Genotypic Prevalence of Cryptosporidium in Egyptian Patients with Liver Cirrhosis

Introduction

Cryptosporidium is a protozoan parasite that infects the epithelial cells of the small intestine causing diarrheal illness in humans. It has a worldwide distribution and is considered an emerging zoonotic parasite with reservoirs in cattle, domestic animals and in fecally contaminated environments.¹

Transmission via the fecal-oral route is due to the ingestion  of  Cryptosporidium oocysts  through  the

consumption of contaminated food or water and through direct person to person or animal-to-person contact. These small-sized oocysts (4-6 μm) are highly resistant to common household disinfectants.^2,3

Diarrhea caused by Cryptosporidium is usually mild and self-limited in patients with good immunity. However, it can become severe, long-standing, and life-threatening diarrhea in immunocompromised patients.⁴

Previous studies demonstrated that severe liver injury and liver failure are closely associated with reduced cellular immunity.⁵ In one study 32% of patients with hepatocellular carcinoma and diarrhea harbored cryptosporidial infection, compared to 22% among patients who had liver cirrhosis without ascites and 36% in patients who had liver cirrhosis with ascites.⁶

The diarrhea is associated with multiple infectious agents; thus, Cryptosporidium diagnosis should rely on laboratory investigations. Despite the major advances in diagnostic techniques, routine cryptosporidial infection diagnosis is still lacking in most clinical laboratories.⁷Based on the characteristic morphology of the oocysts, few microscopic techniques are commonly used for Cryptosporidium detection in feces. However, a complex clinical specimen like feces makes identification a challenging task. It is also time-consuming, tedious and requires great experience.⁸

Neither microscopy nor coproantigen assays can achieve that purpose. Therefore, many PCR-based assays were developed to detect and characterize Cryptosporidium DNA in feces.⁹

The use of molecular epidemiologic tools has provided new insights into the diversity of Cryptosporidium infection among humans and animals.¹⁰ So far, 26 Cryptosporidium spp have been described.¹¹ Most human cases are caused by C.hominis and C. parvum.¹⁰ Several other species are seen in humans at a lower frequency, including C. meleagridis, C.felis, C. canis, C. ubiquitum, and C. cuniculus.¹⁰ Another new species, C. viatorum, has been described in 10 travelers returning to Great Britain from the Indian subcontinent at 2010.¹²

This study was done for detection of the molecular prevalence and genotyping of Cryptosporidium spp. in fecal samples of Egyptian patients suffering from livercirrhosis. Exploration of the possibility of an association between Cryptosporidium spp and grades of liver cirrhosis and assessment of their demographic, environmental and clinical data for risk of infection was done.

Material and Methods

Study population

This cross-sectional study was carried out on a study group of 60 patients (33 males and 27 females) with liver cirrhosis (HCV and HBV positive), and a control group of 60 subjects (32 males and 28 females) free of any known liver diseases as a control group, matched for ethnicity, sex, and age.

Stool samples were collected from September 2015 to January 2017.

The diagnosis of hepatitis C virus (HCV) was made by positive anti-HCV and reverse transcription-polymerase chain reaction (PCR-RT), and the diagnosis of hepatitis B virus (HBV) was made by positive hepatitis B surface antigen (HBsAg) and PCR. Liver cirrhosis was diagnosed by patient history, clinical examination, abnormal liver function tests, and abdominal ultrasound. The patients were further classified according to the modified Child-Pugh score, which consists of five clinical features, and was used to assess the prognosis of liver cirrhosis based on the degree of ascites, the plasma concentrations of bilirubin and albumin, the prothrombin time, and the degree of encephalopathy. The Child-Pugh score corresponds to the total points for each item. According to the sum of these points, patients can be categorized into three grades. A total score of 5 to 6 is considered grade A (well-compensated disease), 7 to 9 is grade B (significant functional compromise), and 10 to 15 is grade C (decompensated disease).

Exclusion Criteria: All known causes of liver diseases rather than (HCV and HBV infection) as well as patients suffering from immunosuppressive diseases (e.g., diabetes mellitus and HIV infection) or with comorbidity were excluded based on analytical, clinical, and epidemiological data.

Participants were recruited from the outpatient clinics of Hepatology, Gastroenterology, and Infectious diseases Department, Al-Azhar University Hospitals, Cairo, Egypt. The study conformed to the medical ethics of the 1975 Declaration of Helsinki, and all participants provided written informed consent. 

Sample and data collection

A single fecal sample was obtained from each subject and was collected in a dry, clean and leak-proof labeled plastic container. A questionnaire containing demographic, clinical and environmental data was obtained with each sample. The designed questionnaire was modified from Da’as ¹³ and Mor et al.¹⁴

Work plan and sample processing

Macroscopic examination of all collected fecal samples was conducted at first regarding physical characteristics, presence of blood, mucus and macroscopic parasite elements. All collected fecal samples were examined coproscopically, then subjected to copro-molecular assays.

Each sample was divided into three portions:

• A small part of each specimen for direct smear examination using a microscope.
• Another part of the specimens was preserved in tight containers using formol-saline 10 % as a fixative for coproscopic examination and staining with acid-fast stain.
• The rest of the specimen was stored at -20^oc in Eppendorf tubes for molecular study.

Coproscopy was carried out in the Diagnostic and Research Unit of Parasitic Diseases (DRUP), Medical Parasitology Department, Kasr Al-Ainy Faculty of Medicine, Cairo University, Egypt. and copro-nPCR assay was held in Lab of Molecular Medical Parasitology (LMMP), Medical Parasitology Department, Kasr Al-Ainy Faculty of Medicine, Cairo University, Egypt.

I-Direct wet smear.¹⁵ Wet smears were examined directly using saline and a drop of iodine was added to another slide.

II. Concentration using modified Ritchie’s biphasic method.

This is a modification of formol ethyl-acetate concentration method according to Garcia.¹⁵

III. Specialized staining of Cryptosporidium spp.Oocysts Cold kinyoun’s AF stain.

All fecal samples were permanently stained to detect cryptosporidial oocysts using cold kinyoun’s AF stain according to Garcia. ¹⁵

IV. Copro-nPCR / RFLP assays.

i. Genomic DNA extraction from stool samples.

This was done using Favor Prep stool DNA isolation Mini Kit (Favorgen Biotech corporation ping-Tung 908, Taiwan, Cat. No. FASTI001) following the manufacturer’s instructions.

ii. Copro n-PCR- RFLP assay. ¹⁶

 Amplification of the extracted genomic DNA was done using the nPCR assay targeting the Cryptosporidium cowpgene. The nPCR was done using the 2 sets of 2 pairs of primers described earlier,¹⁶ where the first set primers were used to produce a DNA template for the nested reaction. The second set of primers was used to anneal internally within the previously obtained amplicon (769 bp) in order to increase the specificity of Cryptosporidium detection. PCR products of 2ry PCR (533 bp) were visualized on 1.5% agarose gel after ethidium bromide staining using gel electrophoresis and visualized on a UV transilluminator.

The PCR products of nPCR were digested by RasI restriction enzyme (Thermo Scientific) and the resulting restriction fragments according to manufacture instruction and resolved using 3% Metaphor agarose after ethidium bromide staining.

Statistical Analysis

Data were collected, revised, coded and entered into the Statistical Package for Social Science (IBM SPSS) version 23. The quantitative data were presented as mean, standard deviations and ranges when parametric. Also, qualitative variables were presented as numbers and percentages. The comparison between groups regarding qualitative data was done by using the Chi-square testand/or Fisher exact test when the expected count in any cell found less than 5.The comparison between two groups regarding quantitative data and parametric distribution was done by using an Independent t-test while more than two groups were compared by using the One-Way ANOVA test.  The confidence interval was set to 95% and the margin of error accepted was set to 5%.

RESULTS

Table 1:The prevalence of Cryptosporidium among liver cirrhotic group and control group using copro-nPCR and AF fecal microscopy.

The previous table showed that the prevalence of cryptosporidial DNA using copro-nPCR was 3.3% (2 cases) among the study group with no statistical significance (P-value was > 0.05), none of them were detected by microscopic examination of AF stained fecal smears. None of the control group were positive molecularly or microscopically for Cryptosporidium DNA or oocysts, respectively.

Table 2:  Distribution of cases of viral hepatitis (HCV and HBV),HCC and Child score among the liver cirrhotic group.

The previous table showed that among liver cirrhotic patients, 85% of patients were HCV positive and 15% were HBV positive, 48.3% (29/60) of them were suffering from HCC. The grades of liver cirrhosis were Child A, Child B and Child C 6.7%, 30%, and 63.3% respectively.

Table 3:  Distribution of Cryptosporidium detected two cases by copro-nPCR among hepatitis  (HCV, HBV) patients of the liver cirrhotic group.

Table 4:   Distribution of Cryptosporidium detected two cases by copro-nPCR among HCC patients of the liver cirrhotic group.

Table 5: Distribution of Cryptosporidium detected two cases by copro-nPCR among hepatitis (HCV, HBV) patients of the liver cirrhotic group.

The previous tables (Tables 3,4 and 5) showed that the detected two Cryptosporidium were among HCV patients, patients with HCC and Child C with no statistical significance (P-value was > 0.05).

Table 6:  Demographic and environmental data of copro-nPCR positive Cryptosporidium cases among the liver cirrhotic group.

Table 7:  Clinical data and macroscopic resultsof copro-nPCR positive Cryptosporidium cases among the liver cirrhotic group.

The previous tables (Tables 6 and 7) showed that none of the demographic, environmental or clinical data of positive Cryptosporidium cases by copro-nPCR among liver cirrhotic patients showed any significant association with Cryptosoriudium infection (P-value > 0.05).