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 Sub clinical intestinal mucosal inflammation in diarrhea predominant Irritable Bowel Syndrome (IBS) in a tropical setting(American Gastroenterological Association(AGA) Institute, Published by Elsevier Inc., 2010) de Silva, A.P.; Manamperi, A.; Hewavisenthi, S.J.de S.; Ariyasinghe, M.P.; Dassanayake, A.S.; Jewell, D.P.; de Silva, H.J.BACKGROUND: There is evidence for potential roles for gut flora and the host immune response in the pathophysiology of IBS, and especially, for low grade colonic mucosal inflammation in the pathophysiology of post-infectious IBS. AIM: To investigate for evidence of sub-clinical intestinal mucosal inflammation in diarrhea- predominant IBS (IBS-D) in a tropical setting. METHODS: In a prospective study over one year, we investigated 49 patients with IBS-D [median age 34 years (range 18-59; M:F 36:13], based on Rome III criteria. None had alarm symptoms: unintentional significant loss of weight, bleeding per rectum or malaena. None were on NSAIDS or proton pump inhibitors. All patients had normal ESR, CRP, TSH and stools reports. 14 individuals with a family history of colon cancer [median age 46.5 years (range 23-56); median 46.5, M:F 6:8] were selected as controls. Stools of patients and controls were tested for calprotectin. During colonoscopy, serial biopsies were obtained from the ileum, caecum, ascending, transverse and descending colon, and rectum. In addition to histology, tissue expression of IL-8 and IL-10 were assessed in biopsy specimens using semi-quantitative RT-PCR. RESULTS: Colono-ileoscopy was macroscopically normal and faecal calprotectin was undetectable in cases and controls. Microscopic colitis not otherwise specified(MNOS) was seen in 10/49 cases and 1/14 controls (p=0.43, Fisher's Exact test). Histology was normal in others. A history suggestive of an episode of infectious diarrhea (ID) was seen in 16/49 cases and 0/14 controls (p=0.013). There was no significant association between ID and the presence of MNOS. Tissue expression of IL-8 was significantly higher and IL-10 significantly lower in cases compared to controls (target/standard cDNA ratio, median (range) IL-8: 1.25 (0.75-2) Vs 0.85 (0.63-1.3), p<0.0001, Mann-Whitney U test; IL-10: 0.33 (0-0.63) Vs 0.55 (0.5-0.7), p<0.0001). There was a significant inverse correlation between IL-8 and IL-10 expression (Pearson Correlation, (-) 0.509; p<0.01). In patients with IBS-D, cytokine abnormalities were not significantly different in those with or without a history of ID or the presence or absence of MNOS. CONCLUSION: There is evidence for subclinical intestinal mucosal inflammation in patients with IBS-D in a tropical setting, whether or not a history of ID or MNOS was present or absent.Item GIS mapping of Lymphatic Filariasis endemic areas in Gampaha district, Sri Lanka; based on the epidemiological and entomological screening(Faculty of Tropical Medicine, Mahidol University, 2009) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Abeyewickreme, W.BACKGROUND: The health issues related to vector borne diseases appear always to be related to space and time. Therefore it is ideal to link Geographical Information Systems (GIS) with epidemiological and entomological data to monitor spread of infection and target control strategies. OBJECTIVE: The objective of this study was to develop a site directed GIS map for lymphatic filariasis (Lf) dispersed areas in Gampaha district, Sri Lanka as a guide to target control activities. METHODOLOGY: Epidemiological and entomological screening of Lf was done in nine pre-identified endemic areas in Gampaha district, using night blood screening and pool-screening PCR-ELISA protocols respectively. RESULTS: Overall, 1073 (286 children, 787 adults) from 9 sites were examined. Mf-positive cases were detected in 2 sites, with a prevalence rate of 10.5% (Hekiththa) and 3.4% (Peliyagoda) with over 30% Mf prevalence in adult mosquito populations. The overall prevalence of mosquitoes with L1-L2 larvae of W. bancrofti ranged from 0%-8.54% by dissection and point estimates of infection prevalence, as assayed by PCR-ELISA, ranged from 0% - 35.4%. According to geographical data, the highest number of cases was found at altitudes between 2.5-3.5 m and highly populated areas where transmission appears to be taken place. Questionnaires indicated limited community awareness can be a reason for the fairly static infection rate prevalent in Peliyagoda sentinel site. DISCUSSION: The maps derived indicate the substantial extent as well as the marked variability in the geographical distribution of Lf in Gampaha, demonstrating site related trends.Item Population data for CSF1PO, TPOX, THO1, D16S539, D7S820, D13S317, FESFPS, vWA and F13B short tandem repeat (STR) polymorphisms in Sri Lanka(Sri Lanka Association for the Advancement of Science, 2004) Manamperi, A.; Gunawardene, Y.I.N.S.; Bandara, K.B.A.T.; Dayanath, M.Y.D.; Hapuarachchi, H.A.C.; Abeyewickreme, W.Item Application of nucleic acid technology (NAT) in the diagnosis of active viral replication in HBV and HCV infections and evidence for HBV surface antigen mutants(Sri Lanka Association for the Advancement of Science, 2008) Manamperi, A.; Gunawardene, Y.I.N.S.; Hapuarachchi, C.; Bandara, A.; Wellawaththage, C.; Abeyewickreme, W.; de Silva, J.Introduction: The community prevalence of Hepatitis B (HBV) and hepatitis C (HCV) infections, although considered low (< 1%) in Sri Lanka based on serological markers, pose a significant health threat to patients in high risk groups. The early diagnosis of active viral infection is crucial in such situations to prevent further transmission and to enable the clinicians to initiate successful therapeutic interventions. Objective: This study was carried out to investigate the usefulness of polymerase chain reaction (PCR) in the diagnosis of active viral replication in HBV and HCV infections. Methodology: All specimens from patients with serological evidence of hepatitis B (HBV surface antigen and/or antibodies for HBV core protein) or hepatitis C (antibodies for hepatitis C core protein-Anti-HCV) and referred to the Molecular Medicine Unit from May 2005 to May 2008 were analyzed by PCR and reverse-transcription PCR (RT-PCR) for HBV DNA (n=130) and HCV RNA (n=95) respectively. Results: Of the 130 patients tested, 57 (44%) were positive for HBV DNA. The positive group of patients included 10 renal transplant patients, 4 multiply transfused patients, 4 paediatric patients with lymphoma, and 1 patient with cirrhosis. Six HBV DNA positive patients had negative HBsAg serology profiles indicating the possibility of surface antigen mutant strains. The HBV DNA negative patients with positive serology profiles indicate sero-converted/ patients with resolved infections or false positive serology results. Of the 95 patients tested, 14 (15%) were positive for HCV RNA and included 3 paediatric patients with thalassaemia. HCV RNA negative, anti-HCV positive profiles reflect either false positive serology results (due to less specific antibody assays) or donors who have been exposed to HCV previously and subsequently resolved their infections. Conclusions: A major proportion of patients with serological markers for HBV have active viral infection whereas only relatively a minor proportion of patients with serological markers for HCV have active viral replication. We have also found the first possible evidence of hepatitis B surface antigen mutant strains. This underlines the importance of the nucleic acid based technology in the diagnosis and assessment of infection with or suspected to have hepatitis B or C infections. We also emphasize the importance of introducing NAT for screening donors for HBV DNA and HCV RNA to substantially lower the risk of acquiring HBV/HCV infection from a transfusion.Item Analysis of genetic polymorphism of Plasmodium vivax duffy binding protein ligand domain of Sri Lankan isolates(Sri Lanka Association for the Advancement of Science, 2008) Premaratne, P.H.; Aravinda, R.; Manamperi, A.; Randeniya, P.V.Interaction of Plasmodium vivax Duffy Binding Protein region II (PvDBPII) critical binding motif (CBM) with its erythrocyte receptor is critical for maintaining blood stage infections, rendering PvDBP a leading vaccine candidate. Since the efficacy of a vaccine based on a polymorphic antigen, such PvDBP, is influenced by the local host immune response, characterization of the GD among local parasite strains is important in specific geographic settings. GD of the PvDBPII-CBM was assessed for the first time among field isolates from Sri Lanka. Forty single clonal P. vivax infections identified from two malaria endemic areas (Anuradhapura and Kataragama) and from a non-endemic area (Colombo), were used to generate nucleotide sequence data of CBM of PvDBPII (aa 285 to 521) by nested PCR amplification followed by direct sequencing. Twenty dimorphic sites, 20 nucleotide polymorphisms and 15 haplotypes (haplotype diversity = 0.890) were identified at the CBM of PvDBPII compared to Sal-1 sequence. Genetic polymorphism in terms of pair wise diversity (p) and Tamura's three parameter model (d) were calculated to be 0.00950 (S.D.=0.00072) and 0.00959 (S.D.= 0.00052), respectively, consistent with published data from world wide isolates. Eighteen non-synonymous(NS) and 02 synonymous(S) mutations were identified, and the ratio of NS (0.01086) to S (0.00458) mutation rates was significantly >1, suggesting that positive selection acts on the CBS of PvDBPII. Residues essential for erythrocyte binding on PvDBPII-CBR were conserved in these 40 isolates. Six polymorphic residues recorded in high frequencies in worldwide isolates were also present among Sri Lankan isolates. Polymorphisms occurring at higher frequencies than Papua New Guinea isolates, of three amino acid residues involved in resistance to binding inhibitory antibodies were also detected. Thus, even under low and unstable transmission conditions prevalent in the island, relatively high allelic diversity and positive selection acting on CBM of PvDBPII, possibly due to immune pressure were detected in Sri Lankan P. vivax field isolates. Financial support by the National Science Foundation (Grant numbers NSF/SCH/2004/07 and NSF/RG/2005/HS/06) and the National Research Council (Grant numbers 05-34) is acknowledged.Item Large-scale entomological assessment of Wuchereria bancrofti transmission by dissection and PCR-ELISA in Gampaha district, Sri Lanka(Sri Lanka Association for the Advancement of Science, 2008) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Hapuarachchi, H.A.C.; Bandara, K.B.A.T.; Abeyewickreme, W.Entomological surveys are important tools for monitoring progress of lymphatic filariasis (Lf) eradication programs. In this study, dissection of Culex quinquefasciatus was compared with a Polymerase Chain Reaction - Enzyme Linked Immunosorbent Assay (PCR-ELISA) for pooled mosquitoes to assess filarial infection levels in the major vector of Wuchereria bancrofti in Gampaha district, following mass-treatment programme with diethylcarbamazine (DEC) and albendazole. Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health (MOH) areas of Gampaha district known for the presence of W. bancrofti transmission. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using Deoxyribonucleic acid (DNA) extracted from mosquito pools (15 body parts/pool) utilizing primers specific for the Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3pools/site) was estimated using the PoolScreenTM algorithm and a novel probability-based method. The prevalence of infected mosquitoes with L1-L2 larvae of W. bancrofti ranged from 0%-8.54% by dissection and point estimates of infection prevalence as assayed by PCR-ELISA, ranged from 0% - 25.4%. Mosquitoes collected from all MOH areas (80%, N = 12), except for Minuwangoda, Dompe and Ragama, were positive for W. bancrofti larvae, with a prevalence rate ranging from 0.78% to 16.97% in both methods. Of 30 sentinel sites, 43.3% (N = 13) were positive for W. bancrofti transmission whereas it was evident in 40% (N = 6) of non-sentinel sites. The proportion of positive pools detected by the PCR-ELISA assay was higher than that obtained by the dissection indicating that PCR-ELISA assay is more sensitive than the dissection method in detecting infected/infective mosquitoes. Also results of this study showed that autochthonous transmission of W. bancrofti continues in the Gampaha district despite completion of the 5 year mass drug administration (MDA) programme. Therefore, we emphasize the use of more sensitive tools such as PCR-ELISA to monitor the impact of the MDA programme on disease transmission. This study also emphasizes that control measures should be further continued until the microfilareamic population is reduced to a level which could interrupt transmission in the area. Financial assistance received from WHO/SEARO/TDR (grant no. SN 1152) and University of Kelaniya (Grant no. RP/03/04/06/01/2006) is acknowledgedItem Comparison of five DNA extraction methods from human blood for the detection of Wuchereria bancrofti by polymerase chain reaction assays(Sri Lanka Association for the Advancement of Science, 2008) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Hapuarachchi, H.A.C.; Gunawardena, N.K.; Abeysundara, S.; Abeyewickreme, W.Introduction: Lymphatic filariasis (Lf) is the second most common vector-borne disease globally. Approximately 90% of global burden of Lf is caused by Wuchereria bancrofti. W. bancrofti is routinely diagnosed by morphological identification of microfilariae (Mf) by microscopy which is a labour intense, low sensitive and time consuming method. Detection of W. bancrofti Deoxyribonucleic acid (DNA) using polymerase chain reaction (PCR) technique has become popular today, because of its high sensitivity and specificity. The overall success of the PCR strategy in detecting a filarial parasite in human blood varies between sample preparation methods. The objective of this study was to compare five DNA extraction methods (Lysis + centrifugation, Chelex method, Mf pellet method, Q1Aamp DNA Mini Kit commercial system, and Phenol-chloroform) with regard to duration of completion, labor involvement and PCR analytical sensitivity in-relation to DNA quality and quantity for the detection of W. bancrofti in human blood. Five blood samples positive for mf of W. bancrofti were tested for each DNA extraction method and were compared with respect to the sensitivity, time and quality/quantity of DNA and also by PCR analysis. Of the 5 methods tested, Mf pellet method was found to be the most simple and effective technique for the isolation of W. bancrofti Mf in human blood. This method was quick (15 min to complete), simple (5 min of manual labor), and very economical. It does not require any organic solvents, and the entire extraction procedure uses only two steps requiring supernatant transfer between tubes, hence minimizing the possibility of cross-contamination. Moreover, the PCR analytical sensitivity of the Mf pellet method was comparable to that of the commercial kit used. No PCR inhibitors were detected, independently of Mf count in the blood. Same method (optimal DNA extraction method) can be also used for the detection of parasite DNA from the field collected Mf positive mosquitoes using a PCR. Therefore, we recommend the Mf pellet method for processing large numbers of blood samples in community surveys aimed at determining the prevalence of W. bancrofti infection.Item Evaluation of an IS 6110 - based PCR assay for laboratory detection of M. tuberculosis complex DNA in clinical samples(Sri Lanka Association for the Advancement of Science, 2008) Palliyaguruge, R.H.; Gunawardene, Y.I.N.S.; Manamperi, A.; Wijekoon, C.N.; Wellawaththage, L.C.; Abeyewickreme, W.Due to the slow growth rate of the causative agent, the diagnosis of Tuberculosis (TB) takes considerable time period leading to the complication and spread of the disease. Towards this end, use of Polymerase Chain Reaction (PCR) technology, has revolutionized diagnosis of TB by reducing the diagnostic time. The aim of the present study was to compare two primer pairs and DNA extraction methods for the PCR based detection of M. tuberculosis complex (MTB) DNA in clinical samples for the routine laboratory diagnosis of TB. Two DNA extraction methods (Modified Boom's method and Roche commercial kit) and two IS 6110-based primer pairs were compared with respect to the sensitivity, time and quality/quantity of DNA. Extra pulmonary and pulmonary specimens from 45 TB suspected patients referred to the Molecular Medicine Unit, University of Kelaniya from February 2007 to April 2008 were analyzed. Results indicated 50 % and 70 % of the samples extracted from modified Boom's method and commercial kit, respectively, had high quality DNA, while 17 % and 67 % of the specimens extracted by the Boom's method and commercial kit, respectively, had over 200 µg/ml DNA. Both primer pairs exhibited similar level of sensitivity (200 fg of MTB DNA). In comparison to the time consuming culture, which takes 4 to 6 weeks, the modified Boom's method and commercial kit combined with PCR takes only 48 and 24 hrs, respectively. Of the 19 positives (42.22%) 11 were males while 17 and 02 were extra-pulmonary and pulmonary TB, respectively. The commonest clinical indication for sending samples was suspected disseminated TB. Presence or absence of fever or presence or absence of very high ESR (>100 mm) did not have a significant positive or negative predictive value for PCR. Moderately high ESR (>50 mm) had a negative predictive value of 0.8 and Mantaux test had a positive predictive value of 0.8. According to the time required for completion, labour, quality/quantity of DNA (statically significant at p=0.05) and reproducibility the commercial kit proved to be an efficient DNA extraction procedure. Both sets of primers elicited similar discriminating power. There was not a single clinical indicator with satisfactory predictive values, which is useful in clinical decision making regarding the need for PCR diagnosis in individual patients. We report a simple, rapid and reproducible PCR assay for routine laboratory diagnosis of MTB DNA from both pulmonary and extra-pulmonary specimens.Item Clinical utility of PCR and real time PCR assays for Cytomegalovirus, hepatitis B and hepatitis C infections.(Sri Lanka Association for the Advancement of Science, 2008) Dassanayake, R.S.; de Silva, P.; Weerasena, J.; Gunawardene, Y.I.N.S.; Manamperi, A.Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Reactivation of cytomegalovirus (CMV), Hepatitis B (HBV) and C (HCV) viruses from the status of latency is seen in immunocompromised individuals and such reactivation is often associated with morbidity and mortality in such individuals. The prevalence of these viral infections in a selected population of patients referred to the Molecular Diagnostic Laboratory at the Durdan's Hospital, Colombo, during the period from August 2007 to May 2008 were studied using qualitative PCR assays. All specimens from patients with suspected clinical diagnoses of either CMV or HBV or HCV infections were analyzed. Of 176 samples analyzed for CMV 78 were positive (37 males, 29 females) and majority of them are patients from a nephrology unit. Out of 40 and 10 samples analyzed from males and females, respectively, 22 and 4 were positive for HBV. Twenty six samples were analyzed for HCV and only 6 were fond to be infected with viruses and all of them were from males. Although PCR detection of these viral DNA/RNA is a sensitive method to detect infection, it lacks specificity for the detection of active viral disease and for monitoring the efficacy of antiviral therapy. Therefore, Real-time PCR (RT-PCR) assays for the detection and quantification of CMV-DNA, HBV-DNA and HCV-RNA were developed using SYBRgreen1 chemistry. The assays developed are capable of detecting viral particles in blood samples and quantifying viral DNA accurately over a broad range of input target copies (102 - 108copies/ml) and therefore, can be used to predict the reactivation of viruses by comparing with published kinetic criteria in clinical guidelines. Post PCR analyses of Real-time PCR products by agarose gel electrophoresis revealed bands having the same intensity for a wide range of target copies (103 -108copies/ml). In contrast, RT-PCR elicited higher cycle threshold for the descending order of concentration of target copies. Therefore, based on these results, it is evident that the intensity of conventional PCR bands should not be used for the assessment of viral reactivation or for monitoring therapeutic intervention and for this purpose RT-PCR is the method of choiceItem Rapid differential diagnosis of dengue and chikungunya infections by multiplex RT-PCR and impact of chikungunya infection on liver biochemical tests(Sri Lanka Association for the Advancement of Science, 2008) Manamperi, A.; de Silva, P.; Ekanayaka, C.; Gunawardene, Y.I.N.S.; de Silva, J.; Weerasena, O.V.D.S.J.; Dassanayake, R.S.