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A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp
Lee D Smythe, Ina L Smith, Greg A Smith, Michael F Dohnt, Meegan L Symonds, Leonie J Barnett, David B McKay
BMC Infectious Diseases , 2002, DOI: 10.1186/1471-2334-2-13
Abstract: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples.The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.Leptospirosis is an emerging infectious disease [1]. Leptospira spp. are endemic to feral and domestic animals that may serve as reservoirs, with rats and rodents recognised as the most important sources [2,3]. Human infections result from contact with contaminated soil, vegetation or water, or with the body fluids of infected animals. The genus Leptospira comprises both pathogenic and non-pathogenic species: L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. meyeri, L. fainei, L. alexanderi, L. santarosai, L. kirschneri, L. inadai; L. biflexa, and L. wolbachii[1]. There is some controversy about the classification or pathogenicity of some members within species such as L. meyeri and L. inadai[2,4,5].The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis [6]. For example, patients presenting with severe fever and haemorrhage have symptoms which are difficult to distinguish from viral haemorrhagic fever [7].There are over 230 known serovars in the genus Leptospira[2]. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which is able to det
Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates  [PDF]
Gustavo M. Cerqueira,Alan J. A. McBride,Rudy A. Hartskeerl,Niyaz Ahmed,Odir A. Dellagostin,Marcus R. Eslab?o,Ana L. T. O. Nascimento
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015335
Abstract: Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.
Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira  [PDF]
Christian A Ganoza equal contributor,Michael A Matthias equal contributor,Devon Collins-Richards,Kimberly C Brouwer,Calaveras B Cunningham,Eddy R Segura,Robert H Gilman,Eduardo Gotuzzo,Joseph M Vinetz
PLOS Medicine , 2006, DOI: 10.1371/journal.pmed.0030308
Abstract: Background Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters. Methods and Findings A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (~103 leptospires/ml versus 0.5 × 102 leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources. Conclusions Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.
Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity  [PDF]
Gustavo M. Cerqueira,Natalie M. Souza,Eduardo R. Araújo,Aline T. Barros,Zenaide M. Morais,Sílvio A. Vasconcellos,Ana L. T. O. Nascimento
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017409
Abstract: Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field.
The OmpL37 Surface-Exposed Protein Is Expressed by Pathogenic Leptospira during Infection and Binds Skin and Vascular Elastin  [PDF]
Marija Pinne ,Henry A. Choy,David A. Haake
PLOS Neglected Tropical Diseases , 2010, DOI: 10.1371/journal.pntd.0000815
Abstract: Pathogenic Leptospira spp. shed in the urine of reservoir hosts into freshwater can be transmitted to a susceptible host through skin abrasions or mucous membranes causing leptospirosis. The infection process involves the ability of leptospires to adhere to cell surface and extracellular matrix components, a crucial step for dissemination and colonization of host tissues. Therefore, the elucidation of novel mediators of host-pathogen interaction is important in the discovery of virulence factors involved in the pathogenesis of leptospirosis. In this study, we assess the functional roles of transmembrane outer membrane proteins OmpL36 (LIC13166), OmpL37 (LIC12263), and OmpL47 (LIC13050), which we recently identified on the leptospiral surface. We determine the capacity of these proteins to bind to host tissue components by enzyme-linked immunosorbent assay. OmpL37 binds elastin preferentially, exhibiting dose-dependent, saturating binding to human skin (Kd, 104±19 nM) and aortic elastin (Kd, 152±27 nM). It also binds fibrinogen (Kd, 244±15 nM), fibrinogen fragment D (Kd, 132±30 nM), plasma fibronectin (Kd, 359±68 nM), and murine laminin (Kd, 410±81 nM). The binding to human skin elastin by both recombinant OmpL37 and live Leptospira interrogans is specifically enhanced by rabbit antiserum for OmpL37, suggesting the involvement of OmpL37 in leptospiral binding to elastin and also the possibility that host-generated antibodies may promote rather than inhibit the adherence of leptospires to elastin-rich tissues. Further, we demonstrate that OmpL37 is recognized by acute and convalescent leptospirosis patient sera and also by Leptospira-infected hamster sera. Finally, OmpL37 protein is detected in pathogenic Leptospira serovars and not in saprophytic Leptospira. Thus, OmpL37 is a novel elastin-binding protein of pathogenic Leptospira that may be promoting attachment of Leptospira to host tissues.
Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction
Romero, Eliete Caló;Blanco, Roberta Morozetti;Yasuda, Paulo Hideki;
Memórias do Instituto Oswaldo Cruz , 2010, DOI: 10.1590/S0074-02762010000800007
Abstract: leptospirosis is a zoonotic disease caused by the pathogenic leptospira spp. the clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. the neurological leptospirosis forms are usually neglected. the aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (pcr). thirty-nine cerebrospinal fluid (csf) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and pcr. leptospira spp dna was detected in 23 (58.97%) of the csf samples. anti-leptospiral antibodies were found in 13 (33.33%) csf samples. twelve csf samples were positive by pcr assay and negative by microscopic agglutination test (mat) assay. two csf samples were positive by mat and negative by pcr. the positive and negative agreement between both tests was 11 and 14, respectively. csf samples from six cases of unknown diagnosis were positive by pcr assay. eight cases showed positive results using pcr and mat. leptospirosis could be detected by pcr assay from the 3rd-26th day after illness onset. the sensitivity of the pcr was assessed with confirmed cases of leptospirosis (by mat) and found to be 89.5%. all csfs were negative by culture. pcr was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. we recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.
Serological prevalence of leptospirosis in cattle slaughtered in the Zango abattoir in Zaria, Kaduna State, Nigeria
Emmanuel O. Ngbede,Mashood A. Raji,Clara N. Kwanashie,Emmanuel C. Okolocha
Veterinaria Italiana , 2012,
Abstract: Leptospirosis is an occupational zoonosis caused by pathogenic leptospires. In this study, the presence and prevalence of antibodies specific to Leptospira spp. serovar Hardjo in 142 cattle slaughtered between June and July 2011 was investigated using the enzyme-linked immunosorbent assay (ELISA). Five (3.50%) of the 142 cattle sampled were seropositive for antibodies to Leptospira spp. serovar Hardjo. Despite the fact that there was no significant difference (p>0.05) in seropositivity between sexes and between breeds sampled, there was a significant difference (p<0.05) in sero-positivity between the different age groups examined. Leptospirosis is present in cattle slaughtered in the Zango abattoir; butchers and abattoir workers are exposed to infected animals and are at risk of being infected by Leptospira spp. serovar Hardjo.
Leptospira and Inflammation  [PDF]
C. F. Gon?alves-de-Albuquerque,P. Burth,A. R. Silva,M. Younes-Ibrahim,H. C. Castro-Faria-Neto,M. V. Castro-Faria
Mediators of Inflammation , 2012, DOI: 10.1155/2012/317950
Abstract: Leptospirosis is an important zoonosis and has a worldwide impact on public health. This paper will discuss both the role of immunogenic and pathogenic molecules during leptospirosis infection and possible new targets for immunotherapy against leptospira components. Leptospira, possess a wide variety of mechanisms that allow them to evade the host immune system and cause infection. Many molecules contribute to the ability of Leptospira to adhere, invade, and colonize. The recent sequencing of the Leptospira genome has increased our knowledge about this pathogen. Although the virulence factors, molecular targets, mechanisms of inflammation, and signaling pathways triggered by leptospiral antigens have been studied, some questions are still unanswered. Toll-like receptors (TLRs) are the primary sensors of invading pathogens. TLRs recognize conserved microbial pattern molecules and activate signaling pathways that are pivotal to innate and adaptive immune responses. Recently, a new molecular target has emerged—the Na/K-ATPase—which may contribute to inflammatory and metabolic alteration in this syndrome. Na/K-ATPase is a target for specific fatty acids of host origin and for bacterial components such as the glycolipoprotein fraction (GLP) that may lead to inflammasome activation. We propose that in addition to TLRs, Na/K-ATPase may play a role in the innate response to leptospirosis infection. 1. Introduction Leptospirosis is a zoonosis of global importance caused by several species and more than 200 different serovars of pathogenic Leptospira spp. The disease affects both animals and humans and has veterinary, economic, and medical relevance [1, 2]. Leptospirosis is still a major public health problem in tropical countries, with epidemic outbreaks occurring in the rainy season and after floods [3–5]. The annual incidence of this disease is estimated at 10–100 per 100,000 in tropical regions and 0.1–1.0 per 100,000 in temperate areas [6]. In recent years, leptospirosis outbreaks have occurred all over the world; thus, an adequate disease notification system would be useful to create surveillance networks [7]. Leptospirosis is transmitted to humans primarily by water contaminated with the urine of either wild or domestic mammals that have been chronically colonized by Leptospira spp [8]. It has recently been reported that Leptospira can persist in certain organs, indicating that people themselves can act as hosts [9]. In developed countries, the transmission mechanism is mainly associated with occupational and recreational activities [10–14]. The infection
Development of a novel ELISA for serodiagnosis of Leptospirosis and additional detection of pathogenic Leptospira by polymerase chain reaction for veterinary routine diagnostics
Theodoridis,Dimitrios; B?hmer,Jan; Homuth,Matthias; Strutzberg-Minde,Katrin;
Revista Cubana de Medicina Tropical , 2005,
Abstract: a multi-serovar elisa based on the outer membrane lipoprotein l41 (lipl41) of pathogenic leptospira was developed to increase sensitivity by using a single test antigen. a sensitivity of 99 % and a specifity of 92 % could be achieved. the established diagnostic polymerase chain reaction is also able to detect fast and reliably pathogenic leptospira in different clinical samples.
Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials  [PDF]
Ahmed Ahmed, Mirjam F. M. Engelberts, Kimberly R. Boer, Niyaz Ahmed, Rudy A. Hartskeerl
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007093
Abstract: Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
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