Effect of temperature and storage duration of Aedes aegypti mosquito specimens artificially infected with dengue-3 virus on the results of immunohistochemical examination

Abstract

To confirm the presence of any dengue viral in a mosquito, mosquito’s head can be squashed ona slide and stained with immunohistochemical staining. The remaining samples then can bestored in the cryo freezer at -80ºC to avoid specimen damage. However, for laboratories withlimited facilities, with only a refrigerator with a temperature range of -20ºC to 4ºC is available,examination to evaluate whether the dengue antigen can still be detected in specimens storedat these temperature is necessary. It was a quasi-experimental study. Three to five-day-old adultfemale Aedes aegypti mosquitoes were injected intrathoracically with dengue-3 (DENV-3) strainH-87 virus and then maintained for about 7 days. The dengue viral antigen on mosquitoes wasidentified using immunohistochemical method after stored at a temperature of 4oC, -20oC, -80oCfor 2, 4 and 8 weeks. Mosquito specimens that were not stored were used as a positive control.Kappa value was counted to analyze level of agreements between two observers. Two-wayAnova was used to analyze mean positive rates. Kappa value showed poor agreement (0.00-0.16) between two observers when the specimens were stored at 4oC for 2-8 weeks, and showedgood agreement (Kappa value of 0.77), when stored at -20oC for 4 weeks. The kappa valueshowed very good agreement (0.90-0.92) when the specimens were stored at -20oC for 2 weeks,and at -80oC for 2-8 weeks. Mean positive rates of the specimens stored at 4oC were significantlylower (p0.05). In conclusion, availability of thedengue viral antigen on mosquito specimens was influenced by temperature and storage durationof the specimens.Keywords: dengue virus - head squash - immunohistochemistry - kappa agreement - temperatur