Tesis

Deteksi gen hsp18 dan esxA pada Spesimen Positif RLEP Mycobacterium leprae untuk Penegakan Diagnosis Klinis Kusta = Detection of hsp18 and esxA Genes in RLEPPositive Mycobacterium leprae Clinical Specimens to Support the Clinical Diagnosis of Leprosy.

Latar Belakang: Pemeriksaan molekuler kusta menggunakan Real-time PCR dengan target gen RLEP memiliki sensitivitas tinggi, namun tidak secara langsung mencerminkan aktivitas metabolik aktif Mycobacterium leprae. Oleh karena itu, penelitian ini dilakukan untuk menilai deteksi gen hsp18 dan esxA pada spesimen kusta dengan hasil Real-time PCR RLEP positif serta menganalisis hubungannya dengan nilai Indeks Bakteri (IB) dan klasifikasi klinis kusta. Metode: Penelitian ini melibatkan 45 pasien kusta yang direkrut dari RSUP dr. Sitanala dan RSUPN Cipto Mangunkusumo dengan desain studi kohort yang menggunakan spesimen klinis kerokan cuping telinga dan lesi pasien kusta. Spesimen diolah dengan pemeriksaan mikroskopis BTA, Real-time PCR RLEP dan deteksi gen hsp18 dan esxA menggunakan metode TaqMan Real-time PCR. Hubungan antara hasil PCR dan nilai IB BTA dianalisis menggunakan uji Mann– Whitney U, sedangkan analisis hubungan nilai Ct dan IB menggunakan uji Spearman. Hasil: Hasil penelitian menunjukkan bahwa gen hsp18 dan esxA dapat terdeteksi pada spesimen klinis kusta. Deteksi gen esxA berhubungan secara bermakna dengan peningkatan nilai median IB dibandingkan kelompok negatif (p=0.001). Walaupun hasil PCR untuk deteksi hsp18 menunjukkan kecenderungan nilai IB yang lebih tinggi, tetapi hubungan tersebut tidak bermakna secara statistik (p=0.106). Pada studi ini menunjukkan bahwa hasil positif PCR RLEP perlu dilanjutkan dengan deteksi gen esxA dan hsp18 sehingga dapat menggambarkan metabolisme aktif M.leprae untuk panduan menegakkan diagnosis klinis secara lebih akurat. Kesimpulan: Uji BTA dapat digunakan untuk menegakkan diagnosis tipe kusta MB (88,2%), tetapi memiliki keterbatasan pada kusta tipe PB (11,8%). Real-time PCR RLEP dapat mendeteksi kusta tipe PB lebih baik dibandingkan dengan pemeriksaan BTA, yaitu secara berurutan sebesar 29,4% dan 11,8%.
Kata kunci : Mycobacterium leprae, RLEP, hsp18, esxA


Background: Molecular examination of leprosy using Real-time PCR targeting the RLEP gene has high sensitivity; however, it does not directly reflect the active metabolic state of Mycobacterium leprae. Therefore, this study was conducted to evaluate the detection of the hsp18 and esxA genes in leprosy specimens with positive Real-time PCR RLEP results and to analyze their associations with the Bacterial Index (BI) and the clinical classification of leprosy. Methods: This study involved 45 leprosy patients recruited from Dr. Sitanala General Hospital and Dr. Cipto Mangunkusumo National Central General Hospital using a cohort study design. Clinical specimens were obtained from slit-skin smears of the earlobes and skin lesions of leprosy patients. The specimens were examined using acid-fast bacilli (AFB) microscopy, Real-time PCR targeting RLEP, and detection of the hsp18 and esxA genes using the TaqMan Real-time PCR method. The association between PCR results and BI values was analyzed using the Mann– Whitney U test, while the correlation between Ct values and BI was assessed using Spearman’s correlation test. Results: The results showed that the hsp18 and esxA genes could be detected in clinical leprosy specimens. Detection of the esxA gene was significantly associated with a higher median BI compared to the negative group (p = 0.001). Although detection of hsp18 showed a tendency toward higher BI values, the association was not statistically significant (p = 0.106). This study demonstrates that positive PCR RLEP results should be followed by detection of esxA and hsp18 genes to better reflect the active metabolic state of M. leprae and to support more accurate clinical diagnosis. Conclusion: AFB microscopy can be used to establish the diagnosis of multibacillary (MB) leprosy (88.2%), but it has limitations in paucibacillary (PB) leprosy (11.8%). Real-time PCR targeting RLEP detects PB leprosy more effectively than AFB microscopy, with detection rates of 29.4% and 11.8%, respectively.
Keywords : Mycobacterium leprae, RLEP, hsp18, esxA