International Postgraduate Research Conference (IPRC)
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Item Evaluation of the Pyrethroid Resistance based on Voltage-Gated Sodium Channel (VGSC) Mutations in Aedes aegypti populations of Colombo, Gampaha and Kandy Districts in Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranathunge, T.; Udayanga, L.; Sarasija, S.; Karunathilaka, S.; Nawarathne, S.; Rathnarajah, H.; Dulficar, F.F.; Shafi, F.N.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.Many countries focus on chemical based vector control strategies to restrict the disease transmissions, where pyrethroid insecticides are widely used as the first line of defense against Ae. aegypti. However, the constant use of insecticides have proven to induce insecticide resistance in mosquitoes. The knockdown resistance (kdr) occurs due to mutations in the Voltage Sensitive Sodium Channel (VSSC) or mutations in the Voltage-Gated Sodium Channel (VGSC), coded by the VSSC gene. Only three kdr mutations namely, the V1016G, S989P, and F1534C have been confirmed as commonly occurring amino acid substitutions among mosquito populations in Southeast Asia. Therefore, to extend this observation, current study was conducted to evaluate the prevalence of V1016G and F1534C mutations among Ae. aegypti mosquito populations in three different geographical regions of Sri Lanka. Immature (both pupae and larvae) stages of Ae. aegypti mosquitoes were collected from Colombo, Gampaha and Kandy districts from March to December 2018 and samples were transported to the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya. A total of 855 Ae. aegypti larvae were collected from all districts and polymerase chain reaction (PCR) assay for molecular genotyping of mutations was performed for collected all Ae. aegypti larvae (III instar), to identify the prevalence of kdr mutations in the three Ae. aegypti populations. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy–Weinberg Equilibrium for each of the point mutation. The Ae. aegypti populations from Colombo, Gampha and Kandy districts showed 40.07% (123/307), 39.58% (114/288) and 19.58% (47/240) of V1016G and F1534C mutations, respectively. The wild type (RR) genotype remained predominant within all the three districts, whereas the homogenous (SS) mutation genotype occurred only in minority. Further, the F1534C was predominant in Ae. aegypti populations of all districts. Among the kdr mutation population, heterogeneous genotyping (RS) for both V1016G and F1534C was prominent, while SS genotyping for V1016G mutation was not observed in the Kandy district. The findings clearly denote that long-term insecticide applications and multiple use of pyrethroids has led to the progression of insecticide resistance among local Ae. aegypti populations. Therefore, evaluation of the prevalence levels of these kdr mutations highlights the necessity for shifting towards novel vector control strategiesItem Assessment of Possible Risk Factors Affecting Transmission of Dengue in the District of Gampaha Based on Reported Dengue Cases(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Perera, E.H.L.; Viswakula, S.; Gunawardene, Y.I.N.S.; Subasinghe, U.; Hapugoda, M.D.Dengue is a fast spreading arboviral infection transmitted by the bite of infected females of Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse). According to the Epidemiology Unit, the second highest number of dengue cases is reported in the District of Gampaha, Sri Lanka over past ten years. Objective of this study was to investigate the entomological and socio-economic risk factors affecting transmission of dengue in laboratory-confirmed dengue case reported stations in the District of Gampaha. Laboratory confirmed positive dengue patients (n=100) by dengue NS1 antigen test during the period of June, 2018-August, 2019 were selected. Entomological surveillance was conducted by visiting to each patient within one week of notification of a positive case. For the collection of socio-economic data, an interviewer-administrated questionnaire was used. Adult Aedes mosquito samples collected using a back-pack aspirator showed, 98.64% (73/74) of Ae. albopictus and 1.35% (1/74) of Ae. aegypti mosquitoes. Larval collection using standard larval surveillance techniques showed 92.96% (185/199) and 7.04% (14/199) of Ae. albopictus and Ae.aegypti larvae respectively. The highest House Index (55.17%-16/29), Container Index (28.89%-13/45) and Breteau Index (44.83%-13/29) were reported in the month of June, 2019. The major Aedes breeding place was identified as plastic buckets/barrels (48.6%-84/173) that being used to discard waste. Piped borne water (88%-88/100) was the major water source of the house-holds. Water source of tube well (9%-9/100) was the next popular water source and 66.67%(6/9) of tube wells were positive breeding places for Aedes larvae. Average homestead of the premises of dengue patients was 16.14 perches. From the 100 dengue cases, 67 cases were from middle of town areas, while 2 were from rural areas. Vegetation coverage of the 78% (78/100) house-holds were grass, bushes and small trees and 3% (3/100) house-holds didn’t have any vegetation coverage. The major mosquito prevention method was usage of mosquito nets (54%-54/100) and among dengue patients 7% (7/100) of dengue patients weren’t using any mosquito prevention method. High density of Ae. albopictus mosquitoes, was reported although Ae. aegypti is the major vector of dengue. Therefore, it is required to draw more attention about the Ae. albopictus breeding sites in dengue control programmes. Participants from the study sites were well aware about the disease but still there is a lack of knowledge on breeding sites and vector control methods. Drawbacks in the waste disposal methods, lack of cleanliness in gardens, unplanned water sources and neglecting preventive actions could be considered as the possible risk factors.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 LankaItem Determination of appropriate positioning of the ovitraps for dengue mosquito surveillance(Faculty of Graduate Studies, University of Kelaniya, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.Three months ovitrap survey was conducted to assess the suitable position in placing the ovitraps for dengue vector mosquito surveillance and this study was initiated due to loss of valuable data from our previous studies as a result of physical damage of the ground kept ovitarps. Thirty four households in the Ragama Medical Officer of Health area in Gampaha District were selected to conduct the ovitrap survey during the period of May to July, 2015 to select the most appropriate positioning of the ovitrap. The conventional black plastic ovitraps (3.2x 2.7 cm) were used in this purpose to collect aquatic stages of Aedes mosquitoes while placing plywood paddle (4 x 0.5 cm) over the upper rim of each coded ovitrap. A total of 136 ovitraps were used in the study site providing four ovitraps (2 each indoor & outdoor) for each house while one of the ovitraps of indoor and outdoor being hung and other being kept on the ground. In positioning ovitraps, the outdoor ones were kept 3m away from the house while leaving indoor ovitraps in the living room in close proximity to racks/hanging clothes or partially shaded places. Following collection of samples at each week, ovitraps were washed thoroughly, refilled with new water and a new paddle, and corresponding data were recorded and analyzed. These analyses revealed that number of larvae and the number of Aedes mosquito eggs present in the two different ovitrap positions (Ground kept vs Hung) were not significantly different; in spite of significant difference (P=0.001) between the outside and inside placements. Further, significantly higher values were observed for both number of mosquito eggs and larvae present in each ovitrap kept outside (60 and 13 respectively) than those placed inside (32 and 3 respectively). Furthermore, slightly higher values were observed for hung ovitraps (49 and 9 respectively) than ones kept on the ground (43 and 7 respectively). Finally, ovitrap placed above the ground level was selected in continuing the routine ovitrap survey, as there was considerable reduction of mechanical damage to the latter thus facilitating continuous data collection.