Tesis

Deteksi Corynebacterium Patogenik dan Analisis Filogenetik Corynebacterium diphtheriae Berdasarkan Gen rpoB Parsial = Detection of Pathogenic Corynebacterium and Phylogenetic Analysis of Corynebacterium diphtheriae based on partial rpoB gene.

Latar Belakang: Patogenitas Corynebacterium pada infeksi difteri, sangat terkait dengan toksin yang dihasilkannya. Indonesia merupakan negara ke dua di dunia yang memiliki kasus difteri terbanyak dengan 1.665 kasus dan 29 kasus kematian di tahun 2018. Toksin difteri dapat dihasilkan oleh 3 spesies penyebab, yaitu C. diphtheriae, C. ulcerans, dan C. pseudotuberculosis yang sulit dibedakan secara mikroskopik dan kultur, sehingga diperlukan deteksi molekuler untuk membedakanya. Karakterisasi C. diphtheriae sebagai spesies penyebab utama, diperlukan untuk mengetahui hubungan kekerabatanya dengan spesies negara lain untuk mencari kemungkinan sumber infeksi. Metode: Sebanyak 108 sampel klinis digunakan dalam penenlitian ini. Teknik kultur dilakukan untuk mendeteksi 3 Corynebacterium patogenik, sedangkan realtime PCR hanya dirancang untuk mendeteksi C. ulcerans dan C. pseudotuberculosis. Pengujian inhibitor, sensitifitas, dan spesifisitas dilakukan pada tahap optimasi. Karakterisasi C. diphtheriae dilakukan dengan metode sekuensing menggunakan gen rpoB parsial pada kultur sampel klinis. Hasil: Limit deteksi real-time PCR untuk C. ulcerans dan C. pseudotuberculosis secara berurutan sebanyak 4,49 dan 1,06 DNA copy number. Uji spesifisitas terhadap 18 mikrorganisme menunjukkan tidak terdapat reaksi silang. Pengujian terhadap 108 sampel klinis memberikan hasil yang sama dengan kultur, tidak ditemukan C. ulcerans dan C. pseudotuberculosis. Pada kultur sampel klinis ditemukan C. diphtheriae sebanyak 10 sampel (9,26%), yang dapat dikelompokkan menjadi 4 clade yaitu clade I, III, IV dan V dengan similaritas 99,2 % sampai 99,7%. Kesimpulan: Kasus suspek difteri dalam studi ini tidak berkaitan dengan infeksi C. ulcerans dan C. pseudotuberculosis, dan hanya positif C. diphtheriae . Hasil karakterisasi gen rpoB pada C. diphtheriae, memperlihatkan hubungan kekerabatan dengan beberapa negara.
Kata kunci: Difteri, Corynebacterium patogenik, deteksi, filogenetik


Background: Pathogenicity of Corynebacterium in diphtheria infection is closely related to toxin production. Indonesia is the second highest country in the world that has the most diphtheria cases with 1,665 cases and 29 deaths in 2018. Diphtheria toxin can be produced by 3 species, such as C. diphtheriae, C. ulcerans and C. pseudotuberculosis which are difficult to distinguish microscopically and culture, therefor molecular detection is needed to differentiate them. Characterization of C. diphtheriae as the main causative species, is needed to determine its relationship with other countries to find possible source of infection. Method: A total of 108 clinical samples were used in this study. Culture techniques were performed to detect 3 pathogenic Corynebacterium and real-time PCR was only designed to detect C. ulcerans and C. pseudotuberculosis. Inhibitor, sensitivity and specificity testing were carried out at the optimization stage. Characterization of C. diphtheriae from culture of clinical samples, was carried out by sequencing method using partial rpoB genes. Result: The real-time PCR detection limits for C. ulcerans and C. pseudotuberculosis were 4.49 and 1.06 DNA copy number, respectively. Specificity test for 18 microorganism showed no cross reaction. Tested on 108 clinical samples gave the same results as culture, there were not found C. ulcerans and C. pseudotuberculosis. In clinical culture samples found 10 (9.26%) C. diphtheriae, which can be grouped into 4 clades namely clades I, III, IV and V with similarities of 99.2% to 99.7%. Conclusion: Suspected diphtheria cases in this study were not related to C. ulcerans and C. pseudotuberculosis infections, and were only positive for C. diphtheriae. The results of the rpoB gene characterization test on C. diphtheriae showed a close relationship with several countries.
Key words: Diphtheria, Pathogenic Corynebacterium, Detection, Phylogenetic