Molecular diagnosis and transmission of dengue virus in Sri Lanka

Abstract

Dengue virus is responsible for Dengue Fever (DF), Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). Four circulating dengue serotypes (DEN 1-4) have been identified. Early diagnosis of dengue viral infection helps monitoring the disease, hospital admission when necessary and reduces case fatality. Detection of dengue viruses in mosquitoes is useful to study on transmission of dengue virus. Study on risk factors for dengue is useful to understand spatial and temporal dynamics of transmission the disease. A novel diagnostic assay, Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) was developed. Amplified products of Non Structural-3 (NS3) gene were hybridized with a mixture of the 4 dengue type specific Deoxyribonucleic Acid (DNA) probes in liquid phase. A high sensitivity of 2 fluorescent focus units of dengue virus/reaction was achieved and the assay was highly specific for dengue virus. This novel assay was first validated for clinical specimens in a preliminary retrospective study using serum specimens known to be dengue positive or negative, according to virus isolation method. Secondly, this assay was validated in a prospective study using serum specimens. As an early (<5 days of fever) laboratory diagnostic method for dengue virus, this assay had 100 percent and 46 percent sensitivity for detection of confirmed and suspected dengue patients respectively. This assay was found to be more sensitive than the other diagnostic techniques: virus isolation, enzyme linked immunosorbant assasy, Haemagglutination inhibition assay and molecular assays based on the same NS3 gene; RT-PCR- mixed phase hybridization, RT-PCR agarose gel electrophoresis (AGE), semi nested PCR-AGE and based on other regions of dengue viruses, for early definitive laboratory diagnosis of primary and secondary dengue infection. Patients with definitive dengue correlated only with few signs and symptoms, indicating that laboratory confirmation is critical to avoid over estimation. Fifty four serum samples were typed; DEN-3 (92 percent) and DEN-2 (7 percent) were found and DEN-3 infected patients in the study population had severe clinical manifestations. This novel technique was also validated to be used for detection of dengue virus in vectors, Aedes aegypti and Ae. albopictus. Wild-caught mosquito samples were collected from 136 dengue case-reported stations during outbreaks and a hot-spot during a period of 31 months. This assay could detect dengue virus in 7 percent of Ae. albopictus specimens. A high density of Ae. albopictus was also associated with the dengue case-reported stations/hot-spots. These results therefore demonstrate that Ae. albopictus acts as an important vector of transmission of dengue in some urban and semi-urban areas. Epidemiological, environmental, entomological and other possible risk factors affecting transmission of dengue were analyzed. Monthly total rainfall, relative humidity and mean temperature having a lag period of 3 months moving average ending with current month can be used to forecast an impending dengue epidemic in a selected hot-spot where a clear seasonal pattern of dengue was evident. Other risk factors affecting transmission are presence of Ae. albopictus, previous dengue patients, untidy garden with mosquito breeding sites and shade of the garden made by vegetation.