Interleukin-6: an early marker of bacterial infection in decompensated cirrhosis

Journal o f Hepatology 1994; 20:819-824 Printed in Denmark. All rights reserved Munksgaard. Copenhagen

Copyright © Journtdof Hepatology 1994

Journal of Hepatology ISSN 0168-8278

Interleukin-6: an early marker of bacterial infection in decompensated cirrhosis Olivier Le M o i n e 1"2, Jacques Devi6re 2, J e a n n e - M a r i e Devaster 2, Alain Crusiaux 2, Francois D u r a n d ~, Jacques B e r n u a u j, Michel G o l d m a n 2 and Jean-Pierre B e n h a m o u I ~Liver Unit, H6pital Beaujon, Clichy. France and "Departments of Gastroenterology & hnmunology. ULB H6pital Erasme. Brussels. Belgium

(Received 31 March 1993)

Fifty-seven patients with decompensated cirrhosis were studied prospectively to assess the sensitivity and specificity of early clinical or biological signs of bacterial infection. Among them, 19 had proven infection on admission (7 spontaneous bacterial peritonitis, 5 bacteraemia, 3 urinary tract infections, 2 pneumonia, 1 dental abscess and 1 cholangitis). Fever, polymorphonuclear cell count, fibrinogen and C-reactive protein levels were found to be of little or no help in diagnosing bacterial infection on admission. Interleukin-6 plasma levels were, however, significantly different between infected (median: 1386 pg/ml, range: 237-20000) and non-infected patients (median: 34 pg/ml, range: 0-4500, p<0.00001). Levels above 200 pg/ml were always found in infected patients, giving a sensitivity of 100% and a specificity of 74%. C-reactive protein correlated weakly with interleukin-6 levels, indicating a defective acute-phase response in cirrhosis. Tumor necrosis factor alpha plasma levels were less sensitive (95%) and specific (68%) for the diagnosis of bacterial infection at a threshold of 50 pg/ml, but were more closely related to a poor patient outcome. In decompensated cirrhosis, interleukin-6 plasma levels on admission provided the most sensitive and specific tool for the diagnosis of bacterial infection. © Journal of Hepatology. Key words: Cytokines; Liver disease; Prognosis

Ascites, jaundice and encephalopathy are the main clinical features of decompensation in cirrhosis. They most frequently result from variceal haemorrhage, bacterial infection, alcoholic hepatitis or the development of hepatocellular carcinoma. Bacterial infections are life-threatening complications of cirrhosis occurring in 25-50% of these patients. On admission, diagnosis of infection is difficult because the usual clinical and biological features such as fever or leucocytosis are often absent in patients with cirrhosis (1). Bacterial cultures require 24 to 48 h to give definitive results and treatment is often delayed. The need for early markers of infection led to several studies that focused on the diagnosis of spontaneous bacterial peritonitis. However, a sensitive and specific marker of infection in cirrhosis is still lacking.

Interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFa) are both produced during bacterial infections and are involved in the initiation of acute-phase response in humans (2). Previous observations in our laboratory showed that IL-6 levels could help in the early diagnosis of infection in cirrhosis (3). The present study aimed to analyse the respective contribution of IL-6, T N F a and acute-phase proteins (C-reactive protein and fibrinogen) in the diagnosis of bacterial infection in decompensated cirrhosis in a large population.

Material and Methods Patients

Fifty-seven patients admitted to the Liver Intensive Care Unit between December 1991 and May 1992 were

Correspondence to." Dr O. Le Moine, Gastroenterology, H6pital Erasme, 808 route de Lennik, 1070 Brussels, Belgium

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O. LE MOINE et al.

included in the study. Inclusion criteria were: histologically proven cirrhosis with a major complication (ascites, jaundice, encephalopathy or variceal haemorrhage), absence of neoplastic disease, and no recent antibacterial therapy. On admission, and before any endoscopic procedure or antibacterial treatment, blood samples for cytokines and C-reactive-protein assays were drawn as well as a complete bacteriological screening for each patient (blood, ascites, urine and if necessary bronchial aspirates for analysis and culture). Patients were divided into two groups based on bacteriological results: patients with or without bacterial infection on admission (Groups 1 and 2, respectively). The clinical and biochemical features of these patients are given in Table 1. A m o n g the 35 patients with alcoholic cirrhosis, 13 had histological signs o f alcoholic hepatitis. A m o n g the other patients, cirrhosis was related to hepatitis C virus (HCV) infection in eight, hepatitis B virus (HBV) infection in seven (with delta coinfection in two), primary biliary cirrhosis in two, sclerosing cholangitis in one and was unknown in four patients. Patients were followed up for 12 weeks after admission.

Diagnosis of #Tfection The following criteria were used to assess the presence of a bacterial infection: spontaneous bacterial peritonitis - a polymorphonuclear cell count of more than 250/mm 3 in the ascitic fluid and a positive culture, bacteraemia at least two blood cultures positive for the same bacteria, urinary tract infection - a polymorphonuclear cell count of more than 10 mm 3 and a positive culture, pneumonia

- X-ray evidence of lobar condensation and positive bronchial aspirate culture. Bacteriological patterns are given in Table 2. Most gram-negative infections were due to E. coli and gram-positive infections to Enterococci or Staphylococci. N o fungal infections were diagnosed on admission and viral infections were not screened.

Blood sampling, cytokines and C-reactive protein assays Five ml of blood were collected on admission in citrated tubes (Terumo Europe, Louvain, Belgium) from a peripheral vein. Plasma was separated immediately by centrifugation (900 g for 15 min) and stored at - 2 5 ° C until assayed (within 2 months of sampling). T N F a and IL-6 were measured using commercially available assays (Medgenix, Fleurus, Belgium). Precision of the assays was 6.0% and 5.6% and reproducibility 7.0% and 7.5% for T N F a and IL-6, respectively. C-reactive protein (reference value: < 10 mg/l) was measured by an immunoturbidimetric method (Tina-quant C-reactive protein, Boehringer-Mannheim, Germany).

Statistical analysis Data are expressed as median with their range. Comparisons between groups were made using the MannWhitney test or the A N O V A test following results of Bartlett's test for homogeneity of variance (4). Correlations were calculated witla the Spearman rank test and significance between survival curves was assessed by the log-rank test. All calculations were performed with a microcomputer. Statistical significance was achieved if p < 0 . 0 5 with two-tail tests.

Results

TABLE 1

Diagnosis of bacterial infection (Table 3)

Population features on admission All n 57 Age (years) 51 (20-74) Aetiology of cirrhosis (n) Alcoholic 35 Viral 12 Other 10 Reason for admission Encephalopathy 20 Haemorrhage 19 Jaundice & ascites 14 Hepatorenal syndrome 4 Child Class (B/C) 23/34 Haemoglobin (gr/dl) Prothrombin time (%) Bilirubin (,umol/l) AST (UI/I) Creatinine (/.tmol/1)

Group 1

Group 2

19 51 (32-69)

38 50 (20-74)

12 4 3

23 8 7

11 9 2* 17 4 10 2 2 7/12 16/22 9.1 (6.1-12) 8.7 (4.2-12.3) 30 (10-63) 36 (13-67) 149 (19-615) 70 (7-657) 66 (21-215) 58 (28-2540) 138 (66-561)* 75 (45-753)

* Significant difference between infected (Group 1) or not infected patients (Group 2) (p<0.05).

The usual markers of infection, namely fever, white blood cell count and fibrinogen levels, were not significantly different in the two groups. Despite an obvious difference in polymorphonuclear cell count and C-reactive protein levels, there was a marked overlap between the

TABLE 2 Results of bacteriological screening in the 19 infected patients n Spontaneous bacterial perito- 7 nitis Bacteraemia 5 Urinary tract infection 3 Pneumonia 2 Others 2 Total 19

Gram negative Gram positive bacteria bacteria 5 3 2 1 1 12

2

IL-6 IN DECOMPENSATED CIRRHOSIS

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TABLE 3

Body temperature (°C) 37.2 (36-39) 37.2 (35.5-39) White blood cells (/mm3) 7980 (1170-19410) 7040 (2570-17640) PMN (%) 77 (40-92)* 70 (41-92) Fibrinogen (mg/dl) 2.6 (0.6-4.9) 2.6 (0.6-4.5) CRP (mg/l) 26 (1-80)* 11 (1-83) IL-6 (pg/ml) 1386 (237-20000)** 34 (0-4500) TNFo~ (pg/ml) 111 (0-1657)** 0 (0-126)

protein nor T N F a favoured the diagnosis of infection. The sensitivity and specificity of IL-6 were 100% and 76% or 95% and 81% for thresholds of plasma levels at 200 pg/ml or 300 pg/ml, respectively. N o difference was found in IL-6 levels for aetiology of cirrhosis, whether the patients were infected or not, or between infected patients concerning the site of infection. T N F a levels were less accurate in diagnosing infection, with sensitivity and specificity of 95% and 68% at a threshold of 50 pg/ml, respectively.

PMN=polymorphonuclear cell count. TNFtx=tumour necrosis factor alpha. IL-6=interleukin-6. CPR=C-reactive protein.

Adequacy of empirical antibacterial therapy

Clinical and blood markers of infection on admission. Results are expressed as median (range); * p<0.05; ** p< 10-5 Group 1 (n= 19)

Group 2 (n= 38)

two groups. There was a highly significant difference in IL-6 and T N F a levels between Groups 1 and 2 and IL-6 was always above 200 pg/ml in case of infection. Receiver operating curves (Fig. 1) give the sensitivity and specificity of each marker. IL-6 is clearly the most powerful assay for early diagnosis of infection, although some overlap may be observed between 200 and 1000 pg/ml (Fig. 2). A m o n g the patients with IL-6 levels above 200 pg/ml and without obvious bacterial infection, neither C-reactive

Antibacterial therapy was given on admission if ascitic fluid analysis met the criteria of spontaneous bacterial peritonitis or on clinical grounds based on the severity of the disease and the clinician's decision. Antibacterial therapy led to adequate treatment, on admission, of 17/19 (89%) of the infected patients but also led to unnecessary treatment in 27/38 (71%) of the patients without infection (Fig. 2).

Acute phase proteins and cytokines In this series, there was only a weak correlation between IL-6 or T N F a and C-reactive protein levels in the whole population: r=0.24, 95% CI - 0 . 0 2 < r < 0 . 4 7 for

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1 - Specificity Fig. 1. Receiver operating curves of po]ymorphonuclear cell count, C-reactive protein, tumour necrosis factor alpha and interleukin-6 levels for the diagnosis of bacterial infection on admission. Each point represents the sensitivity and the specificity of the assay for a given threshold. The area under the curve represents the power of the test.

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Fig. 2. Individual admission values of interleukin-6 (semi-logarithmic scale) in infected (group 1) and non-infected patients (group 2). Horizontal lines show the medians. Triangles represent patients who received empirical antibacterial therapy at the time of admission and squares those who did not. TNFa=tumour necrosis factor alpha. CRP=C-reactive protein. PMN=polymorphonuclear cell count.

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O. LE MOINE et al.

IL-6 and r=0.36, 95% CI 0.I 1
Twenty-two patients died during the follow-up period (22/57=39%). Mortality was 59% (11/19) among the infected patients and 29% (11/38) in the uninfected group. No significant difference was found in mean IL-6 levels (-+SD) between patients who died or did not die during this period (2677___5755 vs 1907___5246 pg/ml, p=0.6). In contrast, mean T N F a levels (_SD) were significantly higher in patients who died (146___340 vs 60_+ 130 pg/ml, p<0.03). A receiver operating curve with different TNFot thresholds showed 50 pg/ml as the most sensitive (18/22= 82%) and specific (23/35=66%) cut-off level. Admission levels above 50 pg/ml were associated with poor survival at 2 months (43% (13/30)) compared to patients with levels below 50 pg/ml (81% (22/27), p<0.01, Fig. 3). This difference was not related to the presence of associated alcoholic hepatitis (6/22 vs 7/35, p>0.9). Discussion

This study emphasises the poor sensitivity of fever, polymorphonuclear cell count and C-reactive protein for the diagnosis of bacterial infection in decompensated liver 100

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disease. It clearly shows that plasma IL-6 levels on admission have the best sensitivity and specificity for the diagnosis of this complication. Several features are probably involved in the marked elevation of IL-6 in chronic liver disease during bacterial infection. Both IL-6 and T N F c t are mainly metabolised at the level of hepatocytes after binding to their receptors (5,6). The reduction in the number of functional hepatocytes is probably responsible for decreased cytokine catabolism in cirrhosis (7-9). On the other hand, monomacrophagic cells from these patients display a preactivation status leading to increased in vitro and in vivo secretion after stimulation with endotoxins (10-12). The marked increase in IL-6 during infection is probably due to both mechanisms. IL-6 is one of the main afferent pathways for hepatocyte acute phase protein synthesis (2). During bacterial infection and other inflammatory conditions, IL-6 and C-reactive protein levels are closely correlated in patients without liver disease (13-15). The present study did not support these observations in the case of cirrhosis. Indeed, it seems that despite high IL-6 levels the diseased liver is unable to produce sufficient amounts of acute phase proteins like Creactive protein and fibrinogen. This defective acute phase protein production in cirrhosis has already been observed after abdominal surgery (15) and is probably linked to the loss of functional hepatocytes. Another hypothesis might be a down-regulation of IL-6 receptors on hepatocytes. Indeed, even without infection, patients with cirrhosis have significantly higher circulating IL-6 levels compared to normals (16,17). Continuous exposure of hepatoma cells to IL6 in vitro leads to their desensitisation and a reduction of acute phase protein production by a down-regulation of IL-6 receptors (18,19). Hepatocytes of patients with cirrhosis chronically exposed to significant levels of IL-6 might then be desensitised and thus respond inadequately to a further increase in IL-6 levels during infection. Finally, several studies in patients without liver disease have shown that a delay of at least 24 h after an IL-6 peak is necessary for a maximal acute phase protein response, therefore making IL-6 an earlier marker of infection than C-reactive protein (20-23). Blood sampling was performed on admission, at the time when a therapeutic decision needed to be taken. From the individual IL-6 levels at this time, the sensitivity of this assay for the diagnosis of bacterial infection reached 100% at a threshold of 200 pg/ml. As immunoenzymatic semi-automated methods for IL-6 measurement become available, admission screening of infected patients will be faster and easier and early appropriate antibacterial therapy can be provided. At the level of 200 pg/ml the specificity of the assay was 74%, and 26% of the patients with levels above the threshold were not infected using our criteria. None of

IL-6 IN DECOMPENSATED CIRRHOSIS these patients had histological evidence o f alcoholic hepatitis, which might result in increased cytokine levels (24-26); thus an occult infection such as paucibacillar or viral infections might have been missed by the usual bacteriological screening in some o f them. Since all o f these patients received empirical antibacterial therapy, the question remains unanswered. In addition, better screening for infection could reduce the misuse o f costly empirical therapy and also reduce the occurrence o f bacterial resistance in these high-risk patients. T u m o u r necrosis factor alpha was less sensitive and specific than IL-6 for the diagnosis o f infection, but was more closely associated with a p o o r clinical outcome. These findings have been reported in alcoholic hepatitis (26,27), and the present study extends this concept to decompensated patients with cirrhosis, whatever the aetiology o f the disease. The role o f T N F a in the cytopathic consequences o f sepsis has been well established (28,29). Levels o f this factor have been shown to be m a r k e d l y increased and sustained for prolonged periods in patients with cirrhosis with infection c o m p a r e d to septic patients without underlying liver disease (3). These observations in addition to the well-known m o r t a l i t y due to infection in cirrhosis could m a k e these patients a particularly interesting p o p u l a t i o n to evaluate the efficacy o f a n t i - T N F a antibodies or o f T N F a inhibiting cytokines such as IL-10 (30) in reducing mortality and/or morbidity. In conclusion, this study illustrates the low diagnostic value o f fever, P M N count, C-reactive protein and fibrinogen levels in patients with decompensated cirrhosis and bacterial infection, whereas IL-6, the main afferent cytokine o f acute phase protein stimulation, is m a r k e d l y increased and appears to be the most sensitive early m a r k e r in these cases.

Acknowledgements This work was supported in part by grants from the F o n d a t i o n Andr6 Loicq (ULB, Brussels, Belgium) and from the Coll~ge de M r d e c i n e des H 6 p i t a u x de Paris (Paris, France). The authors thank Medgenix, Fleurus, Belgium for providing the immunoassays, SmithKline Beecham Belgium for financial support and D r J. Love for reviewing this manuscript. This work was presented in part at the 43rd A A S L D annual meeting N o v e m b e r 2, 1992, Chicago (USA).

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