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Studies on leptospirosis : clinical aspects and pathophysiology
Wagenaar, J.F.P.
Publication date
2010
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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 –
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
Studies on leptospirosis
- clinical aspects and pathophysiology –
ACADEMISCH PROEFSCHRIFTter 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
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?
Trop Med Int Health 2007, 12(1): 111-22.
Chapter 3: Leptospirosis with pulmonary hemorrhage, caused by a new 45 strain of serovar Lai: Langkawi.
J Travel Med 2004, 11(6): 379-81.
Chapter 4: Coagulation disorders in patients with severe leptospirosis 55 are associated with severe bleeding and mortality.
Trop Med Int Health 2010, 15(2): 152-59.
Chapter 5: Bleeding in patients with severe leptospirosis is not associated 73 with activation of endothelial cells.
Submitted.
Chapter 6: Low factor XII and factor XI levels in patients with severe 85 leptospirosis.
Submitted.
Part II: Inflammation
Chapter 7: Long pentraxin PTX3 is associated with mortality and disease 101 severity in severe leptospirosis.
J Infect 2009, 58(6): 425-32.
Chapter 8: Soluble ST2 levels are associated with bleeding in patients 119 with severe leptospirosis.
PLoS Negl Trop Dis 2009, 3(6): e453.
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.
Ann Trop Med & Parasitology 2004, 98(8): 843-50.
Chapter 11: Murine typhus and leptospirosis as a cause of acute 173 undifferentiated fever in Central Java, Indonesia.
Emerg Infect Dis 2009, 15(6): 975-7.
Chapter 12: Summary, discussion and directions for future studies 183
Samenvatting en discussie voor niet-ingewijden 195
Dankwoord 205
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
General Introduction and outline of the thesis 11
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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.
Clinical features
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.
Laboratory diagnosis
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.
Inflammation and coagulation
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.
General Introduction and outline of the thesis 15
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Table:Differential diagnosis of leptospirosis (not complete).
Rickettsioses Typhoid fever Malaria Dengue Yellow fever Chikungunya Hanta fever Meningococcal infection Influenza Viral hepatitis HIV
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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. Leptospira and 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.
General Introduction and outline of the thesis 17
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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
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I
PART
What role do coagulation disorders play in the pathogenesis of Leptospirosis? 21
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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 Netherlands 2 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 Internal Medicine, University ofMaastricht,
Maastricht, the Netherlands
5 Department of virology, Erasmus University, Rotterdam, the Netherlands
Trop Med Int Health 2007; 12(1): 111-122.
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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.
What role do coagulation disorders play in the pathogenesis of Leptospirosis? 23
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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
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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-dimer
Thrombocytopenia ↑ ↓ N N N N
Thrombocytopathy ↑ N N N N N
DIC ↑ ↓ ↑ ↑ ↓ ↑
Factor VII N N ↑ N N N
Factor XI, IX, VIII N N N ↑ N N
Mild hepatic disease N N ↑ ↑ N N
Severe hepatic disease ↑ ↓ ↑ ↑ N N
Vasculopathies ↑ N N ↑ N N
Abbreviations: BT, bleeding time; PT, prothrombin time; APTT, activated partial thromboplastin time; Fib, fibrinogen; DIC, disseminated intravascular coagulation.
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
What role do coagulation disorders play in the pathogenesis of Leptospirosis? 27
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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 Leptospira interrogans 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).
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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 value
Cases Controls
Platelets
Daher 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) 135
Nicodemo et al. 1990 (150- 450 x 10³/mm³) 70
Edwards 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)‡
Prothrombin time
Daher 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)
Activated partial thromboplastin time
Daher de Francesco et al. 2002 (32- 38,4 seconds) 32.7 (SD 2.1)
Jaroonvesama et al. 1975 (55- 69 seconds) 73
Edwards et al. 1990 (no normal values denoted) 28.6 (SD 12.3)
Sitprija et al. 1980 (25-55 seconds) 33.4 (SEM 0.5)
Thrombin time (s)
Daher de Francesco et al. 2002 (9,8- 11 seconds) 11 (SD 1.4)
Jaroonvesama et al. 1975 (5- 6 seconds) 6.9
Fibrinogen
Daher de Francesco et al. 2002 (150- 380 mg/dl) 515 (SD 220)
Jaroonvesama et al. 1975 (306 mg/100ml) 529
Sitprija et al. 1980 (200- 400 mg/dl) 818 (SEM 57.6)
FDP (fibrinogen degradation products)
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) 12.4
Sitprija et al. 1980 (< 0,5 mg/ml) 3.7 (SEM 0.39)
Factor V
Jaroonvesama et al. 1975 (130%) 77
Sitprija et al. 1980 (70- 120%) 90.8 (SEM 0.9)
Factor VIII
Jaroonvesama et al. 1975 (120%) 113
Sitprija et al. 1980 (70- 120%) 88.6 (SEM 1.3)
Factor X
Jaroonvesama et al. 1975 (130%) 113
† All thrombocytopenic patients
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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
What role do coagulation disorders play in the pathogenesis of Leptospirosis? 31
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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.
Primary hemostasis
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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Secondary hemostasisWhen 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 non-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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Anticoagulation pathwaysUpon 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
