Transforming Growth Factor Beta 1 in the Ascitic Fluid as ‎Predictor for Spontaneous Bacterial Peritonitis in Cirrhotic Patients ‎due to Hepatitis C Virus infection in Suez Canal University ‎Hospital

INTRODUCTION

Cirrhosis is considered an immunocompromised state that leads to a variety of infections, accounting for around 30% of all mortality [1]. infections account for a 4-fold increase in mortality among patients with liver cirrhosis [2]. Spontaneous bacterial peritonitis (SBP) is a leading cause of morbidity and mortality in cirrhotic patients with ascites and it is thought to impact 10–30% of cirrhotic patients admitted to the hospital with ascites leading to a death rate approaching 30% [3]. In hospitalized patients with cirrhosis, bacterial infections account for 32% to 34% of them, and infection affects 45% of those with gastrointestinal bleeding [2]. SBP is more common in hospitalized patients who have routine paracentesis (12%), while it is diagnosed in up to 3.5 percent of patients on outpatient visits [2,3]. With early diagnosis and treatment, the death rate has declined to around 20% [4]. On the other hand, patients have a bad prognosis after SBP with a 6-month mortality rate of 50% after the first diagnosis of SBP [5]. Because of the poor prognosis and outcome, it is critical to diagnose SBP as soon as possible [6]. In patients with liver cirrhosis, SBP is diagnosed when the polymorphonuclear ‎‎(PMN) cell count in ascitic fluid reaches 250 cells/mm³, ascitic fluid cultures ‎show only a single organism, and other types of peritonitis have been ruled out ‎‎[7]. ‎ For early detection of SBP,‎  several tests have been created and examined, Calprotectin, Lactoferrin, Hepcidin, lipopolysaccharide-binding protein, and Complement 3 are some of these markers [8]. Unfortunately, their diagnostic accuracies are limited, and their usage depends on laboratory personnel and commercially available reagents/components. Therefore, there is still a clinical need for an accurate and convenient method of rapid diagnosis of SBP [9].

Transforming growth factor-beta (TGF- β) is a group of polypeptide growth factors that have a variety of functions and seems to be a specific inhibitor of hematopoietic cells, according to various studies [10]. It affects the proliferation of peritoneal exudates macrophages and causes apoptosis in human lymphocytes and hepatocytes [11]. TGF- β levels have been associated with a variety of illnesses, including atherosclerosis and fibrous diseases of the kidney, liver, and lungs. The persistence of chronic inflammation in liver illnesses, such as chronic viral hepatitis, is a crucial factor in dictating the shift in the TGF-signaling pathway from tumor suppression to fibrogenesis, which accelerates liver fibrosis and raises the risk of HCC [12]. The role of TGF- β 1 in the progression of liver fibrosis has been widely studied. This cytokine promotes the conversion of hepatic stellate cells to myofibroblast-like cells and the creation and breakdown of extracellular matrix proteins [13]. The present work aimed at assessing the ascitic fluid TGF- β 1 as a reliable diagnostic test marker for diagnosing spontaneous bacterial peritonitis (SBP).

PATIENTS‎ AND METHODS

This study was an analytical cross-sectional study, carried out at the inpatient ward of the Internal Medicine Department of Suez Canal university hospital. The study included 82 Egyptian patients with liver cirrhosis and ascites aging 18 - 65 years of Both female & male gender, Patients were divided into 2 groups:

1-   Group A included 40 Patients with liver cirrhosis and ascites without SBP.

2-   Group B included 42 Patients with liver cirrhosis and SBP based on the clinical presentation and Ascitic fluid (AF) PMNL of 250/mm³ or higher and had not yet begun antibiotic treatment for SBP.

The criteria for liver cirrhosis diagnosis in the patients that were included in this study include clinical and laboratory and radiological, clinical criteria include clubbing, palmar erythema, spider nevi (angiomata), gynecomastia, feminizing hair distribution, testicular atrophy, small irregular shrunken liver, anemia, caput medusae,  drowsiness and asterixis in encephalopathy, Jaundice, ascites, leukonychia, lower limb edema and bruising. Patients were categorized regarding Child-Pugh classification [by three biochemical variables (serum albumin, bilirubin, and prothrombin time (international normalized ratio, INR) in addition to the presence or absence of ascites and clinical signs of encephalopathy]. Patients were scored as follows: 5 – 6 as class A, 7 – 9 as class B, and 10 – 15 as class C. All patients involved in the study were subjected to a thorough medical history and physical examination,  and abdominal ultrasound (liver & presence of hepatic focal lesions, spleen, gallbladder, ascites), and a laboratory test that included a full blood count, liver profile, creatinine, and ascitic fluid analysis (WBCs, protein, bacteriologic culture with sensitivity and TGF-B1 level).  To diagnose SBP, the presence of 250 cells/mL PMN in the ascitic fluid, with or without a positive ascitic fluid culture in the absence of hemorrhagic ascites and secondary peritonitis.

Exclusion criteria included patients who received antibiotic treatment two weeks before hospital admission or treatment that can modify the level of TGF beta 1 (steroids and non-steroidal anti-inflammatory or immunosuppressive drugs). Also, patients with Hepatocellular carcinoma, systemic infection, diabetes mellitus, neoplastic disorders, heart failure, and hematological disorders were ruled out.

Sampling

1. Venipuncture was used to obtain 5 milliliters of blood, 1 milliliter was placed in an EDTA tube for CBC, and 4 milliliters were placed in a plastic tube and allowed to clot. Centrifugation was done to separate non-hemolyzed sera for detecting creatinine and liver functions (ALT, AST, total bilirubin, and albumin).
2. Paracentesis to obtain an ascitic fluid sample from a puncture site in the left or right lower quadrant under aseptic conditions while the patient was supine. All diagnostic samples were obtained at the bedside and processed by laboratory personnel without delay.

Methodology:

1-   Blood glucose, liver profile, and creatinine concentrations were tested using commercially available reagents and an enzyme-based kit on a Dimension Xpand plus chemistry analyzer (Roche Diagnostics, Basel, Switzerland).

2-   TGF beta 1 ELISA Kit was used to assess TGF beta 1 in the ascitic fluid using an enzyme-linked immunosorbent assay.

Statistical analysis[14][15]

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Qualitative data were described using numbers and percentages. The Kolmogorov-Smirnov test was used to verify the normality of distribution Quantitative data were described using range (minimum and maximum), mean, standard deviation, and median. The significance of the obtained results was judged at the 5% level.

RESULTS:

Patient characteristics:

This study includes 82 patients with chronic liver disease and ascites, the final diagnosis (SBP) was made According to ascitic fluid analysis and clinical data, they were divided into a SBP group including 42 patients (29 males and 13 females) and a non-SBP group of 40 patients (24 males and 16 females). 37 (45.1%) of the patients were classed as stage B, and 45 (54.9%) of the patients were classified as stage C, according to the Child-Turcotte-Pugh Score (Table 1).

Laboratory and ascitic fluid analysis:

there was a marked increase in total leukocytic count, bilirubin, creatinine, albumin, ascitic fluid LDH, and ascitic fluid TLC compared to the non-SBP group (10.5 vs. 7.1; 5 vs. 2.6; 2.2 vs. 1.8; 4.0 vs. 2.3; 388.7 vs. 93.9 and 9336.5 vs. 220.9 respectively)

There were no significant differences between hemoglobin (Hb), platelet count, ALT, AST, prothrombin time, and Ascitic fluid Glucose in both groups 10.4 vs. 9.8; 168.4 vs. 125.6; 32.1 vs. 47.7; 62 vs. 97.8; 20.5 vs. 17.1 and 120.6 vs. 135.7 respectively) (Table 2).

Diagnostic value of ascitic TGF β1:

Analyzing the given results through the Receiver Operating Characteristic (ROC) curve; it is revealed that a cutoff point of 151.5 ng/mL was statistically 100% sensitive and 100% specific in diagnosing SBP for patients in the current study (figure 1).

Relation between TGF-β1 and stage of liver disease:

TGF-β1 was higher in child class C patients than in class B patients by (194.7) and (149.6) respectively (figure 2)

Relation between TGF-β1 and SBP:

There was a positive correlation between the level of ascitic fluid TGF-β1 and SBP because TGF-β1 is higher in SBP than in non-SBP by (264.2±40.4) and (80±17.2) respectively (Table 3).

* Statistically significant at p<0.05

DISCUSSION