Molecular and serological characterization of Leptospira kirschneri serogroup Pomona isolated from a human case in a Brazilian rural area

Submitted by Sandra Infurna ([email protected]) on 2017-11-30T11:27:51Z No. of bitstreams: 1 juliana_vitalbrasil_etal_IOC_2017.pdf: 1011010 bytes, checksum: 4ea03b48249a25f91b3c7a098c03779d (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-11-30T11:35:59Z (GMT) No. of bitstreams: 1 juliana_vitalbrasil_etal_IOC_2017.pdf: 1011010 bytes, checksum: 4ea03b48249a25f91b3c7a098c03779d (MD5)Made available in DSpace on 2017-11-30T11:35:59Z (GMT). No. of bitstreams: 1 juliana_vitalbrasil_etal_IOC_2017.pdf: 1011010 bytes, checksum: 4ea03b48249a25f91b3c7a098c03779d (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Centro de Referência Nacional para Leptospirose. World Health Organization/Pan American Health Organization. Centro Colaborador para Leptospirose - Coleção de Leptospira. Rio de Janeiro, RJ. Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Centro de Referência Nacional para Leptospirose. World Health Organization/Pan American Health Organization. Centro Colaborador para Leptospirose - Coleção de Leptospira. Rio de Janeiro, RJ. Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Centro de Referência Nacional para Leptospirose. World Health Organization/Pan American Health Organization. Centro Colaborador para Leptospirose - Coleção de Leptospira. Rio de Janeiro, RJ. Brasil..Fundação Estadual de Produção e Pesquisa em Saúde. Instituto de Pesquisas Biológicas. Laboratório Central. Porto Alegre, RS, Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Centro de Referência Nacional para Leptospirose. World Health Organization/Pan American Health Organization. Centro Colaborador para Leptospirose - Coleção de Leptospira. Rio de Janeiro, RJ. Brasil..Leptospirosis is an important health concern in Brazil. Currently, information on the epidemiology of the disease in the rural areas of the country is lacking

Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing

Submitted by sandra infurna ([email protected]) on 2016-05-10T17:33:26Z No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-05-10T17:49:31Z (GMT) No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5)Made available in DSpace on 2016-05-10T17:49:31Z (GMT). No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5) Previous issue date: 2015Stanford University School of Medicine.Department of Medicine. Division of Infectious Diseases and Geographic Medicine. Stanford, California, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Stanford University School of Medicine.Department of Medicine. Division of Infectious Diseases and Geographic Medicine. Stanford, California, USA / Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Background Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. Methods/Principal Findings 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). Conclusions/Significance This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection

Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies

Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique

Submitted by Sandra Infurna ([email protected]) on 2018-03-13T16:47:33Z No. of bitstreams: 1 rachel_riberio_etal_IOC_2010.pdf: 677571 bytes, checksum: 6a76dba0531d5351c61bda6b4254c354 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-03-13T16:55:10Z (GMT) No. of bitstreams: 1 rachel_riberio_etal_IOC_2010.pdf: 677571 bytes, checksum: 6a76dba0531d5351c61bda6b4254c354 (MD5)Made available in DSpace on 2018-03-13T16:55:10Z (GMT). No. of bitstreams: 1 rachel_riberio_etal_IOC_2010.pdf: 677571 bytes, checksum: 6a76dba0531d5351c61bda6b4254c354 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional para Leptospirose. Centro Colaborador da Organização Mundial da Saúde. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional para Leptospirose. Centro Colaborador da Organização Mundial da Saúde. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional para Leptospirose. Centro Colaborador da Organização Mundial da Saúde. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional para Leptospirose. Centro Colaborador da Organização Mundial da Saúde. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional para Leptospirose. Centro Colaborador da Organização Mundial da Saúde. Rio de Janeiro, RJ. Brasil.A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. Métodos: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. Resultados: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. Conclusões: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. Methods: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. Results: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. Conclusions: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases

<i>Leptospira</i> C_T values in the Lepto-MD assay for patients with recorded day of disease information.

<p><i>Leptospira</i> C_T values in the Lepto-MD assay for patients with recorded day of disease information.</p

Leptospirosis cases and recorded rainfall in Rio de Janeiro by study month.

<p>(A) Leptospirosis cases diagnosed by RT-PCR (blue), MAT (maroon), or both (purple) are displayed by study month. MAT results are shown for all samples from 2008. (B) Rainfall for 2008 (black circles and line) and the average rainfall for the years 2003–2013 (red triangles and line).</p

Patient characteristics associated with 478 samples that tested positive and negative for <i>Leptospira</i>.

<p>Abbreviations: SD, standard deviation</p><p>¹ 65 samples tested positive for <i>Leptospira</i> in the Lepto-MD assay and/or by MAT; 3 samples tested positive by both methods.</p><p>Patient characteristics associated with 478 samples that tested positive and negative for <i>Leptospira</i>.</p

Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

Made available in DSpace on 2015-06-17T12:05:21Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) ilana_balassianoetal_IOC_2014.pdf: 255699 bytes, checksum: 3d36819ae7364a9491227409490d48e9 (MD5) Previous issue date: 2014Stanford University School of Medicine. Department of Medicine. Division of Infectious Diseases and Geographic Medicine. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. WHO/PAHO Centro Colaborador para Leptospirose. Centro de Referência Nacional para Leptospirose. Coleção de Leptospira. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. WHO/PAHO Centro Colaborador para Leptospirose. Centro de Referência Nacional para Leptospirose. Coleção de Leptospira. Rio de Janeiro, RJ, Brasil.Sustainable Sciences Institute. Managua, Nicaragua.Ministry of Health. Centro Nacional de Diagnóstico y Referencia. National Virology Laboratory. Mnagua, Nicaragua.University of California. School of Public Health. Division of Infectious Diseases and Vaccinology. Berkeley, CA, USA.Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens