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Item Assessment of Real Time Polymerase Chain Reaction (PCR) Assay for the Early Diagnosis of Leptospirosis in Humans(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Uduwawala, U.M.H.U.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Chandani, W.L.; Hapugoda, M.Leptospirosis is the most widespread zoonotic disease worldwide having a great impact on health issues in developing countries. It is caused by a pathogenic spirochete of the genus Leptospira where humans become infected through contact with the urine of infected animals. It is often exceptionally under-recognized as the clinical manifestation mimics variety of similar disease conditions that occur in the same environmental and climatologic conditions which accentuate the importance of laboratory diagnosis of leptospirosis. At present, no hospital based facilities are available for acute confirmation of the disease. The existing practice is retrospective confirmation with serological diagnosis. Therefore, the establishment of acute phase diagnosis will help in monitoring the disease, determining when hospital admission is required and reduce case fatalities. The objective of this study was to establish and evaluate a molecular-based assay to provide laboratory confirmation of leptospirosis at the acute phase of the infection (1-5 days of fever). Patients fulfilling clinical criteria stipulated by the accepted case definition were selected for the study and patients who failed to show evidence of sero conversion were considered as true negatives. A real time Polymerase Chain Reaction (PCR) assay with targeting a 203 bp fragment in the secY gene which is conserved among pathogenic serovars of Leptospira was established using a reference DNA sample (L.interrogans serovar Icterohaemorrhagiae strain RGA). Analytical sensitivity and the analytical specificity of the assay were calculated. The accuracy of the real time PCR was determined by a panel of acute blood samples collected from laboratory confirmed leptospirosis patients (n=35) and non-leptospirosis (n=44) patients based on Microscopic Agglutination Test (MAT) and/or IgM immunochromatography. Patients who failed to give positive test results either with MAT or IgM immunochromatography were considered as true negatives. Analytical sensitivity was approximately 314 genome equivalents per reaction and analytical specificity showed no amplification of Leptospira saprophytic sp. and other micro-organisms. The assay could effectively detect Leptospira DNA from clinically diagnosed leptospirosis suspected patients with 60.0% (21/35) diagnostic sensitivity and 77.27% (34/44) diagnostic specificity. This may be attributed to some samples failing laboratory confirmation despite their collection based on clinical suspicion. Therefore, real time PCR established can be used for rapid and definitive diagnosis of leptospirosis during the acute phase of infectionItem Detection of pathogenic Leptospira species in rat blood samples by molecular-based assays(University of Kelaniya, 2013) Denipitiya, D.T.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hapugoda, M.D.Background: Leptospirosis is a worldwide zoonotic infection, caused by pathogenic species of the genus Leptospira. It was traditionally known as ‘rat fever’ in Sri Lanka, because rodents, especially rats, are considered to be the most important reservoirs or maintenance hosts of Leptospira. In 2012, the highest numbers of cases were reported in the District of Gampaha. The objective of this study is to detect pathogenic Leptospira species in rat blood samples by molecular based assays. Method: Rats (n=38) were trapped in a high risk area (Mirigama) in the District of Gampaha, from May 2012 to February 2013 by using live traps. Each rat was anesthetized by using diethyl ether and 2-3 ml sample of blood was collected from each rat. Blood samples collected from all rats were tested by molecular- based assays and a serological assay. Qualitative Polymerase Chain Reaction (PCR), real time PCR and Loop Mediated Isothermal Amplification (LAMP) were used as molecular-based assays which targetted conserved gene regions among pathogenic serovars of Leptospira species. Microscopic Agglutination Test (MAT), the Gold Standard assay for detection of anti Leptospira antibody was used as a serological assay. Results and Discussion: Of the 38 rat blood samples, molecular-based assays confirmed Leptospira infection in 5% (2/38), 16% (6/38) and 11% (4/38) by qualitative PCR, real time PCR and LAMP assay respectively. None of the samples was positive by MAT. After first infection, some Leptospira species live in the host animal as commensal bacteria. Therefore, host does not stimulate antibody production further and that may be below the detection level of the antibody by MAT. Conclusions: Results of molecular based assays showed that Leptospira are circulating among the rats tested in this study, although at the time of collection, their antibody levels were too low to detect by MAT, which had the lowest detection limit of 1:800.Item Risk factors associated with human leptospirosis in the District of Gampaha, Sri Lanka(University of Kelaniya, 2013) Denipitiya, D.T.H.; Athapaththu, M.; Chandrasekharan, N.V.; Abeyewickreme, W.; Hapugoda, M.D.Background & Objective: A large number of leptospirosis cases are recorded in Sri Lanka every year. Increased numbers of cases have been reported in the District of Gampaha in the recent past. The incidence of leptospirosis is often influenced by various socio-economic, occupational, environmental and other factors. To date, a study on potential risk factors has not been conducted in the District of Gampaha. The objective of this study is to identify risk factors involved in transmission of leptospirosis to humans in the District of Gampaha. Methods: Data were collected at the household level, using an interviewer-administered questionnaire and by inspecting the surrounding of laboratory confirmed leptospirosis patients (n=81) and non leptospirosis persons (n=117) during the period of June 2011 to June 2013. The risk factors in the questionnaire were divided into three broad categories: environmental, contact with animals and behavioral/occupational factors. Chi-square test (The SAS System for Windows 9.0) was used for comparison of data from different categories. Results and discussion: 95% of the leptospirosis patients were adult males (77/81) and they had a monthly income of Rs. 10,001-20,000 and 50% of them were agricultural and rental work labourers (40/81). In contrast, 56% of persons not infected with leptospirosis were adult females (66/117) and most of them (48%) were housewives or homemakers (56/117). Data on the type of premises were collected under three categories as poor, moderate and well constructed along with the land use type of the surrounding areas. There were significant statistical associations between the leptospirosis patient with the type of premises (, χ2=23.38, p=0.00), surrounding cleanliness of premises (χ2=45.05, p=0.00), sanitary facilities (χ2=11.66, p=0.00), waste disposal method (χ2=32.23, p=0.00) and age level of patients (χ2=21.07, p=0.00). No significant statistical associations were observed between recorded leptospirosis cases and vegetation coverage in surrounding area of premises (χ2=1.25, p>0.05), source of drinking water (χ2= 0.55, p>0.05) and numbers of persons in family (χ2=0.17, p>0.05). Conclusion: Identification of the potential risk factors would help understand the transmission dynamics of the disease and formulate public health interventions.Item 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.Item Spatial and seasonal analysis of human leptospirosis in the District of Gampaha, Sri Lanka(University of Kelaniya, 2014) Denipitiya, D.T.H.; Abeyewickreme, W.; Hapugoda, M.D.; Chandrasekharan, N.V.Leptospirosis is a zoonostic infectious disease, caused by a pathogenic species of the genus Leptospira. In Sri Lanka, around 1500 human leptospirosis cases are reported annually. Typically, the risk of the disease is seasonal with a small spike occurs in March to May and a large spike occurs during October to December. Objective of this study was to analyze spatial and seasonal pattern of human leptospirosis in the District of Gampaha, Sri Lanka.