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"ABSTRAKLatar belakang: Zoonosis malaria telah menjadi perhatian komunitas kesehatan dunia setelah adanya laporan kasus di Sarawak pada tahun 2004. Penyakit ini disebabkan oleh parasit malaria satwa primata Plasmodium knowlesi dengan inang alami Macaca fascicularis dan M. nemestrina. Baku emas diagnosis parasit malaria masih berdasarkan pada identifikasi mikroskopik. Selain membutuhkan keahlian yang tinggi, teknik ini terkadang sulit menentukan spesies parasit bila terjadi infeksi campuran dan parasitemia yang sangat rendah. Belakangan diusulkan DNA barcoding, suatu metode identifikasi menggunakan penanda gen sitokrom c oksidase subunit I COI DNA mitokondria untuk spesiasi. Penelitian yang dilakukan bertujuan untuk mengembangkan metode identifikasi spesies parasit menggunakan gen COI sebagai penanda molekul dan mengungkap dasar molekul transmisi zoonosis parasit malaria dengan mempelajari peran gen penyandi protein DARC Duffy Antigen Receptor for Chemokines dan DBP Duffy Binding Protein yang berhubungan dengan invasi sel darah merah.Metode: Verifikasi potensi barcode COI sebagai penanda identifikasi spesies parasit malaria dilakukan dengan studi in-silico, sedangkan validasi penggunaan barcode COI dilakukan dengan analisis sensitivitas dan spesifisitas. Teknologi molekuler PCR-Sequencing dilakukan untuk mengaplikasikan barcode COI pada penapisan parasit malaria di populasi manusia dan satwa primata, serta identifikasi variasi genetik gen penyandi protein DARC dan DBP terutama pada daerah pengikatan ligan parasit dan reseptor inang.Hasil: Studi in-silico menunjukkan bahwa DNA barcoding berpotensi sebagai penanda identifikasi parasit malaria. Primer yang dirancang mengamplifikasi daerah COI sepanjang 670 pb berhasil mengidentifikasi parasit malaria dengan sensitivitas 1 ndash; 3 parasit/ l. Pada penapisan parasit malaria di populasi manusia di Kalimantan Tengah ditemukan 3,34 78/2309 kasus malaria, di mana dua diantaranya adalah kasus malaria knowlesi, yang secara statistik berbeda bermakna bila dibandingkan dengan mikroskopik 2,82 dan 18S rRNA 1,82 . Pada daerah yang sama, penapisan parasit malaria di populasi satwa primata, ditemukan 52,01 168/323 sampel orangutan dan 23,25 10/43 sampel monyet Macaca positif malaria. Spesies parasit yang ditemukan pada orangutan adalah P. species tipe parasit ovale, P. species tipe vivax-cynomolgi, P. species tipe vivax-hylobati dan P. species tipe malariae-inui, sedangkan pada monyet Macaca meliputi P. knowlesi, P. coatneyi, P. inui, juga P. spesies tipe malariae-inui, spesies parasit yang sama ditemukan di orangutan. Studi ini juga menemukan keanekaragaman genetik pada gen penyandi protein Duffy Antigen Receptor for Chemokines manusia maupun satwa primata dan Duffy Binding Protein parasit malaria yang memainkan peran penting dalam invasi parasit malaria.Kesimpulan: Barcode COI dapat secara spesifik dan sensitif mengidentifikasi spesies parasit malaria dan dapat diaplikasikan sebagai alat identifikasi zoonosis malaria. Terdapat variasi genetik gen penyandi protein Duffy Antigen Receptor for Chemokines dan Duffy Binding Protein yang berhubungan dengan invasi sel darah merah.

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ABSTRACTBackground Zoonotic case of malaria had just come to the attention of public health communities after the Sarawak study in 2004. Zoonotic malaria is caused by Plasmodium knowlesi, primarily a simian malaria parasite in wild long tail macaque Macaca fascicularis and pig tail macaque M. nemestrina as the reservoir hosts. The diagnosis of malaria parasites has mainly relied on the microscopic examination. However, this method is labor intensive, requires an experienced microscopist and difficult in identifying mixed infections in very low parasitemia cases. Recently, DNA barcoding system, which is based on the PCR amplification of a short and highly conserved region of mitochondrial cytochrome c oxidase sub unit I COI has shown to be an invaluable tool for diagnosing and differentiating the species of wide range of organisms. This study was aimed to develop identification tools of malaria parasite by using mtDNA COI gene as a molecular marker and reveal the molecular basis of zoonotic malaria by identifying the genetic variation of protein coding gene of DARC Duffy Antigen Receptor for Chemokines and DBP Duffy Binding Protein that are related to receptor ligand interaction in red blood cell invasion.Methods In silico study was carried out for verifying the potential of DNA barcoding based on the mtDNA COI gene sequence as a marker identification. Sensitivity and specificity analyses were carried out to validate the use of DNA barcoding for medical diagnosis of parasitic infection. Molecular technology of PCR Sequencing was carried out for screening malaria parasit in human and non human primate population and identifying the genetic variation within protein coding gene of DARC and DBP. Results We have initiated a study to explore the use of DNA barcoding for malaria parasite diagnosis through in silico study. We have thus designed primers spanning a 670 bp fragment of the 5 rsquo region of COI gene that could detect parasite isolates as low as 1 3 parasite per l. DNA barcode was used to detect malaria parasite in human population in Central Kalimantan. Of the 2309 subjects, 78 3.34 subjects were malaria positive of which two samples were determined as P. knowlesi infection. The detection rate of COI barcode was significantly higher as compared to microscopic 2.82 and 18S rRNA 1.82 analyses. Of the 366 non human primate samples that include 323 orangutan and 43 macaque 168 orangutan were found to be positive for either P. species ovale type, P. species vivax cynomolgi type, P. species vivax hylobati type and P. species malariae inui type. In macaque, 10 samples were positive for P. knowlesi, P. coatneyi, P. inui and P. species malariae inui type similar to that found in orangutan. The study has also found genetic variation in both human and non human primates Duffy Antigen Receptor for Chemokines and malaria parasite Duffy Binding Protein.Conclusions The study showed that mtDNA COI can be used to diagnose malaria parasites at very low parasitemia level and applied as a diagnosis tool for identification of zoonotic malaria. There is genetic variation in both human and non human primates Duffy Antigen Receptor for Chemokines and malaria parasite Duffy Binding Protein as major determinants for the invasion of malaria parasite."

"Latar belakang: Intervensi epidemiologi malaria bertujuan mendeteksi dan mengobati reservoir parasit di daerah endemik untuk mengurangi penularan lokal. Gametosit merupakan satu-satunya stadium penular sehingga penelitian ini dilakukan untuk mengevaluasi keberadaan gametosit sebelum dan sesudah intervensi skrining dan pengobatan massal (mass screening and treatment/MST) yang dilakukan selama tahun 2013 di Nusa Tenggara Timur, Indonesia.Metode: RT-qPCR pada transkrip pfs25 dan pvs25 - penanda molekuler gametosit untuk Plasmodium falciparum dan Plasmodium vivax, dilakukan untuk mendeteksi dan mengukur gametosit dalam sampel darah subjek yang terinfeksi P. falciparum dan P. vivax selama studi MST. Keberadaan parasit aseksual dan seksual secara mikroskopis dan submikroskopik pada awal dan akhir MST dibandingkan dengan menggunakan uji proporsi serta uji parametrik dan non-parametrik.Hasil: Prevalensi parasitemia pada P. falciparum tidak menunjukkan perubahan (6%=52/811 versus 7%=50/740, p=0,838), namun sedikit menurun untuk P. vivax (24%=192/811 versus 19%=142/740, p=0,035) antara awal dan akhir MST. Tidak ada perbedaan signifikan yang diamati pada prevalensi gametosit baik untuk P. falciparum (2%=19/803 versus 3%=23/729, p=0,353; OR=1,34; 95%CI=0,69-2,63), atau P. vivax ( 7%=49/744 versus 5%=39/704, p=0,442; OR=0,83; 95%CI=0.52-1.31). Meskipun tidak ditemukan perbedaan yang signifikan, sebagian besar subjek positif parasit pada akhir MST memiliki hasil negatif pada awal MST (P. falciparum: 66%=29/44, P. vivax: 60%=80/134) . Hal ini juga ditunjukkan untuk stadium infektif - dimana mayoritas subjek positif gametosit pada akhir MST menunjukkan hasil negatif pada awal MST (P. falciparum: 95%=20/21, P. vivax: 94%=30/32). Hasil ini tidak tergantung pada pengobatan yang diberikan selama kegiatan MST. Tidak ada perbedaan yang ditunjukkan dalam kepadatan parasit dan gametosit antara awal dan akhir MST baik di P. falciparum atau P. vivax.Kesimpulan: Di daerah penelitian ini, tingkat prevalensi parasit dan gametosit P. falciparum dan P. vivax yang sama sebelum dan sesudah MST, meskipun pada individu yang berbeda, menunjukkan tidak adanya dampak pada reservoir parasit. Pemberian pengobatan berdasarkan parasitemia positif yang diterapkan di MST perlu dievaluasi kembali untuk strategi eliminasi di masyarakat.

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Background

A goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas – an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia.

Methods

RT-qPCR targeting pfs25 and pvs25 transcripts - gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests.

Results

Parasite prevalence remained unchanged for P. falciparum (6%=52/811 versus 7%=50/740, p=0.838), and decreased slightly for P. vivax (24%=192/811 versus 19%=142/740, p=0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2%=19/803 versus 3%=23/729, p=0.353, OR=1.34, 95%CI=0.69-2.63), or P. vivax (7%=49/744 versus 5%=39/704, p=0.442, OR=0.83, 95%CI=0.52-1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66%=29/44, P. vivax: 60%=80/134). This was similarly demonstrated for the transmissible stage - where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95%=20/21, P. vivax: 94%=30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax.

Conclusion

In this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community."