Browsing by Author "Hapuarachchi, C."

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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.
Chikungunya outbreak in 2008 in Ratnapura district, Sri Lanka - clinical and socio-economic analysis
(Sri Lanka Association for the Advancement of Science, 2008) Sumanadasa, S.D.M.; Hapuarachchi, C.; Bandara, K.B.A.T.; Wellawaththage, L.C.; Abeyewickreme, W.
Since 2006, Sri Lanka has experienced several outbreaks of chikungunya fever (CHIK) affecting several thousands of people. Today, CHIK has become one of the most important vector-borne diseases in the country. The objective of this study was to analyse the clinical manifestations and socio-economic status among CHIK patients reported from Pallebedda and Godakawela areas in Ratnapura district during the outbreak in February and March 2008. After obtaining the informed written consent, venous blood samples were collected from 80 suspected patients. A medical officer carried out clinical examination of each patient. Clinical information along with socio economic data of the patients was recorded in an interviewer-administered questionnaire. Serum samples were tested for CHIK by a Reverse-Transcription Polymerase Chain Reaction (RT-PCR) assay. Of eighty patients tested, 51% (n=42) were positive for CHIK. All positive patients had fever for less than 5 days duration. Majority of them (95%, n=40) had severe arthralgia with arthritis of small joints of hands and feet (81%, n=34). Moreover, a generalized, Itchy maculopapular rash was present in 78% (n=33) of them. The appearance of skin rash only after 4-5 days of fever was characteristic in the majority of patients. The mean age of positive patients was 38 years and consisted of 48% (n=20) of males. Many (43%, n=18) of them were farmers having a mean monthly family income of Rs. 4867.00. Analysis of educational status revealed that 60% (n=26) of family members had educated up to G.C.E. O/L whereas only 26% (n=12) had completed G.C.E. A/Ls. Twenty eight (67%) positive patients had at least one or more CHIK infected family members in addition. Moreover, 95% (n=40) of them were surrounded by infected neighbours indicating active, intense transmission in the area. According to the results, the most predominant clinical features of CHIK were fever either with severe arthralgia or arthritis of small joints of hands and feet. Skin rash, though characteristic, appeared to develop 4-5 days after the infection. CHIK has mainly affected the most productive labour force in these areas with majority belonging to the middle class farming community with a low monthly income. Hence, the sources of income of the affected families were severely hampered by the CHIK outbreak. Therefore, non-fatal, CHIK may have a negative impact on the socio-economic status of the affected communities. "The staff of the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Dr Richard Perera and the staff of Godakawela Hospital and Dr. Susanth Kariyawasam and the staff of Pallebadda Hospital are acknowledged".
Confirmation of 2006 chikungunya outbreak in Sri Lanka using RT-PCR
(Malaysian Society of Parasitology and Tropical Medicine, 2007) Abeyewickreme, W.; Bandara, K.B.A.T.; Perera, H.; Dayanath, M.Y.D.; Hapuarachchi, C.
Chikungunya, a mosquito-borne viral infection caused by a single-stranded RNA virus of the family Togaviridae, is considered as a rare, non-fatal disease. During February to October 2006, an epidemic of over 1.3 million suspected cases was reported in India and neighbouring countries causing a significant economic loss due to crippling manifestations of this infection. With the outbreak of many viral fevers including dengue and dengue haemorrhagic fever, in October–November 2006, patients with manifestations suggestive of chikungunya such as high fever, headache, arthralgia and arthiritis (particularly, in ankle, knee and small joints of hands) were reported in many parts of Sri Lanka. As no chikungunya cases had been officially reported in the island since 1969, laboratory investigations for the presence of chikungunya virus was a prime requirement for confirmation of the outbreak. A total of 60 venous blood samples collected from suspected patients from different geographical regions of Sri Lanka were analysed using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique to confirm the presence of chikungunya virus. Viral RNA was extracted from samples collected within 1-4 days of fever by using a Qiagen RNA extraction kit. RT-PCR was performed using chikungunya specific oligonucleotides. Both positive and negative controls were included in each set of reactions. The amplified products (354 bp) were visualized by running in a 1.5% agarose gel followed by ethidium bromide staining. Of the 60 samples, 33 (55%) were positive for chikungunya. They were distributed among almost all the geographical regions, highlighting the presence of a wide-spread epidemic in the country.
Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka
(Asian Pacific Organization for Cancer Prevention, 2013) Wijegunawardana, A.D.; Gunawardene, N.S.; Hapuarachchi, C.; Manamperi, A.; Gunawardena, K.; Abeyewickreme, W.; Latif, B.
OBJECTIVE: To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culex quinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009). METHODS: Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha Districtknown for the presence of W. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for 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 (3 pools/site) was estimated using the PoolScreen™ algorithm and a novel probability-based method. RESULTS: Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods. CONCLUSIONS: Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity
Genetic Polymorphism in Pvmsp-3a. and Pvcs genes in Plasmodium vivax infections in Sri Lanka
(Sri Lanka College of Microbiologists, 2008) Manamperi, A.; Fernando, D.; Mahawithanage, S.; Wickremasinghe, R*.; Bandara, A.; Wellawatta, C.; Hapuarachchi, C.; Abeyewickreme, W.; Wickremasinghe, R.
INTRODUCTION: Plasmodim vivax malaria accounts for about 70% of all malaria infections in Sri Lanka. There is limited information on the genetic heterogeneity of P. vivax parasites in endemic areas of the country. OBJECTIVE: The objective of this study was to assess the potential of two P. vivax genes, Pvmsp-3v. and Pvcs. as genetic markers for their use in genotyping parasites collected from the field. METHOD: DNA was extracted from 12 Geimsa-stained P. vivax positive slides by phenol/chloroform method. A nested polymerase chain reaction (PCR) approach was adopted for both Pvmsp-3a and Pvcs genes. RFLP analysis ofPvmsp-la nested PCR products was carried out with Hha\ restriction enzyme. RESULTS AND DISCUSSION: Nested amplification of the marker genes resulted in 4 size variants for Pvcs (~ 600-750 bp) and 2 size variants for Pvmsp-3a (1.9 kb and 1.1 kb). Further, all PCR-RFLP products of Pvmsp-3a. Gene showed a major size polymorphism. Three samples showed evidence of infections with mixed genotypes and there was also evidence to identify a relapse infection. Analysis of these two genetic markers revealed 11 distinguishable variant types: 4 for Pvcs and 7 for Pvmsp-3a. CONCLUSIONS: The observed PCR and PCR-RFLP profiles of the Pvcs and Pvmsp-3& genes demonstrate that the P. vivax parasites in Sri Lanka were highly diverse despite the prevailing low transmission levels. It could be concluded that these two genes in combination could be considered suitable genetic markers to analyze P. vivax parasite dynamics in Sri Lanka.
Genotyping of Plasmodium vivax infections in Sri Lanka using Pvmsp-3 alpha and Pvcs genes as markers:a preliminary report
(Malaysian Society of Parasitology and Tropical Medicine, 2008) Manamperi, A.; Sanath, M.; Fernando, D.; Wickremasinghe, R.; Anura, B.; Hapuarachchi, C.; Abeyewickreme, W.; Wickremasinghe, A.R.
Plasmodim vivax malaria accounts for more than 90% of malaria cases in Sri Lanka. There is limited information on the genetic heterogeneity of P. vivax in endemic areas of the country. Here we have assessed the potential of two P. vivax genes as genetic markers for their use in genotyping parasites collected from the field. DNA extracted from Geimsa-stained P. vivax positive slides were genotyped at two polymorphic loci: the P. vivax merozoite surface protein 3- alpha (Pvmsp-3alpha) and circumsporozoite protein (Pvcs). Analysis of these two genetic markers revealed 11 distinguishable variant types from the two genetic markers: 4 for Pvcs and 7 for Pvmsp-3alpha. The results indicate that the P. vivax parasite population is highly diverse in Sri Lanka, despite the low level of transmission.
Re-emergence of chikungunya in Sri Lanka: First confirmation of the 2006 outbreak by molecular diagnosis
(Sri Lanka Association for the Advancement of Science, 2007) Perera, E.D.T.; Wijesiriwardena, B.; Hapugoda, M.D.; Bandara, K.B.A.T.; Dayanath, M.Y.D.; Wellawaththage, L.C.; Gunawardena, N.K.; Hapuarachchi, C.; Abeyewickreme, W.
Chikungunya virus infection is clinically similar to many other acute febrile illnesses, such as dengue infection, malaria, west nile fever and leptospirosis. Rapid confirmation of the outbreak by laboratory diagnosis is important to ensure public health safety by appropriate patient management and controlling the disease. Molecular diagnosis by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) assists rapid diagnosis. The objective of the present study was to determine the clinical manifestations of chikungunya confirmed patients in Sri Lanka. Venous blood samples and clinical information were collected from 66 chikungunya suspected patients having fever of less than 4 days from different geographical areas in Sri Lanka during the period September 2006 to February 2007. Serum samples were tested for chikungunya RNA by RT-PCR. Amplified products were visualized by agarose gel electrophoresis. Among 66 suspected patients, 51% (34/66) were positive for chikungunya by RT-PCR assay and 55.9% (19/34) were females. All age groups were affected similarly with the mean age of 41 years (range = 4 months to 80 years). Of the PCR positive 34, all had fever with either arthralgia or arthritis or both. Most of them had only pain in the joints without swelling (arthralgia only); 67.6% (23/34) in knee, 55.9% (19/34) in ankle, 50% (17/34) in wrist, 44.1% (15/34) in elbow and 52.9% (18/34) in small joints. Arthritis of ankle joint 35.2% (12/34) was more frequent compared with arthritis of the knee joint17.6% (6/34). PCR positive patients manifested more symptoms compared with PCR negative patients; 85.3% (29/34) headache, 79.4% (27/34) backache, 58.8% (20/34) nausea and 61.8% (21/34) vomiting. Compared with dengue, most of the chikungunya patients did not have dermatological manifestations. This is the first confirmation of the 2006 chikungunya outbreak in Sri Lanka. Some of the patients who had symptoms suggestive of chikungunya, tested by PCR were negative. These patients were probably suffering from other illnesses such as dengue. Acknowledgements: The International Atomic Energy Agency (TC SRL 6/028) for technical cooperation
STR polymorphisms in Sri Lanka: evaluation of forensic utility in identification of individuals and parentage testing
(Sri Lanka Medical Association, 2009) Manamperi, A.; Hapuarachchi, C.; Gunawardene, N.S.; Bandara, A.; Dayanath, D.; Abeyewickreme, W.
OBJECTIVES: This preliminary study was carried out to determine the allele frequencies and forensic efficiency parameters for the short tandem repeat loci CSF1PO, TPOX, THO1, D16S539, D7S820, D13S317, vWA, FESFPS and F13B in a test sample population of Sri Lankans. DESIGN: Test samples were obtained from 305 non-related individuals originating from all 9 provinces of Sri Lanka. DNA was extracted from whole blood using chelex-100 and amplified by PCR using the GenePrint STR kit and silver stained. Final DNA profiles were analysed for forensic efficiency parameters and paternity indices using PowerStats version 12. Possible divergence from Hardy-Weinberg Equilibrium was tested using the chi-square test and exact test. RESULTS: All common alleles in the allelic ladders were found in the test sample studied. PIC values >0.5 for all 9 STR loci indicate this STR system to be informative and useful for identification purposes. The D13S317, vWA and D7S820 loci were found to be the most polymorphic markers of the system studied. CONCLUSIONS: No deviations from Hardy-Weinberg Equilibrium were found for any of the loci examined. The results indicate that the 9 STR loci system described here is suitable for estimating DNA profile frequencies in human identification and forensic and parentage testing for legal purposes among Sri Lankans.