**2. Laboratory diagnosis**

#### **2.1 Isolation of virus**

The two basic methods for establishing a laboratory diagnosis of dengue fever are: the detection of viruses (egg, culture), or detection of IgM antibodies anti-dengue (serology). Blood samples for viral isolation should be collected within five days from the onset of symptoms. Serum is obtained by centrifugation and stored at -70°C.The inoculation of clinical specimens in adult mosquitoes or larvae in the culture technique is more sensitive for the detection of DENV. In laboratories where colonized mosquitoes are not available, samples can be inoculated in any mosquito cell lines available such as C6/36 (*Aedes albopictus* clone) that has a high sensitivity to DENV and other arboviruses.

#### **2.2 Serological diagnosis**

Sera should be collected from patients from the sixth day of illness and stored at -20°C. The MAC-ELISA is an antibody-capture assay of IgM from sera of patients suspected of DF. Briefy, the plate is sensitized with an anti-human IgM, and after various steps of the assay (blocking, dilution, washing, incubation overnight with the pool of antigen (DENV-1, DENV-2 and DENV-3), the presence of specific IgM antibodies to dengue in the patient serum is shown by color change of the substrate that undergo enzymatic action of the conjugate. The color intensity is directly proportional to the amount of IgM antibodies contained in serum (Kuno et al., 1987).
