Disertasi

Pengandung gametosit berdasarkan marker molekular pfs25 (P. falciparum) dan pvs25 (P. vivax) beserta faktor risikonya, serta status gen terkait resistensi artemisinin pfk13 berkaitan dengan Mass Blood Survey di Kabupaten Belu, NTT. = Gametocyte carriers and risk factors based on molecular markers pfs25 (P. falciparum) and pfs25 (P. vivax), and status of gene associated with artemisinin resistance pfk13 in relation to Mass Blood Survey in Belu District, East Nusa Tenggara.

Latar belakang: Gametosit merupakan stadium infektif Plasmodium spp. yang dapat digunakan sebagai parameter transmisi. Namun deteksi stadium ini membutuhkan pemeriksaan berbasis molekular karena pemeriksaan mikroskopik terbukti kurang sensitif. Penelitian ini bertujuan menganalisis pengandung gametosit dan faktor risikonya selama dilakukan intervensi Mass Blood Survey (MBS) serta mengidentifikasi mutasi gen terkait resistensi terhadap artemisinin. Metode: Penelitian ini merupakan penelitian longitudinal yang bersarang pada penelitian MBS pada tahun 2013 di Kabupaten Belu, NTT. Gametosit dideteksi dengan RT qPCR menggunakan marker pfs25 dan pvs25 pada individu positif P. falciparum atau P. vivax secara mikroskopik dan/atau PCR. Prevalensi parasitemia (mikroskopik dan PCR) serta pengandung gametosit dibandingkan pada MBS awal dan akhir. Nested PCR terhadap gen terkait resistensi terhadap artemisinin, kelch13 (pfk13), dilakukan pada isolat P. falciparum. Faktor risiko pengandung gametosit dinilai dengan berbagai variabel, yaitu usia, jenis kelamin, demam, riwayat demam dalam seminggu terakhir, infeksi paten, dan jumlah DNA plasmodium. Hasil: Pada MBS awal dan akhir prevalensi parasitemia P. falciparum tidak menunjukkan perbedaan bermakna (6%=52/811 vs. 7%=50/740, OR & IK95%=1,06; 0,69-1,61). Sedangkan prevalensi parasitemia P. vivax menunjukkan adanya penurunan bermakna (24%=192/811 vs. 19%=142/740, OR & IK95%=0,77; 0,59-0,98*). Tidak ditemukan perubahan proporsi pengandung gametosit untuk P. falciparum (50%=19/38 vs. 62%=23/37, OR & IK 95%=1,64; 0,60-4,56) maupun P. vivax (39%=48/124 vs. 36%=38/104, OR & IK 95%=0,91; 0,51-1,62). Selain itu, tidak ditemukan perbedaan jumlah gametosit pada MBS awal dan akhir (pfs25: 132,5 vs. 210,9; rasio rerata & IK 95%=0,6; 0,1-4,2; pvs25: 54,0 vs. 68,0; rasio rerata & IK 95%=0,8; 0,2-2,9). Walaupun demikian, 56-87% subjek positif (parasit/gametosit) pada MBS akhir menunjukkan hasil negatif pada MBS awal. Pada kelompok ini jumlah gametositnya adalah 141,1 (39,4-504,7) untuk pfs25 dan 49,7 (18,1-136,6) untuk pvs25. Tidak ditemukan mutasi pfk13 pada isolat P. falciparum yang diperiksa. Kelompok usia < 15 tahun merupakan kelompok berisiko menjadi pengandung gametosit pada P. falciparum maupun P. vivax. Selain itu, riwayat demam juga diidentifikasi sebagai faktor risiko untuk gametositemia P. falciparum, sedangkan densitas parasit tinggi merupakan faktor risiko pada P. vivax. Kesimpulan: Di wilayah penelitian tidak terjadi penurunan prevalensi infeksi dan pengandung gametosit P. falciparum dan P. vivax selama intervensi MBS walaupun tidak ditemukan mutasi pada gen marker terkait resistensi terhadap artemisinin pfk13. Keadaan dinamis parasitemia dan gametosit kemungkinan menyebabkan sulitnya mendeteksi sumber infeksi sehingga perlu dipertimbangkan strategi yang lebih agresif untuk menurunkan jumlah reservoar, misalnya Mass Drug Administration (MDA).
Kata Kunci: Gametosit, pfs25, pvs25, MBS, pfk13, faktor risiko pengandung gametosit.


Background: Gametocytes are the infective stages of Plasmodium spp. which can be used as indicator for transmission. Detection of this stage requires molecular-based examination as microscopic examination was proven less sensitive. This study aims to analyze gametocyte carriers and risk factors during MBS intervention. Moreover, we also seek for any mutation in artemisinin resistance marker gen pfk13. Methods: This is a longitudinal study nested within the MBS trial in 2013 in Belu Regency, NTT. RT qPCR targeting pfs25 and pvs25 marker was utilized to detect gametocyte in P falciparum- and P. vivax-positive subjects based on microscopy and/or PCR. The prevalence of parasite (microscopic and PCR) and gametocyte at baseline and endpoint were compared. Furthermore, nested PCR targeting kelch13 gene (pfk13) was performed to all P. falciparum isolates. Lastly, the risk factors of gametocytes were assessed with various variables, i.e. age, gender, fever, fever history in the past week, patent infection, and Plasmodium DNA numbers. Result: There was no significant difference in the prevalence of P. falciparum infection between baseline and endpoint (6%=52/811 vs. 7%=50/740, OR & 95%CI=1.06, 0.69-1.61). However, P. vivax prevalence showed a significant decrease (24%=192/811 vs. 19%=142/740, OR & 95%CI=0.77, 0.59-0.98*). Furthermore, there was no change in the gametocyte carriage rate between baseline and endpoint either for P. falciparum (50%=19/38 vs. 62%=23/37, OR & 95%CI=1.64, 0.60-4.56) or P. vivax (39%=48/124 vs. 36%=38/104, OR & 95%CI=0.91, 0.51-1.62). No difference was found either in the gametocyte density between the two time points (pfs25: 132.5 vs. 210.9, ratio of geometric mean & 95%CI=0.6, 0.1-4.2; pvs25: 54.0 vs. 68.0, ratio of geometric mean & 95%CI=0.8, 0.2-2.9). Nevertheless, 56-87% of parasite/gametocyte positive subjects at endpoint had been negative at baseline. In this group the number of gametocytes were 141.1 (39.4-504.7) for pfs25 and 49.7 (18.1-136.6) for pvs25. No pfk13 mutations was found in P. falciparum isolates examined. Age < 15 years was identified as risk factor for gametocyte positivity in both species. In addition, history of fever was also found as a risk factor for P. falciparum gametositemia, while high parasite density was a risk factor in P. vivax. Conclusion: In this study area, similar parasite and gametocyte prevalence was observed in P. falciparum and P. vivax during MBS intervention. No mutations were found in artemisinin resistance marker gene, pfk13. The dynamic condition of parasitemia and gametocyte might make it difficult to detect the infection sources, pointing to a need for a more aggressive strategy to reduce the reservoirs pool, e.g. Mass Drug Administration (MDA).
Key word: Gametocyte, pfs25, pvs25, MBS, pfk13, gametocyte risk factors.