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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Composition of malaria vectors and diversity of anopheline breeding habitats in the district of Mannar, Sri Lanka(Central Environmental Authority (CEA), 2016) Gunathilaka, P.A.D.H.N.; Hapugoda, M.D.; Wickremasinghe, A.R.; Abeyewickreme, W.In the malaria elimination phase in Sri Lanka, investigation on biological and ecological factors of malaria vectors are important in planning appropriate vector controlling strategies. Lack of sufficient biological and ecological information on malaria vectors in the Northern Province of the country, a malaria endemic region, is a major constrain in successful implementation of malaria control programmes. Therefore, the objective of this study was to explore the diversity of breeding habitats and species composition of malaria vector mosquitoes in the District of Mannar, Sri Lanka. Potential habitats for Anopheles mosquito larvae were surveyed from June, 2010 to July 20 J2 on a monthly basis in selected sampling sites in the Mannar District: Mannar Town, Vankalai and Silawathiura, within a radius about 20 km. In each site, 4 sub sites were selected A total of 37,788 Anopheles representing ten species was recorded from 12 breeding habitat categories. Built wells and waste water collections were conducive for anopheline breeding. Anopheles subpictus (96.2%, n= 36,351) was the dominant species followed by An. peditaeniatus (1.47%, n= 557), An. barbirostris (1.23%, n= 463), An. nigerriums (0.75%,n = 285), An. varuna (0.19%, n= 74), An. barbumbrosus (0.1%, n= 38), An. vagus' (0.03%, n= 12), An. pallidus (0.01%, n= 4), An.jamesii (0.05%, n= 2) and An. pseudojamesi (0.05%, n= 2). Use of wells and waste water drains as breeding places by potential malaria vectors indicates that both of these habitats act as larval reservoirs during the dry season. Presence of theses habitats in close proximity to human habitats create a potential risk of malaria transmission among humans. Therefore, health authorities need to be vigilant on these new habitats in vector control programmes.Item Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka(Academic Press, Elsevier, 2016) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hartskeerl, C.M.; Hartskeerl, R.A.; Jiffrey, A.M.; Hapugoda, M.D.Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.Item Molecular evidence of hantavirus infection among clinically suspected patients with hae-morrhagic fever with renal syndrome (HFRS)(Sri Lanka College of Microbiologists, 2013) Muthugala, M.A.R.V.; Manamperi, A.A.P.S.; Gunasena, S.; Hapugoda, M.D.; Butch, G.INTRODUCTION: Hantavirus disease is an emerging zoonotic viral infection with high fatality. Transmission is by inhalation of aerosols generated from virus contaminated rodent excreta. There are two major clinical forms, haemorraghic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Clinical features of HFRS, often mimic leptospirosis. Large number of cases of leptospirosis like illness has been reported in Sri Lanka annually. Although there were serological evidence of different types of hantavirus infection in Sri Lanka, diagnosis of hantavirus is not routinely performed. Due to the genetic and antigenic diversity, an assay that could detect a wide range of hantaviruses need to be established. OBJECTIVES: To establish, evaluate and validate a genus specific hantavirus RT-PCR assay. To diagnose hantavirus infection among clinically suspected HFRS patients in three selected hospitals.To describe clinical manifestations of hantavirus infections in the study population. METHODOLOGY: Genus specific conventional RT-PCR assay was established using panhanta primers and evaluated, optimized and validated using synthetic genes of 12 known hantavirus species as reference samples. Assay was able to detect a wide range of hantaviruses at minimum detection limit of 70 copies/ reaction. Molecular diagnosis of hantavirus infection was carried out in three hospitals in Colombo and Gampaha districts. Study was conducted from 01st of January 2011 to 31st of April 2011 and 61 adult patients were recruited to this study. Hantavirus RT-PCR was performed on all collected samples after extraction of RNA by TRIzol® method. RESULTS: Of 61 tested samples, 05 were positive for hantavirus genome. These results were confirmed at reference laboratory as well and species identification result is pending. Of 58 tested samples, 06 samples were positive for hantavirus IgM by in-house ELISA. All PCR positive samples were positive for hanta virus IgM. All patients with hantavirus infection had clinical and biochemical features of liver involvement in addition to fever, thrombocytopenia and renal involvement. CONCLUSION: Established RT-PCR assay was able to detect a wide range of hantaviruses and by using it molecular evidence of hantavirus infection was demonstrated in humans in Sri Lanka. Further studies are required to describe the disease epidemiology and to identify natural hosts in the country.Item Clinical and virological features of dengue in 2010(Sri Lanka College of Microbiologists, 2011) Hapugoda, M.D.; Manamperi, H.; Gunasena, S.; Athapaththu, A.M.M.H.; Premawansa, G.; Wellawaththage, C.; Jayarathna, T.D.S.S.; Abeyewickreme, W.INTRODUCTION: Dengue is an important viral infection in Sri Lanka. All 4 serotypes co-circulate in Sri Lanka. OBJECTIVE: To study the clinical and virological features of dengue in 2010. DESIGN, SETTING AND METHODS: A hospital-based study was carried out at North Colombo Teaching Hospital, Ragama in 2010. Patients clinically suspected of having dengue, with fever less than 5 days were recruited. Acute and convalescent blood samples were collected within 7 days after obtaining informed written consent. Demographic, clinical information and laboratory results were obtained. Acute serum samples were tested using molecular (RT-PCR and Semi-Nested PCR) and serological (ELlSAs and HAI) assays. Convalescent samples were tested by serological assays. RESULTS: Of 209 patients enrolled, 93 % (195/209) were laboratory confirmed as recent positive cases of dengue viral infection; of these, 5% (9/195) were classified as dengue fever; 85%(1G5/195) dengue haemorrhagic fever (DHF) and 0.5% (1/195) dengue shock syndrome. Mean platelet value and packed cell volume (PCV) in laboratory confirmed dengue patients were 56,107/mm3 (range 10,000-306,000) and 42%(range 34-61 %) respectively. Patients infected with DHF showed both primary (n=45) and secondary (n=102) infections. Interestingly, secondary infection was not significantly correlated with DHF (x2-0.3:p=0.6). DEN-1 was responsible for the majority of cases, with a minority due to other three serotypes; all serotypes contributed to severe disease. CONCLUSION: DEN-1 was responsible for the majority of cases in 2010 but it circulated at a low level during previous epidemics. Majority of patients had severe clinical symptoms. In this epidemic, the clinical presentation of dengue differed according to the geographic region and viral serotype. ACKNOWLEDGMENTS: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (lAEA/SRL/5/042) is acknowledged.Item Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody(Sri Lanka College of Microbiologists, 2011) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.INTRODUCTION: Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. OBJECTIVE: To compare two antigens for detection of anti-CHIK antibody. DESIGN, SETTING AND METHODS: In this study, two antigens {viral cell lysate and recombinant protein) were evaluated for detection of anti-1 CH IK antibody by using IgM ELISA. A novel recombinant | protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein, I This protein was expressed in Escherichia coli and resultant protein was affinity purified and ~10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine,] Nagasaki University. RESULTS: Atotal of 64 serum samples confirmed as positives andl 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was] 17% and 100% respectively. Conclusion Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. ACKNOWLEDGMENT: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.Item Identification of risk factors affecting transmission of dengue in the District of Gampaha, Sri Lanka(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Withanage, G.P.W.K.; Abeyewickreme, W.; Hapugoda, M.D.INTRODUCTION: Dengue is the most significant mosquito-borne infectious disease in Sri Lanka, causing more than 35,000 cases annually for last five years. The second highest prevalence of dengue was reported from the District of Gampaha for last 10 years. OBJECTIVE: To identify possible risk factors affecting transmission of dengue in selected high risk areas of dengue in the Gampha District. METHODOLOGY: The study was conducted in four high risk Medical Officer of Health (MOH) areas where the annual number of dengue cases greater than 250 during last ten years. In each MOH area, one Grama Niladhari (GN) division with the highest dengue incidence was selected as study areas. In each study area, a cluster of 150, including house-holds, open areas (barren lands, dump yards, and construction sites), abandoned houses, and religious facilities, was selected. A house-hold and entomological surveys were performed in March, 2015 after obtaining an informed written consent. RESULTS: The selected study areas were Akbar town (Mahara MOH), Eriyawatiya (Kelaniya MOH), Kurana (Negombo MOH), and Welikadamulla (Wattala MOH). The size of study human population was 2,544 in 574 house-holds in the study areas and 53.66% (1,365/2,544) were females. The average size of a homestead was 19.3 perches and more than one house was observed in 21.6% (124/574) of land plots. The type of the house-hold was mostly moderate (67.2%-386/574) and the gender of the house-hold head was male in 77.7% (446/574) house-holds. The average number of dwellers in a house-hold was 4 and 7.35% (187/2,544) of the population has previously infected with dengue. Most of house-holds were individual houses with a small garden (98.9%-568/574) and residential function of the house-hold was mainly residential only (83.4%-479/574). The main source of water was piped water (95.1%-546/574), but 39.5% (227/574) using ground-well or tube-well water daily purposes other than drinking and food processing. The most common mosquito preventive measure was bed nets (31.7%-182/574) and 47.6% (273/574) house-holds were using more than one mosquito preventive measures. Collecting tailors of municipal councils is the main solid waste disposal method, while 16.0% (92/574) using burning, burring in pits, composting or open ground due to lack of proper solid waste collection system. Bushes and small trees were the most common vegetation cover of the homesteads (84.5%-485/574) and potential breeding sites were observed in 97.5% (585/600) premises in the study areas. Main mosquito breeding places were plastic and polythene wares, discarded cans and tins, discarded tyres, plant axils, and aluminum and clay pots. The Breteau Index (BI) for Aedes larvae was 4.88 (28/574). Most prominent adult mosquito species in the areas was Aedes albopictus (92.4%-281/304). Dwellers in the study areas have considerable knowledge of the disease and preventive measures, but they reluctant to pursue preventive measures. DISCUSSION: Possible risk factors for transmission of dengue may be crowded conditions, small house-holds and homesteads, poor water and waste management systems, disfavor to pursue preventive measures, and dependence on government vector control programs. Therefore, immense persuade will be required to control dengue in the areas.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 Enzyme-linked Immunosorbent Assay (ELISA) using recombinant protein antigens for detection of anti-chikungunya antibodies(Faculty of Tropical Medicine, Mahidol University, 2010) Athapaththu, A.M.M.H.; Khanna, N.; Abeyewickreme, W.; Gunasena, S.; Hapugoda, M.D.OBJECTIVES: Chikungunya is a mosquito borne viral infection that has caused great medical and public health problems in South East Asia during last few years. Currently available laboratory diagnostic kits depend on Enzyme-Linked Immunosorbent Assay (ELISA) based on whole viral antigens caused biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. These problems can be avoided by using recombinant protein antigens in ELISAs. METHODOLOGY: Two novel recombinant protein antigens based on Envelope (E) domain, a critical antigenic region of the major structural protein of chikungunya virus were expressed separately in a bacterial expression system (Escherichia coli). Two proteins were purified under denatured conditions. They were evaluated as potential diagnostic intermediates for detection of and-chikungunya antibodies in Immunoglobulin M (IgM) and Immunoglobulin G (IgG) ELISAs separately using a panel of serum samples confirmed by the gold standard assay, Heamagglutination Inhibition (HAI) assayRESULTS: These 2 protein antigens: El and E2 showed more than 60% positivity in IgG ELISAs and IgM ELISAs. A field validation using a large number of serum samples should be done for further confirmation of these results. It can be concluded that these 2 novel recombinant protein antigens can be used as a diagnostic intermediate to detect anti-chikungunya antibodies. ACKNOWLEDGEMENTS: Financial assistance from the International Centre for Genetic Engineering and Biotechnology (1CGEB CRP/ SRI08-02) is gratefully acknowledgedItem Shifting of circulating serotypes in dengue outbreaks during 2009/2010 in Sri lanka(Faculty of Tropical Medicine, Mahidol University, 2010) Manamperi, N.H.; Athapaththu, A.M.M.H.; Premawansa, G.; Wellawaththage, C.; Jayarathna, T. D. S. S.; Abeyewickreme, W.; Hapugoda, M.D.OBJECTIVES: Sri Lanka has experienced explosive outbreaks of dengue infection in 2009 and 2010. It has been identified that DEN- 3 and DEN- 2 were the predominant serotypes with DEN-1 and DEN- 4 circulating at a lower level in previous dengue outbreaks during 2003-2006, Objective of this study was to identify the circulating serotype/s during 2009 - 2010 outbreaks. METHODOLOGY: A prospective study was carried out at North Colombo Teaching Hospital, Sri Lanka during December 2009-August 2010. Clinically suspected dengue patients, with fever less than 5 days were recruited. An interviewer administered questionnaire was filled for each patient, by a Medical Officer. Venous blood samples confirmed for the presence of dengue virus by RT-PCR were typed by Semi-Nested PCR. RESULTS: Out of the 209 patients recruited in the study 80 (38%) were positive for dengue virus by RT-PCR. Of the positives, 43 (54%) were typed and circulation of all 4 serotypes was observed- Of the 43 positives, presence of DEN-1, DEN-2, DEN-3 and DEN-4 serotypes was 34 (79%), 3 (7%), 2 (5%) and 3 (7%) respectively DEN-1 was the predominant serotype in the recent epidemics which was circulating at a low level in previous epidemics. In DEN-1 infected patients, the mean platelet value was 58,588/ rnm3 and the mean PCV value was 41.4%. Associated symptoms such as headache, retro-orbital pain, neck pain and limb pain were present in 94% (32/34), 59% (20/34), 24% (8/34J and 91% (31/34) patients respectively. Bleeding manifestation developed in 47 % (16/34) patients. The mortality rate ranged from 0.7%- 1.0% during the recent outbreaks. Acknowledgement: Financial and technical assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08-02) and the International Atomic Energy Agency (IAEA SRI 5/042) 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.