Browsing by Author "Hapugoda, M."

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Anopheles culicifacies breeding in polluted water bodies in Trincomalee district of Sri Lanka
(BioMed Central, 2013) Gunathilaka, N.; Fernando, T.; Hapugoda, M.; Wickremasinghe, R.; Wijeyerathne, P.; Abeyewickreme, W.
BACKGROUND: Anopheles culicifacies, the major vector of malaria in Sri Lanka, is known to breed in clean and clear water. The main objective of the study was to detect the breeding habitat diversity of An. culicifacies. METHODS:Potential larval habitats for Anopheles mosquitoes were surveyed on a monthly basis for 17 months (January 2011--June 2012) in four different selected sampling sites (Murthankulam, Kommnaimottai, Paranamadawachchiya and Kokmotawewa) in Trincomalee District of Sri Lanka. RESULTS: A total of 2,996 larval specimens representing 13 Anopheles species were reported from 16 different breeding habitats. According to density criterion, An. culicifacies, Anopheles subpictus, Anopheles barbirostris, Anopheles peditaeniatus and Anopheles nigerrimus were dominant. Anopheles nigerrimus, An. subpictus and An. peditaeniatus were observed as constant in relation to their distribution. The most productive breeding site for An. culicifacies was drains filled with waste water in remote areas; the second highest productivity was found in built wells. CONCLUSIONS: These results indicate that An. culicifacies has adapted to breed in a wide range of water bodies including waste water collections although they were earlier considered to breed only in clean and clear water. Copyright © 2013 Gunathilaka et al.; licensee BioMed Central Ltd.
Appearance of Anopheles jeyporiensis James from Sri Lanka; a new species to the mosquito checklist
(The Japan Society of Medical Entomology and Zoology, 2015) Gunathilaka, N.; Abeyewickreme, W.; Hapugoda, M.; Wickremasinghe, A.R.
Previous records of Sri Lankan Anopheles were most imperfect, and even the number and names of the species present were very doubtful. There was no systematic study conducted for Sri Lankan adult anophelines since 1990. Therefore, the objective of this study was to explore the species abundance and morphological variations of anopheline mosquitoes in Sri Lanka. Entomological surveys were conducted on a monthly basis from June 2010 to December 2013 in Trincomalee District, using five entomological techniques. Entomological surveys identified a total of 131,804 mosquito specimens belong to 18 anopheline species. One of which was An. jeyporiensis, a species that was not in the checklist in Sri Lanka. Its basic morphological features are similar to the members in Myzomyia series under the subgenus Cellia. Following characteristics were used to confirm the species as An. jeyporiensis; Centre of the scutum covered with short oblong white scales extending back to scutellum; Vein R 1 usually with accessory pale spot on preapical dark (PD) area; Foretarsomere 1 with apical pale band nearly 2.0 width of tarsomere diameter.
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.
Assessment of Real Time Polymerase Chain Reaction (PCR) Assay for the Early Diagnosis of Leptospirosis in Humans
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Uduwawala, U.M.H.U.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Chandani, W.L.; Hapugoda, M.
Leptospirosis is the most widespread zoonotic disease worldwide having a great impact on health issues in developing countries. It is caused by a pathogenic spirochete of the genus Leptospira where humans become infected through contact with the urine of infected animals. It is often exceptionally under-recognized as the clinical manifestation mimics variety of similar disease conditions that occur in the same environmental and climatologic conditions which accentuate the importance of laboratory diagnosis of leptospirosis. At present, no hospital based facilities are available for acute confirmation of the disease. The existing practice is retrospective confirmation with serological diagnosis. Therefore, the establishment of acute phase diagnosis will help in monitoring the disease, determining when hospital admission is required and reduce case fatalities. The objective of this study was to establish and evaluate a molecular-based assay to provide laboratory confirmation of leptospirosis at the acute phase of the infection (1-5 days of fever). Patients fulfilling clinical criteria stipulated by the accepted case definition were selected for the study and patients who failed to show evidence of sero conversion were considered as true negatives. A real time Polymerase Chain Reaction (PCR) assay with targeting a 203 bp fragment in the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (L.interrogans serovar Icterohaemorrhagiae strain RGA). Analytical sensitivity and the analytical specificity of the assay were calculated. The accuracy of the real time PCR was determined by a panel of acute blood samples collected from laboratory confirmed leptospirosis patients (n=35) and non-leptospirosis (n=44) patients based on Microscopic Agglutination Test (MAT) and/or IgM immunochromatography. Patients who failed to give positive test results either with MAT or IgM immunochromatography were considered as true negatives. Analytical sensitivity was approximately 314 genome equivalents per reaction and analytical specificity showed no amplification of Leptospira saprophytic sp. and other micro-organisms. The assay could effectively detect Leptospira DNA from clinically diagnosed leptospirosis suspected patients with 60.0% (21/35) diagnostic sensitivity and 77.27% (34/44) diagnostic specificity. This may be attributed to some samples failing laboratory confirmation despite their collection based on clinical suspicion. Therefore, real time PCR established can be used for rapid and definitive diagnosis of leptospirosis during the acute phase of infection
Biocontrol potential of six locally available fish species as predators of Aedes aegypti in Sri Lanka
(Elsevier Ltd, 2021) Ranathunge, T.; Kusumawathie, P.H.D.; Abeyewickreme, W.; Udayanga, L.; Fernando, T.; Hapugoda, M.
ABSTRACT: This study was conducted to evaluate the potential of six locally abundant fish species to control Aedes mosquito larvae and thereby manage dengue epidemics in a sustainable, cost-effective and environmentally friendly manner. The biocontrol efficacy of six larvivorous fish species, namely, Poecilia reticulata, Rasbora daniconius, Aplocheilus dayi, Oriochromis mossambicus, O. niloticus and Puntius bimaculatus, was evaluated under laboratory and field conditions. Five size-matched fish (of the same species) were introduced into separate tanks (replicates) containing 2 L of dechlorinated water and 200 third instar larvae of Aedes aegypti (L.). The number of larvae consumed by each fish species was recorded at three-hour intervals for 24 h. Acclimatized fish were introduced into a total of eighteen artificial breeding habitats located in the Gampola Medical Officer of Health (MOH) area at the species level with three replications. In addition, three breeding sites without fish were monitored as controls. Aedes larvae were monitored by dipping and siphoning methods in each breeding habitat at weekly intervals for three months and the number of fish surviving in each habitat was tallied. Over 24 h under laboratory conditions, O. mossambicus showed the highest predation rate, consuming 320.2 ± 14.5 larvae per day, with a predatory efficiency of 87.5 ± 3.5%. In comparison, O. niloticus consumed 264.6 ± 12.2 larvae per day with consumption efficiciency of 78.1 ± 3.7%, whereas R. daniconius had the lowest larval consumption (33.2 ± 2.7 larvae per day) and predatory efficiency (33.2 ± 3.2%). Over 12 weeks of observation under field conditions, breeding sites with Ap. dayi had the lowest Aedes larval counts, followed by Po. reticulata. Considering predation efficiency and survival under field conditions, Ap. dayi and Po. reticulata were considered to be the best potential candidates for biological control of Ae. aegypti. Further studies under field settings are warranted to evaluate the survival and predatory potential of the selected candidates under more varied environmental conditions.
Chracterisation of beta giobin mutations in Sri Lankan patients with betathalassaemia intermedia
(Sri Lanka Medical Association, 2013) Perera, S.; Silva, D.P.S.I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesirwardhena, I.; Efremove, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.P.
INTRODUCTION AND OBJECTIVES: Patients with beta thalassaemia intermedia account for a third of patients attending thalassaemia clinics in Sri Lanka. They show immense phenotypic diversity, the genetic basis for which has not been identified so far. Objective were to characterise beta globin gene mutations in Sri Lankan thalassaemia intermedia patients and to determine how it to influences disease severity. METHODS: We identified 64 thalassaemia intermedia patients from the five main thalassaemia centers; Anuradhapura (n= 6), Kuruncgala (n= 4), Ragama (n= 42), Badulla (n=7) and Chilaw (n=5). Their beta globin DNA sequences were analyzed using ABI PRISM 313lx genetic analyser. RESULTS: Of sixteen patients identified to be homozygous for beta mutations, eleven carried mild beta alleles, IVSI 5 G_C (n= 10) and a rare homozygous promoter mutation - 90 C_T (N=l). Other five were shown to have different types of severe iputations in homozygous state. Nearly half the sample (n=39) was heterozygous for beta mutations. Of them 33 showed mild to severe mutation in one of the alleles IVSI-5 G_C (n=12), IVSI-1 G_A (n= 11) were the commonest. Two patients who were hetcrozygones for beta mutation had a highly unstable Hb variant haemoglobin Mizuho causing severe haemolytic anacma. Hb variants Hb G-Szuhu and Hb G-Coushatta were identified in two patients. CONCLUSIONS: We identified types of beta mutations in some patients with thalassaemia intermedia, which account for the clinical severity.
Clinical and molecular heterogeneity among Beta Thalassaemia Intermedia in Sri Lanka
(Sri lanka Medical Association, 2015) Perera, P.S.; Silva, D.P.S.I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesiriwardena, I.; Efremov, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.
INTRODUCTION AND OBJECTIVES: Patients with beta thalassaemia intermedia (Tl) unrelated to haemoglobin E/beta thalassaemia account for an important minority in thalassaemia clinics in Sri Lanka. We investigated the genotypic/phenotypic diversity of this small group of patients. METHOD: Fifty Tl patients identified from five thalassaemia centers were clinically assessed and divided in to severity groups based on agreed criteria. Genetic analysis was done by PCR based techniques. RESULTS: There were 26 mild, 12 moderate and 12 in the severe groups. Ages ranged from 5-65 years. Mean haemoglobin of the whole group was 7.8g/dl. Age at presentation ranged from 3 months - 57 years (mean 16.8yrs) and varied according to severity; 17.8 years in mild to 4.8 years in severe group. 86% were on intermittent transfusions whilst 14% were never transfused. Mean total transfusion load in the three groups ranged from 6, 28 to 89. Majority (60%) had splenomegaly and 12% were splenectomised. The median spleen size of each severity group was 0, 4.5 and 7.5 cm respectively. Thalassaemicfacial features were not_ demonstrable in the majority (86%). Genetic analysis identified the commonest mechanism for Tl to be coexistence of a single beta mutation with excess alpha genes (56%). None of these patients had severe phenotype. Coexistence of two beta mutations with alpha thalassaemia invariably gave rise to severe phenotype. Other mechanisms gave rise to varying disease severity. CONCLUSION: This study highlights the remarkable phenotypic variations in beta Tl in Sri Lanka and identifies some genetic mechanisms which can explain this variation.
Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(University of Kelaniya, 2011) Athapaththu, A.M.M.; Abeyewickreme, W.; Hapugoda, M.; Khanna, N.; Inouve, S.; Tun, M.M.N.; Gunasena, S.
Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. The objective of this study is to compare two antigens for detection of anti-CHIK antibody. In this study, two antigens (viral cell lysate and recombinant protein) were evaluated for detection of anti-CHIK antibody by using IgM ELISA. A novel recombinant protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein. This protein was expressed in Escherichia coli and resultant protein was affinity purified and 10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine, Nagasaki University. A total of 64 serum samples confirmed as positives and 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was 17% and 100% respectively. Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. Acknowledgment: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.
A Comprehensive analysis on abundance, distribution, and bionomics of potential malaria vectors in Mannar District of Sri Lanka
(Hindawi Publishing Corporation, 2019) Gunathilaka, N.; Hapugoda, M.; Wickremasinghe, R.; Abeyewickreme, W.
BACKGROUND:A detailed knowledge of the distribution of the malaria vectors in Mannar district of Sri Lanka has not been studied after 1927. Past records indicated the presence of only seven species of anophelines, namely, An. culicifacies, An. subpictus, An. barbirostris, An. peditaeniatus, An. nigerrimus, An. Jamesii, and An. maculatus. There have been many changes in terms of distribution of Anopheles in the district over time. METHODS: Entomological surveillance was conducted on a monthly basis, comprising indoor hand collection, window trap collection, cattle-baited net collection, cattle-baited hut collection, and larval survey from June 2010 to June 2012 in 12 study areas under three entomological sentinel sites. The relationship between seven abiotic variables of the breeding habitats was measured. Pearson's correlation coefficients were used to determine the associations between climatic variables and anopheline densities. RESULTS:A total of 74,181 mosquitoes belonging to 14 Anopheles species were recorded. An. subpictus was the most predominant species from all techniques representing 92% (n=68,268) of the total anopheline collection. However, Anopheles culicifacies was not recorded from any site during the study period. Larval surveys identified 12 breeding habitat categories including waste water collections, lagoon water collections, and drains which were not recorded as breeding habitats by previous studies. The mean dissolved oxygen level of waste water collections was 3.45±0.15 mg/l. The mean salinity and conductivity of lagoon water collections were 21105±1344 mg/l and 34734±1974 μs/cm, respectively.CONCLUSION: The present study provides the updated knowledge on anopheline distribution and vector bionomics. Therefore, documentation of the current knowledge would be useful for learners and health authorities to design appropriate vector control measures in the prevention of reintroduction of malaria.
Correlation of genotype with phenotype in beta thalassaemia intermedia in Sri lanka
(Thalassaemia International Federation, 2015) Perera, P.S.; Silva, D.P.S.I.; Hapugoda, M.; Wickramarathne, M.N.; Wijesiriwardena, I.; Efremov, D.G.; Fisher, C.A.; Weatherall, D.J.; Premawardhena, A.
Abstract Available
Corrigendum to "Larvicidal Potential of Five Selected Dragonfly Nymphs in Sri Lanka over Aedes aegypti (Linnaeus) Larvae under Laboratory Settings"
(Hindawi Publishing Corporation, 2019) Samanmali, C.; Udayanga, L.; Ranathunge, T.; Perera, S.J.; Hapugoda, M.; Weliwitiya, C.
ABSTRACT: [This corrects the article DOI: 10.1155/2018/8759459.]. Erratum for, Larvicidal Potential of Five Selected Dragonfly Nymphs in Sri Lanka over Aedes aegypti (Linnaeus) Larvae under Laboratory Settings. BioMed Research International. 2018; 2018:8759459.
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.
Determination of demographic, epidemiological, and socio‑economic determinants and their potential impact on malaria transmission in Mannar and Trincomalee districts of Sri Lanka
(Biomed Central, 2016) Gunathilaka, N.; Abeyewickreme, W.; Hapugoda, M.; Wickremasinghe, R.
BACKGROUND: Malaria was an endemic problem in Mannar and Trincomalee districts of Sri Lanka until the recent past. Currently, no local case has been found since October 2012. Therefore, the present study was conducted to identify existing demographic, epidemiological and socio-cultural factors in Mannar and Trincomalee districts of Sri Lanka, since there is limited information available on the potential influence of above variables responsible for low malaria transmission. METHODS: An analytical cross-sectional survey was carried out on selected demographic, epidemiological and socio-economic variables in 32 localities under eight sentinel sites (Each sentinel with four localities) using a predefined questionnaire during June–September 2012. Household heads of 45 houses from each locality were selected randomly to participate in the present study. Data were analysed using the Paired Chi Square test and Bray–Curtis method. RESULTS: A total of 1440 household heads were interviewed. Both districts indicated statistically acceptable similarities (p > 0.05) in age structure, gender, family size and presence of animals. The knowledge on malaria was observed under “Poor” category. The protective measures against mosquito bites, spraying status of houses and occupation pattern were varied significantly in both districts (p < 0.05). Educational level was statistically similar (p > 0.05) in both districts. Majority of the families were identified as living in “Moderate” house type under low economic condition. Both populations were indicated 85 % similarity according to Bray–Curtis analysis. CONCLUSION: Lack of awareness in these communities about the disease may facilitate to the re-emerge of malaria.
Determination of the foraging behaviour and blood meal source of malaria vector mosquitoes in Trincomalee District of Sri Lanka using a multiplex real time polymerase chain reaction assay
(BioMed Central, 2016) Gunathilaka, N.; Denipitiya, T.; Hapugoda, M.; Abeyewickreme, W.; Wickremasinghe, R.
BACKGROUND: Studies of host preference patterns in blood-feeding anopheline mosquitoes are crucial to incriminating malaria vectors. However, little information is available on host preferences of Anopheles mosquitoes in Sri Lanka. METHODS: Adult Anopheles mosquitoes were collected from five selected sentinel sites in Trincomalee District during June-September 2011. Each blood-fed mosquito was processed on filter papers. DNA was extracted using the dried blood meal protocol of the QIAmp DNA mini kit. A multiplexed, real-time PCR assay targeting eight animals was developed for two panels 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 280 field-caught, freshly engorged female mosquitoes belonging to 12 anopheline species were analysed. The overall HBI and HFI in the present study were low indicating that humans were not the preferred host for the tested anopheline species. Nevertheless, a small proportion engorged Anopheles aconitus, Anopheles culicifacies, Anopheles barbirostris, Anopheles annularis, Anopheles subpictus, Anopheles peditaeniatus, Anopheles pseudojamesi, and Anopheles barbumbrosus contained human blood. CONCLUSION: The presence of human blood in mosquito species indicates the possibility of them transmitting malaria. Further studies on vector competence are needed to determine the role of each of the above anopheline species as efficient vectors of malaria.
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.
Development of recombinant protein antigens using a bacterial expression system for the detection of anti-Chikungunya (CHIK) antibodies
(Sri Lanka College of Microbiologists, 2013) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.
INTRODUCTION AND OBJECTIVE: Laboratory confirmation of Chikungunya (CHIK) virus is very useful as clinical symptoms of CHIK can overlap with other diseases. Chikungunya virus specific antigen, which shows high specificity, sensitivity and low cross reactivity with other related diseases, is required for laboratory confirmation. Objective of this study was to develop and compare two recombinant protein antigens for detection of anti-CHIK antibodies. DESIGN, SETTING AND METHODS: Recombinant CHIK protein antigens were prepared using Envelope (E1 and E2) regions of the CHIK virus. The genes were custom designed and chemically synthesized with a 6X His tag. Bacterial expression systems [BL21 (DE3)] were used to clone and express the recombinant proteins. The recombinant proteins were purified with >95% of purity per liter of culture using Ni-NTA columns under denature conditions. In this study, two antigens were evaluated for detection of anti-CHIK antibody by using novel optimized in-house IgM and IgG ELISAs, using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Center for Viral Reference and Research) Institute of Tropical Medicine, Nagasaki University, Japan. RESULTS: Atotal of 55 serum samples confirmed as positives and 186 confirmed as negatives by HA! test, IgM capture ELISA and indirect IgG ELISA using the purified CHIK antigen were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 148 (E1) or 227 (E2) (148 + extra 79) confirmed ac negatives were used to evaluate the antigens using novel IgG ELISA. The E1 recombinant protein showed 5% (3/ 55) sensitivity and 99% (184/186) specificity for IgM ELISA and 60% (47/78) sensitivity and 63% (94/148) specificity for IgG ELISA. The E2 recombinant protein showed 65% (36/55) sensitivity and 70% (131/186) specificity for IgM ELISA and 83% (65/78) sensitivity and 86% (195/227) specificity for IgG ELISA. CONCLUSION: Recombinant CHIK-E2 protein antigen showed higher specificity and sensitivity in detection of both IgM and IgG antibodies. Thus the E2 recombinant protein antigen used in this study could be expressed in an eukaryotic expression system to achieve much higher results. ACKNOWLEDGMENT: International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (lAEA/SRL/5/042) are gratefully acknowledged.
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.
Effect of γ-radiation on fertility and survival of Aedes albopictus (skuse) males in the laboratory for Sterile Insect Technique
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Harishchandra, J.; Abeyewickreme, W.; Hapugoda, M.; Premaratne, R.G.; Gilles, J.R.S.
BACKGROUND: It is believed that Aedes albopictus, one of the dengue vectors in Sri Lanka can be controlled using Sterile Insect Technique (SIT) when integrated with other conventional control methods. The objective of this study was to determine the effective dose of gamma radiation for producing sterile males of Ae. albopictus mosquitoes. METHODS: A batch of male pupae (n=32) aged 24-48 hours in F1 was irradiated using a gamma-ray irradiator (Gamma 220, Atomic Energy of Canada Ltd., Co60) with 25 Gy in duplicates. Following the same procedure, different doses (30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy) were given to each pupal batch in duplicates. Then they were transferred to laboratory cages (30 cm X 30 cm X 30 cm) for emergence and supplied with 10% sucrose solution. Adult emergence rate and male longevity were recorded. Virgin females from the same cohort (F1) were introduced into each cage for mating and fed blood starting 5 days after emergence. Females were then isolated in to individual tubes and hatching rate of individual egg batches was determined after two weeks of egg maturation in hatching solution containing 0.25 g BNB, 0.05g BY in 700 ml distilled water. Spermachecae of female mosquitoes were dissected and insemination rates were calculated after egg laying. RESULTS: Male mosquito pupae in F1 showed low mortality (0-3.12%) immediately after exposing to radiation (0-48 hours). After 21 days of the irradiation, probability of survival of male mosquitoes in F1 were 0.578, 0.494, 0.453, 0.313, 0.328, 0.381 and 0.219 at 0Gy, 25 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy respectively (Kaplan Meier survival analysis). Log Rank test indicated significant differences of survival of control males with males irradiated at 40Gy, 50 Gy, 60 Gy and 70 Gy. The survival of males irradiated at 25 Gy and 30 Gy did not differ from each other and from the control. The survival of males irradiated at 40 Gy, 50 Gy, 60 Gy were not significantly different among each other. The mean hatching rate (Mean+SE) of the F2 progeny of Ae. albopictus males (F1 progeny) were 10.89+2.76%, 7.36+1.75%, 3.09+0.71%, 0.79+0.27%, 0.66+0.23% and 0% when irradiated at 25 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy respectively. The control group showed a hatching rate of 64.26 + 7.12%. It was found that insemination rate of the irradiated males among different doses and controls were above 90% in F1 based on spermatheca dissection of blood-fed females (F1). CONCLUSION: 50 Gy is recommended as the most suitable γ radiation dose to produce 99% sterility in Ae. albopictus males which has 0.328 survival probability after 21 days of irradiation. This dose can be used to produce sterile males of Ae. albopictus for population suppression.
Entomological investigations on malaria vectors in some war-torn areas in the Trincomalee district of Sri lanka after settlement of 30-year civil disturbance
(Hindawi Publishing Corporation, 2015) Gunathilaka, N.; Hapugoda, M.; Abeyewickreme, W.; Wickremasinghe, R.
Background. Malaria was an endemic problem in Trincomalee District, Eastern Province of Sri Lanka. Very few recent data concerning Anopheles are available which transmit malaria. Therefore, the aim of this study is to identify various Anopheles species and the dynamics of anophelines including malaria vectors in Trincomalee District for effective vector control under the current malaria elimination program embarked in the country. Method. Entomological surveys were conducted on a monthly basis, using five entomological techniques, namely, indoor hand collection (HC), window trap collection (WTC), cattle-baited net collection (CBNC), and cattle-baited hut collection (CBHC) from June 2010 to June 2012 in 32 study areas under five entomological sentinel sites. Results. Seventeen anopheline species were encountered, of which Anopheles subpictus was the predominant species in all sampling methods. It is noted that A. culicifacies and A. subpictus have adapted to breed in polluted water in urban settings which may cause serious implications on the epidemiology of malaria in the country. Conclusions. It is important to determine the abundance, biology, distribution, and relationship with climatic factors of main and secondary malaria vectors in Sri Lanka in order to initiate evidence based controlling programs under the current malaria elimination program in Sri Lanka.
Entomological surveillance with viral tracking demonstrates a migrated viral strain caused dengue epidemic in July, 2017 in Sri Lanka.
(Public Library of Science, 2020) Withanage, G.P.; Hapuarachchi, H.C.; Viswakula, S.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.
BACKGROUND: Dengue is the most important mosquito-borne viral infection disease in Sri Lanka triggering extensive economic and social burden in the country. Even after numerous source reduction programmes, more than 30,000 incidences are reporting in the country every year. The last and greatest dengue epidemic in the country was reported in July, 2017 with more than 300 dengue related deaths and the highest number of dengue incidences were reported from the District of Gampaha. There is no Dengue Virus (DENV) detection system in field specimens in the district yet and therefore the aim of the study is development of entomological surveillance approach through vector survey programmes together with molecular and phylogenetic methods to identify detection of DENV serotypes circulation in order to minimize adverse effects of imminent dengue outbreaks. Entomological surveys were conducted in five study areas in the district for 36 months and altogether, 10,616 potential breeding places were investigated and 423 were positive for immature stages of dengue vector mosquitoes. During adult collections, 2,718 dengue vector mosquitoes were collected and 4.6% (n = 124) were Aedes aegypti. While entomological indices demonstrate various correlations with meteorological variables and reported dengue incidences, the mosquito pools collected during the epidemic in 2017 were positive for DENV. The results of the phylogenetic analysis illustrated that Envelope (E) gene sequences derived from the isolated DENV belongs to the Clade Ib of Cosmopolitan genotype of the DENV serotype 2 which has been the dominant stain in South-East Asian evidencing that a recent migration of DENV strain to Sri Lanka.