Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 30  |  Issue : 4  |  Page : 984--990

Serum level of mannose-binding lectin in cirrhotic patients with spontaneous bacterial peritonitis


Ahmed B Mahmoud1, Azza M Abd El Aziz2, Tawfik M Abd El Motelb2, Nashwa A El Fetoh Shebl3, Radwa H Abdel Sattar Salem2,  
1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufiya University, Menoufiya, Egypt
2 Department of Medical Microbiology and Immunology, National Liver Institute, Menoufiya University, Menoufiya, Egypt
3 Department of Hepatology, National Liver Institute, Menoufiya University, Menoufiya, Egypt

Correspondence Address:
Radwa H Abdel Sattar Salem
Medical Microbiology and Immunology,, National Liver Institute, Menoufiya University, Shebien El Kom, Menoufia
Egypt

Abstract

Objectives To evaluate the role of mannose-binding lectin (MBL) in cirrhotic patients with and without spontaneous bacterial peritonitis (SBP) and to determine its role in the diagnosis of peritonitis. Background MBL deficiency increases the risk of various infections mostly in immunodeficient conditions. SBP is one of the bacterial infections causing morbidity and mortality under liver cirrhotic patients. Materials and methods A total of 90 individuals (66 male and 24 female with the mean age 52.55 ± 9.90 years) were included: 35 cirrhotic patients with ascites and SBP, 35 cirrhotic patients with ascites and no SBP and 20 apparently healthy persons as a control. The studied groups were subjected to full medical history, clinical examination and measurement of serum level of mannose-binding lectin (sr MBL) by enzyme-linked immunosorbent assay. Cirrhotic patients were subjected to liver function tests, and biochemical and bacteriological examination of ascitic fluid including culture and sensitivity testing was performed for all cirrhotic patients with ascetic fluid total leucocytic count (TLC) greater than 500 cells/mm3. Results sr MBL was significantly lower in group 1 than in group 2 (P < 0.001); sr MBL can predicts SBP with a sensitivity of 71.4% at a cutoff point of 1202.5 ng/ml; also, the sr MBL level was significantly lower in culture-positive ascitic fluid samples than in culture-negative samples (P < 0.05). Patients with positive cultures for Gram-negative isolates had a significantly lower sr MBL than those with Gram-positive isolates (P < 0.05). A negative correlation was found between sr mannose-binding lectin (MBL) and sr albumin, ascitic fluid TLC, and glucose, but a positive correlation was found between sr MBL and aspartate aminotransferase, ascitic fluid albumin, and total protein. Conclusion sr MBL could be considered as a predictive marker for SBP in cirrhotic patients.



How to cite this article:
Mahmoud AB, Abd El Aziz AM, Abd El Motelb TM, El Fetoh Shebl NA, Abdel Sattar Salem RH. Serum level of mannose-binding lectin in cirrhotic patients with spontaneous bacterial peritonitis.Menoufia Med J 2017;30:984-990


How to cite this URL:
Mahmoud AB, Abd El Aziz AM, Abd El Motelb TM, El Fetoh Shebl NA, Abdel Sattar Salem RH. Serum level of mannose-binding lectin in cirrhotic patients with spontaneous bacterial peritonitis. Menoufia Med J [serial online] 2017 [cited 2018 Apr 26 ];30:984-990
Available from: http://www.mmj.eg.net/text.asp?2017/30/4/984/229231


Full Text



 Introduction



Mannose-binding lectin (MBL) is a protein from lectins family that is able to recognize carbohydrate patterns formed on the surface of pathogens and host cells, and plays a role in the activation of complement and immune defense. MBL serum deficiency has the ability to decrease complement activation, an action that is extremely important for the humoral immunity functionality[1].

Epidemiological studies show that MBL deficiency influences susceptibility and evolution of very common diseases such as infections, neoplasm and metabolic and cardiovascular diseases[2].

Spontaneous bacterial peritonitis (SBP) is an acute bacterial infection of ascitic fluid; it occurs in both children and adults and is a well-known complication in patients with cirrhosis[3]. SBP is a potentially fatal yet reversible cause of deterioration in patients with advanced liver cirrhosis and ascites that occurs in the absence of any intra-abdominal, surgically treatable source of infection with elevation of polymorphonuclear leukocyte count more than 250/mm 3[4].

Traditionally, three-fourths of SBP infections have been caused by aerobic Gram-negative organisms (50%of these being Escherichia coli), with one-fourth of these infections caused due to aerobic Gram-positive organisms (19% streptococcal species)[4].

In SBP with MBL deficiency, the co-existence of MBL deficiency increases infection susceptibility, allowing further rapidly progressive liver disease, which increases the risk of morbidity and mortality[5].

Both high and low serum levels of functional MBL have been associated with a variety of diseases and their complications. Functioning as a double-edged sword, low MBL serum levels have been shown to enhance the risk for infections. In contrast, high MBL serum levels and high MBL activity have been associated with inflammatory diseases, transplant rejection, and diabetic nephropathy[6].

 Materials and Methods



This study was carried out during the period from February 2012 to April 2013 in the National Liver Institute, Menoufiya University; it was conducted on 90 individuals (66 male and 24 female, with mean age 52.55 ± 9.90 years) who were classified into three groups: group 1: 35 cirrhotic patients with ascites and SBP; group 2: 35 cirrhotic patients with ascites and no SBP (the diagnosis of ascites and SBP was based on clinical examination abdominal ultrasonography and ascetic fluid tapping for examination); group 3: 20 apparently healthy persons of matched age and sex as a control group. Written consent was obtained from each individual.

Exclusion criteria

Patients with secondary bacterial peritonitis and noncirrhotic causes of ascites and those who received systemic antibiotics in the last 2 weeks were excluded.

All patients and controls were matched for age and sex. Informed consent was obtained from every patient and control in accordance with the local ethical committee. All individuals were subjected to complete history taking, complete clinical examination, and abdominal ultrasonography.

For ascetic fluid examination, 15 ml of ascetic fluid was obtained under complete aseptic conditions and was divided as follows: 5–10 ml were introduced into blood culture broth bottles at bedside and incubated at 37°C; 3 ml were introduced into purple top tube for cell count; if TLC greater than 500 cells/mm 3 another 3 ml were introduced into red top tube for routine chemistries [lactate dehydrogenase (LDH), glucose, albumin, and protein].

The growing organism was identified by direct microscopic examination of Gram-stained smear and biochemical tests according to UK Standards for Microbiology Investigations[7]. Antibiotic sensitivity tests were performed by disk diffusion method; also, VITEK 2-compactis (Biomerieux, France - World Headquarters, bioMérieux Marcy l'Etoile, 376, chemin de l'Orme, 69280 Marcy l'Etoile) was used for identification and antibiotic sensitivity[8]. Extended-spectrum β-lactamase (ESBL) production and methicillin-resistant Staphylococcus aureus (MRSA) were determined by the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) performance standards.

Blood samples from patients and control individuals (about 10 ml) were collected by complete aseptic techniques from all individuals using a serum separator tube, and samples were allowed to clot for 30 min before centrifugation for 15 min at 1000g; then, serum was removed and divided into small aliquots: one was stored at −20°C until use for MBL immunoassay and the other was tested for liver function tests (alanine transaminase, aspartate aminotransferase, serum total and direct bilirubin, serum albumin). A second blood sample was added to EDTA for complete blood count and tested immediately.

Serum MBL level was determined by enzyme-linked immunosorbent assay using Human MBL Immunoassay R and D Systems Inc. (Cat. No DMBL00; Minneapolis, Minnesota, USA)[9].

Serum samples required at least a 400-fold dilution, which was achieved by adding 10 μl of sample to 190 μl of calibrator Diluent (France - World Headquarters, bioMérieux Marcy l'Etoile, 376, chemin de l'Orme, 69280 Marcy l'Etoile) RD5-26 (1X). The 400-fold dilution was completed by adding 10 μl of diluted sample to 190 μl of Calibrator Diluent RD5-26 (1X). About 50 μl of Assay Diluent RD1-21, standard, control or sample (after dilution) per well were added and covered with the adhesive strip. It was incubated for 2 h at room temperature on a horizontal orbital microplate shaker (0.12' orbit) set at 500 rpm, Each well was aspirated and washed, and the process was repeated three times for a total of four washes by filling each well with wash buffer (400 μl) using autowasher; 100 μl of the MBL conjugate was added to each well, covered with a new adhesive strip and the plate was incubated for 2 h at room temperature on the shaker; then, the aspiration/wash was repeated, and 100 μl of substrate solution was added to each well. The plate was incubated for 30 min at room temperature on bench top protected from light, and then 100 μl of stop solution was added to each well; the color in the wells changed from blue to yellow and optical density of each well was determined within 30 min using a microplate reader set to 450 nm, with correction set to 540 nm. As the samples were diluted 400-fold, the concentration read from the standard curve were by 400.

Statistical analysis

Quantitative data were statistically described in terms of range, mean, SD, frequencies, and relative frequencies when appropriate. Comparison of quantitative variables between the study groups was performed using t-test for paired samples. A P value less than 0.05 was considered statistically significant. All statistical calculations were performed using computer package statistical package for the social science (SPSS, version 17; SPSS Inc., Chicago, Illinois, USA) for Windows 7 (Microsoft Corporation, New York, New York, USA).

 Results



In this study, we found that there was no significant statistical difference between the studied groups with regard to age and sex [Figure 1].{Figure 1}

The serum level of mannose-binding lectin (sr MBL) was significantly lower in group 1 than in group 2; its level was also significantly higher in group 2 than in group 3, whereas there was no significant difference between group 1 and group 3 [Table 1] and [Figure 2]; about 5.7% of patients in group 1 had absolute MBL deficiency and 71.4% in group 2 had an MBL level above 1000 ng/ml [Table 2]; the sr MBL had a significantly positive correlation with the degree of liver fibrosis according to the Child classification [Figure 3].{Table 1}{Table 2}{Figure 2}{Figure 3}

The sr MBL showed a significant positive correlation with ascitic fluid albumin and total protein levels and a significant negative correlation with TLC, glucose, and LDH levels [Table 2].

The receiver operating characteristic curve of serum Mannose-Binding Lectin (MBL) level for the diagnosis of SBP from non-SBP cases (group 1 and group 2, respectively) showed that the measurement of the sr MBL plays a significant role in the differential diagnosis of SBP from non-SBP cirrhotic patients with sensitivity 71.4%, and specificity 69.4%, at cutoff point 1202.5 ng/ml [Table 3] and [Figure 4].{Table 3}{Figure 4}

Laboratory data of the ascetic fluid of the studied patients revealed that TLC and LDH were significantly higher in group 1 than in group 2, whereas the glucose level in the ascetic fluid was significantly lower in group 1 than in group 2 [Table 4] and [Table 5].{Table 4}{Table 5}

Ascitic fluid cultures showed that 42.9% of group 1 comprised culture-positive cases, of which 80% were caused by Gram-negative organisms (33.3% of them were ESBL-producing organisms); E. coli was the most commonly isolated organism (53.3%), followed by Klebsiella spp. (13.3%), whereas 20% were caused by Gram-positive organism (33.7% were MRSA), while ascitic fluid cultures of group 2 were all negative [Table 6] and [Figure 5].{Table 6}{Figure 5}

On comparison, we found that the MBL serum level was significantly lower in culture-positive SBP patients than in culture-negative cases; also, sr MBL was significantly lower in culture-positive ascitic fluid caused by Gram-negative organisms than in those caused by Gram-positive organisms [Table 7].{Table 7}

 Discussion



Ascites is the most common complication of cirrhosis, and about 60% of the patients with compensated cirrhosis develop ascites within 10 years during the course of their disease[10].

SBP is a very common bacterial infection in patients with cirrhosis and ascites[11]. When first described, its mortality exceeded 90%, but it has been reduced to ~20% with early diagnosis and treatment[12].

MBL is able to recognize carbohydrate patterns formed on the surface of pathogens and host cells, and plays a role in complement activation and activation of immune defense. MBL serum deficiency has the ability to decrease complement activation, an action that is extremely important for the humoral immunity functionality[1].

In the present study, we aimed to evaluate the role of MBL in cirrhotic patients with and without SBP and to determine its role in the diagnosis of peritonitis.

sr MBL was significantly lower in group 1 than in group 2, which proves its role in protection against infections; this finding matches results of Esmat et al.[13] and Yuen et al.[14].

In this study 5.7% (2/35) of patients in group 1 had absolute MBL deficiency, which supports MBL deficiency as a factor for infection. This finding was confirmed by Altorjay et al.[15], who found that in absolute MBL deficiency (MBL level <100 ng/ml), the time to first infection was shorter. The impressive fact was the considerable variations between individuals in the studied groups regarding their sr MBL and this may be explained by the high genetic heterogenecity of MBL-expressing genes[16].

The result of positive ascitic fluid culture in our patients was 42.9% higher than the result obtained in studies by Redwan et al.[4] and Garcia-Tsao et al.[17], which were 21.7 and 23%, respectively. This variation may be due to false-negative cultures due to a low bacterial count[4],[17]. However, Saleh[18] reported that despite the use of sensitive methods, ascites culture was negative in as many as 60% of their patients with clinical manifestations suggestive of SBP and increased ascites neutrophil count. Pelletier et al. and Cholongitas et al. reported higher results ranging from 50 to 70%; this could be attributed to the larger amount of ascetic fluid (10 ml) inoculated into blood culture bottles as reported by these studies[19],[20]. Although the amount inoculated in this study was less than 10 ml, other unexplained factors could have played a role in this difference.

In ascitic fluid cultures, 79% was Gram-negative bacteria, followed by Gram-positive bacteria (21%). E. coli was the predominant organism (53.3%), followed by Klebsiella spp. (13.3%), and this was more or less close to the results of Garcia-Tsao et al.[17] and Altorjay et al.[15], and in contrast to those of Redwan et al.[4]and Jain et al.[21], who noted a predominance of Gram-positive organisms in culture-positive SBP.

Among Gram-positive bacteria, S. aureus, Staphylococcus epidermidis, and Streptococcus pneumonia were isolated at 6.7% each; this is different from Smithson et al.[22], who reported that S. aureus was the most commonly isolated organism from culture-positive ascitic fluid, whereas Song et al.[23] found that 53% of the organisms were Streptococci spp.

This study recorded that 40% of E. coli and Klebsiella isolates were ESBL producing (ESBL-EK); this was higher than the results of Tandon et al.[24], who found that SBP due to ESBL-EK accounted for only 7.5% (26/346) of the SBP due to EK.

The isolate of S. aureus was a methicillin-resistant strain. Eisen et al.[25] reported a high rate of antibiotic-resistant bacterial infections in patients with cirrhosis, as they found 70 culture-positive infections and 33 antibiotic-resistant bacteria; 15% (5/33) were MRSA and 27% (9/33) were ESBL.

sr MBL was significantly lower in culture-positive ascitic fluid than in culture-negative samples; this showed that MBL deficiency plays an important role in susceptibility to bacterial infection[26]; also, in this study, cases with Gram-negative isolates had a significantly lower sr MBL than cases with Gram-positive isolates; this would be due to a variable degree of binding between MBL molecules and bacterial surface; some studies have found an association between infection due to Gram-positive bacteria and MBL deficiency[27]; other studies have observed a link between infection due to Gram-negative bacteria and MBL deficiency[28].

In this study, sr MBL was the highest in group 2 compared with other studied groups, and this can be explained by the fact that MBL is an acute-phase reactant[13],[29].

sr MBL in control group were close to the range reported in the study by Swierzko et al.[30], which was 1466 mg/l in the age group of 41–57 years, and in this study, it was 1271.75 ng/ml. Large interindividual variations in serum MBL levels were documented in healthy persons, but individual values were very stable over time, largely due to their genetic disposition[16].

There was a positive correlation between the sr MBL and Child classification of patients; other studies observed that MBL concentrations have generally been higher in chronic hepatitis C virus patients and the median value for patients was approximately twice that of healthy controls[31]. In contrast, Altorjay et al.[15] found that MBL levels in cirrhotic patients of various etiologies were similar to those in noncirrhotic liver groups[15].

 Conclusion



In conclusion sr MBL are low in cirrhotic patients with SBP with a negative correlation with sr albumin, ascitic fluid TLC, and glucose, whereas a positive correlation with aspartate aminotransferase, ascitic fluid albumin, and total protein. sr MBL could be considered as a predictor of SBP in cirrhotic patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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