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Studies on leptospirosis : clinical aspects and pathophysiology
Wagenaar, J.F.P.
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Download date: 30 Mar 2020
Studies on leptospirosis
- clinical aspects and pathophysiology –
Jiři František Pavel Wagenaar
Studies on leptospirosis - clinical aspects and pathophysiology -
Dissertation, University of Amsterdam, the Netherlands
Copyright ©2010, J.F.P. Wagenaar, Amsterdam, the Netherlands
All rights reserved. No part of this publication may be reproduced or transmitted in
any form by any means, electronic or mechanical, including photocopy, recording or
any information storage and retrieval system, without the prior permission of the
author.
Author: Jiři František Pavel Wagenaar
Cover: Alenka Wagenaar
Layout and Printed by: Gildeprint Drukkerijen – Enschede, the Netherlands
Financial support:
Wagenaar communicatie, ViiV Healthcare, Abbott, Tibotec -een divisie van Janssen
Cilag-, Merck Sharp & Dome, Gilead Sciences, Boehringer Ingelheim, Tomas
beenmode, Cirion, SKWOSZ
ISBN: 978-94-61080-14-1
Studies on leptospirosis
- clinical aspects and pathophysiology –
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde
commissie, in het openbaar te verdedigen in de Agnietenkapel
op woensdag 3 maart 2010, te 14.00 uur
door Jiři František Pavel Wagenaar
geboren te Delft
Promotiecommissie:
Promotor(es): Prof. dr. T. van der Poll
Co-promotor(es): Dr. E.C.M. van Gorp
Dr. R.A. Hartskeerl
Dr. M.H. Gasem
Overige leden: Prof. dr. P. Kager
Prof. dr. P. Speelman
Prof. dr. J.C.M. Meijers
Prof. dr. A.D.M.E. Osterhaus
Dr. C. van ’t Veer
Dr. A.I. Ko
Dr. D.P.M. Brandjes
Faculteit der Geneeskunde
Chapter 1: General Introduction and outline of the thesis 7
Part I: Coagulation and endothelium
Chapter 2: What role do coagulation disorders play in the pathogenesis 21
of leptospirosis?
TropMedIntHealth2007,12(1):111-22.
Chapter 3: Leptospirosis with pulmonary hemorrhage, caused by a new 45
strain of serovar Lai: Langkawi.
JTravelMed2004,11(6):379-81.
Chapter 4: Coagulation disorders in patients with severe leptospirosis 55
are associated with severe bleeding and mortality.
TropMedIntHealth2010,15(2):152-59.
Chapter 5: Bleeding in patients with severe leptospirosis is not associated 73
with activation of endothelial cells.
Submitted.
Chapter 6: Low factor XII and factor XI levels in patients with severe 85
leptospirosis.
Submitted.
Part II: Inflammation
Chapter 7: Long pentraxin PTX3 is associated with mortality and disease 101
severity in severe leptospirosis.
JInfect2009,58(6):425-32.
Chapter 8: Soluble ST2 levels are associated with bleeding in patients 119
with severe leptospirosis.
PLoSNeglTropDis2009,3(6):e453.
CONTENTS
Chapter 9: Innate immune response to pathogenic leptospira is 135
dependent of both TLR2 and TLR4 signaling in human
whole blood.
Submitted.
Part III: Diagnostic and epidemiological aspects
Chapter 10: Rapid serologic assays for leptospirosis are of limited value 161
in southern Vietnam.
AnnTropMed&Parasitology2004,98(8):843-50.
Chapter 11: Murine typhus and leptospirosis as a cause of acute 173
undifferentiated fever in Central Java, Indonesia.
EmergInfectDis2009,15(6):975-7.
Chapter 12: Summary, discussion and directions for future studies 183
Samenvatting en discussie voor niet-ingewijden 195
Dankwoord 205
About the author 207
General Introduction and outline of the thesis
J.F.P. Wagenaar ¹, M.H. Gasem ², R.A. Hartskeerl ³ and E.C.M. van Gorp ¹
1 Department of Internal Medicine, Slotervaart Hospital, Amsterdam, the Netherlands
2 Department of Internal Medicine, Dr. Kariadi Hospital, Diponegoro University, Semarang, Indonesia
3 Royal Tropical Institute (KIT), KIT Biomedical Research, Amsterdam, the Netherlands
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Leptospirosis has been recognized as an emerging infectious disease of global
importance (1). Large outbreaks occur annually in the (sub) tropics where conditions
for its transmission are particularly favourable. Although Leptospira thrive in warm,
humid conditions, the incidence of leptospirosis in temperate regions is also significant
(2;3). Leptospirosis has also emerged as a disease that impacts the adventure
traveller, especially those that participate in water sports (4) and visit tropical
regions where the disease is endemic (5). Several large clusters of leptospirosis cases
that follow hurricanes and excessive rainfall have recently been reported (6;7). In
the light of global warming and related extreme weather events, leptospirosis now
receives more international attention as a climate-sensitive disease.
Microbiology
The disease was first reported by Adolf Weil in 1886. However, it was not until the
second decade of the 20th century that the pathogen was first isolated (8). Leptospira
are spirochetes, a group of bacteria that diverged early in bacterial evolution (9).
Classically, Leptospira are serologically classified into numerous serovars defined by
agglutination after cross-absorption with homologous antigen (10). Serovars that are
antigenically related have been grouped into serogroups. To date, over 200 serovars
have been identified, arranged into 24 serogroups (2). More recently, a molecular
classification has been described, dividing the Leptospira genus into several species,
based on DNA relatedness (1).
Leptospires have a typical double membrane structure, in common with other
spirochetes, see figure 1. The cytoplasmic membrane and peptidoglycan cell wall are
closely associated and are overlain by an outer membrane. Leptospiral LPS differs
from Gram-negative LPS in several biochemical, physical and biological properties
(11). Leptospira contain a periplasmatic situated flagellum, making them highly
motile.
INTRODuCTION
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Figure 1: Schematic depiction of the membrane architecture of Leptospira.
Epidemiology
The incidence rate of leptospirosis is thought to be underestimated, due to
unawareness, misdiagnosis and lack of appropriate diagnostic laboratory facilities.
The World Health Organization (WHO) recognizes leptospirosis as a growing
worldwide public health problem and recently established “The Leptospirosis Burden
Epidemiology Reference Group” to gain more insight into the true burden of the
disease. Currently, it is estimated that, statistically speaking, 0.1 to 1.0 per 100,000
people living in temperate climates are affected by the disease each year, with
the number increasing to 10 or more per 100,000 people living in tropical climates.
During an epidemic, the WHO estimates that the incidence rate can soar to 100 or
more per 100,000 people. Typical risk groups include farmers and sewer and abattoir
workers. In developing countries, leptospirosis is a significant health burden for poor
rural populations (12). And as the rural poor migrate to the cities, leptospirosis has,
in turn, become an urban disease. This is particularly true of urban slums, where
a lack of basic sanitation has produced the ecological conditions for rodent-born
transmission.
The source of infection in humans is usually either direct or indirect contact with
the urine of an infected animal. The portal of entry in humans is through abrasions
or cuts in the skin or via the conjunctiva, usually following water contact. Large
numbers of animals act as carriers, most importantly small mammals like rats and
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mice, but also domestic animals such as dairy cattle, horses, pigs and dogs. The
bacteria are kept alive in nature by the chronic infection of the renal tubules of
their hosts.
Clinicalfeatures
Although potentially fatal, most cases of human leptospirosis are thought to run a
mild course. Most patients will probably not seek medical attention and will develop
a febrile illness of mild severity that resembles many other diseases. The differential
diagnosis is therefore extensive (see table). Symptoms of leptospirosis include chills,
headache, myalgia, abdominal pain, conjunctival suffusion and, less often, a rash.
The resolution of the symptoms may coincide with the appearance of antibodies
and leptospiruria. The fever may however be biphasic, recurring after a remission
of three to four days. Aseptic meningitis may be found in up to a quarter of all
leptospirosis cases.
Severe cases of the disease are often rapidly progressive, with a case fatality
rate ranging from 5 to 25%. The classical presentation, called Weil’s disease, is
characterized by the triad of jaundice, acute renal failure and bleeding. Patients
die from septic shock with multi-organ failure and/or severe bleeding complications,
like leptospirosis severe pulmonary hemorrhage syndrome (SPHS). Leptospirosis SPHS
is now recognized as a widespread public health problem (7;13;13;14). Hemoptysis,
the characteristic sign of SPHS, may not be apparent until patients are intubated.
The severity of the respiratory disease is unrelated to the presence of jaundice.
Thrombocytopenia develops in up to 50% of patients and is associated with poor
outcome (15). Serum bilirubin concentrations may be high, whereas transaminase
levels will only be moderately elevated. Renal failure, reported in between 16 to 40%
of cases (16), has been identified as an independent risk factor for mortality. Other
risk factors for mortality include: respiratory insufficiency, hypotension, arrhythmias
and altered mental status (6;17;18)
The use of antibiotic treatment is strongly recommended for severe leptospirosis (19).
The antibiotic of choice is penicillin, but treatment with ceftriaxone or cefotaxime
and doxycycline in mild cases have shown equivalent efficacy (20;21). Doxycycline
has been shown to be effective for short-term prophylaxis in high-risk environments
and can be prescribed for travelers at risk (22).
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The clinical management of leptospirosis is merely supportive. Severe cases initially
need admission to the intensive care unit for dialysis and mechanical ventilation. To
date, no studies have explored the role of intervention in the coagulation cascade
with, e.g., activated protein C.
Laboratorydiagnosis
Although laborious and expensive, the microscopic agglutination test (MAT) remains
the reference standard for the serological diagnosis of leptospirosis (2). The MAT
detects agglutinating antibodies in serum, using a wide panel of different viable
serovars. The test calls for significant expertise from well-trained laboratory
workers, making the MAT unfeasible in poor-resource settings. Several serologically
based rapid tests have been developed in recent years, all lacking sensitivity in the
first week of illness but relatively cheap and easy to use (23;24). Polymerase chain
reaction (PCR) based assays are available, making it possible to detect Leptospira
early in the disease. Culture is insensitive and slow, but Leptospira can be isolated
from the blood during the first seven to ten days of the illness. Even under optimum
conditions, the bacteria grow slowly and cultures can be reported as negative only
after a minimum of six to eight weeks. The diagnostic test of choice depends largely
on the local setting and infrastructure.
Inflammationandcoagulation
Sepsis with multi-organ failure and hemorrhaging are the biggest threats to patients
suffering from severe leptospirosis. It is now widely thought that the host response
to sepsis involves both exaggerated inflammation and immune suppression (25), see
figure 2. Additionally, evidence is accumulating that microbial virulence and bacterial
load contribute to the severity of sepsis. The interaction between pathogens and
the host is mediated via an interaction between pathogen-associated molecular
patterns (PAMPs) and Toll-like receptors (TLRs). TLR signaling rapidly results in an
inflammatory response that is harmful to the host when excessive. The interaction
of TLRs with damage-associated molecular patterns (DAMPs), which are endogenous
mediators released by damaged tissues, further amplifies this inflammatory process
(26).
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Figure 2: Host immune response to sepsis.
During septic conditions, the blood coagulation system is triggered due to the pro-
inflammatory environment and/or endothelial cell damage. When insufficiently
controlled, this can lead to the syndrome of disseminated intravascular coagulation
(DIC), with bleeding and microvascular thrombosis as the clinical hallmarks. Tissue
factor is regarded as the primary initiator of coagulation in sepsis. Activated
monocytes, endothelial cells, along with circulating microvesicles become sources
of tissue factor during severe sepsis. The impairment of anti-coagulant proteins and
fibrinolysis also results in a net procoagulant state in septic patients. Inflammation
and coagulation act in a bidirectional manner (27). Activated thrombin can promote
the activation of various pro-inflammatory pathways, whereas cytokines, in turn,
can stimulate coagulation. Knowledge of the role of the coagulation and fibrinolysis
system in the pathogenesis of leptospirosis and its interplay with inflammation is
highly limited.
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The overall aim of this thesis is to increase insight into the pathogenesis of leptospirosis
so that we can identify possible new treatment targets. More specifically, we have
focused on hemostasis and inflammation, since bleeding and septic shock are
the most important causes of death. Our key objectives were: (I) to obtain more
insight into both coagulation and fibrinolysis in leptospirosis; (II) to characterize the
innate immune response during leptospirosis; and, (III) to give some insight in the
epidemiology and diagnosis of leptospirosis.
Part I starts with chapter 2, which gives an overview of hemostasis in leptospirosis.
Chapter 3 describes a typical case of severe leptospirosis with pulmonary
hemorrhages. Chapter 4 reports on the activation of coagulation and fibrinolysis in
patients with severe leptospirosis. Chapter 5 addresses the role of endothelial cell
dysfunction in relation to bleeding, while chapter 6 describes the activation of the
contact system in patients with severe leptospirosis.
Part II focuses on inflammation. In chapter 7 we have evaluated the usefulness of
the long pentraxin PTX3 as a biomarker to predict disease severity and poor outcome
in patients suffering from severe leptospirosis. Chapter 8 concentrates on soluble
ST2, a molecule involved in the regulation of the innate immune response. Chapter 9
describes some aspects of the innate immune response, especially Toll-like receptor
involvement, to viable Leptospira in an experimental in-vitro model.
Part III starts with chapter 10, in which we have evaluated a rapid serological assay
to diagnose leptospirosis in a febrile and non-febrile Vietnamese cohort. Chapter 11
reports on the epidemiology of leptospirosis and rickettsiosis in an in-hospital and
outpatient febrile Indonesian cohort. The results and potential implications of the
studies are summarized and discussed in chapter 12.
AIM AND OuTLINE OF THIS THESIS
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Table: Differential diagnosis of leptospirosis (not complete).
RickettsiosesTyphoid feverMalariaDengueYellow feverChikungunyaHanta feverMeningococcal infectionInfluenzaViral hepatitisHIVOther viral hemorrhagic fevers
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(1) Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003 December;3(12):757-71.
(2) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (3) Desai S, van TU, Lierz M, Espelage W, Zota L, Sarbu A et al. Resurgence of field fever
in a temperate country: an epidemic of leptospirosis among seasonal strawberry harvesters in Germany in 2007. Clin Infect Dis 2009 March 15;48(6):691-7.
(4) Morgan J, Bornstein SL, Karpati AM, Bruce M, Bolin CA, Austin CC et al. Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois, 1998. Clin Infect Dis 2002 June 15;34(12):1593-9.
(5) Pavli A, Maltezou HC. Travel-acquired leptospirosis. J Travel Med 2008 November;15(6):447-53.
(6) Ko AI, Galvao RM, Ribeiro Dourado CM, Johnson WD, Jr., Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 1999 September 4;354(9181):820-5.
(7) Trevejo RT, Rigau-Perez JG, Ashford DA, McClure EM, Jarquin-Gonzalez C, Amador JJ et al. Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua, 1995. J Infect Dis 1998 November;178(5):1457-63.
(8) Weil A. Ueber eine eigenthumliche, mit milztumor, icterus und nephritis einhergehende, acute infectionskrankheit. Dtsch Arch Klin Med 1886;39:209.
(9) Paster BJ, Dewhirst FE, Weisburg WG, Tordoff LA, Fraser GJ, Hespell RB et al. Phylogenetic analysis of the spirochetes. J Bacteriol 1991 October;173(19):6101-9.
(10) Faine S, Adler B, Bolin C, Perolat P. Leptospiraand Leptospirosis. MediSci, Melbourne, Australia; 1999.
(11) Schroder NW, Eckert J, Stubs G, Schumann RR. Immune responses induced by spirochetal outer membrane lipoproteins and glycolipids. Immunobiology 2008;213(3-4):329-40.
(12) McBride AJ, Athanazio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis 2005 October;18(5):376-86.
(13) Zaki SR, Shieh WJ. Leptospirosis associated with outbreak of acute febrile illness and pulmonary haemorrhage, Nicaragua, 1995. The Epidemic Working Group at Ministry of Health in Nicaragua. Lancet 1996 February 24;347(9000):535-6.
(14) Park SK, Lee SH, Rhee YK, Kang SK, Kim KJ, Kim MC et al. Leptospirosis in Chonbuk Province of Korea in 1987: a study of 93 patients. Am J Trop Med Hyg 1989 September;41(3):345-51.
(15) Edwards CN, Nicholson GD, Everard CO. Thrombocytopenia in leptospirosis. Am J Trop Med Hyg 1982 July;31(4):827-9.
(16) Abdulkader RC. Acute renal failure in leptospirosis. Ren Fail 1997 March;19(2):191-8. (17) Dupont H, Dupont-Perdrizet D, Perie JL, Zehner-Hansen S, Jarrige B, Daijardin JB.
Leptospirosis: prognostic factors associated with mortality. Clin Infect Dis 1997 September;25(3):720-4.
(18) Panaphut T, Domrongkitchaiporn S, Thinkamrop B. Prognostic factors of death in leptospirosis: a prospective cohort study in Khon Kaen, Thailand. Int J Infect Dis 2002 March;6(1):52-9.
(19) Vinetz JM. A mountain out of a molehill: do we treat acute leptospirosis, and if so, with what? Clin Infect Dis 2003 June 15;36(12):1514-5.
(20) Panaphut T, Domrongkitchaiporn S, Vibhagool A, Thinkamrop B, Susaengrat W. Ceftriaxone compared with sodium penicillin g for treatment of severe leptospirosis. Clin Infect Dis 2003 June 15;36(12):1507-13.
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(21) Suputtamongkol Y, Niwattayakul K, Suttinont C, Losuwanaluk K, Limpaiboon R, Chierakul W et al. An open, randomized, controlled trial of penicillin, doxycycline, and cefotaxime for patients with severe leptospirosis. Clin Infect Dis 2004 November 15;39(10):1417-24.
(22) Takafuji ET, Kirkpatrick JW, Miller RN, Karwacki JJ, Kelley PW, Gray MR et al. An efficacy trial of doxycycline chemoprophylaxis against leptospirosis. N Engl J Med 1984 February 23;310(8):497-500.
(23) Smits HL, Chee HD, Eapen CK, Kuriakose M, Sugathan S, Gasem MH et al. Latex based, rapid and easy assay for human leptospirosis in a single test format. Trop Med Int Health 2001 February;6(2):114-8.
(24) Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Lab Immunol 2001 January;8(1):166-9.
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IPARTCoagulation and endothelium
Wha
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thogen
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What role do coagulation disorders play in the
pathogenesis of Leptospirosis?
J.F.P. Wagenaar¹, M.G.A. Goris², M.S. Sakundarno³, M.H. Gasem³, A.T.A. Mairuhu1, M.D. de Kruif1, H. ten Cate4, R.A. Hartskeerl², D.P.M. Brandjes¹ and
E.C.M. van Gorp¹, 5
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Royal Tropical Institute (KIT), KIT biomedical Research,
Amsterdam, the Netherlands3 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University,
Semarang, Indonesia 4 Department of Internal Medicine, University of Maastricht,
Maastricht, the Netherlands5 Department of virology, Erasmus University, Rotterdam, the Netherlands
TropMedIntHealth2007;12(1):111-122.
2CHAPTER
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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 hemostatic derangements and multi
organ failure eventually leading to death. Although the hemorrhagic 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.
In human leptospirosis however limited data are available. Because of the growing
interest in this field, the impact of leptospirosis, and availability of new therapeutic
strategies, the authors reviewed the present evidenced regarding this topic in
leptospirosis and will provide suggestions for future research.
AbSTRACT
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Leptospirosis is a zoonosis of global importance (1) caused by infection with pathogenic
Leptospira species. Transmission occurs where humans come into direct or indirect
contact with urine of infected animals (2) Due to the longer survival of leptospires
in warm and humid conditions, leptospirosis is predominantly common in the (sub)
tropics but transmission occurs in both industrialized and developing countries (3).
The incidence of leptospirosis during outbreaks and in high exposure risk groups is
estimated to reach over 100 per 100,000 persons per year (WHO). However, this
incidence is probably heavily underestimated due to the lack of diagnostic tools in
endemic areas and the atypical presentation of the disease, resembling many other
illnesses including Dengue and other hemorrhagic fevers (4-6).
The clinical picture of leptospirosis varies from a febrile illness of sudden onset,
to a potentially fatal disease complicated by jaundice, renal failure and serious
haemorrhages. Pulmonary haemorrhage has become recognized among the most
important manifestation of human leptospirosis and is increasingly reported over the
world (7-12). Other bleeding manifestations include: haematuria, haematemesis,
melaena, epistaxis, petechiae, ecchymoses, bleeding from venipuncture sites and
subarachnoid bleeding (13). Pathologist’s findings in autopsies of humans and animals
underline the bleeding tendency, and show widespread haemorrhages throughout
the body (14-17).
Although the haemorrhagic potential of leptospirosis was already noted by Weil in
1886 (18), its pathophysiology is still not clearly elucidated, particularly regarding
the cause and mechanisms of bleeding. Theoretically, bleeding may be the result
of a defect in the primary hemostasis or a dysbalance in secondary hemostasis by
depletion of coagulation proteins due to enhanced coagulation or by activated
fibrinolysis.
Regarding therapy, there is some evidence that antibiotic treatment of leptospirosis
may be beneficial, even given in late stage of disease (19). However there is an urge
to improve therapy and supportive care, since severe leptospirosis still accounts for
many deaths. Novel therapeutic agents intervening with the coagulation and cytokine
cascades may be beneficial. Hence, understanding of the pathogenic mechanisms is
INTRODuCTION
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crucial. In this article the authors review current insights on the involvement of
abnormal hemostasis in the pathophysiology of leptospirosis. What is the evidence
for defects in primary hemostasis and is there any proof for exaggerated coagulation
activation and impaired fibrinolysis?
Clinical features of Leptospirosis
The symptoms seen in human leptospirosis are very diverse. Most infections are mild
and only a minority of infected patients will seek medical attention. These usually
present with a febrile illness and accompanying symptoms may include: chills,
headache, myalgia (especially intense calve pain), gastro-intestinal complaints and
mild hemorrhagic manifestations such as conjunctival suffusion. Skin symptoms
such as rash are seen less often and may be misdiagnosed as scrub-typhus or viral
infections.
The most severe presentation of icteric human leptospirosis, often referred to as
Weil’s disease, is characterised by jaundice, renal failure, extensive haemorrhage and
a high case fatality rate between 5-15% (3). Icteric leptospirosis occurs between 5 and
10% of all leptospirosis cases (20) and is thought not to be the result of hepatocellular
damage but rather sepsis related cholestasis (21). Raises in transaminase levels are
usually moderate and the liver function will restore to normal during recovery.
Thrombocytopenia is often reported but is not directly correlated with a higher
incidence of haemorrhage in leptospirosis. However thrombocytopenia is positive
correlated to the development of acute renal failure and the age of the patients
(22). Thrombocytopenia and renal failure were found not to be associated with
higher mortality in another retrospective study among 60 cases of leptospirosis (23).
The renal failure seen in leptospirosis is unique because it is hypokalemic and usually
non-oliguric (24). When, nonetheless oliguria develops, it is a significant predictor of
mortality (25). Leptospirosis can also severely affect the lungs. Pulmonary symptoms
may include cough, dyspnoea and haemoptysis and may eventually develop into adult
respiratory distress syndrome (ARDS) and severe pulmonary haemorrhage syndrome
(SPHS). Haemoptysis may not be evident until patients are intubated and therefore
clinicians should suspect SPHS in all patients with signs of respiratory distress, also
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without signs of haemoptysis (26). Some evidence suggests that an auto-immune
process is responsible for the damage to the pulmonary endothelium (16;17;26).
The severity of respiratory disease is not related to the presence of jaundice (27).
Other clinical manifestations of leptospirosis reported include aseptic meningitis,
cardiac involvement with ECG alterations and myocarditis and ocular involvement
with autoimmune associated anterior uveitis.
Table 1: Search strategy and selection criteria.
Citations were retrieved from Pub Med and MEDLINE databases, and from locally accessible
files of the KIT Royal Tropical Institute library, Amsterdam, the Netherlands. The single terms
“Leptospirosis”, “Weil disease”, “Hemostasis”, “Coagulation”, “Fibrinolysis”, “Inflammation”,
“Endothelium”, “Thrombocytopenia”, “Coagulation Protein Disorders”, “Disseminated
Intravascular Coagulation”, “Blood Coagulation Disorders”, were used and combinations of
these terms. Titles, abstracts and references were scanned for relevance on the current topic.
Both English and German language papers were reviewed.
Haemorrhagic syndromes in leptospirosis
Infection-associated activation of the coagulation cascade may lead to a wide
spectrum of clinical effects, ranging from clinical insignificant rise in laboratory
markers to severe thrombo-hemorrhagic syndromes such as disseminated
intravascular coagulation (DIC), haemolytic uremic syndrome (HUS), thrombotic
thrombocytopenic purpura (TTP) and vasculitis (28). Patients suffering from these
disorders may present with bleeding, thrombosis or both. Various hemostatic
markers are used to discriminate between these syndromes. Some of these markers
are summarized in Table 2.
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Table 2: Expected results of screening tests in haemorrhagic disorders.
Defect bT Platelet count PT APTT Fib D-dimerThrombocytopenia ↑ ↓ N N N NThrombocytopathy ↑ N N N N NDIC ↑ ↓ ↑ ↑ ↓ ↑
Factor VII N N ↑ N N NFactor XI, IX, VIII N N N ↑ N NMild hepatic disease N N ↑ ↑ N NSevere hepatic disease ↑ ↓ ↑ ↑ N NVasculopathies ↑ N N ↑ N N
Abbreviations:BT,bleedingtime;PT,prothrombintime;APTT,activatedpartialthromboplastin
time;Fib,fibrinogen;DIC,disseminatedintravascularcoagulation.
Is there any proof that leptospirosis patients suffer from thrombo-hemorrhagic
complications? Considering pathological findings, vasculitis with endothelial damage
and inflammatory infiltrates composed of monocytic cells, plasma cells, histiocytes
and neutrophils is thought to be the pathological hallmark in both human and
animal leptospirosis (2). In addition, petechial hemorrhages are commonly found
and may be extensive. Other findings include: pulmonary hemorrhages, intrahepatic
cholestasis, hypertrophy and hyperplasia of Kupffer cells, interstitial nephritis,
coronary arteritis and hemorrhagic necrosis in skeletal muscles (2;2;14;14;15;15;16
;16;17;17;29;29;30). The intense intra-alveolar hemorrhages seem to be unique for
leptospirosis, the morphological features include interstitial inflammatory infiltrates
and extravasations of red blood cells from the capillary bed, which are not seen in
other capillary leakage syndromes such as Dengue hemorrhagic fever and pulmonary
Hanta (17)
Whether thrombocytopenia is in any way due to DIC is a topic of ongoing research
in the field of leptospirosis. DIC is a potentially fatal syndrome where activation
of the coagulation cascade results in microvascular thrombosis together with a
macrovascular bleeding tendency due to depletion from blood cells and proteins,
including fibrinogen. Fibrin deposition is the result of tissue factor mediated
thrombin formation and simultaneous inhibition of anti-coagulant mechanisms
such as the protein C system. In chorus, high levels of PAI-1, a strong inhibitor of
fibrinolysis, and the effects of pro-inflammatory cytokines contribute to enhanced
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fibrin deposition (31). This combination of events may lead to multi organ failure
(MOF) and eventually death. DIC can be caused by several stimuli such as bacteria,
viruses and other invading pathogens.Some animal models showed evidence of DIC in
leptospirosis infected guinea pigs (14;29) and one experimental model showed DIC in
infected dogs (32), while others did not (16;30). Unfortunately, large clinical studies
related to the association of DIC and leptospirosis has not been reported. In DIC
levels of fibrin and its precursor protein fibrinogen, are usually low due to increased
consumption. Several reports concerning leptospirosis demonstrated increased levels
of plasma fibrinogen. These findings probably reflect properties of fibrinogen as an
acute phase reactant, although some authors attributed this phenomenon to severe
tissue damage, vascular endothelial injury or a compensating mechanism by the liver
in response to increased consumption (14;29;33-35).
Endothelial cell injury and vasculitis are generally accepted as major pathological
characteristics of leptospirosis. Vasculitis is a condition characterized by inflammation
of the vessel wall with reactive damage to mural structures causing endothelial cell
injury. The clinical result may lead to intravascular thrombosis, subsequent organ
infarction and dysfunction.
Lung samples of 12 humans, who died from leptospirosis, showed stimulated
vascular endothelium marked by swelling of endothelial cells, an increase in
pinocytotic vesicles, and giant dense bodies in the cytoplasm of these cells (36).
An experimental guinea pig model of pulmonary haemorrhage showed no signs of
systemic vasculitis (16). Pulmonary endothelial cell blebs were the only observation
made. Immunofluorescence showed the presence of IgM, IgG, IgA and C3 along the
alveolar basement membranes of the lungs, possibly causing haemorrhage. Other
guinea pig models showed significant vascular damage with vascular congestion and
detached endothelial cells (14;29;37). A marmoset monkey model, infected with
Leptospirainterrogans serovar Copenhageni, showed microscopic patterns of tissue
reaction comparable to dose seen in the severe forms of human leptospirosis. Besides
intense intra-alveolar hemorrhages, alveolar septal vessels were congested and
contained a higher number of megakaryocytes than the controls. The liver showed
mild interstitial oedema, vascular congestion and focal necrosis (17).
TTP and HUS are only scarcely reported in leptospirosis and probably don’t play a
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major clinical role. However, the diagnosis may be missed due to lack of diagnostic
tools in developing countries, endemic for leptospirosis. The clinical picture of
these syndromes are characterized by both thrombocytopenia and microangiopathic
haemolytic anaemia without any other clinically apparent cause. Some studies
distinguish the two syndromes with predominantly neurological abnormalities
without renal impairment in TTP, and renal failure together with minimal or absent
neurological symptoms in HUS. Laing et al. described in a case report the association
between TTP and leptospirosis (38). The case described concerned a patient
presented with progressive neurological deterioration and hemolysis. Post mortem
histology showed the characteristic hyaline thrombi within small vessels of the brain,
heart, lung and kidney, a finding not seen in DIC.
Impairment of renal functioning is one of the features of HUS and is a commonly found
symptom in leptospirosis. Based on the findings of thrombocytopenia, fragmented
red blood cells, reticulocytosis, high serum FDP’s and renal failure the diagnosis of
HUS in relation to leptospirosis was published as case report (39). Relative to the
frequent occurrence of renal failure in severe disease, the finding of HUS is probably
a rare phenomenon.
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Table 3: Results of tests of coagulation.
Test and study reference Mean value Mean valueCases Controls
PlateletsDaher de Francesco et al. 2002 (decreased: < 150 x 10³/mm³) 69 (SD 65)Edwards et al. 1986 (decreased: < 100 x 109/l)† 42.3 (SD 29.8)† 207.8 (67.2)Jaroonvesama et al. 1975 (129- 230 x 10³/mm³) 135Nicodemo et al. 1990 (150- 450 x 10³/mm³) 70Edwards et al. 1990 (decreased: ≤ 100 x 109/l) 125 (SD 84)Edwards et al. 1982 (decreased: ≤ 100 x 10³/mm³) 46.9 (SD 26.7)† 188.2 (SD 69.4)‡
ProthrombintimeDaher de Francesco et al. 2002 (12-14,4 seconds) 13.3 (SD 0.8)Jaroonvesama et al. 1975 (15-16 seconds) 25.1 Edwards et al. 1990 (no normal values denoted) 13.3 (SD 2.1)Edwards et al. 1982 (13- 15 seconds) 15.8 (SD 2)† 15.9 (SD 1.5)‡Sitprija et al. 1980 (70 -100 %) 78.9 (SEM 0.9)
ActivatedpartialthromboplastintimeDaher de Francesco et al. 2002 (32- 38,4 seconds) 32.7 (SD 2.1)Jaroonvesama et al. 1975 (55- 69 seconds) 73Edwards et al. 1990 (no normal values denoted) 28.6 (SD 12.3)Sitprija et al. 1980 (25-55 seconds) 33.4 (SEM 0.5)
Thrombintime(s)Daher de Francesco et al. 2002 (9,8- 11 seconds) 11 (SD 1.4)Jaroonvesama et al. 1975 (5- 6 seconds) 6.9
FibrinogenDaher de Francesco et al. 2002 (150- 380 mg/dl) 515 (SD 220)Jaroonvesama et al. 1975 (306 mg/100ml) 529Sitprija et al. 1980 (200- 400 mg/dl) 818 (SEM 57.6)
FDP(fibrinogendegradationproducts)Edwards et al. 1986 (< 7,5 μg/ml) 8.1 (SD 4.8)† 9.5 (SD 4.4)Jaroonvesama et al. 1975 (6- 9 μg/ml) 12.4Sitprija et al. 1980 (< 0,5 mg/ml) 3.7 (SEM 0.39)
FactorVJaroonvesama et al. 1975 (130%) 77Sitprija et al. 1980 (70- 120%) 90.8 (SEM 0.9)
FactorVIIIJaroonvesama et al. 1975 (120%) 113Sitprija et al. 1980 (70- 120%) 88.6 (SEM 1.3)
FactorXJaroonvesama et al. 1975 (130%) 113
† All thrombocytopenic patients‡ Non-thrombocytopenic leptospirosis cases
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General aspects of hemostasis and infection induced coagulation activation
Basically hemostasis is a balanced system of procoagulant and anticoagulant
mechanisms, consisting of the following parts: primary hemostasis, coagulation
(secondary hemostasis), anticoagulant mechanisms and fibrinolysis. The formation
of a hemostatic plug by adhesion and aggregation of platelets in close concert
with the endothelium is considered the primary hemostasis. Secondary hemostasis
encompasses a series of protease-zymogen reactions necessary to stabilize this
hemostatic plug with the formation of fibrin strands. This process is counteracted
by anticoagulant mechanisms, including the proteins C and S and the anti-thrombin-
heparin pathway. Finally, the plug is degraded by plasmin mediated cleavage of fibrin
strands during the process of fibrinolysis.
Coagulation activation and fibrin deposition during inflammation can be seen as
an important part of the host defence of the body against for example infectious
organisms, in order to limit the invading antigen and the inflammatory response to a
certain area (40). In humans, severe infection or sepsis invariably leads to systemic
coagulation activation, impairment of anti-coagulant mechanisms and inhibited
fibrinolysis (41;42). In the worst case scenario this may lead to DIC, which may besides
hemodynamic and metabolic derangements, contribute to MOF (31). Membrane
components of virtually all microorganisms are able to induce this syndrome.
The tissue factor pathway is the most important route for activation of the coagulation
cascade in DIC (31). A number of cells express tissue factor throughout the body
(43), for example circulating mononuclear cells when stimulated by proinflammatory
cytokines (44;45). The majority of cells expressing tissue factor are in tissues not in
direct contact with blood, but histological tissue factor appears to be present in al
blood tissue barriers (46). When exposed to blood, tissue factor binds to factor VIIa.
This complex catalyses the conversion of factor X to Xa which eventually leads to
fibrin clot formation.
Inhibitors of coagulation include anti-thrombin, proteins C and S and tissue factor
pathway inhibitor (TFPI). In severe human sepsis anti-thrombin and proteins C and
S plasma levels are markedly reduced (41;47;48). There is some evidence that the
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function of the TFPI system is impaired in patients with DIC (49). Animal models of
severe infection show activation of the fibrinolytic system, which is eventually shut of
by plasminogen activator inhibitor type 1 (PAI-1) activity causing a net procoagulant
state (50-52). This is in concert with severe human sepsis, where non-survivors show
besides derangements in coagulation activation, a more pronounced suppression of
the fibrinolytic system (28;41).
Platelets play an important role in inflammation induced coagulation activation as
well. They can be activated directly by endotoxins and proinflammatory mediators,
such as platelet-activating factor (PAF) (53;54). Once activated platelets start
expressing P-selectin on the membrane which mediates the adherence of platelets
to leucocytes and endothelial cells, but also the enhancement of tissue factor
expression on mononuclear cells (55).
There is an increasing body of evidence that supports the concept of an intensive
cross-talk between inflammation and coagulation. Activated coagulation proteases
have been shown to induce the release of (pro)inflammatory cytokines, whereas
some cytokines (13) elicit procoagulant effects (54).
Current insights in the pathogenesis of abnormal hemostasis in leptospirosis:
The following section will discuss the possible pathological mechanisms behind the
hemostatic changes found in leptospirosis.
Primaryhemostasis
Disorders of primary hemostasis are very common during the course of many
infectious diseases. In this regard thrombocytopenia is a well-documented feature in
leptospirosis, with a high incidence. The underlying mechanism of thrombocytopenia
is not always clear. It and may be the result of decreased thrombopoiesis, increased
platelet consumption due to immune or non-immune causes, thrombocytopathy or a
combination. Some authors suggested that bone marrow suppression, due to a direct
toxic effect of Leptospira could be cause the observed thrombocytopenia (56) or did
not rule out the possibility (57). Concerning non-immune platelet destruction, one
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study explored the possibility of platelet consumption due to DIC as an explanation
for the frequently observed thrombocytopenia in leptospirosis. Based upon laboratory
measurements the authors concluded that there was no causal relationship (58). In
general, it is assumed that thrombocytopenia is mainly immune mediated contributing
to enhanced clearance. For leptospirosis in this regard, some authors postulated
that this phenomenon could be attributed to a yet unknown platelet antibody (59),
however there are technical difficulties in the study of such antibodies (57). One
case report offered some evidence for immune mediated platelet destruction, as was
shown by high titres of surface bound immunoglobulin, the complement factor C3d
and the beneficial response to treatment with methylprednisolone and hydrocortisone
(60). Several other studies demonstrated peripheral platelet destruction by bone-
marrow aspirates, revealing hypercellularity and increased megakaryocytes (60;61).
Another study suggested, based on human post mortem lung fragments, that the
thrombocytopenia was determined by activation, adhesion and aggregation of
platelets to stimulated vascular endothelium (36). Platelet surface receptors with
high affinity for subendothelium adhesion glycoprotein’s (e.g. Von Willebrand) may
facilitate this process. Indeed an amorphous substance was found, interposed between
the endothelial cells and platelets in places where the subendothelial collagen was
not exposed. No fibrin was found in the platelet aggregates. An experimental guinea
pig model by Yang and colleagues (30) showed evidence for platelet activation,
reflected by increased plasma levels 11-dehydrogenate thromboxane B2 (11-DH-
TXB2) which is considered a sensitive marker. Aggregation of platelets and Kupffer
cell phagocytosis of platelets in the liver was another feature found.
Several genes of L.interrogans were found to encode proteins with close homology to
animal proteins which play an important role in hemostasis (62), including a protein
that resembles the mammalian platelet activation factor (PAF) acetylhydrolase
(pafAH) and another protein that showed similarity to von Willebrand factor type A
domains (Vwa). Also an orthologue of paraoxonase (Pon) was found, which hydrolyses
PAF through its arylesterase activity. It is possible that each of these proteins
contribute to hemostatic chances in leptospirosis. In the same study genes were
found encoding for haemolysins and sphingo-myelinase-like proteins. It is not clear
whether these proteins play a significant role in the pathogenesis of leptospirosis.
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Secondaryhemostasis
When mononuclear cells were stimulated in vitro by a virulent or non-virulent
strain of Leptospira interrogans serovar Icterohaemorrhagiae coagulant activity
was observed, measured by one-stage plasma recalcification time (63). There
was a significant difference in degree of induction between the virulent and non-
virulent strains. Cells incubated with the virulent strain developed significantly
higher coagulant activation expressed by shortening of the clotting time, than those
cells incubated with the non-virulent strain. Interestingly, there was no coagulation
activity observed in factor VII deficient blood. Based on these data the authors
concluded that mononuclear cells induced by (non-) virulent strains of Leptospira
expressed tissue factor dependent procoagulant activity. A small Indonesian cohort
study revealed activated coagulation, reflected by increased plasma levels of the
coagulation activation markers thrombin-antithrombin complex (TAT) and fibrin
fragments 1 and 2 (F1 + 2) in severe human subjects (unpublished results). Increased
D-dimer plasma levels showed evidence for active fibrinolysis. In contrast, a guinea
pig model showed a trend of declining thrombin-antithrombin (TAT) complexes after
inoculation of Leptospira(30). This observation is surprising, since TAT is a sensitive
marker of coagulation activation.
In gram negative sepsis, circulating endotoxins play a pivotal role by activating
coagulation via the tissue factor pathway (64-67). Endotoxins are lipopolysaccharide
(LPS) constituents of the outer membrane of gram-negative microorganisms.
Leptospiral LPS has structural, chemical and immunological properties resembling
those of gram-negative bacterial LPS (68). Nevertheless, it is relatively non-toxic to
cells or animals, but large doses can cause hemorrhages in mice (68). The possible
mechanism of coagulation activation in leptospirosis is summarized in figure 1.
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Figure 1: Mechanisms of coagulation activation in leptospirosis.
(1) Amplification loops: a. Activation of factor IX by the tissue factor-factor VIIa complex,
generates additional factor Xa. b. Thrombin induced factor XI activation, leading to additional
factors IXa and Xa. c. Activation of the essential co-factors V and VIII by thrombin.(2) Tissue
factor expression by monocytes and endothelial cells. Leptospiral LPS or other outer membrane
components may induce cytokine release by monocytes and/or endothelial cells. Both may
induce TF expression and thereby induce coagulation activation, inhibition of anticoagulant
pathways or fibrinolysis. (3) The anticoagulant pathways: proteins C and S, antithrombin
and tissue factor pathway inhibitor. (4) The process of fibrinolysis breaks down cross-linked
fibrin molecules. Coagulation activation triggers the activation of the fibrinolytic system by
increasing levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator
(uPA), followed by an increase of PAI-1, a strong inhibitor of the fibrinolysis.
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Anticoagulationpathways
Upon activation of protein C by the thrombin-thrombomodulin complex activated
protein C (APC) is a powerful inhibitor of the coagulation cascade, acting in concert
with protein S. APC is believed to be not only a potent anticoagulant but also an
enzyme that modulates a number of inflammatory processes through direct cellular
or indirect pathways (the latter through interaction with protease activated
receptors (PARs) or the endothelial protein C receptor (EPCR)). The nature and
extent of these inflammatory actions is the subject of ongoing research. While
pro-inflammatory cytokines may reduce the cellular expression of the cofactors
thrombomodulin and EPCR, diminishing the PC mechanism, in infectious disease
in general a second mechanism may undermine this natural anticoagulant system.
In patients with severe leptospirosis and significantly elevated concentrations of
antiphospholipid antibodies (33;69) the function of the protein C system may also be
inhibited (70). Antithrombin (AT), a circulating serine protease inhibitor, is another
important inhibitor of the activated coagulation system. The third anticoagulant
pathway consists of tissue factor pathway inhibitor (TFPI), primarily synthesized in
the microvascular endothelium. Its anticoagulant mechanism is due to quaternary
complex formation with factor X and tissue factor-factor VII, thereby impairing
coagulation. These pathways have never been studied in leptospirosis.
Fibrinolysis
In the circulating blood the process of fibrinolysis is important for limited proteolysis
of cross-linked fibrin molecules. Infection associated coagulation activation is
followed by activation of the fibrinolytic system due to increased levels of tissue
plasminogen activator (tPA) and urokinase plasminogen activator (uPA), followed by
an increase of PAI-1. The degree of rise in systemic PAI-1 concentration determines
whether a net procoagulant state occurs, such as in case of DIC (50). Only a limited
amount of data exists about the activation of the fibrinolytic system in leptospirosis.
Elevated levels of fibrin degradation products (FDP) in leptospirosis were reported in
a number of studies (29;32;33;35;58;71). There are no studies focusing on regulatory
pathways of fibrinolysis, like the PAI-1 protein.
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Leptospirosis and the endothelial cell
As referred earlier, pathological findings points to the endothelial cell to be
important in the pathophysiology of leptospirosis. The endothelial cell plays a crucial
role in the regulation of both coagulation and fibrinolysis (72). Endothelium is able
to express tissue factor, von Willebrand factor and a wide variety of cytokines in
reaction to various pathogens. Endothelial cells exposed to a pathogen lose their
anticoagulant properties, which results in a net procoagulant state (72). Increased
levels of thrombomodulin found in an animal model, may reflect endothelial cell
injury in leptospirosis (30) and point to soluble thrombomodulin as a marker for
endothelial damage. An other experimental model found that intact Leptospires and
leptospiral peptidoglycans activate cultured human endothelial cells, reflected by an
increased adhesiveness for neutrophilic granulocytes (73) This resembles the effects
of LPS on endothelial cells.
The complete genomic sequencing of the virulent serovar Lai identified a colA gene
encoding for microbial collagenase (62) The authors proposed that collagenase
mediated injury of the vascular endothelium may contribute to the loss of hemostasis
in human leptospirosis.
Inflammatory response to Leptospira
Cytokines play an important role in the activation of the coagulation cascade (54).
Surface exposed membrane components, such as LPS, trigger a general host immune
response. Thus far over 260 membrane associated proteins have been identified in
Leptospira, and for most of these the relevance with regard to an immunogenic
reaction remains to be established (74). Six surface exposed lipoproteins have been
identified, of which LipL32 and LipL 21 are of most interest, because they are found in
all pathogenic Leptospira (75;76). Both leptospiral LPS and LipL32 interact with the
Toll-like receptor (TLR)2 and CD14 to signal to activated macrophages (77). LPS of
gram negative bacteria interact predominantly with the TLR4 receptor. In contrast,
another study found a protective role for the TLR4 receptor in an experimental
leptospirosis mouse model (78), which suggest this receptor is of importance for
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leptospiral LPS. Injected tumor necrosis factor (TNF)-a activated the coagulation
system via the tissue factor pathway in healthy volunteers (67;79;80). Elevated
plasma concentrations of TNF-a were found in patients affected by leptospirosis (81)
and higher TNF-a levels were associated with disease severity and mortality (82).
Leptospiral peptidoglycans induce TNF-a release from peripheral blood mononuclear
cells in a dose dependant manner in vitro (83). Other in vitro experiments showed
that the inflammatory response was also mediated primarily by a Th1 response,
involved in cellular immunity. Increased levels of Interferon (INF) -g, interleukin (IL)-
12p40, IL-12 and TNF-a were found after stimulation with heat-killed Leptospira
(84;85). Leptospirainterrogans glycolipoprotein (GLP) was found to induce cellular
production of TNF-a and interleukin-10 in peripheral blood mononuclear cell cultured
from healthy donors (86).
Leptospirosis is one of the most prevalent zoonosis in the world with a clinical
picture varying from mild to a potential life threatening disease in which hemostatic
derangements play a central role. Despite these facts leptospirosis still is a neglected
disease, which explains that many crucial aspects concerning the pathogenesis are
unanswered. Besides antibiotic therapy, which is the cornerstone of treatment,
there is an urgent need to improve supportive treatment, especially in those cases
associated with life threatening bleeding complications.
From the studies reviewed we may conclude that also in leptospirosis, as in other
infectious disease, the bleeding tendency is the result of a dysbalance in the
hemostatic equilibrium, although it is unclear how this dysbalance is triggered and
what inflammatory and coagulation proteins are involved. The hemostatic dysbalance
may lead to DIC, but no human studies with modern, sensitive assays have been
done to elucidate this question. DIC is a clinical syndrome, and only a combination
of laboratory markers can establish or rule out the diagnosis (31). Ascertain such a
syndrome is important to asses the potential role of new treatment options available
in this regard.
DISCuSSION AND CONCLuSIONS
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It would be premature to speculate what is the origin of the bleeding phenomena.
However, in several studies the endothelial cell seems to be one of the target cells
in leptospirosis. After infection the endothelial cell may loose its anti-thrombotic
properties and we hypothesize that this might well be the link to a dysbalanced
coagulation cascade. The endothelial cell may influence hemostasis due to
stimulation of cytokines in concert with e.g. circulating lymphocytes or platelets or
by direct effects of the invading micro-organism, i.e. Leptospira. Ample evidence
stress the importance of the endothelium as the conductor of the orchestra of pro-
and anticoagulant pathways (72;87).
To understand the hemorrhagic complications of leptospirosis, there is an urgent need
for prospective studies. Efforts should be made to enrol both mild and severe cases
in all stages of disease, using case record forms to be able to relate clinical signs and
clinical outcome with laboratory disturbances. Sensitive laboratory tests available,
focusing on the different involved pathways should be used. Studies should address
the role of the endothelium because its possible central role in the pathogenesis of
leptospirosis as posed here. Emphasis should be put on identifying involved (pro)
inflammatory cytokines, given the intensive crosstalk between coagulation and
inflammation. Finally, contributing factors like age, gender and genetic profile
should be taken into account.
Such studies will provide us the data necessary to improve supportive and therapeutic
management strategies to treat this potentially fatal disease.
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(87) Levi M, Cate ten H, Poll van der T. Endothelium: interface between coagulation and inflammation. Crit Care Med 2002 May;30(5 Suppl):S220-S224.
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Leptospirosis with pulmonary hemorrhage,
caused by a new strain of serovar Lai: Langkawi
J.F.P. Wagenaar 1, 2, P.J. de Vries 2 and R.A. Hartskeerl 3
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands
2 Div. Infectious Diseases, Tropical Medicine & AIDS, Academic Medical Center, Amsterdam, the Netherlands
3 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
J.Travel.Med.2004;11(6):379-81.
3CHAPTER
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A 50 year old Dutch male developed fever, eight days after wading through a small
stream, one of the attractions of a package tour to Langkawi Island, Malaysia. On
the sixth day of his disease he developed diffuse pulmonary hemorrhages, severe
jaundice, rhabdomyolysis and a moderate renal insufficiency with circulatory
collapse, requiring intensive care and mechanical ventilation. Leptospirosis was
confirmed by ELISA and the Microscopic Agglutination Test. Four months later a
new Leptospira strain was isolated from blood, collected on admission. Monoclonal
antibody cross agglutination tests and PCR-finger printing techniques showed a close
relationship with serovar Lai. It is proposed to denote the strain as a sub-type of
serovar Lai, type Langkawi, strain Langkawi.
Leptospirosis should be incorporated in the differential diagnosis of fever in the
returning traveler, acknowledging that a history of exposure may be concealed.
AbSTRACT
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Leptospirosis is an important ubiquitous zoonosis, most common in humid (sub-)
tropical regions, caused by pathogenic strains of Leptospirainterrogans. Leptospirosis
is mainly an occupational infection associated with animal handling or contact with
their urine, soil or water. Portals of entry are abraded skin and mucous membranes.
Exposure to fresh water, but also dry agriculture, such as banana or sugar cane
cultivation, are risk factors (1). Adventure activities, such as rafting, are increasingly
the cause of exposure to pathogenic leptospires, but the following case illustrates
that the less adventurous tourist may also become infected.
A 50-year-old Dutch male, developed fever 5 days after returning from a holiday on
Langkawi Island, Malaysia. Additional complaints were myalgia, nausea, vomiting,
and diarrhea. After an additional 5 days he was referred to our hospital, the Academic
Medical Center in Amsterdam, division Infectious Diseases, Tropical Medicine & AIDS.
On physical examination he was ill with a temperature of 39.3° C, blood pressure
120/68 mmHg and pulse rate of 124/min, without dyspnea. Oxygen saturation was
96%. Both sclerae showed peripheral vascular injection without jaundice. There was
a discrete macular exanthema. Abnormal laboratory values were: WBC 16.2 x 109/L
(N 4.2 – 10.6); platelets 115 x 109/L (N 150 – 350); creatinine 174 mmol/L (N 70-
110); total (conjugated) bilirubin 53 (36) mmol/L (N < 18 (7)); AST 243 U/L (N < 40);
ALT 153 U/L (N < 45); CK 5421 U/L (N < 190). Urinalysis showed some erythrocytes,
leukocytes, a few granular casts and a strong reaction for protein and hemoglobin.
This indicated rhabdomyolysis.
Leptospirosis was suspected, but scrub typhus was also considered. Empiric treatment
with doxycycline was started. Although it was a non-adventure package tour, the
patient confirmed participating in a tourist activity, in which he waded through
mangrove forests and a small stream nearby the coast, eight days before developing
fever. Therefore, doxycycline was switched to amoxicillin. The Leptospira IgM-ELISA
INTRODuCTION
CASE REPORT
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was positive on day 2 with a titer of 1:160, and increased to 1:5120 on day 14. The
microscopic agglutination test (MAT) was strongly positive on day 14 for the Leptospira
interrogans serogroups Cynopteri and Icterohaemorrhagiae. During the first day of
admission, his condition deteriorated rapidly. Jaundice developed, followed by signs
of septic shock. He was transferred to the intensive care unit and received IV fluids,
dopamine and furosemide. The circulatory collapse was short-lived and dopamine
was discontinued after 12 hours. But progressive dyspnea developed. A chest X-ray
showed patchy opacifications compatible with pulmonary hemorrhage (Figure 1).
Figure 1: Chest X-ray of a 50-year-old male with pulmonary hemorrhage, caused by
leptospirosis.
Mechanical ventilation was started on day 2. This was complicated by severe
hemoptysis, requiring blood transfusion. The platelet count decreased to a nadir
of 84.109/L, twelve hours after admission; the PTT increased to a maximum value
of 13.7 seconds (N < 13), twelve hours after admission; the APTT remained within
normal limits; bilirubin rose to 258 mmol/L; albumin decreased to 20 g/L; ALT and AST
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increased to 370 U/L and 1110 U/L respectively; creatinine increased to 315 mmol/L
without oliguria. Hemoptysis lasted for approximately two days, and mechanical
ventilation could be discontinued after two and a half days. The patient recovered
gradually and was discharged on the eleventh day after admission. His recovery was
slow but complete. In follow-up there were no complains or signs of respiratory
distress, and a control chest X-ray showed no pulmonary sequelae.
On admission, a blood sample was taken and, according to standard procedures,
inoculated into EMJH culture medium and incubated at 30oC, routinely for 4 - 6
months. After 4 months the culture became positive. For further classification, the
isolate was grown to a density of 2–4 x 108 leptospires/ml. Serogroup determination
was done by the Microscopic Agglutination Test (MAT) using a panel of 43 rabbit anti-
Leptospira reference antisera, representative for 24 pathogenic and 5 saprophytic
serogroups and revealed that the isolate belonged to serogroup Icterohaemorrhagiae.
Subsequent typing of the isolate at the level of serovars was done by MAT using a
panel of 18 monoclonal antibodies (mAbs), characteristically agglutinating serovars
of the serogroup Icterohaemorrhagiae (2). The experiments were repeated twice
with the same result. The agglutination profile resembled that of serovar Lai but
differed in the agglutinations with 2 mAbs. Whereas mAb F52C2 strongly agglutinates
the serovar Lai reference strain, no agglutination was found with the isolate. On
the other hand, mAb F70C7 did agglutinate the isolate but does not agglutinate
serovar Lai. Differences in the agglutination profiles likely reflect differences in the
antigenic composition of the leptospiral LPS between Lai and the isolate.
Interestingly, the agglutination characteristics of the isolate closely resembled those
of strain AF 61, a ‘Lai-like’ isolate from a patient at the Andaman Islands, which in
turn serologically resembles the ‘Lai-like’ strain WH 20, isolated from a patient with
pulmonary haemorrhage in Korea (3). PCR-fingerprinting, using outwards directed
primers deduced from IS1500 and IS1533 (4;5) generated various patterns resembling
the patterns of serovar Lai (results not shown). Apparently, the isolate is genotypically
closely related, but not completely identical to the Lai reference strain. According
to the recommendations of the Subcommittee on the Taxonomy of Leptospira, we
propose that this antigenic and genotypic variant of Lai is denoted as a sub-type of
serovar Lai. We suggest the name serovar Lai type Langkawi, strain Langkawi.
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Figu
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The clinical presentation of leptospirosis can vary from a non-specific febrile illness
to a complicated, potentially fatal, disease after an incubation period of 2 to 30
days (6). An acute leptospiremic phase may be followed by an immune phase.
Symptoms usually start with a sudden onset of fever, chills, headache and myalgia.
Peripheral conjunctival suffusion is often seen. Jaundice is reported in 0-93% of
large confirmed case series, and is typically orange-yellow, caused by reversible
hepatocellular dysfunction. Renal failure is common in complicated disease. Severe
leptospirosis with hepatorenal complications and hemorrhages is often referred
to as Weil’s syndrome. The histopathological hallmark of leptospirosis is capillary
vasculitis. Oliguria is caused by tubular necrosis, and this clinical finding signals a
poor prognosis. Interstitial nephritis may also occur. Lungs are not often affected
but pulmonary hemorrhage (PH) is being reported increasingly all over the world
and several serovars have been implicated (7-10). Severe disease with PH may occur
without jaundice (10). Rhabdomyolysis and myocarditis are rare. Septic meningitis is
an infrequent complication of the late, immune phase of leptospirosis.
Because the pathogenic mechanism underlying this striking feature of leptospirosis
is not yet understood, it would be premature to speculate about the apparent
increase of PH in association with strain variation and increased virulence. Improved
diagnostic tools and increased awareness for leptospirosis with PH after the outbreak
in Nicaragua in 1995, probably contributed to an increase of reported incidence (10).
The differential diagnosis of leptospirosis includes dengue fever, Hantavirus infection,
hemorrhagic fevers, influenza, rickettsial infections, typhoid fever, brucellosis, and
others. Leptospirosis is commonly treated with penicillin, amoxicillin or doxycycline.
There is some evidence that treatment with antibiotics may improve recovery, even
when given in late stage disease (11).
Human leptospirosis is caused by pathogenic strains of Leptospirainterrogans (sensu
lato). Classically, the microscopic agglutination test (MAT) and cross agglutination
absorption test (CAAT) are used to identify serogroups and serovars respectively.
Within the more than 200 serovars, different antigenic profiles can be discriminated
with monoclonal antibodies (2). Genotypic classification shows dissimilarities with
DISCuSSION
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the serological classification, discriminating different pathogenic genomospecies (1).
There is a weak correlation between causative serovar and clinical presentation. Some
serovars, including serovar Lai, were incriminated during outbreaks of leptospirosis
with pulmonary hemorrhage (PH) in Nicaragua, the Seychelles and Korea (7-10). In
an outbreak of leptospirosis with PH on the Andaman Islands, notably flanking the
Malaysian coast near Langkawi, a “Lai –like” strain was isolated, but from a patient
without PH (3). The antigenic profile of this strain, determined by monoclonal
antibodies, is closest to the strain of our patient. Consistent with this finding, DNA
analysis revealed that the strain of our patient belongs to L.interrogans sensu stricto
and represents a newly recognized type of serovar Lai. Therefore, we designate this
strain as L.interrogans, serogroup Icterohaemorrhagiae, serovar Lai, type Langkawi,
strain “Langkawi”. We suspect that this variant of Lai is common in South-East Asia.
In conclusion, leptospirosis should be suspected in the febrile traveler. A history of
exposure should be actively sought for but is not a prerequisite for the diagnosis
leptospirosis. Pulmonary hemorrhage, requiring intensive care, and other
complications such as aseptic meningitis, Weil’s disease, acute renal failure and
cardiac involvement, should be anticipated.
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(1) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (2) Terpstra WJ, Korver H, van LJ, Klatser PR, Kolk AH. The classification of Sejroe group
serovars of Leptospira interrogans with monoclonal antibodies. Zentralbl Bakteriol Mikrobiol Hyg A 1985 July;259(4):498-506.
(3) Sehgal SC, Vijayachari P, Smythe LD, Norris M, Symonds M, Dohnt M et al. Lai-like leptospira from the Andaman Islands. Indian J Med Res 2000 October;112:135-9.
(4) Zuerner RL, Alt D, Bolin CA. IS1533-based PCR assay for identification of Leptospira interrogans sensu lato serovars. J Clin Microbiol 1995 December;33(12):3284-9.
(5) Zuerner RL, Bolin CA. Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays. J Clin Microbiol 1997 October;35(10):2612-7.
(6) Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003 December;3(12):757-71.
(7) Park SK, Lee SH, Rhee YK, Kang SK, Kim KJ, Kim MC et al. Leptospirosis in Chonbuk Province of Korea in 1987: a study of 93 patients. Am J Trop Med Hyg 1989 September;41(3):345-51.
(8) Trevejo RT, Rigau-Perez JG, Ashford DA, McClure EM, Jarquin-Gonzalez C, Amador JJ et al. Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua, 1995. J Infect Dis 1998 November;178(5):1457-63.
(9) Yersin C, Bovet P, Merien F, Clement J, Laille M, van RM et al. Pulmonary haemorrhage as a predominant cause of death in leptospirosis in Seychelles. Trans R Soc Trop Med Hyg 2000 January;94(1):71-6.
(10) Zaki SR, Shieh WJ. Leptospirosis associated with outbreak of acute febrile illness and pulmonary haemorrhage, Nicaragua, 1995. The Epidemic Working Group at Ministry of Health in Nicaragua. Lancet 1996 February 24;347(9000):535-6.
(11) Watt G, Padre LP, Tuazon ML, Calubaquib C, Santiago E, Ranoa CP et al. Placebo-controlled trial of intravenous penicillin for severe and late leptospirosis. Lancet 1988 February 27;1(8583):433-5.
REFERENCES
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Coagulation disorders in patients with severe
leptospirosis are associated with severe
bleeding and mortality
J.F.P. Wagenaar 1, M.G.A. Goris 2, D.L. Partiningrum 3, B. Isbandrio 4, R.A. Hartskeerl 2, D.P.M. Brandjes 1, J.C.M. Meijers 5, M.H. Gasem 3 and E.C.M. van Gorp 1, 6
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
3 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University, Semarang, Indonesia
4 Department of Microbiology, Leptospirosis Laboratory, Diponegoro University, Semarang, Indonesia
5 Depts. Vascular Medicine and Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
6 Department of virology, Erasmus University, Rotterdam, the Netherlands
TropMedIntHealth2010,15(2):152-59.
4CHAPTER
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Background: Leptospirosis is an acute, febrile illness, frequently complicated by
haemostatic abnormalities and high mortality.
Objective: To determine the involvement of the coagulation system in relation to
bleeding and poor outcome in patients with severe leptospirosis.
Methods:In a prospective study, parameters of 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),
decreased 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 that went
on to die. In this regard, PT prolongation was associated with mortality (OR 1.4,
95%CI: 1.0-1.8, p = .04). Bleeding occurred in 31 subjects (60%). Of these, 24 patients
had mild bleedings and 7 had severe haemorrhages. Thrombocytopenia (platelets
≤100x109/L) was significantly associated with clinical bleeding (OR 4.6, 95%CI: 1.3-
16). A subanalysis of patients with and without severe bleeding revealed a more
pronounced dysbalance of the coagulation system in patients with severe bleeding,
as reflected by a significant association with PT (OR 1.4, 95%CI: 1.0-1.8, p = .05)
and the TAT/ PAP ratio (OR 1.3, 95%CI: 1.0-1.6, p = .05), which is an indicator of the
balance between coagulation and fibrinolysis.
Overt disseminated intravascular coagulation (DIC) was found in 10 (22%) out of
the 46 patients for whom the score could be calculated. There was no significant
association between DIC scores, bleeding diathesis or poor outcome.
Conclusion:The coagulation system was strongly activated in leptospirosis patients.
This was more pronounced in the deceased and in patients with severe bleeding than
in the survivors and in those without severe bleeding.
AbSTRACT
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Leptospirosis, caused by pathogenic Leptospira spirochetes, is an acute febrile illness
wherein hemostatic abnormalities play a pivotal role (1). Disease transmission takes
place worldwide, but large outbreaks are usually reported from the (sub-) tropics
following floods, hurricanes and other disasters (2). The incidence of leptospirosis is
estimated to exceed 100 per 100.000 persons per year in areas at risk. Maintenance
hosts, like rats and other mammals spread the spirochete via their urine into the
environment. Humans get infected when Leptospira enter the body via mucosal
membranes, wounds or abraded skin. Hospitalized patients often present with
thrombocytopenia, haemorrhagic symptoms, jaundice and renal failure. Patients
usually die from septic shock complicated by multi-organ failure (MOF) and/or a
bleeding diathesis. Pathological findings reveal widespread haemorrhaging at mucosal
surfaces, muscles, peritoneum and various organs such as heart, lungs and kidneys (3).
Thrombocytopenia is frequently observed and is reported to be associated with poor
outcome (4;5). Despite accumulating knowledge regarding tissue factor-mediated
coagulation activation and impaired fibrinolysis during infection, to date there is
limited data available on the involved haemostatic pathways in human leptospirosis.
Some studies reported elevated fibrin degradation products in human cases (6;7) and
recently a Thai cohort study showed activation of the coagulation system (8).
The aim of this study was to elucidate coagulation, anticoagulant and fibrinolytic
pathways in patients with severe leptospirosis over time and to determine whether
these markers were associated with bleeding and poor outcome. The following
markers were measured on admission and during follow up: prothrombin time (PT),
activated partial thromboplastin time (APTT), fibrinogen, thrombin-antithrombin
complexes (TAT), prothrombin fragment 1+2 (F1+2), cross-linked fibrin degradation
products (D-dimer), protein C (PC), antithrombin (AT), plasminogen activator
inhibitor type -1 (PAI-1) and plasmin-antiplasmin complexes (PAP).
INTRODuCTION
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Inclusionandsamplecollection
A prospective study was carried out at the department of internal medicine of the
Dr. Kariadi hospital, Semarang, Indonesia. Inclusion took place from February 2005
till September 2006. All eligible severe leptospirosis cases were included in the
study. Severe cases were defined as hospitalized patients with clinical suspected
leptospirosis, presenting usually with at least one of the following symptoms or
signs: jaundice, renal failure, thrombocytopenia (platelets <100x109/L) and/or
haemorrhaging. Sepsis was defined using internationally accepted sepsis criteria (9).
Inclusion followed after written informed consent was given by the subject or his/
her caretaker when too ill to consent. The medical ethics committees of the Dr.
Kariadi hospital and the Slotervaart hospital both approved the study protocol.
For coagulation assays, 5 ml citrated blood was obtained in sodium citrate 3.2% tubes
(BD Vacutainer, Plymouth, UK). Blood samples were taken on admission and during
follow up on days 1, 2, 7 and 14. Citrated plasma samples were aliquoted and stored
at -70°C until further testing.The algorithm for overt disseminated intravascular
coagulation (DIC) formulated by the DIC subcommittee of the International Society
on Thrombosis and Haemostasis (ISTH) was used to calculate overt DIC scores (10).
A platelet count <100x109/L was assigned 1 point, <50x109/L 2 points. An elevated
D-dimer between 400 and 4000 µg/L scored 2 points, >4000 µg/L scored 3. One point
was assigned to a prothrombin time prolongation between 3 and 6 seconds and >6
seconds received 2 points. Plasma fibrinogen <1.0 g/L received 1 point. A score ≥5
was compatible with an overt DIC.
Diagnosticprocedures
Clinical diagnosis of leptospirosis was confirmed by either a positive culture,
LeptoTek Dri-Dot assay (Biomérieux), in house PCR, microscopic agglutination test
(MAT) or a combination. For the MAT a panel of 31 serovars was used (28 pathogenic
serovars: Australis, Bratislava, Autumnalis, Rachmati, Ballum, Castellonis, Bataviae,
Benjamini, Whitcombi, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni,
Icterohaemorrhagiae, Lai, Naam, Coxi, Javanica, Panama, Pomona, Proechimys,
METHODS
Coagulationdisordersinsevereleptospirosis
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Pyrogenes, Sarmin, Hardjo, Saxkoebing, Sejroe, Shermani, Tarassovi and 3 non-
pathogenic serovars: Andamana, Patoc and Semaranga). A titre of ≥ 1:320 on a single
sample, seroconversion or a fourfold or higher increase of the titre in paired samples
or a titre of ≥ 1:40 in a single sample from early deceased patients with a clinical
diagnosis of leptospirosis, were considered to be positive. After inclusion, blood was
cultured at the bedside onto EMJH, EMJH + 5 fluoruracil and Fletcher medium and
incubated at 30°C. Cultures were checked every 2 weeks for growth with dark field
microscopy during a maximum of 4 months. All diagnostic tests were performed at
Department of Microbiology, and Center of Biomedical Research (CEBIOR), Faculty
of Medicine, Diponegoro University, Indonesia. Crosschecks were performed at the
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on
Leptospirosis, City, the Netherlands.
Coagulationandfibrinolysisassays
Measurements of F1+2 (Dade Behring, Marburg, Germany), TAT complexes (Dade
Behring), PAP complexes (DRG, Marburg, Germany), D-Dimers (Diagnostica Stago,
Asnières-sur-Seine, France), PAI-1 antigen (Hyphen BioMed, Andrésy, France), were
performed by ELISA. PC was determined using the Coamatic protein C activity kit from
Chromogenix (Mölndal, Sweden). Coagulation times (PT and APTT) were determined
on a Behring Coagulation System according to protocols from the manufacturer (Dade
Behring). The fibrinogen concentration was derived from the change in optical signal
during prothrombin time determination. AT activity was determined with Berichrom
Antithrombin (Dade Behring).
Statisticalanalyses
Relevant patient characteristics are presented as medians with corresponding ranges
or interquartile ranges or as numbers with percentages. In case of categorical data
the Chi square test was used or Fisher’s exact test when the expected cell count
was less than 5. Continuous variables were statistically evaluated using the non-
parametric Mann-Whitney U test. Associations between markers of coagulation and
outcome (dependent factors) were calculated using a univariate binary logistic
regression approach and denoted as odds ratios (OR) with 95% confidence intervals
Chap
ter4
60
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
(CI). In the model we used as dependent factors: bleeding overall, mild bleeding or
severe bleeding and the following covariates: PT, APTT, fibrinogen, thrombocytopenia
(platelets <100x109/L), TAT, F1+2, D-dimer, AT, PC, PAP, PAI and TAT/PAP x 100. All
test were 2 tailed with the a set to 0.05. Analyses were done using SPSS (version
15.0, Chicago, Illinois).
Patients
Fifty-two consecutive patients with severe leptospirosis were enrolled in the study.
Among these were 37 males (71%) and 15 (29%) females. The median age of the
cohort was 45 years (IQR 32-55). Fourteen patients (27%) died during follow up, after
a median of 3 days (IQR 1-5) post-admission.
Twenty-eight patients (54%) fulfilled sepsis criteria and 19 (37%) fulfilled the severe
sepsis criteria on admission. The remaining 5 non-septic patients all survived. All
patients had serological evidence of acute leptospirosis. The MAT results revealed
serovar Icterohaemorrhagiae (n=15) and Bataviae (n=14) as the most prevalent
infecting serovars. Other reactive serovars included: Sejroe, Pyrogenens, Autumnalis,
Ballum and Javanica. Sixteen samples were not classifiable by MAT because of
multiple reactive serovars. Patient characteristics are summarized in table 1.
RESuLTS
Coagulationdisordersinsevereleptospirosis
61
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34Tabl
e 1:
Pat
ient
cha
ract
eris
tics
on
adm
issi
on.
n
All
Pati
ents
Surv
ivor
sN
on-s
urvi
vors
Nor
mal
p-va
lue
Gen
eral
Num
ber
of c
ases
5238
14M
ale
sex
(%)
5237
(71
)29
(58
)8
(57)
0.2
Age,
med
ian
(IQ
R)52
45 (
33-5
5)42
(31
-55)
47 (
40-5
5)0.
3D
PO,
med
ian
(IQ
R)52
6 (5
-8)
7 (6
-8)
6 (5
-8)
0.9
Day
s at
hos
pita
l, m
edia
n (I
QR)
5210
(6-
14)
11 (
10-1
4)3
(2-6
)Se
psis
(%)
5228
(54
)23
(61
)5
(36)
0.1
Seve
re s
epsi
s (%
)52
19 (
37)
10 (
26)
9 (6
4)0.
01
Physicalexamination
Tem
pera
ture
(ºC
), m
edia
n (I
QR)
5238
.0 (
37.7
-38.
5)38
.0 (
37.6
-38.
4)38
.2 (
37.6
-38.
6)0.
5Pu
lse
(BPM
), m
edia
n (I
QR)
5210
0 (9
2-10
8)10
0 (9
2-10
4)10
0 (9
2-10
8)0.
9Sy
stol
ic b
lood
pre
ssur
e (m
mH
g),
med
ian
(IQ
R)52
110
(110
-128
)11
0 (1
09-1
20)
120
(108
-130
)0.
3D
iast
olic
blo
od p
ress
ure
(mm
Hg)
, m
edia
n (I
QR)
5270
(60
-80)
70 (
65-8
0)70
(60
-80)
0.4
Resp
irat
ory
rate
(br
eath
s/m
in),
med
ian
(IQ
R)52
24 (
20-2
8)24
(20
-24)
26 (
20-3
6)0.
04Ja
undi
ce (
%)52
42 (
81)
32 (
84)
10 (
71)
0.4
Olig
uria
(%)
5212
(23
)7
(18)
5 (3
6)0.
3An
uria
(%)
522
(4)
02
(14)
0.07
Hae
mor
rhag
ic m
anif
esta
tion
ove
rall
(%)
5231
(60
)21
(55)
10 (
71)
0.3
Mild
ble
edin
g (%
)24
(46
)18
(47
)6
(43)
0.8
Seve
re b
leed
ing
(%)
7 (1
4)3
(8)
4 (2
9)0.
08
Labo
ratoryresults
Hb
(gr/
dl),
med
ian
(IQ
R)52
11.3
(10
.2-1
2.3)
11.4
(10
.6-1
2.4)
10.9
(9.
1-11
.6)
12.0
-15.
00.
1H
t (%
), m
edia
n (I
QR)
5133
.5 (
30.6
-36.
5)34
.1 (
31.3
-37.
0)31
.4 (
27.0
-34.
1)40
.0-5
4.0
0.1
Leuc
ocyt
es (
109 /
L),
med
ian
(IQ
R)51
15 (
11-1
8)16
(13
-18)
11 (
10-2
0)4-
110.
2Pl
atel
ets
(109 /
L),
med
ian
(IQ
R)51
66 (
35-1
35)
65 (
35-1
61)
76 (
34-9
6)15
0-40
00.
8Th
rom
bocy
tope
nia
≤100
x109 /
L (%
)52
35 (
67)
24 (
63)
11 (
79)
0.3
AST
(U/l
), m
edia
n (I
QR)
3866
(40
-112
)63
(39
-101
)68
(54
-299
)15
-37
0.3
ALT
(U/l
), m
edia
n (I
QR)
3753
(43
-76)
58 (
43-7
8)52
(38
-94)
30-6
50.
9Bi
lirub
in t
otal
(m
g/dl
), m
edia
n (I
QR)
358.
3 (3
.4-2
0.0)
8.3
(3.4
-19.
5)11
.1 (
7.3-
25.5
)0-
1.0
0.3
Crea
tini
n (m
g/dl
), m
edia
n (I
QR)
495.
4 (3
.1-7
.6)
4.7
(2.3
-7.5
)7.
2 (5
.7-9
.7)
0.6-
1.3
0.1
Num
bers
rep
rese
nt m
edia
n (I
QR)
val
ues
of 5
2 le
ptos
piro
sis
pati
ents
on
adm
issi
on.
P-va
lues
rep
rese
nt d
iffe
renc
e be
twee
n su
rviv
ors
and
non-
surv
ivor
s (M
ann-
Whi
tney
U t
est,
Chi
squ
are
test
or
Fish
er’s
exa
ct t
est
whe
n ap
prop
riat
e).
A p-
valu
e of
< .
05 w
as c
onsi
dere
d st
atis
tica
lly s
igni
fican
t.Abb
reviations:IQR,interqu
artileran
ge,DPO
,da
ypo
stonsetsym
ptom
s,BPM
,be
atspe
rminute.
Chap
ter4
62
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Haemorrhagicmanifestationsandglobalcoagulationtests
Thirty-one subjects (60%) showed clinical signs of bleeding. Of these 24 patients
had mild bleeding (petechiae n= 22, ecchymoses n= 3, epistaxis n= 2) and 7 had
severe bleeding (melaena n= 7, heamatemesis n= 2, heamaturia n=1). Bleeding
occurred more frequently in the non-survivors, but this difference was not significant
(p = .3). Thrombocytopenia (platelets <100x109/L) was observed in 35 subjects
(67%), whereas 19 subjects (37%) had a platelet count < 50x10^9/L. Median platelet
counts were significantly lower in patients with bleedings (p = .02). Furthermore,
thrombocytopenia (platelets ≤100x109/L) was significantly associated with clinical
bleeding (OR 4.6, 95%CI: 1.3-16) but not with mortality (OR 2.1, 95%CI: 0.5-9.0). On
admission the median APTT was prolonged (51s), whereas PT was high normal (12.9s).
Twenty-five subjects had a normal PT and a prolonged APTT. Subjects with severe
bleeding showed a prolonged PT compared to those without severe haemorrhagic
manifestations (14.9s vs. 12.9s) but this was not significant. Yet, severe bleeding was
associated with a more increased PT by binary logistic regression (OR 1.4, 95%CI:
1.0-1.8, p = .05). From the patients with severe bleeding, two had a normal PT (2
missing values) and one had a normal APTT (one missing value). Deceased displayed
a significant PT prolongation compared to the survivors (p = .04). In addition,
prolongation of either PT or APTT was associated with mortality (OR 1.4, 95%CI:
1.0-1.8, p = .04; OR 1.1, 95%CI: 1.0-1.2, p = .04). Fibrinogen was elevated during
admission in all subjects (median 9g/L). Massive consumption of fibrinogen (plasma
levels <1g/L) was not found in any of the subjects. Global coagulation tests and
markers of coagulation activation and fibrinolysis are presented in table 2.
Coagulationdisordersinsevereleptospirosis
63
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Tabl
e 2:
Mar
kers
of
coag
ulat
ion
and
fibri
noly
sis
on a
dmis
sion
in p
atie
nts
wit
h se
vere
lept
ospi
rosi
s.
A
ll pa
tien
tsSu
rviv
ors
Non
-sur
vivo
rsN
orm
al r
ange
p-va
lue
Test
n
n
n
Globa
lcoagulationtests
PT
(s)
, m
edia
n (I
QR)
4612
.9 (
12.0
-14.
5)28
12.8
(11
.8-1
3.8)
1014
.7 (
12.3
-17.
1)10
.7-1
2.9
0.04
APTT
(s)
, m
edia
n (I
QR)
4851
(44
-57)
3050
(43
-55)
1157
(48
-74)
25-3
80.
06Fi
brin
ogen
(g/
L),
med
ian
(IQ
R)52
9 (6
-13)
389
(7-1
3)14
9 (5
-14)
1.9-
4.0
0.7
Procoa
gulantactivity
TA
T (µ
g/L)
, m
edia
n (I
QR)
526.
9 (4
.3-1
3.6)
386.
6 4.
(1-9
.4)
149.
8 (6
.9-1
9.4)
<4.6
0.03
F1+2
(pm
ol/L
), m
edia
n (I
QR)
5130
1 (1
46-5
33)
3725
6 (1
46-4
73)
1444
4 (1
64-2
584)
53-2
710.
4D
-dim
er (
µg/L
), m
edia
n (I
QR)
5240
88 (
2831
-602
9)38
4018
(24
27-5
742)
1450
94 (
3122
-828
6)<4
000.
2
Anticoa
gulantactivity
AT
(%)
, m
edia
n (I
QR)
5265
(54
-78)
3867
(57
-81)
1456
(44
-71)
86-1
390.
02PC
(%)
, m
edia
n (I
QR)
5276
(56
-99)
3879
(64
-99)
1462
(53
-74)
70-1
200.
01
(anti-)Fibrinolyticactivity
PA
P (µ
g/L)
, m
edia
n (I
QR)
5186
2 (7
11-1
115)
3787
0 (7
22-1
130)
1480
0 (4
53-1
115)
221-
512
0.4
PAI (
ng/m
l),
med
ian
(IQ
R)52
122
(77-
296)
3811
4 (7
5-26
3)14
206
(100
-532
)10
-70
0.1
TAT/
PAP
rati
o (x
100
)51
0.8
(0.5
-2.0
)37
0.7
(0.4
-1.0
)14
1.0
(0.7
-4.0
)-
0.02
Hem
osta
tic
para
met
ers
of 5
2 le
ptos
piro
sis
pati
ents
mea
sure
d on
adm
issi
on.
P-va
lues
rep
rese
nt d
iffe
renc
e be
twee
n su
rviv
ors
and
non-
surv
ivor
s (M
ann-
Whi
tney
U t
est)
. A
p-va
lue
of <
.05
was
con
side
red
stat
isti
cally
sig
nific
ant.
Abb
reviations:IQR,in
terqua
rtilerange,TAT,thrombin-an
tithrombincomplexes,F1
+2,prothrom
binfragmen
tF1
+2,D-dim
er,cross-link
edfibrin
degrad
ationprod
ucts,PC
,proteinC,AT,antithrom
bin,PAP,plasm
in-antiplasm
incom
plexes,PAI-1,plasm
inogen
activatorinh
ibitortyp
e-1.
Chap
ter4
64
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Procoagulant,anticoagulantandfibrinolyticactivity
Figure 1 shows markers of coagulation and fibrinolysis over time in patients with
severe leptospirosis. Markers reflecting thrombin formation: TAT complexes, F1+2
and D-dimer were elevated on admission. TAT and F1+2 remained elevated until
day 14. The fibrin split product D-dimer showed a decreasing trend over time in the
survivors. On admission, procoagulant activity was not significant different between
subjects with and without bleeding (TAT: 6.8µg/L vs. 7.3µg/L; F1+2: 318pmol/L vs.
244pmol/L; D-dimer: 4674µg/L vs. 4078µg/L). Subjects with severe bleeding had
marked elevations of TAT and F1+2 levels compared to subjects without severe
bleeding (TAT: 16.4µg/L vs. 6.8µg/L; F1+2: 900pmol/L vs. 247pmol/L), but these
values were not statistically different. A binary logistic regression approach revealed
a weak association between severe bleeding and increased TAT levels (OR 1.1, 95%CI:
1.0-1.1, p = .02). In a sub analyses between survivors and non-survivors on admission,
TAT complexes were significantly higher in the deceased group (p = .03).
Anticoagulant markers were decreased (AT: 65%) or low (PC: 76%) on admission. In
the survivors both AT and PC showed an increasing trend over time. Anticoagulant
activity was not different between subjects with or without haemorrhages on
admission (PC: 77% vs. 71%; AT 67% vs. 61%). Both PC (p = .01) and AT (p = .02) were
lower in the deceased group when compared to the survivors.
All subjects had a marked activation of the fibrinolytic system on day 0, reflected by
elevated PAP plasma levels. During follow up PAP levels decreased. PAI-1, a strong
inhibitor of fibrinolysis was elevated on admission and during follow-up. There was
no statistical difference in PAP or PAI-1 concentrations between subjects with or
without hemorrhages (PAP: 880µg/L vs. 852µg/L; PAI-1: 114ng/ml vs. 135ng/ml) or
survivors and patients that went on to die. However, patients with severe bleeding
had significant lower PAP levels then patients without this symptom (695µg/L vs.
884µg/L; p = .04).
To determine the balance between coagulation and fibrinolysis, the TAT/PAP ratio
was calculated. Patients who had severe bleedings displayed a significantly higher
TAT/PAP (x100) ratio (4.0 vs. 1.0; p = .03) compared to subjects without severe
bleedings. A significant association between TAT/PAP ratio and severe bleeding was
found (OR 1.3, 95%CI: 1.0-1.6, p = .05). Moreover, admission TAT/PAP ratios were
higher in the deceased group compared to the survivors (p = .02).
Coagulationdisordersinsevereleptospirosis
65
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
DICscores
The ISTH overt DIC algorithm was scored on admission to find evidence for
consumption coagulopathy and defibrination. Of 6 patients PT values were missing,
hence DIC scores could be calculated for 46 patients. The overall median overt DIC
score was 4 (IQR 3-4), 22% scored ≥5. Non-survivors scored a median of 4 points (IQR
3-5), survivors scored 4 (IQR 3-4) points as well (p = 0.2). DIC scores (number of
patients) were distributed as follows for the survivors: 2 (6), 3 (11), 4 (10), 5 (6), 6
(1) and non-survivors: 3 (4), 4 (5), 5 (2) and 7 (1). There was no significant association
between DIC scores and bleeding diathesis.
Chap
ter4
66
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Figure 1 A-E: Markers of coagulation and fibrinolysis over time in severe leptospirosis
patients.
Scatter dot plot diagrams of hemostatic parameters in patients with severe leptospirosis. Day
0 represents the day of admission. Survivors are compared to non-survivors where the open
symbols demonstrate the survivors and the closed symbols the non-survivors. Bars indicate the
median value.
Abbreviations: TAT, thrombin-antithrombin complexes, F1+2, prothrombin fragment F1+2,
D-dimer, cross-linked fibrin degradation products, PC, protein C, AT, antithrombin, PAP,
plasmin-antiplasmincomplexes,PAI-1,plasminogenactivatorinhibitortype-1.
Leptospirosis contributes importantly to clinical disease in Southeast Asia and
is considered endemic to Indonesia (11). During the rainy season, leptospirosis is
among the most important causes of hospitalisation and death in Semarang. Clinical
bleeding is frequently observed in severe cases. The pathophysiological mechanisms
are largely unknown but a dysbalanced secondary hemostasis might contribute to the
clinical picture.
In the present study we demonstrate elevated markers of coagulation activation,
fibrinolysis and low levels of anticoagulant markers in patients suffering from severe
leptospirosis. Overall, markers were not significantly different between patients with
DISCuSSION
Coagulationdisordersinsevereleptospirosis
67
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
and without bleeding. Thrombocytopenia however, was associated with bleeding,
whereas severe bleeding was associated with PT prolongation, elevated TAT
levels and increased TAT/PAP ratios. These disorders point towards a consumption
coagulopathy with aggregated coagulation activation and hyperfibrinolysis as seen
in other haemorrhagic fevers (e.g. dengue) (12). However, we can not exclude
the possibility of impaired function or synthesis of clotting factors. Interestingly,
14 patients had signs of (severe) bleeding but did not show disorders in either PT
or APTT. This, together with equal hemostatic derangements in patients with and
without bleeding makes, besides impaired secondary hemostatic pathways, another
pathophysiological mechanism likely. One hypothesis for the pathogenesis of the
observed hemostatic diathesis proposes a direct action on endothelial cells by
Leptospira. After rapid dissemination throughout the body during the first phase of
infection, Leptospira might activate or damage, directly or indirectly, the endothelial
surface. A dysbalanced secondary hemostasis, seen in virtually all septic patients
(13), might further contribute to bleeding and organ dysfunction.
Both deceased and survivors showed abnormal coagulation markers. In the deceased
these alterations were more pronounced and consisted of ongoing coagulation and
fibrinolysis activation and impaired anti-coagulation. Impaired synthesis might play a
role in these observations. Higher TAT/PAP ratios in the deceased showed a hemostatic
dysbalance that was shifted to a more procoagulant state. The only markers that
were associated with mortality were PT and to lesser extent APTT. In the survivors a
restoration of plasmatic coagulation was observed during follow-up. It is interesting
to speculate whether a prothrombotic state contributes to death or is just a
reflection of disease severity. In general, sepsis leads almost invariably to hemostatic
abnormalities (13). These disorders can range from an isolated thrombocytopenia or
prolonged global clotting tests to complex disorders such as DIC (14;15). Coagulation
activation during sepsis is largely dependent on the tissue factor-activated factor VII
complex (TF-FVIIa). Experimental work showed that inhibiting this complex during
endotoxemia completely attenuated coagulation activation in human volunteers
(16). Host-derived mediators of inflammation, such as TNF-a, are able to induce TF
expression on mononuclear cells and macrophages (17). There is some evidence that
mononuclear cells stimulated by pathogenic Leptospira are able to express tissue
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factor invitro (18). A recent paper provided evidence for coagulation activation in
patients with leptospirosis reflected by prolonged clotting times and elevated F1+2,
TAT and D-dimer levels (8). Moreover, nearly one half of patients with leptospirosis
had overt DIC as defined by the ISTH. In the present study 22% had a positive overt
DIC score. However, DIC was not associated with mortality in the present work. The
occurrence of DIC in leptospirosis has always been a subject of debate. Previous
experimental work in a leptospirosis model in guinea pigs showed evidence for
DIC in which heparin prolonged life (19;20). Pathology showed haemorrhaging,
deposits of fibrin and scattered foci of necrosis in various organs (20). In contrast,
no fibrin thrombi were found in liver lung or kidney in two leptospirosis pulmonary
haemorrhage guinea pig models (21;22). Human leptospirosis studies in the past
could not confirm DIC (5-7;23;24). However in these studies, disease severity and
used coagulation assays differed largely and were not compatible with the current
ISTH overt DIC algorithm. It has to be noted that this score is designed to define
severity of the haemostatic dysbalance but is not, or less helpful in understanding
disease pathophysiology.
Although the aim of this study was to describe (anti-) coagulation and fibrinolytic
pathways in patients with severe leptospirosis, it is interesting to discuss the possible
clinical consequences. The PT, global test of coagulation, was associated with both
clinical bleeding and poor outcome. This makes it a candidate tool to monitor
leptospirosis patients, especially in resource-poor settings, where such tools may aid
the clinician in allocating the scarce high-care facilities to those patients with high
chances of mortality and bleeding complications. Secondly, thrombocytopenia was
confirmed to be associated with bleeding complications. More studies are warranted
to explore the role of giving platelet concentrates to this group of patients. Last,
one could speculate that providing plasma or specific (recombinant) proteins
that interfere with the coagulation cascade could be beneficial in a subgroup of
leptospirosis patients.
Some issues of the present study merit further comment. Firstly, only patients with
severe leptospirosis were enrolled in the study. Hence our findings are only applicable
to patients with the severest forms of leptospirosis and further studies are required
to investigate hemostatic mechanisms in patients with mild leptospirosis. Next, most
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patients presented in late stage disease which can explain the fulminant clinical
picture and high mortality in this cohort. Last, the number of patients included in the
cohort was relatively limited. Therefore caution should be used when interpreting
this data.
In conclusion, patients with severe leptospirosis showed disorders in both coagulation
and fibrinolytic pathways which was more pronounced in the deceased and in those
with severe bleeding. The absence of marked derangements in patients with mild
bleeding compared to those without any signs of haemorrhaging points to the
involvement of other pathophysiological mechanisms. Future studies should focus on
other hemostatic pathways and new treatment strategies that restore coagulation
in patients suffering from severe leptospirosis with the ultimate goal to reduce
mortality from this potentially lethal disease.
Acknowledgements
We thank the persons and organizations that provided invaluable assistance during
this study: S.M.H. Faradz and personnel (CEBIOR, Diponegoro University, Semarang
Indonesia), residents of the department of Internal Medicine (Dr. Kariadi hospital,
Semarang, Indonesia), W.F. Kopatz (Department of Experimental Vascular Medicine,
AMC, Amsterdam the Netherlands) and A.A. Ahmed (Royal Tropical Institute,
Amsterdam, The Netherlands).
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(1) Wagenaar JF, Goris MG, Sakundarno MS, Gasem MH, Mairuhu AT, Kruif de MD et al. What role do coagulation disorders play in the pathogenesis of leptospirosis? Trop Med Int Health 2007 January;12(1):111-22.
(2) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (3) Arean VM. The pathologic anatomy and pathogenesis of fatal human leptospirosis
(Weil’s disease). Am J Pathol 1962 April;40:393-423. (4) Edwards CN, Nicholson GD, Everard CO. Thrombocytopenia in leptospirosis. Am J Trop
Med Hyg 1982 July;31(4):827-9. (5) Turgut M, Sunbul M, Bayirli D, Bilge A, Leblebicioglu H, Haznedaroglu I.
Thrombocytopenia complicating the clinical course of leptospiral infection. J Int Med Res 2002 September;30(5):535-40.
(6) Edwards CN, Nicholson GD, Hassell TA, Everard CO, Callender J. Thrombocytopenia in leptospirosis: the absence of evidence for disseminated intravascular coagulation. Am J Trop Med Hyg 1986 March;35(2):352-4.
(7) Sitprija V, Pipatanagul V, Mertowidjojo K, Boonpucknavig V, Boonpucknavig S. Pathogenesis of renal disease in leptospirosis: Clinical and experimental studies. Kidney Int 1980 June;17(6):827-36.
(8) Chierakul W, Tientadakul P, Suputtamongkol Y, Wuthiekanun V, Phimda K, Limpaiboon R et al. Activation of the coagulation cascade in patients with leptospirosis. Clin Infect Dis 2008 January 15;46(2):254-60.
(9) American College of Chest Physicians/Society of Critical Care Medicine. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992 June;20(6):864-74.
(10) Toh CH, Hoots WK. The scoring system of the Scientific and Standardisation Committee on Disseminated Intravascular Coagulation of the International Society on Thrombosis and Haemostasis: a 5-year overview. J Thromb Haemost 2007 March;5(3):604-6.
(11) Laras K, Cao BV, Bounlu K, Nguyen TK, Olson JG, Thongchanh S et al. The importance of leptospirosis in Southeast Asia. Am J Trop Med Hyg 2002 September;67(3):278-86.
(12) van Gorp EC, Setiati TE, Mairuhu AT, Suharti C, Cate HH, Dolmans WM et al. Impaired fibrinolysis in the pathogenesis of dengue hemorrhagic fever. J Med Virol 2002 August;67(4):549-54.
(13) Kinasewitz GT, Yan SB, Basson B, Comp P, Russell JA, Cariou A et al. Universal changes in biomarkers of coagulation and inflammation occur in patients with severe sepsis, regardless of causative micro-organism [ISRCTN74215569]. Crit Care 2004 April;8(2):R82-R90.
(14) Levi M, van der Poll T. Coagulation in sepsis: all bugs bite equally. Crit Care 2004 April;8(2):99-100.
(15) Levi M, Opal SM. Coagulation abnormalities in critically ill patients. Crit Care 2006;10(4):222.
(16) de Jonge E., Dekkers PE, Creasey AA, Hack CE, Paulson SK, Karim A et al. Tissue factor pathway inhibitor does not influence inflammatory pathways during human endotoxemia. J Infect Dis 2001 June 15;183(12):1815-8.
(17) Schouten M, Wiersinga WJ, Levi M, van der Poll T. Inflammation, endothelium, and coagulation in sepsis. J Leukoc Biol 2007 November 21.
(18) Miragliotta G, Fumarola D. In vitro effect of Leptospira icterohaemorrhagiae on human mononuclear leukocytes procoagulant activity: comparison of virulent with nonvirulent strain. Can J Comp Med 1983 January;47(1):70-2.
REFERENCES
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(19) Higgins R, Cousineau G. The pathogenesis of leptospirosis I. Hemorrhages in experimental leptospirosis in guinea pigs. Can J Comp Med 1977 April;41(2):174-81.
(20) Pereira da Silva JJ, Netto BA, Lilembaum W, Alvim ME, de Oliveira AV. The hemorrhagic syndrome of leptospirosis: an experimental study in guinea pigs. Rev Soc Bras Med Trop 1995 July;28(3):169-77.
(21) Yang HL, Jiang XC, Zhang XY, Li WJ, Hu BY, Zhao GP et al. Thrombocytopenia in the experimental leptospirosis of guinea pig is not related to disseminated intravascular coagulation. BMC Infect Dis 2006 February 2;6(1):19.
(22) Nally JE, Chantranuwat C, Wu XY, Fishbein MC, Pereira MM, Da Silva JJ et al. Alveolar septal deposition of immunoglobulin and complement parallels pulmonary hemorrhage in a guinea pig model of severe pulmonary leptospirosis. Am J Pathol 2004 March;164(3):1115-27.
(23) Nicodemo AC, Del NG, Amato N, V. Thrombocytopenia and leptospirosis. Rev Inst Med Trop Sao Paulo 1990 July;32(4):252-9.
(24) Daher de Francesco E, Oliveira Neto FH, Ramirez SM. Evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis. Rev Inst Med Trop Sao Paulo 2002 March;44(2):85-90.
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bleeding in patients with severe leptospirosis
is not associated with activation of endothelial
cells
J.F.P. Wagenaar 1, M.H. Gasem 2, J.C.M. Meijers 3, E.C.M. van Gorp 1, 4 and D.P.M. Brandjes 1
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University,
Semarang, Indonesia 3 Depts. Vascular Medicine and Experimental Vascular Medicine Academic Medical Center,
University of Amsterdam, Amsterdam, the Netherlands4 Department of virology, Erasmus University, Rotterdam, the Netherlands
Submitted
5CHAPTER
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Objectives: To investigate endothelial cell involvement in relation to bleeding
manifestations in patients with severe leptospirosis by studying the dynamics of the
endothelial cell specific markers soluble E-selectin (sE-selectin) and von Willebrand
factor (vWF).
Methods: Cases of severe leptospirosis were included in the Dr. Kariadi hospital
Semarang, Indonesia. Blood was taken on admission and during follow up. Twenty
healthy subjects served as controls.
Results:52 patients were included, of which 37 (71%) males. The median age was 45
(IQR 33-55) years. In total 14 patients (27%) did not survive. The mean sE-selectin
(169 ng/ml; SD 73) and vWF (mean: 500%; SD 182) levels were strongly elevated on
admission compared to the controls (sE-selectin: 29 ng/ml, p < .0001; vWF: 91%, p <
.0001). Both markers decreased during follow up, but did not reach normal values.
In a subgroup analysis between patients with bleeding (n=31) and those without
bleeding (n=21), sE-selectin (175 ng/ml vs. 161 ng/ml) and vWF (511% vs. 483%)
levels were not significantly different. There was no statistical difference in both
sE-selectin (157 ng/ml vs. 203 ng/ml) or vWF (489% vs. 530%) plasma concentrations
between patients that survived and those that went on to die.
Conclusion: Markers of endothelial cell activation are strongly elevated in patients
suffering from severe leptospirosis, regardless of bleeding manifestations or outcome.
AbSTRACT
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Leptospirosis is an infectious disease of global importance (1). The disease is caused
by spirochetes that are spread by the urine of infected animals. Mucous membranes
small cuts and abraded skin are the usual ports of entrée. Although the clinical
course varies widely, leptospirosis can be fatal in up to 50% of all cases (2). The more
common mild form of leptospirosis is characterized by non-specific symptoms such as:
acute fever, headache chills and myalgia. In the severest forms, the clinical picture
of leptospirosis encompasses jaundice, renal failure and apparent hemorrhaging of
skin, mucous membranes and/or lungs. Pathological findings confirm widespread
hemorrhaging and endothelial cell damage throughout the body as a clinical hallmark
(3).
Although the pathophysiology of the hemorrhagic diathesis in leptospirosis is unclear,
bleeding might be the result of endothelial dysfunction (4). Under physiological
conditions the vascular endothelium inhibits coagulation, prevents platelet
aggregation and due to low levels of expressed adhesion molecules, it precludes
adherence and migration of leucocytes. Injury or activation of endothelial cells in
response to pathogens or inflammatory cytokines can cause bleeding due to loss of
integrity of the blood vessel or consumption coagulopathy (5).
Currently several biomarkers are available that are thought to be a good reflection
of endothelial cell activation and damage. E-selectin (ELAM-1) is such a molecule
as it is exclusively expressed on endothelial cells (6). Exposure of endothelial cells
to endotoxin or proinflammatory cytokines leads to a massive increase of E-selectin
surface expression (7). An important function of E-selectin is promoting neutrophil
attachment to the endothelial cell surface. Expression of E-selectin requires de novo
protein synthesis. Hence, maximal expression is reached after several hours.
Von Willebrand factor another marker of endothelial cell involvement, is a large
glycoprotein synthesized in the vascular endothelium. Endothelial cells store vWF in
the Weibel-Palade bodies in the form of unusually large vWF multimers. The function
of vWF comprises adhesion of platelets to exposed subendothelium and platelet
aggregation, forming the basis for the hemostatic plug. Hence, low plasma levels of
vWF can cause bleeding. In addition to its function, vWF binds and carries factor VIII,
protecting it from rapid removal from the circulation.
INTRODuCTION
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The aim of this work was to investigate endothelial cell dysfunction in relation to
bleeding in patients suffering from severe leptospirosis using two endothelial specific
markers: sE-selectin and vWF.
Patientsandcontrols
Consecutive cases of severe leptospirosis were included from February 2005 till
September 2006 at the Dr. Kariadi hospital, Semarang, Indonesia. Severe leptospirosis
was defined as hospitalized patients with a high clinical suspicion of leptospirosis,
presenting with at least one of the following symptoms or signs: jaundice, renal
failure, thrombocytopenia and/or bleeding and a positive LeptoTek Dri-Dot assay
(Biomérieux). We defined bleeding as the spontaneous occurrence of: petechiae,
ecchymoses, epistaxis, gum bleeding, haematuria, melaena, haematemesis,
haemoptysis and/or other. Sepsis was defined using standard sepsis criteria (8). This
was a static classification scored on admission; progression from e.g. sepsis to severe
sepsis within days was not taken into account. After written informed consent was
given, blood samples were taken on admission and during follow up at day 1, 2, 7
and 14. Citrated blood was worked up immediately and aliquots were stored at -70°C
until further analyses. As controls, 20 healthy Indonesian volunteers were tested. The
medical ethics committees of the Dr. Kariadi hospital and the Slotervaart hospital
both approved the study protocol.
MeasurementsandAssays
Clinical diagnosis of leptospirosis was confirmed by the microscopic agglutination
test (MAT). For the MAT a panel of 31 serovars was used (28 pathogenic serovars and
3 non-pathogenic serovars). A titre of ≥ 1:320 on a single sample, seroconversion or a
fourfold or higher increase of the titre in paired samples or a titre ≥ 1:80 in a single
sample from early deceased patients, were considered to be positive.
Both endothelial markers, sE-selectin (R&D Systems, Minneapolis, MN, USA) and vWF
(antibodies from Dako, Glostrup, Denmark) were determined by ELISA.
MATERIALS AND METHODS
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Statisticalanalysis
Patient characteristics are presented as medians with corresponding interquartile
ranges (IQR), means with standard deviations or as numbers with percentages when
appropriate. Continuous variables were statistically evaluated using the Students
t-test or analyses of variance (one-way ANOVA). Associations were expressed as odds
ratios (OR) and in case of dichotomous data calculated by cross-tabulation. All test
were 2 tailed with the a set to 0.05. All analyses were done using SPSS (version 15.0,
Chicago, Illinois).
Patients
Fifty-two patients were included in the study, of which 37 (71%) males. The median
age was 45 (IQR 33-55) years. Fourteen patients (27%) died during admission. The
median time from admission to death was 3 days. Bleeding was apparent in 31
(60%) subjects; of these 21 survived and 10 died (difference not significant). We
recognized: petechiae (n= 25), ecchymoses (n= 3), epistaxis (n= 2), melaena (n= 7),
haematemesis (n= 2) and haematuria (n=1). On admission 5 patients were stratified
as non-septic, 28 as septic and 19 as severe septic. Statistically, bleeding was equally
distributed among the non-septic, septic and severe septic patients. The median
plasma creatinine and bilirubin levels were 5.4 mg/dl (IQR 3.1-7.6) and 8.3 mg/
dl (IQR 3.4-20) respectively. Liver enzymes were only mildly elevated: ASAT 66 U/l,
ALAT 53 U/l. Thrombocytopenia (platelets ≤100x109/L) was recognized in 32 (62%)
subjects.
SolubleE-selectinandvWFarenotassociatedwithbleeding
Soluble E-selectin (mean: 169 ng/ml; SD 73) and vWF (mean: 500%; SD 182) levels
in severe leptospirosis patients were strongly elevated on admission. The controls
displayed significant lower mean values: sE-selectin: 29 ng/ml (SD 9) and vWF: 91%
(SD 29) on all time points (figure 1). Both endothelial markers decreased gradually
during follow up, but did not reach normal values on day 14 of follow up. When
RESuLTS
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comparing the subgroup of patients with (n=31) and without (n=21) hemorrhagic
manifestations on admission, no significant difference was observed in sE-selectin
(175 ng/ml vs. 161 ng/ml; p = .50) or vWF (511% vs. 483%; p = .60) levels, see figure
2A-B.
Figure 1A: Plasma levels of soluble E-selectin in patients with severe leptospirosis over time.
Figure 1b: Plasma levels of vWF in patients with severe leptospirosis over time.
Soluble E-selectin and vWF plasma levels over time in patients suffering from severe leptospirosis
(n=52). Results are presented as means with error bars indicating the standard error of the
mean. The values denoted in the bars indicate the number of available samples. * p-value <
0.0001 (significant difference from controls).
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Figure 2A-b: Soluble E-selectin and vWF plasma levels in leptospirosis patients with and
without haemorrhagic manifestations.
Scatter plots show plasma levels sE-selectin and vWF in leptospirosis patients with (B) and
without (nB) bleeding manifestations on admission. Horizontal line indicates the mean.
Endothelialcellmarkersarenotassociatedwithmortalityanddiseaseseverity
Next we analyzed the subgroup survivors and non-survivors. The mean concentration
circulating sE-selectin in the survivors was 157 ng/ml (SD 62) and in the non-survivors
203 ng/ml (SD 89). Plasma levels vWF were in the survivors 489% (SD 182) and in non-
survivors 530% (SD 186). No significant difference in sE-selectin or vWF levels between
these groups was found. The controls showed significant lower sE-selectin (mean 29
ng/ml, SD 10, p < .001) and vWF (mean: 91 %, SD 29, p < .001) levels compared to
both survivors and deceased. In regard to disease severity we compared non-septic,
septic and severe septic patients on admission. Mean sE-selectin levels were: 136 ng/
ml (SD 40), 186 ng/ml (SD 77) and 153 ng/ml (SD 70), respectively. Mean vWF levels
were: 432 % (SD 126), 538 % (SD 209) and 463 % (SD 141). The differences between
non-septic, septic and severe septic patients were not significant.
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This study investigated the patterns of the endothelial specific markers sE-selectin
and vWF in relation to bleeding in patients with severe leptospirosis. Clearly sE-
selectin and vWF were highly expressed and circulating in patients with severe
leptospirosis, reflecting endothelial cell dysfunction. However, no association with
bleeding was observed.
Bleeding can be caused by dysfunction of primary hemostatic pathways that include:
platelet activation, adhesion and aggregation. Von Willebrand factor is crucial for
platelet adhesion, a process primarily mediated by the binding of platelet surface
receptor GP Ib/IX/V complex to vWF in the subendothelial matrix (9). Low levels of
vWF will cause bleeding, as is observed in patients suffering from von Willebrand’s
disease. In the current work bleeding was not the result of reduced levels of vWF.
Although a decreasing trend was observed during follow up, on day 14 vWF levels
were still elevated compared to the controls. We did not find significantly different
vWF levels in the deceased or in the sickest groups. Elevated levels of vWF have been
observed in several inflammatory disease states, including sepsis and septic shock
(10;11). Data that correlate plasma levels of vWF with severity of inflammation and
patient outcome, are inconsistent in the literature (12).
Under physiological conditions the endothelium has anticoagulant properties and
prevents platelet aggregation as well as the adherence and migration of blood
cells due to low expression of adhesion molecules such as E-selectin. Endothelial
cells lose their anticoagulant properties when stimulated by cytokines or other
inflammatory mediators. This transformation to a procoagulant surface is considered
to play a prominent role in all three major pathogenetic pathways associated with
coagulopathy in sepsis: tissue factor (TF) mediated thrombin generation, impaired
anticoagulant pathways and blocked fibrinolysis (7). Activated endothelium can
cause a dysbalance in the ignition of the plasmatic coagulation pathways which can
lead to both thrombosis and bleeding. High levels of E-selectin were observed in
the present study, reflecting endothelial cell activation or damage. Several studies
have indicated that sE-selectin is elevated during septic conditions and that high
levels are associated with poor clinical outcome (13). However, we showed elevated
DISCuSSION
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sE-selectin, regardless of disease severity, bleeding or outcome. More studies are
warranted to elucidate plasmatic coagulation disorders in leptospirosis.
Data on the interaction between the endothelial cell and Leptospira are scarce.
Leptospires were shown to induce endothelial cell adhesiveness for neutrophils in an
experimental model, using cultured human umbilical vein endothelial cells (HUVEC)
and intact or sonicated Leptospira (14). Blocking of E-selectin abrogated the
observed leukocyte adherence. Leptospiral peptidoglycan but not leptospiral LPS,
actively induced the observed endothelial cell adhesiveness for neutrophils. Vieira et
al. reported expression of E-selectin and ICAM-1 by HUVEC when directly stimulated
with the leptospiral protein LIC10365 in a dose-dependant matter (15). Pathological
studies suggest that the hemorrhagic phenomena in leptospirosis patients are due to
capillary wall damage (16;17). One could speculate that Leptospira might be able to
damage (endothelial) cell membranes directly or indirectly by their granular product
when degraded. Indeed it has been shown that pathogenic Leptospira contain different
genes encoding for proteases and other products that can cause host cell membrane
degradation like: sphingomyelinases and phospholipases (18). Several issues of the
present study merit further comment. Current techniques are not able to distinguish
between endothelial damage and activation. Soluble E-selectin has its physiological
activity on the endothelial cell surface, whereas vWF mediates adhesion of platelets
to sites of vascular damage and is important for platelet aggregation by binding the
GP IIb/IIIa platelet receptor. However, both sE-selectin and vWF are released by
proinflammatory mediators such as endotoxin (19;20). Intravenous administration of
endotoxin into humans caused a dose dependent increase of sE-selectin, establishing
its role as a quantitative marker of inflammation induced endothelial activation (21).
Secondly, only patients with severe leptospirosis were enrolled in the study. This
might explain why we did not find a difference between survivors and non-survivors
on admission. In a previous study, sE-selectin was shown to be elevated (mean 93
ng/ml) in 20 Thai patients with mild leptospirosis (22). These levels were higher
(albeit not significantly) compared to patients presenting with murine typhus, scrub
typhus, typhoid, dengue fever or uncomplicated malaria but lower then our results.
Future studies should investigate the role of endothelial cells in mild and severe
leptospirosis. Last, the number of patients studied was limited and therefore caution
should be used when interpreting these data.
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In conclusion, we have demonstrated that markers of endothelial cell activation are
strongly elevated in severe leptospirosis but were not associated with bleeding. No
difference in elevation of these markers was found between the survivors and non-
survivors. More studies are needed to establish the precise role of the endothelial
cell in the pathophysiology of leptospirosis.
Acknowledgements
We gratefully thank S.M.H. Faradz and personnel (CEBIOR, Semarang Indonesia) and
the residents of the department of Internal Medicine (Dr. Kariadi hospital, Semarang,
Indonesia) for their help and assistance during the study.
Leptospirosisan
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(1) Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003 December;3(12):757-71.
(2) McBride AJ, Athanazio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis 2005 October;18(5):376-86.
(3) Arean VM. The pathologic anatomy and pathogenesis of fatal human leptospirosis (Weil’s disease). Am J Pathol 1962 April;40:393-423.
(4) Wagenaar JF, Goris MG, Sakundarno MS, Gasem MH, Mairuhu AT, Kruif de MD et al. What role do coagulation disorders play in the pathogenesis of leptospirosis? Trop Med Int Health 2007 January;12(1):111-22.
(5) Keller TT, Mairuhu AT, Kruif de MD, Klein SK, Gerdes VE, ten CH et al. Infections and endothelial cells. Cardiovasc Res 2003 October 15;60(1):40-8.
(6) Blann AD, Amiral J, McCollum CN. Prognostic value of increased soluble thrombomodulin and increased soluble E-selectin in ischaemic heart disease. Eur J Haematol 1997 August;59(2):115-20.
(7) Levi M, Cate ten H, Poll van der T. Endothelium: interface between coagulation and inflammation. Crit Care Med 2002 May;30(5 Suppl):S220-S224.
(8) American College of Chest Physicians/Society of Critical Care Medicine. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992 June;20(6):864-74.
(9) Clemetson KJ. Platelet GPIb-V-IX complex. Thromb Haemost 1997 July;78(1):266-70. (10) Kayal S, Jais JP, Aguini N, Chaudiere J, Labrousse J. Elevated circulating E-selectin,
intercellular adhesion molecule 1, and von Willebrand factor in patients with severe infection. Am J Respir Crit Care Med 1998 March;157(3 Pt 1):776-84.
(11) Rubin DB, Wiener-Kronish JP, Murray JF, Green DR, Turner J, Luce JM et al. Elevated von Willebrand factor antigen is an early plasma predictor of acute lung injury in nonpulmonary sepsis syndrome. J Clin Invest 1990 August;86(2):474-80.
(12) Reinhart K, Bayer O, Brunkhorst F, Meisner M. Markers of endothelial damage in organ dysfunction and sepsis. Crit Care Med 2002 May;30(5 Suppl):S302-S312.
(13) Reinhart K, Bayer O, Brunkhorst F, Meisner M. Markers of endothelial damage in organ dysfunction and sepsis. Crit Care Med 2002 May;30(5 Suppl):S302-S312.
(14) Dobrina A, Nardon E, Vecile E, Cinco M, Patriarca P. Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes. Infect Immun 1995 August;63(8):2995-9.
(15) Vieira ML, D’Atri LP, Schattner M, Habarta AM, Barbosa AS, de Morais ZM et al. A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells. FEMS Microbiol Lett 2007 November;276(2):172-80.
(16) Arean VM. The pathologic anatomy and pathogenesis of fatal human leptospirosis (Weil’s disease). Am J Pathol 1962 April;40:393-423.
(17) Nicodemo AC, Duarte MI, Alves VA, Takakura CF, Santos RT, Nicodemo EL. Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia. Am J Trop Med Hyg 1997 February;56(2):181-7.
(18) Nascimento AL, Ko AI, Martins EA, Monteiro-Vitorello CB, Ho PL, Haake DA et al. Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacteriol 2004 April;186(7):2164-72.
(19) Kuhns DB, Alvord WG, Gallin JI. Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis 1995 January;171(1):145-52.
REFERENCES
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(20) Schorer AE, Moldow CF, Rick ME. Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells. Br J Haematol 1987 October;67(2):193-7.
(21) Kuhns DB, Alvord WG, Gallin JI. Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis 1995 January;171(1):145-52.
(22) Paris DH, Jenjaroen K, Blacksell SD, Phetsouvanh R, Wuthiekanun V, Newton PN et al. Differential patterns of endothelial and leucocyte activation in ‘typhus-like’ illnesses in Laos and Thailand. Clin Exp Immunol 2008 July;153(1):63-7.
Low factor XII and factor XI levels in patients
with severe leptospirosis
J.F.P. Wagenaar 1, J.W.P. Govers-Riemslag 2, H. ten Cate 2, M.H. Gasem 3 and E.C.M. van Gorp 1, 4
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine
and Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands3 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University,
Semarang, Indonesia 4 Department of Virology, Erasmus MC, Rotterdam, the Netherlands
Submitted
6CHAPTER
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Background: Leptospirosis is an acute, febrile illness, frequently complicated by
severe bleeding.
Objective: To determine the involvement of the contact system in relation to
bleeding and poor outcome in patients with severe leptospirosis.
Methods: In a prospective study, activation of the contact system was determined
by measuring factor XI (FXI), factor XII (FXII) and activated protein-inhibitor (INH)
complexes (FXIa-C1-IH, FXIIa-C1-IH, kallikrein-C1-INH and FXIa-AT-INH) in patients
with severe leptospirosis. In addition, markers of tissue factor (TF) pathway activation
(thrombin-antithrombin complexes (TAT), prothrombin fragment 1+2 (F1+2)) and
fibrinolysis (plasmin-antiplasmin complexes (PAP)) were measured.
Results: Sixty-six consecutive severe leptospirosis patients were recruited, of whom
19 (29%) died during follow up. Bleeding complications were observed in 36 (55%)
patients, of whom 12 had severe bleeding consisting of heamatemesis, melaena, gum
bleeding and/or haemoptysis. Overall, the median prothrombin time (PT) was high
normal, whereas the activated partial thromboplastin time (APTT) was markedly
prolonged. Twenty-six patients had a prolonged APTT and a normal PT. Patients had
significantly lower levels of FXII (p < .001) and FXI (p = .04) on admission compared to
the controls. FXII (rho -.426 p = .001) and FXI (rho -.291, p = .03) levels were inversely
correlated with APTT prolongation. The median FXIIa-C1-IH (0.38%), FXIa-C1-INH
(0.39%) and FXIa-AT-INH (0.48%) complexes were not statistically different from the
controls. Kallikrein-C1-INH complexes were undetectable in all samples. There was
no association between either (severe) bleeding or mortality and contact activation
markers. Marked activation of the TF and fibrinolytic pathway was demonstrated,
reflected by elevated TAT (6.8μg/L), F1+2 (371μg/L) and PAP (862μg/L) complexes.
These derangements were more pronounced in subjects with severe bleedings and
in the deceased.
Conclusion:Patients suffering from severe leptospirosis yielded reduced levels of FXII
and FXI, explaining (isolated) APTT prolongation but not bleeding or poor outcome.
Although the low levels of FXII and FXI determined may indicate consumption of
these coagulation factors, no evidence of contact system activation was observed.
AbSTRACT
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Leptospirosis is now identified as one of the emerging infectious diseases in many
(sub-) tropic regions of the world (1). Outbreaks are frequently reported following
severe floods, a phenomenon not only restricted to tropical regions. The source of
infection in humans is usually either direct or indirect contact with the urine of an
infected animal. The vast majority of infections caused by Leptospira are either sub-
clinical or of mild severity and presenting symptoms are oftentimes non-specific.
Severe disease, in which the clinical course is often very rapidly progressive, usually
presents with jaundice, renal failure and bleeding. In recent years, endemic and
epidemic severe pulmonary haemorrhage has increasingly become recognized as an
important manifestation.
The pathogenesis of bleeding in severe leptospirosis is poorly understood. Bleeding
that is spontaneous and excessive may be the result of a disorder in the complex
interplay between vascular integrity, platelet number and function and number,
coagulation factors and fibrinolysis. It is now well established that in sepsis, systemic
inflammation invariably leads to activation of the coagulation system and inhibition
of anticoagulant mechanisms and fibrinolysis (2). During infection, the pivotal
initiator of inflammation-induced activation of coagulation is tissue factor (TF). In
its most severe form, exaggerated coagulation activation leads to both thrombosis
and bleeding due to consumption coagulopathy, a syndrome known as disseminated
intravascular coagulation (DIC). Although activation of the “extrinsic” route or FVII/
tissue factor (TF) pathway of coagulation and DIC are observed in leptospirosis
patients, these phenomena do not explain all clinical bleeding events (wagenaar et
al. accepted Trop Med Int Health).
In addition to TF mediated coagulation we observed prolonged activated partial
thromboplastin times (APTT) with concurrently normal prothrombin (PT) clotting
times in some of these patients (25 subjects), suggesting altered activity of the
intrinsic route of coagulation.
The initial phase of the intrinsic or contact activation pathway consists of several
plasma proteins including factor XII (Hageman factor; FXII), prekallikrein (Fletcher
factor) and high molecular weight kininogen (HMWK) which are activated by
INTRODuCTION
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contact with negatively charged surfaces. Moreover there is evidence that specific
components of Gram-negative bacteria and Streptococcal M-protein can activate
this proteolytic system (3;4). Activated FXII (FXIIa) activates FXI and henceforth FIX
which in complex with FVIIIa leads to the formation of FXa. The remainder of the
intrinsic pathway uses the same cascade as the extrinsic pathway, the so called
common pathway. Via a positive feed back loop of FXI activation by thrombin,
additional thrombin is formed via activation of FIX and X. Since contact activation
may therefore give a substantial amplification in the levels of thrombin generation
produced, this pathway may theoretically enhance the risk of DIC and associated
bleeding complications.
In the present study we investigated the possible role of the contact activation
pathway in relation to bleeding and outcome in patients with severe leptospirosis.
Patients
Consecutive patients were derived from two observational cohort studies performed
in the Dr. Kariadi Hospital, Diponegoro University, Semarang, Indonesia. The first
cohort was a pilot study that started in 2002. This pilot was followed by a second
cohort (2005-2006) that has been described previously (ref). Both the ethics
committee of the Dr. Kariadi hospital and the Slotervaart hospital, Amsterdam, the
Netherlands, approved both study protocols which were part of an ongoing study on
the pathophysiology of leptospirosis. All eligible leptospirosis patients were included
after written informed consent was given by the patients, or care takers when
too ill to approve. Cases were defined as hospitalized patients with high clinical
suspicion of severe leptospirosis, usually presenting with at least one of the following
symptoms or signs: jaundice, renal failure, thrombocytopenia (platelets <100x109/L),
haemorrhaging and a positive LeptoTek Dri-Dot assay (Biomérieux). Blood samples,
5 ml serum and 5 ml citrated plasma 3.2% (both BD Vacutainer, Plymouth, UK) were
taken on admission and on day 1, 2, 7 and on day 14 only in the second cohort.
After centrifuging, plasma aliquots were stored at -80°C until further testing. Twenty
METHODS
LowfactorXIIa
ndfactorXIlevelsinpatientswithseverelep
tospirosis
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healthy Javanese volunteers served as a normal reference group to establish local
normal laboratory test values.
Diagnosticprocedures
Clinical diagnosis of leptospirosis was confirmed by a positive microscopic agglutination
test (MAT). For the latter a panel of 31 serovars was used (28 pathogenic serovars and
3 non-pathogenic serovars). A titre of ≥ 1:320 on a single sample, seroconversion or a
fourfold or higher increase of the titre in paired samples or a titre ≥ 1:80 in a single
sample from early deceased patients, were considered to be positive. All diagnostic
tests were performed at the department of microbiology, faculty of medicine,
Diponegoro University, Indonesia. Cross-checks were performed at the WHO/FAO/
OIE and National Collaborating Centre for Reference and Research on Leptospirosis,
The Netherlands.
Laboratorymethods
Upon activation in plasma all activated factors of the intrinsic coagulation proteins
are rapidly inhibited in vivo by the formation of inhibitory complexes with endogenous
inhibitors, the dominant inhibitor being C1-inhibitor (INH) (5-7). Activation of the
intrinsic coagulation proteins was determined by measuring these activated protein-
inhibitor complexes formed in the patients and controls. Complexes of Factor XIa,
XIIa, Kallikrein and C1-INH and complexes of Factor XIa and AT-INH were measured
in citrated plasma with enzyme-linked immunosorbent assays (ELISAs), as previously
published (8). Results were expressed in arbitrary units as a percentage of activated
normal pooled plasma. To obtain reference curves for the FXIIa–C1-INH and kallikrein–
C1-INH complex ELISAs, plasma was activated with dextran sulfate (Mr 500 000;
Sigma Chemical Co., St Louis, MO, USA) whereas kaolin was used as plasma activator
for the FXIa–AT and FXIa–C1-INH ELISAs. The detection limits were 0.10% for all four
assays, respectively. FXII and FXI antigen levels were analyzed by commercially
available ELISAs (Affinity Biologicals FXII-EIA en FXI-EIA, Kordia, NL). Measurements
of F1+2 (Dade Behring, Marburg, Germany), TAT complexes (Dade Behring) and PAP
complexes (DRG, Marburg, Germany) were performed by ELISA.
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Statisticalanalyses
Relevant patient characteristics are presented as medians with corresponding
interquartile ranges (IQR) or as numbers with percentages. Continuous variables were
statistically evaluated using the non-parametric Mann-Whitney U test (two-tailed).
Correlations between markers of contact activation and markers of coagulation
activation and fibrinolysis are expressed as Spearman correlation coefficients (rho).
Associations were calculated using a binary logistic regression analysis and were
expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). The
logrank test was performed to calculate differences in survival between patients
with and without bleeding. A p-value of ≤0.05 was considered to indicate statistical
significance. All analyses were done using SPSS (version 15.0, Chicago, Illinois).
Patients
A total number of 66 patients were included: 14 from the first and 52 from the second
cohort. All patients (46 male, 20 female) were confirmed to have acute leptospirosis.
The most frequently identified serovars by MAT were: Icterohaemorrhagiae (n=21)
and Bataviae (n=16). The median (IQR) age of the patients was 45 (32-55) years.
Symptoms had started 6 days (4-8) before admission to the hospital. Nineteen (29%)
patients died during follow up. Baseline characteristics, presented as medians (IQR)
were as follows: pulse 100 beats/minute (92-108), systolic blood pressure 110mmHg
(105-123), respiratory rate 24 breaths/minute (20-28), platelets 66x109/L (35-135),
creatinin 5.4mg/dl (3.0-7.6), total bilirubin 8.3mg/dl (3.4-19.6), AST 66U/L (38-
112), ALT 53U/L (43-76). Signs of bleeding were observed in 36 (55%) patients. Mild
bleeding (petechiae, ecchymoses, epistaxis) was recorded in 24 (36%) subjects,
whereas severe bleeding (heamatemesis, melaena, gum bleeding and haemoptysis)
was recognized in 12 (18%) patients. Bleeding was significantly associated with
mortality (p < .05) as shown in figure 1.
RESuLTS
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ndfactorXIlevelsinpatientswithseverelep
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Figure 1: Comparison of survival in leptospirosis patients with and without bleeding.
Survival curves of patients with mild (n=24), severe (n=12) and no bleeding complications
(n=30). Differences were calculated by the Logrank test. A p-value of <0.05 was considered
statistically significant.
Markersofcontactactivation,coagulationandfibrinolysis
The table shows global markers of contact activation, coagulation and fibrinolysis.
The median prothrombin time was high normal, whereas the APTT was markedly
prolonged. Twenty-six patients had prolonged APTT and a normal PT. Figure 2 shows
the plasma proteins FXI, FXII and the contact phase activation markers: FXIIa-C1-INH,
FXIa-C1-INH and FXIa-AT-INH over time. Patients had significantly lower levels of FXII
(p < .001) and FXI (p = .04) on admission compared to the controls. During follow up
FXII levels showed a rising trend over time in the survivors. FXII (rho -.426 p = .001)
and FXI (rho -.291, p = .03) levels were inversely correlated with APTT prolongation.
No significant difference was recorded between the markers of contact activation:
FXIIa-C1-INH, FXIa-C1-INH and FXIa-AT-INH on admission and the control values.
Kallikrein-C1-INH complexes were undetectable in all samples (results not shown).
Markers of coagulation activation (TAT, F1+2) and fibrinolysis (PAP) were elevated on
admission, reflecting thrombin and plasmin generation, respectively (table). Besides
a correlation between FXII and F1+2 (rho 0.37, p = .002) and FXIIa-C1-IH and F1+2
(rho 0.26, p = 0.04), contact phase markers did not correlate with PT or markers of
thrombin generation (TAT) and fibrinolysis (PAP).
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Tabl
e: G
loba
l mar
kers
of
cont
act
acti
vati
on,
coag
ulat
ion
and
fibri
noly
sis
on a
dmis
sion
.
Test
nA
ll pa
tien
tsn
Surv
ivor
sn
Non
-sur
vivo
rsn
Cont
rols
p-va
lue
Globa
lcoagulationtests
PT (
s),
norm
al:
10.7
-12.
955
12.9
(12
-14.
5)40
12.8
(11
.9-1
3.9)
1514
.5 (
12.3
-16.
3)N
A0.
04AP
TT (
s),
norm
al:
25-3
858
48.5
(39
.7-5
6.1)
4248
.0 (
41.2
-54.
4)16
51.7
(38
.4-7
2)N
A0.
2Plasmazymogen
FXI (
%)65
73.3
(59
.3-1
03.6
)47
73.3
(61
.1-9
9.6)
1876
.7 (
54.0
-109
.1)
2097
.1 (
76.6
-111
.7)*
0.8
FXII
(%)
6538
.1 (
29.7
-48.
8)47
41.2
(30
.1-5
3.0)
1835
.7 (
27.4
-46.
5)20
67.1
(46
.8-8
2.5)
*0.
4Co
ntactactivation
markers
FXIIa
-C1-
INH
com
plex
(%)
650.
38 (
0.25
-0.5
0)47
0.35
(0.
24-0
.53)
180.
44 (
0.26
-0.4
7)20
0.33
(0.
27-0
.38)
0.5
FXIa
-C1-
INH
com
plex
(%)
650.
39 (
0.22
-0.6
4)47
0.42
(0.
25-0
.82)
180.
30 (
0.21
-0.4
5)20
0.34
(0.
25-0
.79)
0.1
FXIa
-AT-
INH
com
plex
(%)
650.
48 (
0.33
-0.6
0)47
0.48
(0.
32-0
.60)
180.
48 (
0.31
-0.6
2)20
0.22
( 0
.16-
0.32
)1.
0Procoa
gulantactivity
TAT
(µg/
L),
norm
al:
<4.6
666.
8 (4
.2-1
2.8)
196.
4 (3
.9-9
.2)
199.
3 (6
.7-1
8.8)
NA
0.03
F1+2
(µg
/L),
nor
mal
: 53
-271
6537
1 (1
83-1
280)
1933
1 (1
66-7
45)
1990
0 (1
83-2
186)
NA
0.1
Fibrinolyticactivity
PAP
(µg/
L),
norm
al:
221-
512
5186
2 (7
11-1
115)
3787
0 (7
22-1
130)
1480
0 (4
53-1
115)
NA
0.4
Hae
mos
tati
c pa
ram
eter
s (m
edia
n, 2
5-75
th p
erce
ntile
) of
66
pati
ents
wit
h le
ptos
piro
sis
mea
sure
d on
adm
issi
on.
P-va
lues
rep
rese
nt d
iffe
renc
e be
twee
n su
rviv
ors
(n=4
7) a
nd n
on-s
urvi
vors
(n=
18)
as c
alcu
late
d w
ith
the
Man
n-W
hitn
ey U
tes
t. A
p-v
alue
of
<0.0
5 w
as c
onsi
dere
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atis
tica
lly
sign
ifica
nt.
* Co
ntro
ls s
igni
fican
tly
diff
eren
t fr
om a
ll pa
tien
ts (
FXI:
p =
.04
; FX
II: p
< .
001)
Abb
reviations:PT,prothrom
bintime;APT
T,activated
partialthrom
boplastintime;FXIIa-C1-INHcom
plex,Fa
ctorXIIa
C1-inhibitorcomplexes;
FXIa-C1-INHcom
plex,Fa
ctorXIaC1-inhibitorcomplexes;FX
Ia-AT-INHcom
plex,Fa
ctorXIa
a1-an
titryp
sin-inhibitorcomplexes;TAT,throm
bin
antithrombincomplexes,F1
+2,prothrom
binfragmen
t1+2;PAP,plasm
in-antiplasm
incom
plexes;NA,no
tavailable.
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Figure 2: Markers of contact activation
over time in patients with severe
leptospirosis.
Scatter dot plot of markers of contact
activation in 66 patients with severe
leptospirosis on admission (day 0) and
follow up. Survivors are compared to
non-survivors, where the open symbols
demonstrate the survivors and the closed
symbols the non-survivors. Controls values (open diamonds) are obtained from healthy
Indonesian volunteers. Bars indicate the median value.
Abbreviations: FXI, factor XI; FXII, factor XII; FXIa-C1-INH complex, Factor XIaC1-inhibitor
complexes; FXIIa-C1-INH complex, FactorXIIaC1-inhibitor complexes; FXIa-AT-INH complex,
FactorXIaa1-antitrypsin-inhibitorcomplexes.
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No significant difference in FXI, FXII, FXIIa-C1-INH, FXIa-C1-INH and FXIa-AT-
INH levels was observed between patients with or without (severe) bleeding on
admission (data not shown). Most clotting indices were not significantly different
among patients that had clinical bleeding and those without clinically significant
bleeding. However, severe bleeding was found to be associated with higher PT
values by binary logistic regression (OR 1.3, 95%CI: 1.0-1.7, p = 0.05). Subjects with
severe bleeding also showed elevated TAT and F1+2 levels compared to subjects
without severe bleeding (TAT: 11.8µg/L vs. 6.8µg/L; F1+2: 1298µg/L vs. 325µg/L).
This difference was significant for F1+2 levels (p = 0.006). PAP levels were significant
lower in patients with severe bleeding compared to those without severe bleeding
(695µg/L vs. 884µg/L, p = 0.04).
No significant differences were demonstrated with regard to FXI, FXII and contact
activation markers between survivors and non-survivors. Thrombin generation was
increased in patients that went on to die, reflected by significant higher TAT levels
(p = .03). No difference was found in fibrinolytic activity reflected by statistically
similar PAP levels.
Leptospirosis importantly contributes to morbidity and mortality in endemic regions.
Hospitalized cases often present with severe bleeding manifestations, which pose
a great challenge for treating physicians. Unfortunately many questions regarding
the pathophysiology of bleeding in leptospirosis remain unanswered. Exaggerated
coagulation activation is likely to contribute to loss of hemostasis when clotting
factors are depleted. In this regard, we previously showed activation of the
coagulation system and the association with bleeding in patients suffering from
severe leptospirosis (Wagenaar et al. accepted TMIH). A subset of these patients
showed a marked prolongation of the APTT in comparison with PT values, which
led us to hypothesize that the contact route of coagulation may be a contributing
mechanism in the course of leptospirosis associated hypercoagulability. Using
a combination of specific immunoassays for activated contact system proteins in
DISCuSSION
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conjunction with conventional zymogen analysis, we assessed the activity of the
contact system vis a vis the other coagulation pathways. We show that low levels of
the plasma zymogens FXII and FXI correlate with APTT prolongation. Previous work
showed contact activation in 7 patients with shock due to invasive Streptococcus
pyogenes infection (9). Five of these patients had isolated APTT prolongation, but no
bleeding tendency. Contact activation has also been documented in cases of (gram-
negative) sepsis (10;11) and has been associated with activation and regulation of
the fibrinolytic system (12).
However, in our study we did not detect any significant activation of the contact
system, as reflected by non elevated FXIIa-C1-INH, FXIa-C1-INH, FXIa-AT-INH levels
and undetectable Kallikrein-C1-INH complexes. These results are in line with studies
of experimental bacteraemia suggesting that the primary route of activation of
coagulation is mainly through the TF pathway (13). In contrast, activation of the
contact system has been described in human meningococcal disease (14) and in
experimental models of bacteraemia in baboons (8). However, in the latter study
blocking of activation of the intrinsic pathway of blood coagulation by means of
a monoclonal antibody to FXIIa did not affect coagulation in experimental sepsis
in baboons (15). Indeed, most individuals with congenital contact-protease
deficiency have no abnormalities of hemostasis but mild bleeding tendency is seen
with deficiency of FXI (16). In the present study FXI plasma levels were low but no
association with bleeding was observed.
It is tempting to speculate what is causing the low FXI and FXII levels in these
patients. Hepatic dysfunction is not likely since severe hepatitis is not observed
in this group of patients. Antibodies to clotting factors are possible to occur. Lupus
antibodies are known to give APTT prolongation in this regard; however a thrombotic
phenotype is expected (17). Proteins, such as curli and fimbriae, on the surface
of Gram-negative bacteria and several bacterial proteases are known to cleave or
bind to FXII (3;18). Although the low levels of FXII and FXI determined may indicate
consumption of these coagulation factors, no evidence of contact system activation
was observed in this cohort. The observation that the contact system is mainly
involved in the induction of inflammatory reactions (19) could not be confirmed,
while the Kallikrein-C1-INH complexes were undetectable in all samples (results not
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shown). One alternative mechanism may involve the depletion of FXI and FXII due to
early onset activation of the contact system since these patients were already quite
ill at the time of admission. It may be possible that formation of contact protease-
inhibitor complexes had occurred prior to admission and that the reduced zymogen
concentrations are still indicative of an activated contact system. The modest but
statistically significant correlations between FXII (activation) and F1 +2 generation
may be indicative of such a mechanism.
Some issues of the present study merit further comment. Firstly, only patients with
severe leptospirosis were enrolled in the study. Hence our findings are only applicable
to patients with the severest forms of leptospirosis and further studies are required
to investigate hemostatic mechanisms in patients with mild leptospirosis. Second,
most patients presented in late stage disease which can explain the fulminant clinical
picture and high mortality in this cohort. Last, the number of patients included in the
cohort was relatively limited. Therefore caution should be used when interpreting
this data.
In conclusion, patients suffering from severe leptospirosis display reduced levels of
FXII and FXI, explaining (isolated) APTT prolongation but not bleeding. While early
activation of the contact system cannot be excluded, at the time of severe disease
this does not seem to contribute to the clinical course in this disease. Bleeding
appears to depend more on extrinsic than intrinsic coagulation pathways. More
studies are warranted to elucidate the cause of bleeding in depletion of clotting
factors in these patients.
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(1) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (2) Schouten M, Wiersinga WJ, Levi M, van der Poll T. Inflammation, endothelium, and
coagulation in sepsis. J Leukoc Biol 2007 November 21. (3) Herwald H, Morgelin M, Olsen A, Rhen M, Dahlback B, Muller-Esterl W et al. Activation
of the contact-phase system on bacterial surfaces--a clue to serious complications in infectious diseases. Nat Med 1998 March;4(3):298-302.
(4) Sriskandan S, Kemball-Cook G, Moyes D, Canvin J, Tuddenham E, Cohen J. Contact activation in shock caused by invasive group A Streptococcus pyogenes. Crit Care Med 2000 November;28(11):3684-91.
(5) de Agostini A, Lijnen HR, Pixley RA, Colman RW, Schapira M. Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor. J Clin Invest 1984 June;73(6):1542-9.
(6) Schapira M, Scott CF, Colman RW. Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma. J Clin Invest 1982 February;69(2):462-8.
(7) Wuillemin WA, Minnema M, Meijers JC, Roem D, Eerenberg AJ, Nuijens JH et al. Inactivation of factor XIa in human plasma assessed by measuring factor XIa-protease inhibitor complexes: major role for C1-inhibitor. Blood 1995 March 15;85(6):1517-26.
(8) Govers-Riemslag JW, Smid M, Cooper JA, Bauer KA, Rosenberg RD, Hack CE et al. The plasma kallikrein-kinin system and risk of cardiovascular disease in men. J Thromb Haemost 2007 September;5(9):1896-903.
(9) Sriskandan S, Cohen J. Kallikrein-kinin system activation in streptococcal toxic shock syndrome. Clin Infect Dis 2000 June;30(6):961-2.
(10) La Cadena RA, Suffredini AF, Page JD, Pixley RA, Kaufman N, Parrillo JE et al. Activation of the kallikrein-kinin system after endotoxin administration to normal human volunteers. Blood 1993 June 15;81(12):3313-7.
(11) Nuijens JH, Huijbregts CC, Eerenberg-Belmer AJ, Abbink JJ, Strack van Schijndel RJ, Felt-Bersma RJ et al. Quantification of plasma factor XIIa-Cl(-)-inhibitor and kallikrein-Cl(-)-inhibitor complexes in sepsis. Blood 1988 December;72(6):1841-8.
(12) Jansen PM, Pixley RA, Brouwer M, de J, I, Chang AC, Hack CE et al. Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase. Blood 1996 March 15;87(6):2337-44.
(13) Levi M, Cate ten H. Disseminated intravascular coagulation. N Engl J Med 1999 August 19;341(8):586-92.
(14) Wuillemin WA, Fijnvandraat K, Derkx BH, Peters M, Vreede W, ten CH et al. Activation of the intrinsic pathway of coagulation in children with meningococcal septic shock. Thromb Haemost 1995 December;74(6):1436-41.
(15) Pixley RA, De La CR, Page JD, Kaufman N, Wyshock EG, Chang A et al. The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons. J Clin Invest 1993 January;91(1):61-8.
(16) Asakai R, Chung DW, Davie EW, Seligsohn U. Factor XI deficiency in Ashkenazi Jews in Israel. N Engl J Med 1991 July 18;325(3):153-8.
(17) Galli M, Barbui T. Antiphospholipid syndrome: clinical and diagnostic utility of laboratory tests. Semin Thromb Hemost 2005 February;31(1):17-24.
(18) Molla A, Yamamoto T, Akaike T, Miyoshi S, Maeda H. Activation of hageman factor and prekallikrein and generation of kinin by various microbial proteinases. J Biol Chem 1989 June 25;264(18):10589-94.
(19) Frick IM, Akesson P, Herwald H, Morgelin M, Malmsten M, Nagler DK et al. The contact system--a novel branch of innate immunity generating antibacterial peptides. EMBO J 2006 November 29;25(23):5569-78.
REFERENCES
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IIPARTInflammation
Long pentraxin PTX3 is associated with
mortality and disease severity in severe
Leptospirosis
J.F.P. Wagenaar 1, M.G.A. Goris 2, M.H. Gasem 3, B. Isbandrio 4, Federica Moalli 5, A. Mantovani 5, 6, K.R. Boer 2, R.A. Hartskeerl 2, C. Garlanda 5 and E.C.M. van Gorp 1, 7
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
3 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University, Semarang, Indonesia
4 Department of Microbiology, Leptospirosis Laboratory, Diponegoro University, Semarang, Indonesia
5 Institute Clinico Humanitas, Lab. Ricerche in Immunologia e Infiammazione, Milan, Italy6 Institute of General Pathology, University of Milan, Milan, Italy
7 Department of virology, Erasmus University, Rotterdam, the Netherlands
J.Infect.2009April;58(6):425-32.
7CHAPTER
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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,
were 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.
AbSTRACT
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Leptospirosis is considered one of the most prevalent zoonosis worldwide and may
run an insidious course (1). The spirochete is usually spread by infected urine of rats,
mice or other mammals entering and infecting humans via abraded skin or mucous
membranes. Most cases of leptospirosis present as mild disease with symptoms such
as fever and malaise. However, patients with severe leptospirosis can suffer from
bleeding and (multiple-) organ failure, which could in turn lead to circulatory shock
and eventually even death. It is thought that an exaggerated immune response may
play a pivotal role in the pathophysiology of leptospirosis. Several cytokines, such
as Interferon (INF) -g, interleukin (IL)-12p40 and TNF-a, have been found to be up
regulated in response to Leptospira (2-4). TNF-a has seen to be associated with
disease severity and death in human leptospirosis cases (5). Other markers may also
be useful to clinicians in determining more severe cases from mild leptospirosis
cases. In other words such markers may be indicators of disease prognosis.
The long pentraxin PTX3 could potentially be such a marker. In previous studies, long
pentraxin PTX3 was found to be elevated in critically ill patients and was correlated
with severity of disease and infection (6;7). Long pentraxin PTX3 may also be
associated with other disease and it has also been suggested that PTX3 be measured
in the blood for early diagnosis and prognosis prediction in diseases such as psoriasis,
unstable angina pectoris, acute myocardial infarction and hearth failure (8-11)
Pentraxins are a superfamily of proteins that are characterized by a multimeric,
usually pentameric structure. Both genes of the classic short pentraxins C-reactive
protein (CRP) and serum amyloid P component (SAP) are localized on chromosome
1. CRP is a widely used indicator for the diagnosis and monitoring of inflammatory
and infectious diseases, whereas SAP is proven to be useful in identifying amyloid
deposits. Both acute phase proteins are mainly produced by the liver in response
to inflammatory signals, most prominently IL-6. The human PTX3 gene is localized
on chromosome 3, band q25. The protein is structurally related to CRP and SAP;
however, it has an unrelated long amino-terminal domain coupled to the carboxy-
terminal pentraxin domain (12). Moreover, PTX3 differs in cellular source and
ligands recognized. Toll-like receptor engagement and cytokines like TNF-α and IL-1α
INTRODuCTION
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provoke a rapid release, particular from mononuclear phagocytes, dendritic cells,
fibroblasts, endothelial cells and epithelial cells (13-20), whereas IL-6 usually seems
not to induce PTX3 (21). Functionally, PTX3 is thought to fulfill an important role in
the innate resistance to microbes and tuning of inflammation (22).
In the present study we investigated plasma PTX3 levels in a series of severe
leptospirosis patients from Indonesia and compared it with CRP and the pro-
inflammatory cytokines interleukin (IL-) 6 and IL-8. The aim of the study was to
evaluate the usefulness of PTX3 in monitoring patients suffering from severe
leptospirosis.
Patients
The study was carried out at the department of internal medicine of Dr. Kariadi
Hospital –Diponegoro University, Semarang, Indonesia from February 2005 to
September 2006. Ethical approval was granted by both the medical ethics committee
of Dr. Kariadi hospital and the Slotervaart hospital, Amsterdam, the Netherlands.
The study protocol was approved the as part of a larger ongoing study on the
pathophysiology of leptospirosis. Potential patients were screened for eligibility and
prior to enrolling written informed consent was collected from the patients or from
their caretakers when patients were too ill to approve themselves. Cases were defined
as hospitalized patients with high clinical suspicion of severe leptospirosis usually
presenting with at least one of the following symptoms or signs: jaundice, renal
failure, thrombocytopenia and/or haemorrhaging and a positive LeptoTek Dri-Dot
assay (Biomérieux). Blood samples were taken on admission and again approximately
14 days later. After centrifuging, aliquots were stored at -70°C for further testing.
Casedefinitiondiseaseseverity
At admission patients were stratified into 3 disease severity categories; non-sepsis,
sepsis and severe sepsis/septic shock (23). Sepsis was defined as having an infection
by the presence of at least two of the four following criteria: fever or hypothermia
MATERIALS AND METHODS
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(temperature >38°C or <36°C), tachycardia (>90 beats per minute), tachypnea (>20
breaths per minute or PaCO2 <32 mm Hg or the need of mechanical ventilation)
and an altered white blood cell count of >12x109/L or <4.0x109/L. Severe sepsis
was defined as sepsis together with at least one manifestation of hypoperfusion or
organ failure: hypoxemia (PaO2 <75 mm Hg), metabolic acidosis (pH <7,3), oliguria
(output <30 ml/hr) or an acute alteration in mental status in the absence of sedation
(reduction by at least 3 points from baseline value using the Glasgow Coma Score).
Septic shock was defined as sepsis accompanied by a sustained decrease in systolic
blood pressure (<90 mm Hg or a drop of 40 mm Hg from baseline) despite fluid
resuscitation to maintain adequate blood pressure. Due to the fact that sepsis can
occur or deteriorate suddenly, we chose to redefine each patient within 2 days of
hospital admission, these were used for the final analysis.
Diagnosticprocedures
Diagnostic tests were performed at the department of microbiology, faculty of
medicine, Diponegoro University, Indonesia. Cross-checks were performed at the
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on
Leptospirosis, The Netherlands. Clinical diagnosis of leptospirosis was confirmed by
a positive microscopic agglutination test (MAT). The MAT included 31 serovars: 28
pathogenic serovars and 3 non-pathogenic serovars. The MAT was considered positive
when: (1) a titre ≥ 1:320 in a single sample, (2) a seroconversion, (3) a fourfold or
higher increase in titres between paired samples or (4) a titre ≥ 1:80 in a single
sample from early deceased patients was detected. All cases (n=52) in the present
study were serologically confirmed by this method.
Laboratorymethods
The Sandwich ELISA for PTX3 was performed as previously described (7). In brief,
ELISA plates (96 well; Nunc Immuno Plate, MaxiSorp; Nunc) were coated with 100
ng/well of rat monoclonal anti-PTX3 antibody (mAb) MNB4 diluted in coating buffer
(15 mM carbonate, Na2CO3 + NaHCO3, buffer pH 9.6) and were incubated overnight
at 4°C. The plates were washed with washing buffer (Dulbecco’s phosphate buffered
containing 0.05% Tween20) and 300 µl of 5% dry milk were added to block non-specific
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binding sites. 50 µl of recombinant human PTX3 standards (100 pg/ml to 2 ng/ml)
and unknown samples were added in duplicate, and incubated for 2 h at 37°C. After
three washes with the washing buffer, 25ng/well of biotin conjugated PTX3 affinity-
purified rabbit IgG were added for 1 h at 37°C. Wells were extensively washed and
incubated with 100 μl of Streptavidin-peroxidase conjugated to dextran backbone
(AmDex, Copenhagen, Denmark) diluted 1:4000 for 1 h at room temperature. After
incubation the plates were washed four times and 100 µl of TMB chromogen (BD,
Pharmingen) were added. Absorbance values were read at 405 nm in an automatic
ELISA reader. PTX3 levels in plasma were analyzed after addition of EDTA 0.35% final
concentration to sample dilution buffer in order to eliminate interference in the
assay by Calcium-dependent factors. The absence of interfering factors in the assay
and its specificity were ruled out by adding known amounts of recombinant PTX3
or mAb MNB4 to the samples. The lower limit of detection was 100ng/ml, whereas
inter-assay variability was 8-10%.
Statisticalanalyses
Relevant patient characteristics are presented as medians with corresponding
interquartile ranges (IQR) or as numbers with percentages. Between survivors and
non-survivors continuous variables were statistically evaluated with the Mann-
Whitney U test, whereas categorical data were evaluated using the Chi square test.
Differences in PTX3, IL-6, IL-8 and CRP levels between: non-sepsis, sepsis and severe
sepsis/septic shock patients were evaluated with the Kruskal-Wallis test. Spearman’s
rho was used to express correlations between PTX3 and the other cytokines. The
association of PTX3, IL-6, IL-8 and CRP with disease severity (dichotomized as non-
sepsis/sepsis vs. severe sepsis/septic shock) and mortality were calculated using
individual univariate binary logistic regression analyses. Since PTX3 and the other
markers were non-parametric, we log transformed the data which resulted in a
parametric distribution (Kolmogorov > .05).
In addition, we used a receiver-operating-characteristic (ROC) approach to
determine the extent of which PTX3, IL-6, IL-8 or CRP predicts either disease
severity (non-sepsis/sepsis vs. severe sepsis/septic shock) or mortality. The ROC
curve demonstrates the trade-off between sensitivity and specificity (any increase
PTX3
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in sensitivity will be accompanied by a decrease in specificity). The area under the
curve with its 95% confidence intervals is a measure of the test accuracy, wherein a
perfect test (one that has zero false positives and zero false negatives) has an area
of 1.00, and a 0.5 indicates that PXT3 is no better than tossing a coin in determining
mortality or disease severity. A p-value of ≤0.05 was considered to indicate statistical
significance. All analyses were done using SPSS (version 15.0, Chicago, Illinois).
Patientsanddiseaseseverity
Fifty-two leptospirosis patients were included in the study; all cases were
serologically confirmed. The median age was 44 years and the cohort consisted
largely of males (71%). Symptoms started at a median of 7 days before admission.
Fourteen patients (27%) died during hospital stay, with a median of 3 days post
admission. On admission 28 patients (54%) fulfilled sepsis criteria, 19 (36%) fulfilled
severe sepsis criteria and 5 (10%) patients fulfilled neither (non-septic patients).
Within 2 days post-admission, one patient progressed from non-sepsis to sepsis,
2 from sepsis to severe sepsis, 3 from sepsis to septic shock and 4 from severe
sepsis to septic shock. This led to 4 (8%) non-septic cases, 24 (46%) septic cases, 17
(33%) severe septic cases and 7 (14%) septic shock cases. Following admission due
to available facilities, only one patient was transferred to the ICU (non-survivor),
one received mechanical ventilation (survivor), 2 received peritoneal dialysis (both
non-survivors) and 7 received vaso-active amines (all septic shock, 5 non-survivors).
Expectedly, when comparing the non-sepsis/sepsis patients with the severe sepsis/
septic shock patients a strong association was found between disease severity at
hospital admission and mortality (OR 32, 95%CI: 3.7-274; p < .0001). All non-sepsis
patients survived. Patient characteristics and laboratory results in accordance to
survivor (n= 38) and non-survivors (n=14) are listed in table 1.
RESuLTS
Chap
ter7
108
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R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Tabl
e 1:
Clin
ical
cha
ract
eris
tics
of
52 p
atie
nts
wit
h se
vere
lept
ospi
rosi
s.
All(52)
Survivors(38)
Non
-survivors(14)
p-value
Gen
eral
Mal
e, (
%)37
(71
)29
(76
)8
(57)
0.2
med
ian
age,
(IQ
R)45
(32
-55)
42 (
31-5
5)47
(40
-55)
0.3
Med
ian
days
at
hosp
ital
, (I
QR)
10 (
6-13
)11
(10
-14)
3 (2
-6)
Disea
seseverity
Jaun
dice
, (%
)42
(81
)32
(84
)10
(71
)0.
4O
ligur
ia,
(%)
8 (1
6)7
(18)
5 (3
6)0.
3An
uria
, (%
)2
(4)
02
(14)
0.07
Blee
ding
(%)
31 (
60)
21(5
5)10
(71
)0.
3N
on-s
epsi
s, (
%)4
(8)
4 (1
1)0
(0)
0.6
Fulfi
l cri
teri
a se
psis
, (%
)24
(46
)23
(61
)1
(7)
0.00
1Fu
lfil c
rite
ria
seve
re s
epsi
s, (
%)17
(33
)9
(24)
8 (5
7)0.
02Fu
lfil c
rite
ria
sept
ic s
hock
, (%
)7
(14)
2 (5
)5
(36)
0.01
Labo
ratoryresults
Tem
pera
ture
(ºC
), (
IQR)
38 (
37.7
-38.
5)38
.0 (
37.6
-38.
4)38
.2 (
37.6
-38.
6)0.
5Pu
lse
(BPM
), (
IQR)
100
(92-
108)
100
(92-
104)
100
(92-
108)
0.9
Syst
olic
BP
(mm
Hg)
, (I
QR)
110
(110
-128
)11
0 (1
09-1
20)
120
(108
-130
)0.
3D
iast
olic
BP
(mm
Hg)
, (I
QR)
70 (
60-8
0)70
(65
-80)
70 (
60-8
0)0.
4Re
spir
ator
y ra
te (
brea
ths/
min
), (
IQR)
24 (
20-2
8)24
(20
-24)
26 (
20-3
6)0.
04H
b (1
2-15
gr/
dl),
(IQ
R)11
(10
-12)
11.4
(10
.6-1
2.4)
10.9
(9.
1-11
.6)
0.1
Ht
(0.4
0-0.
54),
(IQ
R)0.
33 (
0.31
-0.3
7)34
.1 (
31.3
-37.
0)31
.4 (
27.0
-34.
1)0.
1Le
ucoc
ytes
(4-
11x1
09 /L)
, (I
QR)
16 (
11-1
8)16
(13
-18.
0)11
(10
-20)
0.2
Plat
elet
s (1
50-4
00x1
09 /L)
, (I
QR)
70 (
35-1
35)
64.5
(35
-161
)75
.5 (
34-9
6)0.
8AS
T (1
5-37
U/L
), (
IQR)
70 (
40-1
12)
63 (
39-1
01)
68 (
54-2
99)
0.3
ALT
(30-
65 U
/L),
(IQ
R)53
(43
-76)
58 (
43-7
8)52
(38
-94)
0.9
Bilir
ubin
(2-
17 μ
mol
/L),
(IQ
R)14
2 (5
8-34
2)14
2 (5
8-33
4)19
0 (1
25-4
36)
0.3
Crea
tini
n (4
6-99
μm
ol/L
), (
IQR)
477
(274
-672
)41
5 (2
03-6
63)
637
(504
-858
)0.
1
Num
bers
are
med
ians
wit
h co
rres
pond
ing
IQR
or n
umbe
rs w
ith
perc
enta
ges.
Sep
sis
crit
eria
wer
e sc
ored
fro
m a
dmis
sion
in
2 da
y ev
olut
ion.
D
iffe
renc
e be
twee
n su
rviv
ors
and
non-
surv
ivor
s w
ere
calc
ulat
ed u
sing
the
Man
n-W
hitn
ey U
tes
t or
Chi
squ
are
test
whe
n ap
prop
riat
e. A
p-v
alue
of
< 0
.05
was
con
side
red
stat
isti
cally
sig
nific
ant.
Abb
reviations:IQR,25t
h an
d75
thinterqu
artileran
ge;BP
M,be
atspe
rminute;BP,blood
pressure.
PTX3
inseverelep
tospirosis
109
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R9
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R11
R12
R13
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
Tabl
e 1:
Clin
ical
cha
ract
eris
tics
of
52 p
atie
nts
wit
h se
vere
lept
ospi
rosi
s.
All(52)
Survivors(38)
Non
-survivors(14)
p-value
Gen
eral
Mal
e, (
%)37
(71
)29
(76
)8
(57)
0.2
med
ian
age,
(IQ
R)45
(32
-55)
42 (
31-5
5)47
(40
-55)
0.3
Med
ian
days
at
hosp
ital
, (I
QR)
10 (
6-13
)11
(10
-14)
3 (2
-6)
Disea
seseverity
Jaun
dice
, (%
)42
(81
)32
(84
)10
(71
)0.
4O
ligur
ia,
(%)
8 (1
6)7
(18)
5 (3
6)0.
3An
uria
, (%
)2
(4)
02
(14)
0.07
Blee
ding
(%)
31 (
60)
21(5
5)10
(71
)0.
3N
on-s
epsi
s, (
%)4
(8)
4 (1
1)0
(0)
0.6
Fulfi
l cri
teri
a se
psis
, (%
)24
(46
)23
(61
)1
(7)
0.00
1Fu
lfil c
rite
ria
seve
re s
epsi
s, (
%)17
(33
)9
(24)
8 (5
7)0.
02Fu
lfil c
rite
ria
sept
ic s
hock
, (%
)7
(14)
2 (5
)5
(36)
0.01
Labo
ratoryresults
Tem
pera
ture
(ºC
), (
IQR)
38 (
37.7
-38.
5)38
.0 (
37.6
-38.
4)38
.2 (
37.6
-38.
6)0.
5Pu
lse
(BPM
), (
IQR)
100
(92-
108)
100
(92-
104)
100
(92-
108)
0.9
Syst
olic
BP
(mm
Hg)
, (I
QR)
110
(110
-128
)11
0 (1
09-1
20)
120
(108
-130
)0.
3D
iast
olic
BP
(mm
Hg)
, (I
QR)
70 (
60-8
0)70
(65
-80)
70 (
60-8
0)0.
4Re
spir
ator
y ra
te (
brea
ths/
min
), (
IQR)
24 (
20-2
8)24
(20
-24)
26 (
20-3
6)0.
04H
b (1
2-15
gr/
dl),
(IQ
R)11
(10
-12)
11.4
(10
.6-1
2.4)
10.9
(9.
1-11
.6)
0.1
Ht
(0.4
0-0.
54),
(IQ
R)0.
33 (
0.31
-0.3
7)34
.1 (
31.3
-37.
0)31
.4 (
27.0
-34.
1)0.
1Le
ucoc
ytes
(4-
11x1
09 /L)
, (I
QR)
16 (
11-1
8)16
(13
-18.
0)11
(10
-20)
0.2
Plat
elet
s (1
50-4
00x1
09 /L)
, (I
QR)
70 (
35-1
35)
64.5
(35
-161
)75
.5 (
34-9
6)0.
8AS
T (1
5-37
U/L
), (
IQR)
70 (
40-1
12)
63 (
39-1
01)
68 (
54-2
99)
0.3
ALT
(30-
65 U
/L),
(IQ
R)53
(43
-76)
58 (
43-7
8)52
(38
-94)
0.9
Bilir
ubin
(2-
17 μ
mol
/L),
(IQ
R)14
2 (5
8-34
2)14
2 (5
8-33
4)19
0 (1
25-4
36)
0.3
Crea
tini
n (4
6-99
μm
ol/L
), (
IQR)
477
(274
-672
)41
5 (2
03-6
63)
637
(504
-858
)0.
1
Num
bers
are
med
ians
wit
h co
rres
pond
ing
IQR
or n
umbe
rs w
ith
perc
enta
ges.
Sep
sis
crit
eria
wer
e sc
ored
fro
m a
dmis
sion
in
2 da
y ev
olut
ion.
D
iffe
renc
e be
twee
n su
rviv
ors
and
non-
surv
ivor
s w
ere
calc
ulat
ed u
sing
the
Man
n-W
hitn
ey U
tes
t or
Chi
squ
are
test
whe
n ap
prop
riat
e. A
p-v
alue
of
< 0
.05
was
con
side
red
stat
isti
cally
sig
nific
ant.
Abb
reviations:IQR,25t
h an
d75
thinterqu
artileran
ge;BP
M,be
atspe
rminute;BP,blood
pressure.
PTX3andotherbiomarkersareelevatedinsevereleptospirosispatients
Leptospirosis patients showed elevated PTX3 levels on admission with a median (IQR)
of 33.6 (15.7-70.2) ng/ml. The maximum PTX3 concentration observed was 399ng/
ml in a deceased patient. Follow-up data (day 14) displayed a strong decline of PTX3
levels, with a median (IQR) PTX3 concentration of 5.0 (3.0-9.0) ng/ml. To compare
PTX3 levels with known markers of inflammation, CRP, IL-6 and IL-8 were measured.
Elevated plasma levels of CRP, IL-6 and IL-8 were found on admission. A marked
decrease was observed during follow up. Admission PTX3 levels correlated with IL-8
concentrations (rho 0.64, p < .0001). Weaker correlations were found between CRP
(rho 0.41, p = .002) or IL-6 (rho 0.40, p = .003) and PTX3. In addition, the time from
onset symptoms to admission was inversely correlated with PTX3 (rho -0.37, p =
.007), CRP (rho -0.41, p = .002) and IL-8 levels (rho -0.36, p = .01).
PTX3levelsareassociatedwithdiseaseseverityandmortality
Figure 1 shows that the deceased patients displayed marked elevations of PTX3
compared with the survivors (p = .03). The same held true for both IL-6 (p = .005)
and IL-8 (p = .006) levels, although CRP levels were the same in both groups (p =
.95). PTX3, CRP and cytokine levels in patients with severe leptospirosis stratified by
disease severity are shown in figure 2. No statistical differences were found between
the non-sepsis and sepsis patients, whereas PTX3 was significantly elevated in the
severe sepsis/septic shock group when compared with the sepsis patients (p = .005).
This difference was not observed in case of IL-6, IL-8 or CRP.
Next we were interested in whether PTX3 plasma levels were statistically associated
with outcome and disease severity.
The results reported in table 2 indicate that log transformed PTX3, IL-6, IL-8 but not
CRP, were significantly associated with mortality in a logistic regression model. The
ROC curves indicate that PTX3, IL-6 and IL-8 are significantly predictors of mortality.
PTX3, in contrast to the other markers of inflammation, was the only marker that
was significantly associated with disease severity. The ROC curves, demonstrating the
trade-off between sensitivity and specificity of all markers are displayed in figure 3.
Chap
ter7
110
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Figure 1: Pentraxin 3 (PTX3), C-reactive protein (CRP) and cytokine plasma levels in patients
with severe leptospirosis.
Scatter dot diagram with median pentraxin 3 (PTX3), C-reactive protein (CRP), interleukin 6
(IL-6) and interleukin 8 (IL-8) levels on admission in all patients (n=52), survivors (n=38), non-
survivors (n=14) and during follow-up on day 14 (n=28). Patients who went on to die displayed
significantly higher PTX3, IL-6 and IL-8 plasma levels compared to the survivors (*p < .05; Mann-
Whitney U test)
PTX3
inseverelep
tospirosis
111
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Figure 2: Pentraxin 3 (PTX3), C-reactive protein (CRP) and cytokine levels in patients with
severe leptospirosis stratified by disease severity.
Scatter dot diagram with median pentraxin 3 (PTX3), C-reactive protein (CRP), interleukin 6
(IL-6) and interleukin 8 (IL-8) levels on admission in patients stratified into: non-sepsis (n=4),
sepsis (n=24) and severe sepsis/septic shock (24). Asterisks in the figures indicate the difference
between sepsis and severe sepsis/septic shock patients (* p<0.05; Kruskal-Wallis test).
Chap
ter7
112
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Figure 3: Receiver-operating-characteristic (ROC) curve for pentraxin 3 (PTX3), C-reactive
protein (CRP) and cytokine levels in patients with severe leptospirosis stratified by mortality
and disease severity.
ROC curves show the predictive accuracy of pentraxin 3 (PTX3), C-reactive protein (CRP),
interleukin 6 (IL-6) and interleukin 8 (IL-8) levels on admission for mortality or disease severity
which was stratified in two day evolution into a non-sepsis (n=4)/sepsis group (n=24) and a
severe sepsis (n=17)/septic shock (n=7) group.
Table 2: Association between pentraxin 3 (PTX3), mortality and disease severity.
Mortality Disease severityVariable AuC (95%CI) OR (95%CI) p-value AuC (95%CI) OR (95%CI) p-valueLog PTX3 0.70 (0.54-0.86)* 5.6 (1.2-26) 0.03 0.73 (0.60-0.87)* 9.5 (2.0-45) 0.005Log CRP 0.51 (0.30-0.71) 0.8 (0.2-3.4) 0.80 0.53 (0.37-0.70) 1.2 (0.3-4.5) 0.78Log IL-6 0.75 (0.60-0.91)* 3.2 (1.2-8.1) 0.02 0.67 (0.52-0.82)* 2.5 (1.1-5.9) 0.03Log IL-8 0.75 (0.59-0.91)* 6.5 (1.5-28) 0.01 0.64 (0.49-0.80) 3.0 (0.9-9.6) 0.06
The table shows the association between PTX3 and other markers of inflammation with mortality and disease severity. Disease severity was dichotomized into a non-sepsis (n=4)/sepsis group (n=24) and a severe sepsis (n=17)/septic shock (n=7) group. OR with 95% confidence intervals were calculated using a univariate binary logistic regression approach. A p-value of ≤0.05 was considered to indicate statistical significance. * P-value < .05Abbreviations:AUC,areaunderthereceiveroperatingcurve;OR,oddsratio.
PTX3
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tospirosis
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Leptospirosis is presumed to be the most widespread zoonosis in the world (24).
The incidence is high in (sub-) tropical countries; due mainly to the ability of
the spirochetes to survive longer in a warm, humid environment. Although most
leptospirosis infections are mild, severe leptospirosis cases occur and often run a
rapidly progressive course with a high case fatality rate. Therefore early triage of
these severe cases might reduce mortality. Several patient factors are found to be
associated with mortality including age, sex. As well several disease conditions, such
as altered mental status, oliguria and respiratory insufficiency (25) also indicate
more severe leptospirosis. At present, there is limited data available on potential
biomarkers that may aid the clinician in monitoring disease progression and identify
severe disease at an early stage. Hence, in a group of leptospirosis patients, with a
majority of sever cases, we evaluated PTX3 and other candidate biomarkers in their
ability to predict disease severity and outcome.
The results presented here demonstrate that long pentraxin PTX3 is elevated in
patients suffering from severe leptospirosis, with maximum elevations of PTX3
observed in the deceased and the most severely ill. As well, plasma PTX3 levels
correlated with admission CRP, IL-6 and IL-8 levels. Moreover we report an association
between PTX3, IL-6, IL-8 and mortality. The frequently used short pentraxin CRP
failed to demonstrate this association. In regard to disease severity, only PTX3 was
able to distinguish septic patients from severe septic/septic shock patients. This was
in contrast to the other inflammatory markers that showed either comparable levels
(CRP) or a very wide spread (IL-6, IL-8).
Previous studies also investigated the performance of PTX3 in critically ill patients
(6;7;10). Muller et al. reported an association with mortality (Area under ROC: 0.63;
OR 1.38) and with the diagnosis of sepsis (Area under ROC: 0.73; OR not reported).
Another study carried out in dengue patients, showed that PTX3 levels were
significantly further elevated in patients suffering from dengue shock syndrome when
compared to non-shock cases (6). In acute myocardial infarction, PTX3 but not CRP
or other cardiac biomarkers, was able to predict three-month mortality (10). The
highest fourth quartile of plasma PTX3 levels was associated with the highest risk
DISCuSSION
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ter7
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of cardiac events (9.23-fold compared with the first quartile) in patients with heart
failure (11). Moreover PTX3 concentrations were significantly higher in patients with
unstable angina pectoris compared with controls (9).
Different sepsis studies have demonstrated an association between risk of death and
elevated cytokine levels (26-28). In our cohort IL-6 and IL-8 scored best in predicting
mortality, however in terms of AUC values differences were small. Other results
indicated that IL-6 (OR 1.47; Area under ROC: 0.72) scored better than PTX3 and CRP
(7). These conflicting results are in line with observation that the range of cytokine
levels from survivors and non-survivors often overlap, making them of poor prognostic
value (29). In regard to IL-6, it has been shown that persistent elevations appear to
be more important than initial or peak levels in terms of outcome associations (30).
The interpretation of our data is limited by some shortcomings that merit further
comment. Our relatively small cohort consisted only of severally ill patients of which
only 4 patients were non-septic. Hence we were not able to adequately study the
performance of PTX3 in mild cases versus the severe. Secondly we were not able to
compare the expression of PTX3 with the widely used APACHE or SAPS scores, since
most patients were not admitted to the ICU and therefore not scored accordingly.
Instead we stratified disease severity by sepsis scores in 2 days evolution as best
alternative.
In conclusion, elevated PTX3 levels were found in patients with severe leptospirosis.
High concentrations were more likely to occur in the severely ill and deceased group.
The usefulness of PTX3 in monitoring severe leptospirosis deserves to be evaluated in
larger prospective studies with both mild, moderate and severely ill patients.
PTX3
inseverelep
tospirosis
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Acknowledgements
We thank the persons and organizations who provided invaluable assistance during this
study: S.M.H. Faradz and staff (CEBIOR, Faculty of Medicine, Diponegoro University,
Semarang Indonesia), the residents from the department of Internal Medicine (Dr.
Kariadi hospital, Semarang, Indonesia), J. Pater, D.W.M. Kruijswijk (Center for
Experimental and Molecular Medicine, Amsterdam, The Netherlands), M. Limper for
critically reviewing the manuscript and the Cirion Investigators group (Amsterdam,
The Netherlands). This work was funded by Fondazione CARIPLO (Project NOBEL),
Sixth Research Framework Programme of the European Union, (project: MUGEN NoE
www.mugen-noe.org MUGEN LSHG-CT-2005-005203), Telethon (Project: GGP05095).
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Leptospira peptidoglycans induce the release of tumor necrosis factor alpha from human monocytes. FEMS Microbiol Lett 1996 May 1;138(2-3):211-4.
(3) Fost de M, Hartskeerl RA, Groenendijk MR, Van der PT. Interleukin 12 in part regulates gamma interferon release in human whole blood stimulated with Leptospira interrogans. Clin Diagn Lab Immunol 2003 March;10(2):332-5.
(4) Chierakul W, Fost de M, Suputtamongkol Y, Limpaiboon R, Dondorp A, White NJ et al. Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis. Clin Immunol 2004 November;113(2):140-4.
(5) Tajiki H, Salomao R. Association of plasma levels of tumor necrosis factor alpha with severity of disease and mortality among patients with leptospirosis. Clin Infect Dis 1996 November;23(5):1177-8.
(6) Mairuhu AT, Peri G, Setiati TE, Hack CE, Koraka P, Soemantri A et al. Elevated plasma levels of the long pentraxin, pentraxin 3, in severe dengue virus infections. J Med Virol 2005 August;76(4):547-52.
(7) Muller B, Peri G, Doni A, Torri V, Landmann R, Bottazzi B et al. Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. Crit Care Med 2001 July;29(7):1404-7.
(8) Bevelacqua V, Libra M, Mazzarino MC, Gangemi P, Nicotra G, Curatolo S et al. Long pentraxin 3: a marker of inflammation in untreated psoriatic patients. Int J Mol Med 2006 September;18(3):415-23.
(9) Inoue K, Sugiyama A, Reid PC, Ito Y, Miyauchi K, Mukai S et al. Establishment of a high sensitivity plasma assay for human pentraxin3 as a marker for unstable angina pectoris. Arterioscler Thromb Vasc Biol 2007 January;27(1):161-7.
(10) Latini R, Maggioni AP, Peri G, Gonzini L, Lucci D, Mocarelli P et al. Prognostic significance of the long pentraxin PTX3 in acute myocardial infarction. Circulation 2004 October 19;110(16):2349-54.
(11) Suzuki S, Takeishi Y, Niizeki T, Koyama Y, Kitahara T, Sasaki T et al. Pentraxin 3, a new marker for vascular inflammation, predicts adverse clinical outcomes in patients with heart failure. Am Heart J 2008 January;155(1):75-81.
(12) Garlanda C, Bottazzi B, Bastone A, Mantovani A. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol 2005;23:337-66.
(13) Alles VV, Bottazzi B, Peri G, Golay J, Introna M, Mantovani A. Inducible expression of PTX3, a new member of the pentraxin family, in human mononuclear phagocytes. Blood 1994 November 15;84(10):3483-93.
(14) Breviario F, d’Aniello EM, Golay J, Peri G, Bottazzi B, Bairoch A et al. Interleukin-1-inducible genes in endothelial cells. Cloning of a new gene related to C-reactive protein and serum amyloid P component. J Biol Chem 1992 November 5;267(31):22190-7.
(15) Doni A, Michela M, Bottazzi B, Peri G, Valentino S, Polentarutti N et al. Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-gamma. J Leukoc Biol 2006 April;79(4):797-802.
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(16) Goodman AR, Levy DE, Reis LF, Vilcek J. Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages. J Leukoc Biol 2000 March;67(3):387-95.
(17) Han B, Mura M, Andrade CF, Okutani D, Lodyga M, dos Santos CC et al. TNFalpha-induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. J Immunol 2005 December 15;175(12):8303-11.
(18) Lee GW, Goodman AR, Lee TH, Vilcek J. Relationship of TSG-14 protein to the pentraxin family of major acute phase proteins. J Immunol 1994 October 15;153(8):3700-7.
(19) Luchetti MM, Sambo P, Majlingova P, Svegliati BS, Peri G, Paroncini P et al. Scleroderma fibroblasts constitutively express the long pentraxin PTX3. Clin Exp Rheumatol 2004 January;22(3 Suppl 33):S66-S72.
(20) Nauta AJ, de Haij S, Bottazzi B, Mantovani A, Borrias MC, Aten J et al. Human renal epithelial cells produce the long pentraxin PTX3. Kidney Int 2005 February;67(2):543-53.
(21) He X, Han B, Liu M. Long pentraxin 3 in pulmonary infection and acute lung injury. Am J Physiol Lung Cell Mol Physiol 2007 May;292(5):L1039-L1049.
(22) Bottazzi B, Garlanda C, Salvatori G, Jeannin P, Manfredi A, Mantovani A. Pentraxins as a key component of innate immunity. Curr Opin Immunol 2006 February;18(1):10-5.
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(24) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (25) Ko AI, Galvao RM, Ribeiro Dourado CM, Johnson WD, Jr., Riley LW. Urban epidemic
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(27) Casey LC, Balk RA, Bone RC. Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med 1993 October 15;119(8):771-8.
(28) Pinsky MR, Vincent JL, Deviere J, Alegre M, Kahn RJ, Dupont E. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest 1993 February;103(2):565-75.
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Soluble ST2 levels are associated with bleeding
in patients with severe Leptospirosis
J.F.P. Wagenaar 1, M.H. Gasem 2, M.G.A. Goris 3, M. Leeflang 3, R.A. Hartskeerl 3, T. van der Poll 4, C. van ’t Veer 4 and E.C.M van Gorp 1, 5
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Department of Internal medicine, Dr. Kariadi hospital, Diponegoro University,
Semarang, Indonesia 3 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
4 Center for Experimental and Molecular Medicine, Amsterdam, the Netherlands5 Department of virology, Erasmus University, Rotterdam, the Netherlands
PLoSNeglTropDis2009;3(6):e453.
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Background:Severe leptospirosis is featured by bleedings 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 the study was to determine the extent of sST2 and (pro-)
inflammatory cytokine release in patients with severe leptospirosis.
Methodologyandprinciplefindings:In an observational study, 68 consecutive cases
of severe leptospirosis were included. Soluble ST2 and cytokines (TNF-a, 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 aninvitro 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 invitro, no sST2
production could be detected.
Conclusions: Patients with severe leptospirosis demonstrated elevated plasma
sST2 levels. Soluble ST2 levels were associated 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.
AbSTRACT
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Leptospirosis is a worldwide occurring zoonosis (1), reported to be fatal in up to
50% of cases (2). The disease is caused by spirochetes that are spread by the urine
of infected animals, for example rats, mice and cattle amongst others. Survival of
Leptospira is enhanced in a warm and humid environment, where environmental
circumstances are most favourable. Hence prevalence is higher in (sub) tropical
countries. Severe leptospirosis is featured by bleeding complications and multi-organ
failure, which can eventually lead to shock and even death. Necropsy reports confirm
widespread haemorrhaging throughout the body, involving most vital organs and
tissues (3). This haemorrhaging could possibly be the result of capillary wall damage.
Several proinflammatory cytokines, such as TNF-a and IL-12p40 are reported to be
induced during infection with Leptospira (4;5). As well, elevated plasma concentrations
of TNF-a have been associated with lethal outcome amongst leptospirosis patients
(6), In a hamster model, late expression of the anti-inflammatory cytokines IL-4,
TGF-β and IL-10 have been shown (7).
Currently it is assumed that both exaggerated inflammation and immune suppression
contribute to an adverse outcome in sepsis (8). ST2, also designated T1, Fit-1 and
DER4, is thought to play a significant role in tuning the host inflammatory response.
ST2 is a receptor that is present in two main forms, in the soluble secreted form
(sST2) (9) and in a membrane-anchored form (ST2L) (10). Both are encoded from the
ST2gene regulated by different promoters (11) and are members of the IL-1 receptor
family. ST2 gene expression was identified originally in fibroblasts (9;12). Expression
has also been detected in several other cells, including Th2 cells, mast cells and
macrophages (13-16).
ST2L has been reported to attenuate downstream IL-1RI and TLR4 signalling by
sequestering MyD88 and MAL (MyD88 adaptor-like) (17). In contrast, previous work
has demonstrated that interleukin (IL)-33 is able to activate NF-κB and MAP kinases
by signaling through ST2L (18). IL-33/ST2L signalling in mast cells and Th2 cells
results in the production of Th2-associated cytokines, potentially balancing ongoing
inflammatory Th1 responses (18).
INTRODuCTION
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The functional role of soluble ST2 has not yet been fully elucidated. Elevated
concentrations of sST2 have been found in patients with inflammatory disorders
associated with abnormal Th2 mediated responses, in for example, autoimmune
diseases (19), asthma (20), idiopathic pulmonary fibrosis (21) and in patients with
sepsis (22). Moreover sST2 have also been proposed as a biomarker for heart failure
(23) and elevated levels have been seen to be predictive for clinical outcome in
acute myocardial infarction (24).
By using a soluble ST2-Immunoglobulin fusion protein, Sweet et al. demonstrated
that this molecule was able to bind macrophages through a putative ST2 receptor,
the expression of which was enhanced by LPS stimulation (25). Furthermore this
molecule was shown to suppress LPS-induced proinflammatory response (TNF-a, IL-6
and IL-12) in vitro and reduced inflammation and mortality in LPS challenged mice
(25). Administration of soluble ST2-Immunoglobulin fusion protein markedly reduced
proinflammatory cytokine production and lethality in intestinal ischemia/reperfusion
injury in mice (26). The anti-inflammatory effect exerted by sST2-Fc was dependent
on the elevated production of IL-10. Similar results were seen in hepatic ischemia/
reperfusion injury (27). sST2-Fc fusion protein administration also shown to have
beneficial effects in a murine model of collagen-induced arthritis (28). Asthmatic
mice administered with sST2-Fc fusion protein or a soluble ST2 expressing vector
showed attenuated production of the Th2 cytokines IL-4 and IL-5 (29;30), whereas
ST2L signaling resulted in the opposite. sST2 has therefore been proposed to act as a
decoy receptor for IL-33 (31;32).
To the best of our knowledge, in previous literature plasma levels of sST2 have not
been documented in leptospirosis patients. Hence, in the present study we evaluate
sST2, cytokine kinetics and their association with clinical events in a series of severe
leptospirosis patients.
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Ethicsstatement
The study protocol was approved by the medical ethic committees of both the Dr.
Kariadi hospital- University of Diponegoro, Semarang, Indonesia and the Slotervaart
Hospital in The Netherlands. Written informed consent was obtained from all
included subjects.
Patientsanddesign
Consecutive cases of severe leptospirosis were included from February 2005 to
September 2006 at the Dr. Kariadi hospital- University of Diponegoro, Semarang,
Indonesia. Severe leptospirosis was defined as a hospitalized patient with high clinical
suspicion of severe leptospirosis a positive LeptoTek Dri-Dot assay (Biomérieux),
presenting with at least one of the following symptoms or signs jaundice, renal
failure, thrombocytopenia and/or haemorrhaging..Cases were confirmed by further
laboratory testing. Blood samples were taken on hospital admission and during follow
up. Plasma was worked up immediately and aliquots were stored at -70°C for further
analyses. Twenty control (non-leptospirosis patients) samples were collected among
healthy volunteers at the department of internal medicine of the Dr. Kariadi hospital-
University of Diponegoro, Semarang, Indonesia.
MeasurementsandAssays
Soluble ST2 was measured by the commercially available ELISA (R&D systems,
Minneapolis, MN). Tumor necrosis factor (TNF)-a, IL-1ß, IL-6, IL-8, IL-10, and IL-12p70
were determined using a cytometric beads array multiplex assay (BD Biosciences,
San Jose, CA). The detection limits were as follows, TNF-a, IL-10 (2.5 pg/ml); IL-1ß,
IL-6, IL-8 (5 pg/ml); IL-12p70 (10 pg/ml); sST2 (15 pg/ml).
Leptospirosis was confirmed by either a positive culture or microscopic agglutination
test (MAT). Tests were considered positive for the MAT with a titre of ≥ 1:320 on a
single sample, seroconversion or a fourfold or higher increase of the titre in paired
samples or a titre ≥ 1:80 in a single sample from early deceased patients.
METHODS
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Invitroexperiments
To determine whether blood cells are an important source of sST2 during infection, we
undertook aninvitro experiment. We used either human whole blood or peripheral
blood mononuclear cells (PBMCs), which were then stimulated with Leptospira
interrogans serovar Bataviae strain M, as this serovar is commonly found in the region.
This was a fresh, low passage isolate obtained from the Leptospirosis Reference
Center in Amsterdam, The Netherlands. For the invitro experiments, bacteria were
washed 3 times with RPMI 1640 (Gibco) and counted using a Helber Counting Chamber
(Hawksley, Lancing, Sussex, UK) under darkfield microscopy and then resuspended at
concentrations of 2.5 x 107 till 2.5 x 105 bacteria per ml. Shortly before starting the
experiment, heparinized blood was sterilely drawn from multiple healthy donors and
diluted 1:1 in RPMI. PBMC were obtained using Lymphoprep™ (Axis-Shield) according
to the manufacturer’s guidelines. Whole blood (50 µl per well) or PBMC were divided
over each well of a 96-well plate before Leptospira concentrates were added. The
concentration PBMC was equivalent to 50 µl whole blood per well (approximately
0.5x109 monocytes). Plates were incubated for six hours at 37°C, 5% CO2. Following
incubation the plates were centrifuged and supernatant was collected and stored at
–70 °C for further testing. All experiments were performed in quadruplicate. For the
negative controls RPMI without bacteria was used.
Statisticalanalysis
Continuous variables were presented as medians with corresponding interquartile
ranges (IQR) and were statistically evaluated using the non-parametric Mann-Whitney
U test. Correlation between soluble ST2, clinical characteristics and cytokine levels
in patients were determined using the Spearman correlation coefficient (rho).
Associations were calculated using a binary logistic regression analysis and were
expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). An OR
>1 indicates that the risk of a clinical event is higher with the increase of biomarker
plasma levels and an OR of <1 indicates that the risk of a clinical event is lower
with the increase of plasma levels. Associations were further analyzed using the
receiver-operating-characteristic (ROC) approach by calculating the area under the
ROC curve (AUC). The AUC reflects the probability that a patient with higher plasma
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levels has a higher chance of the event than a patient with lower plasma levels. Log
transformation of the original variables was used to improve the goodness of fit of
the model. A p-value of <0.05 was considered to indicate statistical significance. All
analyses were done using SPSS (version 15.0, Chicago, Illinois).
Characteristicsofincludedpatients
In total 68 leptospirosis patients were included, of which 49 (72%) were male. The
median age (IQR) was 45 (34-55) years old. In total 16 patients (24%) did not survive,
with a median (IQR) time to death of 3 days post hospital admission. On average
patient’s symptoms had started at a median of 7 days pre hospital admission. Clinical
manifestations/symptoms included jaundice (75%), thrombocytopenia (platelets
< 100x109: 65%), oliguria (19%) and anuria (4%). Median leucocyte, platelet,
creatinine and bilirubin levels were: 15x109/L, 69x109/L, 412µmol/L and 113µmol/L
respectively. All patients had MAT serologically confirmed leptospirosis, with the
most frequently identified serogroups being Bataviae (19), Icterohaemorrhagiae (18)
and Ballum (2).
All patients showed elevated sST2, IL-6, IL-8 and IL-10 levels on admission,
presented in Table 1. TNF-a, IL-1β and IL-12p70 levels taken at hospital admission
were either very low or undetectable and were not significantly different from the
healthy controls (data not shown). Patients that died from leptospirosis (n=16) had
significantly further elevated plasma levels on admission compared to the survivors,
sST2 (p = .006), IL-6 (p = .003) and IL-8 (p = .003). However, IL-10 (p = .64) was
not found to be significantly elevated. Figure 1 shows the dynamics of sST2, IL-6,
IL-8 and IL-10. Circulating sST2 levels in the survivors showed a peak at day 0 after
which levels gradually decreased and normalized on day 7. However, patients who
died during their hospital stay displayed continuously high plasma levels of sST2
until death occurred. Table 2 shows that admission sST2 levels were significantly
correlated with levels of IL-6 (rho 0.45; p = .001), IL-8 (rho 0.72; p < .0001), IL-
10 (rho 0.56; p < .0001) and CRP (rho 0.50; p < .0001) also taken at admission..
RESuLTS
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Admission sST2 levels resulted in moderate correlations with respiratory rate (rho
-0.25; p = .04), platelets (rho -0.25; p = .04) creatinin (rho 0.33; p = .007), AST (rho
0.46; p = .001) levels and (severe) haemorrhaging (haemorrhaging: rho 0.34; p =
.005, severe haemorrhaging: rho 0.32; p = .008).
Table 1: Soluble ST2 (sST2) and cytokines on admission in patient with severe leptospirosis.
Marker (pg/ml) All (n=68) Survivors (n=52) Non-survivors (n=16) Controls p-value A B C D BCsST2 1480 (502-4378) 1203 (285-2773) 3596 (1452-8590) < 15 0.006IL-6 45 (17-135) 27 (16-74) 133 (52-430) < 5 0.003IL-8 40 (16-98) 32 (51-182) 81 (51-182) < 5 0.003IL-10 7 (4-18) 6 (4-17) 8 (4-37) < 2.5 0.64
Values represent medians with the corresponding IQR range. Statistical difference between survivors and non-survivors was calculated using the Mann-Whitney U test. A p-value < 0.05 was considered significant.Abbreviations:IQR,interquartilerange.
Table 2: Correlation between soluble ST2 (sST2), clinical markers and cytokines on day of
admission.
Serum sST2Variable rho p-value
Pulse 0.20 0.1RR -0.25 0.04Leucocytes -0.05 0.7Platelets -0.25 0.04Creatinin 0.33 0.007AST 0.46 0.001ALT -0.016 0.9CRP 0.50 < 0.0001Haemorrhaging 0.34 0.005
Mild 0.10 0.4Severe 0.32 0.008
TNF-a 0.17 0.2IL-1β 0.14 0.3IL-12p70 0.04 0.8IL-6 0.45 0.001IL-8 0.72 < 0.0001IL-10 0.56 < 0.0001
The correlation coefficient (rho) is calculated by the non-parametric Spearman’s rank correlation test. A p-value < 0.05 was considered significant.Abbreviations:RR,respiratoryrate;CRP,C-reactiveprotein.
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Figure 1: Soluble ST2 and cytokine dynamics in patients with severe leptospirosis.
The bar graphs show mean soluble ST2 and cytokine plasma levels for survivors (white) and non-
survivors (black). The error bars indicate the standard error of the mean (SEM). The horizontal
dotted line represents the detection limit of the assays. Asterisks in the figure indicate the
strength of the statistical difference from healthy controls (* p<0.05, ** p<0.001, *** p<0.0001;
Mann-Whitney U test).
BleedingisassociatedwithincreasedsST2plasmalevels
Since bleeding is an important feature of severe leptospirosis we were interested
whether this event was also associated with sST2 levels. In total 34 patients (50%)
showed signs of bleeding. We found mild haemorrhages (petechiae, ecchymoses
and epistaxis) in 23 cases and severe haemorrhages (gastrointestinal, melaena, gum
bleeding, hemoptysis and heamaturia) in 10 cases. To determine whether elevated
sST2 levels were associated with bleeding, we performed a binary logistic regression
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analysis and calculated the area under the ROC curve (AUC), see Table 3. Elevated
sST2 levels were significantly associated with overall haemorrhaging (mild and severe)
(OR 2.0; 95%CI: 1.2-3.6, p = .01; AUC: 0.70, p = .006) and severe haemorrhaging
(OR 5.1; 95%CI: 1.1-23.8, p = .04; AUC: 0.76, p = .009), but not with mild bleeding
alone (OR 1.3; 95%CI: 0.76-2.2, p = .3; AUC: 0.6, p = .5). As we log-transformed the
original variables, this means that for a ten-fold increase of plasma sST2 levels, the
odds of developing mild or severe bleeding will be 2.0 times higher, and the odds of
developing severe bleeding 5.1 times higher. None of the cytokines were significantly
associated with (severe) haemorrhaging (see table 3).
SolubleST2andcytokinelevelsareassociatedwithmortality
The association between plasma levels sST2, cytokines and mortality was calculated
using a binary logistic regression (OR, 95% CI) and a ROC approach (AUC). The odds
of patients with leptospirosis dying increased by 2.4 (95%CI: 1.0-5.8, p = .05) with a
ten-fold increase of plasma sST2 levels with an AUC of 0.73 (p = .006). As well the
odds of patients with leptospirosis dying increased by 3.2 (95%CI: 1.4-7.7, p = .008)
with an AUC of 0.74 (p = .003), with a ten-fold increase of plasma IL-6 levels. With
a ten-fold increase of plasma IL-8 there was an odds of 6.9 (95%CI: 1.8-27, p = .005)
and an AUC of 0.75 (p = .003). The anti-inflammatory cytokine IL-10 failed to reach
significance (OR 1.3; 95%CI: 0.5-3.9, p = .58; AUC: 0.58, p = .40), see table 3.
SolubleST2isnotreleasedinwholebloodafterstimulationwithviableLeptospira
To evaluate whether blood cells are an important source of sST2 during infection,
we undertook aninvitro experiment. We used a fresh viable pathogenic isolate of
Leptospirainterrogans serovar Bataviae strain M. When either human whole blood
or isolated PBMCs were stimulated with different concentrations bacteria, we could
not detect sST2 production after 6 hours incubation (Figure 2). The same held true
for the negative controls. In contrast, high levels TNF-a were measured as dose
dependent upon stimulation with viable Leptospira.
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Table 3: Association between soluble ST2 (sST2) bleeding and mortality.
Variable OR (95%CI) p-value AuC p-valueBleeding 10-log sST2 2.0 (1.2-3.6) 0.01 0.70 0.00610-log IL-6 1.8 (0.9-3.7) 0.1 0.61 0.110-log IL-8 2.6 (1.0-7.4) 0.06 0.62 0.0810-log IL-10 1.3 (0.5-3.3) 0.6 0.55 0.5Severebleeding 10-log sST2 5.1 (1.1-24) 0.04 0.76 0.00910-log IL-6 2.0 (0.83-4.9) 0.1 0.66 0.110-log IL-8 2.4 (0.77-7.7) 0.1 0.65 0.110-log IL-10 2.0 (0.6-6.6) 0.3 0.61 0.3Mortality 10-log sST2 2.4 (1.0-5.8) 0.05 0.73 0.00610-log IL-6 3.2 (1.4-7.7) 0.008 0.74 0.00310-log IL-8 6.9 (1.8-27) 0.005 0.75 0.00310-log IL-10 1.3 (0.5-3.9) 0.58 0.58 0.4
Associations are presented as OR with 95% confidence interval and AUC values. A p-value < 0.05 was considered significant.Abbreviations:OR,oddsratio;CI,confidenceinterval,AUC,areaundertheROCcurve(receiveroperatingcharacteristic).
Figure 2: In vitro stimulation of whole blood or peripheral blood mononuclear cells (PbMC)
with pathogenic Leptospira.
This bar graph shows mean TNF-a(white) and sST2 (black) levels with standard error of the
mean (SEM) for each group. Either human whole blood or peripheral blood mononuclear cells
(PBMC) were incubated with various concentrations pathogenic Leptospira. Controls were not
incubated with Leptospira. Soluble ST2 (sST2) and TNF-alevels were measured in supernatant
after 6 hours incubation.
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This study reports elevated sST2 levels in patients with severe leptospirosis. Soluble
ST2 levels correlated with other indicators of inflammation. A unique, significant
association between sST2 and bleeding was observed. As well soluble ST2, IL-6 and
IL-8 levels were all associated with poor outcome in leptospirosis patients.
Previous work has reported elevated sST2 plasma levels in fifteen septic patients, but
in this study no association with mortality was found (22). Becerra et al. reported
elevated sST2 levels in patients suffering from dengue fever, but in the convalescent
samples sST2 levels were normalized (33). Since all patients in this study survived and
had only mild disease, no associations with regard to disease severity and outcome
could be found. The data presented here extends these earlier studies, with findings
of elevated sST2 levels during infection in a larger, homogeneous group of patients.
Leptospirosis patients yielded elevated levels of IL-6 and IL-8 associated with
mortality in the present study which were stronger than sST2. From the literature,
several studies have found similar results presenting data on the association between
cytokine levels and poor outcome in septic patients (34;35). However, in these studies
the range of cytokine levels from survivors and non-survivors often overlapped, which
means that although the cytokines are associated with poor outcomes, they are of
little or no prognostic value (36). In a study by Chierakul et al. elevated TNF-a and IL-
12p40 levels were reported in 28 patients with mild leptospirosis (4). The biologically
active IL-12p70 heterodimer was detected in only 4 patients. An other study reported
increased TNF-a levels in four out of eighteen leptospirosis patients (6) . In this small
study, TNF-a was found to be associated with disease severity and poor outcome. In
contrast, in the current work TNF-a, IL-1β and IL-12p70 concentrations were either
very low or undetectable and did not differ from the controls. The fact that we could
not confirm the association between poor outcome and TNF-a can be explained by
the fact that our patients presented in late stage disease and TNF-a is considered to
be an early response cytokine.
It is interesting to speculate why (severe) bleeding was found to be associated with
sST2 plasma levels. Weinberg et al. identified sST2 release in response to myocardial
infarction and suggested that sST2 participates in the cardiovascular response
DISCuSSION
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to injury of the cardiomyocytes (37). Disruption of the endothelial cell barrier, a
possible explanation for the haemorrhages found in severe leptospirosis, exposes
underlying fibroblasts. In this light, sST2 could be an indicative marker for tissue
injury given the fact that serum stimulation of resting fibroblasts results in sST2
release (9). Our invitro experiments with pathogenic Leptospira showed that, at
least in the early phase, blood is not the source of sST2 production. These findings
were in line with previous findings of our group in which we found that membrane
bound ST2 is upregulated on monocytes when whole blood is incubated with LPS,
while sST2 remains undetectable in blood plasma after 24 hour whole blood LPS
stimulation (38).
In conclusion, in patients with severe leptospirosis we demonstrated elevated plasma
sST2 levels that normalized during follow-up and were associated with mortality.
Interestingly sST2 was the only marker that was associated with (severe) bleeding.
More research is warranted to elucidate the function of sST2 in the innate immune
response to Leptospira and to evaluate its value as a marker for tissue damage in
severely ill patients.
Acknowledgements
We gratefully thank the persons and organizations who provided invaluable assistance
during this study: S.M.H. Faradz and staff (CEBIOR, Faculty of Medicine, Diponegoro
University, Semarang Indonesia), the residents from the department of Internal
Medicine (Dr. Kariadi Hospital, Semarang, Indonesia), J. Pater, D.W.M. Kruijswijk
(Center for Experimental and Molecular Medicine, Amsterdam, The Netherlands),
K.R. Boer (Royal Tropical Institute (KIT), KIT Biomedical Research, Amsterdam, The
Netherlands) and the Cirion Investigators group (Amsterdam, The Netherlands).
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spectrum of pulmonary involvement in leptospirosis in a region of endemicity, with quantification of leptospiral burden. Clin Infect Dis 2005 February 1;40(3):343-51.
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(15) Oshikawa K, Yanagisawa K, Tominaga S, Sugiyama Y. ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation. Biochem Biophys Res Commun 2002 November 22;299(1):18-24.
(16) Bergers G, Reikerstorfer A, Braselmann S, Graninger P, Busslinger M. Alternative promoter usage of the Fos-responsive gene Fit-1 generates mRNA isoforms coding for either secreted or membrane-bound proteins related to the IL-1 receptor. EMBO J 1994 March 1;13(5):1176-88.
(17) Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O‘Neill LA et al. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 2004 April;5(4):373-9.
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(27) Yin H, Huang BJ, Yang H, Huang YF, Xiong P, Zheng F et al. Pretreatment with soluble ST2 reduces warm hepatic ischemia/reperfusion injury. Biochem Biophys Res Commun 2006 December 29;351(4):940-6.
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Innate immune response to pathogenic
Leptospira is dependent of both TLR2 and TLR4
signaling in human whole blood
J.F.P. Wagenaar 1*, M.G.A. Goris 2*, R.A. Hartskeerl 2, E.C.M van Gorp 1, J.E. Nally 3, A. M. Monahan 3, T. van der Poll 4, 5 and C. van ’t Veer 4, 5
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
3 Veterinary Sciences Centre, School of Agriculture Food Science & Veterinary Medicine, College of Life Sciences, University College Dublin, Belfield, Dublin, Ireland
4 Center for Experimental and Molecular Medicine, Amsterdam, the Netherlands5 Center for Infection and Immunity Amsterdam (CINIMA), University of Amsterdam,
Amsterdam, the Netherlands
* Both authors equally contributed
Submitted
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Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. Being
spread by the urine of infected animals, the bacteria enter the human body via
abraded skin or mucous membranes and rapidly disseminate throughout the body.
Mostly, the illness 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 Leptospira. Previous work showed that a human monocytic cell line (THP-
1) recognized heat-killed Leptospira and leptospiral LPS through TLR2 instead of
TLR4 as for typical gram-negative LPS. We investigated immune responses to diverse
live leptospiral serovars. Killing and stimulation experiments were done using THP-
1, human PBMC and whole blood. We examined the involvement of TLR2 and TLR4
signaling. Saprophytic and pathogenic reference strains but not fresh, host-adapted
Leptospira were killed by whole blood and serum. Live Leptospira induced a vigorous
serovar dependent cytokine response. PBMC was more sensitive to Leptospira than
THP-1 and whole blood was more sensitive than PBMC. Inhibition experiments with
anti-TLR2 and anti-TLR4 antibodies in whole blood showed involvement of both TLR2
and TLR4. Their signaling was additive in case of reference serovar Bataviae and
synergistic in case of its host-adapted counterpart. This study reveals three findings
with a major impact on the investigation of immune responses against leptospires
infection: (i) both TLR2 and TLR4 are involved in the innate immune response; (ii)
immune responses are (for part) serovar dependent; (iii) fresh isolates are needed to
better mimic natural infection.
AbSTRACT
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Leptospirosis transmission occurs worldwide and comprises all infections caused by
pathogenic spirochetes of the genus Leptospira. Currently over 250 serovars are
known, traditionally grouped into several serogroups. Being spread by the urine of
infected animals, the bacteria enter the human body via abraded skin, conjunctivae
or mucous membranes, after which they rapidly disseminate throughout the body.
The majority of infections are thought to result in a mild illness with rather non-
specific symptoms like fever, myalgia and headache. Some patients develop severe
disease, which is often rapidly progressive and can be fatal in up to 70% of cases (10).
The clinical picture is dominated by hepato-renal impairment and hemorrhages.
Patients usually die from septic shock with multi-organ failure and/or overt
(pulmonary) hemorrhages. Pathological findings reveal widespread haemorrhaging in
virtually all organs and tissues with diffuse inflammatory infiltrates (2). Leptospira
antigen has been identified in many organs, including lungs, liver and kidneys (16,18).
It is believed that Leptospira migrate through intercellular junctions, however
electron microscopy demonstrated bacteria within the cytoplasm, not contained in a
membrane compartment or intercellular junction (14).
Immunity against Leptospira depends on the production of circulating antibodies
directed against serovar specific lipopolysaccharides (LPS). Interestingly, leptospiral
LPS differs from gram-negative LPS in several biochemical, physical and biological
properties (8). Although crucial in early stage infection, not much is known about the
innate immune response to Leptospira. Several cytokines such as interferon (IFN) -g,
interleukin (IL)-12p40 and TNF-a, are released during infection (4,7). A human
monocytic cell line (THP-1), transfected with human CD14 was able to recognize
heat killed Leptospira and leptospiral LPS through toll-like receptor (TLR) 2 but not
TLR4 (30). This finding was unique since TLR4 is considered to be the predominant
receptor mediating a LPS induced cytokine response. Subsequent murine models,
using either heat killed bacteria or LPS, showed evidence that both TLR2 and TLR4
play a role (19,26). However, these previous studies might not be representative
of the immune response to different serovars and viable Leptospira. Hence, in
the present study we investigated the innate immune response to several viable
INTRODuCTION
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leptospiral serovars. Killing assays and stimulation experiments were performed
using a human monocytic cell line (THP-1), human peripheral blood mononuclear
cells (PBMC) and human whole blood. Moreover we examined the involvement of
TLR2 and TLR4 in the cellular responsiveness to leptospiral serovars.
Bacteriaandreagents
Leptospira strains from the WHO/FAO/OIE and National collaborating centre for
reference and research on Leptospirosis, Amsterdam, The Netherlands, were used.
Saprophytic (non-pathogenic) reference strain: Leptospira biflexa serovar Patoc
strain Patoc I. Pathogenic reference strains: Leptospirainterrogans serovar Bataviae
strain Swart and L.interrogans serovar Lai strain Lai. Two fresh isolates from human
patients: L.interrogans serovar Bataviae strain Kariadi-Satu, isolated in 2005 from
an adult male admitted at the Dr Kariadi Hospital in Semarang, Indonesia, identified
by cross-agglutinin absorption test (CAAT) (11) and sequencing (25) for serovar and
species determination, and L.interrogans serovar Lai type Langkawi strain Langkawi
respectively (27). Strain Kariadi-Satu was aliquoted and stored at –70 ºC shortly after
isolation, i.e. at a minimum of invitropassages, to maintain the integrity as human
host-adapted variant of the reference strain. Strain Langkawi was aliquoted and
stored at -70°C one year after isolation (approximately 15 passages). Leptospires
were grown in liquid Ellinghausen McCullough Johnson and Harris medium (EMJH, in
house prepared (9)). Fifty ml of a full grown culture was inoculated into 500 ml of
EMJH at 30°C in a shaking incubator for 5-7 days.
For the experiments, leptospires were washed 3 times with RPMI 1640 (Invitrogen,
Paisley, Scotland, UK) to remove possible LPS contamination from the growth
medium, counted using a Helber Counting Chamber (Hawksley, Lancing, Sussex, UK)
under darkfield microscopy and resuspended at a concentration of 2.5 X 109 bacteria
per ml from which further 10-fold dilutions were made in RPMI 1640. To heat-kill,
washed Leptospirawere subjected to a 30 minutes treatment at 56°C before dilution
in appropriate concentrations.
MATERIALS AND METHODS
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Virulenceassaybyinfectionofguineapigs
The virulence of leptospiral strains was determined by injection of guinea pigs with
Leptospira. The positive control was an isolate of Leptospira interrogans serovar
Copenhageni, designated RJ16441, obtained from a patient suffering from a severe
pulmonary form of leptospirosis (23). Low in vitro passages of this isolate were
passed through guinea pigs and rats to maintain virulence (20).
The saprophyte L.biflexa serovar Patoc strain Patoc I was used as negative control.
Hartley male guinea pigs (Charles River Laboratories, UK) at 3 weeks of age, weighing
250 to 260g were injected intraperitoneal with 107 in vitro cultivated Leptospirain
a final volume of 500 µl media. Animals were monitored daily for signs of illness
including weight loss and loss of mobility, and were euthanized when they appeared
moribund. If no illness developed, they were euthanized on day 7 or 8 post infection.
Liver and kidneys were cultured to detect the presence of leptospires. All study
protocols were approved by the University College Dublin Animal Research Ethics
Committee and conducted under license from the Department of Health and Children.
VirulencetestingbydetectionofLigA
To test virulence of the Leptospira, we performed a LigA Western blot. Only fresh,
pathogenic isolates express this protein (13). Rabbit anti-LigA was raised against
a fragment called LigANI, and includes amino acids 625 – 1224; The LigA positive
control was a truncated form of LigA. Both were generously supplied by the Goncalo
Moniz Research centre, Fundacao Oswaldo Cruz foundation, Salvador. Leptospira
were cultured and enumerated as described above, spun down at 1730g for 30
minutes and then washed twice with PBS+5mM Magnesium Chloride wash buffer to
form pellets with a concentration of 2X108leptospires/ pellet. The pellets were used
for SDS- PAGE in a discontinuous buffer system using a 7.5% polyacrylamide resolving
gel and 5% stacking gel freshly prepared. The Leptospirapellet was resuspended in
20ul reducing sample buffer consisting 0.5M Tris-HCl pH 6.8, 6 % SDS, 3% glycerol,
1,5mL ß-mercaptoethanol and 0,1% bromophenol blue and then boiled at 950C for
5minutes and the ensuing mixture then loaded to stacking gel and electrophoresed at
200V constant. Following electrophoresis, the separated proteins were transferred
to a nitrocellulose membrane using the BIORAD Mini Protean III apparatus. The
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membranes were then placed in blocking solution (4% skimmed milk in 0.05% PBS
Tween 20) for 30 minutes or overnight. Detection was carried out by immunostaining
to identify LigA/LigB in the sample using anti-LigA or anti-LigB as the primary
antibody for 60mins then washed 3 times in 0.05% PBS-Tween 20.This was followed
by 90 minutes incubation with a 1:4000 dilution goat anti-rabbit immunoglobulin
conjugated with horseradish peroxidase, (Jackson ImmunoResearch Laboratories.
Inc). The membrane was again washed 3 times with PBS-Tween 20. The membranes
were then placed in DAB solution until the bands were of an optimum visible intensity
and the reaction stopped with milliQ water. Pictures of the blot were then taken.
LigA expression was identified then a comparison was performed. Interpretation of
results was by visual analysis of the blot.
THP-1monocytes
The THP-1 monocyte cell line was obtained from American Type Culture Collection
(ATCC: TIB 202). Cells were cultured in RPMI 1640 containing 10% FCS (Invitrogen,
Paisley, Scotland, UK), 2 mM glutamin (Lonza, Basel, Switzerland), 40 U/ml penicillin,
40 µg/ml streptomycin and 0.1 µg/ml amphotericin B (Invitrogen, Paisley Scotland UK)
and incubated at 37ºC, 5 %CO2. Four days before the experiment, cells were washed
with RPMI 1640/10%FCS/2mM glutamin medium without antibiotics/antimycotics and
sub-cultured in this medium. Prior to the experiment THP-1 cells were spun down
and resuspended in fresh RPMI 1640/10%FCS/2mM glutamin to approximately 2.0 X
106 cells per ml. For experiments with normal human serum (NHS) filtrated, heat
inactivated NHS (Sanquin, Amsterdam, the Netherlands) was added to the medium
to a final concentration of 2%.
PBMC
Heparinized blood was collected aseptically from multiple healthy donors and
PBMC were obtained using Lymphoprep™ (Axis-Shield, Oslo, Norway) according to
the manufacturer’s guidelines. Briefly, blood was diluted 1:1 with pyrogen free
0.9%NaCl and carefully layered on top of the Lymphoprep solution and centrifuged
20 minutes at 800 g. The distinct band of PBMC was harvested, washed with RPMI and
resuspended in RPMI to the same volume of the original blood.
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WholeBlood
Shortly before starting the experiment, heparinized blood was collected aseptically
from multiple healthy donors.
Experiments
Experiments were performed in quadruplicate. Fifty µl of whole blood or cell
suspensions prepared as described above were put in the wells of a 96 well tissue
culture plate (Greiner Bio-One GmbH, Frickenhausen, Germany, CELLSTAR® 96-Well
Polystyrene). Leptospira were diluted in RPMI 1640 until appropriate concentrations
were reached. Final concentrations ranged from 2.5 X 103 to 2.5 X 108 bacteria/
ml. Leptospira were added to the various cell suspensions to a final volume of 100
µl per well. Concentrations of PBMC were equivalent to 50 µl whole blood per well
(approximately 0.5 X 106/ml monocytes). Plates were incubated for six hours at 37°C,
5% CO2. After incubation plates were centrifuged and supernatant was collected and
stored at -70°C before further testing. When studying TLR engagement, specific
blocking monoclonal antibodies to human TLR2 and TLR4 (InvivoGen, Toulouse,
France, anti-hTLR2-IgA and anti-hTLR4-IgA) were used. Antibodies were diluted
in RPMI 1640 to a final concentration of 2500 ng/ml and 1000 ng/ml respectively.
Control experiments were done using LTA (LTA-S.aureus) and LPS (E.coli ultrapure,
both InvivoGen, Toulouse France), known for their capacity to signal through TLR2 or
TLR4 respectively. Blood cells and antibodies were incubated for 30 minutes on the
microplate shaker before Leptospira were added.
Killingassay
To evaluate the sensitivity of the different Leptospira strains to killing by host
factors or cells, we incubated in quadruplicate 2.5 X 106 Leptospira/ml of all six
strains with whole blood, PBMC, THP-1 and 2% NHS from a healthy donor (without
a known history of leptospiral infection, not heat inactivated) for six hours at
37°C, 5% CO2. Concentrations and final volumes were identical to the incubation
experiments described above. Of every sample 50 µl was transferred into 450µl EMJH
supplemented with 0.02% 5-fluorouracil to avoid contamination. Numbers of viable
bacteria were estimated by serial tenfold dilution of samples. Growth was checked
after 3 weeks incubation at 30 ºC by dark field microscopy.
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TNF-aassay
Levels of TNF-a in culture supernatants were determined by enzyme-linked
immunosorbent assay (Biosource Europe SA Nivelles Belgium, human TNF-a
CytoSetsTM) according to the instructions of the manufacturer.
VirulencetestingofL.interrogansstrainsbyinfectionofguineapigs
Since mice are resistant to L.interrogans, virulence testing of this Leptospira genus
is performed in guinea pigs (3,12,20). The virulence of the L. interrogans strains
used herein of the serovar Batavia and Lai was tested in the standard guinea pig
model (20) by i.p. injection of viable bacteria. The freshly isolated minimally passed
serovar Batavia induced progressive weight loss, caused profound lung pathology, and
successfully infected all organs tested (Fig.1). Thus, the freshly isolated L.interrogans
Batavia strain displayed full virulence in this well accepted invivo reference model.
The serovar Lai type Langkawi isolated from a patient about one year ago displayed
only a low /moderate virulent phenotype. This moderately passaged strain caused
only transient weight loss and failed to induce pathology. However organs were still
culture positive 5 days after injection and clearly indicated a low virulent phenotype
of the serovar Lai type Langkawi isolate. The corresponding multi-passaged serovar
Batavia and Lai reference strains had lost their virulent phenotype as displayed by
failure to induce weight loss or pathology and complete clearance of viable bacteria
of these reference strains in the guinea pig infection model (Fig.1).
RESuLTS
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VirulencetestingbydetectionofLigA
Consistent with the results from the virulence testing in guinea pigs, Western blot
analysis showed that the fresh isolate L.interrogans serovar Bataviae strain Kariadi-
Satu strongly expressed LigA while LigA was only weakly detectable in the moderately
in vitro passaged strain Langkawi. LigA was completely absent in the saprophytic
strain Leptospirabiflexa serovar Patoc strain Patoc I (data not shown).
Figure 1A: Record of guinea pig weights.
Hartley male guinea pigs (3 weeks of age) were injected intraperitoneally with 107 in vitro
cultivated Leptospira. All guinea pigs were between 250-260g on the day of infection (day 0).
Weights were recorded daily until euthanasia.
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Figure 1B: Lung Gross pathology of following infection with various strains os Leptospira.
Lung Gross pathology of guinea pigs following infection with: (a) serovar Copenhageni strain
RJ16441, (b) serovar Patoc strain Patoc I, (c) serovar Lai strain Lai, (d) serovar Bataviae strain
Swart, (e) serovar Lai type Langkawi strain Langkawi, (f) serovar Bataviae strain Kariadi-Satu
(WithcourtesyA.M.Monahan,VeterinarySciencesCentre,SchoolofAgricultureFoodScience
&VeterinaryMedicine,CollegeofLifeSciences,UniversityCollegeDublin,Belfield,Dublin,
Ireland).
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Figure 1C: Culture results of kidney and liver tissue from guinea pigs post mortem.
Leptospira Kidney culture Liver cultureserovar Copenhageni strain RJ16441 + +serovar Patoc strain Patoc I - -serovar Lai strain Lai - -serovar Bataviae strain Swart - -serovar Lai type Langkawi strain Langkawi + +serovar Bataviae strain Kariadi-Satu + +
Differentinnateimmuneresponsetoheat-killedandaliveL.interrogans
Former studies investigating the innate immune response elicited by L.interrogans
were focused on the heat-stable lipopolysaccharide moiety of these bacteria.
However, since host cells encounter living bacteria during infection we compared
the response of human monocytic cells (THP-1 cell line) to heat-killed and viable
preparations of the reference and fresh isolate from serovars Batavia and Lai strains
mentioned above. In figure 2 the THP-1 response is shown to the heat-killed Leptospira.
Consistent with the previous study of Werts (27), the heat-killed highly virulent fresh
Batavia serovar isolate displayed a much lower potency to stimulate THP-1 cells than
the corresponding genetically indistinguishable heat-killed avirulent multi-passaged
Batavia reference strain. In contrast the heat-killed low virulent fresh isolate
serovar Lai type Langkawi and corresponding heat-killed avirulent multipassage Lai
reference strain were equipotent and both displayed the same potency as the heat-
killed avirulent Batavia reference strain. Thus our data are fully consistent with the
notion that highly virulent heat-killed L.interrogans are less potent innate immune
activators than heat-killed low virulent or avirulent L.interrogans. Interestingly,
incubations of living preparations of the low and avirulent strains resulted in a
similar response of the THP-1 cells when compared to the heat-killed response with
these bacteria. However, the viable preparation of the highly virulent fresh Batavia
isolate displayed a remarkable significantly enhanced potency to stimulate THP-
1 cells when compared to the living avirulent Batavia reference strain and both
living Lai preparations. It can be concluded that the minimally passed highly virulent
Batavia strain lost a large part of its potency to activate an innate immune response
upon heat-killing, and that living virulent L.interrogans may exert a relative potent
stimulating effect on human monocytes.
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Since the THP-1 cell line is not always an appropriate model for human monocytes
we performed stimulations with human mononuclear cells (PBMC’s). The response
to living Leptospira by PBMC’s was more vigorous to both avirulent reference strains
when compared to the freshly isolated virulent serovars. Interestingly, in this PBMC
employing test the avirulent Batavia reference strain was most potent, while
this strain displayed equal potency in THP-1 triggering as both Lai strains tested
either heat-killed or alive. The above indicates that the relative potency to trigger
innate immune responses of genetically indistinguishable virulent and avirulent L.
interrogans strains is largely dependent on the way of application (alive or heat-
killed) and the cell type used.
Figure 2: Activation of THP-1 cells with heat-killed and live Leptospira.
Cells were stimulated for 6 h with varying concentrations of Leptospira. TNF-a concentrations
were measured from cell culture supernatants. Data represent the mean values of quadruplicate
experiments. Error bars represent the standard deviation (SD) of the mean. Left panel: Heat
killed Leptospira; Right panel: Alive Leptospira.
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Figure 3: Activation of PMbC with live Leptospira.
Cells were stimulated for 6 h with varying concentrations of Leptospira. TNFa concentrations
were measured from cell culture supernatants. Data represent the mean values of quadruplicate
experiments. Error bars represent the standard deviation (SD) of the mean.
Abbreviations:PBMC,peripheralbloodmononuclearcells.
Wholeblood
During the pathophysiology of leptospirosis the Leptospira encounter and spread
through the blood stream. To explore the proinflammatory potency of this
compartment in response to virulent and avirulent Leptospira we incubated the
different Leptospirastrains in a minimally altered whole blood assay. It appeared that
both avirulent reference strains were less potent at inducing TNF at low bacterial
burden compared to the more virulent fresh isolates. At higher concentrations all
strains displayed the same potency in whole blood. In efforts to characterize the
response in whole blood we inhibited TLR2 and TLR4 in incubations with low, but
physiologically relevant (A.Ahmed, personal communication), concentrations of the
virulent and avirulent serovar Batavia strains. Interestingly, inhibition of TLR2 or
TLR4 had both a small but significant effect on the TNF production by the avirulent
Batavia reference strain. Combined inhibition of TLR2 and TLR4 completely blocked
the TNF production by this avirulent strain. In complete contrast, the majority
of TNF production induced by the virulent Batavia strain in whole blood could be
inhibited with either anti-TLR2 or anti-TLR4. And remarkably, combined inhibition
of both TLR2 and TLR4 did not further reduce the response driven by the virulent
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Leptospira. The apparent non-additive signaling behavior of TLR2 and TLR4 upon
incubation with the virulent strain suggests that these receptors react in synergism
to virulent Leptospira in this whole blood mode
Figure 4: Activation of whole blood with live Leptospira.
Whole blood was stimulated for 6 h with varying concentrations of Leptospira. TNFa
concentrations were measured from cell culture supernatants. Data represent the mean values
of quadruplicate experiments. Error bars represent the standard deviation (SD) of the mean.
Figure 5: TLR2 and TLR4 mediate activation of whole blood in response to live Leptospira.
Whole blood was stimulated with (X 2.5) 105/ml leptospires of reference strain serovar Bataviae
or (X 2.5) 105/ml of the equivalent fresh isolate of serovar Bataviae or (X 2.5) 105/ml in the
presence of anti-TLR2 and anti-TLR4 antibodies. TNF-a was measured in quadruplicate. Error
bars represent the standard deviation (SD) of the mean.
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KillingofthedifferentLeptospirastrainsbyhostfactors
In whole blood the virulent Leptospira seemed more potent in initiating an immune
response than the avirulent Leptospira, at least at low bacterial load. The opposite
was observed in reactions with isolated PBMC’s, which were more responsive to the
avirulent Leptospira. It appeared that the way of exposure of Leptospira to the
TNF producing monocytes is different in whole blood compared to the exposure of
Leptospira to monocytes in a purified mononuclear cell fraction of blood. Of course
the integrity and viability of the different Leptospirastrains may be differentially
altered by either plasma proteins or cellular reactions. This prompted us to test
potential killing of the Leptospira strains by the different cell types, complement,
or whole blood. Thus, the viability of the different Leptospira strains was evaluated
semi-quantitatively by culture of serial dilutions of Leptospira incubated with the
different cells or blood components.
As shown in figure 6 incubations with THP1 cells or PBMC’s did not affect the viability
of any of the used Leptospira strains. Upon incubation with serum and whole blood
the avirulent reference strains were killed, while the virulent strains displayed
complete serum resistance and also survived the 6 hour whole blood incubation.
Incubation with heat-inactivated serum did not result in killing of the avirulent
strains (result not shown) which is consistent with complement mediated killing of
the avirulent Leptospira by normal serum. These observations indicated that the
avirulent Leptospira strains are efficiently and rapidly destroyed by complement
activity while the virulent minimally passed Leptospira strains evade complement
dependent killing and also do not loose their integrity in whole blood.
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Figure 6: Killing of Leptospira.
Leptospira are incubated for 6 hours. 10-fold serial dilutions were made after 6 h in EMJH, and
leptospiral growth was evaluated after 3 weeks incubation at 30°C.
Abbreviations: sv, serovar; PBMC, peripheral bloodmononuclear cells;NHS, normal human
serum.
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Leptospirosis can cause severe human disease, leading to bleeding, multi-organ
failure and septic shock. The disease is caused by strains of the Leptospirainterrogans
bacteria that may differ in their LPS moiety which is used to discriminate the different
Leptospiraserovars (10). Previous reports identified TLR2 is the innate receptor that
recognizes L.interrogans derived LPS as well as heat-killed whole L. interrogans
bacteria (15,27). Furthermore, the LPS from virulent Leptospira was found to be a
weaker activator of monocytes than the LPS from avirulent Leptospira (27). These
findings suggested that TLR4 is not involved in the host response to L.interrogans,
and a potential escape mechanism of virulent bacteria by concealment of the TLR2
activating LPS structure. In order to further elucidate how the innate immune system
reacts to L.interrogans we confirmed the observations with heat-killed bacteria by
Werts using a monocytic cell line and 4 different clinical isolates of L.interrogans.
Indeed, when used heat-killed the most virulent L.interrogans strain was weakest in
stimulating a monocytic cell line, consistent with the earlier report.However when
the same Leptospira were incubated aliveon the monocytic cells the most virulent
strain showed an importantly higher potency to activate immune cells in comparison
to the avirulent strains. These experiments indicated that the virulent strain
displays an important immune activation pathway that is lost upon heat activation
of the bacteria. These observations prompted us to determine the host reaction to
different L.interrogans isolates in a minimally altered system relevant to the shock
like symptoms of patients. For this purpose we conducted whole blood incubations
with viable Leptospira in which we were able to show that virulent L.interrogans
are as potent activators of the innate immune system as avirulent Leptospira.
Furthermore, at low, but physiologically relevant bacterial burden virulent freshly
isolated L. interrogans strains of different serovars (Lai and Batavia) displayed
both an enhanced potency to stimulate the innate immune system compared to the
respective repeatedly-passaged avirulent Batavia and Lai reference strains. The main
difference between the virulent fresh isolates and reference strains was the complete
resistance of the virulent fresh isolates to killing by whole blood and serum while the
avirulent reference strains were rapidly killed in these. Both avirulent and virulent
DISCuSSION
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L.interrogans bacteria induced cytokine release in whole blood that was partially
TLR2 as well as TLR4 dependent. Combined inhibition of TLR2 and TLR4 revealed
that the avirulent Leptospira stimulated cytokine release in whole blood by TLR2
and TLR4 in an additive manner, while in contrast, virulent Leptospira stimulated
cytokine release in a manner that strongly suggested synergistic cooperation of TLR2/
TLR4. Since the avirulent Leptospira are rapidly killed and loose their integrity in
whole blood it may be hypothesized that the fragments or destroyed corpses of these
bacteria become less potent or dilute activators of the innate immune system during
the course of whole blood exposure. In contrast, the viable virulent Leptospiramay
trigger the host response in whole blood by more concentrated pathogen-associated
molecular patterns (PAMP’s) exposed on their intact surface. Since induction of
signaling by human TLR4 through PAMP’s exposed by L.interrogans is unlikely (15,27)
the observed TLR4 involvement is more likely caused by TLR4 activating released
danger-associated molecular patterns (DAMP’s) by host cells upon encounter of living
specimen of this invasive genus of bacteria (17).
To the best of our knowledge, we are the first to show that both TLR2 and TLR4 play a
role in the response to viable pathogenic Leptospira in a human whole blood model.
Furthermore, in contrast to previous results with heat-inactivated bacteria (Werts)
we show here that viable virulent L.interrogans strains are equipotent compared to
their respective identical serovar avirulent reference strains with regard to activation
of the innate immune system in human whole blood. Remarkably, the virulent strains
that resist the killing capacity of whole blood are at low bacterial dose even some
what more potent than the killed avirulent strains. Interestingly, this is associated
with a remarkable difference in use of TLR2/TLR4 signalling by host cells in whole
blood in response to virulent L.interrogans compared to the appropriate avirulent
reference strain.
Basically, human whole blood incubations with viable Leptospira revealed that
virulence characteristics of L.interrogans are associated with complete resistance
to killing by normal whole blood, and not with impaired recognition by the cellular
receptors of the innate immune system.
Our results are in line with previous work showing that pathogenic Leptospira are
able to survive in the non-immune host by evading, to various degree, complement-
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mediated killing (5,16). Although C3 is probably equally deposited on both non-
pathogenic and pathogenic strains, the latter are able to bind factor H on their
surface which is a strong inhibitor of the complement (C) system (16,24). In another
study, Anderson and Johnson (1) showed by electron microscopy that leptospires
belonging to saprophytic strain Patoc I and non-virulent serovar Canicola retained
their shape but lost their outer sheath after incubation with complement and immune
serum. In contrast, leptospires of the virulent serovar Canicola were not affected at
all by complement and antibodies.
Incubation with either THP-1 cells or PBMCs did not result in killing of any of the
viable Leptospirastrains. Indeed, previous studies reported similar findings (28,29),
suggesting that phagocytosis or the release of bactericidal mediators are not major
processes involved in the killing of Leptospira.
Nally et al (21) found in a lethal guinea pig model that in host tissue derived
leptospires the LPS O-antigen content was markedly reduced compared with in
vitro cultured leptospires. However host tissue derived leptospires isolated from
chronically infected rat kidney tubules were indistinguishable from in vitro cultured
leptospires. It was shown that the surface expressed lipoprotein LigB, only present
in pathogenic species (6,15) was attenuated during culture passage (22). The exact
functional consequence of LigB deficiency of Leptospira is not yet elucidated.
The ability of phagocytes to kill pathogenic Leptospiradepends on opsonisation with
specific immune serum (29). In the absence of immune serum pathogenic Leptospira
may interact, but are not ingested or killed by monocytes, macrophages and
neutrophils. Thus, it appears that in the non-immune patient pathogenic Leptospira
are not effectively killed by the innate immune system upon entry via skin or mucosa
abrasions. When living L.interrogans enters the circulation we show here that human
blood cells may react in a way that leads to TLR2 and TLR4 dependent cytokine
production by synergistic actions of these receptors.
Clearly, there are fundamental differences between fresh clinical L. interrogans
isolates and isolates which have been maintained for a long time in vitro. The
data presented herein emphasize the importance to use viable low passage clinical
isolates to study the host response to L. interrogans infection. In conclusion, our
results show that the innate human host response to leptospiraemia may involve
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synergistic actions of TLR2 and TLR4. These studies prompt further research to the
mechanism of action of this observed synergy and studies to unravel whether TLR4 is
involved during human leptospirosis (15).
Acknowledgments
We would like to thank Precious E. Manyenga for setting up the LigA experiments
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1. Anderson, D. L. and R. C. Johnson. 1968. Electron microscopy of immune disruption of leptospires: action of complement and lysozyme. J.Bacteriol. 95:2293-2309.
2. Arean, V. M. 1962. The pathologic anatomy and pathogenesis of fatal human leptospirosis (Weil’s disease). Am.J.Pathol. 40:393-423.
3. Bharti, A. R., J. E. Nally, J. N. Ricaldi, M. A. Matthias, M. M. Diaz, M. A. Lovett, P. N. Levett, R. H. Gilman, M. R. Willig, E. Gotuzzo, and J. M. Vinetz. 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect.Dis. 3:757-771.
4. Chierakul, W., F. M. de, Y. Suputtamongkol, R. Limpaiboon, A. Dondorp, N. J. White, and T. van der Poll. 2004. Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis. Clin.Immunol. 113:140-144.
5. Cinco, M. and E. Banfi. 1983. Activation of complement by leptospires and its bactericidal activity. Zentralbl.Bakteriol.Mikrobiol.Hyg.A 254:261-265.
6. Croda, J., C. P. Figueira, E. A. Wunder, Jr., C. S. Santos, M. G. Reis, A. I. Ko, and M. Picardeau. 2008. Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis. Infect.Immun. 76:5826-5833.
7. de Fost M., R. A. Hartskeerl, M. R. Groenendijk, and T. van der Poll. 2003. Interleukin 12 in part regulates gamma interferon release in human whole blood stimulated with Leptospira interrogans. Clin.Diagn.Lab Immunol. 10:332-335.
8. de Souza. L. and M. C. Koury. 1992. Isolation and biological activities of endotoxin from Leptospira interrogans. Can.J.Microbiol. 38:284-289.
9. Faine, S. 1982. Guidelines for Leptospirosis Control. Geneva: WHO offset Publication 67.
10. Gouveia, E. L., J. Metcalfe, A. L. de Carvalho, T. S. Aires, J. C. Villasboas-Bisneto, A. Queirroz, A. C. Santos, K. Salgado, M. G. Reis, and A. I. Ko. 2008. Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg.Infect.Dis. 14:505-508.
11. Kmety E. and H. Dikken. 1993. Classification of the Species Leptospira Interrogans and History of its Serovars. Groningen: University Press Groningen.
12. Levett, P. N. 2001. Leptospirosis. Clin.Microbiol.Rev. 14:296-326. doi:10.1128/CMR.14.2.296-326.2001.
13. Matsunaga, J., M. A. Barocchi, J. Croda, T. A. Young, Y. Sanchez, I. Siqueira, C. A. Bolin, M. G. Reis, L. W. Riley, D. A. Haake, and A. I. Ko. 2003. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol.Microbiol. 49:929-945.
14. McBride, A. J., D. A. Athanazio, M. G. Reis, and A. I. Ko. 2005. Leptospirosis. Curr.Opin.Infect.Dis. 18:376-386.
15. McBride, A. J., G. M. Cerqueira, M. A. Suchard, A. N. Moreira, R. L. Zuerner, M. G. Reis, D. A. Haake, A. I. Ko, and O. A. Dellagostin. 2009. Genetic diversity of the Leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp. Infect.Genet.Evol. 9:196-205.
16. Meri, T., R. Murgia, P. Stefanel, S. Meri, and M. Cinco. 2005. Regulation of complement activation at the C3-level by serum resistant leptospires. Microb.Pathog. 39:139-147.
17. Merien, F., G. Baranton, and P. Perolat. 1997. Invasion of Vero cells and induction of apoptosis in macrophages by pathogenic Leptospira interrogans are correlated with virulence. Infect.Immun. 65:729-738.
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IIIPARTDiagnostic and epidemiological aspects
Rapid serological assays for leptospirosis are of
limited value in southern Vietnam
J.F.P. Wagenaar 1, T.H.F. Falke 1, N.V. Nam 2, T.Q. Binh 3, H.L. Smits 4, F.G.J. Cobelens 1 and P.J. de Vries 1
1 Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, the Netherlands
2 Binh Thuan Provincial Malaria Station, Phan Thiet, Binh Thuan Province, Vietnam3 Tropical Diseases Clinical Research Center, Cho Ray Hospital, Ho Chi Minh City, Vietnam
4 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
TropMed&Parasitology2004;98(8):843-50.
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Leptospirosis, although ubiquitous and potentially lethal, is often not diagnosed.
The seroprevalence of anti-Leptospiraantibodies and the utility of two rapid tests
for the serodiagnosis of the disease were studied in Binh Thuan, an area in southern
Vietnam with favourable conditions for Leptospira. In an initial survey, blood samples
from 44 patients with undifferentiated fever and 83 healthy subjects were each
examined for anti-Leptospiraantibodies using three tests: an ELISA; a latex card-
agglutination test (Dri Dot©); and a lateral-flow assay (LeptoTek Lateral Flow©). In
the ELISA, samples from 35% of the healthy subjects and 40% of the febrile patients
were found to have titres of anti-LeptospiraIgM of at least 1:80. Only one of the 13
patients checked again, in ELISA, 3 weeks later, showed the marked increase in IgM
titre that is indicative of acute leptospirosis.
In the initial survey, although the positive results of the lateral-flow assay, applied
to whole blood and serum, showed a good agreement with those of the ELISA
(kappa=0.743), the results of the lateral-flow assay were often indeterminate.
The card-agglutination test was more specific. The overall agreement between
the results of the rapid tests and those of the ELISA was generally poor. When the
samples classified as ‘indeterminate’ in the lateral-flow assay were considered
positive, the maximum kappa-value for this assay applied to whole blood was only
0.512. In conclusion, it appears that high seroprevalences of anti-Leptospira IgM
and low incidences of acute leptospirosis limit the diagnostic value of the rapid
tests that were investigated. The lateral-flow assay is not specific enough. The card-
agglutination test is possibly better but, because of the low incidence, its sensitivity
could not be evaluated adequately in the present study.
AbSTRACT
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Although human leptospirosis is ubiquitous and potentially fatal, its clinical diagnosis
may be difficult and this often leads to under-reporting, even in outbreaks of severe
disease (1;2). Treatment with antibiotics, especially if initiated early in the disease,
may be beneficial, although the evidence for this is scanty (3-5). In specialised, well-
equipped laboratories, a diagnosis of leptospirosis can be confirmed by microscopic
agglutination tests (MAT) and the isolation of leptospirae (1). In primary-healthcare
settings, however, even ELISA may be impossible or too laborious, and such limitations
have stimulated the development and marketing of several rapid tests for diagnosing
leptospirosis.
In Vietnam, fever is a common reason for seeking medical care. Its causes may
include leptospirosis, as indicated by the high seroprevalences of anti-Leptospira
antibodies among populations in the Mekong delta (6;7). One aim of the present
study was to use ELISA to estimate the seroprevalence of leptospirosis in febrile
and apparently healthy subjects from rural Binh Thuan, a Vietnamese province with
favourable conditions for leptospirosis. Another aim was to compare the results of
the ELISA with those of two commercially-available rapid tests (a card-agglutination
test and a lateral-flow assay), to evaluate the diagnostic accuracy of the rapid tests.
The lateral-flow test was applied not only to sera but also to samples of whole blood
(which can be collected easily from finger pricks).
StudySite
The province of Binh Thuan lies by the South China Sea, in southern Vietnam. Forested
mountains cover half the province but the other, lowland areas have largely been
developed as rice paddies and orchards in small-scale farms. Most of the farmers also
raise some pigs, cattle or poultry. For the present study, serosurveys were conducted
among apparently healthy subjects from the rural communes of Ham My and Tra Tan
and among febrile patients from Ham My or the malaria clinic in Phan Thiet, the
provincial capital.
INTRODuCTION
SubJECTS AND METHODS
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Subjects
To be considered a febrile patient, an individual had to present with a axillary
temperature of >38°C and to have been febrile for <2 weeks. Patients found smear-
positive for malaria (by microscopy), those with severe disease requiring referral (such
as meningitis or sepsis), or signs and symptoms indicating a specific localised disease
(such as tonsillitis, pneumonia, abscesses, peritonitis, arthritis, pyelonephritis) or
chronic underlying conditions such as renal or hepatic disease, diabetes mellitus,
HIV infection, and those taking immunosuppressive medication were excluded.
Treatment with amoxicillin was initiated upon clinical suspicion of leptospirosis.
Informed consent was obtained from all participants or their caregivers.
The study protocol was approved by the scientific board of Cho Ray Hospital in Ho Chi
Minh City, the Vietnamese Ministry of Health, and the Binh Thuan provincial health
authorities.
Samples
In the initial survey, venous blood was drawn from all participants. A second blood
sample was collected from some of the febrile patients after 3 weeks. Serum was
separated, by immediate centrifugation at the study site, and stored at -20°C.
ELISA
An ELISA for the detection of anti-Leptospira IgM was performed using antigen
prepared
from the Patoc I strain of L.biflexa (8;9). Sera giving titres of 1:80 or higher were
considered positive. A two-fold or greater increase in titres after 3 weeks was
considered indicative of ‘acute leptospirosis’.
LateralFlowassay
Both sera and whole-blood samples were tested using the LeptoTek Lateral Flow®
assay (Organon Teknika, Durham, NC). This one-step, colloidal-gold immuno-assay
is based on the binding of specific IgM antibodies to immobilized antigen from the
Patoc I strain (10). The bound IgM antibodies are detected using anti-IgM antibodies
that areconjugated to mobile, red particles of colloidal gold.
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Serum (5 μl) or whole blood (10 μl) was drawn into a heparinized capillary and
then spotted on the pad in the round sample port before 130 μl of running buffer
were added. Results were read after 10 min. In the presence of a positive control
line, specific anti-Leptospira IgM antibodies are indicated by the appearance of a
distinct coloured line in the test zone. Samples giving hazy lines were classified as
indeterminate.
Card-agglutinationtest
Sera were also tested using the Dri Dot® card-agglutination test (Organon Teknika).
In this test, Leptospira-specific antibodies are detected when they agglutinate blue
latex particles that have been coated with antigen from the Lely 607 strain (11).
A dry spot of these latex particles, affixed to a white card, was mixed with 10 μl
serum, stirring with a spatula. After gently swirling the suspensions for 30 s, each
test spot was scored positive, negative or indeterminate, according to the degree of
agglutination observed.
DataAnalysis
Version 11 of the SPSS software package (SPSS Inc., Chicago, IL) was used for all
the data analysis. The results of the respective tests were compared. Levels of
agreement between the tests were evaluated by calculating Cohen’s kappa (Κ).
Sensitivities, specificities and predictive values were not calculated because ELISA is
not unequivocally the ‘gold standard’ in the diagnosis of leptospirosis.
Forty-five febrile patients — 19 females and 26 males, with a mean age (range) of
23 (8–46) years — were enrolled. Nineteen of the patients were farmers or forest
labourers. Ten patients acknowledged recent contact with fresh water. The mean
duration of illness was 2.8 days (range=0–7 days). In the initial survey, blood samples
were obtained from 44 patients. Three weeks later, 19 patients were re-tested with
the rapid tests and 13 with the ELISA. The 83 apparently healthy subjects were
RESuLTS
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farmers or children of farmers (37 males and 46 females, aged 6-79 years), each
with a history of at least one episode of undifferentiated fever in the previous year.
The seroprevalences of anti-Leptospira IgM, as detected in the ELISA, were high:
35% among the healthy subjects and 40% among the patients. The titres seen in the
initial survey were, however, never higher than 1:320. One patient, a suspected case
of leptospirosis who was treated with amoxicillin, was confirmed as having acute
leptospirosis by a fourfold increase in IgM titre after 3 weeks. This patient, however,
was scored negative and then (3 weeks later) indeterminate in the lateral-flow assay
(both in tests of serum and whole blood), and consistently negative in the card-
agglutination tests.
The full results of the initial survey and the re-testing of some of the patients 3 weeks
later are summarized in the Table. The Figure shows how the results of the rapid
tests compare with those of the IgM-ELISA. The samples scored ‘indeterminate’ in
the rapid tests, which were more common with the lateral-flow assay than the card-
agglutination test, required re-scoring as positive or negative for the data analysis.
Evaluation of the levels of agreement between a positive result in the IgM-ELISA (i.e.
a titre of ≥1:80) and a positive result in a rapid test, expressed as K-values, was
occasionally unfeasible because there were too few positive results. The highest value
of K observed for such a comparison was 0.512 (seen for the comparison between
the ELISA and the lateral-flow assay, when the samples classified as ‘indeterminate’
in the lateral-flow assay were considered positive). For the lateral-flow assay, the
results obtained when whole blood was tested showed reasonable agreement with
those seen when serum was tested, notably after reclassifying the indeterminate
samples as positive (K=0.743).
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Table: Leptospirosis antibodies in sera of healthy subjects and febrile patients.
Lateral-flow assay, based on:
ELISA Serum Whole blood
Card agglutination
test Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21
No. and (%) of samples from healthy subjects
Tested 83 0 83 0 83 0 83 0Scored positive 29 (35) _ 13 (16) _ 8 (10) _ 6 (7) _Scored negative 54 (65) _ 45 (54) _ 52 (63) _ 76 (92) _Scored indeterminate 0 (0) _ 25 (30) _ 23 (28) _ 1 (1) _
No. and (%) of samples from febrile patients
Tested 40 13 37 19 40 14 37 19Scored positive 16 (40) 7 (54) 2 (5) 0 (0) 0 (0) 0 (0) 2 (5) 0 (0)Scored negative 24 (60) 6 (46) 27 (73) 14 (74) 30 (75) 10 (71) 34 (92) 9 (100)Scored indeterminate 0 (0) 0 (0) 8 (22) 5 (26) 10 (25) 4 (29) 1 (3) 0 (0)
Leptospira antibodies in the sera of healthy subjects and febrile patients, as detected by ELISA (detecting anti-Leptospira IgM in sera), the LeptoTek Lateral Flow assay (detecting specific IgM in blood or serum), and the Dri Dot test card-agglutination test (detecting anti-Leptospira agglutinating antibodies in sera). Samples were collected from all the subjects during an initial survey (day 0) and from some of the patients 3 weeks later (day 21)
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Figu
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r th
e pa
tien
ts w
ith
undi
ffer
enti
ated
fev
er w
hen
they
wer
e te
sted
upo
n pr
esen
tati
on a
t a
prim
ary
heal
thca
re f
acili
ty (○
) or
thr
ee w
eeks
late
r (●
)
Rapidserologicalassaysforlep
tospirosisareoflimited
value
insouthe
rnVietnam
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Although the present results indicate a high seroprevalence of anti-LeptospiraIgM
among apparently healthy subjects in Binh Thuan province, only one of the patients
tested twice, with a 3-week interval, appeared to have acute leptospirosis. Similarly,
Van et al. (7) found 19% of their subjects from the Mekong delta to be seropositive
in a Leptospira-specific MAT but saw no indications of acute leptospirosis. This
combination of high seroprevalences but low incidences of acute disease have been
seen elsewhere in South–east Asia (6;12). It is a combination that negates the use
of single-sample IgM-ELISA, despite the confirmed diagnostic value of such tests in
other epidemiological situations (8;9). In the present study area, raising the cut-off
threshold for positivity in the ELISA, to compensate for the low specificity, would
reduce the sensitivity to levels that are useless (see Figure). Although the diagnostic
accuracy of ELISA can be improved by testing both ‘acute’ and ‘convalescent’ sera,
paired sera are often difficult to collect, and delaying diagnosis, until after collection
of the convalescent sample, is likely to have a negative impact on the prognosis (13).
In earlier investigations, in which the results of MAT were used as the ‘gold standard’,
the lateral-flow assay was found to have a sensitivity of 86% and a specificity of 94%,
whereas the card-agglutination test had a sensitivity of 72%–88% and a specificity
of 90% (10;11;14). In the present study, it was perhaps not surprising that the high
frequencies of positivity in the lateral-flow assay paralleled the high seroprevalences
recorded with the IgM-ELISA, since both assays are based on the detection of specific
IgM antibodies (and therefore have relatively low specificities for the diagnosis of
clinical leptospirosis). As is shown in the Figure, the card-agglutination test, which
is based on the detection of specific agglutinating antibodies, was more specific.
The high seroprevalences recorded among the healthy subjects of the present study
indicates repeated exposure of the population to leptospirae, which is plausible
considering the many risk factors — such as wet-rice cultivation, high animal
densities, and the monsoon climate — in Binh Thuan (15). Leptospirosis appears
to be associated with the monsoon season, when the general seroprevalence of
Leptospira-specific IgM also shows a seasonal peak (16). That the present study was
carried out before the monsoon may be one reason why the incidence of acute
DISCuSSION
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leptospirosis observed was low. The low incidence of confirmed, acute disease
may, however, also indicate immunity, perhaps induced by repeated exposure to
relatively non-pathogenic serogroups and preventing complicated disease and major
outbreaks. This scenario, which is comparable with that of holo-endemic malaria
or several parasitic infections of childhood, could be confirmed by demonstrating
decreasing incidence of disease and increasing seroprevalence with increasing age.
Although age-dependent changes in seroprevalence may be more apparent in IgG
antibodies than in IgM, anti-LeptospiraIgM may remain detectable for several years
post-infection (13;17). The possibility that the apparently high seroprevalences of
anti-LeptospiraIgM observed, in the present study and several other investigations,
is the result of cross-reactivity with the pathogens causing other endemic diseases,
such as dengue, cannot be excluded (18;19).
Many difficulties remain for those attempting to diagnose acute leptospirosis,
particularly at the level of primary healthcare in highly endemic areas (20;21). In
industrialized countries where leptospiral infection is rare, and under the optimal
conditions to be found in referral laboratories and serum banks, the rapid tests
have been found to be sufficiently sensitive and specific (10;11;22). The tests have
also been found useful in some endemic areas (14;23;24). In highly endemic areas,
however, leptospirosis serology remains far from optimal, and cut-off levels have
to be raised, thereby threatening sensitivity (25). In the present study, the card-
agglutination test, which detects not only IgM but also IgG, was less affected by the
high seroprevalence than the lateral-flow assay. Whether the card-agglutination test
has adequate sensitivity, especially when cut-off levels have to be increased, needs
further study.
In conclusion, the high seroprevalence of anti-LeptospiraIgM antibodies in southern
Vietnam limited the ability of a recently developed lateral-flow assay to confirm
active leptospirosis. A card-agglutination test was more specific, but its sensitivity,
especially when seroprevalences are high, needs further evaluation.
Rapidserologicalassaysforlep
tospirosisareoflimited
value
insouthe
rnVietnam
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(1) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (2) Plank R, Dean D. Overview of the epidemiology, microbiology, and pathogenesis of
Leptospira spp. in humans. Microbes Infect 2000 August;2(10):1265-76. (3) Guidugli F, Castro AA, Atallah AN. WITHDRAWN: Antibiotics for preventing leptospirosis.
Cochrane Database Syst Rev 2009;(3):CD001305. (4) Kobayashi Y. Clinical observation and treatment of leptospirosis. J Infect Chemother
2001 June;7(2):59-68. (5) Watt G, Padre LP, Tuazon ML, Calubaquib C, Santiago E, Ranoa CP et al. Placebo-
controlled trial of intravenous penicillin for severe and late leptospirosis. Lancet 1988 February 27;1(8583):433-5.
(6) Boqvist S, Chau BL, Gunnarsson A, Olsson EE, Vagsholm I, Magnusson U. Animal- and herd-level risk factors for leptospiral seropositivity among sows in the Mekong delta, Vietnam. Prev Vet Med 2002 March 14;53(3):233-45.
(7) Van CT, Thuy NT, San NH, Hien TT, Baranton G, Perolat P. Human leptospirosis in the Mekong delta, Viet Nam. Trans R Soc Trop Med Hyg 1998 November;92(6):625-8.
(8) Terpstra WJ, Ligthart GS, Schoone GJ. Serodiagnosis of human leptospirosis by enzyme-linked-immunosorrbent-assay (ELISA). Zentralbl Bakteriol A 1980 August;247(3):400-5.
(9) Terpstra WJ, Ligthart GS, Schoone GJ. ELISA for the detection of specific IgM and IgG in human leptospirosis. J Gen Microbiol 1985 February;131(2):377-85.
(10) Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Lab Immunol 2001 January;8(1):166-9.
(11) Smits HL, Chee HD, Eapen CK, Kuriakose M, Sugathan S, Gasem MH et al. Latex based, rapid and easy assay for human leptospirosis in a single test format. Trop Med Int Health 2001 February;6(2):114-8.
(12) Laras K, Cao BV, Bounlu K, Nguyen TK, Olson JG, Thongchanh S et al. The importance of leptospirosis in Southeast Asia. Am J Trop Med Hyg 2002 September;67(3):278-86.
(13) Cumberland P, Everard CO, Wheeler JG, Levett PN. Persistence of anti-leptospiral IgM, IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados 1979-1989. Eur J Epidemiol 2001;17(7):601-8.
(14) Vijayachari P, Sugunan AP, Sehgal SC. Evaluation of Lepto Dri Dot as a rapid test for the diagnosis of leptospirosis. Epidemiol Infect 2002 December;129(3):617-21.
(15) Tangkanakul W, Tharmaphornpil P, Plikaytis BD, Bragg S, Poonsuksombat D, Choomkasien P et al. Risk factors associated with leptospirosis in northeastern Thailand, 1998. Am J Trop Med Hyg 2000 September;63(3-4):204-8.
(16) Ashford DA, Kaiser RM, Spiegel RA, Perkins BA, Weyant RS, Bragg SL et al. Asymptomatic infection and risk factors for leptospirosis in Nicaragua. Am J Trop Med Hyg 2000 November;63(5-6):249-54.
(17) da Silva MV, Nakamura PM, Camargo ED, Batista L, Vaz AJ, Romero EC et al. Immunodiagnosis of human leptospirosis by dot-ELISA for the detection of IgM, IgG, and IgA antibodies. Am J Trop Med Hyg 1997 June;56(6):650-5.
(18) Flannery B, Pereira MM, Velloso Ld, Carvalho Cd, De Codes LG, Orrico Gd et al. Referral pattern of leptospirosis cases during a large urban epidemic of dengue. Am J Trop Med Hyg 2001 November;65(5):657-63.
(19) Levett PN, Branch SL, Edwards CN. Detection of dengue infection in patients investigated for leptospirosis in Barbados. Am J Trop Med Hyg 2000 January;62(1):112-4.
(20) Effler PV, Bogard AK, Domen HY, Katz AR, Higa HY, Sasaki DM. Evaluation of eight rapid screening tests for acute leptospirosis in Hawaii. J Clin Microbiol 2002 April;40(4):1464-9.
REFERENCES
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ter10
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(21) Zochowski WJ, Palmer MF, Coleman TJ. An evaluation of three commercial kits for use as screening methods for the detection of leptospiral antibodies in the UK. J Clin Pathol 2001 January;54(1):25-30.
(22) Sehgal SC, Vijayachari P, Sharma S, Sugunan AP. LEPTO Dipstick: a rapid and simple method for serodiagnosis of acute leptospirosis. Trans R Soc Trop Med Hyg 1999 March;93(2):161-4.
(23) Smits HL, Ananyina YV, Chereshsky A, Dancel L, Lai AFR, Chee HD et al. International multicenter evaluation of the clinical utility of a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human serum specimens. J Clin Microbiol 1999 September;37(9):2904-9.
(24) Smits HL, Hartskeerl RA, Terpstra WJ. International multi-centre evaluation of a dipstick assay for human leptospirosis. Trop Med Int Health 2000 February;5(2):124-8.
(25) Vijayachari P, Sugunan AP, Sehgal SC. Evaluation of microscopic agglutination test as a diagnostic tool during acute stage of leptospirosis in high & low endemic areas. Indian J Med Res 2001 September;114:99-106.
Murine typhus and Leptospirosis as a cause of
acute undifferentiated fever in Central Java,
Indonesia
J.F.P. Wagenaar* 1, M.H. Gasem* 2, M.G.A. Goris 3, M.S. Adi 4, B. Isbandrio 5, R.A. Hartskeerl 3, J.M. Rolain 6, D. Raoult 6 and E.C.M. van Gorp 1, 7
1 Department of Internal medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Department of Internal medicine, Dr. Kariadi hospital,
Diponegoro University, Semarang, Indonesia 3 Royal Tropical Institute (KIT), KIT biomedical Research, Amsterdam, the Netherlands
4 Faculty of Public Health, Diponegoro University, Semarang, Indonesia5 Department of Microbiology, Leptospirosis Laboratory,
Diponegoro University, Semarang, Indonesia6 URMITE CNRS-IRD UMR 6236, Faculté de Médicine et de Pharmacie,
Université de la Méditerranée, Marseille, France7 Department of virology, Erasmus University, Rotterdam, the Netherlands
* Both authors equally contributed
EmergInfectDis2009;15(6):975-7.
11CHAPTER
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To investigate the presence of rickettsioses and leptospirosis in urban residents of
Semarang, Indonesia, we tested the blood of 137 patients with fever. Murine typhus
was found in 9 cases. Another 9 cases showed inconclusive serology. Thirteen subjects
were diagnosed with leptospirosis. No dual infections were detected.
AbSTRACT
Murinetyph
usand
lep
tospirosisascauseofund
ifferentiatedfever
175
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Fever is among the main reasons for seeking medical attention in Indonesia. The
etiology of fever usually remains obscure because of limited laboratory diagnostic
facilities and expertise for performing laboratory confirmation. Favorable
environmental conditions mean that both rickettsiosis and leptospirosis are
considered endemic in Indonesia and may result in clinically indistinguishable cases
of acute undifferentiated fever (AUF). Serosurveys conducted on Java, Sumatra and
islands in eastern Indonesia revealed antibodies to Rickettsiatyphi (murine typhus),
Orientiatsutsugamushi (scrub typhus) and to members of the spotted fever group
rickettsia (SFGR) in healthy individuals (1-3). In addition, several investigations
reported leptospirosis as cause of AUF in Indonesia (4;5).
Murine typhus and leptospirosis are likely to share routes of transmission in an urban
setting were rats are abundant. The main vector for R.typhi is the Asiatic rat flea
Xenopsyllacheopsis. Humans usually become infected when R.typhi infected flea
feces contaminates excoriated skin or is inhaled. Leptospirosis is mainly spread
by rats and other small mammals, shedding the bacteria via their urine into the
environment. Humans are infected through mucous membranes, conjunctivae or
abraded skin. The clinical features of both mild leptospirosis and murine typhus are
non-specific. Classically, murine typhus presents with fever, headache and a rash,
though the latter is often absent. Renal failure, jaundice and hemorrhages are the
classical symptoms of severe leptospirosis, while fever, headache and myalgia may
be the only presenting symptoms of mild disease. Dual infections with murine typhus
are reported to occur in Southeast Asia and may complicate treatment and clinical
course (6;7).
This study attempts to find evidence for acute rickettsial disease, leptospirosis and
dual infections among patients presenting with AUF in Indonesia, where risk factors
for both diseases are likely to be present.
INTRODuCTION
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The study was based in Semarang; a large coastal harbor city in central Java.
Consecutive out-patients were recruited at two primary healthcare centers and
hospitalized patients at a governmental referral centre (Dr. Kariadi university
hospital, department of internal medicine). All eligible AUF patients (≥ 5 years of
age) defined as: fever ≥ 38°C (central), for less than 14 days with no apparent other
disease, were included. After informed consent, a first blood sample was taken. A
convalescent sample was drawn after approximately 14 days. The study was approved
by the local medical ethical committee.
A specific micro-immunofluorescence (IFA) assay for Rickettsia species was performed
in Marseille, France, by using whole-cell antigens of O.tsutsugamushi, R.japonica,
R.heilongjiangensis, R.slovaca, R.honei, R.conorii subsp. indica, Rickettsia “ATI”,
R.helvetica, R.felis, R.typhi and R.prowazekii . The assay was considered positive
when: 1) antibody titers were ≥1/256 for IgG and ≥1/64 for IgM, or 2) a seroconversion
was observed or 3) ≥4-fold increase in titers between the acute and the convalescent
serum was detected. Leptospirosis serology was performed in Semarang, Indonesia.
Crosschecks and PCR were performed in Amsterdam, the Netherlands. Convalescent
samples were screened with the LeptoTek Dri Dot (Biomérieux). All positive cases were
tested by the microscopic agglutination test (MAT) and IgG ELISA (8). Additionally,
a real time PCR (manuscript in preparation) specifically targeting the secY gene of
pathogenic Leptospira (9) was performed on all samples. For the MAT a panel of 31
serovars was used containing 28 pathogenic and 3 non-pathogenic serovars. Patients
with an ELISA or MAT: a titer of ≥1:320 on a single sample, seroconversion, or a ≥4-
fold increase in titers between paired samples as well as any patient with a positive
PCR, irrespective of the serology result, were considered positive. All samples were
run in parallel
From February 2005 to February 2006, 137 AUF patients were included; 67 hospitalized
and 70 out-patients. A convalescent sample was available in 106 (77%) patients. The
main complaints were headache (85%), myalgia (70%), nausea (64%), cough (44%) and
abdominal pain (38%).
THE STuDY
Murinetyph
usand
lep
tospirosisascauseofund
ifferentiatedfever
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Murine typhus and leptospirosis were both found to cause AUF in this clinical series
(Table). In total, nine patients (7%) had evidence of an acute infection with R.typhi,
none showed a rash. In-hospital, murine typhus could be diagnosed in 6 out of 67
patients (9%). Three out of 70 out-patients had acute murine typhus (4%). Another
nine cases (7%) showed inconclusiveR.typhi serology. One case showed evidence of
a past infection with R.typhi (IFA: 128/0 in both sera). Evidence for acute infection
with O.tsutsugamushi or SFGR was not found.
Leptospirosis was diagnosed in 13 out of 137 patients (10%), 2 of these were
positive only by PCR. Eleven leptospirosis patients were recruited in-hospital; two
patients were recruited out-hospital. Consequently, the percentage of AUF caused
by leptospirosis in hospitalized patients was 16% and in out-patients 3%. The most
frequently identified serogroup by MAT was Bataviae (5 cases). No dual infections
were detected; however three leptospirosis patients (23%) showed titers in the R.
typhi IFA.
In this study, we report for the first time murine typhus and leptospirosis as an
important cause of AUF in Semarang, Indonesia. A previous study from rural Thailand
identified both disease in respectively 2.8% and 36.9% of AUF cases (10;11). In the
capital city of Laos, R.typhi was reported to cause fever in 9.6% of investigated
subjects, closely resembling our data (12). Unfortunately leptospirosis was not
investigated. In the present study, we expected leptospirosis as a cause of AUF, since
the Dr. Kariadi hospital admits around 50 severe cases each year. These cases were
not included in the study because of the high clinical suspicion on admission with
jaundice, azotemia and/or bleeding manifestations. A definite diagnosis of murine
typhus and leptospirosis double infections could not be made but in 3 cases this
scenario was plausible. We did not find evidence for scrub typhus as expected,
because O.tsutsugamushi transmission occurs primarily in rural areas (13). Although
SFGR have been reported in Southeast Asia and proof for their presence in Indonesia
is accumulating (14;15), these were not identified as a cause AUF in the present
study.
CONCLuSIONS
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R34 Tabl
e: C
linic
al a
nd la
bora
tory
dat
a of
pat
ient
s w
ith
rick
etts
iose
s an
d le
ptos
piro
sis.
Pati
ent
no.
Age
/ se
xM
onth
of
ons
et
illne
ss
R. t
yphi
IFA
(IgG
/IgM
)M
ATLe
ptos
piro
sis
PCR
Clin
ical
fea
ture
s an
d an
tibi
otic
tr
eatm
ent
Ad
mis
sion
sa
mpl
eCo
nval
esce
nt
sam
ple
Hig
hest
tit
er
path
ogen
ic
sero
var
Puta
tive
infe
ctin
g se
rogr
oup
Murinetyph
us7
49/M
Febr
uary
0/0
1024
/102
4N
AN
Ane
gati
ve4-
day
feve
r, m
yalg
ia,
head
ache
, ch
ills,
cou
gh,
abdo
min
al p
ain;
Rx:
cip
roflox
acin
1045
/MM
arch
512/
1024
512/
1024
NA
NA
nega
tive
7-da
y fe
ver,
mya
lgia
, he
adac
he,
naus
ea,
abdo
min
al
pain
; Rx
: ci
profl
oxac
in
1246
/FM
arch
2048
/512
2048
/512
NA
NA
nega
tive
3-da
y fe
ver,
hea
dach
e, c
hills
, na
usea
, vo
mit
ing,
he
pato
meg
aly;
Rx:
cip
roflox
acin
1815
/MAp
ril
256/
6425
6/64
NA
NA
nega
tive
3-da
y fe
ver,
hea
dach
e, s
ore
thro
at,
abdo
min
al p
ain;
Rx
: ce
fadr
oxyl
2446
/FAp
ril
128/
512
1024
/102
4N
AN
Ane
gati
ve7-
day
feve
r, h
eada
che,
nau
sea,
abd
omin
al p
ain;
Rx:
ci
profl
oxac
in
6368
/FN
ovem
ber
2048
/256
2048
/512
NA
NA
nega
tive
3-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, n
ause
a,
dysp
nea;
Rx:
cip
roflox
acin
1107
13/F
Febr
uary
0/32
1024
/512
NA
NA
nega
tive
2-da
y fe
ver,
hea
dach
e, a
nore
xia;
Rx:
cot
rim
oxaz
ol
1112
25/M
Febr
uary
256/
64N
AN
AN
Ane
gati
ve2-
day
feve
r, m
yalg
ia,
head
ache
, an
orex
ia,
sore
th
roat
, co
ugh,
abd
omin
al p
ain,
cal
ve p
ain;
Rx:
am
oxic
illin
1123
8/F
Mar
ch0/
6451
2/0
NA
NA
nega
tive
1-da
y fe
ver,
hea
dach
e, s
ore
thro
at,
naus
ea,
vom
itin
g, a
bdom
inal
pai
n; R
x: a
mox
icill
in
Possiblem
urinetyph
us2
17/F
Febr
uary
0/0
0/64
NA
NA
nega
tive
5-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, c
ough
and
so
re t
hroa
t; n
o an
tim
icro
bial
dru
g
531
/FFe
brua
ry0/
012
8/0
NA
NA
nega
tive
5-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, n
ause
a; n
o an
tim
icro
bial
dru
g
5322
/FAu
gust
128/
3212
8/64
NA
NA
nega
tive
4-da
y fe
ver,
mya
lgia
, he
adac
he,
naus
ea,
vom
itin
g,
abdo
min
al p
ain,
dia
rrhe
a; n
o an
tim
icro
bial
dru
g
6217
/MN
ovem
ber
128/
6412
8/64
NA
NA
nega
tive
4-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, s
ore
thro
at,
naus
ea,
vom
itin
g, a
bdom
inal
pai
n, p
etec
hiae
; no
an
tim
icro
bial
dru
g
6946
/MD
ecem
ber
0/12
80/
128
NA
NA
nega
tive
9-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, n
ause
a,
abdo
min
al p
ain;
Rx:
cip
roflox
acin
1104
14/M
Febr
uary
128/
6412
8/64
NA
NA
nega
tive
1-da
y fe
ver,
hea
dach
e; R
x: a
mox
icill
in
1105
11/F
Febr
uary
128/
32N
AN
AN
Ane
gati
ve2-
day
feve
r, m
yalg
ia,
head
ache
, na
usea
, vo
mit
ing,
ab
dom
inal
pai
n; R
x: a
mox
icill
in
Murinetyph
usand
lep
tospirosisascauseofund
ifferentiatedfever
179
R1
R2
R3
R4
R5
R6
R7
R8
R9
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R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
1113
70/F
Febr
uary
0/0
0/64
NA
NA
nega
tive
1-da
y fe
ver,
mya
lgia
, he
adac
he,
sore
thr
oat,
cou
gh,
calv
e pa
in;
Rx:
amox
icill
in
1132
7/M
Mar
ch25
6/0
NA
NA
NA
NAb
3-da
y fe
ver,
hea
dach
e, s
ore
thro
at,
naus
ea,
vom
itin
g, c
onju
ncti
val s
uffu
sion
, pe
tech
iae;
Rx:
am
oxic
illin
Leptospirosis
427
/MFe
brua
ryne
gati
vene
gati
ve1:
80 a
Java
nica
nega
tive
2-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s,
naus
ea,
vom
itin
g, a
bdom
inal
pai
n, c
alve
pai
n,
hepa
tom
egal
y; R
x: c
efot
axim
e
1463
/MM
arch
128/
012
8/0
1:12
80H
ebdo
mad
isne
gati
ve3-
day
feve
r, m
yalg
ia,
head
ache
, na
usea
, vo
mit
ing,
ab
dom
inal
pai
n; R
x: c
ipro
flox
acin
2541
/FM
ayne
gati
vene
gati
ve1:
80 a
Bata
viae
nega
tive
2-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, n
ause
a,
vom
itin
g; n
o an
tim
icro
bial
dru
g
2949
/MM
ayne
gati
vene
gati
ve1:
160 a
Bata
viae
nega
tive
4-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, d
iarr
hea,
co
njun
ctiv
al s
uffu
sion
; Rx
: ci
profl
oxac
in a
nd P
P
3240
/MM
ayne
gati
vene
gati
ve1:
320
Bata
viae
nega
tive
5-da
y fe
ver,
mya
lgia
, he
adac
he,
naus
ea,
vom
itin
g,
abdo
min
al p
ain,
pet
echi
ae;
no a
ntim
icro
bial
dru
g
3750
/MM
ayne
gati
vene
gati
ve1:
40 a
ncne
gati
ve5-
day
feve
r, h
eada
che,
chi
lls,
coug
h, n
ause
a,
vom
itin
g, a
bdom
inal
pai
n, d
iarr
hea;
Rx:
PP
4063
/MJu
nene
gati
vene
gati
vene
gati
venc
Posi
tive
2 da
y fe
ver,
mya
lgia
, he
adac
he,
naus
ea,
vom
itin
g,
abdo
min
al p
ain,
cal
ve p
ain;
Rx:
PP
4816
/MJu
nene
gati
vene
gati
ve1:
640
Saxk
oebi
ngne
gati
ve2-
day
feve
r, m
yalg
ia,
head
ache
, ch
ills,
pet
echi
ae;
no a
ntim
icro
bial
dru
g
1152
17/M
Mar
chne
gati
vene
gati
ve1:
160 a
Bata
viae
nega
tive
2-da
y fe
ver,
mya
lgia
, he
adac
he,
chill
s, s
ore
thro
at,
coug
h, n
ause
a; R
x: a
mox
icill
in,
tetr
acyc
line,
co
trim
oxaz
ol
1182
58/F
Janu
ary
nega
tive
nega
tive
1:32
0cnc
Posi
tive
2-da
y fe
ver,
mya
lgia
, he
adac
he,
coug
h;
Rx:
cotr
imox
azol
Possibledou
bleinfection
3338
/MM
ay10
24/0
1024
/01:
320
Icte
roha
emor
rhag
iae
nega
tive
5-da
y fe
ver,
mya
lgia
, he
adac
he,
naus
ea,
abdo
min
al
pain
, di
arrh
ea,
calv
e pa
in;
Rx:
cefo
taxi
me
4339
/FJu
ly0/
00/
256
1:64
0Ba
tavi
aePo
siti
ve5-
day
feve
r, m
yalg
ia,
head
ache
, ch
ills,
nau
sea,
vo
mit
ing,
jau
ndic
e, c
alve
pai
n; R
x: P
P
6037
/MO
ctob
er0/
6464
/64
nega
tive
ncPo
siti
ve5-
day
feve
r, m
yalg
ia,
head
ache
, na
usea
, ab
dom
inal
pa
in;
no a
ntim
icro
bial
dru
g
Abb
reviations:IFA,m
icro-immun
ofluo
rescen
ceassay;M
AT,m
icroscop
icagglutina
tion
test,M,m
ale;F,fem
ale;NA,n
otavailab
le;n
c,notclassifiab
le;
PP,procaine
pen
icillin;Rx,m
edicalprescription.
a Pa
tien
t se
roco
nver
ted
from
neg
ativ
e (<
20)
to a
pos
itiv
e ti
ter
by M
AT o
r EL
ISA
b Th
ere
was
not
eno
ugh
sam
ple
to p
erfo
rm P
CR,
pati
ent
high
ly s
uspe
cted
for
lept
ospi
rosi
sc I
gG E
LISA
res
ult,
the
hig
hest
tit
er b
y M
AT w
as <
40
Chap
ter11
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From epidemiological point of view, Semarang seems to encompass environmental
circumstances that are prerequisites for R. typhi and leptospirosis transmission.
Indeed, previous studies show that murine typhus is particularly prevalent in tropical
port cities where rats are abundant (16;17). For the Indonesian urban situation R.
rattusand R.norvegicus are likely to be the main hosts harboring R.typhi infected
X. cheopsis (18;19). These rats are also likely to be the maintenance hosts for
pathogenic Leptospira in Indonesia. In fact, the identified serogroups are commonly
associated with rats.
Although serology might be hampered by cross-reactions and sensitivity issues, we
believe that our data is representative for the area. The chosen cut-off values are
unlikely to cause false positives in an endemic setting. In regard to leptospirosis
serology, we used a wide panel for the MAT. This panel included WHO recommended
serovars and serovars that were previously isolated in Indonesia. Moreover, most
serogroups were represented in our panel and cross-reactions are likely to pick-up
missing serovars.
Due to non-specific presentation as reported here, both diseases are difficult to
diagnose on clinical grounds only. Misdiagnosis can lead to aberrant use of antibiotics
and other pharmaceuticals. Therefore rapid, cheap and reliable tests are warranted
to support clinical decision making.
Acknowledgements
We gratefully thank S.M.H. Faradz and personnel (CEBIOR, Semarang Indonesia) and
the residents of the department of Internal Medicine (Dr. Kariadi hospital, Semarang,
Indonesia) for their help and assistance during the study. We gratefully thank A.A.
Ahmed (Royal Tropical Institute, Amsterdam, The Netherlands) for performing the
leptospirosis PCR.
Murinetyph
usand
lep
tospirosisascauseofund
ifferentiatedfever
181
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(1) Richards AL, Soeatmadji DW, Widodo MA, Sardjono TW, Yanuwiadi B, Hernowati TE et al. Seroepidemiologic evidence for murine and scrub typhus in Malang, Indonesia. Am J Trop Med Hyg 1997 July;57(1):91-5.
(2) Richards AL, Ratiwayanto S, Rahardjo E, Kelly DJ, Dasch GA, Fryauff DJ et al. Serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae among residents of Gag Island, Indonesia. Am J Trop Med Hyg 2003 April;68(4):480-4.
(3) Van Peenen PF, Koesharjono C, See R, Bourgeois AL, Irving GS. Antibodies against murine typhyus in sera from Indonesians. Trans R Soc Trop Med Hyg 1977;71(4):297-9.
(4) Light RH, Nasution R, Van Peenen PF. Leptospirosis in febrile hospital patients in Djakarta. Southeast Asian J Trop Med Public Health 1971 December;2(4):493-5.
(5) Laras K, Cao BV, Bounlu K, Nguyen TK, Olson JG, Thongchanh S et al. The importance of leptospirosis in Southeast Asia. Am J Trop Med Hyg 2002 September;67(3):278-86.
(6) Ellis RD, Fukuda MM, McDaniel P, Welch K, Nisalak A, Murray CK et al. Causes of fever in adults on the Thai-Myanmar border. Am J Trop Med Hyg 2006 January;74(1):108-13.
(7) Suttinont C, Losuwanaluk K, Niwatayakul K, Hoontrakul S, Intaranongpai W, Silpasakorn S et al. Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study. Ann Trop Med Parasitol 2006 June;100(4):363-70.
(8) Terpstra WJ, Ligthart GS, Schoone GJ. Serodiagnosis of human leptospirosis by enzyme-linked-immunosorrbent-assay (ELISA). Zentralbl Bakteriol A 1980 August;247(3):400-5.
(9) Victoria B, Ahmed A, Zuerner RL, Ahmed N, Bulach DM, Quinteiro J et al. Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira. PLoS ONE 2008;3(7):e2752.
(10) Ellis RD, Fukuda MM, McDaniel P, Welch K, Nisalak A, Murray CK et al. Causes of fever in adults on the Thai-Myanmar border. Am J Trop Med Hyg 2006 January;74(1):108-13.
(11) Suttinont C, Losuwanaluk K, Niwatayakul K, Hoontrakul S, Intaranongpai W, Silpasakorn S et al. Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study. Ann Trop Med Parasitol 2006 June;100(4):363-70.
(12) Phongmany S, Rolain JM, Phetsouvanh R, Blacksell SD, Soukkhaseum V, Rasachack B et al. Rickettsial infections and fever, Vientiane, Laos. Emerg Infect Dis 2006 February;12(2):256-62.
(13) Watt G, Parola P. Scrub typhus and tropical rickettsioses. Curr Opin Infect Dis 2003 October;16(5):429-36.
(14) Ibrahim IN, Okabayashi T, Ristiyanto, Lestari EW, Yanase T, Muramatsu Y et al. Serosurvey of wild rodents for Rickettsioses (spotted fever, murine typhus and Q fever) in Java Island, Indonesia. Eur J Epidemiol 1999 January;15(1):89-93.
(15) Richards AL, Ratiwayanto S, Rahardjo E, Kelly DJ, Dasch GA, Fryauff DJ et al. Serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae among residents of Gag Island, Indonesia. Am J Trop Med Hyg 2003 April;68(4):480-4.
(16) Richards AL, Rahardjo E, Rusjdi AF, Kelly DJ, Dasch GA, Church CJ et al. Evidence of Rickettsia typhi and the potential for murine typhus in Jayapura, Irian Jaya, Indonesia. Am J Trop Med Hyg 2002 April;66(4):431-4.
(17) Dupont HT, Brouqui P, Faugere B, Raoult D. Prevalence of antibodies to Coxiella burnetti, Rickettsia conorii, and Rickettsia typhi in seven African countries. Clin Infect Dis 1995 November;21(5):1126-33.
(18) Ibrahim IN, Okabayashi T, Ristiyanto, Lestari EW, Yanase T, Muramatsu Y et al. Serosurvey of wild rodents for Rickettsioses (spotted fever, murine typhus and Q fever) in Java Island, Indonesia. Eur J Epidemiol 1999 January;15(1):89-93.
(19) Jiang J, Soeatmadji DW, Henry KM, Ratiwayanto S, Bangs MJ, Richards AL. Rickettsia felis in Xenopsylla cheopis, Java, Indonesia. Emerg Infect Dis 2006 August;12(8):1281-3.
REFERENCES
Chap
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Summary, discussion and directions
for future studies
J.F.P. Wagenaar ¹, M.H. Gasem ², R.A. Hartskeerl ³ and E.C.M. van Gorp ¹
1 Department of Internal Medicine, Slotervaart Hospital, Amsterdam, the Netherlands2 Department of Internal Medicine, Dr. Kariadi Hospital,
Diponegoro University, Semarang, Indonesia 3 Royal Tropical Institute (KIT), KIT Biomedical Research, Amsterdam, the Netherlands
12CHAPTER
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Summary,discussionan
ddirectionsforfuturestud
ies
185
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Leptospirosis is an emerging infectious disease of global importance (1). A severe
case manifests itself as a fulminant sepsis with multi-organ failure and hemorrhaging
(2). Although considerable progress has been made in unraveling the mechanisms of
sepsis and related coagulation disorders (3), knowledge on the pathophysiology of
leptospirosis remains extremely limited. This thesis studied the role of secondary
hemostasis and the innate immune response in patients suffering from severe
leptospirosis. In addition, we have also presented some epidemiological data and
throw some light on the usefulness of a rapid diagnostic assay. Most data presented
in this thesis was gleaned from patients who were admitted and treated in the Dr.
Kariadi hospital in Semarang, Indonesia.
Part I of the thesis gives the reader more insight into coagulation disorders
in leptospirosis. In Chapter 2 the literature was reviewed in this regard and we
addressed hemorrhagic syndromes, inflammation, primary and secondary hemostasis
and the fibrinolytic pathways. The endothelial cell receives special attention as
potential target cell. From the studies reviewed we concluded that in leptospirosis
the bleeding tendency might be the result of an imbalance in the hemostatic
equilibrium. However, at that point, detailed studies on how this imbalance is
triggered, and what inflammatory and coagulation proteins are involved, were
lacking. More insight into the pathways involved is given in this thesis. The case
report in Chapter 3 describes a typical case of severe leptospirosis with pulmonary
hemorrhages. From this patient we recovered strain Langkawi that was identified
as a new subtype of serovar Lai, which is the classical serovar associated with lung
bleeding Langkawi. In Chapter 4 we have provided data on coagulation activation
and fibrinolysis in relation to bleeding and poor outcome in patients with severe
leptospirosis. Patients demonstrated strong activation of the coagulation system,
as reflected by high plasma levels of thrombin-antithrombin (TAT) complexes,
prothrombin fragment 1+2 and D-dimer. Prolongation of prothrombin time (PT) and
activated partial thromboplastin time in these patients point towards consumption
coagulopathy. However, we cannot exclude the possibility of impaired function
SuMMARY
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or synthesis of clotting factors. Concurrently, anticoagulant pathways were down
regulated, as reflected by decreased levels of the anticoagulant markers protein C
and antithrombin. Patients also demonstrated evidence of activation and inhibition
of fibrinolysis, which was reflected by elevated plasmin-antiplasmin (PAP) complexes
and plasminogen activator inhibitor -1 plasma levels. These distinctions were more
pronounced in patients with a fatal outcome. A sub analysis between patients with and
without severe bleeding revealed a more pronounced imbalance of the coagulation
system in patients with severe bleeding. This was reflected by a significant association
with PT and the TAT/ PAP ratio, an indicator of the balance between coagulation and
fibrinolysis. Also thrombocytopenia was associated with bleeding. The absence of
marked disorders in patients with mild bleeding compared to those without any signs
of hemorrhaging points to the involvement of other pathophysiological mechanisms.
Overt disseminated intravascular coagulation was apparent in 10 out of 46 patients,
but was associated with neither bleeding nor poor outcome. In Chapter 5 the role
of endothelial cell dysfunction in relation to bleeding is characterized by studying
the dynamics of the endothelial cell-specific markers, soluble E-selectin (sE-selectin)
and the von Willebrand factor (vWF). Soluble E-selectin and vWF levels were strongly
elevated on admission and decreased during follow up, but they did not revert to
norm values. In a subgroup analysis between patients with and without bleeding,
plasma levels were not significantly different. No statistical difference was observed
in either sE-selectin or vWF plasma concentrations between patients that survived
and those that didn’t.We concluded that markers of endothelial cell activation are
strongly elevated in patients suffering from severe leptospirosis, regardless of bleeding
manifestations or outcome. The final chapter of this part of the thesis, Chapter 6,
defines the activation of the contact system in patients with severe leptospirosis. The
activation of the contact system was determined by measuring factor XI (FXI), factor
XII (FXII) and activated protein-inhibitor (INH) complexes (FXIa-C1-IH, FXIIa-C1-IH,
kallikrein-C1-INH and FXIa-AT-INH) in patients with severe leptospirosis. In addition,
markers of coagulation activation (TAT, prothrombin fragment 1+2) and fibrinolysis
(PAP) were measured. Overall, the median prothrombin time (PT) was high normal,
whereas the activated partial thromboplastin time (APTT) was markedly prolonged.
Twenty-six patients had a prolonged APTT and a normal PT. Patients had significantly
Summary,discussionan
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ies
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lower levels of FXII and FXI on admission compared to the controls. FXII and FXI
levels were inversely correlated with APTT prolongation. No evidence for contact
activation was observed since the median FXIIa-C1-IH, FXIa-C1-INH and FXIa-AT-INH
complexes were not statistically different from the controls and kallikrein-C1-INH
complexes were undetectable. There was no association between either (severe)
bleeding or mortality and contact activation markers. Marked activation of the
TF and fibrinolytic pathway was demonstrated, especially in subjects with severe
bleedings and in those who went on to die. In conclusion, patients suffering from
severe leptospirosis yielded reduced levels of FXII and FXI, that explained (isolated)
APTT prolongation but not bleeding or poor outcome. Although the low levels of
FXII and FXI may indicate consumption of these coagulation factors, no evidence of
contact system activation was observed.
The second part, Part II, of this thesis focuses on inflammation. In Chapter 7 the
long pentraxin PTX3 is explored in patients with leptospirosis. Pentraxins are a super
family of proteins characterized by a multimeric, usually pentameric structure.
The aim of the study was to evaluate the usefulness of PTX3 in monitoring patients
suffering from severe leptospirosis. The results were compared with the widely
used, short pentraxin C-reactive protein (CRP) and the pro-inflammatory cytokines
IL-6 and IL-8. Leptospirosis patients showed elevated plasma PTX3 levels. PTX3
correlated with IL-8 and, to a lesser extent, with CRP and IL-6 levels. High plasma
concentrations of PTX3, IL-6 and IL-8 were associated with mortality. Likewise,
PTX3 levels were associated with the severity of the sepsis. This association was not
apparent in any of the other markers. The widely used marker, CRP, appeared to be a
poor predictor for the severity of the disease. PTX3 has a great potential to be used
as a biomarker to monitor the disease severity in severe leptospirosis, or predict
the outcome. In Chapter 8 we investigated soluble ST2 (sST2), a molecule involved
in the regulation of the innate immune response. Toll-like receptors (TLRs) play an
important role in the initiation of an innate immune response. TLRs are inhibited
by negative regulators including the membrane-bound ST2 (mST2) receptor. sST2
can inhibit signaling through mST2. The aim of this study was to determine the
degree of sST2 and (pro) inflammatory cytokine release in patients admitted with
Chap
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severe leptospirosis. All patients had elevated sST2, IL-6, IL-8 and IL-10 levels
on admission. Soluble ST2 levels were found to be correlated with admission IL-
6, IL-8 and IL-10 concentrations. Soluble ST2 levels were significantly associated
with (severe) bleeding. This association was unique, since none of the cytokines
showed this correlation. Furthermore, sST2 was associated with poor outcome.
When either whole blood or isolated peripheral blood mononuclear cells (PBMCs)
were stimulated with Leptospira in vitro, no sST2 production could be detected.
Given the strong association with bleeding in combination with the results of the
in vitro experiments, we concluded that sST2 could be an indicative marker for
tissue damage in patients suffering from severe leptospirosis. Chapter 9 describes
a set of in-vitro experiments focusing on the innate immune response to different
Leptospira serovars (two saprophytic, two pathogenic reference and two host-
adapted virulent strains). We undertook killing and stimulation experiments using a
human monocytic cell line (THP-1), human PBMC and whole blood. Saprophytic and
pathogenic reference strains, but not virulent, host-adapted Leptospira, were killed
by whole blood and serum. Live Leptospira induced a vigorous serovar-dependent
cytokine response with a different pattern, compared to the response obtained with
heat-killed bacteria. The strongest cytokine response to Leptospira was observed
in whole blood, followed by PBMCs and THP-1 cells. Inhibition experiments with
anti-TLR2 and anti-TLR4 antibodies in whole blood showed the involvement of both
TLR2 and TLR4. These findings are unique, since previous work showed that THP-
1 recognized heat-killed Leptospira and leptospiral LPS exclusively through TLR2
instead of TLR4. In addition, our data revealed that TLR signaling was additive in
the case of the pathogenic reference strain and synergistic in the case of the host-
adapted virulent strain.
Part III of this thesis gives some insight into diagnostic and epidemiological aspects.
In Chapter 10 the seroprevalence of anti-Leptospiraantibodies and the utility of
two rapid tests for the serodiagnosis of the disease were studied in Binh Thuan,
an area in southern Vietnam. In an initial survey, blood samples from 44 patients
with acute undifferentiated fever and 83 healthy subjects were examined for anti-
Leptospiraantibodies using three tests: an ELISA; a latex card-agglutination test
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(Dri Dot); and a lateral-flow assay (LeptoTek Lateral Flow). In the ELISA, samples
from 35% of the healthy subjects and 40% of the febrile patients were found to have
titers of anti-LeptospiraIgM of at least 1:80. In the convalescent sample only one
of the febrile patients showed the marked increase in IgM titer that is indicative of
acute leptospirosis. Although the positive results of the lateral-flow assay – applied
to whole blood and serum – corresponded with those of the ELISA, the results of
the lateral-flow assay were often indeterminate. The card-agglutination test was
more specific. The overall agreement between the results of the rapid tests and
those of the ELISA was generally poor. It appeared that high seroprevalences of anti-
Leptospira IgM and low incidences of acute leptospirosis in this part of Vietnam
limit the diagnostic value of the rapid tests that were investigated. The lateral-flow
assay was not specific enough. The card-agglutination test is possibly better, but,
because of the low incidence, its sensitivity could not be evaluated adequately. In
Chapter 11 we studied the occurrence of acute rickettsial disease, leptospirosis and
dual infections among patients with acute undifferentiated fever (AUF) in Semarang,
Indonesia, where risk factors for both diseases are likely to be present. Consecutive
outpatients were recruited at two primary healthcare centers and hospitalized
patients were recruited at a governmental referral centre (Dr. Kariadi University
Hospital, Department of Internal Medicine). The blood of 137 febrile patients, 67
hospitalized patients and 70 outpatients was tested. A convalescent sample was
available in 106 (77%) patients. Nine patients (7%, 9/137) displayed signs of an
acute infection with R.typhi (murine typhus). Murine typhus could be diagnosed in
9% (6/67) of hospitalized patients; for outpatients this percentage was 4% (3/70).
Another nine cases (7%, 9/137) showed inconclusive R. typhi serology. Evidence
of acute infection with Orientia tsutsugamushi (scrub typhus) in members of the
spotted fever group (rickettsia) was not found. Leptospirosis was diagnosed in 13
patients (10%, 13/137), 2 of these were positive only by PCR. Eleven leptospirosis
patients were recruited in-hospital, so that the percentage of AUF caused by
leptospirosis in hospitalized patients was 16% (11/67) and in outpatients 3% (2/70).
No dual infections were detected; however three leptospirosis patients (23%, 3/13)
showed titers in the R.typhi assay. In this study, we have reported for the first time
on how murine typhus and leptospirosis are important causes of AUF in Semarang,
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Indonesia. Both diseases are difficult to diagnose on clinical grounds only, leading to
misdiagnosis and aberrant use of antibiotics and other pharmaceuticals. Therefore
rapid, cheap and reliable tests are warranted, to support clinical decision making.
Table: Questions for future research.
PartI:Coagulationandtheendothelialcell
• Do Leptospira have a direct toxic effect on platelets and are (virulent) Leptospira able to alter platelet function?
• Which mechanism, besides inflammation-induced coagulation activation, contributes to bleeding?
• Are clotting factors depleted and are clotting-factor antibodies present?• Is endothelium damaged or just activated in leptospirosis?• Can Leptospira directly activate or damage endothelial cells?• Are markers for endothelial cell activation/damage also increased in mild leptospirosis?
• What is the role of new drugs that intervene in the coagulation cascade in the treatment of patients with severe leptospirosis?
PartII:Inflammation• What is the role of TLR negative regulatory pathways in leptospirosis?
• To what extent is LPS an important factor in the virulence and pathogenicity of Leptospira?
• Which TLR4 ligands are involved in the innate immune response to virulent Leptospira?• What is the mechanism of TLR4 engagement in vivo?• Are there new immunomodulating strategies to treat severe leptospirosis?PartIII:Diagnosticandepidemiologicalaspects• What is the true burden of disease from leptospirosis worldwide?
• Which preventative measures are most effective in reducing morbidity and mortality of leptospirosis?
• To what extent will prompt diagnosis, early triage of severe cases and better clinical management improve the outcome of leptospirosis in endemic areas?
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The data presented in this thesis will contribute to our knowledge of leptospirosis
but consequently raises many new questions regarding its pathophysiology and
epidemiology. The next section discusses the major findings and addresses some key
questions that await further investigation (see table).
PartI:Coagulationandtheendothelialcell
This thesis demonstrates that patients suffering from severe leptospirosis show
coagulation activation with concurrent down regulation of anticoagulant systems
and fibrinolysis. In deceased patients and in those with severe bleeding, these
disorders are more pronounced. Interestingly, our studies also turned up patients
who suffered from bleeding, but who had normal results from global coagulation
tests. Therefore, in addition to consumption coagulopathy, other pathophysiological
mechanisms are also likely to contribute to the clinical picture. Endothelium was
shown to be highly dysregulated, but no association with bleeding was observed.
From clinical studies it is not possible to differentiate between endothelial cell
damage and activation. Hence we need experimental studies to elucidate the exact
role of the endothelial cell. In this thesis it was also shown that thrombocytopenia is
associated with bleeding, as reported in previous studies (4). In this regard, studying
platelets might be a promising key to elucidate the pathophysiology of bleeding in
severe leptospirosis. Lastly, we should explore novel therapies that intervene in the
coagulation cascade (e.g. activated protein C), with the ultimate goal of reducing
mortality from severe leptospirosis.
PartII:Inflammation
Late recognition of severe leptospirosis cases contributes to the high mortality rate
(2). Biomarkers may be helpful in the early discrimination between severe and non-
severe cases. Adequate discrimination between severe and less-severe cases of
leptospirosis is very important because it helps the clinician in the allocation of high-
care facilities only to those patients with a high likelihood of mortality and (bleeding)
complications. This is particularly important in low-resource environments. Studies
DISCuSSION AND DIRECTIONS FOR FuTuRE STuDIES
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that identify such biomarkers in leptospirosis are greatly lacking. In this thesis
we have identified the long pentraxin PTX3 as a promising candidate marker, as it
was prognostic for both mortality and disease severity. Soluble ST2 was, uniquely,
associated with both bleeding and mortality. Our in-vitro data showed that blood
cells were not the source of sST2, as was observed in previous studies (5). We
hypothesized that sST2 might be a marker for deep tissue damage, which should be
evaluated in future studies.
Little is known about the innate immune response to Leptospira. In a set of experiments
we showed that the cytokine response to Leptospira is serovar dependant. When
using viable bacteria in our experiments, cytokine dynamics changed, emphasizing
the importance of using fresh isolates in future studies. We are the first to report
the involvement of both TLR2 and TLR4 engagement in a leptospirosis human whole
blood model. Whereas the interaction of TLR2 and TLR4 in mediating whole blood
TNF-a release appears to be additive for non-virulent pathogenic Leptospira, we
observed that whole blood TNF-a release induced by virulent Leptospira was boosted
by synergistic actions of TLR2 and TLR4. We hypothesized that viable Leptospira
interact with host cells in whole blood in a TLR2 and disruptive manner such that
endogenous TLR4 ligands are released which effectively boost cytokine production.
Future studies are warranted to reveal the mechanism of action of this observed
synergy and to unravel the involvement of TLR4 during leptospirosis in vivo.
PartIII:Diagnosticandepidemiologicalaspects
The real burden of leptospirosis is unknown. Its incidence rate is thought to be
greatly underestimated due to ignorance, misdiagnosis and lack of appropriate
diagnostic laboratory services (6). Misdiagnosis has become a critical issue in
regions where dengue and other infectious diseases, with overlapping clinical
presentation, are endemic (7;8). Ignorance is largely due to a lack of adequate
laboratory testing facilities, which remains the major barrier for diagnosis and
epidemiologic surveillance. Leptospirosis serology is a flawing method for prompt
diagnosis, especially because a convalescent sample is required and tests are often
too laborious. Consequently, there is an urgent need for reliable, cheap and non-
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laborious, antigen-based diagnostic tests. When combining molecular methods (PCR)
with serological tests more (mild) cases will be identified, as was shown in this
thesis. Indeed, early diagnosis may decrease the case fatality rate since antibiotic
treatment provides the greatest benefit when it is initiated early in the course
of disease (6). More insight into the true epidemiology of leptospirosis will help
convince policy makers to support approaches for focused prevention measures and
will reduce the aberrant use of antibiotics and other pharmaceuticals prescribed by
physicians. What remains is to define clinical guidelines, which are often anecdotal
rather than evidence based.
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(1) Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003 December;3(12):757-71.
(2) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (3) Schouten M, Wiersinga WJ, Levi M, van der Poll T. Inflammation, endothelium, and
coagulation in sepsis. J Leukoc Biol 2007 November 21. (4) Chierakul W, Tientadakul P, Suputtamongkol Y, Wuthiekanun V, Phimda K, Limpaiboon
R et al. Activation of the coagulation cascade in patients with leptospirosis. Clin Infect Dis 2008 January 15;46(2):254-60.
(5) van ‘t Veer C, van den Pangaart PS, van Zoelen MA, de KM, Birjmohun RS, Stroes ES et al. Induction of IRAK-M is associated with lipopolysaccharide tolerance in a human endotoxemia model. J Immunol 2007 November 15;179(10):7110-20.
(6) McBride AJ, Athanazio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis 2005 October;18(5):376-86.
(7) Ko AI, Galvao RM, Ribeiro Dourado CM, Johnson WD, Jr., Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 1999 September 4;354(9181):820-5.
(8) LaRocque RC, Breiman RF, Ari MD, Morey RE, Janan FA, Hayes JM et al. Leptospirosis during dengue outbreak, Bangladesh. Emerg Infect Dis 2005 May;11(5):766-9.
REFERENCES
Samenvatting en discussie voor niet-ingewijden
J.F.P. Wagenaar ¹, M.H. Gasem ², R.A. Hartskeerl ³ and E.C.M. van Gorp ¹
1 Afdeling interne geneeskunde, Slotervaartziekenhuis, Amsterdam, Nederland2 Afdeling interne geneeskunde, Dr. Kariadi ziekenhuis,
Diponegoro universiteit, Semarang, Indonesië 3 Koninklijk Instituut voor de Tropen (KIT), KIT Biomedisch Onderzoek,
Amsterdam, Nederland
12HOOFDSTuK
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Leptospirose is een bacteriële infectieziekte die wereldwijd voorkomt. Ernstige
gevallen presenteren zich met een fulminante sepsis (“bloedvergiftiging”), met
multi-orgaan falen en uitgesproken bloedingen. Hoewel aanzienlijke vooruitgang
is geboekt in het ontrafelen van de mechanismen van sepsis en de aanverwante
bloedingen die optreden, is de kennis over de pathofysiologie van leptospirose zeer
beperkt. In dit proefschrift wordt de bloedstolling (haemostase) en de immuunrespons
in patiënten die lijden aan ernstige leptospirose bestudeerd. Daarnaast bevat dit
proefschrift epidemiologische studies. De meeste onderzoeksgegevens komen van
patiënten die werden opgenomen en behandeld in het Dr. Kariadi ziekenhuis in
Semarang, Indonesië.
Deel I van dit proefschrift geeft de lezer meer inzicht in stoornissen van
de bloedstollingscascade in leptospirose. In hoofdstuk 2 wordt de huidige
wetenschappelijke literatuur samengevat. We beschrijven syndromen die gepaard gaan
met bloedingen, ontstekingsmechanismen, de primaire en secundaire haemostase en
de rol van de vaatwand (endotheel). Uit de huidige studies kan worden geconcludeerd
dat de bloedingsneiging in leptospirose het resultaat kan zijn van een disbalans in de
bloedstolling en schade aan het endotheel. Echter, gedetailleerde studies over hoe
deze dysbalans ontstaat en welke ontstekings- en bloedstollingseiwitten betrokken
zijn ontbreken. In dit proefschrift wordt hier meer opheldering over gegeven. In
hoofdstuk 3 wordt een patiënt met ernstige longbloedingen beschreven, veroorzaakt
door een leptospirose infectie. Uit deze patiënt werd een tot nu toe onbekende stam
geïsoleerd: Langkawi, een nieuw subtype van serovar Lai, wat het klassieke serovar
is dat geassocieerd wordt met longbloedingen. Hoofdstuk 4 beschrijft de activatie
van bloedstollingseiwitten en afbraak van de bloedprop (fibrinolyse) in relatie tot
het optreden van bloedingen en sterfte van patiënten met ernstige leptospirose.
Patiënten toonden een sterke activatie van bloedstollingseiwitten, dat werd
weerspiegeld door hoge bloedwaarden van trombine-antitrombine (TAT) complexen,
protrombine fragment 1 + 2 (F1+2) en D-dimeer. Verlenging van de protrombinetijd
(PTT) en de geactiveerde partiële tromboplastinetijd (APTT) in deze patiënten wijst
SAMENVATTING
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in de richting van consumptie van bloedstollingseiwitten. We kunnen echter de
mogelijkheid van een verminderde functie of synthese van deze stollingsfactoren
niet uitsluiten. Gelijktijdig werden mechanismen die de bloedstolling remmen
geremd, wat weerspiegeld werd door verlaagde proteïne C en antitrombine spiegels
in het bloed. Verder werd bewijs gevonden van activering en remming van de
fibrinolyse, dat werd weerspiegeld door verhoogde plasmine-antiplasmine (PAP)
complexen en plasminogeen activator inhibitor -1 plasmaspiegels. Deze disbalans in
de stollingscascade was meer uitgesproken bij patiënten die stierven en in patiënten
met ernstige bloedingen. In deze laatste groep werd een significante associatie met
zowel de PT als de TAT/PAP-ratio (een indicator van het evenwicht tussen stolling
en fibrinolyse) gevonden. In patiënten met milde bloedingen was de disbalans
minder uitgesproken, wat wijst op de betrokkenheid van andere pathosfysiologische
mechanismen. Tien van de 46 patiënten voldeden aan de internationale criteria
voor diffuus intravasale stolling (DIS). Deze diagnose was echter niet geassocieerd
met het optreden van bloedingen of sterfte. In hoofdstuk 5 wordt de rol van de
vaatwand (endotheel) disfunctie in relatie tot bloeden bestudeerd. Als markers werd
gebruik gemaakt van twee endotheelcel specifieke eiwitten: soluble E-selectine (sE-
selectine) en von Willebrand factor (vWF). Soluble E-selectine en vWF spiegels in
het bloed waren sterk verhoogd in patiënten tijdens presentatie in het ziekenhuis
en daalden gedurende de follow-up. In een subgroepanalyse tussen patiënten met
en zonder bloedingen, waren de plasmaspiegels van deze eiwitten niet significant
verschillend. Ook werd er geen statistisch verschil waargenomen tussen patiënten
die stierven of de ziekte overleefden. We concluderen dat deze markers van
endotheelcel activatie sterk verhoogd zijn in patiënten met ernstige leptospirose,
ongeacht of ze bloeden. In het laatste hoofdstuk van dit deel van het proefschrift,
hoofdstuk 6, wordt de activering van het contact systeem (specifieke eiwitten in de
bloedstollingcascade) bij patiënten met ernstige leptospirose bestudeerd. Activering
van het contact systeem werd bepaald door meting van de stollingsfactoren factor
XI (FXI), FXII en de geactiveerde eiwit-remmer complexen (FXIa, XIIa, kallikreïne-
C1-remmer (INH) en FXIa-AT-INH). Daarnaast werden markers van de “gewone”
activatie van bloedstolling (TAT, F1+2) en fibrinolyse (PAP) gemeten. Patiënten met
leptospirose hadden significant verlaagde FXI en FXII spiegels in het bloed. Deze
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verlaagde waarden correleerden met verlenging van de APTT. Echter er werd geen
activering van het contact systeem waargenomen: kallikreïne-C1-INH complexen
waren niet aantoonbaar, terwijl FXIIa-C1 - IH, FXIa-C1-INH en FXIa-AT-INH complexen
statistisch niet verschilden van de controle groep. Mogelijk duiden deze bevindingen
op vroeg verbruik van FXI en FXII.
Deel II van dit proefschrift richt zich op ontsteking en sepsis. In hoofdstuk 7 wordt
de rol van de lange pentraxine PTX3 onderzocht bij patiënten met leptospirose.
Pentraxines behoren tot een superfamilie van eiwitten die worden gekenmerkt door
een multimere, meestal pentamere structuur. Het doel van deze studie was om PTX3
te evalueren als marker voor ernst van de ziekte. De resultaten werden vergeleken
met de in de kliniek veel gebruikte korte pentraxine C-reactief proteïne (CRP) en
de pro-inflammatoire cytokines (ontstekingseiwitten) IL-6 en IL-8. Leptospirose
patiënten toonden verhoogde plasma PTX3 concentraties in het bloed. PTX3
correleerde met IL-8 en in mindere mate met CRP en IL-6. Hoge plasmaconcentraties
van PTX3, IL-6 en IL-8 waren geassocieerd met sterfte. Ook bleek de concentratie
van PTX3 in het bloed samen te hangen met de ernst van sepsis. Voor de andere
markers was dit niet het geval. De in de kliniek veel gebruikte marker CRP bleek
in alle gevallen een slechte prognostische waarde te hebben. PTX3 heeft een groot
potentieel om te worden gebruikt als biomarker om de ernst van ziekte en het beloop
te voorspellen. In hoofdstuk 8 onderzochten we soluble ST2 (sST2), een molecuul
betrokken bij de regulatie van de immuunrespons. Toll-like receptoren (TLR’s)
spelen een belangrijke rol in de initiatie van deze immuunrespons. De werking van
TLR’s wordt o.a. geantagoneerd door de membraan-gebonden ST2 (MST2) receptor.
Soluble ST2 wordt in staat geacht om signalering via MST2 te remmen. Het doel van
deze studie was om de mate van sST2 en (pro-) inflammatoire cytokine activiteit bij
patiënten met ernstige leptospirose te bepalen. Alle patiënten toonden verhoogde
bloedwaarden van sST2, IL-6, IL-8 en IL-10 bij opname in het ziekenhuis. Soluble
ST2 plasma spiegels correleerden met IL-6, IL-8 en IL-10 concentraties. Soluble ST2
spiegels waren significant geassocieerd met (ernstige) bloedingen. Deze associatie
was uniek, omdat geen van de cytokines deze correlatie toonde. Ook was sST2
geassocieerd met sterfte. Als volbloed of perifeer bloed mononucleaire cellen
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(PBMCs) werden gestimuleerd met Leptospirainvitro(cel experimenten), kon geen
sST2 productie worden aangetoond. Gezien de sterke associatie met bloeden in
combinatie met de resultaten van de invitro experimenten, concluderen we dat sST2
een potentiële marker voor diepe weefsel schade is. Meer onderzoek zal dit moeten
uitwijzen. Hoofdstuk 9 beschrijft een reeks van in vitro experimenten gericht
op de immuunrespons tegen verschillende Leptospira serovars (1 saprofytische,
2 pathogene referentie stammen en 2 virulente stammen). Wij voerden killing en
stimulatie experimenten uit met behulp van een menselijke monocytaire cellijn (THP-
1), humane PBMC’s en volbloed. Het killing experiment toonde dat saprofytische en
pathogene referentie stammen, maar niet de virulente Leptospira, werden gedood in
volbloed en serum. In de stimulatie proeven veroorzaakte de levende Leptospira een
heftige serovar afhankelijke cytokine-respons met een ander patroon in vergelijking
met de immuunreactie op hitte gedode bacteriën. De sterkste cytokine-respons op
Leptospira werd waargenomen in volbloed, gevolgd door PBMCs en THP-1 cellen.
Remming experimenten met anti-TLR2 en anti-TLR4 antilichamen in volbloed toonde
dat zowel TLR2 en TLR4 een rol spelen in het induceren van een immuunrespons. Deze
bevindingen zijn uniek, omdat eerdere studies suggereerden dat de immuunrespons
tegen hitte gedode Leptospira en Leptospira LPS uitsluitend via TLR2 plaatsvindt.
Onze studies suggereren verder dat de TLR-respons additief verliep als gestimuleerd
werd met pathogene referentie stammen en dat de respons synergistisch verliep in
het geval van de virulente bacteriën.
Deel III van dit proefschrift geeft inzicht in diagnostische en epidemiologische
aspecten. In hoofdstuk 10 wordt de seroprevalentie van anti-Leptospira antistoffen
en het nut van twee diagnostische sneltests voor de diagnose van leptospirose
bestudeerd in Binh Thuan, een gebied in het zuiden van Vietnam. In een survey onder
44 patiënten met acute ongedifferentieerde koorts en in 83 gezonde proefpersonen
werd het voorkomen van anti-Leptospira antilichamen onderzocht met behulp van
drie diagnostische testen: een ELISA, een latex card-agglutinatietest (Dri Dot) en
een laterale-flow tests (LeptoTek Lateral Flow). Met de ELISA werden in 35% van
de gezonde proefpersonen en in 40% van de patiënten met koorts anti-Leptospira
IgM antistoffen gedetecteerd. Slechts 1 van de febriele patiënten toonde in het
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follow-up monster een verhoging van de IgM-titer, indicatief voor acute leptospirose.
Hoewel de positieve resultaten van de laterale-flow-test in volbloed en serum
goed overeen kwamen met de ELISA resultaten, bleek de laterale-flow-test vaak
een indeterminate resultaat weer te geven. De kaart-agglutinatietest was meer
specifiek. De overeenkomst tussen de resultaten van de sneltests en die van de
ELISA was over het algemeen slecht. Het bleek dat de hoge seroprevalentie van
anti-Leptospira IgM en de lage incidentie van acute leptospirose in dit deel van
Vietnam de diagnostische waarde van de sneltests ondermijnden. De laterale-flow-
test was niet specifiek genoeg. De kaart-agglutinatietest presteerde beter, maar
vanwege de lage incidentie, kon de gevoeligheid van deze test niet naar behoren
worden geëvalueerd. In hoofdstuk 11 bestuderen we het voorkomen van acute
rickettsia infecties (tyfus), leptospirose en dubbel infecties bij patiënten met acute
ongedifferentieerde koorts in Semarang, Indonesië, waar risicofactoren voor beide
ziekten aanwezig zijn. Poliklinische patiënten werden gerekruteerd in twee primaire
gezondheidszorg centra en gehospitaliseerde patiënten werden gerekruteerd in het
Dr. Kariadi universitair ziekenhuis, afdeling interne geneeskunde. Het bloed van 137
patiënten met koorts, waarvan 67 gehospitaliseerd en 70 poliklinisch, werd getest.
Een follow-up monster werd in 106 (77%) patiënten verkregen. Negen patiënten (7%,
9/137) hadden een acute infectie met R. typhi (muizen tyfus). In het ziekenhuis
werd muizen tyfus gediagnosticeerd in 9% (6/67) van de gevallen, voor poliklinische
patiënten bedroeg dit percentage 4% (3/70). In negen gevallen (7%, 9/137) was
de R. typhi serologie indeterminate. Bewijs voor een acute infectie met Orientia
tsutsugamushi (scrubtyfus) of “spotted fever” groep rickettsia werd niet gevonden.
Leptospirose werd gediagnosticeerd bij 13 patiënten (10%, 13/137), 2 monsters waren
alleen positief met de PCR test. Elf leptospirose patiënten werden gerekruteerd in
het ziekenhuis, zodat het percentage van ongedifferentieerde koorts veroorzaakt
door leptospirose in het ziekenhuis 16% (11/67) was. In de poliklinische patiënten
was dit percentage 3% (2/70). Er werden geen dubbel infecties aangetroffen, echter
drie leptospirose patiënten (23%, 3/13) toonden ook titers in de R. typhi test. In
deze studie rapporteren we voor de eerste keer muizen tyfus en leptospirose als een
belangrijke oorzaak van ongedifferentieerde koorts in Semarang, Indonesië. Beide
ziekten zijn moeilijk te diagnosticeren op klinische gronden alleen, wat leidt tot
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verkeerde diagnose en gebruik van antibiotica. Daarom is het van belang om snelle,
goedkope en betrouwbare tests te ontwikkelen ter ondersteuning van de klinische
diagnose.
De gegevens in dit proefschrift zullen bijdragen aan de kennis over leptospirose,
maar roepen veel nieuwe vragen op met betrekking tot de pathofysiologie en
epidemiologie. De volgende sectie bespreekt de belangrijkste bevindingen uit dit
proefschrift en geeft richting aan toekomstig onderzoek.
DeelI:Bloedstollingenendotheelcellen
Dit proefschrift toont aan dat patiënten die lijden aan ernstige leptospirose een
activatie van de bloedstolling hebben met gelijktijdig een down-regulatie van
antistollingssystemen en fibrinolyse. In de overleden patiënten en bij patiënten
met ernstige bloedingen, zijn deze verstoringen meer uitgesproken. Interessant is
dat onze studies ook aantonen dat patiënten in sommige gevallen geen afwijkingen
vertonen in de algemene stollingstesten. Dus naast een verbruik van stollingseiwitten
(door overdadige stollingsactivatie) is het waarschijnlijk dat ook een ander
pathosfysiologisch mechanisme bijdraagt aan het klinisch beeld. De vaatwand
(endotheel) bleek zeer geactiveerd dan wel beschadigd te zijn, maar een associatie
met bloedingen werd niet waargenomen. Uit klinische studies is het niet mogelijk
om onderscheid te maken tussen endotheelcel schade of activering. Daarom zijn
experimentele studies nodig om de rol van het endotheel te onderzoeken. In dit
proefschrift wordt voorts aangetoond dat een laag aantal bloedplaatjes geassocieerd
was met het optreden van bloedingen, zoals gerapporteerd in eerdere studies. Het
bestuderen van bloedplaatjes is in dit opzicht dus veelbelovend. Als laatste moeten
er nieuwe therapieën worden bestudeerd die ingrijpen in de bloedstollingscascade,
met het ultieme doel om de sterfte door ernstige leptospirose te verminderen.
DISCuSSIE EN RICHTING VOOR TOEKOMSTIGE STuDIES
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DeelII:Ontsteking
Late erkenning van ernstige leptospirose draagt bij aan de hoge sterfte. Biomarkers
kunnen nuttig zijn om onderscheid te maken tussen ernstige en niet-ernstige
gevallen. Vooral in ontwikkelingslanden is dit onderscheid van groot belang. Zo is
de behandelend arts in staat om zijn schaarse middelen in te zetten voor patiënten
die er het meeste baat bij hebben. Weinig onderzoek heeft plaatsgevonden naar
biomarkers in leptospirose. In dit proefschrift wijzen we op PTX3 als veelbelovende
kandidaat-marker, met goede prognostische eigenschappen voor zowel de
sterfte als ziekte ernst. Soluble ST2 was uniek, omdat hoge concentraties in het
bloed zowel met sterfte als met bloedingen werd geassocieerd. Uit onze in vitro
experimenten bleek dat bloedcellen niet de bron van het sST2 waren, zoals ook
werd waargenomen in eerdere studies. We veronderstellen dat sST2 wellicht een
goede marker voor diepe weefsel schade is, hetgeen in toekomstige studies verder
moet worden uitgezocht. Er is niet veel bekend over de immuunrespons tegen
Leptospira. In een reeks experimenten werd aangetoond dat de cytokine-respons
tegen Leptospira, serovar afhankelijk is. Bij gebruik van levende bacteriën zagen we
deze dynamiek veranderen, wat het belang aangeeft van het gebruik van levende
isolaten in toekomstige studies. Wij zijn de eersten die aantonen dat zowel TLR2 en
TLR4 betrokken zijn in de immuunrespons tegen Leptospira in een humaan volbloed
model. Virulente bacteriën bleken op synergistische wijze TLRs te activeren.
Toekomstige studies zullen moeten uitwijzen hoe het werkingsmechanisme van deze
waargenomen synergie is en wat de precieze rol van TLR4 is in de immuunrespons
tegen Leptospira.
DeelIII:Diagnostischeenepidemiologischeaspecten
Het aantal wereldwijde gevallen van leptospirose is onbekend. De incidentie van
leptospirose wordt zeer waarschijnlijk onderschat als gevolg van onwetendheid
van artsen, het stellen van de verkeerde diagnose en het ontbreken van adequate
diagnostische middelen. Het stellen van de verkeerde diagnose is met name een
probleem in regio’s waar knokkelkoorts (dengue) en andere besmettelijke ziekte
voorkomen met een overlappende klinische presentatie. Voorts is misdiagnose te
wijten aan het ontbreken van adequate laboratorium testen. Serologische testen zijn
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niet geschikt voor snelle diagnose, vooral omdat een follow-up monster nodig is en
de test methoden vaak te bewerkelijk en te duur zijn. Bijgevolg is er een dringende
behoefte aan betrouwbare, goedkope en onbewerkelijke diagnostische testen. De
combinatie van het gebruik van moleculaire methoden (PCR) en serologische test
methoden zal meer gevallen van leptospirose identificeren. Een vroege diagnose van
leptospirose kan het sterftecijfer verlagen, omdat snelle behandeling met antibiotica
een gunstig beloop van de ziekte bewerkstelligd. Inzicht in de ware epidemiologie
van leptospirose zal bijdragen beleidsmakers er van te overtuigen om te investeren
in preventie en voorkoming van verkeerd antibiotica gebruik. Verder moeten er
richtlijnen worden ontwikkeld om deze potentieel dodelijke ziekte te behandelen.
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Dit proefschrift zou nooit verschenen zijn zonder de inzet van vele mensen, te
weten: patiënten, arts-assistenten, co-assistenten, studenten, verpleegkundigen,
secretaresses, klinisch chemici, analisten, internisten, collega’s, vrienden, mijn
dierbare familie en mijn liefste vriendin. Een aantal mensen wil ik in het bijzonder
bedanken: mijn co-promotores: Eric van Gorp, Rudy Hartskeerl en Hussein Gasem.
Mijn promotor Tom van der Poll en mijn counterpart Marga Goris. For all my Indonesian
colleagues: terimakasih!
DANKWOORD
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Jiři František Pavel Wagenaar (Delft 1976) graduated from the Atheneum in 1996.
Following this he received his medical training at the University of Amsterdam
(Amsterdam, the Netherlands), with additional clinical and research courses in
Vietnam and Indonesia. He started his PhD project at the Slotervaart hospital in 2004
under the supervision of E.C.M. van Gorp and D.P.M. Brandjes in close cooperation
with R.A. Hartskeerl (Royal Tropical Institute (KIT); Amsterdam), M.H. Gasem (Dr.
Kariadi hospital, Diponegoro University; Semarang, Indonesia) and Prof. T. van der
Poll (Center for Experimental and Molecular Medicine, University of Amsterdam).
During his PhD, he worked as HIV physician at the patient clinic Internal Medicine
of the Slotervaart hospital under the supervision of J.W. Mulder. Currently he works
as a resident in internal medicine at the Department of Medicine at the Slotervaart
hospital and the Academic Medical Centre.
Jiři František Pavel Wagenaar (Delft 1976) studeerde af aan het Atheneum in
1996. Hierna startte zijn medische opleiding aan de Universiteit van Amsterdam
(Amsterdam, Nederland), met aanvullend klinisch onderzoek en cursussen in Vietnam
en Indonesië. Hij begon zijn PhD project in het Slotervaart ziekenhuis in 2004 onder
supervisie van ECM van Gorp en D.P.M. Brandjes en in nauwe samenwerking met
R.A. Hartskeerl (Koninklijk Instituut voor de Tropen (KIT), Amsterdam), MH Gasem
(Dr. Kariadi ziekenhuis, Diponegoro University; Semarang, Indonesië) en prof. T. van
der Poll (Centrum voor Experimentele en Moleculaire Geneeskunde, Universiteit van
Amsterdam). Tijdens zijn promotie werkte hij als HIV arts op de polikliniek Interne
Geneeskunde van het Slotervaart ziekenhuis onder begeleiding van J.W. Mulder.
Momenteel werkt hij als arts assistent interne geneeskunde in opleiding in het
Slotervaart ziekenhuis en het Academisch Medisch Centrum.
AbOuT THE AuTHOR