IPRC - 2019
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Item Dengue Encephalitis with Concurrent Infections by Multiple Dengue Virus Serotypes(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Jayarathne, J.A.J.C.; Wijesinghe, W.A.T.A.; Mendis, S.; Waidyasekara, J.; Gunathilaka, P.A.D.H.N.; Gunasekara, D.Dengue is a serious public health problem caused by an arbovirus. Abnormal-neurological presentations associated with dengue infection is rare. Herewith, we report a case of a patient with dengue encephalitis who was concurrently infected with multiple serotypes. A 36-year-old, male resident from Poogoda presented to North Colombo Teaching Hospital with a history of generalized tonic-clonic seizures (GTCS) and unconsciousness. On admission, he displayed only a persisting headache. He had no significant illnesses in the past. Detailed history revealed that he has had a fever and left the hospital against the medical advice on the same day. On clinical examination, he was afebrile and blood pressure was 110/80 mmHg. He was well oriented with a Glasgow Coma Scale (GCS) of 15/15 and there was no focal neurological deficit. The funduscopic examination was normal. His initial full blood count revealed a rise in white blood cells with a predominant elevation of neutrophils of 12.44 x 103/uL. His platelet count was reported to be 306 x103/uL and, he was treated for a bacterial infection. On the following day, the patient started developing a fever and GTCS. Aggressive behavior, confusion, and delirium were also demonstrated. Neck stiffness was not observed. Non-contrast computed tomography (NCCT) scan, contrast-enhanced computed tomography (CECT) scan, chest X-ray scan and magnetic resonance image (MRI) of the brain were normal. Electroencephalography (EEG) revealed encephalopathy with non-epileptic discharge. Lumbar puncture was performed on the third day of admission and analysis of cerebrospinal fluid (CSF) was unremarkable. IgM antibody for the dengue virus (DENV) was detected in CSF. All the other viral and parasitological studies were normal. The patient was treated with anti-epileptic, anti-psychotic, antibacterial and antiviral drugs. High fever spikes and seizures were continued. GCS started to deteriorate from 9/15 to 3/15. Urine out-put started to decline. Ultra-sound scanning revealed mild ascites. Blood picture analysis showed evidence of having a severe bacterial infection. On day 9, both CSF and serum specimens were referred to Molecular Diagnostic Laboratory, Faculty of Medicine, Ragama for dengue viral investigations. The presence of NS-1 antigen, IgM/IgG antibodies, and the occurrence of concurrent infections with serotype 2 and 3 in the serum sample confirmed that the patient was infected with DENV. The presence of IgM/IgG antibodies were observed in the CSF sample. On day 10, the patient was transferred to the medical intensive care unit and monitored thoroughly. As the condition was deteriorated, he was ventilated and incubated for 11 days in the intensive care unit. He started to recover gradually after the 31st day. On day 36, the patient was discharged without any residual neurological symptoms. The patient is currently being followed up at the neurological clinic. Dengue encephalitis is a rare and deleterious manifestation of dengue infection. It is believed that concurrent infections with multiple serotypes and serotype replacement may cause disease severity. Therefore, reporting uncommon cases will provide insights for the physicians in early diagnosis and effective managementItem Mosquito Breeding Habitat Diversity and Distribution of Species in Selected Areas in the Districts of Kurunegala, Gampaha, Kegalle and Kandy in Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranasinghe, H.A.K.; Gunathilaka, P.A.D.H.N.; Amarasinghe, L.D.; Udayanga, N.W.B.L.Sri Lanka has been suffering from mosquito-borne diseases since ancient times with the high prevalence of malaria, filariasis and Japanese Encephalitis (JE). As a result of successive efforts in control programs, Sri Lanka has received remarkable achievements. However, with all these successful efforts some arbovirus infections majorly the dengue has increased rapidly over last few decades. Some control programs only target the specified vectors for that disease and report of other vector species is ignored. The prevalence of different mosquito species from surveillance has been limitedly document is Sri Lanka which is grossly inadequate in providing evidence for potential health risks. The present investigation attempts to cater for this knowledge gap by documenting the prevalence of mosquito species in four selected districts of Sri Lanka. Entomological surveys were conducted from a total of 160 temporary and permanent mosquito breeding habitats identified in selected areas, in the districts of Kurunegala, Kegalle, Kandy and Gampaha from June 2017 to October 2018. Mosquito immature stages were sampled using standard dipping, siphoning or pipetting methods according to the nature of breeding habitat and collected specimen were identified up to the species level. Chi-square test of independence was used to evaluate the significance in the distribution of different mosquito species among different breeding sites in the studied districts. The diversity indices for breeding habitats were subjected to a Kruskal Wallis test followed by Dunn’s Multiple Comparison to identify the significance in the variations of diversity indices across breeding sites and studied districts. A total of 4663 mosquito larvae belonging to seven genera and fifteen species of mosquitoes were collected from natural and artificial water holding macro and micro habitats located in study sites. The relative distribution of different mosquito species differed significantly among the four studied districts (X2=143.248; df= 33; P< 0.001). Mosquito assemblages in the districts of Gampaha and Kandy indicated a similarity of 83.16%, while the district of Kegalle shared a similarity of 75.45% with the above cluster. A total of 21 permanent/temporary key breeding sites were found from the study areas. As suggested by the Kruskal Wallis statistics (P < 0.05 at 95% of significance), all the diversity indices for immature stages of mosquitoes varied significantly across different breeding sites recorded during the study. Meanwhile, none of the diversity indices indicate significant differences in terms of locality. The highest values of Pielou's Index (2.01 ± 0.6), Menhinik's Index (3.34 ± 0.83) and Margelef Index (0.59 ± 0.19) and highest species richness (4.0 ± 2.82) in terms of mosquito larvae were shown by paddy fields within the studied four districts. Findings of the current study would be useful to identify the entomological potential for disease transmission and facilitate implementation of appropriate vector control interventionsItem Bacterial Diversity in the Midgut of Field Caught Culex tritaeniorhynchus(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranasinghe, H.A.K.; Gunathilaka, P.A.D.H.N.; Amarasinghe, L.D.; Rodrigo, W.W.P.Ingestion of blood meal by female mosquitoes triggers a series of physiological processes in midgut where symbiotic microbes also exist. These symbiotic microbes can be engineered to produce molecules that inhibit pathogens; through paratransgenic approach. Little is known about the midgut microbes of Culex mosquitoes and no attempt has been made so far in Sri Lanka. Cx. tritaeniorhynchus mosquitoes were separated from the entomological surveys conducted at Kelaniya Medical officer of Health (MOH) area from June – August 2019. Unfed adult female mosquitoes were sacrificed using a cold shock and were surface sterilized using 70% ethanol followed by rinsing with phosphate buffer saline (PBS). Midgut of mosquitoes were dissected and midgut of ten mosquitoes were pooled in sterile PBS (250 μL) to make a homogenized lysate. A dilution series (100- 10-7) was made from lysate. 100 μL from each dilution was plated on Plate Count Agar (PCA) and were incubated for 48 hours at 37 0C. Pure cultures for each microbe were obtained from the primary plates using streak plate method, sub culturing in Nutrient Agar. The experiment set up was repeated 25 times with ten mosquito pools at each effort. Colony separation was done based on phenotypical differences and basic biochemical tests. Stab cultures of isolates were sequenced for 16S ribosomal RNA partial gene. To identify the closest related sequence, obtained sequences were analyzed by Bioedit software package and completely aligned sequences were compared with the BLAST database. The evolutionary history was inferred using the neighbor-Joining method and the evolutionary analyses were conducted in MEGA X. A total of eight bacterial strains namely; Staphylococcus pasteuri, Bacillus megaterium, Staphylococcus cohnii, Pantoea dispersa, Staphylococcus chromogenes, Bacillus aquimaris, Staphylococcus arlettae, Staphylococcus scuiri was isolated from Cx. tritaeniorhynchus (n=250). All of these species were belonged to two phyla; Firmicutes and Proteobacteria. Phylum Firmicutes was the dominant phyla which include seven species. The evolutionary distances which were computed using Tajima-Nei method were used to infer the phylogenetic tree. It represented a close relationship between the species of two genera; Staphylococcus and Bacillus while the relationship was distant for genus Pantoea. The present data strongly encourage further investigations to explore the potential usage of these microbes through the paratransgenic approach which is a novel eco-friendly vector control strategyItem Susceptibility among Dengue Vectors to Malathion and Deltamethrin Insecticides in Three Selected Medical Officer of Health (MOH) Areas in the District of Gampaha, Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Dalpadado, C.P.R.D.; Amarasinghe, L.D.; Gunathilaka, P.A.D.H.N.Dengue has become the most important public health problem in Sri Lanka at present. As there is no effective vaccine or drug up to now, vector control is the main arm of disease control. Of these approaches, insecticide based control is used widely. However, excess use of chemicals and inappropriate application without determining the discriminative doses has given rise to the insecticide resistance among vector mosquitoes. Entomological surveys (Ovitap) were conducted in Negombo, Attanagalla and Dompe Medical Officer of Health (MOH) areas representing Urban, Sub urban and rural conditions, respectively from December 2016 December to June 2019. A total of 100 ovitraps each was placed outdoor and indoor in randomly followed by the collection after 5 - 7days. Eggs collected from ovitraps were reared under laboratory condition the laboratory (Temperature; 27 ± 10C: Relative Humidity 70 - 80%). The reared larvae were identified using standard taxonomic keys. Identification was further confirmed by identification of adults emerged from larvae . Adult mosquitoes were reared under optimized insectary conditions provided with sugar feeding. Batch of 140 mosquitoes from Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse) was taken separately from the colony 24 hour after a sugar feeding. Each batch of mosquito was exposed to malathion (0.8%) and Deltamethrin (0.03%) insecticide susceptibility following the World Health Organization (WHO) defined procedures. The experiment setup was repeated for 2 times. Mortality and corrected morality was calculated using Abbortt’s formula after 24 hours’ exposure period. The results indicate that both dengue vectors have evolved high level of insecticide resistance to malathion (0.8%) and deltamethrin (0.03%) in the District of Gampaha except Ae. albopictus mosquitoes in rural areas. Ae. albopictus mosquitoes were found fully susceptible for 0.03% deltamethrin in Dompe (rural) MOH area and emergence of resistance (95.9%) was observed in Attanagalla (suburban) area. It was found resistant in Negombo area (83.8%) which is highly populated. Ae. albopictus mosquitoes were found fully resistant for 0.8% malathion in both urban (71.5%) and Sub urban (85.3%) areas while emergence of resistance was observed in Dompe MOH area (90.8%). Ae. aegypti was found resistance to both 0.03% deltamethrin and 0.8% malathion in Negombo area (88.1%, 53.5% respectively) and resistance was suggested for 0.03% deltamethrin in Attanagalla MOH area (91.8%) while it was found resistant for 0.8% malathion (89.8%) in the same area. Ae. aegypti was not recorded in Dompe MOH area during the study period. Since both the dengue vectors have developed resistance to WHO diagnostic discriminative doses for Malathion and deltamethrin intensity bioassays (five times and ten times of discriminative dose) are needed to be done to confirm the level of resistance in these vectors. Therefore, results highlight the requirement of continuous insecticide resistance monitoring incorporated with the surveillance and control efforts. Hence, use of evidence based discriminative doses of insecticide for each area should be vital to delay the development of resistance among natural population