Browsing by Author "Handunnetti, S."

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Clinical and laboratory associations of severity in a Sri Lankan cohort of patients with serologically confirmed Leptospirosis - a prospective study
(Sri Lanka Medical Association, 2015) Rajapakse, S.; Weeratunga, P.N.; Rodrigo, C.; Sriharan, S.; Niloofa, M.J.R.; Fernando, N.; de Silva, H.J.; Karunanayake, L.; Premawansa, S.; Handunnetti, S.
INTRODUCTION AND OBJECTIVES: Leptospirosis is a zoonotic infection of significant morbidity and mortality. This study elucidates the markers of severity in a cohort of Sri Lankan patients with serologically confirmed leptospirosis. METHOD: Prospectively recruited patients presenting to three healthcare institutions in the Western province of Sri Lanka with serological confirmation of leptospirosis with the microscopic agglutination test were included. Data regarding the socio-deruographic profile, clinical presentation, complications and biochemical parameters were recorded. Univariate associations and subsequent multivariate logistic regression models were constructed with severity as the dependent variable. RESULTS: A total of 232 patients were included. Majority were male (86.6%). Severe disease was noted in 68.5%. Significant clinical associations of severe disease included fever > 38.8°C on presentation (p=0.008), age>40 yrs; (p = 0.033), muscle tenderness (p=0.04) and tachycardia on admission (p=0.05). Laboratory associations of severe disease were highest white cell count > 12,350/mm3 (p<0.001) and < 7900/mm3 (p = 0.009), highest neutrophil percentage > 84% {p < 0.001). Hemoglobin > 11.2g/dL (p<0.001) and < 10.2 (p<0.001), packed cell volume > 33.8% (p <0.001) and <29.8% (p <0.001), lowest platelet count <63,500/mm3 (p = 0.01), highest ALT > 70 IU/L {p = 0.02) and hyponatremia with sodium <131mEq/L (p=0.004) On multivariate analysis, PCV < 29.8 (P = 0.011; adjusted OR =3.750; Cl = 1.394 - 10.423), ALT >70 P =0.044 adjusted OR =2.639; Cl =1.028-6.774 and hyponatremia< 131 (p=0.019 adjusted OR=6.413; Cl=1.353 -30.388) were found to be independent associations of severe disease. CONCLUSION: Severity associations were demonstrated with both clinical and laboratory parameters.
Clinical and laboratory associations of severity in a Sri Lankan cohort of patients with serologically confirmed leptospirosis: a prospective study
(Oxford : Oxford University Press, 2015) Rajapakse, S.; Weeratunga, P.; Niloofa, M.J.; Fernando, N.; Rodrigo, C.; Maduranga, S.; de Silva, N.L.; de Silva, H.J.; Karunanayake, L.; Handunnetti, S.
BACKGROUND: Leptospirosis results in significant morbidity and mortality. This study elucidates markers of severity in a cohort of Sri Lankan patients. METHODS: Patients presenting to three healthcare institutions in the Western province of SriLanka with leptospirosis serological confirmed by the microscopic agglutination test (MAT) were included. Prospective data regarding demographic, clinical and laboratory parameters was extracted. Univariate associations and subsequent multivariate logistic regression models were constructed. RESULTS: The study included 232 patients, with 68.5% (159) demonstrating severe disease. Significant associations of severe disease at a significance level of p<0.05 were fever >38.8°C on presentation, age >40 years, muscle tenderness, tachycardia on admission, highest white cell count >12 350/mm(3) and <7900/mm(3), highest neutrophil percentage >84%, haemoglobin >11.2 g/dL and <10.2 g/dL, packed cell volume (PCV) >33.8% and <29.8%, lowest platelet count <63 500/mm(3), highest alanine transaminase (ALT) >70 IU/L and hyponatremia with sodium <131mEq/L. On multivariate analysis, PCV <29.8% (p=0.011; OR 3.750; CI: 1.394-10.423), ALT >70 IU/L (p=0.044; OR 2.639; CI: 1.028-6.774) and hyponatremia <131mEq/L (p=0.019; OR 6.413; CI: 1.353-30.388) were independent associations of severe disease. CONCLUSIONS: Severity associations were demonstrated with both clinical and laboratoryparameters. There is a need for novel biomarkers for prediction of severity in leptospirosis. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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.
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.
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.
Is Total Serum Nitrite and Nitrate (NOx) Level in Dengue Patients a Potential Prognostic Marker of Dengue Hemorrhagic Fever?
(Hindawi Pub. Corp., 2018) Mapalagamage, M.; Handunnetti, S.; Premawansa, G.; Thillainathan, S.; Fernando, T.; Kanapathippillai, K.; Wickremasinghe, R.; de Silva, A.D.; Premawansa, S.
Potential use of total nitrite plus nitrate (NOx) and nitrite (NO2-) separately as surrogate markers for serum nitric oxide in severe dengue and their longitudinal changes along with the progression of infection was studied. Deproteinized sera from confirmed dengue fever (DF, n = 145) and dengue hemorrhagic fever (DHF, n = 74) patients on admission-A, critical-C, discharge-D, and convalescence-CON stages and from age-gender matched healthy individuals (HC, n = 77) were taken to assess NO2- and NOx levels using Griess and modified Griess assays. Serum NOx in DHFA was significantly lower compared to DFA (p < 0.001). HC had the lowest NOx and NO2- compared to all patient categories (p < 0.001) except NO2- in DF-CON and DHF-CON and NOx in DHF-CON. Serum NOx and NO2- in DHF patients admitted on fever day 3 (DHFA-3) was significantly lower compared to DFA-3 (p < 0.05). Cut-off values of 4.46 μM for NOx (91.3% sensitivity and 80.1% specificity) and 1.25 μM for NO2-(75.0% sensitivity and 73.3% specificity) were obtained for day 3 of fever. Serum NOx may be used as potential prognostic marker of DHF in patients presenting with DF in the early stage (on day 3 of fever) of the disease.
Low serum total nitrite and nitrate levels in severe leptospirosis
(BioMed Central, 2013) Kalugalage, T.; Rodrigo, C.; Vithanage, T.; Somaratne, P.; de Silva, H.J.; Handunnetti, S.; Rajapakse, S.
BACKGROUND: The relationship between inducible nitric oxide synthatase activity and disease severity in leptospirosis is unclear. Nitric oxide is converted to nitrites and nitrates, thus nitrite and nitrate levels (NOx) in serum are considered surrogate markers for nitric oxide. NOx are excreted through the kidneys, and elimination is diminished in renal impairment. We assessed the correlation of NOx with disease severity in patients with leptospirosis, compared with healthy controls and non-leptospirosis fever patients. METHODS: All patients admitted over a two-month period to the National Hospital, Colombo, Sri Lanka with a clinical picture suggestive ofleptospirosis were included. Leptospirosis was confirmed by the microscopic agglutination test (titre ≥ 400). Severe leptospirosis was defined by the presence of two or more of the following criteria: jaundice (bilirubin> 51.3 μmol/l), oliguria (urine output < 400 ml/day), serum creatinine> 133 μmol/l or blood urea > 25.5 mmol/l, or the presence of organ dysfunction. Non-leptospirosis fever patients and healthy volunteers were used as control groups. NOx levels were measured using a modified Griess reaction. RESULTS: Forty patients were confirmed as having leptospirosis and 26 of them had severe disease. NOx levels were significantly higher in confirmed leptospirosis patients compared to healthy controls, MAT equivocal patients and non-leptospirosis fever patients (p<0.001). NOx concentrations were also significantly higher in patients with severe compared to mild leptospirosis (p<0.001). Once NOx levels were corrected for renal function, by using the ratio NOx/creatinine, NOx levels were actually significantly lower in patients with severe disease compared to other patients, and values were similar to those of healthy controls. CONCLUSIONS: We postulate that high NOx levels may be protective against severe leptospirosis, and that finding low NOx levels (when corrected for renal function) in patients with leptospirosis may predict the development of severe disease and organ dysfunction. © 2013 Kalugalage et al.; licensee BioMed Central Ltd.
Protection against malaria in toque mokeys immunized with p.cynomolgi MSP1inv in alum
(Sri Lanka Association for the Advancement of Science, 2000) Amaratunga, C.; Nandasiri, K.; Weerasinghe, S.; Manamperi, A.; Holm, I.; Longacre, S.; Handunnetti, S.
Immunization of toque monkeys with baculovirus-expressed, His-tagged recombinant plasmodium cynomoigi ceylonensis (Pcc) C-terminal 19 kDa proteins of the major merozoite surface protein 1 (MSP1inv) with Freunds adjuvant, mediates long-term protection against homologous and heterologous p. cynomolgi blood-stage challenge infection. However, Freunds adjuvant is unsuitable for use in humans, which necessitates the testing of alternative adjuvants. Antigen-alum formulation and binding was optimized for the new preparation of matalloaffinity-purified MSP1inv antigen, under conditions acceptable for human trails, including 800ug aluminium per dose, in accordance with the permitted FDA maximum. This formulation was used in an immunization trial using the p. cynomolgi-toque monkeys system, which is analogous to the p.vivax-human system. Group 1 comprising 4 animals were immunized with alum+MSP1inv , and group 2 comprising 3 monkeys received alum alone. Four doses of immunization were given intramuscularly at 0,1,3 and 4 month intervals. After immunization, the anti-MSP1inv antibody titres of immunized animals reached 2.8x104 . all animals were given a homologous Pcc challenge infection one month after the last dose of immunization. One of the four immunized animals was completely protected while the other 3 animals showed low patent parasitaemia, resulting In an overall partially protective effect (p=0.02). immunization with alum did not result in sterile immunity, as seen with Freunds, where antibody titres range from 106 - 107 . Following treatment, the animals were given a second heterologous, blood-stage challenge infection of p.cynomolgi Gombak, (PcG) four months after the first challenge infection. Sequence analysis of PcG DNA reveald a single amino acid differing from that of Pcc. The substitution which occurs at the nt 207 position in the C-terminal 19-kDa sequence changes the amino acid glutamate into lysine. Statistically significant partial protection was observed in the immunized animals (p=0.04), despite having a lower titre of antibodies (3.3x103) at the time of re-challenge. Together with sequence data, this documents the ability of recombinant MSG1inv to protect against a heterologous infection.
Protection against malaria in toque monkeys immunized with P. cynomolgi MSPI-19 in alum
(The royal society of tropical medicine and hygiene, British society for parasitology, The American society of tropical medicine and hygiene, 2000) Amaratunga, C.; Nandasiri, K.; Weerasinghe, S.; Manamperi, A.; Longacre, S.; Holm, I.; Handunnetti, S.
In a pre-clinical trail of a vaccine against P.vivax malaria, toque monkeys were immunized with matalloaffinity-purified baculovirus-expressed. His-tagged recombinant Plasmodium cynomolgy ceylonensis (Pcc) C-terminal 19 kDa protiens of the major merozoite surface protein 1 (MSP1p19) with alum, under conditions acceptable for clinical trials. The P. cynomolgi-toque monkey system is highly analogous to P.vivax in humans. Eight animals were immunized with alum+MSP1p19 and 3 monkeys received alum alone. After four doses, the anti-MSP1p19 antibody titres of immunized animals reached 2.8x104. all animals were challenged with Pcc asexual blood stage parasites. The immunized animals showed significant, partial protection (p=0.0024). Four of these animals were given a second heterologous challenge infection of p.cynomolgi Gombak, (PcG) four months after the first challenge infection. Sequence analysis of PcG DNA revealed a single amino acid challenge differing from that of Pcc. The substitution which occurs at the nt 207 position in the C-terminal 19-kDa sequence changes the amino acid glutamate into lysine. Statistically significant protection was observed in the immunized animals (p=0.04), despite having a lower titre of antibodies at the time of re-challenge. This documents the ability of recombinant MSP1p19 with alum to protect against Pcc and PcG infections.