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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Efficacy of hydroxyurea in reducing the erythropoietic stress of ineffective erythropoiesis in transfusion dependent beta thalassaemia: A randomised, double-blind placebo-controlled clinical trial(Sri Lanka Association for the Advancement of Science, 2021) Yasara, N.; Premawardhena, A.; Perera, P.; Manamperi, A.; Mettananda, S.The unbalanced synthesis and accumulation of a-globin chains due to impaired synthesis of 0- globin results in the destruction of red blood cells (RBC) and erythroid precursors of patients with P-thalassaemia. This leads to an increased erythropoietic activity and ineffective erythropoiesis in the bone marrow of these patients. Hydroxyurea is a licenced medication that decreases the RBC destruction of patients with p-thalassaemia. However, its effect on erythropoietic stress is unclear. In this study, our objective was to evaluate the effect of hydroxyurea on erythropoietic stress of ineffective erythropoiesis in transfusion-dependent (TD) p-thalassaemia. This experimental study was carried out at the Thalassaemia Unit of Colombo North Teaching Hospital as part of a randomised, double-blind placebo-controlled clinical trial that evaluates the efficacy of hydroxyurea. We recruited 24 patients with TD p-thalassaemia who were taking hydroxyurea IQ- 20 mg/kg/day and 16 patients who were receiving a placebo. The erythropoietic stress of ineffective erythropoiesis was assessed by measuring serum soluble transferrin receptor (sTfR) levels before and six months after taking either hydroxyurea or placebo. Levels of sTfR were measured using a validated enzyme-linked immunosorbent assay. Paired t-test was used in the statistical analysis. Nineteen (79%) out of 24 patients who received hydroxyurea showed a reduction in sTfR level, of which 8 (33%) and 6 (25%) showed >25% and 10-25% reductions, respectively. A significant reduction in mean sTfR level was observed after hydroxyurea treatment (72.3±SD25.9) compared to pre-treatment levels (89.6士SD22.9), (pv0.01). Conversely, no difference in sTfR levels was seen in patients who received the placebo pre・(91.9土SD24.7) and post-treatment (96.4±SD19.4), (p=0.17). In conclusion, oral hydroxyurea significantly reduced the erythropoietic stress of ineffective erythropoiesis in patients with TD p-thalassaemia showing a promise as a treatment modality.Item In vitro study to identify effects of fetal haemoglobin inducing agents on primary human erythroid cells(Sri Lanka Association for the Advancement of Science, 2021) Yasara, N.; Manamperi, A.; Mettananda, S.Beta-thalassemia is an autosomal, recessively inherited monogenic disorder characterized by defective beta-globin synthesis. Deposition of excess alpha-globin in erythrocytes and their precursors due to unbalanced globin synthesis is the main underlying pathophysiology. Clinical data show that induction of fetal haemoglobin (HbF) in erythroid cells ameliorates the disease severity and associated complications. Hence, improving HbF via up-regulating gamma-globin gene expression has been identified as a novel treatment. The aim of our in vitro study was to identify the effects of fetal haemoglobin inducing agents in human erythroid cells. Haematopoietic stem cells (HSC) from umbilical cord blood samples collected from three healthy human placentas were isolated. Firstly, mononuclear cells were separated from the interface after fractionation on Histopaque®-1077 Hybri-Max and CD34+ HSCs were isolated using positive selection by magnetic activated cell sorting. HSCs were then expanded and differentiated into mature erythroid cells using a three-phase liquid culture protocol. Primary human erythroid cells at day 7 of the culture were incubated with hydroxyurea (20 pM), butyric acid (10 ^M), 5-azacystidine (10 pM), decitabine (5 pM), busulfan (30 pM), vorinostat (2.5 pM) and valproic acid (1000 pM) for 72 hours. Effects of these compounds on cell expansion, viability, morphology, as well as a-, P- and y-globin mRNA levels were measured using standard laboratory methods. Negative controls were tested in parallel. Compared to other compounds, hydroxyurea and butyrate treated erythroid cells displayed a significantly high mean fold expansion and viability. Significantly higher gamma-globin mRNA levels were observed in hydroxyurea treated cells (Mean relative expression: 186 ± SEM16) compared to negative control cells (Mean relative expression: 137 ± SEM14). Highest y/p globin mRNA ratios were observed in busulfan (12.6 ± SEM2.9) and decitabine (12.1 ± SEM3.3) treated erythroid cells. In conclusion, hydroxyurea induces gamma-globin expression and decitabine and busulfan favourably alter the y/P-globin mRNA ratios in vitro in human erythroid cells.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 Hydroxyurea for transfusion dependent β-thalassaemia: A randomized double-blind placebo-controlled clinical trial(Sri Lanka Medical Association, 2021) Yasara, N.; Wickramarathne, N.; Silva, I.; Hameed, N.; Attanayaka, A.M.K.R.; Jayasinghe, V.L.; Gunathilaka, P.A.C.K.; Wickramasinghe, N.; Rodrigo, R.; Perera, L; Perera, P.S.; Mettananda, K.C.D.; Manamperi, A.; Premawardhena, A.; Mettananda, S.Introduction and objectives Hydroxyurea induces fetal haemoglobin in vitro however, its clinical usefulness in β-thalassaemia is unclear. Here, we aim to assess the efficacy and safety of oral hydroxyurea in patients with transfusion dependent β-thalassaemia. Methods A phase 3 randomized double-blind placebo-controlled clinical trial was conducted at Colombo North Teaching Hospital in 2019/20. Sixty patients with transfusion dependent β-thalassaemia were randomized into hydroxyurea (10-20mg/kg/day) or placebo groups. Transfused blood volume, pre-transfusion haemoglobin, fetal haemoglobin and adverse effects were monitored during 6-month treatment and post-treatment periods. The study was approved by the ethics committee of University of Kelaniya and registered in Sri Lanka Clinical Trials Registry (SLCTR/2018/024). Results Fifty-four (hydroxyurea-27; placebo-27) patients completed the trial. Mean pre-transfusion haemoglobin (8.2±0.8g/ dLvs8.0±0.88g/dL, p=0.43) and fetal haemoglobin levels (7.9±11.2%vs4.6±4.3%, p=0.17) were higher in hydroxyurea group compared to placebo. Also, transfused blood volume was lower in the hydroxyurea group (94±29ml/kgvs102±28ml/kg, p=0.34). However, none were statistically significant. Based on elevation of fetal haemoglobin (>1.5% from baseline), we identified 12/27 patients who respond well to hydroxyurea (hydroxyurea-responders). Hydroxyurea-responders required significantly lower blood volume (77±27ml/kg) compared to non-responders (108±24ml/kg, p<0.01) and placebo group (102±28ml/kg, p<0.05). HbE β-thalassaemia sub-type (p<0.01) and Xmn1 polymorphism of γ-globin gene (p<0.05) were significant predictors of response to hydroxyurea. No serious side effects due to hydroxyurea were reported. Conclusion Over 40% of patients with transfusion dependent β-thalassaemia- specifically those with HbE β-thalassaemia and Xmn1 polymorphism of γ-globin gene- responded to hydroxyurea and required 25% less blood compared to controls. No serious adverse effects were reported following hydroxyurea treatment.Item Efficacy and Safety of Oral Hydroxyurea in Patients with Transfusion Dependent β Thalassaemia: a Randomized Double-Blind Placebo-Controlled Clinical Trial(Sri Lanka Medical Association, 2020) Yasara, N.; Wickramarathne, N.; Silva, I.; Hameed, N.; Attanayaka, A.M.K.R.; Jayasinghe, V.L.; Wickramasinghe, N.; Rodrigo, R.; Perera, L.; Mettananda, K.C.D.; Manamperi, A.; Premawardhena, A.; Mettananda, S.INTRODUCTION AND OBJECTIVES: Patients with β- thalassaemia require blood transfusions and iron chelation for life. Hydroxyurea is a licenced medication for sickle cell disease but its usefulness in transfusion dependent β-thalassaemia is unclear. Here, we aim to assess the efficacy and safety of oral hydroxyurea in patients with transfusion dependent β-thalassaemia. METHODS: A phase III randomized double-blind placebo-controlled clinical trial was conducted at Thalassaemia Unit of Colombo North Teaching Hospital in 2019. Forty-one patients with transfusion dependent β-thalassaemia were randomized into hydroxyurea (10-20mg/kg/day) or placebo (pharmaceutically inert capsule identical to hydroxyurea) groups. Transfused blood volume, pre-transfusion haemoglobin, haemoglobin F level and side effects were monitored monthly during 6- month treatment and 6-month follow-up periods. Adverse events were assessed by trained medical officers. The study was approved by ethics committee of University of Kelaniya and registered in Sri Lanka Clinical Trials Registry (SLCTR/ 2018/024). RESULTS: Of the 41 (hydroxyurea-20; placebo-21) patients, three discontinued treatment due to thrombocytopenia (hydroxyurea-2) and rash (placebo-1). Baseline characteristics of two groups were similar. Mean pre-transfusion haemoglobin (8.52+0.57 vs 8.38+0.55, p=0.45) and haemoglobin F levels (4.3+7.1% vs 3.1+1.9%, p=0.48) were higher in hydroxyurea group compared to placebo. Also, transfused blood volume was lower in hydroxyurea group (102+24ml/kg vs 111+27ml/kg, p=0.3). However, none were statistically significant. Based on elevation of haemoglobin F (>1.5% from baseline), we identified 6/18 patients as hydroxyurea responders. Hydroxyurea responders required significantly lower blood volume (87+13ml/kg) compared to non-responders (110+25ml/kg, p=0.05) and placebo group (111+27ml/kg, p<0.05) while maintaining higher pre-transfusion haemoglobin level (8.6+0.5 vs 8.4+0.5 and 8.3+0.5). No serious side effects were reported. CONCLUSIONS: One-third of patients with transfusion dependent β-thalassaemia responded to hydroxyurea treatment requiring 20% less blood compared to controls. No serious side effects were reported following hydroxyurea treatment.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 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 Introduction of Recombinase Polymerase Amplification assay based mobile suitcase laboratory as a point of need tool to diagnose cutaneous leishmaniasis in Sri Lanka(Sri Lanka Medical Association, 2018) Gunaratna, G.P.S.; Ranasinghe, P. H. K. I. S.; Manamperi, A.; Pathirana, N.; Pathirana, H.; Wickremasinghe, R.; de Silva, N.R.; Sooriyarachchi, M.; Ahmed, A.E.W.INTRODUCTION AND OBJECTIVES: Cutaneous leishmaniasis (CL) caused by the vector-borne protozoan parasite is now endemic in Sri Lanka. Microscopy of Giemsa stained slit skin smears (SSS), lesion aspirates or scrapings for the presence of amastigotes, is widely used for laboratory confirmation of CL, although the reported sensitivity is low. Facilities for more sensitive culture and molecular techniques are available only in reference laboratories. A newly developed, Recombinase Polymerase Amplification (RPA) assay based Mobile Suitcase Laboratory (MSL) is a promising, molecular point of care test with high sensitivity and specificity for diagnosis of both post-kala• azar dermal leishmaniasis and visceral leishmaniasis. Objective was to assess RPA based MSL as a point of need tool to diagnose CL in Sri Lanka.METHODS: Twenty seven army personnel at Mullaitivu Army camp clinically suspected of having CL were recruited for this pilot study. Two slit skin smears and two punch biopsy specimens were obtained from each of them. Slit skin smears were stained with Giemsa in the field and examined for the presence of amastigotes and RPA was carried out at the point of collection. PCR was performed at the Parasitology Department, Sri Jayewardenepura University. RESULTS: Fifteen patients were confirmed by PCR as having CL and 14 of them were also positive by RPA based MSL conducted in the field (93.33% sensitivity). Only 3/15 were positive with microscopy of SSS (20% sensitivity). CONCLUSION: This pilot study shows RPA based MSL as a promising tool to diagnose CL at point of need.Item Genetic origin and clinical variability of sickle cell disease in Sri Lanka(Sri Lanka Medical Association, 2018) Darshana, L.G.T.; Bandara, W.D.M.S.; Nawaratna, U.S.B.; Costa, Y. J.; Nizri, A.H.M.; Silva, D.P.S. l.; de Silva, T.U.N.; Pushpakumara, K.P.C.; Pathirage, S.P.; Manamperi, A.; Premawardhena, A.P.INTRODUCTION AND OBJECTIVES: Sickle cell disease (SCD) is found at a low prevalence In Sri Lanka. A recent hospital based survey identified 60 patients in the country The clinical spectrum of SCD in Sri Lanka has not been studied and its genetic origin remains unknown.We envisaged to study the genetic origin and to carry out a clinical description ofSCD in Sri Lankan patients. METHODS: Patients were recruited from Ragama, Anuradhapura Hambantota and Kurunegala thalassaemia centres. All patients were examined and clinical details recorded. Genetic analyses were performed to identify the haplotype of HbS, Xmn I polymorphism gene deletions and mutations. RESULTS: A total of 49 SCD patients were studied Ages ranged from 5 - 47 years (mean 20.4) 91.8% of the patients were Sinhalese and the rest were Muslims. 87.7% of the patients had sickle-thalassaemia (SBT) and 6 were homozygous (HbSS) Joint pains were the commonest symptom in patients with SBT Clinical presentations in SBT varied from those with none to frequent crises Clinical management varied with only 42.86% of patients being on hydroxyurea. Most patients had not inherited disease ameliorating genetic factors with 95 9% not having gene deletions and 89 8% not having Hb F up-regulators Three Sickle haplotypes were identified including; Arab• Indian, Benin and Bantu.CONCLUSION: There appears to be at-least three genetic origins of HbS in Sri Lanka SCD is extremely clinically variable in Sri Lanka. The reason for this variation needs further study as most patients seem not to have common inherited modifiersItem Handbook on general laboratory procedures, safety considerations and housekeeping in a DNA laboratory environment(Authour, 2009) Manamperi, A.No abstract available