Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Detection of pathogenic Leptospira with rapid extraction followed by recombinase polymerase amplification (RPA) and quantitative polymerase chain reaction (qPCR) assay-A comprehensive study from Sri Lanka(Public Library of Science, 2024) Uduwawala, H.; Manamperi, A.; Gunaratna, G.P.S.; Karunanayake, L.; Ceruti, A.; Wahed, A.A.E.; Fernando, L.; Premaratna, R.; Hapugoda, M.Leptospirosis is the most widespread zoonosis in the world. The disease is more prevalent in tropical regions where the majority of developing countries are located. Leptospirosis is considered a protean manifestation zoonosis with severity of the disease ranging from a mild febrile illness to a severe and life-threatening illness. Clinical symptoms of leptospirosis overlap with other tropical febrile illnesses. Early, rapid, and definitive diagnosis is important for effective patient management. Since Polymerase Chain Reaction (PCR)-based assays are not readily available in most clinical settings, there is a need for an affordable, simple, and rapid diagnostic test. Quantitative PCR (qPCR) and Recombinase Polymerase Amplification (RPA) were implemented at the Faculty of Medicine, University of Kelaniya, and a prospective study to evaluate RPA for diagnosis of acute phase of leptospirosis was conducted. Results indicate that RPA and qPCR were positive in 81% (98/121) of the total positive and acute clinical samples. Of the 81 positive MAT confirmed patients 60 (74%) and 53 (65%) were positive with qPCR and RPA respectively. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity-70% and specificity-87%) of RPA compared to MAT as the reference gold standard. Results further suggest that there is no significant difference between the two assays, qPCR and RPA-SwiftX (P = 0.40). Laboratory procedures for the extraction and detection by qPCR in the laboratory have been optimized to obtain results within 6 hours. However, the RPA-SwiftX method under field conditions took 35 minutes. The RPA-SwiftX method could replace the qPCR which shows similar sensitivity and specificity. Therefore, RPA established under the current study presents a powerful tool for the early and rapid diagnosis of leptospirosis at point-of-care.Item Development of an in-house ELISA as an alternative method for the serodiagnosis of leptospirosis(Elsevier, 2021) Niloofa, R.; Karunanayake, L.; de Silva, H.J.; Premawansa, S.; Rajapakse, S.; Handunnetti, S.BACKGROUND: Leptospirosis is most often clinically diagnosed and a laboratory test with high diagnostic accuracies is required. METHODOLOGY: IgM and IgG-ELISAs using Leptospira antigens were established and evaluated in relation to the Microscopic Agglutination Test (MAT). Antigen preparation consisted either saprophytic Leptospira biflexa to detect genus specific antibodies (genus-specific ELISA) or a pool of five most prevalent Leptospira interrogans serovars in Sri Lanka to detect serovar specific antibodies (serovar-specific ELISA). IgM and IgG immune responses in severe and mild leptospirosis patients (n = 100 in each group) were studied. RESULTS: ELISAs showed high repeatability and reproducibility. Serovar-specific IgM-ELISA showed sensitivity of 80.2% and specificity of 89%; genus-specific IgM-ELISA showed sensitivity of 83.3% and specificity of 91%. Serovar and genus-specific IgG-ELISA showed sensitivities of 73.3% and 81.7%, and specificities of 83.33%. Commercial IgM-ELISA showed sensitivity and specificity of 79.2% and 93% respectively. Commercial IgG-ELISA showed sensitivity, specificity of 50% and 96.7% respectively. IgM levels observed in mild and severe leptospirosis (ML & SL) patients were significantly higher than healthy control (HC) group, having absorbance mean of 0.770, 0.778 and 0.163 respectively. In contrast, SL patients had significantly higher mean anti-leptospiral IgG levels compared to both ML and HC groups (0.643, 0.358 and 0.116 respectively; ANOVA, P < 0.001). Presence of anti-leptospiral IgG above OD 0.643 optical density (OD) at 1:100 could predict high risk of severe disease. CONCLUSION: Serovar-specific In-House ELISAs could be used for the laboratory diagnosis of leptospirosis in endemic settings. Observed high levels of anti-leptospiral IgG suggest its value as a predictor for disease severity. KEYWORDS: IgM and IgG antibodies; In-House ELISA; Leptospirosis; Sero-diagnosis.Item Leptospirosis: challenges in diagnosis, and predictors of severity(Ceylon College of Physicians, 2016) Rajapakse, S.; Fernando, N.; Niloofa, M.J.R.; de Silva, H.J.; Karunanayake, L.; Premawansa, S.; Handunnetti, S.M.No Abstract availableItem A Diagnostic scoring model for Leptospirosis in resource limited settings(Public Library of Science, 2016) Rajapakse, S.; Weeratunga, P.; Niloofa, R.; Fernando, N.; de Silva, N.L.; Rodrigo, C.; Maduranga, S.; Nandasiri, N.; Premawansa, S.; Karunanayake, L.; de Silva, H.J.; Handunnetti, S.Leptospirosis is a zoonotic infection with significant morbidity and mortality. The clinical presentation of leptospirosis is known to mimic the clinical profile of other prevalent tropical fevers. Laboratory confirmation of leptospirosis is based on the reference standard microscopic agglutination test (MAT), direct demonstration of the organism, and isolation by culture and DNA detection by polymerase chain reaction (PCR) amplification. However these methods of confirmation are not widely available in resource limited settings where the infection is prevalent, and reliance is placed on clinical features for provisional diagnosis. In this prospective study, we attempted to develop a model for diagnosis of leptospirosis, based on clinical features and standard laboratory test results. METHODS: The diagnostic score was developed based on data from a prospective multicentre study in two hospitals in the Western Province of Sri Lanka. All patients presenting to these hospitals with a suspected diagnosis of leptospirosis, based on the WHO surveillance criteria, were recruited. Confirmed disease was defined as positive genus specific MAT (Leptospira biflexa). A derivation cohort and a validation cohort were randomly selected from available data. Clinical and laboratory manifestations associated with confirmed leptospirosis in the derivation cohort were selected for construction of a multivariate regression model with correlation matrices, and adjusted odds ratios were extracted for significant variables. The odds ratios thus derived were subsequently utilized in the criteria model, and sensitivity and specificity examined with ROC curves. RESULTS: A total of 592 patients were included in the final analysis with 450 (180 confirmed leptospirosis) in the derivation cohort and 142 (52 confirmed leptospirosis) in the validation cohort. The variables in the final model were: history of exposure to a possible source of leptospirosis(adjusted OR = 2.827; 95% CI = 1.517-5.435; p = 0.001) serum creatinine > 150 micromol/l (adjusted OR = 2.735; 95% CI = 1.374-4.901; p = 0.001), neutrophil differential percentage > 80.0% of total white blood cell count (adjusted OR 2.163; 95% CI = 1.309-3.847; p = 0.032), serum bilirubin > 30 micromol/l (adjusted OR = 1.717; 95% CI 0.938-3.456; p = 0.049) and platelet count < 85,000/mm3 (adjusted OR = 2.350; 95% CI = 1.481-4.513; p = 0.006). Hosmer-Lemeshow test for goodness of fit was 0.931. The Nagelkerke R2 was 0.622. The area under the curve (AUC) was noted as 0.762. A score value of 14 reflected a sensitivity of 0.803, specificity of 0.602, a PPV of 0.54, NPV of 0.84, a positive LR of 2.01 and a negative LR of 0.32. CONCLUSIONS: The above diagnostic model for diagnosis of leptospirosis is suggested for use in clinical settings. It should be further validated in clinical practice.Item Protein Carbonyl as a biomarker of oxidative stress in severe Leptospirosis, and its usefulness in differentiating Leptospirosis from Dengue Infections(Public Library of Science, 2016) Fernando, N.; Wickremesinghe, S.; Niloofa, R.; Rodrigo, C.; Karunanayake, L.; de Silva, H.J.; Wickremasinghe, A.R.; Premawansa, S.; Rajapakse, S.; Handunnetti, S.M.Pathogenesis of disease severity in leptospirosis is not clearly understood whether it is due to direct damage by pathogen or by adverse immune responses. Knowledge on biomarkers of oxidative stress which could be used in identifying patients with severe illness has shown to be of great value in disease management. Thus, the main aim of this study was to assess the damage to serum proteins and lipids, and their significance as biomarkers of oxidative stress in severe leptospirosis. In regions endemic for both leptospirosis and dengue, leptospirosis cases are often misdiagnosed as dengue during dengue epidemics. Therefore, the second aim was to assess the potential of the oxidative stress markers in differentiating severe leptospirosis from critical phase dengue. We measured serum antioxidants (uric acid and bilirubin), total antioxidant capacity (AOC), protein carbonyl (PC) and lipid hydroperoxide (LP) in patients with severe leptospirosis (n = 60), mild leptospirosis (n = 50), dengue during the critical phase (n = 30) and in healthy subjects (n = 30). All patient groups had similar total antioxidant capacity levels. However, the presence of significantly high uric acid and total bilirubin levels may reflect the degree of renal and hepatic involvement seen in severe leptospirosis patients (p<0.02). Serum PC and LP levels were significantly higher in leptospirosis patients compared to critical phase dengue infections (p<0.005). Moreover, high serum PC levels appear to differentiate SL from DC [area under the curve (AUC) = 0.96; p<0.001]. Serum PC may be a reliable biomarker of oxidative damage to serum proteins to identify severe leptospirosis patients (AUC = 0.99) and also to differentiate severe leptospirosis from mild cases (AUC = 0.78; p<0.005) indicating its contribution to pathogenesis. Use of serum PC as an indicator of leptospirosis severity and as an oxidative stress biomarker in differentiating leptospirosis from dengue would provide the opportunity to save lives via prompt patient management.Item Changes in full blood count parameters in leptospirosis: a prospective study(BioMed Central, 2014) de Silva, N.L.; Niloofa, M.; Fernando, N.; Karunanayake, L.; Rodrigo, C.; de Silva, H.J.; Premawansa, S.; Handunnetti, S.M.; Rajapakse, S.BACKGROUND: Leptospirosis presents diagnostic challenges to clinicians, in settings where other acute febrile illness are prevalent. The patterns of serial changes in haematological parameters in leptospirosis has not been evaluated previously. METHODS: Clinical and laboratory data were collected prospectively from patients with leptospirosis in two hospitals in Sri Lanka. Leptospirosis was diagnosed based on WHO clinical criteria with confirmation using Microscopic Agglutination Test titre > 400 or 4 fold rise between acute and convalescent samples. Full blood count parameters were analysed up to the 14th day of illness. RESULTS: Data from 201 patients with leptospirosis were available. Leukocyte counts and absolute neutrophil counts showed a decline over the first 5 days of illness, then rose until the end of the second week. On day 3 of fever, the majority (75%) had normal leukocyte counts, and by day 5, leukocytosis was seen only in 38.1%; leucopenia was an uncommon finding. Lymphopenia was seen in over half on day 5, declining to just under a quarter of patients by day 10. Platelets declined over the first 6 days and then gradually rose. Thrombocytopenia was seen in nearly three-fourths of patients by day 5. Haemoglobin and haematocrit levels declined over the course of illness. Total white cell and neutrophil counts were higher, and haemoglobin and haematorcrit were significantly lower, in patients with severe disease. CONCLUSIONS: Neither leukocytosis nor lymphopenia were prominent features, while thrombocytopenia was seen during the 3rd to 5th day of illness, with dropping haemoglobin levels. Neutrophilia and low haemoglobin levels appear to predict severe disease. These findings may be of use to clinicians in differentiating leptospirosis from other acute infections like dengue, and could help in predicting severe leptospirosis.Item Diagnosis of leptospirosis: comparison between microscopic agglutination test, IgM-ELISA and IgM rapid immunochromatography test(Public Library of Science, 2015) Niloofa, R.; Fernando, N.; de Silva, N.L.; Karunanayake, L.; Wickramasinghe, H.; Dikmadugoda, N.; Premawansa, G.; Wickremasinghe, A.R.; de Silva, H.J.; Premawansa, S.; Rajapakse, S.; Handunnetti, S.BACKGROUND: Leptospirosis is diagnosed on clinical grounds, and confirmed by microscopic agglutination test (MAT). IgM-ELISA (Serion-Virion) and immunochromatography test (Leptocheck-WB) are two immunodiagnostic assays for leptospirosis. Their sensitivity, specificity and applicability in Sri Lanka have not been systematically evaluated. METHODS: Clinically diagnosed leptospirosis patients (n = 919) were recruited from three hospitals in the Western Province of Sri Lanka, during June 2012 to December 2013. MAT, IgM-ELISA and Leptocheck-WB were performed on all patient sera. MAT titer of ≥400 in single sample, four-fold rise or seroconversion ≥100 in paired samples were considered as positive for MAT. For diagnostic confirmation, MAT was performed during both acute and convalescent phases. Anti-leptospiral IgM ≥20 IU/ml and appearance of a band in the test window were considered as positive for IgM-ELISA and Leptocheck-WB test respectively. Patients with an alternative diagnosis (n = 31) were excluded. Data analysis was performed using two methods, i) considering MAT as reference standard and ii) using Bayesian latent class model analysis (BLCM) which considers each test as imperfect. RESULTS: MAT, IgM-ELISA and Leptocheck-WB positivity were 39.8%, 45.8% and 38.7% respectively during the acute phase. Acute-phase MAT had specificity and sensitivity of 95.7% and 55.3% respectively, when compared to overall MAT positivity. IgM-ELISA and Leptocheck-WB had similar diagnostic sensitivity when compared with acute-phase MAT as the gold standard, although IgM-ELISA showed higher specificity (84.5%) than Leptocheck-WB (73.3%). BLCM analysis showed that IgM-ELISA and Leptocheck-WB had similar sensitivities (86.0% and 87.4%), while acute-phase MAT had the lowest sensitivity (77.4%). However, acute-phase MAT had high specificity (97.6%), while IgM-ELISA and Leptocheck-WB showed similar but lower specificity (84.5% and 82.9%). CONCLUSIONS: Both IgM-ELISA and Leptocheck-WB shows similar sensitivities and specificities. IgM-ELISA may be superior to MAT during the acute phase and suitable for early diagnosis of leptospirosis. Leptocheck-WB is suitable as a rapid immunodiagnostic screening test for resource limited settings.