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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Epidemiological evidence of acute transmission of zika virus infection in dengue suspected patients in Sri-Lanka(Elsevier, 2023) Ngwe, T.M.M.; Raini, S.K.; Fernando, L.; Gunawardene, Y.I.N.S.; Inoue, S.; Takamatsu, Y.; Urano, T.; Muthugala, R.; Hapugoda, M.; Morita, K.Background: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. Methods: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. Results: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. Conclusion: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these virusesItem Development of the sterile Insect technique to control the dengue vector Aedes aegypti (Linnaeus) in Sri Lanka(Public Library of Science,San Francisco, 2022) Ranathunge, T.; Harishchandra, J.; Maiga, H.; Bouyer, J.; Gunawardene, Y.I.N.S.; Hapugoda, M.Background: The Sterile Insect Technique (SIT) is presently being tested to control dengue in several countries. SIT aims to cause the decline of the target insect population through the release of a sufficient number of sterilized male insects. This induces sterility in the female population, as females that mate with sterilized males produce no offspring. Male insects are sterilized through the use of ionizing irradiation. This study aimed to evaluate variable parameters that may affect irradiation in mosquito pupae. Methods: An Ae. aegypti colony was maintained under standard laboratory conditions. Male and female Ae. aegypti pupae were separated using a Fay and Morlan glass sorter and exposed to different doses of gamma radiation (40, 50, 60, 70 and 80 Gy) using a Co60 source. The effects of radiation on survival, flight ability and the reproductive capacity of Ae. aegypti were evaluated under laboratory conditions. In addition, mating competitiveness was evaluated for irradiated male Ae. aegypti mosquitoes to be used for future SIT programmes in Sri Lanka. Results: Survival of irradiated pupae was reduced by irradiation in a dose-dependent manner but it was invariably greater than 90% in control, 40, 50, 60, 70 Gy in both male and female Ae. aegypti. Irradiation didn't show any significant adverse effects on flight ability of male and female mosquitoes, which consistently exceeded 90%. A similar number of eggs per female was observed between the non-irradiated groups and the irradiated groups for both irradiated males and females. Egg hatch rates were significantly lower when an irradiation dose above 50 Gy was used as compared to 40 Gy in both males and females. Irradiation at higher doses significantly reduced male and female survival when compared to the non-irradiated Ae. aegypti mosquitoes. Competitiveness index (C) scores of sterile and non-sterile males compared with non-irradiated male mosquitoes under laboratory and semi-field conditions were 0.56 and 0.51 respectively at 50 Gy. Signification: Based on the results obtained from the current study, a 50 Gy dose was selected as the optimal radiation dose for the production of sterile Ae. aegypti males for future SIT-based dengue control programmes aiming at the suppression of Ae. aegypti populations in Sri Lanka.Item Assessment of developmental and reproductive fitness of dengue-resistant transgenic Aedes aegypti and Improvement of fitness using antibiotics(Hindawi Pub. Co., 2021) Ramyasoma, H.P.B.K.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.; Dassanayake, R.S.BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.Item Development of an alternative low-cost larval diet for mass rearing of Aedes aegypti mosquitoes(Hindawi Publishing, 2020) Senevirathna, U.; Udayanga, L.; Ganehiarachchi, G.A.S.M.; Hapugoda, M.; Ranathunge, T.; Gunawardene, N.S.BACKGROUND: Aedes aegypti is a major vector of arboviruses that may be controlled on an area-wide basis, using novel approaches such as Sterile Insect Technique (SIT) and Incompatible Insect Technique (IIT). Larval diet is a critical factor to be considered in mass rearing of Aedes mosquitoes for SIT and IIT programs. Therefore, the current study is aimed at evaluating the effects of two novel diets developed from dry fish powder on the growth and development of immature stages and adult fitness-related characteristics of Ae. aegypti in Sri Lanka. METHOD: Three batches of the first instar Ae. aegypti larva, each containing 250 larvae, were exposed to three different larval diets as standard dry fish powder (D1), dry fish powder meal and brewer’s yeast (D2), and International Atomic Energy Agency- (IAEA-) recommended diet (D3), separately. Morphometric and developmental parameters of the 4th instar larvae, pupae, and adult mosquitoes reared under different dietary treatments were measured. The entire experimental setup was replicated thrice. A General Linear Model (GLM) in the form of two-way ANOVA was used for the statistical analysis. RESULTS: Significant diet-based variations were observed in the head length, head width, thoracic length, thoracic width, abdominal length, abdominal width, and total length (; ) of Ae. aegypti larvae. The highest pupation success and the larval size were observed from the larvae fed the D2 diet, while the lowest was reported from D1. All adult morphometric parameters of adult male and female Ae. aegypti mosquitoes also denoted significant dietary variations, reporting the best-sized adults from the D2 diet (; ). Further, significantly higher fecundity and male longevity were also shown by the adult Ae. aegypti (; ) mosquitoes reared under diet D2. CONCLUSION: Based on all the growth and developmental parameters, the D2 diet tends to perform similar to the IAEA-recommended diet in mass rearing of Ae. aegypti mosquitoes, while being more inexpensive. Therefore, larval diet D2 could be suggested as the ideal diet for mass rearing of Ae. aegypti for IIT and SIT-based vector control in Sri Lanka.Item Larvicidal potential of five selected Dragonfly Nymphs in Sri Lanka over aedes aegypti (Linnaeus) larvae under laboratory settings.(Hindawi Pub. Co., 2018) Samanmali, C.; Udayanga, L.; Ranathunge, T.; Perera, S.J.; Hapugoda, M.; Weliwitiya, C.INTRODUCTION: Limitations in breeding source reduction practices, development of insecticide resistance in mosquitoes, and ill effects of chemical controlling methods on human and ecosystem health have motivated Sri Lankan authorities working for dengue control to seek for alternative, ecofriendly, and sustainable approaches for controlling of Aedes vectors, to manage dengue epidemics. The present study attempted to investigate the predation efficiency of locally available dragonfly nymphs over Aedes aegypti under laboratory conditions, aiming to evaluate the potential of using dragonflies as biocontrol agents against dengue. METHODS: Nymphal stages of five locally abundant dragonfly species were collected from different stagnated water bodies in Belihuloya area. After morphological identification, a well grown individual of each species was starved for 12 hours and introduced into a glass tank containing 1L of pond water with 200 larvae (4th instar) of Aedes aegypti. Number of larvae survived in the tank was enumerated hourly up to 48 hours. In case where >75% of larvae are consumed by dragonfly nymphs, additional Ae. aegypti larvae were introduced into such tanks. Experiment was repeated for five times. Same procedure was followed with different stages of growth of the dragonfly nymphs characterized by the highest predation rate. General Linear Model followed by Tukey's pairwise comparison was used for statistical analysis. RESULTS: The predation rates of different dragonfly species varied significantly (p<0.05), whereby Anax indicus (110±7.14 per day) indicated the highest, followed by Pantala flavescens (54.07±5.15) and Gynacantha dravida (49.00±11.89), while Tholymis tillarga (23.47±2.48) had the lowest. Further, significant variations in the larval predation were found among different maturity stages (10-20; 25-35; and 35-45 mm in body length) of Ana. indicus (p<0.05). Regardless of statistical significance, a relatively higher larvicidal activity was observed at dusk than in dawn. Conclusion. Ana. indicus, which is characterized by the highest predation rate, and P. flavescens that has the widest geographical distribution within Sri Lanka along with a notable predation efficacy could be recommended as potential candidates for field trials in biological control of dengue outbreaks via suppression of Ae. aegypti larvae.Item Mitochondrial metabolic genes provide phylogeographic relationships of global collections of Aedes aegypti (Diptera: Culicidae)(Public Library of Science, 2020) Fernando, H.S.D.; Hapugoda, M.; Perera, R.; Black Iv, W.C.; de Silva, B.G.D.N.K.ABSTRACT: Phylogeographic relationships among global collections of the mosquito Aedes aegypti were evaluated using the mitochondrial Cytochrome C Oxidase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4) genes including new sequences from Sri Lanka. Phylogeographic analysis estimated that Ae. aegypti arose as a species ~614 thousand years ago (kya) in the late Pleistocene. At 545 kya an "early" East African clade arose that continued to differentiate in East Africa, and eventually gave rise to three lineages one of which is distributed throughout all tropical and subtropical regions, a second that contains Southeast Asian/Sri Lankan mosquitoes and a third that contains mostly New World mosquitoes. West African collections were not represented in this early clade. The late clade continued to differentiate throughout Africa and gave rise to a lineage that spread globally. The most recent branches of the late clade are represented by South-East Asia and India/Pakistan collections. Analysis of migration rates suggests abundant gene flow between India/Pakistan and the rest of the world with the exception of Africa.Item Multiple dengue virus serotypes resistant transgenic Aedes aegypti fitness evaluated under laboratory conditions.(Landes Bioscience, 2020) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Hapugoda, M.; Capurro, M.L.; Silva Gunawardene, Y.I.N.ABSTRACT:Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes. KEYWORDS: RNA interference; aedes aegypti; dengue disease; multiple miRshRNA; piggyBacItem Gene flow patterns among Aedes aegypti (Diptera: Culicidae) populations in Sri Lanka(MDPI AG, 2020) Fernando, H.S.D.; Hapugoda, M.; Perera, R.; Black Iv, W.C.; de Silva, B.G.D.N.K.ABSTRACT:In Sri Lanka, dengue is the most serious arboviral disease. Recent increases in dengue cases suggest a higher infection rate and spread of the disease to new areas. The present study explores gene flow patterns of Ae. aegypti, the main vector of dengue disease, among 10 collection sites including major ports and inland cities using variations at 11 microsatellite loci. Discriminant analysis of principal components (DAPC) and k-means clustering estimated eight genetic clusters. Analysis of Molecular Variance (AMOVA) estimated equal variances among cities and among collections in Colombo, Sri Lanka. Significant evidence, although weak, was detected for isolation by distance. Analysis of gene flow rates and directions using MIGRATE-n indicated that populations throughout the island served as a source of immigrants for Colombo with abundant gene flow among major commercial cities in Sri Lanka, which appear to receive migrant mosquitoes from throughout Sri Lanka. The observed patterns probably arise through human movement of Ae. aegypti during commerce from throughout Sri Lanka into Colombo increasing the risk of spread. The patterns uncovered in this study are significant for global health as Sri Lanka is situated along a key international shipping route. KEYWORDS: Aedes aegypti; Sri Lanka; gene flow patterns; population structure.Item Genotype-phenotype association analysis identifies the role of α globin genes in modulating disease severity of β thalassaemia intermedia in Sri Lanka(Nature Publishing Group, 2019) Perera, S.; Allen, A.; Silva, I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesiriwardena, I.; Allen, S.; Rees, D.; Efremov, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.β thalassaemia intermedia (βTI) are a heterogeneous group of disorders known to be extremely phenotypically diverse. This group is more complex to manage as no definitive treatment guidelines exist unlike for β thalassaemia major (βTM). There are only a few studies looking at genotype phenotype associations of βTI outside the Mediterranean region. The reasons for the diverse clinical phenotype in βTI are unknown. We categorized fifty Sri Lankan patients diagnosed with βTI as mild, moderate or severe according to published criteria. DNA samples were genotyped for β thalassaemia mutations, α globin genotype and copy number and known genetic modifiers of haemoglobin F production. There were 26/50 (52.0%) in mild group and 12/50 (24.0%) each in moderate and sever categories. 18/26 (69.2%) classified as mild were β heterozygotes and 17/18 (94.4%) had excess α globin genes. 11/12 (91.6%) classified as moderate were β heterozygotes and 8/11 (72.2%) had excess α globin genes. In contrast, 8/12 (66.7%) classified as severe were β homozygotes and 7/8(87.5%) had α globin gene deletions. In Sri Lanka, co-inheritance of either excess α globin genes in β thalassaemia heterozygotes or α globin gene deletions in β thalassaemia homozygotes is a significant factor in modulating disease severity
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