**2.4 Virological diagnosis**

84 Current Topics in Tropical Medicine

Americas (De Melo et al., 2009). The introduction of this virus in Manaus is probably due to the fact that this is a city with eco-tourism development, and possess an Industrial Center (Free Zone of Manaus), with over 450 factories of large, medium and small size (PORTAL AMAZÔNIA) and many of them are of Asian origin (Figueiredo, 2008). It is possible that the virus has been introduced from an Asian-infected visitor or vector

Natural co-infection with dengue virus can occur in highly endemic areas where several serotypes have been transmitted for many years. Cases of simultaneous infection by more than one species of arboviruses in mosquito or human host were reported (Meyers& Carey, 1967; Gubler et al., 1985). In Brazil, one case of co-infection by DENV-1 and DENV-2 was reported in the patient with classic dengue fever (DF) from Southeastern region through immunofluorescence and RT-PCR (Santos et al., 2003). Another case of co-infection by DENV-2 and DENV-3, was observed in 2005 in the Northeastern region from a patient with DF (Araújo et al., 2006). Simultaneous infection by different strains of dengue virus in mosquitoes and humans underscores the potential for recombination (Santos et al., 2003). Although recombination is rarely recorded in positive-stranded RNA viruses (Lai, 1992), recombination occurrence in picornavirus, coronavirus, and alphavirus have been suggested. The latter viruses are also transmitted by mosquitoes (Hahn et al., 1998.Variations in dengue virus and the occurrence of co-infections with different DENV serotype may lead to genetic exchange between strains increasing the likelihood of

During an outbreak of dengue in São José do Rio Preto, State of São Paulo, 365 samples were positives to DENV-3, 5 samples were to DENV-2, and 8 to Saint Louis encephalitis flavivirus (SLEV). Among the positive samples, one co-infection was detected for DENV-2 and DENV-3. Co-infection of each distint DENV serotype or other flavivirus during dengue outbreaks

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* 

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,

*albopictus* clone) that has a high sensitivity to DENV and other arboviruses.

*A.aegypti*-infected.

**1.5 Co-infection** 

recombination (Kuno, 1997).

**2. Laboratory diagnosis** 

**2.2 Serological diagnosis** 

**2.1 Isolation of virus** 

seems to be common (Terzian et al., 2010).

The technique used to isolate DENV is continuous cell lines of mosquito cells C6/36 (*Aedes albopictus* clone) (Igarashi, 1978), grown in 25 cm flask with growth medium L-15 plus 5% of fetal bovine serum. To 1.5 mL of theL-15 plus 5% of fetal bovine serum containing the C6/36 cells in disposable falcon tubes (15 mL), 70 µl of serum from patients suspected of DF in acute phase was added and incubated at 28° C for two week. Culture media is changed two times per week and incubated. After 10 days of incubation, immunofluorescence (IF) technique is used for the identification of serotypes of dengue.
