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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Hantavirus infection with pulmonary symptoms in north central part of Sri Lanka(Elsevier Ltd, 2021) Muthugala, R.; Dheerasekara, K.; Harischandra, N.; Wickramasinghe, D.; Abeykoon, M.; Dasanayake, D.; Manamperi, A.; Gunasena, S.; Galagoda, G.BACKGROUND: Classical hantavirus infections present as haemorrhagic fever with renal syndrome (HFRS) in Euro-Asia and as hantavirus pulmonary syndrome (HPS) in America. Mixed clinical features have been reported from certain novel hantavirus infections. In the north-central part of Sri Lanka, clusters of patients with fever and non-cardiogenic pulmonary edema have been reported in recent years.OBJECTIVES: To detect hantavirus infection among clinically suspected patients and to describe clinical and demographic features of hantavirus infection in north-central Sri Lanka. STUDY DESIGN: Clinically suspected patients with HFRS and HPS like illness admitted to two leading hospitals in the north-central part of the country from December 2013 to November 2015 and from March 2016 to February 2018 were included in the study. Acute phase blood samples were tested for the presence of anti-hantavirus IgM. Convalescent blood samples were taken from available cases and both acute and convalescent sera were subjected to IgG titre detection. RESULTS: Seventy-two patients were included in the study. Twenty-nine (40.28%) were positive for hantavirus IgM. Of them, 20 (68.97%) presented with pulmonary symptoms with no or mild nephritis. Five (17.24%) had pulmonary symptoms with prominent nephritis and 04 (13.79%) had classic features of HFRS. CONCLUSION: In the north-central part of Sri Lanka, most hantavirus infection was associated with pulmonary symptoms complicated with non-cardiogenic pulmonary edema, which was different from clinical presentation reported previously from other parts of the country. HPS like hantavirus infection in the study area could be due to a Puumala-like virus or a novel virus.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 Development of recombinant protein antigens using a bacterial expression system for the detection of anti-Chikungunya (CHIK) antibodies(Sri Lanka College of Microbiologists, 2013) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.INTRODUCTION AND OBJECTIVE: Laboratory confirmation of Chikungunya (CHIK) virus is very useful as clinical symptoms of CHIK can overlap with other diseases. Chikungunya virus specific antigen, which shows high specificity, sensitivity and low cross reactivity with other related diseases, is required for laboratory confirmation. Objective of this study was to develop and compare two recombinant protein antigens for detection of anti-CHIK antibodies. DESIGN, SETTING AND METHODS: Recombinant CHIK protein antigens were prepared using Envelope (E1 and E2) regions of the CHIK virus. The genes were custom designed and chemically synthesized with a 6X His tag. Bacterial expression systems [BL21 (DE3)] were used to clone and express the recombinant proteins. The recombinant proteins were purified with >95% of purity per liter of culture using Ni-NTA columns under denature conditions. In this study, two antigens were evaluated for detection of anti-CHIK antibody by using novel optimized in-house IgM and IgG ELISAs, using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Center for Viral Reference and Research) Institute of Tropical Medicine, Nagasaki University, Japan. RESULTS: Atotal of 55 serum samples confirmed as positives and 186 confirmed as negatives by HA! test, IgM capture ELISA and indirect IgG ELISA using the purified CHIK antigen were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 148 (E1) or 227 (E2) (148 + extra 79) confirmed ac negatives were used to evaluate the antigens using novel IgG ELISA. The E1 recombinant protein showed 5% (3/ 55) sensitivity and 99% (184/186) specificity for IgM ELISA and 60% (47/78) sensitivity and 63% (94/148) specificity for IgG ELISA. The E2 recombinant protein showed 65% (36/55) sensitivity and 70% (131/186) specificity for IgM ELISA and 83% (65/78) sensitivity and 86% (195/227) specificity for IgG ELISA. CONCLUSION: Recombinant CHIK-E2 protein antigen showed higher specificity and sensitivity in detection of both IgM and IgG antibodies. Thus the E2 recombinant protein antigen used in this study could be expressed in an eukaryotic expression system to achieve much higher results. ACKNOWLEDGMENT: 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) are gratefully acknowledged.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 Molecular diagnosis and transmission of dengue in Sri Lanka(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Hapugoda, M.; Abeyewickreme, W.; de Silva, N.R.; Gunasena, S.; Meegoda, D.; Manamperi, N.BACKGROUND: Dengue virus is an important vector-borne viral infection in Sri Lanka. Laboratory confirmation of suspected dengue cases is important for over/under estimation of cases. Early rapid 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 for studies on transmission of dengue virus. Study on risk factors for dengue is useful to understand spatial and temporal dynamics of transmission the disease. METHODOLOGY: A novel molecular-based assay; Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) was developed and validated for detection of dengue virus in clinical and mosquito specimens. Severity of dengue with circulating serotype was also analyzed. Wild-caught mosquito samples were collected from 236 dengue case-reported stations during outbreaks and a hot-spot during a period of 31 months. Epidemiological, environmental and entomological and other possible risk factors affecting transmission of dengue were analyzed. RESULTS: As an early (<5 days of fever) laboratory diagnostic method for dengue virus, the novel assay had 100% and 46% sensitivity for detection of confirmed and suspected dengue patients respectively. The assay developed in this study was found to be more sensitive than the other diagnostic techniques for early definitive laboratory diagnosis of dengue infection. Patients with definitive dengue correlated only with few signs and symptoms, indicating that laboratory confirmation is critical to avoid over estimation. A high sensitivity of 2 fluorescent focus unit of dengue virus/reaction was achieved and the assay was highly specific for dengue virus. The assay could detect dengue virus in 7% of field-caught Aedes albopictus specimens. A high density of Ae. albopictus was also associated with the dengue case-reported stations/hot-spots. Both vector species were susceptible to the 4 dengue serotypes under the laboratory conditions and DEN-3 under the field conditions. Geographical Information System (GIS) based risk mapping and database including epidemiological, climatic condition and entomological surveillance information were developed for the hot-spot in Kurunegala during the period of 2000-2003, which can be used as an early warning system. In depth study on socio-economic and other related factors affecting transmission of dengue was studied in the District of Gampaha. CONCLUSION: Ae. albopictus acts as an important vector of transmission of dengue in some urban and semi-urban areas. GIS-based risk maps developed are important to predict impending epidemics so that limited resources could be utilized in a cost effective manner to control the disease. Some socio-economic factors directly affecting transmission of dengue.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 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.