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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Detection of pathogenic Leptospira with rapid extraction followed by recombinase polymerase amplification (RPA) and quantitative polymerase chain reaction (qPCR) assay-A comprehensive study from Sri Lanka(Public Library of Science, 2024) Uduwawala, H.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Ceruti, A.; Wahed, A.A.E.; Fernando, L.; Premaratna, R.; Hapugoda, M.Leptospirosis is the most widespread zoonosis in the world. The disease is more prevalent in tropical regions where the majority of developing countries are located. Leptospirosis is considered a protean manifestation zoonosis with severity of the disease ranging from a mild febrile illness to a severe and life-threatening illness. Clinical symptoms of leptospirosis overlap with other tropical febrile illnesses. Early, rapid, and definitive diagnosis is important for effective patient management. Since Polymerase Chain Reaction (PCR)-based assays are not readily available in most clinical settings, there is a need for an affordable, simple, and rapid diagnostic test. Quantitative PCR (qPCR) and Recombinase Polymerase Amplification (RPA) were implemented at the Faculty of Medicine, University of Kelaniya, and a prospective study to evaluate RPA for diagnosis of acute phase of leptospirosis was conducted. Results indicate that RPA and qPCR were positive in 81% (98/121) of the total positive and acute clinical samples. Of the 81 positive MAT confirmed patients 60 (74%) and 53 (65%) were positive with qPCR and RPA respectively. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity-70% and specificity-87%) of RPA compared to MAT as the reference gold standard. Results further suggest that there is no significant difference between the two assays, qPCR and RPA-SwiftX (P = 0.40). Laboratory procedures for the extraction and detection by qPCR in the laboratory have been optimized to obtain results within 6 hours. However, the RPA-SwiftX method under field conditions took 35 minutes. The RPA-SwiftX method could replace the qPCR which shows similar sensitivity and specificity. Therefore, RPA established under the current study presents a powerful tool for the early and rapid diagnosis of leptospirosis at point-of-care.Item Hantavirus Hemorrhagic Fever with Renal Syndrome (HFRS) - Suspected cases in Sri Lanka; clinical picture and epidemiology from 2013-2021(National Institute of Infectious Diseases, 2022) Muthugala, R.; Dheerasekara, K.; Manamperi, A.; Gunasena, S.; Galagoda, G.Hantavirus; Hemorrhagic fever with renal syndrome (HFRS) is an emerging zoonotic disease in Euro-Asia which is clinically indistinguishable from leptospirosis. A total number of 1032 patients were included in the analysis from March 2013 to March 2021 with the clinical suspicion of HFRS-like illness. Of them, 168 patients were positive for hantavirus IgM antibodies. Thirty-one patients out of 35 patients had given a four-fold rise IgG antibody titre with paired serum confirming the acute hantavirus infections. Detected antibodies showed a diverse pattern, strongly cross-reacting with Seoul, Hantaan and Puumala virus antigens. All the IgM positive patients had no serological evidence of acute dengue or leptospirosis and had classical features of HFRS; fever, thrombocytopenia and renal involvement. More than 90% of patients had a history of rodent exposure 2-3 weeks prior to the onset of the fever. The highest number of positive cases were diagnosed from the Western and North-Central Provinces of Sri Lanka during the paddy harvesting seasons. A significant number of patients had developed severe complications with a high mortality rate. Therefore, hantavirus infection should be considered as a differential diagnosis for leptospirosis-like illness in Sri Lanka.Item A randomised double-blind placebo-controlled clinical trial of oral hydroxyurea for transfusion-dependent β-thalassaemia(Nature Publishing Group, 2022) Yasara, N.; Wickramarathne, N.; Mettananda, C.; Silva, I.; Hameed, N.; Attanayaka, K.; Rodrigo, R.; Wickramasinghe, N.; Perera, L.; Manamperi, A.; Premawardhena, A.; Mettananda, S.Hydroxyurea is an antimetabolite drug that induces fetal haemoglobin in sickle cell disease. However, its clinical usefulness in β-thalassaemia is unproven. We conducted a randomised, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of hydroxyurea in transfusion-dependent β-thalassaemia. Sixty patients were assigned 1:1 to oral hydroxyurea 10-20 mg/kg/day or placebo for 6 months by stratified block randomisation. Hydroxyurea treatment did not alter the blood transfusion volume overall. However, a significantly higher proportion of patients on hydroxyurea showed increases in fetal haemoglobin percentage (89% vs. 59%; p < 0.05) and reductions in erythropoietic stress as measured by soluble transferrin receptor concentration (79% vs. 40%; p < 0.05). Based on fetal haemoglobin induction (> 1.5%), 44% of patients were identified as hydroxyurea-responders. Hydroxyurea-responders, required significantly lower blood volume (77 ± SD27ml/kg) compared to hydroxyurea-non-responders (108 ± SD24ml/kg; p < 0.01) and placebo-receivers (102 ± 28ml/kg; p < 0.05). Response to hydroxyurea was significantly higher in patients with HbE β-thalassaemia genotype (50% vs. 0%; p < 0.01) and Xmn1 polymorphism of the γ-globin gene (67% vs. 27%; p < 0.05). We conclude that oral hydroxyurea increased fetal haemoglobin percentage and reduced erythropoietic stress of ineffective erythropoiesis in patients with transfusion-dependent β-thalassaemia. Hydroxyurea reduced the transfusion burden in approximately 40% of patients. Response to hydroxyurea was higher in patients with HbE β-thalassaemia genotype and Xmn1 polymorphism of the γ-globin gene.Item Efficacy and safety of oral hydroxyurea in transfusion-dependent β-thalassaemia: a protocol for randomised double-blind controlled clinical trial(BMJ Publishing Group Ltd., 2020) Yasara, N.; Wickramarathne, N.; Mettananda, C.; Manamperi, A.; Premawardhena, A.; Mettananda, S.INTRODUCTION: Despite being one of the first diseases to be genetically characterised, β-thalassaemia remains a disorder without a cure in a majority of patients. Most patients with β-thalassaemia receive only supportive treatment and therefore have a poor quality of life and shorter life spans. Hydroxyurea, which has shown to induce fetal haemoglobin synthesis in human erythroid cells, is currently recommended for the treatment of sickle cell disease. However, its clinical usefulness in transfusion-dependent β-thalassaemia is unclear. Here, we present a protocol for a randomised double-blind controlled clinical trial to evaluate the efficacy and safety of oral hydroxyurea in transfusion-dependent β-thalassaemia. METHODS AND ANALYSIS: This single-centre randomised double-blind placebo-controlled clinical trial is conducted at the Thalassaemia Centre of Colombo North Teaching Hospital, Ragama, Sri Lanka. Adult and adolescent patients with haematologically and genetically confirmed transfusion-dependent β-thalassaemia are enrolled and randomised into the intervention or control group. The intervention group receives oral hydroxyurea 10-20 mg/kg daily for 6 months, while the control group receives a placebo which is identical in size, shape and colour to hydroxyurea without its active ingredient. Transfused blood volume, pretransfusion haemoglobin level, fetal haemoglobin percentage and adverse effects of treatment are monitored during treatment and 6 months post-treatment. Cessation or reduction of blood transfusions during the treatment period will be the primary outcome measure. The statistical analysis will be based on intention to treat. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Ethics Committee of Faculty of Medicine, University of Kelaniya (P/116/05/2018) and the trial is approved by the National Medicinal Regulatory Authority of Sri Lanka. Results of the trial will be disseminated in scientific publications in reputed journals.Item Sickle cell disease in Sri Lanka: clinical and molecular basis and the unanswered questions about disease severity(BioMed Central., 2020) Darshana, T.; Bandara, D.; Nawarathne, U.; de Silva, U.; Costa, Y.; Pushpakumara, K.; Pathirage, S.; Basnayake, S.; Epa, C.; Dilrukshi, P.; Wijayawardena, M.; Anthony, A. A.; Rodrigo, R.; Manamperi, A.; Smith, F.; Allen, A.; Menzel, S.; Rees, D.; Premawardhena, A.BACKGROUND: Though case reports and limited case series of Sickle cell disease in Sri Lanka have been reported previously, no attempt has been made hitherto to undertake a comprehensive genotypic-phenotypic analysis of this "rare" group of patients. RESULTS: All accessible Sickle cell disease patients, totaling 60, including, 51 Sickle β-thalassaemia and 9 homozygous sickle patients were enrolled from seven thalassaemia treatment centres between December 2016-March 2019. The majority of patients were of Sinhalese ethnicity (n = 52, 86.67%). Geographically, two prominent clusters were identified and the distribution of Sickle haemoglobin in the island contrasted markedly with the other haemoglobinopathies. 3/ 9 homozygous sickle patients and 3/ 51 Sickle β-thalassaemia patients were receiving regular transfusion. Joint pain was the commonest clinical symptom among all sickle cell disease patients (n = 39, 65.0%). Dactylitis was significantly more common in homozygous sickle patients compared with the Sickle β-thalassaemia groups (p 0.027). Two genetic backgrounds sickle mutation were identified namely, Arab Indian and Benin. Among the regulators of Foetal hemoglobin in Sickle patients of the present study rs1427407 G > T seemed to be the most prominent modifier, with a significant association with Foetal haemoglobin levels (p 0.04). CONCLUSIONS: Overall, the clinical course of the Asian version of Sickle cell disease in Sri Lanka appears to be milder than that described in India. KEYWORDS: Clinical; Genetic; Severity; Sickle cell; Sri Lanka.Item Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka(BioMed Central, 2018) Gunaratna, G.; Manamperi, A.; Bohiken-Fascher, S.; Wickremasinghe, R.; Gunawardena, K.; Yapa, B.; Pathiana, N.; Pathirana, H.; de Silva, N.; Sooriyaarachchi, M.; Deerasinghe, T.; Mondal, D.; Ranasinghe, S.; Abd EI Wahed, A.BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.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.