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 on
a slide and stained with immunohistochemical staining. The remaining samples then can be
stored in the cryo freezer at -80ºC to avoid specimen damage. However, for laboratories with
limited 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 stored
at these temperature is necessary. It was a quasi-experimental study. Three to five-day-old adult
female Aedes aegypti mosquitoes were injected intrathoracically with dengue-3 (DENV-3) strain
H-87 virus and then maintained for about 7 days. The dengue viral antigen on mosquitoes was
identified using immunohistochemical method after stored at a temperature of 4oC, -20oC, -80oC
for 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-way
Anova 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 showed
good agreement (Kappa value of 0.77), when stored at -20oC for 4 weeks. The kappa value
showed 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 significantly
lower (p<0.005) than stored at -20oC and -80oC, but there were no significant differences
between specimens stored at -20oC and -80oC (p>0.05). In conclusion, availability of the
dengue viral antigen on mosquito specimens was influenced by temperature and storage duration
of the specimens.

Keywords: dengue virus - head squash - immunohistochemistry - kappa agreement - temperature