Molecular Diagnosis of Dengue.

Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.

Serological Diagnosis of Dengue.

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).

Impact of the emergence and re-emergence of different dengue viruses' serotypes in Rio de Janeiro, Brazil, 2010 to 2012.

BACKGROUND: Rio de Janeiro (RJ) has been of major importance for the epidemiology of dengue viruses (DENVs) in Brazil. After the DENV 1-4 introductions in 1986, 1990, 2000 and 2011, respectively, the state has suffered explosive epidemics. We aimed to describe laboratorial, epidemiological and clinical aspects due to the emergence and re-emergence of distinct DENV in a 2-year period. METHODS: Suspected dengue cases (n=2833), including 190 fatal cases, were submitted to virus isolation, RT-PCR and non-structural 1 (NS1) antigen capture ELISA, IgM antibody-capture (MAC)-ELISA and IgG-ELISA. RESULTS: Case confirmation was 47.5%. MAC-ELISA confirmed 32.6% of the cases, RT-PCR confirmed 56.3%; DENV was recovered in 33.1% of samples inoculated and NS1 ELISA confirmed 27.5% of the cases. DENV-2 was prevalent in 2010, DENV-1 in 2011 and DENV-4 in 2012. Individuals infected by DENV-3 and over 65 years-old, and children 15 years-old and under infected by DENV-2 had a significantly higher risk of developing a severe disease. Fatal cases confirmed (n=67) were due to DENV-1 (26.8%), DENV-2 (14.9%), DENV-3 (2.9%) and DENV-4 (7.4%). CONCLUSIONS: It has been shown here that viral emergences or re-emergences may play different roles in the disease epidemiology, especially when many serotypes co-circulate.