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Abstrak :
Latar belakang. Meningkatnya kasus HIV-AIDS human immunodeficiency virus-acquired 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 diiringi dengan peningkatan kasus resistensi terhadap antibiotik. 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 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, rentang usia terbanyak 31-40 tahun 35, dominan 80 pada kisaran sel limfosit T CD4 200-349 sel/L. 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.

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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 of a tough infection to be cured due to low sensitivity of its diagnostic method following the escalation of PjP resistance against antibiotics. There is no demografic, molecular epidemiology nor antibiotics resistance data were 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 dihydropteroat syinthetase and mtLSU mitochondrial large subunit genes represent genoype 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 was done through DHPS and mtLSU genes amplification followed by restriction fragment length polymorphism RFLP and DNA sequencing analysis. Virulence of the hot spot area are of the mtLSU gene was analyzed by PCR method and DNA sequencing. Results. The prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta was 20.0, male 75 within 31-40 y.o 35, dominant 80 from patients with CD4 T-lymphocytes of 200-349 cells/L. Molecular real-time PCR methods give five times sensitivity higher than Giemsa stain. Twelve patients showed 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 show 30 positive strains which dominated by variant-3. The patients immune status is more related to the resistance genotype compared to the variant type. Conclusion. The developed 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. Resistance genotypes are more related to the clinical condition of patients compared to variant types.

Abstrak :
Identifikasi Pneumocystis jirovecii pada pasien dengan koinfeksi tuberkulosis (TB) paru masih menjadi tantangan karena gambaran klinis maupun radiologis keduanya yang mirip dan P. jirovecii tidak dapat dikultur. Identifikasi P. jirovecii di Indonesia masih berdasarkan pemeriksaan mikroskopik yang seringkali kurang sensitif. Oleh karena itu, dikembangkan teknik real time PCR yang lebih sensitif dan spesifik dengan gen target mitochondrial large subunit (mtLSU) dan mitochondrial small subunit (mtSSU) rRNA. Penelitian ini bertujuan untuk mengoptimasi deteksi gen mitochondrial large dan small subunit rRNA dalam mengidentifikasi P. jirovecii pada pasien terkait TB paru. Penelitian ini menggunakan 26 sampel sputum pasien terkait TB paru. Optimasi teknik real time PCR berupa optimasi konsentrasi primer, probe, suhu penempelan, volume cetakan DNA dan uji reaksi silang dilakukan untuk mendapatkan kondisi yang optimal dalam amplifikasi gen mtLSU dan mtSSU rRNA. Hasil penelitian menunjukkan hasil optimasi deteksi kedua gen tersebut dapat mengidentifikasi P. jirovecii 1 dari 26 sampel (3,84%). Uji real time PCR yang telah di optimasi dalam penelitian ini dapat mendeteksi P. jirovecii pada sampel klinis pasien terkait TB paru. ......Identification of Pneumocystis jirovecii in patients with co-infected pulmonary tuberculosis (TB) is still a challenge because the clinical and radiological features of both are similar and P. jirovecii cannot be cultured. Identification of P. jirovecii in Indonesia is still based on microscopic examination which is often less sensitive. Therefore, a more sensitive and specific real time PCR technique was developed with mitochondrial large subunit (mtLSU) and mitochondrial small subunit (mtSSU) rRNA target genes. This study aimed to optimize the detection of mitochondrial large and small subunit rRNA genes to identify P. jirovecii in pulmonary TB-related patients. A total of 26 sputum samples of pulmonary TB-related patients were collected. Real time PCR technique optimization including the optimization of primer and probe concentrations, annealing temperature, DNA template volume and cross-reaction testing, was carried out to obtain optimal conditions for mtLSU and mtSSU rRNA gene amplification.  The results showed that the optimization of detection for both genes could identify P. jirovecii in 1 out of 26 samples (3.84%). The optimized real time PCR test in this study can detect P. jirovecii in clinical samples of pulmonary TB-related patients.