Conferences and Symposia

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Awareness on dengue and solid waste management among selected school students in Gampaha District of Sri Lanka
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Radhika, N.M.L.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Kasturiratne, K.T.A.A.
BACKGROUND: Community mobilization is often respected as a powerful tool in ensuring the effective controlling of dengue via promoting effective and sustainable dengue vector control strategies. The current study attempts to investigate the level of awareness on dengue and solid waste management, among selected school students in Gampaha District which is the second highest dengue reported district in Sri Lanka. METHODOLOGY: Awareness programmes on dengue (transmission, vector ecology and preventive measures) and solid waste management were conducted in 10 schools belong to Kelaniya Medical Officer of Health (MOH) area selecting a minimum group of 200 students from each school within the age of 13 - 15 years. Tools such audio-visual presentations, leaflets and videos were used for the awareness programmes. A questionnaire was given to each student prior to the awareness and after the awareness programs as pre and post evaluations respectively in order to assess the general awareness on dengue and solid waste management before and after the awareness programme. One-Way ANOVA, paired t-test and multiple regression techniques were utilized in evaluation of the degree of awareness and key factors affecting the level of awareness, prior and after the conducting the awareness programmes. RESULTS: The degree of initial awareness on dengue and solid waste management of the target school community was recognized to be significantly characterized by the grade of the students and the Education Zonal Category [EZU] (into which their school belongs to) in accordance with One-Way ANOVA (p < 0.05). A significant increment in post awareness of the target school community was recognized due to the conducting of awareness programmes in accordance with the statistics of paired t-test (p < 0.05). Furthermore, based on the multiple linear regression modelling, the post awareness was significantly influenced by the grade of the students and the initial awareness (p < 0.05), regardless of the EZU (p > 0.05). CONCLUSIONS: The awareness of school students on dengue and solid waste management is significantly affected by the grade of the students and EZCs. Awareness programmes are an effective media of raising the awareness of school communities on dengue and solid waste management, while motivating them to actively contribute for the controlling of dengue, via establishing a strong inter sectorial network among schools and other relevant institutions within the Gampaha District.
Designing of immunogenic peptides from Dengue Virus NS1 region for production of monoclonal antibodies as diagnostic intermediates
(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Munasinghe, M.M.E.; Chandrasekharan, N.V.; Korbakis, D.; Soosaipillai, A.; Diamandis, E.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
BACKGROUND: Small peptide antigens have become an essential tool for antibody production in the recent life science research applications. The immunogenicity of peptide antigens is a critical factor to induce the immune response in order to produce desired antibodies. METHODS: In the current study, we have previously determined four Dengue (DEN) serotype specific peptides, containing 28 Amino Acid (AA) residues were re-designed. The peptides were re-designed considering many factors, for instance, sequence of the Sri Lankan isolates, abundance of Cysteine residues, solubility and the length of the peptide, carrier protein to be used and several other factors such as the N-terminal and C- terminal AAs and multiple AA residues. The peptide sequences were analysed using Antigen Profiler Peptide Tool (Thermo-scientific), Peptide Property Calculator (Genscript) and Swiss-Model (Biozentrum). RESULTS: The protein sequence of the peptides were changed according to the Sri Lankan isolates (AEB98757.1, ACS32038.1, AHG23239.1 and AHN50410.1). Oxidation of Cysteine residues results in significant conformational changes. Replacement of Cysteine with Serine prevents such oxidation reactions and it often retains full biological activity. Generally, peptides with a high number of hydrophobic AA (>50%) may result insoluble peptides. Similarly, to obtain a soluble peptide, it is important to contain at least one charged AA in every five AAs. Hence, the number of hydrophobic residues in the peptides were maintained below 50% and ensured that one out of every five amino acids is charged. The length of the peptide is an important factor as long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic. In order to obtain both highly conserved and variable regions among four serotypes, the peptide length was determined as 29 residues. A terminal Cysteine was added to allow peptide conjugation with carrier protein. Keyhole Limpet Hemocyanin was selected as the carrier protein due to its higher immunogenicity. N-terminal Glutamine or Aspargine and C-terminal Proline or Glycine in the sequences were avoided. Finally, the peptides sequences were determined as: DEN1; CPESSDDQRA WNIWEVEDYGFGIFTTNIW,DEN2; CAESPN TNRA WNSLEVEDYGFGVFTTNIW, DEN3;CPESPSASRAWNVWEVEDYGFGVFTTNIW and .DEN4;CSESPNERRAWNSLEVEDYGFGMFTTNIW. CONCLUSION: These peptides have a high potential to be used as peptide antigens for Monoclonal Antibody production.
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.
Emerging spatio-temporal trends of dengue incidence in Colombo and Kandy Districts, Sri Lanka
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Udayanga, N.W.B.A.L.; Gunathilaka, P.A.D.H.N.; Iqbal, M.C.M.; Kusumawathie, P.H.D.; Abeyewickreme, W.
BACKGROUND: Dengue has become a prominent challenge to the health sector in Sri Lanka. Prior identification/prediction of possible risk areas with high susceptibility to dengue, could be of major importance in ensuring the effective regulation of dengue epidemics. Therefore, the current study attempts to investigate the spatio-temporal trends in dengue incidence and priority risk factors that characterize dengue epidemics, aiming to stratify the Colombo and Kandy Districts into high, moderate and low risk areas via development of dengue risk maps by devising a statistical and geo-informatics based approach. METHODOLOGY: Socio-economic data of dengue patients and healthy volunteers in selected high risk MOH areas of Colombo (5) and Kandy (5) Districts were obtained through an interviewer administrated questionnaire. Previously reported dengue cases from 2009 to 2014 in each MOH were acquired. The spatio-temporal trends in dengue incidence of studied MOHs, were investigated at the Grama Niladhari Division (GND) level by using Arc GIS (version 10.2). The impacts of gender and age of the community on the disease prevalence were investigated by using normal Chi square test and Paired-Chi square test in SPSS (version 23). RESULTS: At present, 95% and 81% of the overall socio-economic surveillance has been completed in Kandy and Colombo Districts, respectively. Spatial maps on disease outbreaks have been developed for Akurana, Doluwa, Gampola, Gangawatakorale, Kandy Municipal Council (KMC), Kundasale and Poojapitiya MOH areas. According to results of the Paired-Chi square test, the emergence of dengue outbreaks indicated a significantly declining trend in recorded dengue cases in most of the GNDs within the studied MOHs, during the recent years. The Percentage Infected Male:Female Ratio (PIMFR) remained significantly altered throughout the period of study (p=0.001<0.05 at 95% of significance). Within the studied MOHs, males tend to indicate relatively high susceptibility for dengue infection than females (with 58.6: 41.5 of average PIMFR). The age group of 11 – 20 and 21 – 30 years were identified as with highest vulnerability to dengue, while the age > 61 years indicated the least vulnerability in all the MOHs. According to the Paired-Chi square test, the vulnerability of age groups was found to shift significantly throughout the study period [>Χ2 (7, 0.95) = 14.067]. CONCLUSIONS: The KMC and Gampola MOHs indicate relatively high susceptibility to dengue within the district of Kandy. Within all the studied MOHs, males tend to indicate relatively high susceptibility to dengue. Age groups of 11 - 20 and 21 – 30 denote relatively higher vulnerability to dengue, while age group of > 61 emerge as the least vulnerable group for the infection of dengue in the studied MOHs.
Encapsulation of essential oil blend in β-CD nanoparticles as a repellent for slow release
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayaweera, V.; Eliyapura, A.; Gunasekara, D.; Weerasinghe, L.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Karunaratne, V.
BACKGROUND: Malaria, dengue, and other tropical diseases carried by arthropods such as mosquitoes, ticks and fleas have greatly affected life expectancy, infant mortality, quality of life and work productivity. Essential oils such as citronella, lemon eucalyptus, peppermint, lemon, eucalyptus, lemongrass, geranium, and lavender are known for their mosquito repellency. Due to high volatility of essential oils, these are needed to be reapplied to the skin every 30–60 minutes. Therefore, development of a long lasting mosquito repellent is needed for effective repellent activity. The aim of this experiment is to develop a new blend which encompasses citronella, lemon eucalyptus, lemon and lavender. This oil blend is encapsulated using β-Cyclodextrin (β-CD) to decrease the volatility of the oil blend and increases the long-lasting repellency. METHODS: Citronella, lemon eucalyptus, lemon and lavender were obtained in 15% v/v, 15% v/v, 1% v/v and 1% v/v respectively for a total volume of 100 ml. These oils were mixed with 0.2% w/v of β-Cyclodextrin for encapsulation. The blend was sonicated for 20 min and dissolved in ethanol. This final encapsulated mixture was subjected to bioassays using 110 adult Aedes albopictus mosquitoes against the control. Sitting and biting mosquito behavior patterns were studied against the releasing time. RESULTS: The bioassay results indicated that the encapsulated repellent product is effective against the mosquitos up to 6 hours with zero sitting and biting comparatively to the control. In the 7th hour showed single mosquito sitting with zero biting. CONCLUSION: The encapsulated repellent is effective against the Ae, albopictus mosquitoes for 6 hour time period. Therefore, it is a long-lasting repellent that protect against dengue fever caused by mosquitoes.
Engineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Kajan, M.; Abeyewickreme, W.
BACKGROUND: The genus Flavivirus of the family Flaviviridae includes several vector-borne viruses to which the four serotypes of dengue viruses (DENV-1,-2,-3 and 4) belong to and DENV viruses have a messenger like positive polarity, single-stranded RNA genome approximately 11kb in length which encodes three structural proteins (C-prM-E) and seven Non-Structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). RNA interference (RNAi) and its properties as a tool has heralded a new era in functional genomics and short double stranded RNAs mediated by RNAi has become a powerful tool for post transcriptional gene silencing. Therefore, this study took the advantage the latter biological phenomenon and designed a multiple miR-shRNA (multi-mir-shRNA) molecule using bioinformatic approach to be effective to block the replication of all dengue serotypes of Sri Lanka. METHODS: Genome sequences of DENV strains belonged to serotypes 1 and 3 isolated from Sri Lanka deposited in GenBank were analyzed for potential sequences for the best siRNA target sites and identified two such sites from DENV 1 and DENV 3 from non structural protein coding sequence of NS5 and structural protein coding sequence of prM consensus regions, respectively. Two more siRNA targets reported from previous study chosen from upstream and downstream of non coding region effective for silencing all DENV serotypes were also used in designing mir-shRNA sequences. The stem region of miR1175 pre-miRNA sequence (miRBase ref: MI0013470) of Aedes aegypi was then replaced with each selected siRNA targets to generate the DENV effective miR-shRNAs and generated miR-shRNAs connected together by placing restriction endonuclease sites between each other to obtain multi-mir-shRNA containing four loop and stem structures. The transgenic gene cassette containing Ae. aegypti carboxypeptidase A promoter, multi-miR-shRNAs and poly adenylation signal of Simian Virus 40 Major Capsid VP1 was then constructed. Expression of this effecter molecule can be achieved by the induction of Ae. aegypti carboxypeptidase A (AeCPA) promoter following blood meal which then ensures activation of RNAi at the time of virus enter into the midgut of mosquito. RESULTS: Folding patterns of the transcript of the designed multi-mir-shRNA cluster were analyzed using online bioinfomatic tool, mfold and the secondary structure of this transcript shown to have optimum endogenous miRNA cleavge/processing with the lowest -ΔG indicating the ability of this design to exert RNAi in mosquito Ae. aegypti. CONCLUSION: Designing multi-miR-shRNA in bioinformatic means an effective way to construct the effector molecule that could exert the maximum RNAi against DENV. However, the effect of design will have to be demonstrated first by transforming to Ae. aegypti mosquitoes and then by evaluating the inhibition of DENV replication in mosquito.
Identification of risk factors affecting transmission of dengue in the District of Gampaha, Sri Lanka
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Withanage, G.P.W.K.; Abeyewickreme, W.; Hapugoda, M.D.
INTRODUCTION: Dengue is the most significant mosquito-borne infectious disease in Sri Lanka, causing more than 35,000 cases annually for last five years. The second highest prevalence of dengue was reported from the District of Gampaha for last 10 years. OBJECTIVE: To identify possible risk factors affecting transmission of dengue in selected high risk areas of dengue in the Gampha District. METHODOLOGY: The study was conducted in four high risk Medical Officer of Health (MOH) areas where the annual number of dengue cases greater than 250 during last ten years. In each MOH area, one Grama Niladhari (GN) division with the highest dengue incidence was selected as study areas. In each study area, a cluster of 150, including house-holds, open areas (barren lands, dump yards, and construction sites), abandoned houses, and religious facilities, was selected. A house-hold and entomological surveys were performed in March, 2015 after obtaining an informed written consent. RESULTS: The selected study areas were Akbar town (Mahara MOH), Eriyawatiya (Kelaniya MOH), Kurana (Negombo MOH), and Welikadamulla (Wattala MOH). The size of study human population was 2,544 in 574 house-holds in the study areas and 53.66% (1,365/2,544) were females. The average size of a homestead was 19.3 perches and more than one house was observed in 21.6% (124/574) of land plots. The type of the house-hold was mostly moderate (67.2%-386/574) and the gender of the house-hold head was male in 77.7% (446/574) house-holds. The average number of dwellers in a house-hold was 4 and 7.35% (187/2,544) of the population has previously infected with dengue. Most of house-holds were individual houses with a small garden (98.9%-568/574) and residential function of the house-hold was mainly residential only (83.4%-479/574). The main source of water was piped water (95.1%-546/574), but 39.5% (227/574) using ground-well or tube-well water daily purposes other than drinking and food processing. The most common mosquito preventive measure was bed nets (31.7%-182/574) and 47.6% (273/574) house-holds were using more than one mosquito preventive measures. Collecting tailors of municipal councils is the main solid waste disposal method, while 16.0% (92/574) using burning, burring in pits, composting or open ground due to lack of proper solid waste collection system. Bushes and small trees were the most common vegetation cover of the homesteads (84.5%-485/574) and potential breeding sites were observed in 97.5% (585/600) premises in the study areas. Main mosquito breeding places were plastic and polythene wares, discarded cans and tins, discarded tyres, plant axils, and aluminum and clay pots. The Breteau Index (BI) for Aedes larvae was 4.88 (28/574). Most prominent adult mosquito species in the areas was Aedes albopictus (92.4%-281/304). Dwellers in the study areas have considerable knowledge of the disease and preventive measures, but they reluctant to pursue preventive measures. DISCUSSION: Possible risk factors for transmission of dengue may be crowded conditions, small house-holds and homesteads, poor water and waste management systems, disfavor to pursue preventive measures, and dependence on government vector control programs. Therefore, immense persuade will be required to control dengue in the areas.
Laboratory studies on larvivorous potential of five fish species against Aedes ageypti (Linnaeus) and Ae. albopictus (Skuse)
(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Kusumawathie, P.H.D.; Jayasooriya, G.A.J.S.K.; Gunathilaka, P.A.D.H.N.; Hapugoda, M.; Abeyewickreme, W.
BACKGROUND: The dengue vectors, namely, Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse) are container breeders, thus, container management is a key element for elimination of vector breeding sites. Use of predator fish is a potential tool for dengue vector control in some container habitats. However, in order to select suitable fish species, larvivorous potential of different fish species needs to be determined. Therefore, the objective of the study was to determine the larvivorous potential of some fresh water fish species against the dengue vectors. METHODOLOGY: Five fish species, namely: Poecilia reticulata, Aplocheilus dayi, Oreochromis niloticus, Rasbora daniconius and Puntius bimaculatus were collected from their natural breeding places during the field surveillance conducted from January-October, 2015. Each fish species was placed in separate fish stock cement tanks at the Regional Office of the Anti- Malaria Campaign in Kandy until they were used for the study. In order to determine the larvivorous potential of the fish, each species was placed in separate enamel trays containing de-chlorinated water and allowed to acclimatize for 30 minutes. A batch of 25 Ae. aegypti and Ae. albopictus larvae was introduced to each tray superlatively and the number of larvae remaining was counted after 24 hours of observation period. Once all the batch of larvae consumes by the fish species, another batch of 25 larvae from respective species was introduced during the observation period. The whole experiment was repeated for 6 times. RESULTS: The mean values of Ae. aegypti larvae consumed by one fish of O. niloticus, P. reticulata, A. dayi, R. daniconius and P. bimaculatus were 97.6, 66.9, 49.3, 62.5 and 47.5 respectively while, one fish of O. niloticus, P. reticulata, A. dayi, R. daniconius and P. bimaculatus consumed 98.4, 62.3, 51.2, 62.5 and 45.4 of mean Ae. albopictus larvae respectively. CONCLUSIONS: All five fish species showed larvivorous activity. However, O. niloticus showed the highest larvivorous potential followed by P. reticulata. Therefore, these two fish species may be considered as a supplementary method to control immature stages of dengue vectors.
Maternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Ruanareerate, T.; Kittayapong, P.; Abeyewickreme, W.
BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.
Molecular diagnosis and transmission of dengue in Sri Lanka
(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Hapugoda, M.; Abeyewickreme, W.; de Silva, N.R.; Gunasena, S.; Meegoda, D.; Manamperi, N.
BACKGROUND: Dengue virus is an important vector-borne viral infection in Sri Lanka. Laboratory confirmation of suspected dengue cases is important for over/under estimation of cases. Early rapid diagnosis of dengue viral infection helps monitoring the disease, hospital admission when necessary and reduces case fatality. Detection of dengue viruses in mosquitoes is useful for studies on transmission of dengue virus. Study on risk factors for dengue is useful to understand spatial and temporal dynamics of transmission the disease. METHODOLOGY: A novel molecular-based assay; Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) was developed and validated for detection of dengue virus in clinical and mosquito specimens. Severity of dengue with circulating serotype was also analyzed. Wild-caught mosquito samples were collected from 236 dengue case-reported stations during outbreaks and a hot-spot during a period of 31 months. Epidemiological, environmental and entomological and other possible risk factors affecting transmission of dengue were analyzed. RESULTS: As an early (<5 days of fever) laboratory diagnostic method for dengue virus, the novel assay had 100% and 46% sensitivity for detection of confirmed and suspected dengue patients respectively. The assay developed in this study was found to be more sensitive than the other diagnostic techniques for early definitive laboratory diagnosis of dengue infection. Patients with definitive dengue correlated only with few signs and symptoms, indicating that laboratory confirmation is critical to avoid over estimation. A high sensitivity of 2 fluorescent focus unit of dengue virus/reaction was achieved and the assay was highly specific for dengue virus. The assay could detect dengue virus in 7% of field-caught Aedes albopictus specimens. A high density of Ae. albopictus was also associated with the dengue case-reported stations/hot-spots. Both vector species were susceptible to the 4 dengue serotypes under the laboratory conditions and DEN-3 under the field conditions. Geographical Information System (GIS) based risk mapping and database including epidemiological, climatic condition and entomological surveillance information were developed for the hot-spot in Kurunegala during the period of 2000-2003, which can be used as an early warning system. In depth study on socio-economic and other related factors affecting transmission of dengue was studied in the District of Gampaha. CONCLUSION: Ae. albopictus acts as an important vector of transmission of dengue in some urban and semi-urban areas. GIS-based risk maps developed are important to predict impending epidemics so that limited resources could be utilized in a cost effective manner to control the disease. Some socio-economic factors directly affecting transmission of dengue.
Pathogenesis of Leishmania donovani induced cutaneous leishmaniasis: role of Tumor Necrosis Factor α
(Faculty of Medicine, University of Kelaniya, Sri Lanka, 2016) Manamperi, N.H.; Oghumu, S.; Pathirana, K.P.N.; Munidasa, U.A.D.D.; Somaratne, K.K.V.N.; Rathnayake, R.M.D.I.; Thilakaratne, S.M.; de Silva, M.V.C.; Pathmeswaran, A.; Abeyewickreme, W.; Satoskar, A.R.; Karunaweera, N.D.
BACKGROUND: Cutaneous leishmaniasis (CL) in Sri Lanka is caused by the usually visceralizing Leishmania donovani. Host immune response plays a key role in the clinical presentation of leishmaniasis. Role of cytokines in pathogenesis of local lesions has not been studied. OBJECTIVE: To describe tissue cytokine expression with lesion progression with time in CL due to Leishmania donovani. METHODS: Skin biopsies from fifty eight patients with parasitologically or histopathologically confirmed CL and 30 healthy controls were analyzed for local tissue expression of Interleukin (IL)-12A, IL-4, IL-10, Interferon-gamma (IFNg) and Tumor Necrosis Factor-alpha (TNF-α). Cytokine mRNA was quantified by real-time RT- PCR using SYBR green. Relative copy numbers were calculated for each gene by 2-ΔΔCt method using β-actin as the reference gene and healthy controls as the calibrator. Spearman correlation was used to determine the correlation between cytokines and duration of active skin lesions. RESULTS: The study group consisted of 37 males (63.8%) and 21 females (36.2%) with a mean age of 35 (SD=12.05) years which ranged between 18-66 years. Mean duration of lesions was 6.8 (SD=9.10) months with a range of 1-48 months. The Spearman correlation coefficient for relative copy numbers and lesion duration was 0.220, 0.077, 0.073, 0.235 and 0.295 for IL-12A, IL-4, IL-10, IFNg and TNF-α respectively. There was a significant positive correlation between expression of TNF-α and lesion duration (p= 0.024). CONCLUSIONS: Localized expression of TNF-α increases with time in CL due to L. donovani in Sri Lanka, which indicates development of a pro-inflammatory immune response at the site of infection as the disease progresses.
Status of dengue vector breeding in pineapple plantations and environmental factors affecting their breeding
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Dassanayaka, D.M.T.; Aryaprema, V.S.; Jayasooriya, H.T.R.; Abeyewickreme, W.
BACKGROUND: Pineapple plantations are believed to have contributed to the transmission of dengue fever in pineapple growing areas in Sri Lanka. Hence, there is a need to introduce effective control methods to reduce dengue vector breeding in leaf axils of pineapple plants, a characteristic larval habitat of dengue vectors. The current study was designed primarily to obtain baseline information required for a much longer study planned to evaluate the use of pyriproxyfen and temephos in leaf axils of pineapple. METHOD: The study was carried out in five pineapple plantations in Medical Health Office area Meerigama in the Gampaha District from December 2014 to June 2015. Ninety plants from the five plantations were selected randomly and the volume of water in each plant and number of leaf axils positive with immature stages of mosquitoes was recorded twice a month. The water contents of leaf axils positive for mosquito larvae were transported to the laboratory and larvae were identified using standard keys. Chemical parameters: pH and temperature were measured in situ in the same samples and a few selected chemical parameters (concentrations of nitrates, phosphates, potassium and total dissolved solids) were measured in the laboratory using standard methods. Climatic data were obtained from the Meteorological Department of Sri Lanka. RESULTS: Eight mosquito species belonging to five genera were identified in leaf axil water of pineapple. Four species of Aedes including Aedes albopictus, the secondary vector of dengue, were identified while the primary vector Aedes aegypti was not recorded during the entire study period. The water volume per plant varied from 2.34 – 25.27 ml and it showed a significant positive correlation with the vector density (r=0.54, p=0.001). The number of rainy days in each month (r=0.76, p=0.000) and the monthly mean rainfall (r=0.53, p=0.001) showed significantly positive correlations with monthly vector density. Although monthly mean pH value showed no correlation with the monthly mean water temperature correlated negatively with the vector density (r= -0.5, p=0.004). None of the chemical factors except the concentration of nitrate (r=-0.45, p=0.016) in leaf water were associated with the vector density. CONCLUSIONS: Ae. albopictus was the most abundant species in all months except in January. The study revealed that the water volume in leaf axils, number of rainy days per month, monthly mean rainfall, water temperature and nitrate concentration of axil water seemed to affect the Ae. albopictus breeding, which are the important data useful in evaluating the use of pyriproxyfen and temephos in leaf axils of pineapple in the future. However, a continuation of the study for another six months to observe seasonal variation within a year is recommended.
Synthesis of Fe2O3 nanoparticles for the development of a rapid diagnostic test kit for dengue detection
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayarathna, I.P.L.; Gunathilaka, P.A.D.H.N.; Athapaththu, A.M.M.H.; Abeyewickreme, W.
BACKGROUND: Hybrid nanoparticles have great potential for biotechnological and biomedical applications. It was recently proposed that biopolymer/co-shell nano-materials could be easily obtained by adapting traditional routes used in pharmaceutical science to design drug delivery system. Most of the case, super-paramagnetic nanoparticles of iron oxides, magnetite (Fe3O4) and maghemite (ᵧ -Fe2O3) have been employed and these interesting magnetic properties are due to finite-size effects and high surface/volume ratio. METHOD: Iron oxide was synthesized by using modified co-precipitation method and resulting particles were characterized using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Diffused Reflectance Fourier Transform-Infrared Spectroscopy (DRIFT-IR). RESULTS: The XRD pattern matches well that of ᵧ -Fe2O3. Six characteristic peaks for ᵧ -Fe2O3 (2θ = 31.7°, 36.7°, 41.1°, 53.4°, 57.0° and 62.6°) marked by their Miller indices [(220), (311), (400), (422), (511) and (440)] were observed for sample. The TEM images reveal that the particles are in 5 – 20 nm range, and well fitted with spaniel cubic structure, but when particles are dry it prefer to agglomerate with neighboring particles to reduce their surface charges. The spectrum of ᵧ -Fe2O3 nanoparticles shows a characteristic broad band at 3410 cm-1 is due to the stretching vibration of H2O molecules. The band corresponding to the bending vibrations of H2O molecules is positioned at 1633 cm-1. Two intense IR bands at 627 and 451 cm-1 are typical for ferrihydrite or ᵧ -Fe2O3 nanoparticles. The spectrum of this sample showed the presence of carbonate groups on the basis of IR bands at 1508, 1340 and 1069 cm-1. The presence of carbonate groups is due to the adsorption of atmospheric carbon dioxide by ᵧ -Fe2O3 nanoparticles. The bands of Fe–O stretching vibrations of ᵧ -Fe2O3 appeared at 627 and 451 cm-1 and the bands at 892 and 796 cm-1 can be assigned to Fe-OH···H bending vibrations. CONCLUSION: Magnetic ᵧ -Fe2O3 nanoparticles were synthesized by the co-precipitation method and this work confirmed that magnetic ᵧ -Fe2O3 nanoparticles are in nano-scale and well matches with spaniel cubic structure
Vector prevalence and insecticide resistance status of Aedes sp. in dengue high and low risk areas in the Colombo District
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Janaki, M.D.S.; Aryapreme, V.S.; Jayasooriya, H.T.R.; Abeyewickreme, W.
BACKGROUND: In areas where dengue transmission is high, space spraying against adult Aedes sp. is practiced to reduce the density of infected mosquitoes and thereby reduce transmission within a very short time. Repeated use of insecticides in this manner, in dengue high risk areas, lead to the development of insecticide resistance in vector populations. The aim of the present study was to determine the prevalence and status of insecticide resistance of Aedes vectors in dengue high risk and low risk areas in the Colombo District. METHODS: The study was carried out in the area of Regional Director of Health Services, Colombo (RDHS area Colombo) and the Colombo Municipal Council area (CMC area), from December 2014 to June 2015. One each of comparatively high risk and low risk MOH area were selected from each study site based on the reported number of dengue cases during last five years. MOH areas Kolonnawa and D3 (Borella) were selected as high risk areas while Padukka and D5(Wellawatta) were selected as low risk areas respectively from the RDHS area Colombo and the CMC area. Ovitrap survey method was used to determine the vector prevalence and to collect Aedes eggs for bio assay tests. Larval and adult bio assay tests were conducted according to WHO guidelines. RESULTS: Both Ae. aegypti and Ae. albopictus were recorded from all MOH areas, except Padukka where there was only Ae. albopictus present. The vector prevalence in terms of Ovitrap Index and Egg Density Index was higher in Padukka (p=0.001 and p=0.029 respectively) than those in Kolonnawa in the RDHS area Colombo. In the CMC area, there was no such significant difference in the low risk and the high risk areas. Vector prevalence and the number of reported dengue cases showed no significant correlation in any of the MOH area. Aedes larvae in all four MOH areas have acquired resistance to diagnostic dosage of temephos (0.012 mg/l). Ae. aegypti in Kolonnawa, Ae. albopictus in Padukka and Ae. aegypti in D3 (Borella) showed possible resistance to 0.025% Deltamethrin while, Ae. aegypti in D5 (Wellawatta) has already acquired resistance. Ae. albopictus in D5 (Wellawatta) was still susceptible to the same insecticide. CONCLUSION: The vector prevalence does not significantly correlate with disease incidence, but further correlations with patients in localities within MOH and the vector densities will reveal reliable information. Despite the comparative status of high and low risk, all MOH areas have acquired resistance to insecticides, specifically to temephos and deltamethrin. Study reveals the emerging trends in resistance development of Aedes sp. in the Colombo District which has to be taken into account when designing further studies on resistance monitoring with a view to design effective control strategies.