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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Polymerase Chain Reaction and mosquito dissection as tools to monitor filarial Infection levels following mass treatment in Gampaha District, Sri Lanka(Elsevier, 2008) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Bandara, K.B.A.T.; Liyanage, T.; Abeyewickreme, W.BACKGROUND: Mass Drug Administration (MDA)-based Global Lymphatic filariasis (Lf) eradication programmes are aimed at stopping transmission of Wuchereria bancrofti by its mosquito vector. The study was designed to compare one year post treatment (mass distribution of Diethylcarbamazine-Albendazole) infection rates of Wuchereria bancrofti in Culex quenquifaciatus, the main vector of Lf in Sri Lanka using Conventional dissection techniques and a Polymerase Chain Reaction (PCR) assay based on parasite specific Ssp1 repeat which amplifies a fragment of 188 bp. METHODS: Field study was conducted in 45 sites in all Medical Officer of Health (MOH) areas in the Gampaha district, Sri Lanka; identified by the Anti Filariasis Campaign (AFC) as having high-risk for bancroftian filariasis transmission. Indoor-resting mosquitoes were collected by aspiration from 20 houses per each site. Part of the mosquitos were used for dissection and the remainder was used for PCR to detect the filarial parasites in mosquito. RESULTS: Mosquito dissection data revealed 42.22% (19/45) of the sites were infested with mosquitoes positive for Wuchereria bancrofti, indicating 8 transmission active MOH areas (53.33%; 8/15). An infection rate of 5.26% was observed among the mosquitoes caught from households and the larval density was 8.7 per positive mosquito. PCR investigation revealed that 46.67% (21/45) of the sites were positive for W. bancrofti DNA, indicating 11 transmission active areas (73.33%; 11/15). The PCR was found to be more sensitive compared to microscopy in detecting the filarial parasite in field collected mosquito samples with respect to the MOH areas. CONCLUSION: The PCR technique employed offers scope for detection of the filarial parasites with higher sensitivity and specificity; is efficient and rapid. This technique applied for the first time in Sri Lanka, can be adopted as a diagnostic tool for the detection of filarial parasites in the vector population in surveillance to enable effective control of filariasis in the country. Acknowledgements: Authors acknowledge the WHO/SEARO/TDR (grant no. SN 1152) and University of Kelaniya (Research grant no. RP/03/04/06/01/2006) and to Ms. H.M.Renuka and Mr. H.P.Anura U. Pathirana, Mr. M.I.M.Peris and Mr. Y.L.Rassapana for their support during field study activities. © 2008 Elsevier Inc.Item Application of nucleic acid technology (NAT) in the diagnosis of active viral replication in HBV and HCV infections and evidence for HBV surface antigen mutants(Sri Lanka Association for the Advancement of Science, 2008) Manamperi, A.; Gunawardene, Y.I.N.S.; Hapuarachchi, C.; Bandara, A.; Wellawaththage, C.; Abeyewickreme, W.; de Silva, J.Introduction: The community prevalence of Hepatitis B (HBV) and hepatitis C (HCV) infections, although considered low (< 1%) in Sri Lanka based on serological markers, pose a significant health threat to patients in high risk groups. The early diagnosis of active viral infection is crucial in such situations to prevent further transmission and to enable the clinicians to initiate successful therapeutic interventions. Objective: This study was carried out to investigate the usefulness of polymerase chain reaction (PCR) in the diagnosis of active viral replication in HBV and HCV infections. Methodology: All specimens from patients with serological evidence of hepatitis B (HBV surface antigen and/or antibodies for HBV core protein) or hepatitis C (antibodies for hepatitis C core protein-Anti-HCV) and referred to the Molecular Medicine Unit from May 2005 to May 2008 were analyzed by PCR and reverse-transcription PCR (RT-PCR) for HBV DNA (n=130) and HCV RNA (n=95) respectively. Results: Of the 130 patients tested, 57 (44%) were positive for HBV DNA. The positive group of patients included 10 renal transplant patients, 4 multiply transfused patients, 4 paediatric patients with lymphoma, and 1 patient with cirrhosis. Six HBV DNA positive patients had negative HBsAg serology profiles indicating the possibility of surface antigen mutant strains. The HBV DNA negative patients with positive serology profiles indicate sero-converted/ patients with resolved infections or false positive serology results. Of the 95 patients tested, 14 (15%) were positive for HCV RNA and included 3 paediatric patients with thalassaemia. HCV RNA negative, anti-HCV positive profiles reflect either false positive serology results (due to less specific antibody assays) or donors who have been exposed to HCV previously and subsequently resolved their infections. Conclusions: A major proportion of patients with serological markers for HBV have active viral infection whereas only relatively a minor proportion of patients with serological markers for HCV have active viral replication. We have also found the first possible evidence of hepatitis B surface antigen mutant strains. This underlines the importance of the nucleic acid based technology in the diagnosis and assessment of infection with or suspected to have hepatitis B or C infections. We also emphasize the importance of introducing NAT for screening donors for HBV DNA and HCV RNA to substantially lower the risk of acquiring HBV/HCV infection from a transfusion.Item Large-scale entomological assessment of Wuchereria bancrofti transmission by dissection and PCR-ELISA in Gampaha district, Sri Lanka(Sri Lanka Association for the Advancement of Science, 2008) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Hapuarachchi, H.A.C.; Bandara, K.B.A.T.; Abeyewickreme, W.Entomological surveys are important tools for monitoring progress of lymphatic filariasis (Lf) eradication programs. In this study, dissection of Culex quinquefasciatus was compared with a Polymerase Chain Reaction - Enzyme Linked Immunosorbent Assay (PCR-ELISA) for pooled mosquitoes to assess filarial infection levels in the major vector of Wuchereria bancrofti in Gampaha district, following mass-treatment programme with diethylcarbamazine (DEC) and albendazole. Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health (MOH) areas of Gampaha district known for the presence of W. bancrofti transmission. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using Deoxyribonucleic acid (DNA) extracted from mosquito pools (15 body parts/pool) utilizing primers specific for the Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3pools/site) was estimated using the PoolScreenTM algorithm and a novel probability-based method. The prevalence of infected mosquitoes with L1-L2 larvae of W. bancrofti ranged from 0%-8.54% by dissection and point estimates of infection prevalence as assayed by PCR-ELISA, ranged from 0% - 25.4%. Mosquitoes collected from all MOH areas (80%, N = 12), except for Minuwangoda, Dompe and Ragama, were positive for W. bancrofti larvae, with a prevalence rate ranging from 0.78% to 16.97% in both methods. Of 30 sentinel sites, 43.3% (N = 13) were positive for W. bancrofti transmission whereas it was evident in 40% (N = 6) of non-sentinel sites. The proportion of positive pools detected by the PCR-ELISA assay was higher than that obtained by the dissection indicating that PCR-ELISA assay is more sensitive than the dissection method in detecting infected/infective mosquitoes. Also results of this study showed that autochthonous transmission of W. bancrofti continues in the Gampaha district despite completion of the 5 year mass drug administration (MDA) programme. Therefore, we emphasize the use of more sensitive tools such as PCR-ELISA to monitor the impact of the MDA programme on disease transmission. This study also emphasizes that control measures should be further continued until the microfilareamic population is reduced to a level which could interrupt transmission in the area. Financial assistance received from WHO/SEARO/TDR (grant no. SN 1152) and University of Kelaniya (Grant no. RP/03/04/06/01/2006) is acknowledgedItem Comparison of five DNA extraction methods from human blood for the detection of Wuchereria bancrofti by polymerase chain reaction assays(Sri Lanka Association for the Advancement of Science, 2008) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Manamperi, A.; Hapuarachchi, H.A.C.; Gunawardena, N.K.; Abeysundara, S.; Abeyewickreme, W.Introduction: Lymphatic filariasis (Lf) is the second most common vector-borne disease globally. Approximately 90% of global burden of Lf is caused by Wuchereria bancrofti. W. bancrofti is routinely diagnosed by morphological identification of microfilariae (Mf) by microscopy which is a labour intense, low sensitive and time consuming method. Detection of W. bancrofti Deoxyribonucleic acid (DNA) using polymerase chain reaction (PCR) technique has become popular today, because of its high sensitivity and specificity. The overall success of the PCR strategy in detecting a filarial parasite in human blood varies between sample preparation methods. The objective of this study was to compare five DNA extraction methods (Lysis + centrifugation, Chelex method, Mf pellet method, Q1Aamp DNA Mini Kit commercial system, and Phenol-chloroform) with regard to duration of completion, labor involvement and PCR analytical sensitivity in-relation to DNA quality and quantity for the detection of W. bancrofti in human blood. Five blood samples positive for mf of W. bancrofti were tested for each DNA extraction method and were compared with respect to the sensitivity, time and quality/quantity of DNA and also by PCR analysis. Of the 5 methods tested, Mf pellet method was found to be the most simple and effective technique for the isolation of W. bancrofti Mf in human blood. This method was quick (15 min to complete), simple (5 min of manual labor), and very economical. It does not require any organic solvents, and the entire extraction procedure uses only two steps requiring supernatant transfer between tubes, hence minimizing the possibility of cross-contamination. Moreover, the PCR analytical sensitivity of the Mf pellet method was comparable to that of the commercial kit used. No PCR inhibitors were detected, independently of Mf count in the blood. Same method (optimal DNA extraction method) can be also used for the detection of parasite DNA from the field collected Mf positive mosquitoes using a PCR. Therefore, we recommend the Mf pellet method for processing large numbers of blood samples in community surveys aimed at determining the prevalence of W. bancrofti infection.Item Evaluation of an IS 6110 - based PCR assay for laboratory detection of M. tuberculosis complex DNA in clinical samples(Sri Lanka Association for the Advancement of Science, 2008) Palliyaguruge, R.H.; Gunawardene, Y.I.N.S.; Manamperi, A.; Wijekoon, C.N.; Wellawaththage, L.C.; Abeyewickreme, W.Due to the slow growth rate of the causative agent, the diagnosis of Tuberculosis (TB) takes considerable time period leading to the complication and spread of the disease. Towards this end, use of Polymerase Chain Reaction (PCR) technology, has revolutionized diagnosis of TB by reducing the diagnostic time. The aim of the present study was to compare two primer pairs and DNA extraction methods for the PCR based detection of M. tuberculosis complex (MTB) DNA in clinical samples for the routine laboratory diagnosis of TB. Two DNA extraction methods (Modified Boom's method and Roche commercial kit) and two IS 6110-based primer pairs were compared with respect to the sensitivity, time and quality/quantity of DNA. Extra pulmonary and pulmonary specimens from 45 TB suspected patients referred to the Molecular Medicine Unit, University of Kelaniya from February 2007 to April 2008 were analyzed. Results indicated 50 % and 70 % of the samples extracted from modified Boom's method and commercial kit, respectively, had high quality DNA, while 17 % and 67 % of the specimens extracted by the Boom's method and commercial kit, respectively, had over 200 µg/ml DNA. Both primer pairs exhibited similar level of sensitivity (200 fg of MTB DNA). In comparison to the time consuming culture, which takes 4 to 6 weeks, the modified Boom's method and commercial kit combined with PCR takes only 48 and 24 hrs, respectively. Of the 19 positives (42.22%) 11 were males while 17 and 02 were extra-pulmonary and pulmonary TB, respectively. The commonest clinical indication for sending samples was suspected disseminated TB. Presence or absence of fever or presence or absence of very high ESR (>100 mm) did not have a significant positive or negative predictive value for PCR. Moderately high ESR (>50 mm) had a negative predictive value of 0.8 and Mantaux test had a positive predictive value of 0.8. According to the time required for completion, labour, quality/quantity of DNA (statically significant at p=0.05) and reproducibility the commercial kit proved to be an efficient DNA extraction procedure. Both sets of primers elicited similar discriminating power. There was not a single clinical indicator with satisfactory predictive values, which is useful in clinical decision making regarding the need for PCR diagnosis in individual patients. We report a simple, rapid and reproducible PCR assay for routine laboratory diagnosis of MTB DNA from both pulmonary and extra-pulmonary specimens.Item Rapid differential diagnosis of dengue and chikungunya infections by multiplex RT-PCR and impact of chikungunya infection on liver biochemical tests(Sri Lanka Association for the Advancement of Science, 2008) Manamperi, A.; de Silva, P.; Ekanayaka, C.; Gunawardene, Y.I.N.S.; de Silva, J.; Weerasena, O.V.D.S.J.; Dassanayake, R.S.