Summary

Aims

Spontaneous bacterial peritonitis (SBP) is one of the most frequent complications of liver cirrhosis. Ascitic fluid lactoferrin has been proved to be a good diagnostic tool for SBP. However, lactoferrin in ascites may be checked after antibiotic treatment in these patients. Our study aims to assess the utility of ascitic fluid lactoferrin levels for the diagnosis of SBP after antibiotic treatment.

Materials and methods

Twenty-two ascites samples were collected from patients with cirrhosis. Samples were examined for bacterial culture, lactoferrin concentration, and polymorphonuclear leukocyte count. Clinical symptoms and indications for ascitic paracentesis were obtained from medical records. The diagnosis of SBP was based on an elevated ascitic fluid polymorphonuclear leukocyte count of ≥250 cells/mm3 .

Results

Four (18.1%) samples fulfilled the diagnostic criteria for SBP. Three ascites samples showed a positive result for bacterial culture. Patients who received antibiotics for treatment of SBP constituted Group B (n  = 9), whereas those who did not receive any antibiotics comprised Group A (n  = 9). Lactoferrin concentration was significantly elevated (mean: 261.69 ± 145.5 ng/mL) in the three cases with a positive bacterial culture compared to those without SBP, in both Group A (mean: 19.64 ± 6.32 ng/mL, p  = 0.002) and Group B (mean: 23.64 ± 9.53 ng/mL, p  = 0.001).

Conclusion

After systemic antibiotic treatment, elevated lactoferrin levels in the ascites of cirrhotic patients appear to be a promising predictor for the presence of SBP having positive ascitic bacterial culture.

Keywords

Antibiotic treatment ; Ascites ; Lactoferrin

Introduction

Liver cirrhosis represents a terminal clinical stage of chronic liver disease. Patients with liver cirrhosis are susceptible to a variety of complications. Ascites or accumulation of fluid within the peritoneal cavity is one of the most common complications [1] . Spontaneous bacterial peritonitis (SBP) is the infection of pre-existing ascitic fluid without evidence of a secondary infection. The diagnosis of SBP is based on a polymorphonuclear leukocyte (PMN) count in ascitic fluid of ≥250 cells/mm3 , irrespective of whether the ascitic fluid indicates positive results on bacterial culture [2] .

SBP leads to hospitalization of 10–30% of cirrhotic patients, and the mortality rate in this group approaches 30% [3]  ;  [4] . Owing to the high mortality rate, patients with SBP should be started on empiric, broad-spectrum antibiotics immediately. According to the 2012 American Association for the Study of Liver Disease guidelines, in patients with suspected SBP, empiric therapy should be initiated promptly to maximize patient survival [5] .

Lactoferrin is an iron-binding protein found in human mucosal secretions as well as in the specific granules of PMNs [6] . A previous study demonstrated the sensitivity and specificity of fecal lactoferrin concentration for the detection of intestinal inflammation [7] . In addition, measurement of ascitic fluid lactoferrin levels is a reliable biomarker for the presence of PMNs and detection of SBP in patients with cirrhosis [8] . However, as prompt antibiotic treatment is recommended when SBP is suspected, the therapy may be initiated prior to abdominal paracentesis for the collection of ascitic fluid in most cases, which can adversely affect the lactoferrin concentration. Therefore, the diagnostic role of ascitic lactoferrin levels after antibiotic treatment may be compromised and is unclear. Moreover, patients with advanced cirrhosis are prone to develop bacterial infections other than SBP, such as pneumonia or urinary tract infection, and may require antibiotic treatment [9] . In the present study, we aimed to identify the lactoferrin level after systemic antibiotic treatment for SBP and other systemic infections.

Materials and methods

Patients

The inclusion criteria were a known diagnosis of cirrhosis and the presence of ascites. The major indication for abdominal paracentesis was symptom relief to minimize the feeling of abdominal fullness. Exclusion criteria included the presence of other causes of neutrocytic ascites, such as rupture of hepatocellular carcinoma, peritoneal carcinomatosis, and hemorrhagic ascites.

Paracentesis

Paracentesis was performed under aseptic conditions, with the patient in supine position and the puncture site in the left or right lower quadrant. Ultrasonography was performed prior to needle insertion to visualize the intra-abdominal structures. No immediate or delayed complications such as bleeding or hollow organ perforation were noted after the abdominal puncture procedure. Concurrent collection of an ascites fluid sample was performed. The ascites samples were used to measure total cell counts, PMN count, and albumin levels, and for performing aerobic and anaerobic cultures.

Methods

A total of 22 patients with cirrhosis and ascites, who were admitted to Chang Gung Memorial Hospital, Taoyuan, Taiwan and fulfilled the inclusion criteria, were assessed. The diagnosis of liver cirrhosis and ascites was based on clinical and biochemical data, and ultrasonography findings. The diagnosis of SBP was based on the PMN count during the first paracentesis, when SBP was suspected, as the symptoms such as fever, abdominal pain, or sepsis existed and the samples were examined for cell counts and bacterial culture. All the ascites samples collected during the second paracentesis, which was performed for symptom relief, were also examined for cell counts, bacterial culture, and lactoferrin concentration. Patients were classified into three groups: nine patients with an ascitic fluid PMN count of <250 cells/mm3 , who did not initially receive systemic antibiotic treatment (Group A); nine patients with an ascitic fluid PMN count of <250 cells/mm3 , who initially received antibiotic treatment for reasons other than SBP (Group B); and four patients with an ascitic fluid PMN count of ≥250 cells/mm3 (Group C). Group C was further subdivided into culture-negative SBP (defined as Group Cn) and culture-positive SBP (defined as Group Cp) groups. Bacteriological culture was performed using aerobic and anaerobic standard blood culture bottles. The medical history of all patients was recorded, and all patients underwent a clinical examination, routine laboratory investigations, and an abdominal ultrasonographic examination. Aspirated ascitic fluid samples were immediately processed for cell counts (including total and differential cell counts), bacterial cultures, and biochemical assays. The lactoferrin levels in ascitic samples were quantified using a human lactoferrin enzyme-linked immunosorbent assay kit (ELISA) according to the manufacturers instructions (Bethyl Laboratories Inc., Montgomery, TX, USA). The lactoferrin concentrations in the test samples were then quantified by interpolating their absorbance from the standard curve generated in parallel with the samples. After factoring for sample dilutions, lactoferrin concentrations in the original sample were calculated. Lactoferrin levels were expressed as ng/mL of the ascitic fluid.

Statistical analyses

All statistical analyses were performed using the SPSS software package, version 18.0 (SPSS Inc., Chicago, IL, USA). Descriptive statistics were computed for all variables. These included medians and percentiles for continuous factors, and frequencies for categorical factors. A p value <0.05 indicated statistical significance. Data were presented as mean ± standard deviation for continuous variables, and as frequency and percentage for categorical variables. Differences between the groups were evaluated by an independent sample t test and a chi-square test.

Results

A total of 22 ascites samples were evaluated. Thirteen patients received antibiotics prior to paracentesis (13/22, 59%). Cirrhosis was related to chronic viral hepatitis alone in 54.5% (hepatitis B virus = 8, hepatitis C virus = 4) of patients, alcoholism alone in 18.2% (n  = 4), and a combination of alcoholism and chronic viral hepatitis in 18.2% (hepatitis B virus = 2, hepatitis C virus = 2). In 9.1% (n  = 2) of patients, cirrhosis was caused by other factors. Demographic and clinical characteristics of the patients are presented in Table 1 and laboratory characteristics examined are shown in Table 2 . The mean lactoferrin concentration in patients who did not receive antibiotics (Group A, n  = 9) was 19.64 ± 6.32 ng/mL. Group A represented the control group.

Table 1. Baseline characteristics of patients with liver cirrhosis (N  = 22).
Group A (n  = 9)a Group B (n  = 9)b Group C (n  = 4)c
Age (y) 55 ± 10.5 63 ± 17.7 56 ± 11.0
Sex (M:F) 6:3 8:1 1:3
Child–Pugh score 9 ± 2.0 10 ± 1.7 10 ± 2.8
Fever 0 (0/9) 11.1 (1/9) 50 (2/4)
Abdominal pain 0 (0/9) 0 (0/9) 50 (2/4)
Encephalopathy 11.1 (1/9) 22.2 (2/9) 50 (2/4)

Data are presented as % (n /N ) or mean ± SD, unless otherwise indicated.

PMN = polymorphonuclear leukocyte; SBP = spontaneous bacterial peritonitis.

a. An ascitic fluid PMN count of <250 cells/mm3 in patients who did not initially receive systemic antibiotic treatment.

b. An ascitic fluid PMN count of <250 cells/mm3 in patients who initially received antibiotic treatment for reasons other than SBP.

c. An ascitic fluid PMN count of ≥250 cells/mm3 .

Table 2. Laboratory characteristics of study groups.
Group Aa Group Bb Group Cc Group Cpd Group Cn
Leucocyte count (/mm3 )
 Total count 43.1 ± 17.7 125 ± 151.7 653.5 ± 603.5 863.7 ± 556.1 690
 PMN 3.72 ± 3.39 39.5 ± 42.8 285.8 ± 188.6 386.7 ± 106.2 662.4
Albumin level (mg/dL)
 Ascitic 0.84 ± 0.66 0.49 ± 0.50 0.39 ± 0.40 0.49 ± 0.42 0.10
 Serum 2.85 ± 0.45 2.71 ± 0.39 2.58 ± 0.30 2.67 ± 0.31 2.34
 SAAG 2.20 ± 0.45 2.09 ± 0.56 2.19 ± 0.61 2.15 ± 0.71 2.24
Ascitic lactoferrin level (ng/mL) 19.64 ± 6.32 23.64 ± 9.53 197.7 ± 167.7 e 261.69 ± 145.5 6.057

Data are presented as mean ± SD.

PMN = polymorphonuclear leukocyte; SAAG = serum-ascites albumin gradient; SBP = spontaneous bacterial peritonitis.

a. An ascitic fluid PMN count of <250 cells/mm3 in patients who did not initially receive systemic antibiotic treatment.

b. An ascitic fluid PMN count of <250 cells/mm3 in patients who initially received antibiotic treatment for reasons other than SBP.

c. An ascitic fluid PMN count of ≥250 cells/mm3 .

d. An ascitic fluid PMN count of ≥250 cells/mm3 in patients with positive ascitic culture.

e. High variance may relate to the difference in lactoferrin levels between positive and negative ascitic cultures.

Patients who received antibiotics for the treatment of conditions other than SBP

In Group B (n  = 9), indications for antibiotic use among the patients included prophylactic antibiotic for gastrointestinal bleeding, pneumonia, urinary tract infection, and infective endocarditis. SBP was excluded by immediate evaluation of ascites on arrival to the emergency room. The mean lactoferrin concentration in Group B was 23.64 ± 9.53 ng/mL, and was not significantly different from that of Group A (p  = 0.38). However, in patients with clinical sepsis (systemic inflammatory response syndrome and clinical evidence of infection; n  = 5), the ascitic PMN count was higher than that in the control group (3.30 ± 3.61/mm3 vs. 51.18 ± 57.11/mm3 , p  = 0.02). The mean lactoferrin concentration was 32.05 ± 8.25 ng/dL. The lactoferrin concentration was significantly elevated in the patients with clinical sepsis due to reasons other than SBP, compared with the control group (Group A, p  = 0.048).

Patients diagnosed with SBP

Four patients fulfilled the criteria for SBP on admission to the emergency room. Table 3 shows the laboratory findings and bacterial culture results of these patients. The lactoferrin concentration was checked after 3–7 days of antibiotic treatment. Three of the four patients exhibited positive results on bacterial culture. Lactoferrin concentration of the three patients with positive results on bacterial culture (Group Cp) was much higher than that of the patient with a negative result on bacterial culture. The lactoferrin concentration was also significantly elevated (261.69 ± 145.5 ng/mL) in the cases with positive results on bacterial culture compared to the cases without SBP, in both Group A (p  = 0.002) and Group B (p  = 0.001). There was no difference in the lactoferrin concentrations between the negative culture ascites (6.057 ng/mL) of Groups A and B.

Table 3. Laboratory data and bacterial culture results of patients with spontaneous bacterial peritonitis.
PMN count (/mm3 ) at baseline PMN count (/mm3 )after antibiotic treatment Lactoferrin level (ng/mL) Bacterial culture result/causative organism
1 662.4 1.4 6.057 Negative
2 2925 263.4 184.996 Enterococcus faecalis
3 10,450 522.5 134.665 Staphylococcus lugdunensis
4 496.8 356.2 465.397 Escherichia coli

PMN = polymorphonuclear leukocyte.

Discussion

Lactoferrin is an iron-binding protein that is present mainly in external secretions such as breast milk and in polymorphonuclear neutrophils. The presence of lactoferrin in body fluids is proportional to the flux of neutrophils, and its assessment can provide a reliable biomarker for inflammation [10] . Moreover, lactoferrin is widely used as a biomarker in gastrointestinal disease. Fecal lactoferrin concentration is a good marker for the differentiation of inflammatory bowel disease from conditions associated with noninflammatory diarrhea, such as irritable bowel disease, and for the evaluation of disease activity of inflammatory bowel disease [11] . According to a previous study, lactoferrin levels in the ascitic fluid can serve as a sensitive and specific initial screening test for the detection of SBP in cirrhotic patients with ascites [8] .

Patients with cirrhosis have an increased risk of developing bacterial infection, followed by sepsis and death. Infection either is present at admission or develops during hospitalization in approximately 25–35% of patients [12] . SBP, urinary infections, and pneumonia are the most frequent infections in such patients. In immunocompromised patients with liver cirrhosis, antibiotics should be promptly administered in cases of suspected clinical infection. A delay in antibiotic therapy entails a high mortality rate, particularly in patients with SBP.

A positive result on bacterial culture is obtained in some of the patients with SBP, but the results can be delayed for several days [13] . The most common organisms isolated in patients with SBP include Escherichia coli , Gram-positive cocci (mainly Streptococcus species), and enterococci. These organisms account for approximately 70% of all cases of SBP [14] . Ascitic fluid with a PMN count of >250/mm3 and a negative result on bacterial culture is used to indicate culture-negative neutrocytic ascites [15] . According to a previous study, SBP with a positive result on ascitic fluid bacterial culture is associated with a higher mortality than culture-negative neutrocytic ascites [16] .

In the present study, ascitic fluid lactoferrin levels were assessed in patients with cirrhosis after systemic antibiotic treatment. We noted that patients who received antibiotic treatment for conditions other than SBP, such as gastrointestinal bleeding, did not show significantly elevated lactoferrin levels compared to those who did not receive antibiotic treatment and did not have SBP; however, when considering only the patients who were treated for sepsis, lactoferrin levels were significantly elevated compared to those who did not receive antibiotic treatment and did not have SBP, which is consistent with the results of a previous study [17] .

The current study confirmed that ascitic fluid lactoferrin concentration remains elevated in SBP patients with a positive result on bacterial culture, even after systemic antibiotic treatment. However, the lactoferrin concentration in the patient with culture-negative neutrocytic ascites was not elevated compared to the control group. A possible explanation for the differential lactoferrin concentrations between patients with positive and negative results on bacterial culture is that the increased number of bacterial colonies present in the patients with positive results on culture may cause a greater extent of inflammation. A previous study revealed that patients infected with Clostridium difficile with positive results for stool toxins had significantly higher fecal lactoferrin levels, indicative of increased intestinal inflammation [18] . Another study demonstrated that bacterial-related diarrhea (diagnosed by the presence of bacterial colonies on specially prepared agar plates) also led to higher fecal lactoferrin levels [19] .

There are several medications that reduce the concentration of lactoferrin, especially anti-inflammatory medications that were used when sepsis or systemic inflammation was suspected. In another study assessing stool lactoferrin concentration among patients with Crohns disease showed that the use of nonsteroidal anti-inflammatory drugs or steroids caused the anti-inflammatory effect [20] . In our study, the elevation of ascitic fluid lactoferrin level is a good predictive tool for positive ascitic culture. However, larger prospective studies are required to prove that there is a reduction in ascitic fluid lactoferrin concentration after antibiotic treatment.

The major limitation of this study is the small sample size. A larger prospective study is required to determine the cutoff level and the decline in the rate of ascitic fluid lactoferrin concentration after systemic antibiotic treatment.

In conclusion, elevated lactoferrin levels in the ascites of cirrhotic patients with SBP and positive ascitic bacterial culture appears to be a promising diagnostic predictor, even after systemic antibiotic treatment.

Conflicts of interest

The authors declare no conflicts of interest.

References

  1. [1] P. Gines, E. Qunitero, V. Arroyo, J. Terés, M. Bruguera, A. Rimola, et al.; Compensated cirrhosis: natural history and prognostic factors; Hepatology, 7 (1987), pp. 122–128
  2. [2] J. Fernandez, T.M. Bauer, M. Navasa, J. Rodés; Diagnosis, treatment and prevention of spontaneous bacterial peritonitis; Baillieres Best Pract Res Clin Gastroenterol, 14 (2000), pp. 975–990
  3. [3] A. Rimola, G. Garcia-Tsao, M. Navasa, L.J. Piddock, R. Planas, B. Bernard, et al.; Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consensus document. International Ascites Club; J Hepatol, 32 (2000), pp. 142–153
  4. [4] P.J. Thuluvath, S. Morss, R. Thompson; Spontaneous bacterial peritonitis—in-hospital mortality, predictors of survival, and health care costs from 1988 to 1998; Am J Gastroenterol, 96 (2002), pp. 1232–1236
  5. [5] B.A. Runyon; Introduction to the revised American Association for the Study of Liver Diseases Practice Guideline management of adult patients with ascites due to cirrhosis 2012; Hepatology, 57 (2013), pp. 1651–1653
  6. [6] S. Iyer, B. Lonnerdal; Lactoferrin, lactoferrin receptors and iron metabolism; Eur J Clin Nutr, 47 (1993), pp. 232–241
  7. [7] S.V. Kane, W.J. Sandborn, P.A. Rufo, A. Zholudev, J. Boone, D. Lyerly, et al.; Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation; Am J Gastroenterol, 98 (2003), pp. 1309–1314
  8. [8] M.A. Parsi, S.N. Saadeh, N.N. Zein, G.L. Davis, R. Lopez, J. Boone, et al.; Ascitic fluid lactoferrin for diagnosis of spontaneous bacterial peritonitis; Gastroenterology, 135 (2008), pp. 803–807
  9. [9] C. Guarner, G. Soriano; Spontaneous bacterial peritonitis; Semin Liver Dis, 7 (1997), pp. 203–217
  10. [10] T. Hayakawa, C.X. Jin, S.B. Ko, M. Kitagawa, H. Ishiguro; Lactoferrin in gastrointestinal disease; Intern Med, 48 (2009), pp. 1251–1254
  11. [11] J. Dai, W.Z. Liu, Y.P. Zhao, Y.B. Hu, Z.Z. Ge; Relationship between fecal lactoferrin and inflammatory bowel disease; Scand J Gastroenterol, 42 (2007), pp. 1440–1444
  12. [12] J. Fernandez, T. Gustot; Management of bacterial infections in cirrhosis; J Hepatol, 56 (Suppl. 1) (2012), pp. S1–S12
  13. [13] B.A. Runyon; Strips and tubes: improving the diagnosis of spontaneous bacterial peritonitis; Hepatology, 37 (2003), pp. 745–747
  14. [14] G. Garcia-Tsao; Current management of the complications of cirrhosis and portal hypertension: variceal hemorrhage, ascites, and spontaneous bacterial peritonitis; Gastroenterology, 120 (2001), pp. 726–748
  15. [15] B.A. Runyon, J.C. Hoefs; Culture-negative neutrocytic ascites: a variant of spontaneous bacterial peritonitis; Hepatology, 4 (1984), pp. 1209–1211
  16. [16] L. Kamani, K. Mumtaz, U.S. Ahmed, A.W. Ali, W. Jafri; Outcomes in culture positive and culture negative ascitic fluid infection in patients with viral cirrhosis: cohort study; BMC Gastroenterol, 8 (2008), pp. 59–64
  17. [17] I. Berkestedt, H. Herwald, L. Ljunggren, A. Nelson, M. Bodelsson; Elevated plasma levels of antimicrobial polypeptides in patients with severe sepsis; J Innate Immun, 2 (2010), pp. 478–482
  18. [18] J.H. Boone, J.R. Dipersio, M.J. Tan, S.J. Salstrom, K.N. Wickham, R.J. Carman, et al.; Elevated lactoferrin is associated with moderate to severe Clostridium difficile disease, stool toxin, and 027 infection  ; Eur J Clin Microbiol Infect Dis, 32 (2013), pp. 1517–1523
  19. [19] C.C. Chen, C.J. Chang, T.Y. Lin, M.W. Lai, H.C. Chao, M.S. Kong; Usefulness of fecal lactoferrin in predicting and monitoring the clinical severity of infectious diarrhea; World J Gastroenterol, 17 (2011), pp. 4218–4224
  20. [20] R. Sidhu, D.S. Sanders, P. Wilson, L. Foye, S. Morley, M.E. McAlindon; Faecal lactoferrin, capsule endoscopy and Crohns disease. Is there a three way relationship? A pilot study; J Gastrointestin Liver Dis, 19 (3) (2010 Sep), pp. 257–260
Back to Top

Document information

Published on 15/05/17
Submitted on 15/05/17

Licence: Other

Document Score

0

Views 0
Recommendations 0

Share this document

claim authorship

Are you one of the authors of this document?