Characterization of Leptospira isolates from humans and the environment in Uruguay.

Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

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

INTRODUCTION:: 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. METHODS:: Serological and molecular techniques were used to characterize a clinical isolate of Leptospira. RESULTS:: The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok. CONCLUSIONS:: This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.

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

Abstract INTRODUCTION: 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. METHODS: Serological and molecular techniques were used to characterize a clinical isolate of Leptospira. RESULTS: The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok. CONCLUSIONS: This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.

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

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.

Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

BACKGROUND: Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. METHODOLOGY/PRINCIPAL FINDINGS: For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. CONCLUSIONS/SIGNIFICANCE: The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis.

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.

Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic surveillance.

The aim of this study was to develop an immunocapture polymerase chain reaction (IC-PCR) protocol for leptospirosis. For the standardization of IC-PCR, polyclonal (AS) and monoclonal (MAb) antibodies against different serogroups and serovars of Leptospira were coupled to polystyrene plates. Human sera were artificially contaminated with leptospires and incubated on plates. The bacterial DNA was obtained and used in a multiplex PCR. Sensitivity was tested using sera contaminated with crescent concentrations of leptospires, while specificity was established using sera contaminated with different bacterial genera and sera obtained from patients positive for viral infections. IC-PCR using AS was able to recognize specific serogroups, although some cross-reactions have been observed. No cross-reactions were observed when MAbs were used; however, the sensitivity in this case was lower than that of IC-PCR using AS. IC-PCR proved to be specific to Leptospira and is a promising tool for early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup.

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]. / Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis.

INTRODUCTION: 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.

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.

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis / Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique

INTRODUÇÃO: 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.

INTRODUCTION: 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.