Isolation and Serotyping of Dengue Viruses by Mosquito Inoculation and Cell Culture Technique: An Experience in Bangladesh

Dengue virus isolation and serotyping was carried out in 105 clinically-suspected cases of dengue fever by the mosquito inoculation and cell culture techniques to find a suitable virus isolation technique for a resource-constrained country like Bangladesh and to identify the serotypes responsible for the dengue outbreak in 2000. Virus was identified by direct fluorescent antibody technique (DFAT) using FITC-conjugated anti-flavivirus antibody and serotyping was done by indirect fluorescent antibody technique (IFAT) using serotype-specific monoclonal anti-dengue antibody. Of the 105 cases, 97 (92.4%) were found sero-positive for anti-dengue IgM by ELISA and considered as having dengue infection. Dengue virus was isolated from 44 (45.4%) of the 97 anti-dengue IgM positive cases by Aedes aegypti mosquito inoculation and from 24 (24.7%) cases by C6/36 cell culture. The dengue virus isolation rate was significantly higher (p<0.01) by the mosquito inoculation than the cell culture inoculation technique. The isolation rate was also higher in patients who presented on the first day of the fever than in those who presented later. Dengue virus was isolated from patients who had a significantly higher mean body temperature (p<0.01) and the mean anti-dengue IgG level was significantly lower in those from whom virus was isolated (p<0.01). The serotyping revealed isolation of DEN-3 from 31 (70.5%) cases, which was the predominant serotype, followed by the isolation of DEN-1 from 6 (13.6%), DEN-4 from 4 (9.09%) and DEN-2 from 3 (6.8%) cases out of the total 44 positive cases. This indicates the hyperendemicity of dengue in Dhaka with epidemic potential, which needs careful monitoring by laboratory-based surveillance in order to predict the nature of future outbreaks. This is the first report of dengue virus isolation by mosquito inoculation and C6/36 cell culture in Bangladesh. It was revealed from the study that, for routine surveillance of dengue infection, isolation of virus by mosquito inoculation is a more sensitive, economical, easy and sustainable method. However serotyping by IFAT is more convenient in cell culture.