Browsing by Author "Siriphan Boonsilp"

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    Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand
    (2011-04-01) Piengchan Sonthayanon; Wirongrong Chierakul; Vanaporn Wuthiekanun; Janjira Thaipadungpanit; Thareerat Kalambaheti; Siriphan Boonsilp; Premjit Amornchai; Lee D. Smythe; Direk Limmathurotsakul; Nicholas P. Day; Sharon J. Peacock; Mahidol University; Organisation Mondiale de la Sante; Nuffield Department of Clinical Medicine; University of Cambridge
    There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity. Copyright © 2011 by The American Society of Tropical Medicine and Hygiene.
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    Comparison of two multilocus sequence based genotyping schemes for Leptospira species
    (2011-11-01) Ahmed Ahmed; Janjira Thaipadungpanit; Siriphan Boonsilp; Vanaporn Wuthiekanun; Kishore Nalam; Brian G. Spratt; David M. Aanensen; Lee D. Smythe; Niyaz Ahmed; Edward J. Feil; Rudy A. Hartskeerl; Sharon J. Peacock; Royal Tropical Institute - KIT; Mahidol University; Faculty of Medicine, Siriraj Hospital, Mahidol University; University of Hyderabad; Imperial College London; Queensland Health; University of Malaya; University of Bath; University of Cambridge
    Background: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. Methods and Findings: A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5) vs. 93.5 (95% CI 88.6-98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. Conclusions: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages. © 2011 Ahmed et al.
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    Comparison of two multilocus sequence based genotyping schemes for Leptospira species.
    (2011-11) Ahmed, Ahmed; Janjira Thaipadungpanit; จันทร์จิรา ไทยผดุงพานิช; Siriphan Boonsilp; ศิริพรรณ บุญศิลป์; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Nalam, Kishore; Spratt, Brian G.; Aanensen, David M.; Smythe, Lee D.; Ahmed, Niyaz; Feil, Edward J.; Hartskeer, Rudy A.; Peacock, Sharon J.; Ahmed, Ahmed; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.; Mahidol Univeristy. Faculty of Medicine Siriraj Hospital. Medical Proteomics Unit, Office for Research and Development.
    BACKGROUND: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. METHODS AND FINDINGS: A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5) vs. 93.5 (95% CI 88.6-98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. CONCLUSIONS: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.
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    Leptospirosis outbreak in Sri Lanka in 2008: Lessons for assessing the global burden of disease
    (2011-09-01) Suneth B. Agampodi; Sharon J. Peacock; Vasanthi Thevanesam; Danaseela B. Nugegoda; Lee Smythe; Janjira Thaipadungpanit; Scott B. Craig; Mary Ann Burns; Michael Dohnt; Siriphan Boonsilp; Thamarasi Senaratne; Athula Kumara; Paba Palihawadana; Sahan Perera; Joseph M. Vinetz; University of Colombo; University of California, San Diego, School of Medicine; Mahidol University; University of Cambridge; University of Peradeniya; Organisation Mondiale de la Sante; University of the Sunshine Coast; Ministry of Health Colombo
    Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions. Copyright © 2011 by The American Society of Tropical Medicine and Hygiene.
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    Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis
    (2011-12-13) Siriphan Boonsilp; Janjira Thaipadungpanit; Premjit Amornchai; Vanaporn Wuthiekanun; Wirongrong Chierakul; Direk Limmathurotsakul; Nicholas P. Day; Sharon J. Peacock; Mahidol University; Faculty of Medicine, Siriraj Hospital, Mahidol University; Nuffield Department of Clinical Medicine; University of Cambridge
    Background: Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.Methods: We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed.Results: 39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species).Conclusion: Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this. © 2011 Boonsilp et al; licensee BioMed Central Ltd.
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    PublicationOpen Access
    Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis
    (2011-12-13) Siriphan Boonsilp; Janjira Thaipadungpanit; จันทร์จิรา ไทยผดุงพานิช; Premjit Amornchai; เปรมจิตต์ อมรชัย; Vanaporn Wuthiekanun; Wirongrong Chierakul; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Day, Nicholas P.; Peacock, Sharon J.; Janjira Thaipadungpanit; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit
    BACKGROUND: Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium. METHODS: We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed. RESULTS: 39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species). CONCLUSION: Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.
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    Short report: Leptospira species in floodwater during the 2011 floods in the bangkok metropolitan region, Thailand
    (2013-10-01) Janjira Thaipadungpanit; Vanaporn Wuthiekanun; Narisara Chantratita; Surapon Yimsamran; Premjit Amornchai; Siriphan Boonsilp; Wanchai Maneeboonyang; Prapin Tharnpoophasiam; Natnaree Saiprom; Yuvadee Mahakunkijcharoen; Nicholas P.J. Day; Pratap Singhasivanon; Sharon J. Peacock; Direk Limmathurotsakul; Mahidol University; Nuffield Department of Clinical Medicine; University of Cambridge
    Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.
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    A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species
    (2013) Siriphan Boonsilp; ศิริพรรณ บุญศิลป์; Janjira Thaipadungpanit; จันทรจิรา ไทยผดุงพานิช; Premjit Amornchai; เปรมจิตร อมรชัย; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Narisara Chantratita; นริศรา จันทราทิตย์; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.; Janjira Thaipadungpanit; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology
    BACKGROUND: The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. METHODOLOGY AND FINDINGS: We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. CONCLUSION: The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.
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    A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species
    (2013-01-01) Siriphan Boonsilp; Janjira Thaipadungpanit; Premjit Amornchai; Vanaporn Wuthiekanun; Mark S. Bailey; Matthew T G Holden; Cuicai Zhang; Xiugao Jiang; Nobuo Koizumi; Kyle Taylor; Renee Galloway; Alex R. Hoffmaster; Scott Craig; Lee D. Smythe; Rudy A. Hartskeerl; Nicholas P. Day; Narisara Chantratita; Edward J. Feil; David M. Aanensen; Brian G. Spratt; Sharon J. Peacock; Mahidol University; Heartlands Hospital; Wellcome Trust Sanger Institute; Chinese Center for Disease Control and Prevention; National Institute of Infectious Diseases; Hokkaido University; National Center for Emerging and Zoonotic Infectious Diseases; Queensland Health; Royal Tropical Institute - KIT; Nuffield Department of Clinical Medicine; University of Bath; Imperial College London; University of Cambridge
    Background: The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings: We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion: The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.
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    Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway
    (2010-12-01) Araya Dharmkrong at Chusattayanond; Siriphan Boonsilp; Jitra Kasisit; Atsadang Boonmee; Saradee Warit; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology
    A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba. © 2010 Elsevier Ireland Ltd.