Molecular Detection Mutation of rpoB Gene Mycobacterium leprae in Relapse and Default of Leprosy Patient in Jayapura City, Papua

Abstract

Abstrak
 Latar belakang: Lepra masih menjadi masalah kesehatan di Papua terutama di Kota Jayapura.Banyaknya kasus relaps dan default juga menjadi tantangan dalam eliminasi lepra di Jayapura. Kasusrelaps dan riwayat default pada beberapa penelitian berkaitan dengan resistensi terhadap multi drugtreatment (MDT). Tujuan penelitian ini adalah mendeteksi keberadaan mutasi gen rpoB M. leprae padapasien relaps, default dan pasein yang kurang peka terhadap terapi MDT di Kota Jayapura.
 Metode: Sampel diperoleh dari pasien yang terdiagnosis lepra dengan kriteria pasien relaps, default danpasien yang terus bergejala setelah terapi MDT sebanyak 34 sampel. Sampel diambil dalam bentuk insisikulit (skin silt) daun telinga. DNA diekstraksi dengan menggunakan kit Qiagen. Gen rpoB diamplifikasimelalui teknik PCR dan analisis nukleotida dilakukan melalui sekuensing. Analisis mutasi dilakukanmelalui BLAST dengan basis data GenBank.
 Hasil: Sebanyak 34 sampel yang diperiksa, 9 diantaranya positif BTA sedangkan 25 yang lainnya negatifBTA. Pada hasil PCR, sampel yang berhasil teramplifikasi sebanyak 31 sampel, dan 3 sampel tidakteramplifikasi. Hasil BLAST menunjukkan bahwa tidak ditemukan adanya mutasi pada gen rpoB yangdapat menyebabkan resistensi terhadap rifampisin.
 Kesimpulan: Kesimpulan dari penelitian ini adalah gen rpoB Mycobacterium leprae asal Jayapuratidak mengandung mutasi yang dapat menyebabkan terjadinya resistensi terhadap rifampisin. Dengandemikian rifampisin masih sensitif untuk pengobatan lepra di Kota Jayapura.
 Kata kunci: Lepra, gen rpoB, rifampisin, Mycobacterium leprae.
 AbstractBackground: Leprosy remains a prominent health problem in Papua especially in Jayapura City. Numerouscases of relapse and default are also challenges in leprosy elimination in Jayapura. Studies in Relapsecases and history of defaults revealed some resistance related to multi-drug treatment (MDT). The purposeof this study was to detect the presence of mutation in rpoB M. leprae gene in patient relapse, default andpatients who are less sensitive to MDT therapy in Jayapura City.
 Method: Samples were obtained from patients diagnosed with leprosy with criteria of relapse, defaultand symptomatic patients after receiving MDT therapy. A total of 34 samples were taken in the form ofskin incision (skin silt) of the earlobe. DNA was extracted using Qiagen kit. rpoB gene from extractedDNA was amplified through PCR method followed by nucleotide sequences. Analysis of mutation waselaborated using BLAST according to GenBank database.
 Result: 34 samples were examined, and 9 were positive for Ziehl-Neelsen (ZN)staining, while the 25 werenegative. In the PCR results, the samples that successfully amplified were 31 samples, and 3 samples werenot amplified. The results of BLAST indicated that no mutations in the rpoB gene found in which able toinitiate resistance to rifampicin.
 Conclusion: The conclusion of this study is the rpoB Mycobacterium leprae gene from Jayapura did notcontain any mutations that could trigger resistance to rifampicin. Thus rifampicin is still sensitive forleprosy treatment in Jayapura City.
 Keywords: Leprosy, rpoB gene, rifampicin, Mycobacterium leprae

Mycobacterium leprae folP1 gene mutations among dapsone resistant patients in Sudan

“…The remaining tissue on the blade was placed in 1.5 ml of 70% alcohol and used in DNA isolation, PCR detection of M. leprae and sequence analysis of the folP1 gene. The smears were stained by modified ZN before and after combined therapy with dapsone 100 mg and rifampicin 600 mg, and examined microscopically (Figure 1), DNA was extracted according to Maladan et al 7 The DNA concentration and purity were measured using a spectrophotometer at wavelength of 260 nm. S13 and S62 primers were used for the detection of M. leprae (Table 1).…”

“…Another primer set targeting the folP1 gene was amplified, followed by sequence analysis of the PCR product for the presence or absence of mutations at amino acid positions 53 and 55 as relevant indicator for resistance to dapsone (Table 2). 8 The PCR reaction was carried according to Maladan et al 7 The PCR products were sequenced and mutation analyses was performed on the sequence raw data that initially subjected to trimming step using Finch TV program (version: 1.4.0), followed by NCBI blast (https://blast.ncbi.nlm .nih.gov/Blast.cgi) analysis to compare and contrast the similarity between their sequences to those previously submitted for the folP1 gene on the database.…”