Tesis

Uji Diagnostik Rickettsiosis menggunakan Teknologi Real-Time PCR = Diagnostic Evaluation of Rickettsiosis Using Real-Time PCR Technology.

Pendahuluan: Rickettsiosis merupakan penyakit infeksi zoonosis yang sering tidak terdiagnosis di fasilitas kesehatan primer maupun sekunder di Indonesia. Gejalanya yang menyerupai penyakit infeksi lain seperti demam berdarah dan tifoid, serta keterbatasan pemeriksaan serologi seperti IFA (immunofluorescence assay) yang memerlukan fasilitas khusus, menjadi hambatan utama dalam diagnosis dini. Metode: Penelitian ini merupakan studi potong lintang menggunakan 143 sampel plasma darah tersimpan dari pasien dengan demam akut di fasilitas pelayanan kesehatan di Jakarta. Pemeriksaan dilakukan dengan dua metode, yaitu real-time PCR yang menargetkan gen rrs (16S rRNA) dan gltA dan metode IFA sebagai baku emas. Proses optimasi PCR mencakup penyesuaian primer, probe, suhu annealing, serta uji sensitivitas dan spesifisitas. Data dianalisis menggunakan uji diagnostik dan statistik deskriptif. Hasil: Real-time PCR menunjukkan batas deteksi (LoD) hingga 1 salinan DNA, menandakan sensitivitas analitik yang sangat tinggi. Namun, dalam penerapannya pada sampel klinis, sensitivitas diagnostik PCR terhadap IFA hanya sebesar 27,8%, sedangkan spesifisitasnya mencapai 99,2%. Tidak ditemukan perbedaan signifikan pada parameter darah lengkap dan hitung jenis leukosit antara pasien Rickettsia dan nonRickettsia (p > 0,05). Kesimpulan: Real-time PCR memiliki spesifisitas yang baik, tetapi sensitivitasnya masih terbatas dalam penerapan klinis, kemungkinan karena kualitas sampel tersimpan dan waktu pengambilan darah yang tidak optimal. Metode ini tetap menjanjikan sebagai alat diagnosis rickettsiosis, terutama bila dikembangkan lebih lanjut dengan target gen tambahan.
Kata kunci : Rickettsiosis, real-time PCR, IFA


Background: Rickettsiosis is a zoonotic infectious disease that often remains undiagnosed in Indonesia’s primary and secondary healthcare settings. Its clinical presentation closely resembles other febrile illnesses such as dengue fever and typhoid, while serological tests like the immunofluorescence assay (IFA), which require specialized facilities, further hinder early diagnosis. Methods: This cross-sectional study utilized 143 archived plasma samples from patients with acute febrile illness in Jakarta. Two diagnostic methods were evaluated: real-time PCR targeting the rrs (16S rRNA) and gltA genes, and IFA as the reference standard. The PCR protocol was optimized by adjusting primers, probes, and annealing temperatures, and diagnostic performance was assessed through sensitivity, specificity, and descriptive statistics. Results: The real-time PCR method demonstrated an analytical limit of detection (LoD) of as low as 1 DNA copy, indicating very high sensitivity. However, in clinical sample testing, the diagnostic sensitivity relative to IFA was only 27.8%, while specificity reached 99.2%. No significant differences were observed in complete blood count and leukocyte differential parameters between Rickettsia-positive and -negative patients (p > 0.05). Conclusion: Real-time PCR showed high specificity but limited sensitivity in clinical application, likely due to the quality of stored samples and suboptimal timing of blood collection. Nonetheless, this method remains promising for the early detection of rickettsiosis, especially if further developed with additional genetic targets.
Keywords: Rickettsiosis, real-time PCR, IFA