duplex PCR assay optimalization gave results as follow: annealing temperature and time 54°C and 60 seconds, CTand NG primer concentration were 0,7μM and0,5μM, centrifugation time 10 minutes, and elution volume 60 μl. Detection limit of duplex PCR to CTand NG 0.927 and 1.19 pg / PCR reaction, respectively. Duplex PCR to detect NG has ensitivity, specificity, positive predictive value and negative predictive value were 100%, 61.9%, 20%, and 100% in endocervical specimens, respectively and 75%, 40%, 50%, and 66.67%, in male urethral specimens respectively. Duplex PCR to detect CT was compared with chlamydial antigen detection test were show positive results higher both in endocervical and male urethral specimens (10:3 and 1:0). Development of detection system of CT and NG using duplex PCR assay is applicable to endocervical and male urethral swab specimens.

Keywords : sexual transmitted infections (STIs), genital discharge, Duplex PCR in-house assay,Chlamydia trachomatis, Neisseria gonorrhoeae

Abstrak

Insiden infeksi menular seksual (IMS) dengan duh genital di Indonesia pada kelompok berisiko tinggi memberikan pola dominan infeksi Chlamydia trachomatis (CT) dan Neisseriagonorrheae (NG). Perubahan pathogenesis CT dan NG menyebabkan diagnosis rutin menggunakan pendekatan sindrom tidak lagi akurat. Untuk mengembangkan system deteksi CT dan NG menggunakan polymerase chain reaction (PCR) dupleks untuk duh genital pada pasien dengan infeksi menularseksual. Terdapat tiga langkah penelitian yang dilakukan. Pertama adalah optimalisasi PCR terhadap waktu dan suhu annealing, konsentrasi primer, waktu sentrifugasi dan volume elusi. Kedua, uji spesifisitas dan ketiga aplikasi PCR dupleks untuk specimen klinis. Optimalisasi PCR dupleks: suhu annealing 54 ° C, waktu annealing 60 detik, konsentrasi primer CT baik reverse maupun forward 0,7μM dan NG baik reverse maupunforward 0, 5μM, waktu sentrifugasi adalah 10 menit dan volume elusi 60 ml. Batas deteksi PCR duplex untuk CTadalah 0,927 pg / reaksi PCR, dan NG adalah 1,19 pg/ reaksi PCR. PCR dupleks untuk mendeteksi NG memiliki sensitivitas, spesifisitas, nilai prediksi positif dan nilai prediksi negative berturut-turut adalah100%, 61,9%, 20%, dan 100% pada spesimenen do serviks dan 75%, 40%, 50%, dan 66,67% pada specimen uretra laki-laki. PCR dupleks untuk mendeteksi CT dibandingkan dengan tes deteksi antigen klamidia yang menunjukkan hasil positif yang lebih tinggi baik dalam specimen endoserviks dan laki-laki uretra (10: 3 dan 1: 0). Pengembangan system deteksi CT dan NG menggunakan PCR dupleks dapat diaplikasikan pada specimen swab uretra laki-laki dan swab endoserviks.

Kata kunci: Infeksi Menular Seksual (IMS), duh genital, PCR dupleksin-house, Chlamydia trachomatis, Neisseria gonorrhoeae