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. 2021 May 6;45(4):1049–1054. doi: 10.1007/s12639-021-01351-8

Prevalence of Toxoplasma gondii 36-KDa antigen and chronic Hepatitis C: another evidence of an Association

Basma M Mohamed 1, Mohamed M Omran 2, Mohamed A Abdelrazek 1, Abdelfattah M Attallah 1,✉, Mohamed El-Far 3
PMCID: PMC8556424  PMID: 34789988

Abstract

In chronic hepatitis C (CHC), Toxoplasma gondii infection can lead to more severe diseases and is capable of changing the disease course. Former studies were concerning anti-T. gondii IgG/IgM seroprevalence in CHC patients regardless the antigenic proteins that are associated with active infection. Therefore, this study aimed to evaluate association between prevalence of 36-KDa T. gondii antigen (TAg) and both CHC progression and liver and viral biochemical parameters. One hundred-twenty five CHC patients (65 with fibrosis and 60 with cirrhosis) and forty healthy controls constituted this study. Demographics and clinical data were collected. Both TAg and HCV-NS4 were identified using ELISA. In contrast to healthy controls (0%), both seropositivity (P = 0.043) and mean serum level (P = 0.025) of TAg were higher in cirrhotic patients (43.3 %; 1.2 ± 0.2 ng/mL) compared to fibrotic patients (26.2 %; 0.7 ± 0.1 ng/mL). T. gondii infection was significantly (P < 0.05) associated with liver and viral biochemical parameters including increased ALT and AST activities, total bilirubin and AFP levels and decreased albumin and platelets count levels. Interestingly, TAg positivity were associated with elevated HCV-NS4 level compared to negative TAg patients (212.5 ± 25.3 vs. 133.9 ± 17.4 µg/mL (P = 0.026); r = 0.559 (P < 0.0001)). In conclusion, this study highlighted association between T. gondii parasitemia and CHC progression since TAg was more prevalent among cirrhotic than fibrotic patients and healthy controls. The presence of TAg was associated with impaired liver functions and increased HCV-NS4 levels. Further studies are needed to define the mechanism of this association.

Keywords: Toxoplasma gondii, 36-KDa T. gondii antigen, Chronic hepatitis C, Progression, Fibrosis, Cirrhosis

Introduction

Globally, hepatitis C virus (HCV) chronic infection affected about 71 million people, and annually about 400,000 die from HCV-related liver diseases (Tsertsvadze et al. 2020). HCV has emerged as a leading cause of advanced fibrosis, cirrhosis and hepatocellular carcinoma. Epidemiologic and clinical studies have identified many risk factors associated with chronic hepatitis C (CHC) progression including older age, alcohol consumption, diabetes mellitus, obesity, aflatoxin and hepatitis B coinfection (Abbas and Abbas 2018).

Toxoplasma gondii (T. gondii) is protozoan parasite that was discovered over 100 years ago. It caused a zoonotic infection called toxoplasmosis. Worldwide, it is considered as the most successful parasitic organism that able to infect all warm blooded animals including about 2.3 billion people (Mose et al. 2020). Although it is known that toxoplasmosis rarely assist to various hepatic pathologies, like granulomatous hepatitis, CHC patients particularly who have liver cirrhosis are subjected to several immunity and cellular disorders. Thus, toxoplasmosis can lead to more severe and frequent diseases in CHC patients and is capable of changing the disease course (Alvarado-Esquivel et al. 2011; El-Nahas et al. 2014). According to previous comparatively small clinical and epidemiological studies, some results highlighted potential association between T. gondii infection and liver pathologies, such as cirrhosis, jaundice, necrosis, hepatitis, granuloma and hepatomegaly (Weitberg et al. 1979; Karasawa et al. 1981; Botterel et al. 2002; Shapira et al. 2012).

In CHC patients, there is limited information from few studies about the association of HCV and toxoplasma infections (El-Nahas et al. 2014). Also, these studies concerned with determination of anti-T. gondii IgG/IgM seroprevalence in CHC patients without taking it into account T. gondii antigenic proteins that are associated with active infection (Alvarado-Esquivel et al. 2011; El-Nahas et al. 2014). Thus, this study aimed to evaluate the association between a 36-KDa T. gondii antigen (TAg) (Attallah et al. 2006) prevalence and CHC progression in patients with liver fibrosis and cirrhosis related to CHC. We also aimed to evaluate the association of T. gondii antigenic levels and liver biochemical parameters and HCV viral parameters.

Materials and methods

Study population

This study included 125 CHC patients, 65 with liver fibrosis (non-cirrhotic/early-stage) and 60 with cirrhosis (late-stage), and 40 healthy individuals as controls matched by gender and age. Patients were recruited from Tropical medicine unit, Mansoura University Hospital, Egypt. Clinical examination for all subjects was performed. Appropriate written informed consent and institutional ethical clearance was obtained from each patient.

Samples collection and laboratory assays

Within two hours after blood withdrawal, serum samples were processed and stored at − 20 °C until use. Another blood part was added to KEDTA tubes for platelets count determination using automated hematology analyzer (Sysmex Corporation, Kobe, Japan). Serum samples were tested for alanine and aspartate transaminases (ALT, AST), total bilirubin, and albumin on an automated biochemistry analyzer (A15, Biosystem, Spain). Alpha-fetoprotein (AFP) serum levels were measured by using commercial ELISA kits (Biomedica, Sorin, Italy). Samples were also tested for HCV-NS4 according to Attallah et al. (2012).

Detection ofT. gondiiantigen

Detection of TAg using indirect ELISA was previously discussed in details by Attallah et al. (2006). Briefly: at 4 °C overnight, polystyrene microtiter plates were coated with diluted serum samples (1:800) in 50 mM carbonate buffer (pH 9.6). After washing, microtiter plates were incubated for one hour at room temperature with 200 mL/well of bovine serum albumin (0.5 %) in phosphate buffer saline (PBS; pH 7.4). After washing, specific diluted (1:100 in PBS) anti-T. gondii antibody (ABC Diagnostics, New Damietta, Egypt) was added and incubated at 37 °C for two hours. After washing, diluted (1:400 in 0.2 % BSA in PBS) anti-rabbit IgG alkaline phosphatase conjugate (Sigma, USA) was added and incubated at 37 °C for 1 h. After washing, the amount of coupled conjugate was determined by incubation with 1 mg/mL p-nitrophenyl phosphate in substrate buffer for 30 min at 37 °C. The reaction was stopped by adding NaOH (3M) and the absorbance was read at 405 nm.

Statistical analysis

Both GraphPad Prism (version 6.0) and SPSS Inc., Chicago (version 17.0) were used for all statistical analyses. Data was expressed as mean ± standard deviation (SD), whereas categorical variables were expressed as numbers. Differences between independent groups were compared with Pearson’s two-tailed Χ2, ANOVA, or student t tests as appropriate. For correlation evaluations, Pearson’s correlation coefficient was used.

Results

Biochemistry of CHC patients and controls at baseline

Healthy controls were age- and gender-matched with CHC patients. As shown in Table 1, compared with the normal controls, CHC patients had higher activities of ALT (P = 0.0001) and AST (P = 0.0001) and serum levels of total bilirubin (P = 0.002) and AFP (P = 0.003). Whereas CHC patients had lower (P = 0.001) serum albumin levels and platelets count.

Table 1.

Characteristics at baseline of patients and controls

Variables Normal Fibrosis Cirrhosis P value
Age (years) 42.8 ± 5.1 45.8 ± 8.2 54.7 ± 8.3 0.237
Gender (male/female) 24/16 45/20 38/22 0.606
AST (U/L) 34.2 ± 5.4 54.1 ± 12.9 68.9 ± 29.6 0.001
ALT (U/L) 35.1 ± 6.1 56.1 ± 13.9 71.5 ± 26.4 0.0001
Albumin (g/dL) 4.1 ± 0.2 3.6 ± 0.8 2.9 ± 0.3 0.001
Bilirubin (mg/dL) 0.7 ± 0.1 1.4 ± 0.2 2.2 ± 0.3 0.002
Platelet count (×109/L) 242.7 ± 77.6 176.3 ± 63.7 127.5 ± 53.4 0.001
AFP ( U/L) 2.2 ± 0.2 16.5 ± 4.1 19.9 ± 6.4 0.003
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Data were expressed as mean ± SD. Significant difference was determined using X2 or ANOVA tests as appropriate. P < 0.05 is significant. AST aspartate aminotransferase, ALT alanine aminotransferase, AFP alpha fetoprotein

T. gondiiparasitemia was associated with CHC progression

The potential T. gondii impact on CHC progression was assessed by detecting circulating TAg, using ELISA, in cirrhotic patients compared to fibrotic patients and healthy controls. In contrast to healthy controls (0 %), TAg was detected in 43.3 % (26/60) of cirrhotic patients compared to 26.2 % (17/65) of fibrotic patients (P = 0.043; Fig. 1a). Quantitatively, the mean TAg serum level in cirrhotic patients (1.2 ± 0.2 ng/mL) was also significantly (P = 0.025) higher than fibrotic patients (0.7 ± 0.1 ng/mL) (Fig. 1b). Interestingly, T. gondii parasitemia was significantly (P < 0.05) associated with liver biochemical parameters including increased ALT and AST activities, increased total bilirubin and AFP and decreased albumin serum levels and platelets count (Table 2).

Fig. 1.

Fig. 1

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Active T. gondii infection and progression of CHC. In contrast to normal controls, TAg a detection rate and b serum levels were elevated in patients with liver cirrhosis than patients with liver fibrosis. Seropositivity of TAg was associated with c the increase in age but d there was no significant difference between males and females

Table 2.

Impact of T. gondiiparasitemia in laboratory characteristics of CHC patients

Variables CHC patients P value
Negative T. gondii Positive T. gondii
AST (U/L) 46.2 ± 18.3 70.4 ± 25.1 0.006
ALT (U/L) 44.7 ± 17.9 72.8 ± 27.8 0.005
Albumin (g/dL) 3.9 ± 0.1 2.9 ± 0.3 0.011
Bilirubin (mg/dL) 1.3 ± 0.1 2.3 ± 0.2 0.016
Platelet count (x109/L) 216.5 ± 39.1 111.5 ± 15.9 0.002
AFP (U/L) 16.2 ± 1.8 28.2 ± 5.4 0.025
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Data were expressed as mean ± SD. Significant difference was determined using student t test. P < 0.05 is significant. CHC chronic hepatitis C, AST aspartate aminotransferase, ALT alanine aminotransferase, AFP alpha fetoprotein

Elevated TAg levels were associated with old age and HCV-NS4 load

Among CHC patients, T. gondii parasitemia was significantly (P = 0.005) increase with the increase in patients ages (Fig. 1c). The seropositivity of circulating TAg was found to be higher in females (38.1 %) than males (32.5 %) but without significant difference (P = 0.536; Fig. 1d). Interestingly, it was found patients positive for TAg were associated with elevated HCV-NS4 level compared to negative TAg patients (212.5 ± 25.3 vs.133.9 ± 17.4 µg/mL; P = 0.026) as shown in Fig. 2a. Additionally, there was strong positive significant correlation between T. gondii positivity and HCV-NS4 level (r = 0.559 and P < 0.0001; Fig. 2b).

Fig. 2.

Fig. 2

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Active T. gondii infection and HCV-NS4 load. a HCV-NS4 serum levels increased in T. gondii infected than non-infected patients. b There was significant positive correlation between HCV-NS4 and TAg serum levels.Significant difference was determined using student t test. P < 0.05 is significant. Correlation was evaluated using Pearson correlation coefficient

Discussion

Owing to depressed immune system, patients with chronic liver diseases, including CHC, are often highly susceptible to parasitic, viral and bacterial infection (El-Shazly et al. 2015). T. gondii seroprevalence among immunocompromised patients is high and latent infections reactivation in them can be life-threatening (Abd El-Rehim El-Henawy et al. 2015). Attallah et al. (2006) demonstrated the presence of highly reactive TAg (36-kDa) in sera of women with toxoplasmosis. This study aimed to evaluate the association between prevalence of this TAg and CHC progression and to evaluate the association of antigenic levels with liver and viral biochemical parameters.

Our results confirmed the potential T. gondii impact on CHC progression as TAg was detected in 43.3 % of cirrhotic patients compared to 26.2 % of fibrotic patients (P = 0.043). Also, mean TAg serum level in cirrhotic patients (1.2 ± 0.2 ng/mL) was significantly (P = 0.025) higher than fibrotic patients. In immunodeficient patients, T. gondii diagnosis based on antibodies detection has important limitations, as underlying immunosuppression alters antibody production and its kinetics (El-Sayed et al. 2016). Therefore, TAg demonstration in blood providing definitive disease proof.

Liver diseases are accompanied by depression of both cell-mediated and humoral immunity, with significant inability of invading pathogens management. During T. gondii parasitemia, the liver is one of the most important organs affected and involved (Geng et al. 2000). Former studies reported the presence of tissue cysts and tachyzoites within the sinusoidal hepatic capillaries and inside the hepatocytes. When such parasite invades hepatic cells, it can lead to its DNA damage, shape distortion and disturbances in metabolic activity (Sukthana et al. 2003; Ribeiro et al. 2004). It is known that T. gondii locates the liver and leads to pathological changes that progress to necrosis, hepatitis, granuloma and hepatomegaly (Montoya and Liesenfeld 2004). Further, significant relationship between T. gondii antigens and hepatic stellate cells (HSCs) number, which may represent active HSCs role in the pathobiology of T. gondii-related hepatitis (Atmaca et al. 2013).

Interestingly in this study, T. gondii parasitemia was significantly (P < 0.05) associated with alteration of liver related biochemical parameters (ALT, AST, bilirubin, albumin, AFP and platelets count). These findings were in agreement with previous reported findings (Dawood and Mahmood 2012; El-Sayed et al. 2016). Liver injury is well-established toxoplasmosis complication, as this infection can cause liver cells focal necrosis, swollen endothelial cells, cholestasis and round cell infiltration in the portal areas. Protein fractions of liver enzymes (ALT, AST) varied based on inflammation intensity induced by T. gondii infection (El-Sayed et al. 2016). T. gondii mechanisms to induce hepatic histological changes and damage could be due to direct parasite proliferative effect on liver tissues, causing cell death and tissue damage, or due to infection indirect effect because of excessive parasite immunological response (Ferro et al. 1999).

In this study, T. gondii infection was significantly (P = 0.005) increase with the increase in patients ages. Although there are other studies reported such association between T. gondii seropositivity and age (Mendy et al. 2015; Sadiqui et al. 2018), further studies are needed to assess in details whether, is there any significant association exists between age and toxoplasmosis infection. Interestingly, we found that patients positive for TAg were associated with elevated HCV-NS4 level compared to negative TAg patients (212.5 ± 25.3 vs. 133.9 ± 17.4 µg/mL; P = 0.026). Some studies reported significant correlation between T. gondii specific antibodies (IgG, IgM) and HCV viral loads in terms of HCV- antibodies and RNA (El-Nahas et al. 2014). In the line with these findings, we reported here, for the first time, that serum TAg levels were significantly (P < 0.0001) correlated with serum HCV-NS4 (r = 0.559).

Conclusions

This study highlights the association between T. gondii parasitemia and chronic liver diseases progression. For the first time, prevalence of a 35-KDa TAg was evaluated in CHC patients and TAg was more prevalent among cirrhotic than in fibrotic patients and healthy controls. This comorbidity was confirmed since existence of TAg was associated with impaired liver functions, decreased serum albumin, decreased platelets count, increased AFP and increased HCV-NS4 levels. Further studies are needed to define the mechanism of this association.

Acknowledgements

All authors would like to thank Prof. Khaled Farid, Faculty of Medicine, Mansoura University, Mansoura, Egypt who help in providing samples and data. Also, we would like to thank all Biotechnology Research Centre participants who cooperated in the experimental work.

Ethical standards

This study is presented and designed by authors in accordance with the ethical standards.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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