Tesis

Deteksi Mycobacterium leprae dengan Real-time Polymerase Chain Reaction pada Spesimen Kerokan Jaringan Kulit dan Jaringan Biopsi Kulit Pasien Kusta Pausibasilar dengan Basil Tahan Asam Negatif = Detection of Mycobacterium leprae Using Real-time Polymerase Chain Reaction in Slit Skin Smear and Skin Biopsy Specimens of Paucibacillary Patient with Negative Acid-fast Bacilli.

Latar Belakang : Penyakit kusta masih menjadi masalah kesehatan di dunia terutama di Indonesia sebagai negara dengan prevalensi tertinggi ketiga di dunia. Gejala klinis dan pemeriksaan penunjang yang ada sulit untuk menegakkan diagnosis definitif kusta pausibasilar (PB) dengan basil tahan asam (BTA) negatif. Diperlukan uji alternatif seperti real-time polymerase chain reaction berbasis probe TaqMan yang sensitif dan spesifik untuk deteksi Mycobacterium leprae. Tujuan : Mendeteksi M. leprae pada pasien kusta PB dengan basil tahan asam negatif, dengan menggunakan real-time PCR berbasis probe TaqMan pada spesimen kerokan jaringan kulit dan jaringan biopsi kulit. Metode : Penelitian dilakukan dalam 3 tahap. Tahap 1 adalah optimasi konsentrasi primer, probe, suhu penempelan, volume elusi akhir dan volume cetakan. Tahap 2 adalah uji sensitivitas (ambang batas deteksi DNA) dan uji spesifisitas (reaksi silang) dari TaqMan real-time PCR dan tahap 3 adalah penerapan uji TaqMan realtime

PCR yang sudah dioptimasi terhadap spesimen kerokan jaringan kulit dan jaringan biopsi kulit. Hasil : Hasil optimasi TaqMan real-time PCR yang didapatkan adalah sebagai berikut : suhu penempelan 55 o C, konsentrasi primer forward, primer reverse dan probe masing-masing sebesar 0,2 µM, volume elusi kerokan jaringan kulit cuping telinga dan lesi kulit sebesar 50 µl dan 40 µl dan volume cetakan untuk kerokan jaringan kulit cuping telinga dan lesi kulit sebesar 5 µl dan 6 µl. Ambang batas deteksi DNA terendah adalah 11 sel M. leprae. Uji TaqMan real-time PCR ini tidak bereaksi silang terhadap Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Candida sp., Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, virus Herpes Simpex dan virus Varicella Zooster. Uji TaqMan real-time PCR memberikan positivitas sebesar 20,8%, 25% dan 95,8% pada spesimen kerokan jaringan kulit cuping telinga, lesi kulit dan pada jaringan biopsi kulit. Positivitas hasil pemeriksaan patologi anatomi (PA) yang menunjukkan kusta sebesar 79,2%. Kesimpulan : Real-time PCR berbasis probe TaqMan bersifat sensitif dan spesifik untuk mendeteksi M. leprae pada kusta PB dengan BTA negatif dan dapat meningkatkan kemampuan diagnosis sebesar 16,6% dari pemeriksaan PA yang selama ini menjadi pemeriksaan baku emas kusta. Spesimen yang dianjurkan untuk digunakan adalah jaringan biopsi kulit.
Kata kunci : Kusta PB dengan BTA negatif, TaqMan real-time PCR, M. leprae, pemeriksaan PA



Background : Leprosy is still considered as a major health problem worldwide especially in Indonesia as the third highest prevalence in the world. Clinical symptoms which often similar with other skin diseases and the current available laboratory methods to establish definitive diagnosis of paucibacillary leprosy with negative acid-fast bacilli is difficult. Therefore, it is important to apply an alternative assay such as real-time polymerase chain reaction-based TaqMan probe for sensitive and specific detection of Mycobacterium leprae. Objective : Detection of M. leprae in paucibacillary leprosy with negative acid-fast bacilli using real-time PCR-based TaqMan probe in slit skin and skin biopsy tissue specimens. Methods : This study was conducted in three phases. The first is optimization of primers and probe concentrations, annealing temperature, final elution of DNA extraction and volume of PCR template. The second is determination of sensitivity (minimal detection of DNA) and specificity test (cross-reaction) of the optimized TaqMan real-time PCR and the third is application of the TaqMan real-time PCR in slit skin and skin biopsy clinical specimens. Results : TaqMan real-time PCR assay optimization showed results as follow : annealing temperature at 55 o C, concentration of primer both forward and reverse and probe concentration were 0,2 µM, final elution of DNA extraction for slit skin and skin biopsy tissue was 50 µl and 40 µl and volume of PCR templates for slit skin and skin biopsy tissue was 5 µl and 6 µl. Limit detection of the DNA was 11 cells of M. leprae. The TaqMan real-time PCR was not cross reacted to Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Candida sp., Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Herpes Simplex Virus and Varicella Zooster Virus. The TaqMan real-time PCR showed 20,8%, 25% and 95,8% positivity in slit skin of earlobes, slit skin lession and skin biopsy tissue respectively. Histopatology examination result showed 79,2% positivity of leprosy. Conclusion : Real-time PCR-based TaqMan probe is sensitive and specific for detecting M. leprae in paucibacillar leprosy with negative acid-fast bacilli and could improve the diagnosis by 16,6% compared to histopathology which considered as gold standard. Specimen recommended to be used is skin biopsy tissue.
Keywords : Paucibasillary leprosy with negative acid-fast bacilli, the TaqMan realtime PCR, M. leprae, Histopathology examination