Assessment of Fungal Infection of Peritoneal Fluid in Cirrhotic Ascites

Patients and Methods: Between September 2016 and April 2017, 50 Patients were classified into (SP group) and (non SP group) all patients were subjected to history taking, clinical examination, complete blood count, ESR,CRP, liver profile, kidney function tests, serological tests for viral hepatitis markers, abdominal ultrasonography and diagnostic abdominal paracentesis. The ascitic fluid was subjected to the following: microscobic examination, biochemical examination and microbiological cultures (bacterial and fungal).


INTRODUCTION
Spontaneous peritonitis, bacterial (SBP), is a common and life-threatening complication in patients with liver cirrhosis and ascites, with a mortality of 10 to 50%.It should be suspected in patients with ascites who develop fever, abdominal pain, ileus, and worsening hepatic encephalopathy, though 13% of patients may lack any signs or symptoms of SBP at the time of presentation [1].The diagnosis is made if the polymorphonuclear cell (PMN) count in the ascites fluid is ≥250 cells/mm3, culture results are positive and secondary causes of peritonitis are excluded [2].
The impact of fungal infections may be underestimated in clinical routine since the rate of sepsis was higher in patients with fungascites than in those with bacterascites [3].
The aim of this work is to evaluate the occurrence of fungal infection of peritoneal fluid in cirrhotic patients with ascites.

PATIENTS AND METHODS
This study was conducted on 50 patients with decompensated chronic liver diseases, child B and C cirrhosis with ascites admitted to Al-Shabrawishi hospital Patients were classified into two groups:  Group I: the first 25 consecutive patients with ascitic fluid polymorphonuclear leucocyte (PMN) count ≥ 250 cell/ mm 3 . Group II: Included the first 25 consecutive patients with ascitic fluid polymorphnuclear leucocyte (PMN) count > 250 cell /mm 3 .
All patients were subjected to the following: I-history taking with particular attention to: Manifestations suggesting SP and history of previous attack of SBP, refractory ascites and drug history.

II-Full general and local examination:
Looking for signs of liver disease such as palmar erythema, spider naevi, liver size, spleen size, presence of ascites, jaundice and encephalopathy.
Signs of SP such as fever, hypotension, tachycardia and abdominal pain.

IV-Abdominal ultrasonography.
V-Diagnostic abdominal paracentesis: Thirty ml of the ascitic fluid were aspirated from each patient.The technique of paracentesis was explained to the patient and it was done under aseptic precautions using a wide bore needle.The needle was introduced in the right lower quadrant while the patient lies in supine position.

"Z Tracking" technique was used to avoid post paracentesis leaks [4].
This technique is accomplished by displacing (with one gloved hand) the skin approximately 2 cm downward and then slowly inserting the paracentesis needle mounted on the syringe held in the other hand.The skin is not released until the needle has penetrated the peritoneum and fluid flows.They were subjected to the following: (A) Physical examination: -Color. -Aspect.

II-Identification of Candida species:
Colonies grown on Sabouraud's dextrose agar were stained with Gram stain and microscopically examined.Yeast colonies were subjected to: CHROM agar medium: subculture on CHROM agar medium at c37°C and examined after 24-48 hours, then colony color was examined and different species of yeast were identified according to the range of color.

RESULTS
The results of this study were tabulated and statistically analyzed in tables and figures.

This table shows:
 There was highly statistically significant difference between 2 groups regarding ascitic fluid aspect, total leucocytic count, PMNs, lymphocytes, LDH and total protein in ascitic fluid.
 There was highly statistically significant difference between 2 groups regarding bacterial culture of ascitic fluid.

Report of the first case of spontaneous fungal peritonitis:
A 52-year-old female was admitted to the hospital complaining of progressive weakness, increased abdominal distension, abdominal pain, nausea, vomiting, diarrhea and difficult breath.The patient was hospitalized 1 month prior to this admission for hepatic encephalopathy.During that hospitalization, she received anti-coma measures.Her past medical history was significant for end-stage liver disease secondary to hepatitis C induced cirrhosis.Her HIV status was known to be negative and she had no past history of corticosteroid or immunosuppressive therapy.She had a past history of recent antibiotic use, ceftriaxone, and previous attack of SBP with no prophylaxis for it.Also, she had a past history of refractory ascites and regular tapping.On physical examination, the patient was icteric and febrile (39°C) with normal other vital signs.Further examination demonstrated scleral icterus, lower limb edema and flapping tremors.Abdominal examination revealed tense ascites with splenomegaly but no signs of guarding or rebound tenderness.
Diagnostic and therapeutic abdominal paracentesis were done.Ascitic fluid was turbid and yellow.Ascitic fluid analysis revealed leukocytic count of 900 cells/ml³ and polymorphonuclear neutrophil count of 350 cells/ml³; total protein was 2.6 g/dL; LDH was 251 u/l and glucose was 131 mg/dL.She was diagnosed as spontaneous bacterial peritonitis and she was started cefotaxime 2 gm/12 hours till the culture results appeared.Ascitic fluid bacterial culture was negative.Seven days later, the patients ascitic fluid fungal culture grew Candida albicans.
Abdominal ultrasound revealed shrunken cirrhotic liver with dilated portal vein, splenomegaly, normal kidneys and massive ascites.Chest radiograph showed mild right pleural effusion.
The patient was switched to fluconazole 6 mg/kg intravenously after culture results were obtained.Unluckily, on the 8th day, she died following a massive attack of hematemesis.

Report of the second case of spontaneous fungal peritonitis:
A 76-years-old male was admitted to the hospital complaining of progressive weakness, increased abdominal distension, abdominal pain, nausea, hematemesis, melena, and difficult breath.The patient was hospitalized 2 months prior to this admission for hepatic encephalopathy.During that hospitalization, he received anti-coma measures.His past medical history was significant for end-stage liver disease secondary to hepatitis C induced cirrhosis.His HIV status was known to be negative and he had no past history of corticosteroid or immunosuppressive therapy.He had a past history of recent antibiotic use; ceftriaxone, and no previous attacks of SBP with no prophylaxis for it.Also, he had a past history of refractory ascites, regular tapping, DM, hypertension and renal impairment.On physical examination, the patient was febrile (39°C) with normal other vital signs.Further examination demonstrated lower limb edema and flapping tremors.Abdominal examination revealed moderate ascites with splenomegaly but no signs of guarding or rebound tenderness.
Diagnostic and therapeutic abdominal paracentesis were done.Ascitic fluid was turbid and yellow.Ascitic fluid analysis revealed leukocytic count of 1500 cells/ml³; polymorphonuclear neutrophil count of 900 cells/ml³ and lymphocytes 600 cells/ml³; total protein was 2.7 g/dL; LDH was 210 u/l and glucose was 167 mg /dL.He was diagnosed as spontaneous bacterial peritonitis and he was started cefotaxime 2 gm/12 hours till the culture results appeared.Ascitic fluid bacterial culture was negative.Five days later, the patients ascitic fluid fungal culture grew Candida albicans.
Abdominal ultrasound revealed cirrhotic liver, splenomegaly, grade I nephropathy and moderate ascites.Chest radiograph showed mild right pleural effusion.The patient was switched to fluconazole 6 mg/kg intravenously after culture results were obtained.Unluckily, on the 6th day, he died after cardio-pulmonary arrest.

DISCUSSION
The relationship between immune dysfunction and infection in cirrhosis, known as cirrhosis-associated immune dysfunction syndrome (CAIDS), has been investigated extensively.This syndrome is a multifactorial state of dysimmunoregulation, which is comprised of a reduction in serum bactericidal activity, opsonic activity, and complement and fibronectin levels [5].

Spontaneous peritonitis is a common condition.
It is usually seen in patients with end-stage liver disease and ascites.The most common underlying cause for spontaneous peritonitis is bacterial infection; however, very rarely fungal infections can be the causative agents [6].
Infective peritonitis is categorized into primary, secondary, or tertiary according to pathologic etiology.Primary peritonitis also known as spontaneous (bacterial) peritonitis, typically occurs in the setting of ascites without an obvious source of infection.In this condition, it is theorized that organisms seed the peritoneum through a complex process involving intestinal bacterial overgrowth, translocation of bacteria across the bowel wall, and impaired host defenses.Secondary peritonitis results from a breach in the integrity of the gastrointestinal or genitourinary tract and may involve bacterial or fungal organisms.Tertiary peritonitis occurs after treatment of secondary peritonitis has failed, and signs and symptoms of peritonitis persist [7].
Fungal infection is usually overlooked, with a delay in diagnosis, or passes without being noticed.This low index of suspicion might be due to a lack of signs of infection such as fever, or to the absence of the classic picture of peritonitis.Sometimes, clinical presentations of spontaneous fungal peritonitis are largely indistinguishable from those of spontaneous bacterial peritonitis.Further factors contributing to fungal cultures not being performed routinely are the long period required for fungal growth and suboptimal microscopic evaluations and culture [8].
This study was conducted on 50 patients with decompensated chronic liver disease, admitted to Al-Shabrawishi hospital.Their ages ranged from 36 to 76 years with a mean age of 60.96±9.08.Twenty-nine (58%) of them were males and twenty-one (42%) were females.These patients were classified into two groups:  Group I: Included 25 patients with cirrhotic ascites had spontaneous peritonitis (SP group). Group II: Included 25 patients with cirrhotic ascites had no spontaneous peritonitis (non SP group).
Spontaneous peritonitis is diagnosed when PMN count in ascitic fluid >250/ml 3 in the absence of data compatible with secondary peritonitis [9].
The present study revealed that, SP was more in males (56%) than females (44%) and it was not influenced by age.This result was in agreement with the study done in Saudi Arabian patients with non-alcoholic liver cirrhosis and they stated that SBP was frequent in males and was not affected by the age [10].
Analysis of the results showed that abdominal pain, hepatic encephalopathy and melena were the only significant clinical presentations (chief complaint) in patients with SP compared to non SP group (52%, 40% and 8% respectively).These results are not in cop with a study which stated that melena was 60% from clinical presentation [11], this may be due to the wide spread prophylactic band ligation of esophageal varices nowadays.
Fever was detected in 76% of SP group and this was highly statistically significant as a symptom suggestive of SP.One study reported that, fever was detected in 66% of SBP cases [12], which was different to the result of the present study.
Abdominal pain was detected in 52% of SP group.This was lower than the results elicited by a study which stated that, abdominal pain was detected in 75% As regard gastrointestinal bleeding, it was found that, 20% of SP patients had an attack hematemsis and melena before the episode of SP with no significant difference from non SP group (24%).
Our results are in agreement with the study which stated that spontaneous fungal peritonitis associated with cirrhosis to be a rare process, as evidenced by the small number of cases reported in the literature, this low frequency of fungal infection in cirrhotic patients may be due to the lack of prolonged neutropenia and the presence of adequate numbers of functional neutrophils required for normal fungal host defense in the circulation [19].
We found no significant differences between patients with and without fungal infection as regards the clinical judgment, which is in agreement with one study [8].
In this study, increased ascitic fluid lymphocytes were seen among patients with fungal infections.This is in agreement with one study [20] which added that lymphocytes predominance may be a clue to non-bacterial causes, with consideration of a possible spontaneous fungal peritonitis.
Also, we showed that the two cases of spontaneous fungal peritonitis had a clinical picture of spontaneous bacterial peritonitis but with neutrocytic negative bacterial culture ascites, supporting that this variant of spontaneous bacterial peritonitis is also a feature of fungal peritonitis.
We found no significant correlation between the fungal infection of ascitic fluid and laboratory investigation, ascitic fluid parameters, MELD score and prophylactic antibiotics.This was in contrast with a study which reported that the use of prophylactic antibiotics, low ascitic fluid protein (<1 g/dl), a higher MELD score were found to be independently associated with fungal infections [9].Moreover, patients with multiple risk factors were more predisposed to such infections.It has been postulated that these prophylactic antibiotics suppress or change the normal intestinal flora leading to fungal overgrowth; these organisms then migrate across the intestinal wall to reach the peritoneal cavity and cause fungal peritonitis and dissemination.This may be explained by the few number of SFP cases.
The 3-months survival rate was lower in the SP group especially SFP cases than NON SP group (44%, 88%) respectively.This is in agreement with the study which showed a higher mortality rate in cirrhotic patients who developed spontaneous fungal peritonitis [21].
Advanced hepatic cirrhosis, disseminated fungal infection, and delayed diagnosis may contribute to these high mortality rates.Thus, there should be an increased awareness of fungal infection in patients with advanced cirrhosis, as delayed diagnosis and treatment may lead to serious consequences [9].The small number of patients in this series is one of the limitations of our study.Further studies with larger populations are needed on fungal infections, with special attention given to high-risk patients with ESLD.

Conflicts of interest: None
Ethical approval: The protocol of the study was approved by the ethical committee of Faculty of Medicine, Benha University.Informed consents were obtained from all patients.

Table (3): Comparison between non SP and SP group regarding 3 months survival rate.
Comparison between non SP and SP group regarding ascitic fluid analysis and culture.
*:Chi-square test [13]P cases[11].This difference could be explained by a study which summarized that, the clinical picture of SP is extremely broad and very variable and a very high degree of clinical suspicion is required for diagnosis[13].Ascitic fluid fungal culture was positive in only two cases of SP group and negative in all cases of non SP group.The isolated organism was Candida albicans.The ascitic fluid bacterial culture of the two cases of spontaneous fungal peritonitis was negative (culture negative neutrocytic ascites).