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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2(BioMed Central, 2023) Herath, H.M.L.P.B.; de Silva, W.R.M.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Jayasingha, J.R.P.; Gayashan, M.K.; Afonso, L.O.B.; de Silva, K.M.N.BACKGROUND In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfgured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. RESULTS The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fuorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specifc metals, Fe3+ (0.0373), AsO4 3− (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH=7.0 in the medium, resembling the characteristics of wildtype E.coli. CONCLUSION Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions.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 Molecular characterization of a reemergent Brugia malayi parasite in Sri Lanka, suggestive of a novel strain(Hindawi Pub. Co., 2021) Mallawarachchi, C.H.; Chandrasena, T.G.A.N.; Withanage, G.P.; Premaratna, R.; Mallawarachchi, S.M.N.S.M.; Gunawardene, Y.I.N.S.; Dasanayake, R.S.; Gunarathna, D.; de Silva, N.R.ABSTRACT: Sri Lanka achieved elimination status for lymphatic filariasis in 2016; still, the disease remains a potential public health issue. The present study is aimed at identifying a subperiodic Brugia sp. parasite which has reemerged in Sri Lanka after four decades via molecular-based analysis. Polymerase chain reaction performed with pan-filarial primers specific for the internal transcribed spacer region-2 (ITS-2) of the rDNA of Brugia filarial parasites isolated from human, canine, and feline blood samples yielded a 615 bp band establishing the species identity as Brugia malayi. Comparison of the ITS2 sequences of the reemerged B. malayi isolates with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi with similar phylogenetic evidence. However, the mean interspecies Kimura-2-parameter pairwise divergence between the generated Brugia sequences with B. malayi and B. pahangi was less than 3%. During the analysis of parsimony sites of the new ITS2 sequences, substitutions at A36T, A296G, T373A, and G482A made the sequences different from both B. pahangi and B. malayi suggesting the possibility of a new genetic variant or a hybrid strain of B. malayi and B. pahangi. Mosquito dissections and xenomonitoring identified M. uniformis and M. annulifera as vectors of this novel strain of B. malayi circulating among cats, dogs, and humans 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 Use of novaluron-based autocidal gravid ovitraps to control Aedes dengue vector mosquitoes in the district of Gampaha, Sri Lanka(Hindawi Pub. Co., 2020) Withanage, G.P.; Viswakula, S.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.ABSTRACT:Dengue is the most important mosquito-borne viral infection in Sri Lanka causing an enormous social and economic burden in the country. In the absence of therapeutic drugs and the developed vaccines are under investigation, vector control is the best strategy to reduce the disease transmission. Therefore, the development of novel tools to control dengue vector mosquitoes has become the need of the hour. Novaluron is a recently developed Insect Growth Regulator (IGR) which inhibits chitin synthesis in immature stages of insects. The aim of the study was to identify the efficacy of a simple and cost-effective Autocidal Gravid Ovitrap (AGO) developed using Novaluron to control dengue outbreaks in the District of Gampaha, Sri Lanka. Laboratory and semifield experiments were performed to identify the activity range, optimum field dosage, and residual effects of Novaluron following the World Health Organization guidelines, and field experiments were performed in the Ragama Medical Officer of Health (MOH) area. Two study areas 800 m apart were selected and assigned as treated and control areas randomly. In each study area, 30 households were selected randomly. Each household was given two ovitraps, one placed indoors and the other placed outdoors. Mortality and survival counts were recorded separately for one-year time period and data were analyzed using a two-way repeated measures analysis of variance model. During the laboratory experiments, the adult emerging inhibition was 100% in all tested concentrations. The optimum field dosage was 2 ppm and the residual effect was 28 days. In the field experiments, significantly higher mortality counts were recorded in treated areas both indoor- and outdoor-placed AGOs. Two-factor repeated measures ANOVA followed by Tukey's test confirmed that the mean mortality count is high for the developed AGOs both indoor and outdoor settings. The developed AGO can be deployed to control both indoor and outdoor dengue vector mosquito populations, and in dengue-risk areas, the ovitrap will be supportive to local health authorities to enhance the efficiency of future vector control programs.Item Evaluation of the effects of Aedes vector indices and climatic factors on dengueincidence in Gampaha District, Sri Lanka(Hindawi Publishing Corporation, 2019) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Udayanga, N.W.B.A.L.; Abeyewickreme, W.Constant monitoring of Aedes vector indices such as Aedes mosquito abundance and ovitrap data is important for the control of dengue epidemics. Therefore, the current study attempted to evaluate the effect of larval and climatic factors on the incidence of dengue outbreaks in the Gampaha district. Based on the distribution of previously reported dengue cases, 34 households in Narangodapaluwa PHI area, Ragama, Sri Lanka, were selected randomly, and entomological surveillance was done fortnightly using adult mosquito catches and larval surveillance techniques for a period of two years. Further, weekly ovitrap surveillance was conducted for one year, by maintaining four ovitraps in a single house, two indoors and two outdoors at ground and at a height of 1.5-2 m. Based on the findings, larval indices, namely, Breteau index (BI), House index (HI), and Container index (CI), were calculated, along with the Ovitrap index (OI). The study area was positive for Ae. albopictus with an adult capturing range of 1~15/34 households. BI initially remained < 3%, which subsequently decreased up to 0. No significant difference in OI was found between the ovitraps placed at ground level and at a height of 1.5-2m (p>0.05), 95% level of confidence. The OI varied from 56.9% to 94.7% during the study period of 12 months, indicating two peaks at the monsoons. Statistics of one-way ANOVA revealed a significant difference in the monthly OI during the study period (p≤0.001) with two peaks representing the monsoonal rainfall patterns. Pearson's correlation analysis revealed that the association between dengue cases and larval indices (BI, CI, HI, and OI) and meteorological parameters was not significant (p<0.05). Migration of mosquitoes and patients could be considered as possible factors affecting the absence of a significant relationship.Item Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference(BioMed Central, 2018) Somarathne, M.B.C.L.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Ellepola, A.N.B.; Dassanayake, R.S.BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.Item A preliminary survey of filarial parasites in dogs and cats in Sri Lanka(Public Library of Science, 2018) Mallawarachchi, C.H.; Chandrasena, T.G.A.N.; Wickramasinghe, S.; Premaratna, R.; Gunawardene, Y.I.N.S.; Mallawarachchi, N.S.M.S.M.; de Silva, N.R.Human brugian filariasis has re-emerged in Sri Lanka after a quiescent period of four decades. This study investigated the prevalence of canine and feline filarial parasites in three localities with human sub-periodic brugian filariasis, in order to determine their potential reservoir status. All reachable dogs and cats, both stray and domestic, within a 350m radius of an index case of brugian filariasis in three locations (Madampe, Wattala and Weliweriya) were screened for microfilariae using Giemsa stained thick blood smears. A representative sample of canine and feline blood samples positive for Brugia spp. microfilariae by microscopy, from each of the three locations, were further analyzed by PCR with specific primers for internal transcribed spacer region 2 (ITS2) of the ribosomal DNA. A total of 250 dogs and 134 cats were screened. The overall microfilaraemia rates were high among both dogs (68.8%) and cats (47.8%). The prevalence of microfilaraemia was significantly higher among dogs than cats (p<0.05). Two filarial species were identified based on morphology of microfilariae: Dirofilaria (Nochtiella) repens (dogs, 54.4% and cats, 34.3%) and Brugia spp. (dogs, 51.6% and cats, 30.6%). PCR analysis of canine (n = 53) and feline (n = 24) samples elicited bands in the region of 615bp, which confirmed Brugia malayi infection. Co-infection with D.(N.) repens was detected by PCR with an additional band at 484bp, in 36 canine and 17 feline samples. Overall microfilaraemia rates of dogs (81.8%) and cats (75%) in Madampe (rural) were significantly higher than in urbanized Wattala (dogs, 62.4% and cats, 26.0%) (p<0.05). High rates of zoonotic filarial infections strongly implicate dogs and cats as potential reservoirs for human dirofilariasis and brugian filariasis in Sri Lanka.Item Detection of Dengue Viral Migration to Sri Lanka(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Withanage, G.P.; Hapuarachchi, H.C.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.Dengue is one of the most important mosquito-borne viral infectionsin Sri Lanka.The causative agent is Dengue Viruses (DENV) and the primary vector of the virus is Aedesaegypti(Linnaeus) while Ae. albopictus (Skuse) is the subsidiary vector. The current research was focused on the detection of DENV serotypes and genotypes circulating in mosquitoes during the dengue epidemic in June and July, 2017 in the EriyawetiyaGramaNiladhari division, where one of the dengue high-risk area in Kelaniya Medical Officer of Health (MOH) area in the District of Gampaha, Sri Lanka. Aedesmosquitoes were collected following WHO guidelinesandthe field-caught mosquitoes were transported to the laboratory for species identification and subsequent analysis. Head and thorax of each mosquito was removed and mosquito samples were pooled separately. Total RNA was extracted from mosquito samples and semi-nested Polymerase Chain Reaction (PCR) was performed to identify DENV serotypes present in the mosquito samples. The results of the PCR indicated the presence of DENV2 in both Ae. aegypti (1/5) and Ae. albopictus (1/27) mosquitoes. Then complete Envelope (E) gene was amplified with DENV2 specific primers for genotyping of the virus which is required to identify the molecular evolution of the DENV2. Prior to sequencing the PCR products were purified and sequencing results were analyzed usingLaserGene software. The generated sequences were aligned with retrieved DENV2 sequences available at NCBI database and the phylogenetic trees were developed using MEGA7 software with General Time Reversible (GTR) substitution model with gamma distributed rates. The robustness of clades was determined by using bootstrap analysis of 500 replicates. The result of the phylogenetic analysis illustrates that the E gene sequences of DENV2 obtained from two DENVpositive mosquito poolsbelong to DENV2 Cosmopolitan Clade Ib, which has been the dominant strain in South-East Asia, specially Singapore, Indonesia, Malaysia, and China since August, 2015.The evidence suggests recent introduction of this DENV strain into Sri Lanka
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