Disertasi

KARAKTERISASI MOLEKULER GEN RESISTENSI DHPS (DIHIDROPTEROAT SINTASE) DAN POLIMORFISME GEN mtLSU (MITOCHONDRIAL LARGE SUBUNIT) Pneumocystis jirovecii PADA ODHA TERDUGA PNEUMONIA DI JAKARTA

Latar belakang. Meningkatnya kasus HIV-AIDS (human immunodeficiency virusacquired immunodeficiency syndrome) secara global memicu kewaspadaan akan peningkatan infeksi oportunistik, salah satunya infeksi Pneumocystis jirovecii yang mengakibatkan pneumonia (PjP). Infeksi PjP merupakan kasus yang sulit ditangani terkait rendahnya sensitivitas uji diagnostik yang ada diiringi dengan peningkatan kasus resistensi P. jirovecii terhadap antibiotik. Hingga saat ini di Indonesia belum terdapat data demografis, epidemiologi molekuler maupun data resistensi mengenai kasus infeksi PjP. Mengantisipasi masalah tersebut, dalam penelitian ini dikembangkan uji diagnostik PjP pada ODHA (Orang Dengan HIV-AIDS) terduga pneumonia melalui pendekatan secara molekular terhadap gen MSG (major surface glycoprotein) disertai dengan karakterisasi gen DHPS (dihidropteroat sintase) dan gen mtLSU (mitochondrial large subunit) yang berkorelasi dengan genotipe resisten dan virulensi P. jirovecii. Tujuan penelitian. Memperoleh suatu uji deteksi infeksi PjP, data genotipe resistensi dan virulensi PjP melalui pendekatan secara molekuler yang dapat dimanfaatkan sebagai dasar data demografi dan epidemiologi molekuler PjP di Indonesia. Metode penelitian. Pengembangan uji diagnosis molekuler PjP terhadap gen MSG dilakukan dengan metode real- time PCR yang diujikan terhadap 100 sampel sputum. Pola genotipe resistensi dilakukan melalui amplifikasi gen DHPS dilanjutkan dengan restriction fragment length polymorphism (RFLP). Virulensi daerah hot spot gen mtLSU dianalisis dengan metode PCR dan sekuensing DNA. Hasil. Secara demografi, diketahui prevalensi PjP pada ODHA terduga pneumonia di Jakarta mencapai 20,0 %, laki-laki 75%, dengan rentang usia terbanyak 31-40 tahun (35%), dominan (80%) pada kisaran sel limfosit T CD4+ 200-349 sel/µL. Uji realtime PCR yang dikembangkan meningkatkan jumlah positif lebih dari 5 kali dibandingkan uji mikroskopik Giemsa. Sebanyak 12 pasien menunjukkan gen DHPS positif, lima pasien (41,66%) merupakan genotipe wild type (WT) dan 7 pasien lainnya (58,32%) merupakan genotipe resisten, terdiri dari 16,67% genotipe-3 dan 41,66% genotipe campuran (WT dan genotipe 1). Analisis virulensi berdasarkan gen mtLSU diperoleh 30 strain PjP positif yang didominasi oleh variasi-3. Status imun pasien lebih berkaitan dengan genotipe resistensi dibandingkan dengan jenis varian. Kesimpulan. Uji real-time PCR yang dikembangkan mampu memberikan nilai diagnostik yang lebih baik dibandingkan pewarnaan Giemsa. Terdapat 3 genotipe gen resistensi (WT, genotipe 1 dan 3 ) dan 7 varian P. jirovecii yang bersirkulasi di Jakarta. Genotipe resistensi lebih berkaitan terhadap kondisi klinis pasien dibandingkan dengan jenis varian.
Kata kunci : real-time PCR, Giemsa, HIV, Pneumocystis jirovecii, DHPS, mtLSU


Background. The global rise of HIV-AIDS cases increase the alertness against oportunistic infections, one of them is Pneumocystic jirovecii pneumonia (PjP). PjP infection is a one among several infection to be cured due to low sensitivity of its diagnostic method following the escalation of PjP resistance against antibiotics. Until now, there is no demographic, molecular epidemiology nor antibiotics resistance data available related to PjP infection in Indonesia. Thus, this study was conducted to develop a molecular test to diagnose PjP infection in HIV-AIDS suspected pneumonia patients based on MSG (major surface glycoprotein) gene detection, followed by characterization of DHPS (dihydropteroate synthetase) and mtLSU (mitochondrial large subunit) genes representing genotype resistance and P. jirovecii virulence. Research objective. To obtain a molecular test in diagnosing PjP infection and information of P. jirovecii genotype resistance and virulence based on molecular characteristics, which can be used further as demographic and molecular epidemiology basis data of PjP in Indonesia. Research methods. Molecular diagnostic test aimed for MSG gene of P. jirovecii detection was done through real-time PCR against 100 sputum samples. Genotype resistance and P. jirovecii polymorphism patterns were done through DHPS and mtLSU genes amplification followed by restriction fragment length polymorphism (RFLP) and DNA sequencing analysis. Virulence of the hot spot area of the mtLSU gene was analyzed by PCR method and DNA sequencing. Results. Demographic data show that the prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta is 20.0%, male 75% within 31-40 y.o (35%), dominant (80%) from patients with CD T-lymphocytes of 200-349 cells/µL. Molecular real-time PCR methods give five times sensitivity higher than Giemsa stain. Twelve patients show positive DHPS gene, five patients (41.67%) were wild type (WT) genotypes and 7 other patients (58.32%) were resistant genotypes, with 16.66% was genotype-3 and other 41.66% was mixed genotypes (WT and genotype 1). Virulence analysis based on mtLSU gene showed 30 positive strains which were dominated by variant-3. The patients immune status was more related to the resistance genotype compared to the variant type. Conclusion. The development of the real-time PCR method is proven to able to give better diagnostic value than Giemsa stain. There are 3 genotypes of resistance genes (WT, genotypes 1 and 3) and 7 variants of P. jirovecii circulating in Jakarta. Resistant genotypes are more related to the clinical condition of patients compared to variant types.
Keywords : real-time PCR, Giemsa, HIV, Pneumocystis jirovecii, DHPS, mtLSU