Browsing by Author "Hapugoda, M.D."

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Anopheles subpictus s.l. breeding in polluted water bodies in Vankalai area in the Mannar District
(Sri Lanka Association for the Advancement of Science, 2014) Ranathunge, R.M.T.B.; Gunathilaka, P.A.D.H.N.; Kannangara, D.N.; Abeyewickreme, W.; Hapugoda, M.D.
Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka
(Academic Press, Elsevier, 2016) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hartskeerl, C.M.; Hartskeerl, R.A.; Jiffrey, A.M.; Hapugoda, M.D.
Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.
Application of a Real Time Polymerase Chain Reaction (PCR) for Detection of Pathogenic Leptospira in Clinical Samples
(University of Kelaniya, 2012) Denipitiya, D.T.H.; Jiffrey, A.M.; Abeyewickreme, W.; Wellawaththge, C.; Hapugoda, M.D.
Leptospirosis, is a zoonotic disease with worldwide distribution, caused by pathogenic species of the genus Leptospira. It has the greatest impact on health in developing countries where it is often grossly under-recognized. Clinical features are similar to a range of other infectious diseases that occur in the same environmental and climatologic conditions. Therefore, laboratory confirmation is essential for proper management of leptospirosis patients. Molecular assays offer definitive laboratory confirmation of leptospirosis at the early phase of infection (1-5 days of fever) within a few hours. The objective of this study was to establish and evaluate potential use of a real time- PCR assay for early, definitive laboratory confirmation of leptospirosis patients. A SYBR green-based real time PCR assay targeting a 203 bp fragment on the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (Leptospira interrogans strain RGA). Analytical specificity of the assay was tested with the DNA from pathogenic and non-pathogenic Leptospira spp. and five other micro organisms. Analytical sensitivity of the assay was tested using serial dilutions of the reference sample. A panel of acute blood samples (n=150) collected during early phase of infection (1-5 days of fever) from leptospirosis suspected patients was used for evaluation of real time PCR vs qualitative PCR. The results show, real time PCR assay with high analytical specificity (100%) was established and the assay shows 100 times higher sensitivity over qualitative PCR assay (1.3 pg/ml). Real time PCR and qualitative PCR could diagnose current leptospirosis infection in 37.3% (56/150) and 19.3% (29/150) suspected patients respectively. These results indicate high sensitivity of real time PCR over qualitative PCR for diagnosis of leptospirosis patients. In conclusion, this study shows that real time PCR has the potential to facilitate rapid and sensitive diagnosis of acute leptospirosis during early phase of infection.
Assessment of Possible Risk Factors Affecting Transmission of Dengue in the District of Gampaha Based on Reported Dengue Cases
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Perera, E.H.L.; Viswakula, S.; Gunawardene, Y.I.N.S.; Subasinghe, U.; Hapugoda, M.D.
Dengue is a fast spreading arboviral infection transmitted by the bite of infected females of Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse). According to the Epidemiology Unit, the second highest number of dengue cases is reported in the District of Gampaha, Sri Lanka over past ten years. Objective of this study was to investigate the entomological and socio-economic risk factors affecting transmission of dengue in laboratory-confirmed dengue case reported stations in the District of Gampaha. Laboratory confirmed positive dengue patients (n=100) by dengue NS1 antigen test during the period of June, 2018-August, 2019 were selected. Entomological surveillance was conducted by visiting to each patient within one week of notification of a positive case. For the collection of socio-economic data, an interviewer-administrated questionnaire was used. Adult Aedes mosquito samples collected using a back-pack aspirator showed, 98.64% (73/74) of Ae. albopictus and 1.35% (1/74) of Ae. aegypti mosquitoes. Larval collection using standard larval surveillance techniques showed 92.96% (185/199) and 7.04% (14/199) of Ae. albopictus and Ae.aegypti larvae respectively. The highest House Index (55.17%-16/29), Container Index (28.89%-13/45) and Breteau Index (44.83%-13/29) were reported in the month of June, 2019. The major Aedes breeding place was identified as plastic buckets/barrels (48.6%-84/173) that being used to discard waste. Piped borne water (88%-88/100) was the major water source of the house-holds. Water source of tube well (9%-9/100) was the next popular water source and 66.67%(6/9) of tube wells were positive breeding places for Aedes larvae. Average homestead of the premises of dengue patients was 16.14 perches. From the 100 dengue cases, 67 cases were from middle of town areas, while 2 were from rural areas. Vegetation coverage of the 78% (78/100) house-holds were grass, bushes and small trees and 3% (3/100) house-holds didn’t have any vegetation coverage. The major mosquito prevention method was usage of mosquito nets (54%-54/100) and among dengue patients 7% (7/100) of dengue patients weren’t using any mosquito prevention method. High density of Ae. albopictus mosquitoes, was reported although Ae. aegypti is the major vector of dengue. Therefore, it is required to draw more attention about the Ae. albopictus breeding sites in dengue control programmes. Participants from the study sites were well aware about the disease but still there is a lack of knowledge on breeding sites and vector control methods. Drawbacks in the waste disposal methods, lack of cleanliness in gardens, unplanned water sources and neglecting preventive actions could be considered as the possible risk factors.
Assessment of possible social and entomological risk factors affecting transmission of dengue in the District of Gampaha
(Faculty of Graduate Studies, University of Kelaniya, 2015) Withanage, G.P.W.K.; Abeyewickreme, W.; Hapugoda, M.D.
Dengue is the most important mosquito-borne viral infection transmitted to humans in Sri Lanka causing more than 30,000 cases annually. The objective of the study was to identify possible social and entomological risk factors affecting transmission of dengue in Gampaha district where the second highest number of dengue cases recorded during last ten years. Four Medical Officer of Health (MOH) areas were selected based on annual number of dengue cases greater than 250 for last ten years. One GramaNiladhari (GN) division was selected from each MOH area as a study area where the highest number of dengue incidents reported. In each study area, a cluster of 150 households was selected and household and entomological surveys were conducted. The selected areas were Eriyawatiya (Kelaniya MOH), Welikadamulla (Wattala MOH), Akbar town (Mahara MOH), and 3-Kurana (Negombo MOH) GN divisions. There were 2577 population in 600 households in the study areas and 53.5% were females. Average size of a homestead was 17 perches and most of the households (98%-588/600) were individual house. Main vegetations in the homesteads were grass and bushes (97%-583/600) and potential breeding places of dengue vector mosquitoes were observed in 96.1% (577/600) households. Main dengue vector mosquito present in the captured Aedes adult mosquitoes was Aedes albopictusis (92.9%-183/197). Most prominent breeding places were discarded bottles and tins (4.0%-15/371), plant axils (9.7%-36/371), plastic containers (26.4%-98/371), and roof gutters (4.3%-16/371) in the observed premises and 9.2% (34/371) were Aedes larvae positive. Most frequent mosquito preventive measure was bed-nets (30.3%-182/600), but mosquito coils (30.8%-185/600) and vaporizers (17.6%- 106/600) were frequently used. Participants have adequate knowledge about the disease, but they neglect preventive measures and highly depend on government vector control programs. Unplanned households, crowded conditions, poor waste management systems, and negligence to pursue preventive measures may be possible risk factors and therefore, frequent encouragement is needed to control dengue transmission.
Blood-feeding patterns of Anopheles mosquitoes in malaria-endemic areas of Sri Lanka
(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.
Background: Studies on host preference patterns in blood-feeding of anopheline mosquitoes are crucial for incriminating them as malaria vectors. However, little information is available on the host preferences of Anopheles mosquitoes in Sri Lanka. Therefore, the objective of the present study was to determine the hematophagic tendency of the anophelines. Methods: Adult Anopheles mosquitoes were collected using Cattle Baited Trap Collection (CBTC), Cattle Baited Net Collection (CBNC), Window Trap Collection (WTC), and Hand Collection (HC) from selected sentinel sites in Mannnar (3) and Trincomalee (5) Districts during June 2011- June 2012. Each blood fed mosquito was processed in to 9 cm whatman filter papers within 24 hours after blood meal has taken. DNA was extracted using the dried blood meal protocol of the QIAmp DNA mini kit. A multiplexed, Real Time Polymerase Reaction (RT- PCR) assay targeting 8 animals was developed for two panels (Panel 1: Bovine, cat, pig, monkey: Panel 2: Human, rat, dog, chicken) to identify the host meal of Anopheles. Human Blood Index (HBI), Forage Ratio (FR) and Host Feeding Index (HFI) were calculated. Results: A total of 216 field caught freshly engorged females mosquitoes belonging to 12 Anopheles species was analyzed. The host preference of anophelines observed in this study was bovine (86.17%), human (1.84%), cat (0.46%) and pig (0.46%). Only 6.91 % was positive for both human and bovine. In addition 5.0 % of the total samples tested were unknown. The overall HBI and HFI in the present study were low indicating the humans were not the preferred host for the tested anopheline species. Nevertheless, a small proportion engorged An. aconitus (0.37), An. culicifacies (0.27), An. barbirostris (0.2), An. annularis (0.125) and An. subpictus (0.12), An. peditaeniatus (0.08), An. pseudojamesi (0.04) and An. barbumbrosus (0.04) contained human blood, The FRs for human were <1.0 for most of the anophelines, except An. aconitus (1.04). Conclusion: The presence of human blood, in mosquito species indicates the possibility of them transmitting malaria. Hence, further studies on vector competence are needed to determine the role of each of the above anopheline species currently as efficient vectors of malaria.
Breeding habitat diversity and species composition of Anopheles mosquitoes in Trincomalee district, Sri Lanaka
(HABITATS Conservation Initiative, 2014) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.
Entomological studies on the abundance of malaria vector Anopheles mosquitoes have not been studied in some malaria endemic areas of Sri Lanka over past 30 years in view of the security situation. The aim of this study was to explore the habitat diversity and distribution of anopheline species in Trincomalee District in order to prioritize vector breeding habitats for developing timely and cost effective larval controlling measures. Potential larval habitats for Anopheles mosquitoes were surveyed from June 2010 - December 2010, in selected sampling sites in the Trincomalee District; Gomarankadawala, Echchallampaththu, Mollipothana, Thoppur and Padavisiripura, within a radius about 20 km on weekly basis. The species distribution and density were calculated. A total of 3,701 larval specimens representing twelve Anopheles species were reported form 19 breeding habitats (Tank margin, main canal, paddy field, vegetative canal, lake, built well, burrow pit, distribution canal, pond, rock pool, canal, un-built well, common well, river margin, sand pool, animal foot print, rain water collection, quarry pit and marshy land). Ten habitats were categorized under structurally complex group based on the presence of biotic communities. Only An. subpictus can be regarded as constant according to Distribution (C) (C= 80.1-100%).An. nigerrimus, An. peditaeniatus, An. pallidus and An. vagus were frequent (C= 60.1 – 80%). An. varuna, An. barbirostris, An. annularis and An. barbumbrosus were shown as infrequent species (C= 20.1 – 40%) and other namely An. aconitus, An. culicifacies and An. jamesii can be categorized under sporadic appearance (C= 0 – 20%). According to Density (D) criterion, five species (An. subpictus, An. nigerrimus, An. varuna, An. pallidus, An. barbumbrosus) were within the dominant class (D > 5%). Four species (An. vagus, An. peditaeniatus, An. annularis, An. aconitus) were in the subdominant class (1< D <5%). Only An. jamesii and An. culicifacies were the satellite species (D < 1%).
Breeding of Anopheles culicifacies in different waterbodies in the district of Trincomalee
(University of Kelaniya, 2012) Gunathilaka, P.A.D.H.N.; Fernando, M.A.S.T.; Hapugoda, M.D.; Wijeyerathne, P.; Wickremasinghe, A.R.; Abeyewickreme, W.
Introduction: Anopheles culicifacies (Diptera: Culicidae), the major vector of malaria in Sri Lanka is known to breed in clean and clear water. This study was focused to understand the larval habitats of the major malaria vector with the eco system changes in the Trincomalee district of the Eastern Province. Method: Potential larval habitats for Anopheles mosquitoes were surveyed on a monthly basis for 17 months (January 2011 –June 2012) in 4 different selected sampling sites (Murthankulam, Kommnaimottai, Paranamadawachchiya and Kokmotawewa). Collected larvae were identified using standard taxonomic keys. The species Distribution (C) and Density (D) were calculated. Results: A total of 2996 larval specimens representing 13 Anopheles species were reported from 16 different breeding habitats namely, waste water (n= 635), built well (n= 1229), earth well (n=149), agricultural well (n=9), rain water collection (n=89), animal hoof print (n=17), burrow pit (n=256), rock pool (n=10), canal (n=15), irrigation canal (n=27), lake margin (n=27), tank margin (n=448), pond margin (n=15), marshy land (n=13), paddy field (n=15) and slow moving water (n=42). An. culicifacies was observed as the most predominant species throughout the survey. According to Density criterion, An. culicifacies (44.0%), An. subpictus (19.2%), An. barbirostris (13.2%), An. peditaeniatus (10.28%) and An. nigerrimus (8.7%) were within the dominant class; (D > 5%). Two species (An. vagus, An. pallidus) were in the subdominant class (1< D <5%). Only An. annularis, An. varuna, An. barbumbrosus, An. pseudojamesi, An. jamesii and An. tessellatus were the satellite species (D < 1%). An. nigerrimus, An. subpictus and An. peditaeniatus can be regarded as constant according to distribution (C= 80.1-100%). Only An. vagus was the most frequently reported (C= 60.1 – 80%) species. All other Anopheles including An. culicifacies were observed as infrequent species (C= 20.1 – 40%) and no species was identified as sporadic appearance (C= 0 – 20%). Most productive breeding site for An. culicifacies were drains covered with waste water (Density= 81.57%) in remote areas. Interpretation & conclusion: These results indicate that An. culicifacies has adapted to breed in a wide range of water bodies including waste water collections although they are considered to breed in clean and clear water. The survival of the major vector mosquito in widespread water bodies could be responsible for the increase in the incidence of malaria in the future.
A Case report of dengue and chikungunya co-infection in Sri Lanka
(The Parasitology and Tropical Medicine Association of Thailand, 2008) Abeyewickreme, W.; Hapuarachchi, H.A.C.; Bandara, K.B.A.T.; Hapugoda, M.D.; Williams, S.
Dengue fever and chikungunya are arboviral diseases transmitted by Aedes mosquitoes. Though dengue has been an important communicable disease in Sri Lanka for many years, chikungunya has not been reported in Sri Lanka since late 1960s. However, in November 2006, an outbreak suggestive of chikungunya erupted in the country. We report here the first laboratory confirmed case of dengue and chikungunya co-infection in Sri Lanka. The objective is to confirm the co-infection of dengue and chikungunya in a clinical case reported in November 2006. Clinical history of high fever, severe headache, nausea, loss of appetite, severe arthralgia and mild oedema of knees, small joints of hands and feet for 3 days suggested the possibility of dengue and chikungunya in a 70 year old male. There was no skin rash or bleeding manifestations. Laboratory investigations performed included total white blood corpuscle count/differential count (WBC/DC), platelet count (PLT), serum, haemoglobin (Hb%) and packed cell volume levels (PCV). Reverse Transcription- Polyrnerase Chain Reaction (RT-PCR) technology was used to confirm the presence of either dengue or chikungunya. Viral RNA was extracted from serum samples collected during the first five days of infection using QiAmp Viral RNA Kits and amplified products were visualized by 2% agarose gel electrophoresis and ethidium bromide staining. WBC/DC analysis showed a leucopaenia (WBC count 3.04 x 103 per μl) with relative lymphocytosis (51.0%). The total PLT was 115 x 103 per μl. Hb% was 14.3 g/dl with a PCV of 43.8%. The presence of both infections was confirmed by RT-PCR which amplified 225 bp and 354 bp products for dengue and chikungunya respectively. This was the first laboratory confirmed case of dengue and chikungunya co-infection, which was also the first confirmed report of chikungunya since 1969 in Sri Lanka. As clinical and biochemical manifestations of this patient suggested the probability of a mixed infection of dengue and chikungunya, the confirmation was achieved by a RT-PCR assay. This report highlights the importance of using molecular assays to confirm mixed viral infections during their early stages, especially infections such as dengue which can result in fatal complications.
Characterization of Anopheline larval habitats and species composition of aquatic macro-invertebrates in Trincomalee District, Sri Lanka
(Faculty of Graduate Studies, University of Kelaniya, 2015) Ranathunga, R.M.T.B.; Gunathilaka, P.A.D.H.N.; Kannangara, D.N.; Abeyewickreme, W.; Hapugoda, M.D.
Malaria control methods that aim to reduce adult vector populations by targeting their aquatic immature stages. A better fundamental understanding of the biology and ecology of these essential stages could contribute to the implementation of current control methods and to the development of novel strategies. Objective of this study was to examine breeding habitat diversity and analysis of richness, diversity and geographical distribution of Anopheline larvae and the species composition of aquatic macro-invertebrates in their oviposition sites in Trincomalee District. Fifteen major permanent breeding places in five possible malaria sensitive sites (Gomarankadawala, Ichchallampaththu, Mollipothana, Padavisiripura and Thoppur) in Trincomalee District were selected. Anopheles larvae and macro-invertebrates were collected using standard methods for 16 months (April, 2013-July, 2014) and they were identified microscopically. The Shannon diversity index (H') was used to characterize species diversity at the five study sites by its abundance and evenness of the species present. ANOVA were used to analyze the correlation between macro-invertebrates and mosquito larval abundance. In total, 4478 including 11 species of Anopheles larvae were identified. An. subpictus, An. nigerrimus and An. peditaeniatus (71%) were the most abundant and widely-distributed species. Anopheline larval diversity was highest in Mollipothana (H‘=1.986). Whereas in Gomarankadawala, Ichchallampaththu and Thoppur where H‘=1.721, H‘= 0.857 and H‘=0.762 respectively. In total, 28 species of aquatic macro-invertibrates were identified and highest diversity was recorded in Mollipothana, Gomarankadawala and Ichchallampaththu (H‘=3.14-2.56). This deduces that the species richness and diversity of Anopheles mosquitoes and macro-invertebrates are higher in these areas. The presence of permanent breeding places may be the factors for this phenomenon. This study represents the first systematic update to the distribution of macro-invertebrates associated with Anopheles mosquito oviposition sites in Trincomalee District. Knowledge generated on the ecology of Anopheles mosquitoes will help to eliminate malaria vectors in the country.
Climatic factors affecting density of Anopheles vector mosquitoes in Ampara District, Sri Lanka
(University of Kelaniya, 2014) Kannangara, D.N.; Ranathunge, R.M.T.B.; Abeyewickreme, W.; Hapugoda, M.D.; Subasinghe, S.M.C.U.P.
Background: Apart of many vector-borne diseases malaria played a major role during past decades in Sri Lanka. Controlling strategies had effectively addressed this issue so that there were no malaria patients recently. However it has been observed that abundance of vector mosquitoes in districts like Ampara is high, which signifies a potential of spreading of malaria in the area in future. Identification of the relationship between the climatic factors and vector density could be a cost effective way in controlling the mosquito instead of costly strategies currently followed. This study attempts to identify the relationship exists between climatic factors and the vector density in Ampara District.
Clinical and virological features of dengue in 2010
(Sri Lanka College of Microbiologists, 2011) Hapugoda, M.D.; Manamperi, H.; Gunasena, S.; Athapaththu, A.M.M.H.; Premawansa, G.; Wellawaththage, C.; Jayarathna, T.D.S.S.; Abeyewickreme, W.
INTRODUCTION: Dengue is an important viral infection in Sri Lanka. All 4 serotypes co-circulate in Sri Lanka. OBJECTIVE: To study the clinical and virological features of dengue in 2010. DESIGN, SETTING AND METHODS: A hospital-based study was carried out at North Colombo Teaching Hospital, Ragama in 2010. Patients clinically suspected of having dengue, with fever less than 5 days were recruited. Acute and convalescent blood samples were collected within 7 days after obtaining informed written consent. Demographic, clinical information and laboratory results were obtained. Acute serum samples were tested using molecular (RT-PCR and Semi-Nested PCR) and serological (ELlSAs and HAI) assays. Convalescent samples were tested by serological assays. RESULTS: Of 209 patients enrolled, 93 % (195/209) were laboratory confirmed as recent positive cases of dengue viral infection; of these, 5% (9/195) were classified as dengue fever; 85%(1G5/195) dengue haemorrhagic fever (DHF) and 0.5% (1/195) dengue shock syndrome. Mean platelet value and packed cell volume (PCV) in laboratory confirmed dengue patients were 56,107/mm3 (range 10,000-306,000) and 42%(range 34-61 %) respectively. Patients infected with DHF showed both primary (n=45) and secondary (n=102) infections. Interestingly, secondary infection was not significantly correlated with DHF (x2-0.3:p=0.6). DEN-1 was responsible for the majority of cases, with a minority due to other three serotypes; all serotypes contributed to severe disease. CONCLUSION: DEN-1 was responsible for the majority of cases in 2010 but it circulated at a low level during previous epidemics. Majority of patients had severe clinical symptoms. In this epidemic, the clinical presentation of dengue differed according to the geographic region and viral serotype. ACKNOWLEDGMENTS: 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 (lAEA/SRL/5/042) is acknowledged.
Co-existence of double serotypes of dengue in patients of Gampaha District
(Sri Lanka Association for the Advancement of Science, 2007) Jayasooriya, D.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.; Premaratna, R.; Manamperi, A.; de Silva, H.J.; Abeyewickreme, W.
Dengue virus (DENV) known to cause a productive cytolytic infection in humans exists in four different serotypes Dengue 1 (D1), Dengue 2 (D2), Dengue 3 (D3) and Dengue 4 (D4). Among 4 serotypes of DENV, D 3 thought to be associated with explosive DHF epidemics and severe disease in many countries. Our objective was to determine the prevalence of dengue serotypes in Gampaha District and to correlate them with disease severity. Serum samples were collected from patients who were within 4 days of onset of fever and clinically suspected of dengue according to WHO criteria. Total viral RNA extracted from each serum sample was subjected to RT-PCR followed by a semi-nested PCR using specific primers. Out of 91 samples collected between Nov 2005 and Dec 2006, 16 samples were confirmed positive for DENV RNA by RT-PCR. Our results of multiplex semi-nested PCR indicated that 9/16 (56.25 %) of the positive cases were co-infected with serotype 2 and 3 (D2 & D3), while 4/16 (25%) were infected with D 3 and 3/16 (18.75 %) with D 2. 3/4 of D 3 cases had DHF , 1/3 of D2 cases were DHF while there were no DHF cases among the D2 and D3 co-infected patients. The mean Packed cell Volume (PCV) values of D3, D2 and D2 & D3 co-infected were 53.8 %, 48 % and 39.6% respectively while the mean platelet values of those were 66,000 mm3, 123,000 mm3 and 174.000 mm3 , respectively. Dengue infection by a single serotype is common among patients. Although few cases of co-infection by more than one serotype had been previously reported in a few other countries, this is the first description of simultaneous co-infection by D2 and D3 in Gampaha district. In this limited study we have observed a reduction of disease severity in D2 and D3 simultaneously co-infected patients. Could simultaneous co-infection by more than one serotype or a combination of two particular serotypes have lead to a decrease in disease severity among dengue patients is a matter yet to be studied. Further studies are needed to support these conjectures and to establish the clinical implications of simultaneous co-infection on the prevalence of DHF and disease severity. Acknowledgement: NSF (grant SIDA/2006/BT/02) & IAEA (SRL TC 6/028)
A Comparative field study of novel commercial Antigen Detection Enzyme-Linked Immunosorbent Assay (ELISA) with Reverse Transcription Polymerase Chain Reaction (RT- PCR) assay for early definitive laboratory diagnosis of dengue viral infection in Sri Lanka
(Sri Lanka Association for the Advancement of Science, 2007) Hapugoda, M.D.; Jayasooriya, D.H.S.W.; Gunawardene, Y.I.N.S.; Wellawaththage, C.; Premaratna, R.; Abeyewickreme, W.
Dengue is an important mosquito borne viral infection in South East Asia. Early definitive laboratory diagnosis of infection would help in management of patients and reducing the case fatality rate. The objective of this study was to determine the accuracy of novel commercial Antigen Detection Enzyme-Linked Immunosorbent Assay (ELISA) using Non Structural protein 1 (NS1) (Bio Rad) for early definitive laboratory diagnosis of dengue infection under field conditions in Sri Lanka. A panel of acute serum samples collected from 99 patients clinically suspected of having dengue fever (<5 days) warded at the North Colombo Teaching Hospital, Ragama, Sri Lanka were used for the present study. Serum samples were tested using Antigen Detection ELISA according to the method described by the manufacturer. Results of this novel assay were compared with RT-PCR assay using Chi-squared test. Two variables were analyzed at a 95% confidence interval and P value <0.05 was considered as significant. Twenty two and 65 patients were positive and negative, respectively, for dengue infection by both assays. Nine patients were confirmed as dengue by the Antigen Detection ELISA only. Three patients were confirmed as dengue by RT-PCR assay only. Antigen detection ELISA showed 88% of agreement with the RT-PCR assay. According to the Chi-squared test, there was no significant difference between the two assays for early diagnosis of dengue infection (?2=46, P=0.0000). Novel commercial Antigen Detection ELISA kit (Bio-Rad 72830) can be used for early definitive laboratory diagnosis of dengue infection in Sri Lanka under field conditions. Acknowledgement: the International Atomic Energy Agency (SRL 06/28) for technical co-operation and APCOT Marketing LTD, Sri Lanka for supplying Antigen detection ELISA kits.
A Comparative retrospective study of novel Reverse-Transcription Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with Polymerase Chain Reaction (PCR) amplification, virus isolation and serological techniques for early, definitive laboratory diagnosis of dengue infection
(Malaysian Society of Parasitology and Tropical Medicine, 2007) Hapugoda, M.D.; de Silva, N.R.; Khan, B.; Gunasena, S.; Dayanath, M.Y.D.; Abeyewickreme, W.
Dengue is an important vector borne viral infection in South East Asia. Dengue virus is responsible for dengue fever, dengue haemorrhagic fever and dengue shock syndrome. Early diagnosis of infection helps in monitoring the disease, determining when hospital admission is necessary and in reducing case fatalities. The objective of the study was to carry out a comparative retrospective study of a novel Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of Non Structural-3 gene were hybridized with a mixture of the 4 dengue type-specific Deoxyribonucleic Acid (DNA) probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 88 patients with dengue confirmed by Haemagglutination Inhibition (HAI) assay. The assay was highly specific for diagnosis of dengue infection. As an early (<5 days of fever) laboratory diagnostic method, this assay had 100% sensitivity for detection of dengue patients confirmed by HAI assay. A high analytical sensitivity of 2 fluorescent focus units of dengue virus/reaction was achieved. Novel RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection at the time during which serological methods cannot be used.
A Comparative retrospective study of RT-PCR-based liquid hybridization assay for early, definitive diagnosis of dengue
(Oxford University Press, 2010) Hapugoda, M.D.; de Silva, N.R.; Khan, B.; Dayanath, M.Y.D.; Gunesena, S.; Prithimala, L.; Abeyewickreme, W.
Dengue is an important flaviviral infection in tropical and subtropical regions. Early diagnosis of dengue infection helps in monitoring the disease, determining when hospital admission is necessary and reducing case fatalities. The objective of this study was to carry out a retrospective comparison of an RT-PCR-based liquid hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of non-structural 3 gene were hybridized with a mixture of four dengue type-specific DNA probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 119 fever patients with or without dengue, confirmed by haemagglutination inhibition (HAI) assay, the gold standard assay for diagnosis of dengue infection. The RT-PCR-LH assay was highly specific for dengue and, as an early laboratory diagnostic method, had 100% sensitivity in detecting dengue patients confirmed by HAI assay. A high analytical sensitivity of two fluorescent focus units of dengue virus/reaction was achieved. This RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection when serological methods are of little value.
A Comparative study of molecular-based diagnostic assays for early definitive diagnosis of human leptospirosis
(Sri Lanka Association for the Advancement of Science, 2013) Jiffriy, A.M.; Abeyewickreme, W.; Denipitiya, D.T.H.; Hapugoda, M.D.
Comparison of different RNA extraction methods for Dengue Reverse Transcription -Polymerase Chain Reaction (RT-PCR)
(Sri Lanka Association for the Advancement of Science, 2011) Adihetty, D.D.; Wellawaththage, C.; Abeyewickreme, W.; Abeywickrema, K.; Hapugoda, M.D.
Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(Sri Lanka College of Microbiologists, 2011) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.
INTRODUCTION: Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. OBJECTIVE: To compare two antigens for detection of anti-CHIK antibody. DESIGN, SETTING AND METHODS: In this study, two antigens {viral cell lysate and recombinant protein) were evaluated for detection of anti-1 CH IK 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, I 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. RESULTS: Atotal of 64 serum samples confirmed as positives andl 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. Conclusion 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.
Composition of malaria vectors and diversity of anopheline breeding habitats in the district of Mannar, Sri Lanka
(Central Environmental Authority (CEA), 2016) Gunathilaka, P.A.D.H.N.; Hapugoda, M.D.; Wickremasinghe, A.R.; Abeyewickreme, W.
In the malaria elimination phase in Sri Lanka, investigation on biological and ecological factors of malaria vectors are important in planning appropriate vector controlling strategies. Lack of sufficient biological and ecological information on malaria vectors in the Northern Province of the country, a malaria endemic region, is a major constrain in successful implementation of malaria control programmes. Therefore, the objective of this study was to explore the diversity of breeding habitats and species composition of malaria vector mosquitoes in the District of Mannar, Sri Lanka. Potential habitats for Anopheles mosquito larvae were surveyed from June, 2010 to July 20 J2 on a monthly basis in selected sampling sites in the Mannar District: Mannar Town, Vankalai and Silawathiura, within a radius about 20 km. In each site, 4 sub sites were selected A total of 37,788 Anopheles representing ten species was recorded from 12 breeding habitat categories. Built wells and waste water collections were conducive for anopheline breeding. Anopheles subpictus (96.2%, n= 36,351) was the dominant species followed by An. peditaeniatus (1.47%, n= 557), An. barbirostris (1.23%, n= 463), An. nigerriums (0.75%,n = 285), An. varuna (0.19%, n= 74), An. barbumbrosus (0.1%, n= 38), An. vagus' (0.03%, n= 12), An. pallidus (0.01%, n= 4), An.jamesii (0.05%, n= 2) and An. pseudojamesi (0.05%, n= 2). Use of wells and waste water drains as breeding places by potential malaria vectors indicates that both of these habitats act as larval reservoirs during the dry season. Presence of theses habitats in close proximity to human habitats create a potential risk of malaria transmission among humans. Therefore, health authorities need to be vigilant on these new habitats in vector control programmes.