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Studies on leptospirosis : clinical aspects and pathophysiology

Wagenaar, J.F.P.

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Citation for published version (APA):Wagenaar, J. F. P. (2010). Studies on leptospirosis : clinical aspects and pathophysiology.

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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.


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.

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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.

(25) van der Poll T, Opal SM. Host-pathogen interactions in sepsis. Lancet Infect Dis 2008 January;8(1):32-43.

(26) Rittirsch D, Flierl MA, Ward PA. Harmful molecular mechanisms in sepsis. Nat Rev Immunol 2008 October;8(10):776-87.

(27) Levi M, van der Poll T, Buller HR. Bidirectional relation between inflammation and coagulation. Circulation 2004 June 8;109(22):2698-704.

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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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(7) Wagenaar JF, de Vries PJ, Hartskeerl RA. Leptospirosis with pulmonary hemorrhage, caused by a new strain of serovar Lai: Langkawi. J Travel Med 2004 November; 11(6):379-81.

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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.

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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.




(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.


(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


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


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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

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(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


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.


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).


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


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

Chap

ter4

68

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

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.

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(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.


(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.


(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

LowfactorXIIa

ndfactorXIlevelsinpatientswithseverelep

tospirosis

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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

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healthy Javanese volunteers served as a normal reference group to establish local

normal laboratory test values.


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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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).

Chap

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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

d st

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

Chap

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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


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


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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.


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

Chap

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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

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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

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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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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 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

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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

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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

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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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R17

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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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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.

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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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(1) Vinetz JM. Leptospirosis. Curr Opin Infect Dis 2001 October;14(5):527-38. (2) Cinco M, Vecile E, Murgia R, Dobrina P, Dobrina A. Leptospira interrogans and

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.


(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.

REFERENCES

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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.


(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

of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 1999 September 4;354(9181):820-5.

(26) Calandra T, Gerain J, Heumann D, Baumgartner JD, Glauser MP. High circulating levels of interleukin-6 in patients with septic shock: evolution during sepsis, prognostic value, and interplay with other cytokines. The Swiss-Dutch J5 Immunoglobulin Study Group. Am J Med 1991 July;91(1):23-9.

(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.

(29) Angus DC, Wax RS. Epidemiology of sepsis: an update. Crit Care Med 2001 July;29(7 Suppl):S109-S116.

(30) Song M, Kellum JA. Interleukin-6. Crit Care Med 2005 December;33(12 Suppl):S463-S465.

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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

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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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(1) Levett PN. Leptospirosis. Clin Microbiol Rev 2001 April;14(2):296-326. (2) Segura ER, Ganoza CA, Campos K, Ricaldi JN, Torres S, Silva H et al. Clinical

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.


(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) Estavoyer JM, Racadot E, Couetdic G, Leroy J, Grosperrin L. Tumor necrosis factor in patients with leptospirosis. Rev Infect Dis 1991 November;13(6):1245-6.


(7) Vernel-Pauillac F, Merien F. Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans. Infect Immun 2006 July;74(7):4172-9.

(8) van der Poll T, Opal SM. Host-pathogen interactions in sepsis. Lancet Infect Dis 2008 January;8(1):32-43.

(9) Tominaga S. A putative protein of a growth specific cDNA from BALB/c-3T3 cells is highly similar to the extracellular portion of mouse interleukin 1 receptor. FEBS Lett 1989 December 4;258(2):301-4.

(10) Yanagisawa K, Takagi T, Tsukamoto T, Tetsuka T, Tominaga S. Presence of a novel primary response gene ST2L, encoding a product highly similar to the interleukin 1 receptor type 1. FEBS Lett 1993 February 22;318(1):83-7.

(11) Iwahana H, Yanagisawa K, Ito-Kosaka A, Kuroiwa K, Tago K, Komatsu N et al. Different promoter usage and multiple transcription initiation sites of the interleukin-1 receptor-related human ST2 gene in UT-7 and TM12 cells. Eur J Biochem 1999 September;264(2):397-406.

(12) Yanagisawa K, Tsukamoto T, Takagi T, Tominaga S. Murine ST2 gene is a member of the primary response gene family induced by growth factors. FEBS Lett 1992 May 4;302(1):51-3.

(13) Xu D, Chan WL, Leung BP, Huang F, Wheeler R, Piedrafita D et al. Selective expression of a stable cell surface molecule on type 2 but not type 1 helper T cells. J Exp Med 1998 March 2;187(5):787-94.

(14) Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R. The IL-1 receptor-related T1 antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J Immunol 1998 November 1;161(9):4866-74.

(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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(20) Oshikawa K, Kuroiwa K, Tago K, Iwahana H, Yanagisawa K, Ohno S et al. Elevated soluble ST2 protein levels in sera of patients with asthma with an acute exacerbation. Am J Respir Crit Care Med 2001 July 15;164(2):277-81.

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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.

(28) Leung BP, Xu D, Culshaw S, McInnes IB, Liew FY. A novel therapy of murine collagen-induced arthritis with soluble T1/ST2. J Immunol 2004 July 1;173(1):145-50.

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(37) Weinberg EO, Shimpo M, De Keulenaer GW, MacGillivray C, Tominaga S, Solomon SD et al. Expression and regulation of ST2, an interleukin-1 receptor family member, in cardiomyocytes and myocardial infarction. Circulation 2002 December 3;106(23):2961-6.

(38) 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.

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


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.


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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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18. Merien, F., D. Portnoi, P. Bourhy, F. Charavay, A. Berlioz-Arthaud, and G. Baranton. 2005. A rapid and quantitative method for the detection of Leptospira species in human leptospirosis. FEMS Microbiol.Lett. 249:139-147.

19. Nahori, M. A., E. Fournie-Amazouz, N. S. Que-Gewirth, V. Balloy, M. Chignard, C. R. Raetz, G. Saint, I, and C. Werts. 2005. Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J.Immunol. 175:6022-6031.

20. Nally, J. E., C. Chantranuwat, X. Y. Wu, M. C. Fishbein, M. M. Pereira, J. J. Da Silva, D. R. Blanco, and M. A. Lovett. 2004. Alveolar septal deposition of immunoglobulin and complement parallels pulmonary hemorrhage in a guinea pig model of severe pulmonary leptospirosis. Am.J.Pathol. 164:1115-1127.

21. Nally, J. E., E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett. 2005. Changes in lipopolysaccharide O antigen distinguish acute versus chronic Leptospira interrogans infections. Infect.Immun. 73:3251-3260.

22. Palaniappan, R. U., Y. F. Chang, S. S. Jusuf, S. Artiushin, J. F. Timoney, S. P. McDonough, S. C. Barr, T. J. Divers, K. W. Simpson, P. L. McDonough, and H. O. Mohammed. 2002. Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect.Immun. 70:5924-5930.

23. Silva, J. J., M. O. Dalston, J. E. Carvalho, S. Setubal, J. M. Oliveira, and M. M. Pereira. 2002. Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Rev.Soc.Bras.Med.Trop. 35:395-399.

24. Verma, A., J. Hellwage, S. Artiushin, P. F. Zipfel, P. Kraiczy, J. F. Timoney, and B. Stevenson. 2006. LfhA, a novel factor H-binding protein of Leptospira interrogans. Infect.Immun. 74:2659-2666.

25. Victoria, B., A. Ahmed, R. L. Zuerner, N. Ahmed, D. M. Bulach, J. Quinteiro, and R. A. Hartskeerl. 2008. Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira. PLoS.One. 3:e2752.

26. Viriyakosol, S., M. A. Matthias, M. A. Swancutt, T. N. Kirkland, and J. M. Vinetz. 2006. Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden. Infect.Immun. 74:887-895.

27. Wagenaar, J. F., P. J. de Vries, and R. A. Hartskeerl. 2004. Leptospirosis with pulmonary hemorrhage, caused by a new strain of serovar Lai: Langkawi. J.Travel.Med. 11:379-381.

28. Wang, B., J. Sullivan, G. W. Sullivan, and G. L. Mandell. 1984. Interaction of leptospires with human polymorphonuclear neutrophils. Infect.Immun. 44:459-464.

29. Wang, B., J. A. Sullivan, G. W. Sullivan, and G. L. Mandell. 1984. Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages. Infect.Immun. 46:809-813.

30. Werts, C., R. I. Tapping, J. C. Mathison, T. H. Chuang, V. Kravchenko, G. Saint, I, D. A. Haake, P. J. Godowski, F. Hayashi, A. Ozinsky, D. M. Underhill, C. J. Kirschning, H. Wagner, A. Aderem, P. S. Tobias, and R. J. Ulevitch. 2001. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat.Immunol. 2:346-352.

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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

Rapidserologicalassaysforlep

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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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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.



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.


(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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ter11

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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

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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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ter11

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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

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lep


ifferentiatedfever

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R18

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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

Chap

ter11

178

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R14

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R17

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R19

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R23

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R28

R29

R30

R31

R32

R33

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


ifferentiatedfever

179

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

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

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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


ifferentiatedfever

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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.


(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

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Summary, discussion and directions

for future studies


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

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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



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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

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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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(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.


(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


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

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