Conference Papers
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This collection contains abstracts of conference papers, presented at local and international conferences by the staff of the Faculty of Medicine
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Item In situ immune response to cutaneous leishmaniasis in Sri Lanka(Sri Lanka Medical Association, 2017) Manamperi, N.H.; Oghumu, S.; Pathirana, N.; de Silva, M.V.C.; Abeyewickreme, W.; Satoskar, A.R.; Karunaweera, N.D.INTRODUCTION & OBJECTIVES: Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani-MON 37, known to cause visceral leishmaniasis elsewhere. Localized immune response may play a role in disease outcome with T helper (Th) 1 response favouring lesion healing and Th2 response leading to disease progression in animal models. This study describes the localized host immune response to CL in Sri Lanka. METHOD: Skin punch biopsies from 58 patients with parasitologically confirmed CL and 25 healthy controls were quantified for cytokine gene expression of Th1 cytokines interferon (IFN)-γ, interleukin (IL)-12A and tumour necrosis factor (TNF)-α and Th2 cytokines, IL-4 and IL-10 by real-time RT-PCR. Relative copy numbers were calculated using the 2-ΔΔCt method. Non-parametric Mann-Whitney U test and the Spearman’s correlation test were used for statistical analysis. RESULTS: Study group consisted of 37 (63.8%) males and 21 (36.2%) females with a mean age of 35.0 years (SD=12.1, range=18-66), mean lesion duration of 6.75 ±9.1 months (range: 1-48) and a mean size of 176.59±185.76 mm2 (range: 12.6–908.3 mm2). Significant up regulation of IFN-γ (p<0.001) and down regulation of IL-4 (p<0.001) were seen in patients compared to healthy controls. Time taken for lesions to heal correlated significantly with in situ expression of IL-4 (Spearman’s r=0.321, p=0.034). CONCLUSION: Immune response to L. donovani induced CL in Sri Lanka tends to follow the typical Th1/Th2 convention with a Th2 biased milieu favouring poor responsiveness to antimony and delayed lesion healing.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 Study on Phlebotomine sand flies in selected areas in Sri Lanka(Sri Lanka College of Microbiologists, 2006) Senanayake, S.A.S.C.; Karunaweera, N.D.; Abeyewickreme, W.Sandflies are the known vectors of disease leishmaniasis. Though there are three clinical entities (viceral, mucocutaneous and cutaneous),^.only cutaneous form of the disease is seen in Sri Lanka. Presence of sandflies belonging to six species has been reported from various parts of the country since 1910. But the first indigenous case of cutaneous leishmaniasis was recorded in 1922. The number of cases rapidly increased during past few years and it is now been considered as an established disease. The causative organism of the disease is the protozoan parasite Leishmania donovani MON37. The vector of the Sri Lankan cutaneous leishmaniasis is still unknown. This study was carried out in two selected areas in Kurunegala and Matara ditricts where considerable number of patients was reported to the Department of Parasitology, Faculty of Medicine, Colombo. The objectives of the study were to identify the prevalent sandfly species in selected areas and establish the potential vector(s). The adult sandflies were collectedTrom four different sites in selected areas. Three different methodologies were used (cattle-baited net traps, CDC light traps, manual collection and mechanical aspirators). Collections were done for 18 months from Sep2004. Collected sandflies were dissected under dissecting microscope and mounted on glass slides. The specimens were examined under both light and phase contrast microscopes. A subset of collected samples were sent to CDC, Atlanta for molecular based identification. Blood fed females were subjected to gut dissection to demonstrate the presence of leishmania parasites within the vector. Real time PCR analysis was carried out with a subset of samples using Leishmania donovani primers. Two species of sandfies were identified in both areas. They were Phlebotomus argentepes and Sergentomiya zeylanica, two species which had been reported previously. P. argentepes is the established vector of visceral leishmaniasis in India and latter is a non human disease transmitter. A total of 3587 sandfies were examined (1756 from Matara and 1731 from Kurunegala) Male to female ratio of the collection was 6:1(3075 males and 522 females). Only 88 ( 5.01%) P. argentepes were found in Matara and rest 1168 (94.99%) were S. zeylanica. How ever, Kurunegala collection resulted with 1549 (89.48%) P. argentepes and 192 S. zeylanica. None of the method use to demonstrate leishmania parasites in sandflies gave positive results. Financial assistants by National Science Foundation for research Grant 2005 /HS/07 is 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 Genetic Polymorphism in Pvmsp-3a. and Pvcs genes in Plasmodium vivax infections in Sri Lanka(Sri Lanka College of Microbiologists, 2008) Manamperi, A.; Fernando, D.; Mahawithanage, S.; Wickremasinghe, R*.; Bandara, A.; Wellawatta, C.; Hapuarachchi, C.; Abeyewickreme, W.; Wickremasinghe, R.INTRODUCTION: Plasmodim vivax malaria accounts for about 70% of all malaria infections in Sri Lanka. There is limited information on the genetic heterogeneity of P. vivax parasites in endemic areas of the country. OBJECTIVE: The objective of this study was to assess the potential of two P. vivax genes, Pvmsp-3v. and Pvcs. as genetic markers for their use in genotyping parasites collected from the field. METHOD: DNA was extracted from 12 Geimsa-stained P. vivax positive slides by phenol/chloroform method. A nested polymerase chain reaction (PCR) approach was adopted for both Pvmsp-3a and Pvcs genes. RFLP analysis ofPvmsp-la nested PCR products was carried out with Hha\ restriction enzyme. RESULTS AND DISCUSSION: Nested amplification of the marker genes resulted in 4 size variants for Pvcs (~ 600-750 bp) and 2 size variants for Pvmsp-3a (1.9 kb and 1.1 kb). Further, all PCR-RFLP products of Pvmsp-3a. Gene showed a major size polymorphism. Three samples showed evidence of infections with mixed genotypes and there was also evidence to identify a relapse infection. Analysis of these two genetic markers revealed 11 distinguishable variant types: 4 for Pvcs and 7 for Pvmsp-3a. CONCLUSIONS: The observed PCR and PCR-RFLP profiles of the Pvcs and Pvmsp-3& genes demonstrate that the P. vivax parasites in Sri Lanka were highly diverse despite the prevailing low transmission levels. It could be concluded that these two genes in combination could be considered suitable genetic markers to analyze P. vivax parasite dynamics in Sri Lanka.Item Histopathological spectrum in acute and chronic cutaneous leishmaniasis in Sri Lanka(Sri Lanka College of Microbiologists, 2015) Manamperi, N.H.; de Silva, M.V.C.; Fernando, C.; Pathirana, K.P.N.; Abeyewickreme, W.; Karunaweera, N.D.OBJECTIVES: To describe the histological spectrum of acute and chronic cutaneous leishmaniasis. METHOD: Patients from Sri Lanka army were recruited by active and passive case detection methods and punch biopsies were obtained. Skin biopsies of 35 patients with smear positive for Leishmania amastigotes were processed routinely for histopathology, examined at a conference microscope and classified into 4 groups using modified Ridley criteria for Leishmaniasis as: I - parasitized macrophages with variable lymphocytes and plasma cells; II - parasitized macrophages with lymphocytes, plasma cells and ill formed histiocytic granulomata; III -a mixture of macrophages (with or without parasites), lymphocytes, plasma cells and epithelioid granulomata; IV - epithelioid granulomatous response with a few lymphocytes and plasma cells but no amasigotes. Lesions were categorized as acute (<6 months) or chronic (> 6 months). RESULTS: Study group composed of males with a mean age of 32.6 years (range 22-47) and lesion duration of 5.6 months (range 1-24). Twenty nine (82.9%) were also positive by histopathology. Twenty two (62.9%) were acute and 13 (37.1%) chronic. Group I, II, III and IV patterns were seen in 14 (40%), 12 (34.3%), 5 (14.3%) and 4 (11.4%) respectively and 9 (40.9%), 9 (40.9%), 2 (9.1%) and 2 (9.1 %) of acute lesions and 5 (38.5%), 3 (23.1 %), 3 (23.1 %) and 2 (15.4%) of chronic lesions respectively. CONCLUSION: Histology of cutaneous leishmaniasis shows marked inflammatory cell infiltrate with or without granuloma formation. Majority of patients presenting with either acute or chronic cutaneous leishmaniasis belong to histological groups I or II. ACKNOWLEDGEMENTS: Financial assistance from the University Grants Commission, Sri Lanka (UGC/VC/DRIC/PG/2013/KLN/ 03) and University of Kelaniya (RP/03/04/06/01/2014) are acknowledged. An abstract based on similar work was presented at the 128"1 Anniversary International Medical Congress of the Sri Lanka Medical Association, 5th to 8th July 2015.Item Histopathological spectrum in acute and chronic Cutaneous Leishmaniasis in Sri Lanka(Sri lanka Medical Association, 2015) Manamperi, N.H.; Fernando, C.; Pathirana, K.P.N.; Karunaweera, N.D.; Abeyewickreme, W.; de Silva, M.V.C.INTRODUCTION AND OBJECTIVES: Histological spectrum in cutaneous leishmaniasis (CL) is wide and varied. The objective of this study is to describe the histological spectrum of acute and chronic CL. METHOD: Skin biopsies of 35 patients with smear positive for Leishmania amastigotes were processed routinely for histopathology, examined at a conference microscope and classified into 4 groups using modified Ridley criteria for Leishmaniasis as: I- parasitized macrophages with variable lymphocytes and plasma ceils; 1! - parasitized macrophages with lymphocytes, plasma cells and ill formed histiocytic granulomata; 111 - a mixture of macrophages (with or without parasites), lymphocytes, plasma cells and epithelioid granulomata; IV - epithelioid granulomatous response with a few lymphocytes and plasma cells but no amastigotes. Lesions were categorized as acute (< 6 months) or chronic (> 6 months). RESULTS: Study group composed of all males with a mean age of 32.6 years (range 22 - 47) and lesion duration of 5.6 months (range 1-24). Twenty nine (82.9%) were also positive by histopathology. Twenty two (62.9%) were acute and 13 (37.1%) chronic. Group I, II, Ml and IV patterns were seen in 14 (40%), 12 (34.3%), 5 (14.3%) and 4 (11.4%) respectively and 9 (40.9%), 9 (40.9%), 2 (9.1%) and 2 (9.1%) of acute lesions and 5 (38.5%), 3 (23.1%), 3 (23.1%) and 2 (15.4%) of chronic lesions respectively. CONCLUSION: Histology of CL shows marked inflammatory cell infiltrate with or without granuloma formation. Majority of patients presenting with either acute or chronic CL belong to histological groups I or II.Item Comparison of methods for diagnosis of bancroftian filariasis(Sri Lanka Medical Association, 2000) Chandrasena, T.G.A.N.; Premaratna, B.A.H.R.; Abeyewickreme, W.; de Silva, N.R.OBJECTIVE: Evaluate a rapid format immuno-chromatographic card test (ICT Diagnostics, Australia) in the diagnosis of bancroftian filariasis. METHOD: Thick night blood films (TBF), Nuclepore membrane filtration (NMF) and ICT were performed on venous blood collected from 226 individuals selected from highly endemic localities in Colombo [n~153 (63%)] and Gampaha [n=73 (32.3%)] districts. Blood was collected between 20.00 and 23.00 hours. 60ul of non-heparinised blood, 1ml and lOOpl of heparinised blood were used in TBF, NMF and ICT tests respectively. A self-administered questionnaire (expert validated) was used to screen for clinical manifestations. RESULTS: The mean age of the study population was 34-8yrs (range 14-76, SD 16.78); the male: female ratio was 98: 128. NMF was positive in 66/226 (29%), with a mean microfilariae count of 343/ml (range 9-1782, SD 422). All 66 were positive by ICT (sensitivity = 100%) but only 63 by TBF (sen.sitivity=95%). 59/226 (26.1%) had one or more filariasis specific symptoms (lymphoedema, hydrocoele, lymphadenitis, lymphangitis, fever, night cough and red spots). Of the 59, 25 (42.3%) were positive by the ICT, 24 (40.6%) were positive by NMF. The other 34 were negative in both tests. Out of the 166 asymptomatics, 42 were positive in both NMF and ICT, but there were 13 more positives with ICT. CONCLUSIONS: ICT card test was more sensitive in detecting microfilaria compared to venous thick night blood film. Both ICT and NMF were positive in only in about 40% of individuals with symptoms suggestive of filariasis.Item Paediatric rota-virus diarrhoea in Sri Lanka: a preliminary report(Sri Lanka Medical Association, 2007) Chandrasena, T.G.A.N.; Rajindrajith, S.; Ahmed, K.; Pathmeswaran, A.; Abeyewickreme, W.; Nakagomi, O.OBJECTIVE: To determine the prevalence, severity and molecular epidemiology of group A rotavirus infections among children hospitalized with diarrhoea in Sri Lanka. DESIGN, SETTINGS AND METHODS: A prospective hospital-based study was conducted in the paediatric units of the Colombo North Teaching Hospital from April 2005 to February 2006. Stool samples of children admitted with diarrhoea were analysed for Group A rotavirus antigen by enzyme linked immunosorbent assay (ELISA) (Rotaclone ®). Samples positive for rotavirus were characterised by electropherotyping (PAGE) and serotyping (reverse transcription-polymerase chain reaction (RT-PCR) respectively. Severity of diarrhoea was assessed by the Vesikari severity score. RESULTS: A total of 341 children [(204 males, mean age 25.7 months (range 1-144)] were studied. Sixty seven (19.6%) had rotavirus diarrhoea. RT-PCR and PAGE were done on 58 rotavirus positive samples. Thirty one samples were PAGE positive with 6 different electropherotypes. RT-PCR revealed the presence of serotypes Gl, G2, G3, G4 and G9 in 7 (12.1%), 16 (27.6%), 2 (3.4%), 2 (3.4%) and 11 (19.0%) samples respectively. Twenty samples (34.5%) were untypable. Severity score assessed in 326 patients revealed a mean score of 13.3 and 11.4 in rotavirus positive and negative diarrhoeas respectively (p<0.05). Presence, frequency and duration of vomiting and duration of diarrhoea were significantly higher in rotavirus infections (p<0.05). CONCLUSIONS: Rotavirus is an important agent of severe paediatric diarrhoea in Sri Lanka. Molecular analysis indicates genetic diversity among group A rotavirus. This study reports for the first time G9 type rotavirus infection in Sri Lanka.