Annual Research Symposium (ARS)
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Item Role of Aedes albopictus in transmitting dengue virus in some endemic areas in Kurunegala District(University of Kelaniya, Sri Lanka, 2003) Hapugoda, M.D.; de Silva, N.R.; Abesundara, S.; Bandara, K.B.A.T.; Dayanath, M.Y.D.; Abeyewickreme, W.Abstract AvailableItem Detection of pathogenic Leptospira species in rat blood samples by molecular-based assays(University of Kelaniya, 2013) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hapugoda, M.D.Background: Leptospirosis is a worldwide zoonotic infection, caused by pathogenic species of the genus Leptospira. It was traditionally known as ‘rat fever’ in Sri Lanka, because rodents, especially rats, are considered to be the most important reservoirs or maintenance hosts of Leptospira. In 2012, the highest numbers of cases were reported in the District of Gampaha. The objective of this study is to detect pathogenic Leptospira species in rat blood samples by molecular based assays. Method: Rats (n=38) were trapped in a high risk area (Mirigama) in the District of Gampaha, from May 2012 to February 2013 by using live traps. Each rat was anesthetized by using diethyl ether and 2-3 ml sample of blood was collected from each rat. Blood samples collected from all rats were tested by molecular- based assays and a serological assay. Qualitative Polymerase Chain Reaction (PCR), real time PCR and Loop Mediated Isothermal Amplification (LAMP) were used as molecular-based assays which targetted conserved gene regions among pathogenic serovars of Leptospira species. Microscopic Agglutination Test (MAT), the Gold Standard assay for detection of anti Leptospira antibody was used as a serological assay. Results and Discussion: Of the 38 rat blood samples, molecular-based assays confirmed Leptospira infection in 5% (2/38), 16% (6/38) and 11% (4/38) by qualitative PCR, real time PCR and LAMP assay respectively. None of the samples was positive by MAT. After first infection, some Leptospira species live in the host animal as commensal bacteria. Therefore, host does not stimulate antibody production further and that may be below the detection level of the antibody by MAT. Conclusions: Results of molecular based assays showed that Leptospira are circulating among the rats tested in this study, although at the time of collection, their antibody levels were too low to detect by MAT, which had the lowest detection limit of 1:800.Item Risk factors associated with human leptospirosis in the District of Gampaha, Sri Lanka(University of Kelaniya, 2013) Denipitiya, D.T.H.; Athapaththu, M.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hapugoda, M.D.Background & Objective: A large number of leptospirosis cases are recorded in Sri Lanka every year. Increased numbers of cases have been reported in the District of Gampaha in the recent past. The incidence of leptospirosis is often influenced by various socio-economic, occupational, environmental and other factors. To date, a study on potential risk factors has not been conducted in the District of Gampaha. The objective of this study is to identify risk factors involved in transmission of leptospirosis to humans in the District of Gampaha. Methods: Data were collected at the household level, using an interviewer-administered questionnaire and by inspecting the surrounding of laboratory confirmed leptospirosis patients (n=81) and non leptospirosis persons (n=117) during the period of June 2011 to June 2013. The risk factors in the questionnaire were divided into three broad categories: environmental, contact with animals and behavioral/occupational factors. Chi-square test (The SAS System for Windows 9.0) was used for comparison of data from different categories. Results and discussion: 95% of the leptospirosis patients were adult males (77/81) and they had a monthly income of Rs. 10,001-20,000 and 50% of them were agricultural and rental work labourers (40/81). In contrast, 56% of persons not infected with leptospirosis were adult females (66/117) and most of them (48%) were housewives or homemakers (56/117). Data on the type of premises were collected under three categories as poor, moderate and well constructed along with the land use type of the surrounding areas. There were significant statistical associations between the leptospirosis patient with the type of premises (, χ2=23.38, p=0.00), surrounding cleanliness of premises (χ2=45.05, p=0.00), sanitary facilities (χ2=11.66, p=0.00), waste disposal method (χ2=32.23, p=0.00) and age level of patients (χ2=21.07, p=0.00). No significant statistical associations were observed between recorded leptospirosis cases and vegetation coverage in surrounding area of premises (χ2=1.25, p>0.05), source of drinking water (χ2= 0.55, p>0.05) and numbers of persons in family (χ2=0.17, p>0.05). Conclusion: Identification of the potential risk factors would help understand the transmission dynamics of the disease and formulate public health interventions.Item Species succession of malaria vector mosquitoes and physicochemical factors affecting their abundance in rice ecosystems in Trincomalee, Sri Lanka(University of Kelaniya, 2013) Gunathilaka, P.A.D.H.N.; Ranathunge, R.M.T.B.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.Background: Mosquito-borne diseases are a major public health threat in Asia. The present investigation was carried out to study the ecology of vector mosquitoes in Sri Lanka, in order to explore effective mosquito control strategies in rice ecosystems from the ecological point of view. Method: As part of investigations on potential linkages between irrigation and malaria transmission, all surface water bodies in and around 20 selected villages along an irrigation distributary in the District of Trincomalee, Sri Lanka, were surveyed for anopheline mosquito larvae (Diptera: Culicidae) from January 2012 - June 2013. Breeding sites were characterized according to exposure to sunlight, substratum, and presence of vegetation, fauna, inorganic matter and physical water condition (clear/turbid). Dissolved Oxygen (DO), conductivity, salinity, pH, temperature, Total Dissolved Solids (TDS) and turbidity were recorded. Results: A total of 3914 Anopheles larvae of twelve morphological types were collected from 685 breeding habitats. Anopheles peditaeniatus (n= 1041), was the most abundant, followed by An. subpictus (n= 893), An. nigerrimus (n= 808), An. barbirostris (n= 581), An. pallidus (n= 184), An. annularis (n=124), An. jamesii (n= 80), An. varuna (n= 76), An. vagus (n= 56), An. barbumbrosus (n= 48). An. culicifacies (n= 22) and An. aconitus (n= 1). The four most abundant species were significantly associated with waterlogged paddy fields. Anopheles culicifacies was noted only from irrigational canals with vegetation. Conductivity, salinity, and TDS were positively correlated with larval densities of An. peditaeniatus (Pearson correlations= 0.04, 0.17, 0.33: P= 0.94, 0.77, 0.58), An. subpictus (Pearson correlations= 0.6, 0.68, 0.56: P= 0.19, 0.20, 0.31), and An. vagus (Pearson correlations= 0.6, 0.42, 0.17: P= 0.28, 0.47, 0.77). However all physicochemical variables were negatively correlated with An. culicifacies densities. Conclusion: Major malaria vector of An. culicifacies complex occurred at relatively low densities, mainly in irrigated and waterlogged fields. These findings support understanding of mosquito ecology and will support future mosquito control strategies in rice ecosystems in Sri Lanka.Item Resting behavior of malaria vectors in Mannar District of Sri Lanka with reference to zooprophylaxis(University of Kelaniya, 2013) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.Background: The most important factors for effective zooprophylaxis in reducing malaria transmission are the presence of predominate populations of a strongly zoophilic mosquitoes and domestic animals for their blood-meal. Methods: Resting behavior of anopheline mosquitoes was evaluated using indoor and outdoor mosquito collection techniques. Mosquitoes were captured using 389 outdoor cattle-baited traps and indoor hand catches in 5672 houses at 12 possible malaria sensitive areas situated 12 km apart; Pesali, Keeri, Erukkallampeddi, Moor, Vankalai, Narawallikulam, Vanchiyankulam, Arvanum, Arrippu, Kokkupadayan, Koolankulam and Pandaraveilli for 12 months (June 2012 to June 2013) on a weekly basis. These areas had mostly homogenous characteristics in vegetations and house type. All collected mosquitoes were identified using standard taxonomic keys. A hypothesis test was conducted to determine whether there is a difference between indoor and outdoor collections. Results: A total of 3986 anopheline mosquitoes were collected from both indoor (n= 3122) and outdoor (n= 864) collection techniques. Anopheles subpictus was found to account for over 97.5% (n= 3887) of Anopheles species collected in the study areas. Other species were; 1.4% An. peditaeniatus (n= 57), 0.6% An. varuna (n= 22), 0.3% An. pallidus (n= 10), 0.2% An. nigerrimus (n= 7), 0.05% An. tessellatus (n= 2), and 0.03% An. aconitus (n= 1). Only 3 species namely, An. subpictus (n= 485), An. tessellatus (n= 2) and An. aconitus (n= 1) were recorded from indoor hand catches. In experimental cattle-baited hut trials, An. subpictus (n= 3026) was found to have the highest exophilic tendency (96.9%) compared to other anophelines encountered. The hypothesis test suggests that there was a significant difference of An. subpictus attracted to cattle-baited traps and human dwellings (P = 0.008). Although the main vector An. culicifacies was absent, the presence of the secondary vector An. subpictus was observed in high numbers in the District. Conclusion: Exophilic and zoophilic tendencies of An. subpictus are conducive for zooprophylaxis. The study further indicates that zooprophylaxis may be a potential strategy for malaria control. It is recommended that in areas with a predominant An. subpictus population, cattle could be kept close to human dwellings to maximize the effects of zooprophylaxis.Item Breeding of Anopheles culicifacies in different waterbodies in the district of Trincomalee(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.Introduction: Anopheles culicifacies (Diptera: Culicidae), the major vector of malaria in Sri Lanka is known to breed in clean and clear water. This study was focused to understand the larval habitats of the major malaria vector with the eco system changes in the Trincomalee district of the Eastern Province. Method: Potential larval habitats for Anopheles mosquitoes were surveyed on a monthly basis for 17 months (January 2011 –June 2012) in 4 different selected sampling sites (Murthankulam, Kommnaimottai, Paranamadawachchiya and Kokmotawewa). Collected larvae were identified using standard taxonomic keys. The species Distribution (C) and Density (D) were calculated. Results: A total of 2996 larval specimens representing 13 Anopheles species were reported from 16 different breeding habitats namely, waste water (n= 635), built well (n= 1229), earth well (n=149), agricultural well (n=9), rain water collection (n=89), animal hoof print (n=17), burrow pit (n=256), rock pool (n=10), canal (n=15), irrigation canal (n=27), lake margin (n=27), tank margin (n=448), pond margin (n=15), marshy land (n=13), paddy field (n=15) and slow moving water (n=42). An. culicifacies was observed as the most predominant species throughout the survey. According to Density criterion, An. culicifacies (44.0%), An. subpictus (19.2%), An. barbirostris (13.2%), An. peditaeniatus (10.28%) and An. nigerrimus (8.7%) were within the dominant class; (D > 5%). Two species (An. vagus, An. pallidus) were in the subdominant class (1< D <5%). Only An. annularis, An. varuna, An. barbumbrosus, An. pseudojamesi, An. jamesii and An. tessellatus were the satellite species (D < 1%). An. nigerrimus, An. subpictus and An. peditaeniatus can be regarded as constant according to distribution (C= 80.1-100%). Only An. vagus was the most frequently reported (C= 60.1 – 80%) species. All other Anopheles including An. culicifacies were observed as infrequent species (C= 20.1 – 40%) and no species was identified as sporadic appearance (C= 0 – 20%). Most productive breeding site for An. culicifacies were drains covered with waste water (Density= 81.57%) in remote areas. Interpretation & conclusion: These results indicate that An. culicifacies has adapted to breed in a wide range of water bodies including waste water collections although they are considered to breed in clean and clear water. The survival of the major vector mosquito in widespread water bodies could be responsible for the increase in the incidence of malaria in the future.Item Susceptibility of malaria vectors to insecticides in Ampara, Batticaloa, Trincomalee & Mannar districts of Sri Lanka(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.Background: The current status of insecticide resistance was studied for potential malaria vectors in four districts namely Ampara, Batticaloa, Mannar and Trincomalee of Sri Lanka. Methods: Insecticide-susceptibility tests were carried out using WHO standard kits against various chemical compounds at ambient room temperature of 27+1oC and relative humidity of 75-80% for adult and larvae separately. A total of 3629 adult female Anopheles mosquitoes belong to nine species (An. subpictus, An. vagus, An. nigerrimus, An. peditaeniatus, An. pallidus, An. annularis, An. jamesii, An. pseudojamesi and An. barbirostris) were exposed to cyfluthrin- 0.15%, etofenofrox- 0.5%, bendiocarb- 0.01%, malathion – 5%, deltamethrin – 0.05%, permethrin – 0.75%, λ- cyhalothrin– 0.05%, fenitrothion – 1.0% and propoxur – 0.1%. A total of 610 larvae belong to five Anopheles species (An. subpictus, An. vagus, An. barbirostris and An. peditaeniatus) were exposed to 0.0025, 0.005, 0.125 and 0.625 mg/l of viz. temephos (Abate). Results: All of the mosquito larvae or adult species used for the study were susceptible for selected insecticides (Mortality 98-100%). Adult Anopheles species showed a possibility of developing resistance to some chemicals (Mortality 97- 80%): An. nigerrimus (lambdacyhalothrin and permethrin), An. peditaeniatus (malathion, deltamethrin and cyfluthrin) An. subpictus (deltamethrin, permethrin, propoxur, cyfluthrin, and etofenprox), An. vagus (deltamethrin, permethrin and etofenprox) and An. pallidus (deltamethrin). Anopheles larvae of An. subpictus, An. barbirostris and An. peditaeniatus showed the potential of developing resistance (Mortality 97-80%). Interpretation & conclusion: As a result of resettlements and expanding commercial agriculture there can be a potentiality of developing insecticide resistance in mosquito individuals. Therefore, proper use of chemicals as pesticides should be adopted in these areas.Item Blood-feeding patterns of Anopheles mosquitoes in malaria-endemic areas of Sri Lanka(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.Background: Studies on host preference patterns in blood-feeding of anopheline mosquitoes are crucial for incriminating them as malaria vectors. However, little information is available on the host preferences of Anopheles mosquitoes in Sri Lanka. Therefore, the objective of the present study was to determine the hematophagic tendency of the anophelines. Methods: Adult Anopheles mosquitoes were collected using Cattle Baited Trap Collection (CBTC), Cattle Baited Net Collection (CBNC), Window Trap Collection (WTC), and Hand Collection (HC) from selected sentinel sites in Mannnar (3) and Trincomalee (5) Districts during June 2011- June 2012. Each blood fed mosquito was processed in to 9 cm whatman filter papers within 24 hours after blood meal has taken. DNA was extracted using the dried blood meal protocol of the QIAmp DNA mini kit. A multiplexed, Real Time Polymerase Reaction (RT- PCR) assay targeting 8 animals was developed for two panels (Panel 1: Bovine, cat, pig, monkey: Panel 2: Human, rat, dog, chicken) to identify the host meal of Anopheles. Human Blood Index (HBI), Forage Ratio (FR) and Host Feeding Index (HFI) were calculated. Results: A total of 216 field caught freshly engorged females mosquitoes belonging to 12 Anopheles species was analyzed. The host preference of anophelines observed in this study was bovine (86.17%), human (1.84%), cat (0.46%) and pig (0.46%). Only 6.91 % was positive for both human and bovine. In addition 5.0 % of the total samples tested were unknown. The overall HBI and HFI in the present study were low indicating the humans were not the preferred host for the tested anopheline species. Nevertheless, a small proportion engorged An. aconitus (0.37), An. culicifacies (0.27), An. barbirostris (0.2), An. annularis (0.125) and An. subpictus (0.12), An. peditaeniatus (0.08), An. pseudojamesi (0.04) and An. barbumbrosus (0.04) contained human blood, The FRs for human were <1.0 for most of the anophelines, except An. aconitus (1.04). Conclusion: The presence of human blood, in mosquito species indicates the possibility of them transmitting malaria. Hence, further studies on vector competence are needed to determine the role of each of the above anopheline species currently as efficient vectors of malaria.Item Prevalence of Anopheline species in Ampara district, Sri Lanka(University of Kelaniya, 2012) Fernando, M.A.S.T.; Gunathilaka, P.A.D.H.N.; Hapugoda, M.D.; de Silva, B.G.D.N.K.; Wijeyerathne, P.; Abeyewickreme, W.Introduction: Investigating the presence of primary and secondary vectors of malaria in the selected areas where no entomological surveillance was carried out for about 30 years due to ethnic conflict. Objective: To study prevalence of malaria vector in Ampara District and to assess the risk of malaria in the area. Method: Surveillance was preformed from January 2011 to June 2012 in 4 selected areas (i.e. Panama, Thirukkovil, Mahaoya and Dehiattakandiya) in Ampara District. From each area 4 localities (total 16 localities) were selected for the sample collection in order to ensure full coverage of the District. Cattle Baited Hut Collection (CBHC) and Cattle Baited Net Collection (CBNC) were performed as sample collecting methods on monthly basis throughout the surveillance period. Results: Mosquito densities for each collected Anopheles species were calculated as density per hut or net for CBHT and CBNT respectively. 14 Anopheles species were recorded from CBHC with high prevalence for An. subpictus (68.58%), An. nigerrimus (14.02%) and An. vagus (6.73%). 16 Anopheles species were recorded from CBNC with high prevelance of An. nigerrimus (50.07%), An. peditaeniatus (16.12%), An. pseudojemesi (9.21%) and An. subpictus (7.68%). An. culicifacies (primary malaria vector in Sri Lanka) recorded with lesser densities but An. subpictus, the secondary vector for malaria in Sri Lanka was predominant thorough out this study. Conclusions: The presence of primary and secondary malaria vectors in the area may cause a malaria epidemic in these areas. Hence, it is essential to study the seasonal prevalence of Anopheles species in order to initiate timely controlling measures in Ampara District.Item Application of a Real Time Polymerase Chain Reaction (PCR) for Detection of Pathogenic Leptospira in Clinical Samples(University of Kelaniya, 2012) Denipitiya, D.T.H.; Jiffrey, A.M.; Abeyewickreme, W.; Wellawaththge, C.; Hapugoda, M.D.Leptospirosis, is a zoonotic disease with worldwide distribution, caused by pathogenic species of the genus Leptospira. It has the greatest impact on health in developing countries where it is often grossly under-recognized. Clinical features are similar to a range of other infectious diseases that occur in the same environmental and climatologic conditions. Therefore, laboratory confirmation is essential for proper management of leptospirosis patients. Molecular assays offer definitive laboratory confirmation of leptospirosis at the early phase of infection (1-5 days of fever) within a few hours. The objective of this study was to establish and evaluate potential use of a real time- PCR assay for early, definitive laboratory confirmation of leptospirosis patients. A SYBR green-based real time PCR assay targeting a 203 bp fragment on the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (Leptospira interrogans strain RGA). Analytical specificity of the assay was tested with the DNA from pathogenic and non-pathogenic Leptospira spp. and five other micro organisms. Analytical sensitivity of the assay was tested using serial dilutions of the reference sample. A panel of acute blood samples (n=150) collected during early phase of infection (1-5 days of fever) from leptospirosis suspected patients was used for evaluation of real time PCR vs qualitative PCR. The results show, real time PCR assay with high analytical specificity (100%) was established and the assay shows 100 times higher sensitivity over qualitative PCR assay (1.3 pg/ml). Real time PCR and qualitative PCR could diagnose current leptospirosis infection in 37.3% (56/150) and 19.3% (29/150) suspected patients respectively. These results indicate high sensitivity of real time PCR over qualitative PCR for diagnosis of leptospirosis patients. In conclusion, this study shows that real time PCR has the potential to facilitate rapid and sensitive diagnosis of acute leptospirosis during early phase of infection.