Jiri F. P. Wagenaar

Long pentraxin PTX3 is associated with mortality and disease severity in 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.

Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis

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-α, IL-1β, 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.

Intracellular and plasma steady‐state pharmacokinetics of raltegravir, darunavir, etravirine and ritonavir in heavily pre‐treated HIV‐infected patients

AIM To study the steady-state plasma and intracellular pharmacokinetics of raltegravir, etravirine, darunavir and ritonavir in heavily pre-treated patients. METHODS Patients on a salvage regimen containing raltegravir, etravirine, darunavir and ritonavir were eligible for inclusion. During a 12 h dosing interval plasma and peripheral blood mononuclear cells were collected. Drug concentrations were measured using a validated LC-MS/MS assay and pharmacokinetic analysis was performed using non-linear mixed effect modelling. RESULTS Irregular absorption was observed with raltegravir and darunavir, which may be caused by enterohepatic cycling. Relative bioavailability of ritonavir was low, when compared with other ritonavir regimens. Raltegravir plasma pharmacokinetics showed wide interpatient variability, while intracellular raltegravir concentrations could not be detected (<0.001 mg l(-1) in cell lysate). The intracellular to plasma ratios for etravirine, darunavir and ritonavir were 12.9, 1.32 and 7.72, respectively, and the relative standard error of these estimates were 16.3%, 12.3% and 13.0%. CONCLUSIONS The observed distinct intracellular accumulation indicated that these drugs have different affinity for the cellular compartment. The relatively high intracellular accumulation of etravirine may explain its efficacy and its previously described absence of PK-PD relationships in the therapeutic concentration range, when compared with other non-nucleoside reverse transcriptase inhibitors. Lastly, the intracellular concentrations of ritonavir seem sufficient for inhibition of viral replication in the cellular compartment in PI-naive patients, but not in patients with HIV harbouring PI resistance.

Prospective Evaluation of Three Rapid Diagnostic Tests for Diagnosis of Human 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 1st 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 1st 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.

What role do coagulation disorders play in the pathogenesis of leptospirosis

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.

Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood

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 PBMCs 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 PBMCs 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.

Establishment of Valid Laboratory Case Definition for Human Leptospirosis

textabstractLaboratory case definition of leptospirosis is scarcely de ned by a solid evaluation that determines cut-off values in the tests that are applied. This study describes the process of determining optimal cut-off titers of laboratory tests for leptospirosis for a valid case definition of leptospirosis. In this case the tests are the microscopic agglutination test (MAT) and an in-house IgM enzyme-linked immunosorbent assay (ELISA) both on single serum and paired samples using a positive culture as the reference test in the Dutch population. The specificity was assessed using panels of sera from healthy donors, cases with known other diseases and non-leptospirosis cases with symptoms compatible with leptospirosis. Cases were divided into three periods corroborating the acute phase (1-10 days post onset of illness (DPO)), the early convalescent (11-20 DPO) and the late convalescent phase (>20 DPO). Cut-off titers for MAT and IgM ELISA were determined as 1:160 and 1:80 respectively for all three periods. These cut-off titers combined 100% specificity with a sensitivity that changed according to the stage of disease for both tests. The low sensitivities in the early acute phase are consistent with the dynamics of the humoral immune response. IgM ELISA yielded higher sensitivities compared to MAT in the acute and early convalescent stages. Moreover, the optimal sensitivity of MAT, the gold standard was < 82%, implying that a significant part of global cases is missed by this recommended test. MAT and IgM ELISA manifested partly complementary, resulting in a higher sensitivity when combining the results of these two tests. The availability of paired samples and of adequate clinical and epidemiological data are other parameters that will significantly increase the sensitivity of laboratory confirmation. This study enables fine-tuning of the current laboratory definition towards an improved case finding and implies that solid validation of laboratory parameters for case definition will improve both the diagnosis for individual patient care and for estimating the disease burden at a worldwide scale.

Coagulation disorders in patients with severe leptospirosis are associated with severe bleeding and mortality

Objective  To determine the involvement of coagulation in bleeding and poor outcome in patients with severe leptospirosis.

Rodent-borne hemorrhagic fevers: under-recognized, widely spread and preventable – epidemiology, diagnostics and treatment

This review presents an overview of the most important rodent-borne hemorrhagic fever pathogens directly transmitted from rodents to humans, namely Leptospira and hantaviruses, together with the New- and Old-World arenaviruses. These zoonotic diseases frequently share clinical symptoms, transmission routes and other epidemiological features and often have an emerging pattern. Differential diagnostics could benefit from a syndrome-based approach grouping these pathogens. In this review extensive descriptions of the epidemiology, clinical symptoms, diagnostics and treatment are provided including a practical overview, listing clinical features, diagnostics and risk factors for each selected rodent-borne hemorrhagic fever pathogen.

PTX3 predicts severe disease in febrile patients at the emergency department

OBJECTIVES The long pentraxin PTX3 is a promising marker of disease severity in severely ill patients. In order to identify patients warranting critical care as quickly as possible, we investigated the value of PTX3 as a biomarker for disease severity in patients presenting with fever at the emergency department. METHODS Levels of PTX3 were measured in 211 febrile patients at the emergency and the levels were linked to markers of disease severity including admittance to a special care unit, bloodstream infection and congestive heart failure. RESULTS In comparison to median baseline levels of 2.30 ng/ml (interquartile range 1.66-3.67 ng/ml), levels of PTX3 were significantly elevated in patients admitted to the intensive-/medium care unit (median value 44.4 ng/ml, interquartile range 13.6-105.9 ng/ml) and in patients referred to the ward (median value 14.2 ng/ml, interquartile range 7.01-25.1 ng/ml). In addition, PTX3 was associated with duration of hospital stay and acute congestive heart failure. The levels were predictive for bloodstream infection (AUC=0.71; 95% CI 0.62-0.81). CONCLUSIONS PTX3 may be a useful marker for differentiation of patients with severe disease in patients presenting with fever to the emergency department.