Intestinal Parasitic Infections among Egyptian Patients with Chronic Liver Diseases at Zagazig University Hospital

lamblia copro-antigens detections was performed. Results: Parasitic infections among both studied groups were (47%), comprising (58%) in CLD and (36%) in non-CLD as follow: Cryptosporidium (28%, 14%), Giardia (15%, 12%), mixed (5%, 3%), E.histolytica spp. (5%, 4%), Blastocystis hominis (3%, 1%) and H. nana (1%, 2%) respectively. The sensitivity, specificity, PPV, NPP and accuracy of ELISA regarding Giardia and Cryptosporidium infections were (100% and 89.6%, 97.5% and 100%, 87.9% and 100%, 100% and 96.6%, 97.9% and 98.4%) respectively. In GI, most giardial cases had normal ALT and AST levels (74%, 63%), but elevated in cryptosporidial infection (59%, 66%) respectively, with statistically significant difference. Conclusion: Presence of intestinal parasitic infections; mainly Cryptosporidia and Giardia protozoa among CLD patients was striking when compared to diarrheic non-CLD control group and this may be attributed to impaired immune status.


INTRODUCTION
Chronic liver diseases (CLD) encompass a major health problem worldwide.It is one of the major causes of death that has a year-onyear rising incidence [1].CLD  It is well-known that the liver has a chief role in immunity, so patients with CLD usually suffer from defects in the immune system and are highly susceptible for various microbial infections; viral, bacterial, and parasitic infections.Diminished immune response in CLD may increase the vulnerability to catch enteric parasitic infections.Both humoral and cellmediated immunity are depressed which increase with the advance of the diseases.Those patients typically have alterations in the enteric flora, reduction in serum bactericidal, opsonic activity, complements, and fibronectin levels [7].
Patients with CLD are susceptible to a wide spectrum of parasitic infections such as opportunistic protozoa as Giardia lamblia, Entamoeba spp., Blastocystis hominis, Cryptosporidium spp., Cyclospora, Isospora belli and Microsporidia.Patients with parasitic infections suffer mostly from diarrhea, nausea, vomiting that may be aggravated to dehydration and oedema due to hypovolemia.Most of these manifestations are sharable with advanced liver disease.However, enteric protozoa as Cryptosporidium species and Giardia lamblia are the most conjoint worldwide intestinal protozoa affecting man.In developing countries cryptosporidiosis; as an opportunistic infection, hits mainly immunocompromised hosts having direct impact on chronic debilitating diseases, extending from causing only watery diarrhea to biliary tract affection and dysplastic changes of liver parenchyma [11,12,13].Giardiasis causes fatty, offensive, foamy diarrhea, frequently with nausea, gurgling, bloating and weight loss resulting from fat and sugar malabsorption.Chronicity may accomplish different degrees of severity of diarrhea [14].Successful recognition and management of parasitic infections result in avoidance of the complications of diarrhea; as electrolyte disturbance, dehydration, and the expected biliary tract involvement which worsen liver status [11,13].
The current study focuses on the most striking parasitic infections among non-admitted patients with CLD in comparison with non-CLD; as a cause of diarrhea or other gastrointestinal tract (GIT) complaints that attract our attention to compile conventional microscopic with confirmatory RIDA-ELISA diagnosis for cryptosporidiosis and giardiasis.

Parasitological study:
In total, 3 successive fresh stool specimens from each case of 190 enrolled-patients were examined at the Parasitology laboratory of Medical Parasitology Department, Faculty of Medicine, Zagazig University.Stool samples were collected in clean sterile cups.After macroscopic examination of fecal samples including color, odor, special characters, and consistency (watery, fatty 'foamy', soft 'lacking fibers' ,mucoid, or bloody) and for the presence of living adult worms, proglottids of cestoda or undigested food, each stool sample was divided into two portions for the following investigations: (1) Microscopy: The first portion was examined microscopically by direct saline; wet mount preparation and/or iodine mounts to detect ova, cysts or trophozoites of parasites; and then concentration by formolethyl acetate technique (2) ELISA: The second portion was divided into 2 parts and preserved frozen at −20 °C for further processing of the most striking parasitic infections detected by microscopy; the copro-antigens of both Giardia RIDASCREEN®Art.No.: C1101; and Cryptosporidium RIDASCREEN® Art.No.: C1201 [R-Biopharm, Germany] parasites using RIDA-ELISA enzyme immunoassay [21].The test procedures were performed according to the instructions of the manufacturer.On adding stool specimens, antigens bound to microplates coated with Giardia and Cryptosporidium specific purified monoclonal antibodies followed by addition of conjugate.If any of these 2 protozoa were present in the specimen, a sandwich complex was formed and on addition of the substrate, there was a change in the color of the well of the plate from colorless to blue.After adding the stop reagent, the color changed from blue to yellow.The absorbance of the fecal samples was read within about 10 minutes at a wave length 450 nm using an ELISA micro-titer plate reader [Immunoskan-MS, Biological Diagnostic Supplies Limited, UK].The cut-off value of the test was measured by adding 0.15 absorbance units to the measured absorption of the negative control.Samples were considered positive if their absorbance values were more than 10% above the calculated cut-off value.The samples were considered negative if the absorbance value was lower than 10% below the calculated cut-off value.

Laboratory investigations:
Measuring the liver enzymes mainly alanine transaminase (ALT) and aspartate transaminase (AST) as their elevations denote hepatocellular injuries.Normal reference range for (ALT) is 0 to 45 IU/L and (AST) is 0 to 35 IU/L [22].

Abdominal Ultra-Sonography (U/S):
Ultrasound used for assessment of the liver status, diagnosis of cirrhosis, detection of ascites and exclusion of hepatic focal lesions.

Statistical analysis:
All data were collected, tabulated and statistically analyzed using SPSS 22.0 for windows.Continuous Quantitative variables were expressed as the mean ± SD and categorical qualitative variables were expressed as absolute frequencies ''number'' & relative frequencies (percentage).Categorical data were compared using the Chi-square (χ 2 ) test.Probability (P value) was considered statistically significant when <0.05 [23].

RESULTS
Concerning the demographic criteria of the studied groups, there were insignificant statistical differences between them regarding the gender, age and residence.Male gender represented the major percentage of both groups and also, most of them were rural inhabitants, Table (1).
Most cases of Giardia lamblia infection in CLD group had normal levels of ALT and AST (74%, 63%), respectively while most cases of Cryptosporidium spp.infection had elevated ALT and AST (59%, 66%) respectively, with significant difference between the two infected groups, Table (8).
Concerning the RIDA-ELISA results, the negative control reading for Giardia lamblia was 0.042.So, the cut-off value was (0.042+ 0.15) =0.192.Result was considered positive if the reading was 10% more than the cut-off value = 0.192+0.019=0.211.The negative control reading for Cryptosporidium was 0.05.So, the cut-off value was 0.2.Result was considered positive if the reading was more than (0.2+0.02) =0.22.
RIDA-ELISA detected more cases of Giardia lamblia infection than microscopy, 4 cases were not detected by microscopy, while in Cryptosporidium spp.infection ELISA failed to detect five cases which were detected by microscopy.So, the sensitivity, Specificity, PPV, NPP and diagnostic accuracy of ELISA regarding Giardia and Cryptosporidium infections were as follow (100% and 89.6%, 97.5% and 100%, 87.9% and 100%, 100% and 96.6%, 97.9% and 98.4%) respectively, Table (9).In the present study, after history taking, clinical, sonographic and laboratory confirmation, nearly half of CLD non admitted cases were represented by liver cirrhosis (45%) followed by chronic hepatitis C [HCV] (25%), and chronic hepatitis B [HBV] in (14%), while non-alcoholic fatty liver (11%) and autoimmune hepatitis (5%).These results were not in accordance with that reported by Sabah and El-Lessi [27] who found that liver cirrhosis was represented in 36% of adult patients with CLD while non-alcoholic fatty liver was represented in 64% of those patients.Also, our results disagree with that obtained by Elshazly et al. [28] who reported that the etiologies of CLD in 50 children were biliary atresia in (24 %), autoimmune hepatitis in (22 %), chronic HCV in (10 %).Also Larrosa-Haro et al.Gastrointestinal symptoms are common in CLD and their pathophysiology may be related to many factors as the severity of liver disease, psychological distress, and GIT dysfunction.Delayed small bowel transit in patients with liver cirrhosis may lead to overgrowth of microorganisms, which could contribute to the symptoms of abdominal pain and diarrhea [33].
In our study, all GIT manifestations were significantly different between CLD and non-CLD patients except for abdominal pain.This agreed with Fritz and Hammer [34] who found a significant difference between the cirrhotic patients and the control group regarding the GIT manifestations.The most common GIT symptoms reported were abdominal bloating, abdominal pain and diarrhea.
Human intestinal parasitic infections are series of public health problems in developing countries [35].Even in communities where adequate sanitary conditions and higher educational levels are present, some of these intestinal parasites play an important role in causing diseases especially in specific groups such as immunocompromised patients [10].This study found an intimate relationship between the occurrence of chronic hepatic insult and the presence of intestinal parasitic infection in such patients.We found that CLD patients had higher rates of enteric parasitic infections than non CLD patients this could be explained by the major role of the liver in protection against infection.However, the most evident parasitic infections were Cryptosporidium spp.and Giardia lamblia protozoa.This may be attributed to the extension of cryptosporidiosis pathology to the biliary tract and liver especially in immunocompromised [13], and the duodenal habitat of giardiasis, though superficial, but it may aggravate biliary tract and even hepatic affection by causing dehydration and fatty diarrhea.

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and Delghani et al. [30] found that biliary atresia was the most evident etiology of CLD in pediatric patients (27.8 and 27.7%, respectively).Moreover, Chaabouni et al. [31] documented biliary cirrhosis with different etiologies where biliary atresia was the most evident cause (40 %) then (17 %) for each of post-hepatitic and metabolic cirrhosis.These differences in the disease distribution among the previous mentioned studies may be attributed to the different etiologies of CLD among the different ages (children, adolescents and adults) [32].

Table ( 1
): Demographic data of all patients in CLD and Non-CLD studied groups.
*Significant differences at P-value ˂ 0.05.

Table ( 6
): Characters of diarrheic stool among diarrheic cases in the studied groups: *Significant differences at P-value ˂ 0.05.

Table ( 7
): Distribution of intestinal parasites among the studied groups:
*Significant differences at P-value ˂ 0.05.