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PEMERIKSAAN VIRUS DENGUE-3 PADA NYAMUK Aedes aegypti YANG DIINFEKSI SECARA INTRATHORAKAL DENGAN TEKNIK IMUNOSITOKIMIA MENGGUNAKAN ANTIBODI DSSE10 Widiastuti, Dyah; Umniyati, Sitti Rahmah; Wijayanti, Nastiti
Balaba Vol 8, No 1 Jun (2012)
Publisher : Prima Offset

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ABSTRACTDengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infectedAedes mosquitoes. The most important vector of Dengue virus is the mosquito Ae.aegypti, which should be the main targetof surveillance and control activities. Virologic surveillance for dengue viruses in its vector has been used as an earlywarning system to predict outbreaks. Detection of Dengue virus antigen in mosquito head squash usingimmunocytochemical streptavidin biotin peroxidase complex (SBPC) assay is an alternative method for dengue vectorsurveillance. The study aimed to develope immunocytochemical SBPC assay to detect Dengue virus infection in headsquash of Ae.aegypti. The study design was experimental. Artificially-infected adult Ae. aegypti mosquitoes of DENV 3were used as infectious samples and non-infected adult Ae. aegypti mosquitoes were used as normal ones. Theimmunocytochemical SBPC assay using monoclonal antibody DSSE10 then was applied in mosquito head squash todetect Dengue virus antigen. The results were analyzed by descriptive analysis. The immunocytochemical SBPC assaycan detect Dengue virus antigen in mosquito head squash at day 2 postinfection. There are some false positive resultsfound in immunocytochemical SBPC assay.Key Word: Dengue, immunocytochemistry, DSSE10
Sensitivity and specificity of immunocytochemical assay for detection of Dengue virus 3 infection in mosquito Widiastuti, Dyah; Yunianto, Bambang; Umniyati, Sitti Rahmah; Wijayanti, Nastiti
Health Science Journal of Indonesia Vol 2, No 2 Des (2011)
Publisher : Badan Penelitian dan Pengembangan Kesehatan

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Abstract

Latar belakang: Survei virologi pada nyamuk vektor dapat digunakan sebagai Sistem Kewaspadaan Dini untuk mencegah penularan Demam dengue di suatu daerah. Pemeriksaan laboratoris untuk deteksi virus Dengue pada nyamuk seperti isolasi virus, Polymerase Chain Reaction (PCR) dan Direct Fluorescent-Antibody (DFA) memerlukan keahlian yang tinggi, peralatan yang mahal dan waktu yang lama. Suatu metode berdasarkan imunositokimia menggunakan antibody monoclonal DSSE10 memiliki beberapa kelebihan. Tujuan penelitian ini untuk mengevaluasi sensitifitas dan spesifitas pemeriksaan imunositokimia dibandingkan metode Reverse Transcription-Polymerase Chain Reaction (RT-PCR) untuk mendeteksi infeksi Virus Dengue 3. Metode: Penelitian eksperimental dilakukan di laboratorium Parasitologi Fakultas Kedokteran Universitas Gajah Mada (UGM) pada bulan Mei 2009-Oktober 2010. Sebanyak 22 Ae. aegypti yang diinfeksi virus Dengue 3 digunakan sebagai  kelompok  infeksius dan  35  nyamuk  yang  tidak  diinfeksi  sebagai  kelompok  non infeksius.  Pemeriksaan imunositokimia  Streptavidin  Biotin Peroxidase  Complex  (SBPC)  menggunakan  antibodi  monoklonal DSSE10 dilakukan pada sediaan head squash Ae .aegypti untuk mendeteksi antigen virus Dengue 3.  Pemeriksaan RT-PCR sebagai baku emas diaplikasikan pada toraks nyamuk.Hasil: Nilai Kappa menunjukkan kesepakatan yang baik antara dua orang pemeriksa (0,63). Imunositokimia mendeteksi antigen virus Dengue-3 dengan sensitivitas yang sama dengan RT-PCR (sensitivitas 100%). Namun spesifisitas IC lebih rendah dibanding RT-PCR (spesifisitas 91%) karena beberapa hasil positif palsu muncul pada pemeriksaan ini. Kesimpulan: Metode IC memiliki nilai sensitivitas dan spesifisitas yang tinggi dibandingkan dengan metode RT-PCR. Metode IC ini dapat digunakan untuk surveilans virus Dengue pada nyamuk vektor. (Health Science Indones 2011;2:87-91).AbstractBackground: Virological  surveillance  provides  an  early warning  sign  for  the  risk  of  transmission  in  an area. Laboratory  tests  for  dengue  virus  infection  on mosquitoes  include  isolation  of  the  virus,  Polymerase Chain Reaction  (PCR)  and  Direct  Fluorescent-Antibody (DFA)  requires  a  high  level  of  technical  skill, expensive equipment,  and  time-consuming.  A  method based  on  immunocytochemical  (IC)  using  monoclonal antibody DSSE10 has several advantages. This study aimed to evaluate sensitivity and specificity IC assay compared with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) as gold standard to detect Dengue Virus (DENV)-3 infections in mosquito Aedes aegypti.Methods: An experimental study was conducted in laboratory of Medical Parasitology, Faculty of Medicine, Universitas Gadjah Mada (UGM) in May 2009 until October 2010. A total of 22 artificially-infected adult Ae.  aegypti  mosquitoes  of  DENV 3  were  used  as  infectious  samples  and  35  non-infected adult  Ae.  aegypti mosquitoes  were  used  as  normal  ones. The  IC  Streptavidin  Biotin  Peroxidase  Complex  (SBPC) assay  using monoclonal antibody DSSE10 was applied in mosquito head squash to detect Dengue virus antigen. RT-PCR as a gold standard was applied in mosquito thorax.Results:  The  kappa  value  showed  a  good  agreement between  two  observers  (kappa  value  0.63).  IC could detect dengue virus antigen as sensitive as RT-PCR (sensitivity 100%). But IC was less specific than RT-PCR (specificity 91%) because some false positive results were found in this method.Conclusion: The IC method has a high sensitivity and high specificity compared with RT-PCR. This IC method may  be useful  for  virological  surveillance  of  dengue  infected Aedes  mosquitoes.  (Health  Science  Indones 2011;2:87-91). 
Aktivitas Sitotoksik dan Apoptosis Ekstrak Spons Spesies A Anggota Ordo Astroporida terhadap Sel HeLa (Cervical Cancer Cell Line) Nuriliani, Ardaning; Agus Ariyanto, Ibnu; Ria Santi, Mei; Mahendra, Andi; Erly Sintya Dewi, Ni Wayan; Nurul Huda, Arif Luthfi; Wijayanti, Nastiti
Biota Biota Volume 18 Nomor 1 Tahun 2013
Publisher : PBI Yogyakarta

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Abstract

Spons merupakan fauna laut yang diketahui memiliki berbagai senyawa bioaktif. Senyawa tersebut berpotensi sebagai antibakteri, antivirus, dan antikanker. Penelitian ini bertujuan mempelajari aktivitas sitotoksik dan apoptosis ekstrak spons spesies A anggota ordo Astrophorida terhadap sel HeLa. Pada penelitian ini pengujian aktivitas sitotoksik ekstrak etanolik, metanolik, dan kloroform spons spesies A terhadap sel HeLa dilakukan menggunakan MTT assay dan uji apoptosis menggunakan double staining, yaitu etidium bromida-acridine orange. Deteksi golongan senyawa yang terkandung di dalam spons spesies A dilakukan menggunakan Kromatografi Lapis Tipis (KLT). Hasil uji sitotoksisitas menunjukkan bahwa ekstrak etanolik, metanolik, dan kloroform spons spesies A masing-masing memiliki nilai IC50 sebesar 18,25; 27,87; dan 13,87 µg/mL. Ekstrak etanolik, metanolik, dan kloroform spons spesies A pada konsentrasi 31,25 µg/mL dapat menginduksi kematian sel melalui apoptosis masing-masing sebesar 35,3 ± 11,16%; 82,64 ± 16,21%; dan 86,76 ± 9,27%. Berdasarkan uji menggunakan KLT diketahui bahwa spons spesies A menggandung golongan senyawa alkaloid, flavonoid, fenol, dan terpenoid. Oleh karena itu, dapat disimpulkan bahwa ekstrak spons spesies A berpotensi untuk dikembangkan sebagai obat antikanker.Kata kunci: ekstrak spons spesies A, sitotoksik, apoptosis, sel HeLa
Molecular Detection of Taura Syndrome Virus Infections in White Shrimp (Litopenaeus vannamei) and Giant Prawn (Macrobrachium rosenbergii) Willisiani, Fariha; Rohmah, Nur; Rahmawati, Irma Nur; Wijayanti, Nastiti
Jurnal Sain Veteriner Vol 31, No 2 (2013)
Publisher : Fakultas Kedokteran Hewan

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Abstract Giant prawn (Macrobrachium rosenbergii) and vaname shrimp (Litopenaeus vannamei) are types of shrimp that became excellent commodities in the fisheries sector. However, one of the obstacles in the vaname shrimp aquaculture is a disease caused by the infection of Taura Syndrome Virus (TSV). One of the consequence of raising the vaname shrimp in Indonesia is the possibility of spreading TSV infection in another shrimp species. TSV infection in giant prawns in Indonesia has not been reported. The aims of this study were: 1. To determine the resistance of giant prawns toward TSV infection and 2. To detect molecularly using RT-PCR technique the presence of viruses TSV on vaname shrimp or giant prawns infected with 3 different doses (0.05 ml; 0.10 ml and 0.15 ml) of TSV inoculum using a pair of specific primers for TSV 9992F (5-AAG CTT GCG TAG ACA GCC-3) and 9195R (5-TCA AGA ATG GCT TCC TGG-3). The research results showed that vaname shrimp mortality infected by 0.05 ml; 0.10 ml and 0.15 ml TSV inoculums were 14.28%, 42.86%, and 57.14%, respectively. Whereas the giant prawns mortality that were infected using the same dose of TSV inoculums were 0%, 8.33%, and 8.33%, respectively. Positive result was detected molecularly only from haemolymph of vaname shrimp infected using 0.15 ml of TSV inoculum. On the other hand, positive results were detected in pleopod and the gill of vaname shrimp infected using 0.05 ml; 0.10 ml or 0.15 ml of TSV inoculums. In giant prawns, infection using 3 different doses of TSV inoculums causes negative result molecularly. Based on all of the facts, it can be concluded that, giant prawns has the higher resistance to TSV infection than that of vaname shrimp.
Epidemiologi, Stadium, dan Derajat Diferensiasi Kanker Kepala dan Leher Wijayanti, Nastiti; Fachiroh, Jajah
Biogenesis Vol 3, No 1 (2015)
Publisher : Jurusan Biologi UIN Alauddin Makassar

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Head and neck cancer is one of the deadly types of cancer in Indonesia. The main cause of this cancer is the consumption of alcohol and cigarettes. Head and neck cancer attacks the lips, mouth, palate, pharynx and larynx. Studies about head and neck cancer have been carried out in westerncountries, while in Indonesia is still limited. Earlier studies in western countries expressed that head and neck cancer is more common in men than women. Purpose of research were to determine the epidemiology of head and neck cancer in Indonesia related to patient ratio of men and women andthe correlation of stage and differentiation level of head and neck cancer. Data were obtained from the Rumah Sakit Kanker Dharmais-Pusat Kanker Nasional Jakarta and then analyzed descriptively. The data were analyzed came from 36 patients with head and neck cancer. The results showed, menwith head and neck cancer as much as 52,77% and females 47,22%. There were 16 cases with stage IV cancer, 9 with stage III, 8 with stage II and 2 with stage I. There were 6 cases of stage IV cancer with better differentiation, and there were 2 cases of stage II cancer with a poor differentiation. Head and neck cancer is more common in men than women. There was no correlation between the degree of differentiation-stage head and neck cancer. It was influenced by immunity of each person.Keywords: differentiation level, epidemiology, head and neck cancer, stage
PREPARATION OF LYMPHOCYTE CULTURE CELL FROM PERIPHERIAL BLOOD OF NASOPHARYNGEAL CARCINOMA PATIENTS Haryanto, Aris; Mubarika, Sofia; Wijayanti, Nastiti
Jurnal Sain Veteriner Vol 18, No 1&2 (2000)
Publisher : Fakultas Kedokteran Hewan

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Lymphocyte is leukocyte component that difficult to culture in vitro. Several viruses need lymphocytes as host cell in order to proliferate and growth in this media such as Epstein-Barr virus (EBV). This virus was associated with malignant disease like nasopharyngeal carcinoma (NPC). The objectives of this research are to develop and to prepare lymphocyte cell culture as material source of DNA for molecular analysis of virus. Peripheral blood was collected from NPC patients which is histopatologically and serologically positive of EBV. Lymphocytes were separated from the other blood components using ficcol-histopaque. Lymphocytes were diluted using RPMt medium then they were cultivated into 96 microwell plate with concentration of 106 cell/ml. The medium consist of 10% FBS, RPMI, Penstrep and FK 506. Culture of lymphocytes were incubated in 5% CO2 at 37°C. The lymphocyte cultures developed and grew confluendy during the first week. Only B cells whith EBV would be well establish. After 50 cell generations, lymphocytes were transformed and immortalized to be lymphoblastoid cell fine (LCL).
Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR) 2 Primers Siregar, Abdul Rahman; Wibawa, Tri; Wijayanti, Nastiti
Indonesian Journal of Biotechnology Vol 16, No 2 (2011)
Publisher : Universitas Gadjah Mada

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Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3). This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM) with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.
Effect of Nuclear Export Inhibitor Leptomycin B on the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells Haryanto, Aris; Wijayanti, Nastiti; Kann, Michael
Indonesian Journal of Biotechnology Vol 15, No 2 (2010)
Publisher : Universitas Gadjah Mada

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Abstract

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of manyregulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants.In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cell
Effect of the HBV Capsid Assembly Inhibitor Bayer 41-4109 on the Intracellular Localization of EGFP-Core Fusion Proteins Haryanto, Aris; Wijayanti, Nastiti; Kann, Michael
Indonesian Journal of Biotechnology Vol 12, No 2 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Bayer 41-4109 is heteroarylpyrimidine (HAP) which has been identified as potent of HBV capsid assemblyinhibitor. The present study was to study effect of Bayer 41-4109 treatment on the intracellular localization ofEGFP-Core fusion proteins into HepG2 cells. Three recombinant plasmids of pEGFP-Core with single, double andtriple NLS of HBV core (EGFP-Core 1C, 2C and 3C ) and two recombinant plasmids with single and triple NLS ofSV-40 (EGFP-Core 1 and 3 SV-40) were used in this work. After transient transfected into HepG2 cells and treatedwith Bayer 41-4109, the intracellular localization of expressed fusion proteins from all plasmid constructions weredetermined and quantified under confocal laser microscope. Results shown that Bayer 41-4109 treatment in HepG2cells inhibited the nuclear localization of EGFP-Core with single of triple HBV core NLS. As well as the constructionsof expressed fusion protein with single and triple SV-40 NLS (EGFP-Core 1 and 3 SV-40 NLS) showeddecreasing the nuclear localization after treated with Bayer 41-4109, even not as strong as EGFP-Core 1C and 3CNLS. Bayer 41-4109 has been identified as a potent inhibitors of HBV replication which has multiple effects on HBVcapsid assembly. It may inhibit virus replication by inducing assembly inappropriately and by misdirectingassembly decreasing the stability of normal capsids.Keywords: HBV capsid, Bayer 41-4109, EGFP-Core fusion protein, HepG2 cell
Molecular Genotyping of HBV by using Nested PCR-RFLP among Hepatitis B Patients in Daerah Istimewa Yogyakarta Province and Surrounding Area Haryanto, Aris; Mulyani, Nenny Sri; Widowati, Titis; Wijayanti, Nastiti; Hadi, Purnomo
Indonesian Journal of Biotechnology Vol 13, No 2 (2008)
Publisher : Universitas Gadjah Mada

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Hepatitis B virus (HBV) can be classified into 8 genotypes, genotype A to H. Genotype of HBV is important for clinical and etiological investigations. Research for HBV genotyping, HBV transmission study using nested PCR and HBV genotyping based on RFLP using restriction enzymes have been reported. However, both of those methods have been not applied for HBV genotyping study among hepatitis B patients in endemic area, like Indonesia yet. Molecular genotyping of HBV will describe epidemiology, pathogenesis and clinical implication of HBV. Combination of nested PCR and RFLP (nested PCR-RFLP) method to determine HBV genotype in Indonesia is still less information. The objectives of study were to develop a system for HBV genotyping by nested PCR combined with RFLP (nested PCR-RFLP) method based on nucleotide sequence of surface protein encoding</div><div>gene (S gene) in HBV genome and to confirm HBV genotypes which predominantly found among hepatitis B patients in Daerah Istimewa Yogyakarta Province and surrounding area. Total of 149 sera from chronic hepatitis B patients from Daerah Istimewa Yogyakarta and surrounding areas were collected for in this work. Viral DNA were extracted from sera of hepatitis B patients and used as template for first round nested PCR amplification using outer primers set. Amplicons of first round PCR were used as template for second round amplification using inner primers set. Then, amplicons of second round nested PCR were restriction digested by Sty I and Bsr I enzymes. For HBV genotyping then the restriction products were analyzed by RFLP based on restriction pattern. Results showed that the first round nested PCR amplification generated DNA fragments of whole S gene in length 1.233 bp, and in second round nested PCR amplification using inner primer set generated DNA fragments 585 bp in length. Genotype analysis for all samples using nested PCR-RFLP methods by restriction digested of Sty I and Bsr I enzymes found only 2 HBV genotypes among hepatitis B patients, namely genotype B and C. Quantification</div><div>data showed that most of hepatitis B patients found infected by HBV genotype B (92,8%), genotype C (3,6%) and unidentified genotype (3,6%). Nested PCR-RFLP methods for HBV genotyping is simple and inexpensive for clinical diagnostic in large scale.