Sitti Rahmah Umniyati
Department of Parasitology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

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PEMERIKSAAN VIRUS DENGUE-3 PADA NYAMUK Aedes aegypti YANG DIINFEKSI SECARA INTRATHORAKAL DENGAN TEKNIK IMUNOSITOKIMIA MENGGUNAKAN ANTIBODI DSSE10

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

Gambaran Leukosit dan Histologi Hepar, Ren, dan Lien Mencit BALB/c (Mus musculus) yang Terinfeksi Virus Dengue 3 (DEN-3) dengan Imunohistokimia

Jurnal Biomedika Vol 9 No 1 (2016): Jurnal Biomedika
Publisher : Fakultas Ilmu Kesehatan Universitas Setia Budi

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Pengamatan efek infeksi virus dengue 3 (DEN-3) terhadap leukosit dan histologi dari organ dalam (hepar, ren dan lien) dilakukan untuk mendukung pengembangan vaksin dan antiviral virus DEN 3. Penelitian dilakukan pada mencit BALB/c dengan pertimbangan mencit merupakan hewan coba yang secara anatomi dan fisiologi mirip dengan manusia dan hewan ini relatif mudah ditangani dan mudah berkembang biak. Untuk serotipe virusnya dipakai serotipe DEN-3, karena serotipe ini merupakan serotipe yang dominan (50 % kasus DBD di Indonesia disebabkan oleh serotipe ini) dan banyak berhubungan dengan kasus berat. Design penelitian ini adalah eksperimental. Pengamatan yang dilakukan antara lain : keberadaan antigen dengue pada darah (leukosit) dan organ (hepar, ren dan lien) beserta perhitungan infection rate-nya, dan histopatologi organ (hepar, ren dan lien). Infeksi dilakukan secara intra vena. Keberadaan antigen dengue diamati dengan metode Imunohistokimia (IHC). Pengamatan histopatologi dilakukan dengan pewarnaan Hemotoxylin Eosin. Penelitian untuk keberadaan antigen dengue, membuktikan bahwa virus DEN-3 dapat ditemukan pada darah (leukosit) dan organ-organ mencit (hepar, ren dan lien). One-Sample Kolmogorov-Smirnov Test digunakan untuk melihat data terdistribusi normal atau tidak. Hasilnya menunjukkan nilai p=0,289, ini menunjukkan data rerata infection rate terdistribusi normal (p>0,05), sehingga analisis dilanjutkan dengan uji Oneway anova untuk melihat ada tidaknya perbedaan yang signifikan diantara data. Hasil Anova menunjukkan ada perbedaan yang signifikan pada rerata infection rate leukosit (p=0,045; p<0,05). Perhitungan infection rate pada darah mencit yang dianalisa menggunakan Multiple Comparisons Post Hoc, diperoleh hasil perbedaan paling signifikan terlihat pada rerata infection rate hari ke 10 p.i. (pasca infeksi) dengan rerata infection rate hari ke 13 p.i. (p=0,007), ini menunjukkan rerata infection ratepaling tinggi terlihat pada hari ke 10 p.i. dan paling rendah pada hari ke 13 p.i. Pada pengamatan histopatologi hepar mencit perlakuan menunjukkan adanya keadaan patologis berupa nekrosis, degenerasi melemak pada hepatosit, infiltrasi limfosit disertai perdarahan dan banyak sel binukleat. Pada ren mencit perlakuan terdapat infiltrasi limfosit, degenerasi melemak dan perdarahan. Pada lien mencit perlakuan menunjukkan pulpa merah yang lebih luas dan eritrosit dengan jumlah lebih banyak dibanding lien mencit kontrol. Pada organ-organ mencit kontrol tidak ditemukan keadaan patologis seperti mencit perlakuan. Virus dengue 3 (DEN-3) dapat ditemukan pada darah (leukosit) dan organ-organ mencit (hepar, ren dan lien). Pada pengamatan histopatologi hepar, ren dan lien mencit perlakuan menunjukkan adanya keadaan patologis diantaranya berupa nekrosis, degenerasi melemak, infiltrasi limfosit dan perdarahan.

Sensitivity and specificity of immunocytochemical assay for detection of Dengue virus 3 infection in mosquito

Health Science Journal of Indonesia Vol 2, No 2 Des (2011)
Publisher : Badan Penelitian dan Pengembangan Kesehatan

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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). 

UJI AKTIVITAS REPELAN MINYAK ATSIRI JAHE EMPRIT (Zingiber officinale Roxb. “Cochin Ginger”) DAN JAHE MERAH (Zingiber officinale Roxb. var rubrum) DENGAN BASIS MINYAK WIJEN DAN MINYAK KELAPA TERHADAP NYAMUK Aedes aegypti

Majalah Obat Tradisional Vol 19, No 2 (2014)
Publisher : Faculty of Pharmacy, Universitas Gadjah Mada

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Nyamuk Aedes aegypti merupakan vektor penyakit Demam Berdarah Dengue (DBD). Penggunaan repelan sintetik menimbulkan masalah bagi kesehatan. Penelitian ini menggunakan minyak atsiri jahe emprit (A) dan minyak atsiri jahe merah (B), sebagai repelan nyamuk Aedes aegypti. Peningkatan aktivitas repelan diupayakan dengan memformulasikan (A) dan (B) dengan basis minyak wijen dan minyak kelapa. Tujuan penelitian adalah membandingkan aktivitas repelan antara (A) dengan (B), mengetahui pengaruh minyak basis terhadap aktivitas repelan (A) dan (B), mengetahui konsentrasi efektif minyak atsiri dalam minyak basis sebagai repelan. Isolasi minyak atsiri menggunakan destilasi air dan uap air. Pemeriksaan komponen minyak atsiri secara kualitatif dan kuantitatif menggunakan GC-MS. Aktivitas repelan diuji melalui 3 tahap yaitu : inisiasi, penentuan konsentrasi efektif, dan aktivitas repelan konsentrasi efektif. Dilakukan pengolesan minyak atsiri konsentrasi tertentu (100%, 50% dalam basis, konsentrasi efektif hasil Simplex Lattice Design) dan minyak sereh Caplang® (kontrol positif) pada punggung tangan. Tangan dimasukkan ke dalam sangkar berukuran (20x20x20) cm3 berisi 25 ekor nyamuk Aedes aegypti betina. Jumlah nyamuk hinggap dicatat untuk menghitung daya proteksi. Waktu pertama kali nyamuk menggigit dicatat sebagai lama proteksi. Analisis data menggunakan One-way ANOVA dilanjutkan Post Hoc Test Tukey HSD (Homogeneous subsets) atau Kruskal-Wallis dilanjutkan Mann-Whitney Test. Hasil penelitian (A) dan (B) mengandung kamfen, mirsen, 1,8-sineol, l-linalool, l-borneol, neral, geraniol, sitral, dan α-kurkumen (hanya pada A), yang secara kuantitatif berbeda. Hasil uji statistik menunjukkan bahwa (B) memiliki aktivitas repelan yang lebih tinggi dibandingkan dengan (A). Basis minyak wijen memberikan pengaruh yang lebih baik terhadap aktivitas repelan minyak atsiri jahe dibandingkan dengan basis minyak kelapa. Konsentrasi efektif minyak jahe merah dan jahe emprit dalam basis minyak wijen, minyak kelapa, berturut-turut adalah 75, 85, 85 dan 90%.

Standardization of Immunocytochemical method for the diagnosis of Dengue Viral Infection in Aedes aegypti Linn Mosquitoes (Diptera Culicidane)

Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 41, No 01 (2009)
Publisher : Universitas Gadjah Mada

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methods for virus detection in the mosquito, such as the direct fluorescent-antibody test on head squashes. However, it has the disadvantages of being labor-intensive and requiring fluorescent microscope as well as cryofreezer. Newer methods involving enzyme conjugates such as peroxidase in conjunction with either polyclonal or monoclonal antibodies are greatly improved. With new methods of immunocytochemistry, it is now possible to detect dengue viral antigen in a variety of tissues. Objective: This study was aimed to standardize an immunocytochemical streptavidin-biotin-peroxidasecomplex assay for diagnosis of dengue infection in Aedes aegypti using monoclonal antibody DSSC7. Methods: The infected mosquitoes were held in small cylindrical cages covered with mosquito netting, and incubated at 27:t 1°C ami at relative humidity of 88:t 6 %. The specificity of the immunocytochemical procedure was validated by negative and positive controls showing that the antibody was bound to an appropriate structure. The sensitivity and specificity were also evaluated based on Herrmanns Formula. The presence of dengue antigen on head squash preparation was detected based on ISBPC assay using monoclonal antibody against dengue. The validity and reliability of the measurement were evaluated based on kappa values, according to Landis and Koch. Result: Positive result was detected as discrete brownish granular deposits throughout most visual fields of brain tissue. Dengue viral antigen was immunolocalized to the cytoplasm of brain cells. The immunocytochemical test under light microscope at magnification of 400x was 86.67% sensitive, 96.00% specific, and the kappa value is 0.64. Meanwhile the kappa value between two observers was 0.92, with sensitivity and specificity of 96% and 97% respectively at magnification of 1000x. Conclusion: The monoclonal antibody DSSC7 was sensitive, specific, valid, and reliable as primary antibody to detect dengue viral infection in Ae. aegypti head squash preparation based on immunocytochemical streptavidin-biotin-peroxidase-complex assay under light microscope. Key words: antigen - denguevirus - Aedes aegypti - immunocytochemistry - monoclonal antibody DSSC7

Head Lice Infestation and Its Relationship with Hygiene and Knowledge among Urban School Children in Yogyakarta

Tropical Medicine Journal Vol 1, No 1 (2011): Tropical Medicine Journal
Publisher : Fakultas Kedokteran bekerjasama dengan PETRI

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Introduction: Pediculosis capitis is a common infestation in school children and is a worldwide public health concern. Despite this worldwide spread, incidence studies remain few. There is also a misconceptionthat head lice infestation is rarely found in the urban environment.Objectives: The objective of this study is to determine the proportion of head lice infestation among urban school children in Yogyakarta and study its relationship with hygiene and knowledge.Methods: This is a cross-sectional study conducted upon 2 schools in Yogyakarta on 154 pupils. Children range from ages 9 to 13 years. Data was collected from the schools by trained persons. A detailed questionnaire was fi lled for each pupil prior to hair examination.Results: In this research, we report the proportion of head lice infestation among urban school children in Yogyakarta and assess its risk factors. Nineteen pupils (12.3%) were infected with head lice, 5 boys(6.3%) and 14 girls (18.7%). There was a signifi cant relationship between head lice infestation and sex (P<0.05). However, no signifi cant relationship was found between head lice infestation with hygiene andknowledgeConclusion: The results show a total proportion of 12.3% among urban school children in Yogyakarta.Keywords: pediculosis capitis, urban, hygiene, knowledge, school children.

Variations in Dilution of DSSE 10 Antibody in Immunocytochemistry Technique to Detect Dengue-3 Virus in Aedes aegypti Mosquitoes

Tropical Medicine Journal Vol 1, No 1 (2011): Tropical Medicine Journal
Publisher : Fakultas Kedokteran bekerjasama dengan PETRI

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Introduction: Dengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes mosquitoes. The most important vector of Dengue virus is the mosquito Ae. aegypti, which should be the main target of surveillance and control activities. Virologic surveillance for Dengue viruses in its vector has been used as an early warning system to predict outbreaks. Detection of Dengue virus antigen in mosquito head squash using immunocytochemical streptavidin biotin peroxidase complex (SBPC) assay is an alternative method for Dengue vector surveillance.Objectives: The study was aimed to compare several variations of MAb DDSE10 dilutions used in immunocytochemical SBPC assay to detect Dengue virus infection in head squash of Ae. aegypti.Methods: The study design was experimental. Artifi cially-infected adult Ae. aegypti mosquitoes of DENV 3 were used as infectious samples and uninfected adult Ae. aegypti mosquitoes were used as normal ones. The immunocytochemical SBPC assay using monoclonal antibody DSSE10 with 4 variations of dilution (1:5, 1:10, 1:20, and 1:50) was applied on mosquito head squash to detect Dengue virus antigen. The results were analyzed descriptively.Results: All variants of MAb DSSE10 dilutions in immunocytochemical SBPC assay showed positive imunoreaction in infected mosquito head squash. All variants of MAb DSSE10 dilutions in immunocytochemicalSBPC assay showed negative immunoreaction in uninfected mosquito head squash.Conclusion: Monoclonal antibody DSSE10 could be used in immunochemistry technique to detect Dengue-3 virus antigen in Aedes aegypti infected intrathoracally, with 1:50 dilution.Keywords: Aedes aegypti, Dengue virus, Immunocytochemical, SPBC, Monoclonal Antibody DSSE-10

Transovarial Transmission Index of Dengue Virus on Aedes aegypti and Aedes albopictus Mosquitoes in Malalayang District in Manado, North Sulawesi, Indonesia

Tropical Medicine Journal Vol 1, No 2 (2011): Tropical Medicine Journal
Publisher : Fakultas Kedokteran bekerjasama dengan PETRI

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Introduction: Dengue Hemorrhagic Fever (DHF) is an infectious vector-borne disease caused by Aedes sp mosquitoes still cause serious health problem in Indonesia. Based on Manado Health Office Report, Malalayang was identified as dengue-endemic areas. In 2010, number of DHF cases in Malalayang is 211 cases with Incidence Rate (IR) 328 per 100,000 populations. Dengue viruses (DENV) survive in nature by two mechanisms; by horizontal transmission through infected vertebrates and mosquitoes, and by vertical (transovarial) transmission in the mosquitoes. Transovarial transmission is assumed as an important aspect in the maintanance of DENV during inter epidemic, but this problem has not been studied in Malalayang District, Manado. An effort to prevent and control DHF requires knowledge of an Aedes sp Dengue virus transovarial infection.Objectives: To prove the existence of Dengue virus transmission in Ae. aegypti and Ae. albopictus mosquitoes and its relationship with the incidence of DHF in Malalayang District in Manado, North Sulawesi, Indonesia.Methods: The method of this research was an observational analytic study with cross-sectional design. Study samples were unbloodfed Aedes aegypti and Aedes albopictus mosquitoes on the F1 generation from ovitrap placed in five selected villages based on the number of cases in the District Malalayang. The secondary data of DHF patients from Malalayang district was obtained from Health Office Manado and the Community Health Center in 2010. The presence of dengue antigen in head squashes preparation were detected using monoclonal antibody against dengue (DSSE10) based on immunohistochemical streptavidin biotin peroxidase complex (ISBPC) technique to confirm the presence of transovarial transmission of dengue virus both in Ae. Aegypti and Ae. Albopictus, and to obtain the data of transovarial transmission index. Fisher’s Exact test and Pearson correlation are used to analyze those data.Results: Transovarial transmission of Dengue virus in Aedes sp was found from 5 villages in Malalayang district with Transovarial Transmission Index (TTI) ranges 6.1%-17.1%. Statistic test showed significant differences in positive rate (p-value=0.00<0.05) on Ae. aegypti higher than Ae. albopictus. It is also known that there is no statistically significant correlation (p-value=0.528>0.05) between the Aedes sp. Dengue virus TTI and DHF IR in Malalayang district.Conclusion: This study demonstrates the existence of Dengue virus transovarial transmission in Aedes sp in Malalayang district. Ae. aegypti’s TTI is higher than that of Ae. Albopictus, and no significant correlation between TTI and DHF IR in Malalayang district.Keywords: DHF, transovarial transmission, Ae. aegypti, Ae. albopictus

The Differences of the Prevalences and Serotypes of Dengue Virus on Aedes Aegypti Mosquitoes from Pagutan and Pagutan Timur in the Sub District of Mataram

Tropical Medicine Journal Vol 2, No 1 (2012): Tropical Medicine Journal
Publisher : Fakultas Kedokteran bekerjasama dengan PETRI

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Introduction: Sub district of Mataram is one of endemic area of Dengue Haemoraghagic Fever in the West of Nusa Tenggara province, the number of dengue cases are reported increasing every years. Pagutan is a village that has been reported as higth case incidance area and Pagutan Timur as low case insidance area.Objectives: The aims of this study is to determine the density of the Aedes aegypti mosquitoes and to know the prevalence and dominant serotypes of dengue virus in the Aedes aegypti mosquitoes from the village of Pagutan and Pagutan Timur Mataram sub district.Methods: The Collection of Aedes aegypti mosquitoes were conducted by ovitrap provided indoor and outdoor of randomly selected houses. The mosquitoes density was determined by calculating the average number of mosquitoes emerged from eggs collected each houses. The prevalence of dengue virus were calculated by the percentage of mosquitoes that containing of dengue virus as examined by immunocytochemistry method, using the monoclonal antibody anti-dengue DSSE10, while dengue virus serotypes determined by Reverse Transcriptase-Polymerase Chain Reaction using Lanciotti specific primers.Results: The density of Aedes aegypti mosquitoes from Pagutan is significantly higher than Pagutan Timur Village (p<0.05). The confirmed statistical analysis with Paired t test were obtained. The prevalence of dengue virus in Pagutan and Pagutan Timur are 18.4% and respectively 14.3%. The serotypes of dengue virus in Pagutan areas is dengue 1, dengue 2 and dengue 3, whereas in Pagutan Timur areas is dengue 1.Conclusion: There is diffences of mosquitoes density, prevalence and the serotypes of dengue virus found in Aedes aegypti mosquitoes from Pagutan and Pagutan Timur in the sub district of Mataram.Keywords: Mosquito density, Aedes aegypti, dengue virus, prevalence and serotype

Laboratory Testing of Transovarial Transmission in Aedes Aegypti Mosquito

Tropical Medicine Journal Vol 2, No 1 (2012): Tropical Medicine Journal
Publisher : Fakultas Kedokteran bekerjasama dengan PETRI

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Introduction: Ae. aegypti is one of the vectors causing Dengue Hemorrhagic Fever (DHF) that feeds on human blood suffering from DHF and transmit it to another healthy human. The ability of Dengue Virus (DENV) to survive in nature is due to 2 mechanisms i.e. horizontal transmission among viremic vertebratae infected by Aedes mosquitos and vertical (transovarial) transmission from infected female mosquito to another generation.Objectives: To know the differences between next generation’s viral carriage, percentage ofmosquito descendants carrying DENV, and the distribution of DENV in its infected mosquitos.Methods: Study design was a quasi experimental posttest-only design with control group. The study was conducted on 53 second-generation female Ae. aegypti mosquitos which are grouped into treatment and control groups. Dengue Viral antigen in both groups were detected by immunohistochemistry method using Streptavidin Biotin Peroxidase Complex (SBPC) in paraffin embedding preparation of mosquitos aged 1-7 days. Fisher exact test was used to compare proportion of virus detected in moaquitos between 2 groups.Results: The virus detected in second generation of DEN-infected and uninfected mosquitos were 100% and 85.7%, respectively. The virus was distributed from day 1 to 7 in all organs such as brains, thorax, salivary gland, gastric epithelial cells, and ovarium of the infected mosquitos. Similar distribution was not detected in control mosquitoes.Conclusion: There was no difference in next mosquito generation’s transovarial infection among DENV-infected and uninfected Ae. aegypti mosquitos.Keywords: Ae. aegypti mosquito; Dengue virus (DENV); transovarial transmission; immunohistochemistry method, Streptavidin Biotin Peroxidase Complex (SBPC).