Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Detection of dengue virus in Aedes albopictus mosquitoes by Reverse Transcription Polymerase-Chain Reaction-Liquid Hybridization (RT-PCR-LH) based assay.(Sri Lanka College of Microbiologists, 2003) Hapugoda, M.D.; Gunasekera, M.B.; de Silva, N.R.; Gunasena, S.; Prithimala, L.D.; Dayanath, M.Y.D.; Abeyewickreme, W.Dengue is an important public health problem. In this study an RT-PCR-LH assay was developed for the detection of dengue virus in Ae.albopictus, a vector of dengue. Laboratory bred Ae.albopictus (adults inoculated with dengue prototypes were tested by RT-PCR-LH assay. RT-PCR products of NS3 gene of 4 dengue prototypes were hybridized in liquid phase with 32P) labelled cocktail of dengue serotype-specific ologonucliotides. Semi-Nested-PCR agarose gel electrophoresis (Semi-Nested-PCR-AGE) assay with dengue type specific oligonucliotides was carried out for typing of RT-PCR products. Wild-caught Ae.albopictus (larvae (n=89 pools) and adults (n=69 pools) collected from dengue case reported stations during the period of 1999-2002 were also tested by RT-PCR-LH and typed by Nested-PCR-AGE assay). A DNA band (470bp) specific for dengue virus was observed in all pools of Ae.albopictus (inoculated with dengue prototypes in RT-PCR-LH assay. When RT-PCR products of dengue prototypes inoculated mosquitoes were typed by Semi-Nested-PCR-AGE assay, bands of 169,362, 265, 426 bp sizes corresponding to DEN1, DEN2, DEN3 and DEN4 respectively were observed. The DNA band specific for dengue virus (470bp) was also observed in 6 pools of wild-caught adults in RT-PCR-LH assay. They were found to be infected with DEN3 (265bp DEN3 specific DNA band was detected) by Semi-Nested-PCR-AGE assay. None of the wild-caught larvae showed dengue specific DNA band (470bp) in RT-PCR-LH assay). RT-PCR-LH with Semi-Nested-PCR-AGE assays are useful for the detection and typing of dengue virus in Ae.albopictus. Ae.albopictus (in Sri Lanka is competent in transmitting DEN3 and possibly other serotypes. Detection of dengue virus for the first time in Ae.albopictus in Sri Lanka confirms earlier observations that it may play an important role in transmitting dengue). Acknowledgements: Financial assistance by the International Atomic Energy Agency (Technical Co¬operation grant no SLR/ 06 / 024) and University of Kelaniya (Research grant no RP/03/04/06/01/00) is gratefully acknowledged.Item Tetravalent dengue specific domain III based chimeric recombinant protein as dengue diagnostic intermediates for the detection of both anti-dengue immunoglobulin M(IGM) and imunoglobulin G(IGG) antibodies in human serum samples.(International Water Management Institute, 2006) Hapugoda, M.D.; Abeyewickreme, W.; Gunasena, S.; Khanna, N.BACKGROUND: Dengue infection is an important mosquito borne viral infection caused by four serotypes of dengue virus with explosive outbreaks occurring in many tropical areas. Laboratory diagnosis of the disease mainly depends on Enzyme-Linked Immunosorbent Assay (ELISA) based on whole viral antigens which cause biohazard risk, high production cost and cross reactivity with other flaviviruses. OBJECTIVES: To produce a recombinant protein antigen to overcome problems associated with whole dengue viral antigen/lysate or recombinant whole envelope protein. STUDY DESIGN: We have designed and expressed a single recombinant tetravalent protein antigen which contains Domain III of envelope protein from all four serotypes of dengue virus, linked with each other through penta glycine linkers. This synthetic gene was expressed in Escherichia coli and protein was purified using a single affinity chromatographic step. We developed Immunoglobulin M (IgM) and Immunoglobulin G (IgG) ELISAs using this novel protein as the capture antigen. The antigen was validated as a diagnostic reagent on serum samples. RESULTS: 30 mg of recombinant protein per litre of culture could be purified. Both ELISAs developed using this novel recombinant protein showed an excellent agreement with a commercially available IgM ELISA (MRL diagnostic) and haernagglutination inhibition assay respectively. Conclusions: Findings of this study suggests that this single dengue specific tetravalent recombinant protein antigen can be used as a diagnostic intermediate for detection of dengue infection.Item Dengue as-a public health problem in Sri Lanka(La Fondation pour l’Université de Lyon, 2009) Hapangama, H.A.D.C.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.; Premaratna, R.; Manamperi, A.; Gunasena, S.; Abeyewickreme, W.Dengue infection is an important global public health problem and an increasing number of persons from the South Asian region have been directly or indirectly affected by the disease. In Sri Lanka, dengue has become a major threat to public health in many urban and sub-urban' areas during past three decades. Rapid unplanned urbanization and increasing human population has increase the rate of infection and the frequency. The study area, Gampaha District is the second most populous district in the country having a population density of 1 539 persons per km2 and was the district reporting the second highest incidence of dengue in 2008. Therefore, current research efforts are focused on dengue transmission, examining the presence of sub-clinical infections, role of vector mosquitoes and Knowledge, Attitude and Practices (KAP) of the community on dengue infection in an effort to contain the disease. In the present study, dengue antibodies were detected in samples collected from clinically suspected patients and as well as in samples collected from volunteers. Volunteer sera collected around the confirmed cases had a 23.6% sero-positive rate for dengue IgM antibodies. The rate of asymptomatic recent infections was calculated to be 16.9%. In present study we have serologically confirmed the presence of subclinical infections and according to the published data this is the first confirmation of asymptomatic dengue infections in Sri Lanka. According to the entomological investigations carried out, the common breeding places for Aedes vectors were found to be discarded small containers. Even though Ae. Aegypti has been considered as the principal vector transmitting dengue fever, current studies highlighted the predominant ro!e of Ae. albopictus in the disease transmission. A previous study in Sri Lanka also suggested that prevalence and .presence of high-density of Ae. albopictus should be considered as a risk factor for endemic/epidemic dengue. In view of the above, the spread of dengue by Ae. albopictus should be a matter of great concern. Findings of KAP survey revealed that the community possessed substantially higher knowledge on the spread of dengue, vectors, vector breeding and also seriousness of the infection. However it was observed that good knowledge does not necessarily lead to good practices. Since the attitudes of the respondents were found to be good and most of them were supportive of control measures; next effort of the present study is to see how a novel community mobilized solid waste management system will be effective in dengue vector control.Item Silent transmission of the dengue fever in Gampaha District, Sri Lanka(Malaysian Society of Parasitology and Tropical Medicine, 2007) Hapangama, H.A.D.C.; Gunawardene, Y.I.N.S.; Gunasena, S.; Hapugoda, M.D.; Premaratna, R.; Wellawaththage, L.C.; Abeyewickreme, W.Dengue fever is a major infectious disease in Sri Lanka. Silent transmission of dengue virus has been suggested as a possible risk factor for the increasing incidence of dengue. The present study was carried out in the District of Gampaha using cluster investigation method. A cluster consisted of a minimum of 20 volunteers (family members and immediate neighbours) of a hospitalized serologically/molecular biologically confirmed dengue patient. Serum samples were collected from 148 volunteers in 7 clusters. Samples were tested for anti-dengue antibodies using Dengue Duo IgM and IgG Rapid Strip Test. Of these, positives were further tested for anti-dengue IgG antibody by Haemagglutination Inhibition (HAI) assay, the gold standard test for serological diagnosis of virus infection. Of the 148, 41 had evidence of exposure to dengue virus by Dengue Duo IgM and IgG Rapid Strip Test [positive for IgM: 28(68.4%), IgM & IgG: 7(17%) and IgG: 6(14.6%)]. Of that 41, paired sera were collected from 36 volunteers and tested by HAI assay which confirmed dengue virus infection in 4(11.1%) [confirmed secondary-4(100%)]. Additional 32(88.9%) were diagnosed as recent dengue infections [probable secondary-17(53.1%), probable dengue- 15(46.9%)]. Out of 36 volunteers, 12(33.3%) were symptomatic [confirmed secondary-1(8.3%), probable secondary-10(83.4%), probable dengue-1(8.3%)] and 24(66.7%) were asymptomatic [confirmed secondary-3(12.5%), probable secondary-7(29.2%), probable dengue-14(58.3%)]. Presence of dengue vectors, Aedes aegypti and/or Aedes albopictus were detected around all 7 clusters. The present study serologically confirms the persistence of silent transmission of dengue virus with a trend towards clustering around cases. Presence of vector species in the area further supports this phenomenon.Item A Comparative retrospective study of novel Reverse-Transcription Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with Polymerase Chain Reaction (PCR) amplification, virus isolation and serological techniques for early, definitive laboratory diagnosis of dengue infection(Malaysian Society of Parasitology and Tropical Medicine, 2007) Hapugoda, M.D.; de Silva, N.R.; Khan, B.; Gunasena, S.; Dayanath, M.Y.D.; Abeyewickreme, W.Dengue is an important vector borne viral infection in South East Asia. Dengue virus is responsible for dengue fever, dengue haemorrhagic fever and dengue shock syndrome. Early diagnosis of infection helps in monitoring the disease, determining when hospital admission is necessary and in reducing case fatalities. The objective of the study was to carry out a comparative retrospective study of a novel Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of Non Structural-3 gene were hybridized with a mixture of the 4 dengue type-specific Deoxyribonucleic Acid (DNA) probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 88 patients with dengue confirmed by Haemagglutination Inhibition (HAI) assay. The assay was highly specific for diagnosis of dengue infection. As an early (<5 days of fever) laboratory diagnostic method, this assay had 100% sensitivity for detection of dengue patients confirmed by HAI assay. A high analytical sensitivity of 2 fluorescent focus units of dengue virus/reaction was achieved. Novel RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection at the time during which serological methods cannot be used.