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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty

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Author: search.filters.author.de Silva, N.R.Subject: search.filters.subject.Dengue-diagnosis

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    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.
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    Correlation between clinical and laboratory diagnosis of dengue in Sri Lanka.
    (Faculty of Tropical Medicine, Mahidol University, 2007) Hapugoda, M.D.; Khan, B.; de Silva, N.R.; Gunasekera, J.; Abeyewickreme, W.
    BACKGROUND: In Sri Lanka, diagnosis of dengue mainly depends on clinical signs and symptoms. Very few suspected patients from the state and private sector health institutions are tested by laboratory diagnostic assays compared to the number of dengue cases recorded all over the island. OBJECTIVES: To correlate clinical parameters with laboratory diagnosis in confirmation of dengue. RESEARCH DESIGN: Patients, clinically suspected of having dengue (n=201) were selected based on WHO criteria. Serum samples were tested using major 3 types of laboratory diagnostic assays; molecular, virus isolation and serology. Differences in clinical and laboratory data were analyzed on the basis of the final diagnosis assigned as dengue or non-dengue. Chi-square test was used for comparison of data. RESULTS: The proportion of laboratory diagnosed dengue patients were 80% (162/201). Mean platelet value and PCV in laboratory confirmed dengue patients were 92 247/mm3 (range 20 000-318 000) and 45% (range 31-59%) respectively. On comparison of the presence of clinical features that are used by the WHO for diagnosis of dengue, headache (129/162 vs 18/39, x2=23, p=0.00), limb pain (107/162 vs 18/39, x2=4.56, p=0.03) and external bleeding (67/162 vs 00/39, X2=27, p=0.00) showed significant association, with dengue infection. The infection was confirmed as definitive dengue in 75% (121 /162) and probable dengue in 25% (41/162). DISCUSSION: Surveillance based on clinical diagnosis may result in over estimation of the disease as clinical diagnosis is not specific enough. Laboratory confirmation of dengue suspected patients is important to measure the real incidence of the disease is needed in country like Sri Lanka.
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    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.
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