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Journal : Berkala Ilmu Kedokteran

Standardization of Immunocytochemical method for the diagnosis of Dengue Viral Infection in Aedes aegypti Linn Mosquitoes (Diptera Culicidane) Umniyati, Sitti Rahmah
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 41, No 01 (2009)
Publisher : Universitas Gadjah Mada

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Abstract

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
Standardization of Immunocytochemical method for the diagnosis of Dengue Viral Infection in Aedes aegypti Linn Mosquitoes (Diptera Culicidane) Umniyati, Sitti Rahmah
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 41, No 01 (2009)
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

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
Organophosphate insecticide susceptible test and transovarial transmission detection of dengue virus on Aedes aegypti in Kendari Saranani, Muhaimin; Umniyati, Sitti Rahmah; Satoto, Tri Baskoro Tunggul
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 45, No 04 (2013)
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (968.716 KB) | DOI: 10.19106/JMedScie004504201303

Abstract

Dengue Fever (DF) is a disease caused by the dengue virus that transmitted by Aedes aegypti(Ae. Aegypti) and Ae. albopictus. Dengue fever is now one of the most important public healthproblems in Indonesia. Vector control using insecticides is the most important strategy to controlthe DF. Massal fogging and selective abatisation have implementedd intensively to control Ae.aegypti. However after its a long time implementation, mosquitoes resistance and transovarialtransmission have been reported. The aim of the study was to evaluate the susceptibility statusof Ae. aegypti to organophosphate and its ability to transovarial transmit degue virus in KendariCity, South East Sulawesi. This was a observational study using cross-sectional design conductedin high endemic areas (Kadia Village) and low endemic areas (Kambu Village). Susceptibilitystatus of Ae. aegypti larvae from F1’s egg generation was evaluated by biochemic assay, whereasthe transovarial transmission of dengue virus of adult female Ae. aegypti was evaluated byimmunohistochemistry method using head squash preparation. The results showed that the Ae.aegypti larvae resistant was higher in high endemic areas (Kadia Village) (83.33%) than in lowendemica areas (Kambu Village) (60.00%). In addition, transovarial transmission index (TTI) ofAe. aegypti dengue virus in the high endemic areas (26%) was significantly higher than in thelow endemic areas (12%) (p<0.05). In conclusion, the Ae. aegypti larvae resitance toorganophosphate as well as the TTI in high endemic areas is higher than in low endemic area inKendari, Sout East Sulawesi.
Insecticide resistance and mechanisms of aedes aegypti (Diptera: Culicidae) in Yogyakarta, Indonesia Mulyaningsih, Budi; Umniyati, Sitti Rahmah; Satoto, Tri Baskoro Tunggul; Diptyanusa, Ajib; Nugrahaningsih, Dwi Aris Agung; Selian, Yahiddin
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 50, No 1 (2018)
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (344.745 KB) | DOI: 10.19106/JMedSci005001201803

Abstract

For several decades, applications of organophosphates and pyrethroids insecticides have been extensively used to control Aedes aegypti as the primary dengue vector. Hence it is important to study dengue vector resistance status and its mechanisms in relation to long term use of insecticides. This study aimed to determine the resistance status and to characterize mechanisms of Ae. aegypti to organophosphates and pyrethroids using biological, biochemical and molecular assays. Larvae and puppae of Ae. aegypti were collected in the field of Plosokuning, Minomartani, Sleman, Yogyakarta, Indonesia. The biological assay was carried out using CDC Bottle Bioassay to test the resistant status to malation and cypermetrin. The biochemical assay was conducted using microplate assay with substrate α-naphthyl acetate to test the presence of esterase elevated activity, and the molecular assay was done using PCR with AaSCF7 and AaSCR7 primer to detect of point mutation at 1534 site, that located in the area of segment 6 of domain III. The biological assay showed Ae. aegypti suggests the possibility of resistance to malathion (mortality 82%) that needs to be confirmed further and already resistant to cypermetrin (mortality 76%). The biochemical assay of Ae. aegypti showed the presence of non-specific esterase elevated activity. The PCR method showed specific DNA bands were formed with the size of 748bp, and with sequencing showed there has been F1354C point mutation of voltage gated sodium chanel gene in the area of segment 6 of domain III. Long term use of insecticides did not successfully eliminate the targeted dengue vector, because Ae. aegypti mosquitoes were resistant to both insecticides. The results demonstrate the importance of designing better health policies regarding insecticide usage