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Keywords:

  • leptospirosis;
  • Leptospira;
  • coagulation;
  • fibrinolysis;
  • sepsis;
  • disseminated intravascular coagulation
  • leptospirose;
  • Leptospira;
  • coagulation;
  • fibrinolyse;
  • septicémie;
  • coagulation intravasculaire disséminée
  • Leptospirosis;
  • Leptospira;
  • coagulación;
  • fibrinólisis;
  • sepsis;
  • coagulación intravascular diseminada

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

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

Methods  In a prospective study, parameters of the coagulation system were measured on admission and during follow-up in 52 consecutive patients with severe leptospirosis.

Results  All patients showed coagulation disorders, such as prolonged prothrombin time (PT) and activated partial thromboplastin time, marked procoagulant activity [thrombin–antithrombin (TAT) complexes, prothrombin fragment 1+2, D-dimer], reduced levels of anticoagulant markers (protein C, antithrombin) and increased (anti-) fibrinolytic activity [plasmin–antiplasmin (PAP) complexes, plasminogen activator inhibitor-1]. These disorders were more pronounced in patients who died eventually. PT prolongation was associated with mortality (OR 1.4, 95% CI: 1.0–1.8, P = 0.04). Bleeding occurred in 31 subjects (60%). Of these, 24 had mild bleeding and seven had severe haemorrhages. Thrombocytopenia (platelets ≤100 × 109/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 imbalance 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 = 0.05) and the TAT/PAP ratio (OR 1.3, 95% CI: 1.0–1.6, P = 0.05), which is an indicator of the balance between coagulation and fibrinolysis. Overt disseminated intravascular coagulation (DIC) was found in 10 (22%) 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 patients with leptospirosis. This was more pronounced in the deceased and in patients with severe bleeding than in than the survivors and in those without severe bleeding.

Les troubles de la coagulation chez des patients atteints de leptospirose sévère sont associés à de graves hémorragies et à la mortalité

Objectif:  Déterminer l’implication du système de la coagulation dans l’hémorragie et un mauvais pronostic chez les patients atteints de leptospirose sévère.

Méthodes:  Dans une étude prospective, les paramètres du système de coagulation ont été mesurés à l’admission et durant le suivi, chez 52 patients consécutifs atteints de leptospirose sévère.

Résultats:  Tous les patients présentaient des troubles de la coagulation tels que: une prolongation du temps de prothrombine (TP) et du temps de la céphaline partielle activée, une activité pro-coagulante marquée (complexes thrombine-antithrombine (TAT), fragments 1 + 2 de la prothrombine, dimères D), des taux réduits des marqueurs anticoagulants (protéine C, antithrombine) et une augmentation de l’activité des complexes (anti-) fibrinolytique (plasmine-antiplasmine (PAP), activateur/inhibiteur-1 du plasminogène).

Ces troubles étaient plus prononcés chez les patients décédés par la suite. La prolongation du TP était associée à la mortalité (OR: 1,4, IC95%: 1,0-1,8; p  =  .04). Des saignements sont survenus chez 31 sujets (60%). Parmi eux, 24 avaient des saignements légers et 7 avaient des hémorragies graves. La thrombocytopénie (numération plaquettaire ≤ 100x109/L) était significativement associée à un saignement clinique (OR: 4,6; IC95%: 1,3-16). Une sous-analyse des patients avec ou sans saignement grave a révélé un déséquilibre plus marqué du système de coagulation chez les patients présentant une hémorragie sévère, reflété par une association significative avec le TP (OR: 1,4; IC95%: 1,0-1,8; p = 0.05) et le rapport TAT/PAP (OR: 1,3; IC95%: 1,0-1,6; p  =  .05), qui est un indicateur de l’équilibre entre la coagulation et la fibrinolyse. De façon manifeste, une coagulation intravasculaire disséminée (CIVD) a été trouvée chez 10 (22%) des 46 patients pour lesquels le score pouvait être calculé. Il n’y avait aucune association significative entre les scores CIVD, la diathèse hémorragique ou un mauvais pronostic.

Conclusion:  Le système de la coagulation était fortement activé chez les patients atteints de leptospirose. Cela était plus prononcé chez les personnes décédées et chez les patients présentant une hémorragie sévère que chez les survivants et les personnes sans saignement grave.

Los desórdenes de coagulación en pacientes con leptospirosis severa están asociados con sangrado severo y mortalidad

Objetivo:  Determinar el papel de la coagulación en el sangrado y un mal resultado en pacientes con leptospirosis severa.

Métodos:  En un estudio prospectivo, se midieron los parámetros de coagulación en 52 pacientes consecutivos con leptospirosis severa en el momento de la admisión y durante el seguimiento,.

Resultados:  Todos los pacientes tenían desórdenes de coagulación, tales como un tiempo de protrombina prolongado (TP), una actividad procoagulante marcada (complejos trombina-antitrombina, fragmento 1 + 2 protrombina, dímero-D), niveles reducidos de marcadores de anticoagulantes (proteína C, antitrombina) y actividad (anti-) fibrinolítica aumentada (complejos plasmina-antiplasmina (PAP), inhibidor del activador del plasminógeno-1). Estos desórdenes eran más pronunciados en los pacientes que finalmente murieron. El PT prolongado estaba asociado con mortalidad (OR 1.4, 95%IC: 1.0-1.8, p  =  .04). El sangrado ocurrió en 31 sujetos (60%). De estos, 24 tenían un sangrado leve y 7 tenían hemorragias severas. La trombocitopenia (plaquetas ≤100x109/L) estaba asociada de forma significativa con el sangrado clínico (OR 4.6, 95%IC: 1.3-16). El subanálisis de pacientes con y sin sangrado severo reveló un desbalance más pronunciado del sistema de coagulación en pacientes con sangrado severo, tal y como se refleja en la asociación significativa con TP (OR 1.4, 95%IC: 1.0-1.8, p  =  .05) y la proporción TAT/ PAP (OR 1.3, 95%IC: 1.0-1.6, p  =  .05), la cual es un indicador del balance entre coagulación y fibrinólisis. Se encontró una coagulación intravascular diseminada (CID) declarada en 10 (22%) de los 46 pacientes de los que se había podido calcular la puntuación. No había una asociación significativa entre la puntuación CID, diátesis hemorrágica o un mal resultado.

Conclusión:  El sistema de coagulación estaba muy activado en pacientes de leptospirosis, siendo más pronunciado entre aquellos que murieron y en los que tenían sangrado severo comparado con los que sobrevivieron o aquellos sin sangrado severo.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

Leptospirosis, caused by pathogenic Leptospira spirochaetes, is an acute febrile illness wherein haemostatic abnormalities play a pivotal role (Wagenaar et al. 2007). Disease transmission takes place worldwide, but large outbreaks are usually reported from the (sub-) tropics after floods, hurricanes and other disasters (Levett 2001). The incidence of leptospirosis is estimated to exceed 100 per 100 000 persons per year in areas at risk. Maintenance hosts, such as rats and other mammals, spread the spirochaete 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 and/or a bleeding diathesis. Pathological findings reveal widespread haemorrhaging at mucosal surfaces, muscles, peritoneum and various organs such as heart, lungs and kidneys (Arean 1962). Thrombocytopenia is frequently observed and associated with poor outcome (Edwards et al. 1982; Turgut et al. 2002). 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 (Sitprija et al. 1980; Edwards et al. 1986), and recently a Thai cohort study showed activation of the coagulation system (Chierakul et al. 2008). 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.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

Inclusion and sample collection

A prospective study was carried out at the Department of Internal Medicine of 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 suspected leptospirosis, presenting usually with at least one of the following symptoms or signs: jaundice, renal failure, thrombocytopenia (platelets < 100 × 109/l) and/or haemorrhaging. Sepsis was defined using internationally accepted criteria (American College of Chest Physicians/Society of Critical Care Medicine 1992). Inclusion followed written informed consent by the subject or his/her caretaker when too ill to consent. The medical ethics committees of both the Dr. Kariadi hospital and the Slotervaart hospital 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 (Toh & Hoots 2007). A platelet count <100 × 109/l was assigned 1 point, <50 × 109/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 (PT) prolongation between 3 and 6 s and >6 s received 2 points. Plasma fibrinogen <1.0 g/l received 1 point. A score ≥5 was compatible with an overt DIC.

Diagnostic procedures

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, Pyrogenes, Sarmin, Hardjo, Saxkoebing, Sejroe, Shermani, Tarassovi and three 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 was considered to be positive. After inclusion, blood was cultured at the bedside onto EMJH, EMJH+5 fluorouracil 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 the Department of Microbiology, and Center of Biomedical Research (CEBIOR), 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, City, the Netherlands.

Coagulation and fibrinolysis assays

Measurements of F1+2 (Dade Behring, Marburg, Germany), TAT complexes (Dade Behring), plasmin–antiplasmin (PAP) complexes (DRG, Marburg, Germany), D-Dimers (Diagnostica Stago, Asnières-sur-Seine, France), plasminogen activator inhibitor type-1 (PAI-1) antigen (Hyphen BioMed, Andrésy, France), were performed by ELISA. Protein C (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 PT determination. AT activity was determined with Berichrom Antithrombin (Dade Behring).

Statistical analyses

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 (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 < 100 × 109/l), TAT, F1+2, D-dimer, AT, PC, PAP, PAI and TAT/PAP × 100. All test were two-tailed with the α set to 0.05. Analyses were performed using spss (version 15.0, Chicago, Illinois).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

Patients

In this study, 52 consecutive patients with severe leptospirosis were enrolled. Of them, 71% were men; the median age of the cohort was 45 years (IQR 32–55) and 27% died during follow-up, after a median of 3 days (IQR 1–5) post-admission.

Sepsis criteria were fulfilled by 54% and severe sepsis criteria by 37% on admission. The remaining five 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 the following: Sejroe, Pyrogenes, Autumnalis, Ballum and Javanica. Sixteen samples were not classifiable by MAT because of multiple reactive serovars. Patient characteristics are summarized in Table 1.

Table 1.   Patient characteristics on admission
 nAll patientsSurvivorsNon-survivorsNormalP value
  1. Numbers represent median (IQR) values of 52 patients with leptospirosis on admission. P values represent difference between survivors and non-survivors (Mann–Whitney U-test, chi-square test or Fisher’s exact test when appropriate). A P value of <0.05 was considered statistically significant.

  2. IQR, interquartile range; DPO, day post onset symptoms; BPM, beats per minute.

General
 Number of cases 523814  
 Male sex (%)5237 (71)29 (58)8 (57) 0.2
 Age, median (IQR)5245 (33–55)42 (31–55)47 (40–55) 0.3
 DPO, median (IQR)526 (5–8)7 (6–8)6 (5–8) 0.9
 Days at hospital, median (IQR)5210 (6–14)11 (10–14)3 (2–6)  
 Sepsis (%)5228 (54)23 (61)5 (36) 0.1
 Severe sepsis (%)5219 (37)10 (26)9 (64) 0.01
Physical examination
 Temperature (ºC), median (IQR)5238.0 (37.7–38.5)38.0 (37.6–38.4)38.2 (37.6–38.6) 0.5
 Pulse (BPM), median (IQR)52100 (92–108)100 (92–104)100 (92–108) 0.9
 Systolic blood pressure (mmHg), median (IQR)52110 (110–128)110 (109–120)120 (108–130) 0.3
 Diastolic blood pressure (mmHg), median (IQR)5270 (60–80)70 (65–80)70 (60–80) 0.4
 Respiratory rate (breaths/min), median (IQR)5224 (20–28)24 (20–24)26 (20–36) 0.04
 Jaundice (%)5242 (81)32 (84)10 (71) 0.4
 Oliguria (%)5212 (23)7 (18)5 (36) 0.3
 Anuria (%)522 (4)02 (14) 0.07
 Haemorrhagic manifestation overall (%)5231 (60)21(55)10 (71) 0.3
  Mild bleeding (%) 24 (46)18 (47)6 (43) 0.8
  Severe bleeding (%) 7 (14)3 (8)4 (29) 0.08
Laboratory results
 Hb (gr/dl), median (IQR)5211.3 (10.2–12.3)11.4 (10.6–12.4)10.9 (9.1–11.6)12.0–15.00.1
 Ht (%), median (IQR)5133.5 (30.6–36.5)34.1 (31.3–37.0)31.4 (27.0–34.1)40.0–54.00.1
 Leucocytes (109/l), median (IQR)5115 (11–18)16 (13–18)11 (10–20)4–110.2
 Platelets (109/l), median (IQR)5166 (35–135)65 (35–161)76 (34–96)150–4000.8
 Thrombocytopenia ≤100 × 109/l (%)5235 (67)24 (63)11 (79) 0.3
 AST (U/l), median (IQR)3866 (40–112)63 (39–101)68 (54–299)15–370.3
 ALT (U/l), median (IQR)3753 (43–76)58 (43–78)52 (38–94)30–650.9
 Bilirubin total (mg/dl), median (IQR)358.3 (3.4–20.0)8.3 (3.4–19.5)11.1 (7.3–25.5)0–1.00.3
 Creatinine (mg/dl), median (IQR)495.4 (3.1–7.6)4.7 (2.3–7.5)7.2 (5.7–9.7)0.6–1.30.1

Haemorrhagic manifestations and global coagulation tests

Thirty-one subjects (60%) showed clinical signs of bleeding. Of them, 24 had mild bleeding (petechiae n = 22, ecchymoses n = 3, epistaxis n = 2) and seven had severe bleeding (melaena n = 7, haematemesis n = 2, haematuria n = 1). Bleeding occurred more frequently in the non-survivors, but this difference was not significant (P = 0.3). Thrombocytopenia (platelets < 100 × 109/l) was observed in 35 subjects (67%), whereas 19 subjects (37%) had a platelet count <50 × 109/l. Median platelet counts were significantly lower in patients with bleeding (P = 0.02). Furthermore, thrombocytopenia (platelets ≤100 × 109/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 (51 s), whereas PT was high normal (12.9 s). 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 (14.9 s vs. 12.9 s) 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 = 0.05). From the patients with severe bleeding, two had a normal PT (two missing values) and one had a normal APTT (one missing value). Deceased displayed a significant PT prolongation compared to the survivors (P = 0.04). In addition, prolongation of either PT or APTT was associated with mortality (OR 1.4, 95% CI: 1.0–1.8, P = 0.04; OR 1.1, 95% CI: 1.0–1.2, P = 0.04). Fibrinogen was elevated during admission in all subjects (median 9 g/l). Massive consumption of fibrinogen (plasma levels < 1 g/l) was not found in any of the subjects. Global coagulation tests and markers of coagulation activation and fibrinolysis are presented in Table 2.

Table 2.   Markers of coagulation and fibrinolysis on admission in patients with severe leptospirosis
TestnAll patientsnSurvivorsnNon-survivorsNormal rangeP value
  1. Haemostatic parameters of 52 patients with leptospirosis measured on admission. P values represent difference between survivors and non-survivors (Mann–Whitney U-test). A P value of <0.05 was considered statistically significant.

  2. IQR, interquartile range; TAT, thrombin–antithrombin complexes; F1+2, prothrombin fragment F1+2; D-dimer, cross-linked fibrin degradation products; PC, protein C; AT, antithrombin; PAP, plasmin–antiplasmin complexes; PAI-1, plasminogen activator inhibitor type-1.

Global coagulation tests
 Prothrombin time (s), median (IQR)4612.9 (12.0–14.5)2812.8 (11.8–13.8)1014.7 (12.3–17.1)10.7–12.90.04
 APTT (s), median (IQR)4851 (44–57)3050 (43–55)1157 (48–74)25–380.06
 Fibrinogen (g/l), median (IQR)529 (6–13)389 (7–13)149 (5–14)1.9–4.00.7
Procoagulant activity
 TAT (μg/l), median (IQR)526.9 (4.3–13.6)386.6 4.(1–9.4)149.8 (6.9–19.4)<4.60.03
 F1+2 (pmol/l), median (IQR)51301 (146–533)37256 (146–473)14444 (164–2584)53–2710.4
 D-dimer (μg/l), median (IQR)524088 (2831–6029)384018 (2427–5742)145094 (3122–8286)<4000.2
Anticoagulant activity
 AT (%), median (IQR)5265 (54–78)3867 (57–81)1456 (44–71)86–1390.02
 PC (%), median (IQR)5276 (56–99)3879 (64–99)1462 (53–74)70–1200.01
(anti-) Fibrinolytic activity
 PAP (μg/l), median (IQR)51862 (711–1115)37870 (722–1130)14800 (453–1115)221–5120.4
 PAI (ng/ml), median (IQR)52122 (77–296)38114 (75–263)14206 (100–532)10–700.1
 TAT/PAP ratio (×100)510.8 (0.5–2.0)370.7 (0.4–1.0)141.0 (0.7–4.0)0.02

Procoagulant, anticoagulant and fibrinolytic activity

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 falling trend over time in the survivors. On admission, procoagulant activity was not significantly different between subjects with and without bleeding (TAT: 6.8 vs. 7.3 μg/l; F1+2: 318pmol/l vs. 244pmol/l; D-dimer: 4674 vs. 4078 μg/l). Subjects with severe bleeding had more marked elevations of TAT and F1+2 levels than subjects without severe bleeding (TAT: 16.4 μg/l vs. 6.8 μg/l; F1+2: 900 vs. 247 pmol/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 = 0.02). In a sub analysis between survivors and non-survivors on admission, TAT complexes were significantly higher in the deceased group (P = 0.03).

image

Figure 1.  (a–e) Scatter dot plot diagrams of haemostatic 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. TAT, thrombin–antithrombin complexes; F1+2, prothrombin fragment F1+2; D-dimer, cross-linked fibrin degradation products; PC, protein C; AT, antithrombin; PAP, plasmin–antiplasmin complexes; PAI-1, plasminogen activator inhibitor type-1.

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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 = 0.01) and AT (P = 0.02) 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 haemorrhages (PAP: 880 vs. 852 μg/l; PAI-1: 114 vs. 135 ng/ml) or survivors and patients who died. However, patients with severe bleeding had significant lower PAP levels then patients without this symptom (695 vs. 884 μg/l; P = 0.04).

To determine the balance between coagulation and fibrinolysis, the TAT/PAP ratio was calculated. Patients who had severe bleeding displayed a significantly higher TAT/PAP (×100) ratio (4.0 vs. 1.0; P = 0.03) compared to subjects without severe bleeding. A significant association between TAT/PAP ratio and severe bleeding was found (OR 1.3, 95% CI: 1.0–1.6, P = 0.05). Moreover, admission TAT/PAP ratios were higher in the deceased group compared to the survivors (P = 0.02).

Disseminated intravascular coagulation scores

The ISTH overt DIC algorithm was scored on admission to find evidence for consumption coagulopathy and defibrination. Of six 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 four 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.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

Leptospirosis contributes importantly to clinical disease in Southeast Asia and is considered endemic to Indonesia (Laras et al. 2002). During the rainy season, leptospirosis is among the most important causes of hospitalization and death in Semarang. Clinical bleeding is frequently observed in severe cases. The pathophysiological mechanisms are largely unknown but a dysbalanced secondary haemostasis 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 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) (van Gorp et al. 2002). 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 abnormal PT or APTT. This, together with equal haemostatic derangements in patients with and without bleeding makes, besides impaired secondary haemostatic pathways, another pathophysiological mechanism likely. One hypothesis for the pathogenesis of the observed haemostatic 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 haemostasis, seen in virtually all septic patients (Kinasewitz et al. 2004), 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 anticoagulation. Impaired synthesis might play a role in these observations. Higher TAT/PAP ratios in the deceased showed a haemostatic 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 haemostatic abnormalities (Kinasewitz et al. 2004). These disorders can range from isolated thrombocytopenia or prolonged global clotting tests to complex disorders such as DIC (Levi & van der Poll 2004; Levi & Opal 2006). 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 (de Jonge et al. 2001). Host-derived mediators of inflammation, such as TNF-α, are able to induce TF expression on mononuclear cells and macrophages (Schouten et al. 2007).

There is some evidence that mononuclear cells stimulated by pathogenic Leptospira are able to express tissue factor in vitro (Miragliotta & Fumarola 1983). A recent study provided evidence for coagulation activation in patients with leptospirosis reflected by prolonged clotting times and elevated F1+2, TAT and D-dimer levels (Chierakul et al. 2008). 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 (Higgins & Cousineau 1977;Pereira da Silva et al. 1995). Pathology showed haemorrhaging, deposits of fibrin and scattered foci of necrosis in various organs (Pereira da Silva et al. 1995). In contrast, no fibrin thrombi were found in liver lung or kidney in two leptospirosis pulmonary haemorrhage guinea pig models (Nally et al. 2004; Yang et al. 2006). Human leptospirosis studies in the past could not confirm DIC (Sitprija et al. 1980; Edwards et al. 1986; Nicodemo et al. 1990; Daher de Francesco et al. 2002; Turgut et al. 2002). 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 patients with leptospirosis, 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 patients with leptospirosis.

Some issues of the present study merit further comment. 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 haemostatic mechanisms in patients with mild leptospirosis. Most patients presented with late stage disease, which can explain the fulminant clinical picture and high mortality in this cohort. Finally, the number of patients included in the cohort was relatively small. Therefore, caution should be used when interpreting this data.

In conclusion, patients with severe leptospirosis showed disorders in both coagulation and fibrinolytic pathways, which were more pronounced in the deceased and in those with severe bleeding. The absence of marked disorders 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 haemostatic 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

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

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).

Disclosure of conflict of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflict of interest
  9. References
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