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Background: Early diagnosis of leptospirosis may contribute to the effectiveness of antimicrobial therapy and early outbreak recognition. Nucleic acid and antigen detection tests have the potential for early diagnosis of leptospirosis. With this systematic review, we assessed the sensitivity and specificity of nucleic acid and antigen detection tests., Objectives: To determine the diagnostic test accuracy of nucleic acid and antigen detection tests for the diagnosis of human symptomatic leptospirosis., Search Methods: We searched electronic databases including MEDLINE, Embase, the Cochrane Library, and regional databases from inception to 6 July 2018. We did not apply restrictions to language or time of publication., Selection Criteria: We included diagnostic cross-sectional studies and case-control studies of tests that made use of nucleic acid and antigen detection methods in people suspected of systemic leptospirosis. As reference standards, we considered the microscopic agglutination test alone (which detects antibodies against leptospirosis) or in a composite reference standard with culturing or other serological tests. Studies were excluded when the controls were healthy individuals or when there were insufficient data to calculate sensitivity and specificity., Data Collection and Analysis: At least two review authors independently extracted data from each study. We used the revised Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2) to assess risk of bias. We calculated study-specific values for sensitivity and specificity with 95% confidence intervals (CI) and pooled the results in a meta-analysis when appropriate. We used the bivariate model for index tests with one positivity threshold, and we used the hierarchical summary receiver operating characteristic model for index tests with multiple positivity thresholds. As possible sources of heterogeneity, we explored: timing of index test, disease prevalence, blood sample type, primers or target genes, and the real-time polymerase chain reaction (PCR) visualisation method. These were added as covariates to the meta-regression models., Main Results: We included 41 studies evaluating nine index tests (conventional PCR (in short: PCR), real-time PCR, nested PCR, PCR performed twice, loop-mediated isothermal amplification, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, immunochromatography-based lateral flow assay, and dipstick assay) with 5981 participants (1834 with and 4147 without leptospirosis). Methodological quality criteria were often not reported, and the risk of bias of the reference standard was generally considered high. The applicability of findings was limited by the frequent use of frozen samples. We conducted meta-analyses for the PCR and the real-time PCR on blood products.The pooled sensitivity of the PCR was 70% (95% CI 37% to 90%) and the pooled specificity was 95% (95% CI 75% to 99%). When studies with a high risk of bias in the reference standard domain were excluded, the pooled sensitivity was 87% (95% CI 44% to 98%) and the pooled specificity was 97% (95% CI 60% to 100%). For the real-time PCR, we estimated a summary receiver operating characteristic curve. To illustrate, a point on the curve with 85% specificity had a sensitivity of 49% (95% CI 30% to 68%). Likewise, at 90% specificity, sensitivity was 40% (95% CI 24% to 59%) and at 95% specificity, sensitivity was 29% (95% CI 15% to 49%). The median specificity of real-time PCR on blood products was 92%. We did not formally compare the diagnostic test accuracy of PCR and real-time PCR, as direct comparison studies were lacking. Three of 15 studies analysing PCR on blood products reported the timing of sample collection in the studies included in the meta-analyses (range 1 to 7 days postonset of symptoms), and nine out of 16 studies analysing real-time PCR on blood products (range 1 to 19 days postonset of symptoms). In PCR studies, specificity was lower in settings with high leptospirosis prevalence. Other investigations of heterogeneity did not identify statistically significant associations. Two studies suggested that PCR and real-time PCR may be more sensitive on blood samples collected early in the disease stage. Results of other index tests were described narratively., Authors' Conclusions: The validity of review findings are limited and should be interpreted with caution. There is a substantial between-study variability in the accuracy of PCR and real-time PCR, as well as a substantial variability in the prevalence of leptospirosis. Consequently, the position of PCR and real-time PCR in the clinical pathway depends on regional considerations such as disease prevalence, factors that are likely to influence accuracy, and downstream consequences of test results. There is insufficient evidence to conclude which of the nucleic acid and antigen detection tests is the most accurate. There is preliminary evidence that PCR and real-time PCR are more sensitive on blood samples collected early in the disease stage, but this needs to be confirmed in future studies.

Leptospira interrogans serovar Bataviae was recently identified as one of the persistent Leptospira serovars in Malaysia. Here, we report the draft genome sequence of the L. interrogans serovar Bataviae strain LepIMR 22 isolated from kidney of a rodent in Johor, Malaysia., (Copyright © 2016 Amran et al.)

In the Netherlands, 97 human leptospirosis cases were notified in 2014. This represents a 4.6-fold increase in autochthonous cases (n = 60) compared with the annual average between 2010 and 2013. Most cases had symptom onset between June and November. This marked increase in humans coincided with an increase of leptospirosis in dogs. In 2014, 13 dogs with leptospirosis were reported, compared with two to six dogs annually from 2010 to 2013. The majority of the autochthonous cases (n = 20) were linked to recreational exposure, e.g. swimming or fishing, followed by occupational exposure (n = 15). About sixty per cent (n = 37) of the autochthonous cases were most likely attributable to surface water contact, and 13 cases to direct contact with animals, mainly rats. A possible explanation for this increase is the preceding mild winter of 2013-2014 followed by the warmest year in three centuries, possibly enabling rodents and Leptospira spp. to survive better. A slight increase in imported leptospirosis was also observed in Dutch tourists (n = 33) most of whom acquired their infection in Thailand (n = 18). More awareness and early recognition of this mainly rodent-borne zoonosis by medical and veterinary specialists is warranted.

Leptospirosis is a neglected zoonosis of global importance with a complex epidemiology that affects humans, domestic and wild mammals. However, due to the diversity of clinical signs and difficulties of establishing a confirmatory laboratory diagnosis, the disease remains poorly investigated, particularly in the developing world. In Morocco, a descriptive study of the seroprevalence of Leptospira infection in animals has never been undertaken. To fill this gap, the current study was conducted on a subset of animals in north-west Morocco as a preliminary step towards understanding the epidemiological patterns of animal leptospirosis in the country. The study was conducted on 289 serum samples collected between January and April 2012 from dogs, cattle, sheep, goats and donkeys in the areas of Rabat-Temara, Sidi Kacem and Oulmes. All serum samples were tested by the MAT with 14 reference strains of the most prevalent pathogenic serovars of Leptospira and two serovars of non-pathogenic Leptospira. The overall seroprevalence of Leptospira in cattle, sheep, goats, dogs and donkeys was 15%, 18%, 20%, 21% and 20%, respectively. The most prevalent serogroups found in each species were Ballum, Sejroe, and Australis in cattle, Ballum, Australis and Sejroe in sheep, Australis and Ballum in goats, Javanica and Australis in donkey and Australis, Ballum and Canicola in dogs. Of all the serogroups tested in this study, Icterohaemorrhagiae, the only serogroup which has been previously reported in humans in Morocco, was rarely reactive. The majority of reactive sera were collected from low land areas. A large number of sera samples classified as seronegative when tested against pathogenic leptospires were positive when tested against non-pathogenic leptospires; this is suggestive of possible novel, as yet unclassified, Leptospira serovars in Morocco. Eleven of thirteen sheep urine samples were positive by real-time PCR confirming their role as Leptospira carriers in Morocco., (© 2014 Blackwell Verlag GmbH.)

Background: Leptospirosis, a spirochaetal zoonosis, occurs in diverse epidemiological settings and affects vulnerable populations, such as rural subsistence farmers and urban slum dwellers. Although leptospirosis can cause life-threatening disease, there is no global burden of disease estimate in terms of Disability Adjusted Life Years (DALYs) available., Methodology/principal Findings: We utilised the results of a parallel publication that reported global estimates of morbidity and mortality due to leptospirosis. We estimated Years of Life Lost (YLLs) from age and gender stratified mortality rates. Years of Life with Disability (YLDs) were developed from a simple disease model indicating likely sequelae. DALYs were estimated from the sum of YLLs and YLDs. The study suggested that globally approximately 2.90 million DALYs are lost per annum (UIs 1.25-4.54 million) from the approximately annual 1.03 million cases reported previously. Males are predominantly affected with an estimated 2.33 million DALYs (UIs 0.98-3.69) or approximately 80% of the total burden. For comparison, this is over 70% of the global burden of cholera estimated by GBD 2010. Tropical regions of South and South-east Asia, Western Pacific, Central and South America, and Africa had the highest estimated leptospirosis disease burden., Conclusions/significance: Leptospirosis imparts a significant health burden worldwide, which approach or exceed those encountered for a number of other zoonotic and neglected tropical diseases. The study findings indicate that highest burden estimates occur in resource-poor tropical countries, which include regions of Africa where the burden of leptospirosis has been under-appreciated and possibly misallocated to other febrile illnesses such as malaria.

Objectives: Previous studies concluded that haemorrhage is one of the most accurate prognostic factors of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease severity in severe leptospirosis., Methods: Prospective cohort study of severe leptospirosis patients. Plasma levels of sE-selectin and Von Willebrand factor (VWF) were determined. Consequently, an in vitro endothelial cell model was used to assess endothelial activation after exposure to virulent Leptospira. Finally, immune activation, as a potential contributing factor to endothelial cell activation, was determined by soluble IL2-receptor (sIL-2r) and soluble Fas-ligand (sFasL) levels., Results: Plasma levels of sE-selectin and VWF strongly increased in patients compared to healthy controls. Furthermore, sE-selectin was significantly elevated (203 ng/ml vs. 157 ng/ml, p < 0.05) in survivors compared to non-survivors. Endothelial cells exposed to virulent Leptospira showed increased VWF expression. E-selectin and ICAM-1 expression did not change. Immunohistochemistry revealed the presence of intracellular Leptospira and qPCR suggested replication. In vivo analysis showed that increased levels of sFasL and sIL-2r were both strongly associated with mortality. Furthermore sIL-2r levels were increased in patients that developed bleeding and significantly correlated to duration of hospital stay., Discussion: Markers of endothelial activation and immune activation were associated with disease severity in leptospirosis patients., (Copyright © 2015 The British Infection Association. All rights reserved.)

Background: Leptospirosis is an emerging zoonotic infection worldwide, possibly due to climate change and demographic shifts. It is regarded as endemic in Sub-Saharan Africa; however, for most countries scarce epidemiological data, if any, exist. The primary objectives were to describe the prevalence of leptospirosis in countries in Sub-Saharan Africa, and to develop options for prevention and control in the future., Methods: A systematic review was conducted to determine the prevalence of leptospirosis in Sub-Saharan Africa; the PRISMA guidelines were followed. Medline/PubMed, Embase, The Cochrane Library, Web of Science, BIOSIS Previews, the African Index Medicus, AJOL, and Google Scholar were searched., Results: Information about the prevalence and incidence of leptospirosis in humans is available, but remains scarce for many countries. Data are unavailable or outdated for many countries, particularly those in Central Africa. Most data are available from animals, probably due to the economic losses caused by leptospirosis in livestock. In humans, leptospirosis is an important cause of febrile illness in Sub-Saharan Africa. It concerns numerous serogroups, harboured by many different animal carriers., Discussion: A wide variety of data was identified. Prevalence rates vary throughout the continent and more research, especially in humans, is needed to reliably gauge the extent of the problem. Preventive measures need to be reconsidered to control outbreaks in the future.

Leptospirosis and haemorrhagic fever with renal syndrome (HFRS) are hard to distinguish clinically since these two important rodent-borne zoonoses share hallmark symptoms such as renal failure and haemorrhage. Leptospirosis is caused by infection with a spirochete while HFRS is the result of an infection with certain hantaviruses. Both diseases are relatively rare in the Netherlands. Increased incidence of HFRS has been observed since 2007 in countries that border the Netherlands. Since a similar rise in incidence has not been registered in the Netherlands, we hypothesise that due to overlapping clinical manifestations, hantavirus infections may be confused with leptospirosis, leading to underdiagnosis. Therefore, we tested a cohort of non-travelling Dutch patients with symptoms compatible with leptospirosis, but with a negative diagnosis, during 2010 and from April to November 2011. Sera were screened with pan-hantavirus IgG and IgM enzyme-linked immunosorbent assays (ELISAs). Sera with IgM reactivity were tested by immunofluorescence assay (IFA). ELISA (IgM positive) and IFA results were confirmed using focus reduction neutralisation tests (FRNTs). We found hantavirus-specific IgG and/or IgM antibodies in 4.3% (11/255) of samples taken in 2010 and in 4.1% (6/146) of the samples during the 2011 period. After FRNT confirmation, seven patients were classed as having acute Puumala virus infections. A review of hantavirus diagnostic requests revealed that at least three of the seven confirmed acute cases as well as seven probable acute cases of hantavirus infection were missed in the Netherlands during the study period.

The microscopic agglutination test (MAT) is the gold standard for sero-diagnosis of leptospirosis because of its unsurpassed diagnostic specificity. It uses panels of live leptospires, ideally recent isolates, representing the circulating serovars from the area where the patient became infected. A dilution series of the patient's serum is mixed with a suspension of live leptospires in microtiter plates. After incubating for about 2 hr at 30°C, results are read under the dark-field microscope. The titer is the last dilution in which ≥ 50% of the leptospires have remained agglutinated. Seroconversion or ≥ 4-fold titer rise in paired sera is consistent with current leptospirosis. The significance of a titer in a single sample depends on the frequency of residual titers due to past infections and cross-reacting other diseases in the population. Full standardization of the MAT is not possible, but quality assurance can be achieved by participation in the international MAT proficiency testing scheme., (Copyright © 2014 John Wiley & Sons, Inc.)

Background: Leptospirosis is a global zoonotic disease. Although important for the assessment of the burden of leptospirosis, data on the duration of the illness and the occurrence of post-leptospirosis complaints are not well documented. Hence the main objective of this study was to estimate the occurrence of persistent complaints and duration of hospital stay in laboratory confirmed leptospirosis patients in the Netherlands during 1985 to 2010. Additionally, several risk factors potentially impacting on the occurrence of post-leptospirosis complaints were investigated., Methods/principal Findings: The duration of the acute phase of leptospirosis was 16 days (IQR 12-23); 10 days (IQR 7-16) were spent hospitalized. Eighteen fatal cases were excluded from this analysis. Complaints of leptospirosis patients by passive case investigations (CPC) derived from files on ambulant consultations occurring one month after hospital discharge, revealed persistent complaints in 108 of 236 (45.8%) laboratory confirmed cases. Data on persistent complaints after acute leptospirosis (PCAC), assessed in 225 laboratory confirmed leptospirosis cases collected through questionnaires during 1985-1993, indicated 68 (30.2%) PCAC cases. Frequently reported complaints included (extreme) fatigue, myalgia, malaise, headache, and a weak physical condition. These complaints prolonged in 21.1% of the cases beyond 24 months after onset of disease. There was no association between post-leptospirosis complaints and hospitalization. However, individuals admitted at the intensive care unit (ICU) were twice as likely to have continuing complaints after discharge adjusting for age and dialysis (OR 2.0 95% CI 0.8-4.8). No significant association could be found between prolongation of complaints and infecting serogroup, although subgroup analysis suggest that infection with serogroups Sejroe (OR 4.8, 95%CI 0.9-27.0) and icterohaemorrhagiae (OR 2.0, 95%CI 0.9-4.3 CI) are more likely to result in CPC than infections with serogroup Grippotyphosa., Conclusion/significance: In addition to the acute disease, persistent complaints have an impact on the burden of leptospirosis.

Background: Diagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively., Methodology: During 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1(st) submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness., Results: The overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1(st) submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB)., Conclusions: All three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.

To increase knowledge of leptospirosis in the Netherlands and identify changing trends of this disease over time, we analyzed historical passive surveillance reports for an 84-year period (1925-2008). We found that 2,553 mainly severe leptospirosis cases were diagnosed (average annual incidence rate 0.25 cases/100,000 population). The overall case-fatality rate for patients with reported leptospirosis was 6.5% but decreased over the period, probably because of improved treatment. Ninety percent of reported leptospirosis cases were in male patients. Most autochthonous leptospirosis infections were associated with recreational exposures, but 15.5% of the cases were attributed to accidents that resulted in injury and to concomitant water contact. Since the end of the 1950s, the proportion of imported infections gradually increased, reaching 53.1% of the total during 2005-2008. Most (80.1%) imported infections were associated with sporting and adventurous vacation activities.

Unlabelled: Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), despite treatment with conventional or antiangiogenic drugs. We wished to determine whether the high levels of VEGF receptor (VEGFR) within the tumor vasculature could be leveraged for intracellular delivery of therapeutically significant doses of scVEGF/(177)Lu, a novel radiopharmaceutical based on a recombinant single-chain (sc) derivative of VEGF, in orthotopic breast cancer models., Methods: scVEGF-PEG (polyethylene gycol)-DOTA conjugates containing 2.0-, 3.4-, or 5.0-kDa PEG linkers site-specifically conjugated to a cysteine-containing tag (Cys-tag) in scVEGF were radiolabeled with (177)Lu (scVEGF/(177)Lu) for in vivo studies. Human MDA231luc and mouse 4T1luc cell lines were injected orthotopically to establish breast carcinoma tumors in immunodeficient and immunocompetent hosts, respectively. The effects of scVEGF/(177)Lu were defined by analysis of changes in tumor growth and immunohistochemical staining for the endothelial markers CD31 and VEGFR-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for intratumoral apoptosis., Results: Biodistribution assays and dosimetric calculations established that scVEGF/(177)Lu with a 3.4-kDa PEG linker delivered the highest dose of radiation to tumors (69.9 cGy/MBq/g of tissue) and the lowest dose to the kidneys (33.3 cGy/MBq/organ). Total doses below 40 MBq/mouse of scVEGF/(177)Lu did not affect renal function, and 3 divided doses of 6.3 MBq/mouse or a bolus dose of 18.9 MBq/mouse induced only transient lymphopenia and weight loss (<10% baseline weight). In mice with orthotopic mammary breast carcinoma, intravenous injections of well-tolerated bolus and fractionated doses of scVEGF/(177)Lu in the range from 6.3 to 18.9 MBq/mouse (25-76 MBq/m(2)) resulted in dose-dependent tumor growth inhibition. Immunohistochemical analysis of tumors at 4-5 wk after single injections of scVEGF/(177)Lu indicated dose-dependent regression of tumor vasculature and widespread intratumoral apoptosis. A single dose of 7.4 MBq/mouse of scVEGF/(177)Lu given before a course of bevacizumab or sunitinib treatment enhanced the antiangiogenic effects of both drugs., Conclusion: Selective targeting of VEGFR in tumor vasculature with well-tolerated doses of scVEGF/(177)Lu is effective in orthotopic breast cancer models. As high levels of VEGFR expression in the tumor vasculature are a common feature in a variety of cancers, targeting tumor angiogenesis with scVEGF/(177)Lu warrants further exploration.

Background: Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires., Methodology/principal Findings: We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires., Conclusions/significance: Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

Objective: To determine the involvement of coagulation in bleeding and poor outcome in patients with severe leptospirosis., Methods: In a prospective study, parameters of the coagulation system were measured on admission and during follow-up in 52 consecutive patients with severe leptospirosis., Results: All patients showed coagulation disorders, such as prolonged prothrombin time (PT) and activated partial thromboplastin time, marked procoagulant activity [thrombin-antithrombin (TAT) complexes, prothrombin fragment 1+2, D-dimer], reduced levels of anticoagulant markers (protein C, antithrombin) and increased (anti-) fibrinolytic activity [plasmin-antiplasmin (PAP) complexes, plasminogen activator inhibitor-1]. These disorders were more pronounced in patients who died eventually. PT prolongation was associated with mortality (OR 1.4, 95% CI: 1.0-1.8, P = 0.04). Bleeding occurred in 31 subjects (60%). Of these, 24 had mild bleeding and seven had severe haemorrhages. Thrombocytopenia (platelets

Background: Severe leptospirosis features bleeding and multi-organ failure, leading to shock and death. Currently it is assumed that both exaggerated inflammation and immune suppression contribute to mortality in sepsis. Indeed, several proinflammatory cytokines are reported to be induced during leptospirosis. Toll-like receptors, which play an important role in the initiation of an innate immune response, are inhibited by negative regulators including the membrane-bound ST2 (mST2) receptor. Soluble ST2 (sST2) has been implicated to inhibit signaling through mST2. The aim of this study was to determine the extent of sST2 and (pro-) inflammatory cytokine release in patients with severe leptospirosis., Methodology and Principal Findings: In an observational study, 68 consecutive cases of severe leptospirosis were included. Soluble ST2 and cytokines (TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10) were repeatedly measured. To determine whether blood cells are a source of sST2 during infection, we undertook an in vitro experiment: human whole blood and peripheral blood mononuclear cells (PBMC) were stimulated with viable pathogenic Leptospira. All patients showed elevated sST2, IL-6, IL-8, and IL-10 levels on admission. Admission sST2 levels correlated with IL-6, IL-8, and IL-10. Thirty-four patients (50%) showed clinical bleeding. Soluble ST2 levels were significantly associated with bleeding overall (OR 2.0; 95%CI: 1.2-3.6) and severe bleeding (OR 5.1; 95%CI: 1.1-23.8). This association was unique, since none of the cytokines showed this correlation. Moreover, sST2 was associated with mortality (OR 2.4; 95%CI: 1.0-5.8). When either whole blood or isolated PBMCs were stimulated with Leptospira in vitro, no sST2 production could be detected., Conclusions: Patients with severe leptospirosis demonstrated elevated plasma sST2 levels. Soluble ST2 levels were associated with bleeding and mortality. In vitro experiments showed that (white) blood cells are probably not the source. In this regard, sST2 could be an indicative marker for tissue damage in patients suffering from severe leptospirosis.

Objective: To evaluate the long pentraxin PTX3 in patients with severe leptospirosis and to compare the results with the widely used short pentraxin C-reactive protein and the pro-inflammatory cytokines IL-6 and IL-8., Methods: This observational cohort study was carried out in Semarang, Indonesia, where leptospirosis is endemic and mortality is high. Consecutive patients with severe leptospirosis were sampled on admission and during follow-up., Results: A total number of 52 patients entered the study, the mortality was 27%. Severe leptospirosis patient yielded elevated plasma PTX3 levels. PTX3 correlated with IL-8 and to a lesser extent with CRP and IL-6 levels. High levels of PTX3, IL-6 and IL-8 were associated with mortality (OR 5.6, 95%CI: 1.2-26; OR 3.2, 95%CI: 1.2-8.1; OR 6.5, 95%CI: 1.5-28). Moreover, PTX3 levels were associated with disease severity (OR 9.5; 95%CI: 2.9-45). This association was unique, since none of the other markers showed this relation. C-reactive protein was not able to differentiate the severe from the severest cases., Conclusions: The long pentraxin PTX3 is elevated in patients with severe leptospirosis and is associated with fatal disease and disease severity. PTX3 may be used as a marker to monitor disease severity in severe leptospirosis or predict outcome.

To investigate rickettsioses and leptospirosis among urban residents of Semarang, Indonesia, we tested the blood of 137 patients with fever. Evidence of Rickettsia typhi, the agent of murine typhus, was found in 9 patients. Another 9 patients showed inconclusive serologic results. Thirteen patients received a diagnosis of leptospirosis. No dual infections were detected.

Leptospirosis is a worldwide distributed zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The basic taxon of Leptospira is the serovar. Currently, nearly 300 serovars have been identified. Leptospirosis is particularly prevalent in warm and humid tropical regions where conditions for transmission and survival of pathogenic leptospires in the environment are optimal. Leptospirosis probably constitutes a serious veterinary and public health problem in Central America but solid figures are missing. To determine distribution of leptospirosis in Costa Rica and to identify locally circulating pathogenic serovars, we performed a sentinel-based study, isolating and characterizing leptospires from patients attending hospitals. Strain MAVJ 401 was isolated from a hospitalized patient in the Alajuela province. The isolate produced agglutination titers notably with reference rabbit antisera against serovars of serogroup Javanica but appeared serologically unique in the standard Cross Agglutinin Absorption Test. Therefore, MAVJ 401 was considered to represent a new serovar, designated Arenal, of the serogroup Javanica. Genotypic analysis revealed that strain MAVJ 401 belongs to Leptospira santarosai, a species that almost exclusively occurs in Latin America. This is not a unique finding of an exotic serovar. Recent isolates from severely ill patients in the same region appeared to be identical to Arenal. We have identified a novel highly virulent serovar from a patient in Costa Rica that is common in this area, thus posing a threat for the local public and veterinary health.

Leptospirosis is a zoonosis of worldwide distribution, spread by the urine of infected animals. It is a major public health problem, especially in developing countries, where circumstances for transmission are most favourable. The clinical picture varies from mild disease to a severe illness with haemostatic derangements and multiorgan failure eventually leading to death. Although the haemorrhagic complications of severe disease are serious, the pathophysiology is scarcely elucidated. The complex mechanisms involved in inflammation-induced coagulation activation are extensively studied in various infectious diseases, i.e. Gram-negative sepsis. Tissue factor-mediated coagulation activation, impairment of anticoagulant and fibrinolytic pathways in close concert with the cytokine network are thought to be important. But for human leptospirosis, data are limited. Because of the growing interest in this field, the impact of leptospirosis, and the availability of new therapeutic strategies, we reviewed the evidence regarding the role of coagulation in leptospirosis and provide suggestions for future research.

Objective: To estimate the seroprevalence of human leptospirosis in southern Vietnam., Methods: All pupils (n=961) of two primary schools in two communes in southern Vietnam were screened for the presence of serum Leptospira immunoglobulin (Ig)G. Leptospira IgM was tested in 92 randomly selected samples. IgM and IgG were tested with a commercially available enzyme-linked immunosorbent assay (ELISA)., Results: Leptospira IgG was found in 123 (12.8%, 95% CI: 10.8-15.1) children with a male:female ratio of 1.5:1. At the age of 7 years the prevalence was 11%. Log-linear binary regression of the IgG seroprevalence by age showed a mean seroconversion rate of 1.5% (95% CI: 1.23-1.75) per year. Five (5.4%, 95% CI: 2.3-12.1) samples were definitely positive for IgM. Presence of serum Leptospira IgG antibodies was significantly associated with swimming in rivers (RR: 1.745, 95% CI: 1.117-2.724) and wading through water (RR 1.793, 95% CI: 1.181-2.722)., Conclusion: Our data emphasize the importance of leptospirosis among children in Vietnam, despite the absence of severe disease, and stress the need for adequate and cheap diagnostics.

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.

The prevalence of leptospirosis is generally high in domestic animals and rodents in Tanzania. Identification of Leptospira isolates from cattle was carried out to establish prevalent Leptospira serovars. Serological typing was done based on monoclonal antibodies and the standard cross-agglutination absorption test. Molecular typing involved pathogenic- and saprophytic-specific PCRs and a PCR specifically amplifying DNA from the species Leptospira kirschneri. DNA fingerprinting with primers derived from sequences of insertion elements IS1500 and IS1533 was carried out. Both serological and molecular characterization indicated that one of the Leptospira isolates, coded RM1, represents a new serovar of the species L. kirschneri of serogroup Icterohaemorrhagiae. The serovar name Sokoine is proposed for this new Leptospira isolate.

Two identical leptospiral isolates coded Sh9 and Sh25 obtained from the urine of captive African giant pouched rats (Cricetomys gambianus), destined for use as biodetector of antipersonnel landmines were typed as serovar Kenya using cross-agglutination absorption test and DNA fingerprinting with the insertion element sequences IS1533 and IS1500 derived primers. The two isolates were previously characterized using cultural and serological-microagglutination test as pathogenic leptospires of the serogroup Ballum, closely related to serovars Kenya and Peru. To our knowledge, this is the first reported in-depth characterization of leptospira isolates from Tanzania.

Application of a dipstick assay for the detection of Salmonella typhi-specific IgM antibodies on samples collected from S. typhi or S. paratyphi culture-positive patients at the day of admission to the hospital revealed the presence of specific IgM antibodies in 43.5%, 92.9%, and 100% for samples collected 4-6 days, 6-9 days, and > 9 days after the onset of fever, respectively. The mean sensitivity for samples collected an average of 6.6 days after the onset of fever was 65.3%. Culture was positive in 65.9% of the cases with a final clinical diagnosis of typhoid fever. Testing of paired serum samples from culture negative patients with a final clinical diagnosis of typhoid fever resulted in staining of the dipstick in 4.3% of the samples collected at the day of admission to the hospital and in 76.6% of the samples collected one week later, thereby provided strong supporting evidence of typhoid fever by demonstrating seroconversion in a large proportion of the patients. The dipstick assay may thus also be useful for the serodiagnosis of culture-negative patients with clinical signs and symptoms consistent with typhoid fever. The advantages of the dipstick assay are that the result can be obtained on the same day allowing a prompt treatment, that only a small volume of serum is needed, and that no special laboratory equipment is needed to perform the assay. The stability of the reagents of the dipstick and the simplicity of the assay allows its use in places that lack laboratory facilities.

Leptospirosis is an often severe disease which requires prompt treatment. Laboratory testing is required to reach a valid diagnosis. An agglutination assay for the detection of Leptospira-specific antibodies consisting of individually wrapped agglutination cards containing a stable, dried detection reagent is evaluated. The assay is simply performed by suspending the dried reagent with a drop of serum. The result is obtained within 30 s. The sensitivity of the assay varied with the stage of the disease and was 72.3% for samples collected during the first 10 days of the illness and 88.2% for samples collected at a later stage. The specificity was 93.9% and 89.8%, respectively. These characteristics make the test ideal for use in areas where the disease is common and where laboratory support is not routinely available.

A newly developed latex agglutination assay for the detection of genus-specific Leptospira antibodies in human sera was evaluated. The assay is performed by mixing, on an agglutination card, serum with equal volumes of stabilized antigen-coated, dyed test and control latex beads and is read within 2 min. The latex agglutination test was evaluated with groups of serum samples from patients with leptospirosis and control patients from Hawaii, the Seychelles, Thailand, and The Netherlands. The mean overall sensitivity was 82.3%, and the mean overall specificity was 94.6%. The assay is easy to perform and does not require special skills or equipment. The reagents have a long shelf life, even at tropical temperatures. Together, these factors make the assay suitable for use even at the peripheral level of a health care system as a rapid screening test for leptospirosis.

A dipstick assay for the detection of brucella-specific immunoglobulin M antibodies was evaluated with 707 sera from 247 laboratory-confirmed brucellosis patients and 342 control sera from brucellosis-free individuals. These sera were collected from six different countries. The assay was found to be highly sensitive and specific. In addition, the test is easy to use and does not require specialized training or equipment, and the components are stable without a requirement for refrigeration. All of these factors make the test ideal for developing countries and rural settings.

We performed a multicenter evaluation of a robust and easily performed dipstick assay for the serodiagnosis of human leptospirosis. The assay is aimed at the detection of Leptospira-specific immunoglobulin M (IgM) antibodies. The study involved 2,665 serum samples collected from 2,057 patients with suspected leptospirosis in 12 countries on five continents with different levels of endemicity and different surveillance systems. The patients were grouped as laboratory-confirmed leptospirosis case patients and noncase patients based on the results of culturing and the microscopic agglutination test. Paired samples from 27.7% of the subjects were tested. Of the 485 case patients, 87.4% had a positive dipstick result for one or more samples. Of the 1,513 noncase patients, only 7.2% had a positive result. Whereas most (88.4%) of the positive samples from the case patients showed moderate to strong (2+ to 4+) staining in the dipstick assay, most (68.1%) of the positive samples from the noncase patients showed weak (1+) staining. The sensitivity of the dipstick assay increased from 60.1% for acute-phase serum samples to 87.4% for convalescent-phase samples. The specificities for these two groups of samples were 94.1 and 92.7%, respectively. The dipstick assay detected a broad variety of serogroups. The results of the dipstick assay were concordant (observed agreement, 93.2%; kappa value, 0.76) with the results of an enzyme-linked immunosorbent assay for the detection of specific IgM antibodies, a test which is often used in the laboratory diagnosis of current or recent leptospirosis. This study demonstrated that this easily performed dipstick assay is a valuable and useful test for the quick screening for leptospirosis; has a wide applicability in different countries with different degrees of endemicity; can be used at all levels of the health care system, including the field; and will be useful for detecting and monitoring outbreaks of leptospirosis.

We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.