Molecular diagnosis and epidemiology of dengue virus infection

Abstract

Early diagnosis of dengue fever contributes towards appropriate management of the disease and its potentially severe complications. Better and more rapid molecular procedures are increasingly available for detecting dengue viral RNA, antibodies and antigens. Using consensus primers based on the conserved non-structural-3 gene, the reverse transcription-polymerase chain reaction (RT-PCR) technique can amplify all four dengue virus types as well as certain flaviviruses. Consensus primers used together with four type-specific downstream primers in single-step or semi-nested RT-PCR formats can discriminate the specific dengue virus type by virtue of the diagnostic size of the RT-PCR target fragment on agarose gel electrophoresis. Alternatively, RT-PCR products may be labelled with digoxigenin and allowed to hybridise with individual biotinylated type-specific PCR primers which act as capture probes immobilised on solid phase via streptavidin-coated tubes. With automated instrumentation for enzyme-linked immunosorbent assay (ELISA), the hybridised RT-PCR products can be quantitated spectrophotometrically via anti-digoxigenin antibodies conjugated with an enzyme which reacts with colourimetric substrate. While RT-PCR is highly sensitive, specific and successfully identifies the dengue virus type in clinical serum samples and adult Aedes mosquitoes, it generally yields positive results in viraemic sera collected within 2 to 5 days of pyrexia. Sera obtained after the period of viraemia are more likely to be positive by serological tests such as IgM capture ELISA or the commercial Dengue Blot kit. The RT-PCR primers can also be utilised for direct cycle dideoxy DNA sequencing to monitor the molecular epidemiology and evolution of geographically and temporally separated virus strains. To exemplify this, nucleotide and amino acid sequence data as well as phylogenetic trees of several strains of dengue 1 and 2 viruses from patients and field-caught Aedes mosquitoes are presented.