Browsing by Author "Athapaththu, A.M.M.H."

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Clinical and virological features of dengue in 2010
(Sri Lanka College of Microbiologists, 2011) Hapugoda, M.D.; Manamperi, H.; Gunasena, S.; Athapaththu, A.M.M.H.; Premawansa, G.; Wellawaththage, C.; Jayarathna, T.D.S.S.; Abeyewickreme, W.
INTRODUCTION: Dengue is an important viral infection in Sri Lanka. All 4 serotypes co-circulate in Sri Lanka. OBJECTIVE: To study the clinical and virological features of dengue in 2010. DESIGN, SETTING AND METHODS: A hospital-based study was carried out at North Colombo Teaching Hospital, Ragama in 2010. Patients clinically suspected of having dengue, with fever less than 5 days were recruited. Acute and convalescent blood samples were collected within 7 days after obtaining informed written consent. Demographic, clinical information and laboratory results were obtained. Acute serum samples were tested using molecular (RT-PCR and Semi-Nested PCR) and serological (ELlSAs and HAI) assays. Convalescent samples were tested by serological assays. RESULTS: Of 209 patients enrolled, 93 % (195/209) were laboratory confirmed as recent positive cases of dengue viral infection; of these, 5% (9/195) were classified as dengue fever; 85%(1G5/195) dengue haemorrhagic fever (DHF) and 0.5% (1/195) dengue shock syndrome. Mean platelet value and packed cell volume (PCV) in laboratory confirmed dengue patients were 56,107/mm3 (range 10,000-306,000) and 42%(range 34-61 %) respectively. Patients infected with DHF showed both primary (n=45) and secondary (n=102) infections. Interestingly, secondary infection was not significantly correlated with DHF (x2-0.3:p=0.6). DEN-1 was responsible for the majority of cases, with a minority due to other three serotypes; all serotypes contributed to severe disease. CONCLUSION: DEN-1 was responsible for the majority of cases in 2010 but it circulated at a low level during previous epidemics. Majority of patients had severe clinical symptoms. In this epidemic, the clinical presentation of dengue differed according to the geographic region and viral serotype. ACKNOWLEDGMENTS: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (lAEA/SRL/5/042) is acknowledged.
Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(Sri Lanka College of Microbiologists, 2011) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.
INTRODUCTION: Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. OBJECTIVE: To compare two antigens for detection of anti-CHIK antibody. DESIGN, SETTING AND METHODS: In this study, two antigens {viral cell lysate and recombinant protein) were evaluated for detection of anti-1 CH IK antibody by using IgM ELISA. A novel recombinant | protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein, I This protein was expressed in Escherichia coli and resultant protein was affinity purified and ~10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine,] Nagasaki University. RESULTS: Atotal of 64 serum samples confirmed as positives andl 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was] 17% and 100% respectively. Conclusion Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. ACKNOWLEDGMENT: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.
Correlation of clinical presentation and laboratory confirmation of dengue patients
(Sri Lanka Association for the Advancement of Science, 2010) Manamperi, N.H.; Athapaththu, A.M.M.H.; Premawansa, V.; Wellawaththage, C.; Jayarathna, T.D.S.S.; Abeyewickreme, W.; Hapugoda, M.D.
Dengue is one of the most important arthropod-borne diseases in the world and it has become a very important disease in Sri Lanka, today. In Sri Lanka, diagnosis of dengue depends mainly on clinical signs and symptoms. Only a few suspected patients are confirmed by laboratory assays based on aetiological agents. The objective of this study was to determine the correlation between clinical presentation and laboratory confirmation of dengue patients. Acute serum samples (n=100) collected from patients clinically suspected of having dengue fever ("'ª-"ý¦> 5 days) warded at the North Colombo Teaching Hospital, Ragama were used for the present study. Serum samples were collected after obtaining informed written consent frompatients and samples were tested by RT-PCR which has high sensitivity (10 FFU/reaction) and specificity. Final diagnosis as dengue or non-dengue was assigned based on the results of RT-PCR assay. Differences in clinical and laboratory data were analyzed in dengue and non dengue patients. Chi-square test was used for comparison of data. The proportion of laboratory confirmed dengue patients were 56% (56/100). Mean platelet count and PCV in laboratory confirmed dengue patients were 60 269/mm 3 (range 3000-306000) and 41% (range 27-61%) and in non dengue patients were 106 318/mm 3 (range 5000-290000) and 41.6% (range 29-53%). Based on WHO criteria for diagnosis of dengue, heada (48/56 vs 41/44, ÝÖ 2 =0.7, p=0.38), retro-orbital pain (30/56 vs 14/44, ÝÖ 2 =3.8, p=0.04), limb pain (51/56 vs 30/44, ÝÖ 2 =7, p=0.00) and external bleeding (29/56 vs 4/44, ÝÖ 2 =18, p=0.00) showed significant association with dengue. Neck pain (10/56 vs 09/44, ÝÖ 2 =0.01, p=0.94), and lymphadenopathy (3/56 vs 02/44, ÝÖ 2 =0.08, p=0.78) did not show significant association with dengue. The infection was confirmed as dengue fever in 11% (6/56) and dengue hemorrhagic fever in 89% (50/56) based on WHO criteria. Surveillance based on clinical diagnosis may result in over estimation of the disease as clinical diagnosis is not specific enough. Laboratory confirmation of dengue suspected patients is important to measure the real incidence of the disease which leads implementation of control measures. Further, thisis important for efficient management of patients.Acknowledgements: Financial and technical assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08-02) and International Atomic Energy Agency (IAEA SRI 5/042)
Designing of immunogenic peptides from Dengue Virus NS1 region for production of monoclonal antibodies as diagnostic intermediates
(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Munasinghe, M.M.E.; Chandrasekharan, N.V.; Korbakis, D.; Soosaipillai, A.; Diamandis, E.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
BACKGROUND: Small peptide antigens have become an essential tool for antibody production in the recent life science research applications. The immunogenicity of peptide antigens is a critical factor to induce the immune response in order to produce desired antibodies. METHODS: In the current study, we have previously determined four Dengue (DEN) serotype specific peptides, containing 28 Amino Acid (AA) residues were re-designed. The peptides were re-designed considering many factors, for instance, sequence of the Sri Lankan isolates, abundance of Cysteine residues, solubility and the length of the peptide, carrier protein to be used and several other factors such as the N-terminal and C- terminal AAs and multiple AA residues. The peptide sequences were analysed using Antigen Profiler Peptide Tool (Thermo-scientific), Peptide Property Calculator (Genscript) and Swiss-Model (Biozentrum). RESULTS: The protein sequence of the peptides were changed according to the Sri Lankan isolates (AEB98757.1, ACS32038.1, AHG23239.1 and AHN50410.1). Oxidation of Cysteine residues results in significant conformational changes. Replacement of Cysteine with Serine prevents such oxidation reactions and it often retains full biological activity. Generally, peptides with a high number of hydrophobic AA (>50%) may result insoluble peptides. Similarly, to obtain a soluble peptide, it is important to contain at least one charged AA in every five AAs. Hence, the number of hydrophobic residues in the peptides were maintained below 50% and ensured that one out of every five amino acids is charged. The length of the peptide is an important factor as long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic. In order to obtain both highly conserved and variable regions among four serotypes, the peptide length was determined as 29 residues. A terminal Cysteine was added to allow peptide conjugation with carrier protein. Keyhole Limpet Hemocyanin was selected as the carrier protein due to its higher immunogenicity. N-terminal Glutamine or Aspargine and C-terminal Proline or Glycine in the sequences were avoided. Finally, the peptides sequences were determined as: DEN1; CPESSDDQRA WNIWEVEDYGFGIFTTNIW,DEN2; CAESPN TNRA WNSLEVEDYGFGVFTTNIW, DEN3;CPESPSASRAWNVWEVEDYGFGVFTTNIW and .DEN4;CSESPNERRAWNSLEVEDYGFGMFTTNIW. CONCLUSION: These peptides have a high potential to be used as peptide antigens for Monoclonal Antibody production.
Development of recombinant protein antigens using a bacterial expression system for the detection of anti-Chikungunya (CHIK) antibodies
(Sri Lanka College of Microbiologists, 2013) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.
INTRODUCTION AND OBJECTIVE: Laboratory confirmation of Chikungunya (CHIK) virus is very useful as clinical symptoms of CHIK can overlap with other diseases. Chikungunya virus specific antigen, which shows high specificity, sensitivity and low cross reactivity with other related diseases, is required for laboratory confirmation. Objective of this study was to develop and compare two recombinant protein antigens for detection of anti-CHIK antibodies. DESIGN, SETTING AND METHODS: Recombinant CHIK protein antigens were prepared using Envelope (E1 and E2) regions of the CHIK virus. The genes were custom designed and chemically synthesized with a 6X His tag. Bacterial expression systems [BL21 (DE3)] were used to clone and express the recombinant proteins. The recombinant proteins were purified with >95% of purity per liter of culture using Ni-NTA columns under denature conditions. In this study, two antigens were evaluated for detection of anti-CHIK antibody by using novel optimized in-house IgM and IgG ELISAs, using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Center for Viral Reference and Research) Institute of Tropical Medicine, Nagasaki University, Japan. RESULTS: Atotal of 55 serum samples confirmed as positives and 186 confirmed as negatives by HA! test, IgM capture ELISA and indirect IgG ELISA using the purified CHIK antigen were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 148 (E1) or 227 (E2) (148 + extra 79) confirmed ac negatives were used to evaluate the antigens using novel IgG ELISA. The E1 recombinant protein showed 5% (3/ 55) sensitivity and 99% (184/186) specificity for IgM ELISA and 60% (47/78) sensitivity and 63% (94/148) specificity for IgG ELISA. The E2 recombinant protein showed 65% (36/55) sensitivity and 70% (131/186) specificity for IgM ELISA and 83% (65/78) sensitivity and 86% (195/227) specificity for IgG ELISA. CONCLUSION: Recombinant CHIK-E2 protein antigen showed higher specificity and sensitivity in detection of both IgM and IgG antibodies. Thus the E2 recombinant protein antigen used in this study could be expressed in an eukaryotic expression system to achieve much higher results. ACKNOWLEDGMENT: International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (lAEA/SRL/5/042) are gratefully acknowledged.
Development of recombinant proteins as diagnostic intermediates for chikungunya infection
(Sri Lanka Association for the Advancement of Science, 2010) Athapaththu, A.M.M.H.; Khanna, N.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.
Chikungunya is an important disease with explosive outbreaks occurring in many South East Asian countries. As the clinical symptoms associated with chikungunya viral infections are often indistinguishable from those of many other viral, bacterial and parasitic infections confirmation of chikungunya outbreaks is important for clinicians for proper management of patients and for vector control programmers. Laboratory diagnosis of chikungunya in Sri Lanka is hindered by the non-availability of reliable commercial diagnostic kits and inaccessibility to reagents. There is a need to develop an assay that can confirm chikungunya, produced at low cost and easily standardized for the use in field settings. Currently available laboratory diagnostic kits depend on ELISA based on whole viral antigens which cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Therefore, a diagnostic intermediate using a single recombinant protein antigen to overcome problems associated with whole viral antigen/lysate is important. The objective of this study was to assist laboratory confirmation of outbreaks through developing competencies for a rapid laboratory diagnostic method using recombinant protein antigens for chikungunya infection. We have designed 2 novel recombinant protein antigens based on Envelope domain (E), a critical antigenic region of the major structural protein of chikungunya virus. They were expressed in Escherichia coli separately, and resultant proteins were affinity purified and obtained ~5mg and 10mg respectively and protein of >95% purity per liter of culture. These 2 proteins were evaluated as potential diagnostic intermediates in ELISA separately for the detection of anti-chikungunya Immunoglobulin M (IgM) antibody using a panel of well characterized serum samples. E1 and E2 showed 60% and 67% positivity respectively. Specificity proteins were tested using serum from healthy volunteers and infected with other viral diseases. Two proteins could detect only anti-chikungunya IgM antibodies. We demonstrated that these 2 novel recombinant protein antigens can function as diagnostic intermediates. Acknowledgements: Financial assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08 02) and International Atomic Energy Agency (IAEA SRI TC 5-042) are gratefully acknowledged
Development of recombinant protiens as diagnostic intermediates for chikungunya infection
(University of Kelaniya, 2013) Athapaththu, A.M.M.H.
INTRODUCTION: Chikungunya (CHIK) is an important disease with explosive outbreaks occurring in Sri Lanka. Confirmation of CHIK outbreaks is important for clinicians for proper clinical management of patients and epidemiological studies. There is a need to develop a laboratory diagnostic assay which can be discriminated CHIK from other diseases showing similar symptoms, produced at low cost and easily standardized for use in field settings. At present Enzyme-Linked Immunosorbent Assay (ELISA) is widely used for laboratory diagnosis of CHIK infection because of its rapidity, cost effectiveness and ability to standardize easily for field settings. These currently available ELISAs depend on whole viral lysate antigens which cause biohazard risk, high initial production cost and cross reactivity with other organisms of the same genus/family. A diagnostic intermediate using a single recombinant protein antigen to detect both Immuno globulin (Ig) M and G antibodies of CHIK is important to overcome problems associated with whole viral lysate antigen in ELISA. Overall objective of this study was to assist for clinical management of patients and confirmation of CHIK outbreaks through developing rapid laboratory diagnostic assays. Methodology: Surface proteins of Ribonucleic Acid (RNA) viruses are the targets of neutralizing antibodies. Envelope (E) region of the Chikungunya Virus (CHIKV) is the most immunodominant region of the virus. These protein antigens were prepared using the E region of the Virus. Synthetic genes of CHIK named Envelope 1 (El) and Envelope 2 (E2) were custom designed and chemically synthesized and resulted proteins were expressed in both bacteria (Escherichia coli} and yeast (Pichia pastoris) vector expression systems. Resulted recombinant proteins were purified using a single affinity chromatographic step using Ni- Nitrilotriacetic Acid (NTA) columns and evaluated to be used as a diagnostic intermediate using panels of well characterized serum samples. A total of 55 serum samples confirmed as positives and 186 confirmed as negatives for CHIK IgM antibodies were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 227 samples confirmed as negatives for CHIK IgG antibodies were used to evaluate the antigens using novel IgG ELISA. These samples were tested by Heamagglutination Inhibition (HAI) test, IgM Antibody capture (MAC) ELISA and indirect IgG ELISA. RESULTS: The recombinant proteins were purified using Ni-NTA columns under the denature conditions. For the detection of anti-CHIK IgM antibodies, the El recombinant protein expressed in E. coli showed 5% (3/55) sensitivity and 99% (184/186) specificity while the E2 recombinant protein expressed in E. coli showed 65 % (36/55) sensitivity and 70% (131/186) specificity when compared with MAC ELISA using total viral lysate as the antigen. The El recombinant protein expressed in P. pastoris showed 15% (8/55) sensitivity and 97% (181/186) specificity and the E2 recombinant protein expressed in P. pastoris showed 49% (27/55) sensitivity and 78% (146/186) specificity compared with MAC ELISA using total viral lysate as the antigen for detection of IgM antibodies. For the detection of anti-CHIK IgG antibodies, the El recombinant protein expressed in E. coll showed 60 % (47/78) sensitivity and 64% (94/148) specificity while the E2 recombinant protein expressed hi E. coli showed 83 % (65/78) sensitivity and 86% (195/227) specificity compared with the HAI test and indirect IgG ELISA using purified CHIKV. The El recombinant protein expressed in P. pastoris showed 86% (67/78) sensitivity and 61% (90/ 148) specificity and the E2 recombinant protein expressed in P. pastoris showed 76% (59/78) sensitivity and 81% (183/227) specificity compared with the HAI test and indirect IgG ELISA using purified CHIKV for the detection of IgG antibodies. Discussion: When considering El and E2 recombinant antigens, E2 recombinant proteins have showed better performance in identifying both anti-CHIK IgM and IgG antibodies in ELISAs, Here CHIK E2 protein is positioned in a way that it buries much of its partner El in the virus structure giving a CHIK El a less chance of exposure to the human immune system explaining the lower sensitivity and specificity towards detecting anti-CHIK antibodies. The E2 recombinant protein antigens expressed in bacterial expression system have performed better than the E2 recombinant antigens expressed in yeast system. In bacterial expression system the expressed recombinant proteins must have aggregated in cytoplasm in such a way, when denatured, most of the immuno dominant epitopes became linearized and exposed to the outside. CONCLUSION: Recombinant CHIK-E2 protein antigen expressed in E. coli showed higher specificity and sensitivity in detection of both IgM and IgG anti-CHIK antibodies. This type of study on development of diagnostic intermediates have a significant effect for rapid confirmation of outbreaks and take necessary steps to limit the spread of such outbreaks from one geographical area to another. Further, confirmation of disease outbreaks will allow the physicians to provide the necessary treatments rapidly and thereby avoid loss of working hours and socio-economical impact on individuals and government.
Enzyme-linked Immunosorbent Assay (ELISA) using recombinant protein antigens for detection of anti-chikungunya antibodies
(Faculty of Tropical Medicine, Mahidol University, 2010) Athapaththu, A.M.M.H.; Khanna, N.; Abeyewickreme, W.; Gunasena, S.; Hapugoda, M.D.
OBJECTIVES: Chikungunya is a mosquito borne viral infection that has caused great medical and public health problems in South East Asia during last few years. Currently available laboratory diagnostic kits depend on Enzyme-Linked Immunosorbent Assay (ELISA) based on whole viral antigens caused biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. These problems can be avoided by using recombinant protein antigens in ELISAs. METHODOLOGY: Two novel recombinant protein antigens based on Envelope (E) domain, a critical antigenic region of the major structural protein of chikungunya virus were expressed separately in a bacterial expression system (Escherichia coli). Two proteins were purified under denatured conditions. They were evaluated as potential diagnostic intermediates for detection of and-chikungunya antibodies in Immunoglobulin M (IgM) and Immunoglobulin G (IgG) ELISAs separately using a panel of serum samples confirmed by the gold standard assay, Heamagglutination Inhibition (HAI) assayRESULTS: These 2 protein antigens: El and E2 showed more than 60% positivity in IgG ELISAs and IgM ELISAs. A field validation using a large number of serum samples should be done for further confirmation of these results. It can be concluded that these 2 novel recombinant protein antigens can be used as a diagnostic intermediate to detect anti-chikungunya antibodies. ACKNOWLEDGEMENTS: Financial assistance from the International Centre for Genetic Engineering and Biotechnology (1CGEB CRP/ SRI08-02) is gratefully acknowledged
Optimization of the cell culture media to obtain the most effective nutrient concentrations in the medium for the growth and maintenance of the Myeloma cells
(University of Peradeniya, 2015) Munasinghe, M.M.E,; Athapaththu, A.M.M.H.; Gunathilaka, H.N.; Abeyewickreme, W.
Cell culture can be described as the removal of cells, tissues or organs from an animal or a plant and their subsequent placement into an artificial environment. Basically, proper temperature, substrate for cell attachment, appropriate growth medium and correct pH and osmolality in the medium should be properly maintained in order to achieve a better growth in the cells. Typically, a culture medium is composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. The objective of this study is to optimize culture media in order to obtain the most effective nutrient concentrations in the medium for the growth/maintenance of NSO Myeloma cell. Myeloma cells for the monoclonal antibody production were prepared using Dulbecco's Modified Eagle Medium (DMEM) as the growth media for the NSO cell culture. In this study, the culture media was optimized in order to obtain the most effective concentrations in the media. Primarily, in order to culture the cells soon after thawing, 10% growth media was used and then the grown cells were transferred in to a nutrition rich media- Hypoxanthine Thymidine (HT) medium. The growth of the Primary cell culture, soon after thawing, was observed within 2 days of culturing. A 60% of the bottom of the culture flask was covered with the healthy NSO myeloma cells. The transferred cells were also grown to a rate of 60% within 2 days of transferring. The 10% growth media comprises with 422 mL of DMEM with added 4500 mg/L glucose without L-glutamine and sodium pyruvate, 50 mL of Fetal Clone Serum, IZ5 mL of 1M HEPES buffer (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 5 m of 200 mM Glutamin, 5 mL of 100X Non-essential amino acids, 5 mL of 100 mM Sodium pyruvate and 0.5 mL of 55mM jj-mercaptoethanol. The HT medium comprises with 366.5 422 mL of DMEM with added 4500 mg/L glucose without L- glutamine and sodium pyruvate, 100 of mL FetalClone serum, 12.5 mL of 1M HEPES buffer, 5 mL 100X HT supplement, 5 mL of 200 mM Glutamin, 5 mL of 100X Non-essential amino acids, 5 mL of 100 mM Sodium pyruvate, 0.5 mL of 50 mg/mL Genatamicin, 0.5 mL of 55 mM p-mercaptoethanol and 25 uL of Interlukin-6. Since both the culture media showed optimum growth of the Myeloma cells, the above protocol with the provided concentrations of the nutrients could be used to maintain/ grow NSO myeloma cell line in the laboratory.
Shifting of circulating serotypes in dengue outbreaks during 2009/2010 in Sri lanka
(Faculty of Tropical Medicine, Mahidol University, 2010) Manamperi, N.H.; Athapaththu, A.M.M.H.; Premawansa, G.; Wellawaththage, C.; Jayarathna, T. D. S. S.; Abeyewickreme, W.; Hapugoda, M.D.
OBJECTIVES: Sri Lanka has experienced explosive outbreaks of dengue infection in 2009 and 2010. It has been identified that DEN- 3 and DEN- 2 were the predominant serotypes with DEN-1 and DEN- 4 circulating at a lower level in previous dengue outbreaks during 2003-2006, Objective of this study was to identify the circulating serotype/s during 2009 - 2010 outbreaks. METHODOLOGY: A prospective study was carried out at North Colombo Teaching Hospital, Sri Lanka during December 2009-August 2010. Clinically suspected dengue patients, with fever less than 5 days were recruited. An interviewer administered questionnaire was filled for each patient, by a Medical Officer. Venous blood samples confirmed for the presence of dengue virus by RT-PCR were typed by Semi-Nested PCR. RESULTS: Out of the 209 patients recruited in the study 80 (38%) were positive for dengue virus by RT-PCR. Of the positives, 43 (54%) were typed and circulation of all 4 serotypes was observed- Of the 43 positives, presence of DEN-1, DEN-2, DEN-3 and DEN-4 serotypes was 34 (79%), 3 (7%), 2 (5%) and 3 (7%) respectively DEN-1 was the predominant serotype in the recent epidemics which was circulating at a low level in previous epidemics. In DEN-1 infected patients, the mean platelet value was 58,588/ rnm3 and the mean PCV value was 41.4%. Associated symptoms such as headache, retro-orbital pain, neck pain and limb pain were present in 94% (32/34), 59% (20/34), 24% (8/34J and 91% (31/34) patients respectively. Bleeding manifestation developed in 47 % (16/34) patients. The mortality rate ranged from 0.7%- 1.0% during the recent outbreaks. Acknowledgement: Financial and technical assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08-02) and the International Atomic Energy Agency (IAEA SRI 5/042) is gratefully acknowledged.
Synthesis of Fe2O3 nanoparticles for the development of a rapid diagnostic test kit for dengue detection
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayarathna, I.P.L.; Gunathilaka, P.A.D.H.N.; Athapaththu, A.M.M.H.; Abeyewickreme, W.
BACKGROUND: Hybrid nanoparticles have great potential for biotechnological and biomedical applications. It was recently proposed that biopolymer/co-shell nano-materials could be easily obtained by adapting traditional routes used in pharmaceutical science to design drug delivery system. Most of the case, super-paramagnetic nanoparticles of iron oxides, magnetite (Fe3O4) and maghemite (ᵧ -Fe2O3) have been employed and these interesting magnetic properties are due to finite-size effects and high surface/volume ratio. METHOD: Iron oxide was synthesized by using modified co-precipitation method and resulting particles were characterized using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Diffused Reflectance Fourier Transform-Infrared Spectroscopy (DRIFT-IR). RESULTS: The XRD pattern matches well that of ᵧ -Fe2O3. Six characteristic peaks for ᵧ -Fe2O3 (2θ = 31.7°, 36.7°, 41.1°, 53.4°, 57.0° and 62.6°) marked by their Miller indices [(220), (311), (400), (422), (511) and (440)] were observed for sample. The TEM images reveal that the particles are in 5 – 20 nm range, and well fitted with spaniel cubic structure, but when particles are dry it prefer to agglomerate with neighboring particles to reduce their surface charges. The spectrum of ᵧ -Fe2O3 nanoparticles shows a characteristic broad band at 3410 cm-1 is due to the stretching vibration of H2O molecules. The band corresponding to the bending vibrations of H2O molecules is positioned at 1633 cm-1. Two intense IR bands at 627 and 451 cm-1 are typical for ferrihydrite or ᵧ -Fe2O3 nanoparticles. The spectrum of this sample showed the presence of carbonate groups on the basis of IR bands at 1508, 1340 and 1069 cm-1. The presence of carbonate groups is due to the adsorption of atmospheric carbon dioxide by ᵧ -Fe2O3 nanoparticles. The bands of Fe–O stretching vibrations of ᵧ -Fe2O3 appeared at 627 and 451 cm-1 and the bands at 892 and 796 cm-1 can be assigned to Fe-OH···H bending vibrations. CONCLUSION: Magnetic ᵧ -Fe2O3 nanoparticles were synthesized by the co-precipitation method and this work confirmed that magnetic ᵧ -Fe2O3 nanoparticles are in nano-scale and well matches with spaniel cubic structure