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314 DETECTION OF LYMPHATIC WUCHERERIA BANCROFTI IN CARRIERS AND LONG-TERM STORAGE BLOOD SAMPLES USING SEMI- NESTED PCR (NO. 828) P. Kanjanavas 1 , P. Tan-ariya 2 , P. Khawsak 1 , A. Pakpitcharoen 1 , S. Phantana 3 , K. Chansiri 1 1 Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand; 2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3 Filariasis Division, Ministry of Public Health, Nontaburi, Thailand. Key words: Wuchereria bancrofti; Semi-nested PCR The semi-nested PCR was conducted for detection of Wuchereria bancrofti in patients’ blood. The results demonstrated that the semi-nested PCR could detect as little as 0.001 fg of parasite DNA. In addition, the primers showed no PCR amplification from human and other hemoparasites such as Brugia malayi, Plasmodium falciparum and P. vivax DNAs. This technique was used for detection of 18 W. bancrofti infected blood samples with a long-term storage, the data revealed that all samples were positive, the results obtained from this study clearly indicated that the semi-nested PCR is specific. (Molecular and Cellular Probes 19, (2005); 169-172) GENOTYPIC STUDY OF PHEUMOCYSTIS JIROVECII IN HUMAN IMMUNODEFICIENCY VIRUS-POSITIVE PATIENTS IN THAILAND (NO. 829) Suradej Siripattanapipong 1 , Jeerapun Worapong 2 , Mathirut Mungthin 3 , Saovanee Leelayoova 3 and Peerapan Tan-ariya 1 1 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2 Department of Biotechnology, Institute of Science and Technology of Research and Development, Mahidol University, Salaya, Nakornpratom 73170, Thailand; 3 Department of Parasitology, Phramongkutklao College of Medicine, Ratchawithi Rd., Bangkok 10400, Thailand. Key words: Pneumocystis jirovecii, genotypic, (HIV)-infected patients Pneumocystis jirovecii is one of the common opportunistic infections in human immunodeficiency virus (HIV)-infected patients in Thailand. Information regarding genotypic and epidemiological of this organism in Thai patients is not available. We analyzed the genotypes of 28 Pneumocystis jirovecii-positive specimens from bronchoalveolar lavage and sputum samples from HIV-infected Thai patients based on nucleotide variations of the internal transcribed spacer regions 1 and 2 of the rRNA gene. Thirteen genotypes were the same as previously reported outside Thailand. Ten genotypes, which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb and Qq, were new. Ir and Rp were unique and dominant types observed in HIV-infected Thai patients. Thirteen specimens (46.4%) were infected with a single type of Pneumocystis jirovecii, and fifteen (53.6%) mixed infections. These differences may be used as genotypic markers for studying the epidemiology and transmission of Pneumocystis jirovecii in the Thai population. (Clinical Microbiology, Vol.43, No.5 (2005); 2104-2110 Supported by the Thailand-Tropical Diseases Research Program (T-2) Faculty of Science 3-NITROPROPIONIC ACID (3-NPA), A POTENT ANTIMYCOBACTEDRIAL AGENT FRO ENDO- PHYTIC FUNGI : IS 3-NPA IN SOME PLANTS PRODUCED BY ENDOPHYTES? (NO. 830) Porntep Chomcheon 1 , Suthep Wiyakrutta 2 , Nongluksna Sriubolmas 3 , Nattaya Ngamrojanavanich 1 , Duangnate Isarangkul 4 , and Prasat Kittakoop 5 1 Program of Biotechnology and Department of Chemistry, Faculty of Science, Chulalongkorn University, Thailand; 2 Department of Microbiology, Faculty of Science, Mahidol University, Thailand; 3 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand; 4 Center for Biotechnology, Institute of Science and Technology for Research and Development, Mahidol University, Thailand; and 5 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand. Key words: 3-nitropropionic acid, antimycobacterial agent, endophytic fungi 3-Nitropropionic acid (3-NPA) was found in extracts of several strains of endophytic fungi. 3-NPA exhibited potent antimycobacterial activity with the minimum inhibition concentration of 3.3 uM, but was inactive against NCI-H187, BC, KB, and Vero cell lines. Endophytes were found to produce high levels of 3-NPA, and therefore 3-NPA accumulated in certain plants may be produced by the associated endophytes. 3-NPA may be used as a chemotaxonomic marker for endophytic fungi. The structure of 3-hydroxypropionic acid, a nematicidal agent, should be revised to 3-NPA. (Published in Journal of Natural Products 68, (2005); 1103-1105) MIXED LEPTOSPIRE INFECTION OF SMALL MAMMALS IN THAILAND (NO. 831) Doungchawee G. 1* , Phulsuksombat D. 2 , Naigowit P. 3 , Khoaprasert Y. 4 , Sangjun N. 2 , Chalermsanyakorn P. 5 , Smythe L. 6 1 Department of Pathobiology, Science, Mahidol University, Bangkok, Thailand. 2 Thai Component, Armed Force Research Institute of Medical Sciences, Bangkok, Thailand. 3 Research Center for Leptospira Laboratory, National Institute of Health, Nonthaburi, Thailand. 4 Agricultural Zoology Research Group,Department of Agriculture,Bangkok, Thailand. 5 Department of Pathology, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. 6 WHO/FAO Collaborating Centre for Reference and Research on Leptospirosis, WHO Western Pacific Region and Queensland Scientific Services, Australia. Key words : Direct Immunofluorescence, pathogenic Leptospira, Rodent leptospirosis Background: In an attempt to identify the source of leptospires pathogenic to humans, small wild mammals were trapped during leptospirosis outbreaks in a rural area of the Country in 1999- 2000 and subsequent study of an urban area in 2002. Methods: All rodents from the rural source were examined using kidney isolation and those found positive for leptospires were approximately 15% (190 of 1310). A developed direct PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 315 immunofluorescent assay (DFA) was considered as an alternative method to leptospire culturing. The DFA evaluated in this study was effective for the detection of acute leptospirosis and exhibited high sensitivity (100%) and specificity (94%). Results: Among the ten different species of wild animals captured, R. norvegicus had the highest leptospire infection rate as revealed by both detection methods. Moreover, more than a half the proportion of cases positive by DFA had evidence of mixed infection with three or more different serovars of pathogenic Leptospira spp. in the kidneys.From 190 isolates of rural rodents,137 were serotyped by cross agglutinin absorption and microscopic agglutination tests,showing some similarities and variations to the serovar level when compared with the DFA. The detection of leptospires in the kidneys of urban rats and shrews was 33 % (14 of 42), twice that of rural rodents. Overall, the most commonly infecting serovars were autumnalis and canicola in both the rural and urban areas. Conclusion: This finding suggests that wild rodents and shrews play an important source of multiple pathogenic leptospires in both areas and need active surveillance measure to evaluate the epidemiology of the disease. (Oral Presentation : At the the fourth scientific meeting of the International Leptospirosis Society (ILS).The Central Duangtawan Hotel, Chiang Mai, Thailand. November 14-16, 2005.) INVESTIGATION ON EXTRACTED OUTER MEMBRANE SHEATH OF 5 LEPTOSPIRAL SEROVARS BY ELECTRON MICROSCOPY AND IMMUNOBLOT ASSAY (NO. 832) Prukswan Chetanachan, 1 Galayanee Doungchawee, 2 Umaporn Seena, 3 Mayurachat Biaklang, 3 Suchada Geawduanglek 2 and Pimjai Naigowit 4 1 National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 11000, Thailand. 2 Department of Pathobiology, Science, Mahidol University, Bangkok, 10400, Thailand.; 3 Medical Biotechnological Center, Department of Medical Sciences, Ministry, Ministry of Public Health, Nonthaburi, 11000, Thailand. 4 Technical Office, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 11000, Thailand. The purpose of this study was to identify the structure and specificity of Outer Membrane Sheath (OMS) extracts of 5 different serovars of Leptospira spp.: autumnalis, bratislava, sejroe, icterohaemorrhagiae and patoc. The leptospiral outer membrane fraction (OMS) was prepared by 10% NaCl and 0.01% SDS extraction. Then, this OMS was observed under transmission electron microscope (TEM). The SDS separated antigens were transferred and immunostained with reference rabbit antisera raised against each serovars of interest. Under TEM, the swollen leptospiral cells were observed after 10% NaCl absorption and thin fragments were found detached from an axial filament after 0.01% SDS extraction. Whereas, immunoblot results showed these fragments were reactive with each serovar specific antisera and representative of outer membrane components. (Oral Presentation : At the 2005 Annual meeting of the Medical Biotechnology Center, by Department of Medical Science, Ministry of Public Health,at Rose Garden Hotel ,Nakorn Pathom Province. August 16-17, 2005.) COMPARISON IMMUNOBLOTTING ANALYSIS OF LEPTOSPIRA FOR SEROTYPING COMPA- TIBLE TO MICROSCOPIC AGGLUTINATION METHOD (NO. 833) Doungchawee Galayanee, Naigowit Pimjai and Kongtim Suraphol Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Immunoblotting analysis of reference Leptospira using rabbit antiserum generated against leptospiral serovars, representatives of locally important serogroups,the results showed that all leptospires demonstrable with distinct multiband patterns varied with serovars. A major reactivity on immunoblot equivalent to MAT typing of leptospires is a diffuse broad banding of molecular masses between 20-30 kDa. In addition,a number of genus associated or Leptospira common bands exhibited on the immunoblot when reacted with any serovar specific polyclonal antiserum included multiple discrete bands (range between 14-20 kDa) and singlet or duplet band of 36-37 kDa. Whereas bands of 23 ,32 and 41 kDa were only detected in those pathogenic Leptospira investigated. Overall,both pathogenic Leptospira interrogans and non-pathogenic L. biflexa have characteristic immunoblotting patterns useful for differentiation and typing of Leptospira serovar and/or serogroup as compared with the MAT results. The advantages of immunoblotting using polyclonal antibodies are more flexible than MAT method at low risk to prepare non-viable leptospires as antigens and the resulting blots can be kept and tested at any convenience,which is considerable simplicity and accuracy of use . (Oral Presentation : At the 2005 Annual meeting of the Medical Biotechnology Center, by Department of Medical Science, Ministry of Public Health,at Rose Garden Hotel ,Nakorn Pathom Province. August 16-17, 2005.) IMMUNODISTINCTION OF REFERENCE LEPTOSPIRES ON IMMUNOBLOTS (NO. 834) Doungchawee G. 1 *, Naigowit P. 2 , Ekpo P. 3 , Sirawaraporn,W 4 ., Kongtim S. 1 1 Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400. 2 Research Center for Leptospira Laboratory, National Institute of Health, Nonthaburi Province, Thailand. 3 Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University. 4 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words: Immunoblotting, Smearlike reactivity, Serovar differentiation Background : The determination of serovar and serogroup status of various reference leptospires is conventionally based on the use of microscopic agglutination test (MAT). Accordingly,this test is required a number of reference antisera to react with the live leptospires, which known to be time-consuming, difficult for maintaining of organisms until optimal for the test. Considering the tediousness and complexity of the MAT,this would be simplified by the use of immunoblotting as the alterative. PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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