Journal/Magazine Articles
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This collection contains original research articles, review articles and case reports published in local and international peer reviewed journals by the staff members of the Faculty of Medicine
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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 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 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 Optimization of Aedes albopictus rearing procedures for combined sterile insect techniques (SIT) and Wolbachia-based laboratory studies in Sri Lanka(Springer Science and Business Media Deutschland GmbH, 2020) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Abeyewickreme, W.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Manamperi, A.The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of the F1 progeny of individuals collected as free-living larvae in Narahenpita (Western Province), Sri Lanka. The mean temperature of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recommended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility, larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.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 The p.H63D allele of the HFE gene protects against low iron stores in Sri Lanka.(Academic Press, 2019) Allen, A.; Premawardhena, A.; Allen, S.; Rodrigo, R.; Manamperi, A.; Perera, L.; Wray, K.; Armitage, A.; Fisher, C.; Drakesmith, A.; Robson, K.; Weatherall, D.In hereditary hemochromatosis, iron overload is associated with homozygosity for the p.C282Y mutation. A second mutation, p.H63D, occurs at significant frequencies in Europe, North Africa, the Middle East and Asia. Early studies in Sri Lanka indicated that the variant had arisen independently, suggesting that it had been the subject of selective pressure. However, its role in iron absorption is unclear. In a survey of 7526 Sri Lankan secondary school students, we determined hemoglobin genotype and measured red cell indices, serum ferritin, transferrin receptor, iron zinc protoporphyrin and hepcidin. These variables were compared according to the presence or absence of the p.H63D variant in a subset of 1313 students for whom DNA samples were available. Students were classified as having low red cell indices if they had an MCV <80 fl and/or MCH <27 pg. Hetero and/or homozygosity for the p.H63D variant was more common in students with normal than low red cell indices (16.4% and 11.9% respectively; p = 0.019). Iron biomarkers and red cell indices were greater in children with the p.H63D variant than in normal and this was statistically significant for MCV (p = 0.046). Our findings suggest that selective pressure by mild iron deficiency contributes to the high frequencies of the p.H63D variant.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 Haemoglobin variants, iron status and anaemia in Sri Lankan adolescents with low red cell indices: A cross sectional survey(Academic Press, 2018) Rodrigo, R.; Allen, A.; Manamperi, A.; Perera, L.; Fisher, C.A.; Allen, S.; Weatherall, D.J.; Premawardhena, A.Iron deficiency complicates the use of red cell indices to screen for carriers of haemoglobin variants in many populations. In a cross sectional survey of 7526 secondary school students from 25 districts of Sri Lanka, 1963 (26.0%) students had low red cell indices. Iron deficiency, identified by low serum ferritin, was the major identifiable cause occurring in 550/1806 (30.5%) students. Low red cell indices occurred in iron-replete students with alpha-thalassaemia including those with single alpha-globin gene deletions. Anaemia and low red cell indices were also common in beta-thalassaemia trait. An unexpected finding was that low red cell indices occurred in 713 iron-replete students with a normal haemoglobin genotype. It is common practice to prescribe iron supplements to individuals with low red cell indices. Since low red cell indices were a feature of all forms of α thalassaemia and also of iron deficiency, in areas where both conditions are common, such as Sri Lanka, it is imperative to differentiate between the two, to allow targeted administration of iron supplements and avoid the possible deleterious effects of increased iron availability in iron replete individuals with low red cell indices due to other causes such as α thalassaemia.
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