Prevalence of Parasitic Infections Among Patients with Autoimmune Disorders in Menoufia University Hospitals, Egypt

focus on developing a parasite component that may treat autoimmune illnesses. Consequently, the goal of the study was to identify the prevalence of parasitic infections among individuals with various autoimmune disorders and how such infections affect the pathophysiology of these diseases. In the current study, A total of 154 adult patients who had a confirmed diagnosis of autoimmune disease recruited from Menoufia University Hospitals, Egypt, were included. A cross-sectional study design was conducted on the participants who were chosen for history taking, clinical examination, laboratory investigations, and ultrasonography. The results revealed that intestinal parasites such as Enterobius vermicularis , Entamoeba histolytica , Giardia lamblia , and Ascaris lumbricoides were found to be prevalent in 56.5% of the subjects in this study. Immune thrombocytopenia (ITP) patients had intestinal parasites in 100% of cases while parasites were present in 52.6% of autoimmune hemolytic anemia (AIHA) and 47.6% in rheumatoid arthritis (RA) patients. Seroprevalence of toxoplasmosis in the studied cases revealed 15.6% positive IgM and 35.1% positive IgG. The IgM-positive cases were perceived in patients with systemic lupus erythematosus (SLE), Hashimoto thyroiditis (HT), and ITP. Whereas, occurrences of IgG positivity were mainly presented in SLE then AIHA, ITP, and RA patients. Less clinical manifestations were observed in autoimmune disorder patients having intestinal parasitic infections. In conclusion, high parasitic prevalence is common in autoimmune disease patients, which alters the pathogenesis and clinical course of these illnesses.


INTRODUCTION
According to the World Health Organization, over 1.5 billion individuals, or 24% of the world's population have intestinal parasites.Tropical and subtropical regions are home to a large number of those parasites where Africa, China, the Americas, and East Asia having the highest concentrations (WHO, 2020).
The helminthic species like Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, hookworm, and Taenia species as well as protozoan species like Entamoeba histolytica and Giardia lamblia are the main parasites of worldwide public health (Haque, 2007;Lozano et al., 2012).
Many people around the world are impacted by Toxoplasma gondii.In immunocompetent hosts, the primary infection is followed by a self-limited febrile illness, but in immunocompromised individuals, such as those with HIV, it is generally known that it puts individuals at risk for developing severe toxoplasmosis (Robert-Gangneux et al., 2018).
Autoimmunity is characterized as the immune system's inability to distinguish between infections and self-antigens, which results in injury to healthy tissue (Gutierrez-Arcelus et al., 2016).
Self-reactive immune components are what distinguish autoimmune illness from autoimmunity, which also includes pathology.In many areas of the world, autoimmunity and autoimmune illnesses are both sharply on the rise.This is probably due to changes in how exposed we are to environmental variables (Miller, 2023).
In the United States, tens of millions of people are affected by autoimmune illnesses, which are thought to be the third leading cause of morbidity in the developed world (Chang et al., 2023).In Egypt, the estimated adult SLE prevalence rate was 6.1/100,000 people (Gheita et al., 2021).
Helminthic infection is typically associated with diminished cellular immunological responses and a switch from T-cell to Th2 immune responses.Additionally, a negative correlation between the prevalence of helminthic infections and autoimmune diseases was shown, raising the possibility that "helminthic therapy" may be helpful for those with autoimmune diseases.With few adverse effects, the clinical signs of autoimmune diseases can be reduced by helminthic treatment, which can also lessen the intensity of inflammatory responses.Some intracellular protozoan infections, on the other hand, skew immune responses towards the T helper 1 type and may result in the emergence of an autoimmune disease (Abdoli et al., 2022).Some inflammatory disorders have been related to a loss of helminths such as inflammatory bowel disease, asthma, atopic eczema, type 1 diabetes, multiple sclerosis (MS), and rheumatoid arthritis (RA) (Maizels, 2020).

MATERIALS AND METHODS Study Area and Period:
In this study, 154 adult participants who had confirmed diagnosis of autoimmune diseases; were recruited from Menoufia University Hospitals, Shebin El-Kom, Egypt during the period from December 2022 to May 2023.

Study Design:
A cross-sectional study design was conducted among all participants who were chosen for history intaking, clinical examination, and laboratory investigations (blood, serum, stool, and urine examination) during the research period.

Sample Size Estimation:
According to Shapira et al. (2012), it was documented that in 42% of autoimmune illness patients, serum ATxA IgG was positive, at alpha margin 0.05 and 95% confidence interval and according to the following formula [Z 2 α/2 P(P-1) /D 2 ] Z 2 α/2 = the Z score corresponding to the degree of confidence P = prevalence of the indicator D= the desired precision The estimated sample size was 154 autoimmune patients Ethical Consideration: All procedures were completed in accordance with global ethical standards.After receiving approval from the Institutional Ethical Committee of Menoufia University's Faculty of Medicine (IRB: 12-2022 PARA20).

Inclusion Criteria:
All patients (> 18 years), clinically and laboratory-confirmed autoimmune diseases according to; systemic lupus erythematosus (SLE) (Aringer et al, 2019) Simple vacutainer tubes and needles were used aseptically to take the individuals' venous blood, yielding around 2mL.All samples were delivered to Faith Alive Foundation in ice boxes from various healthcare facilities.Prior to analysis, serum was kept at -80°C after being centrifuged at 14000 rpm for 10 minutes.According to the manufacturer's instructions, kits for ELISA tests (Nantong Voyage Medical Co., Ltd., Jiangsu, China) were used to analyze serum samples for IgM and IgG antibodies against T. gondii.The approach recommended by the manufacturer was used to compute the cutoff point.A negative response was considered to be an indication that there were no detectable Toxoplasma antibodies.A positive Toxoplasma IgG/IgM response was considered to be a sign of either a current or prior infection, depending on the case (Nwachukwu et al., 2023).

Stool Sample Collection and Process:
Fresh stool samples were taken for examination to check for parasites.About 2 g of fresh feces were collected from each participant after sufficient instruction, clean dry labeled collection containers, and applicator sticks were provided.The following information was collected for each subject at the time of collection: the sample date, the participant's name, age, and sex.The stool sample was transferred to the laboratory after being preserved in 10% formalin.Following the steps outlined in the WHO standards, a 1g sample of each feces was processed and intestinal parasites were identified and detected microscopically utilizing direct wet-mount and formal-ether concentration procedures (WHO, 1992).

Study Outcomes:
The outcomes of this study, which were obtained through evaluating medical records, largely included the following: Statistical Analysis: Statistical Package for Social Science (SPSS) (version 20, SPSS Inc., Illinois, Chicago, USA) was used to enter, classify, and analyze data.
Mean and standard deviation were the terms used to describe quantitative data.Comparatively, qualitative data was expressed as numbers and percentages.Chisquared test was used to compare qualitative variables.Quantitative variables were compared between the four studied groups by analysis of variance (ANOVA test) (for data that are normally distributed) and the Kruskal-Wallis test (for data that are not normally distributed).P-value <0.05 was considered statistically significant.

RESULTS
This study documented a prevalence of intestinal parasites (56.5%) and a prevalence of toxoplasmosis (15.6% positive toxoplasmosis IgM & 35.1% positive toxoplasmosis IgG) (Table 1 & Fig. 1).The positivity of toxoplasmosis IgM among the studied cases with autoimmune disease was significantly related to the female sex, toxoplasmosis IgM positive cases were detected in SLE (37.5%),ITP (20.8%) and Hashimoto thyroiditis (20.8%).Regarding the presenting symptoms, positive cases were presented as 25% recurrent abortion, 20.8% fatigue, 20.8% ecchymosis &, purpura, and 16.7% menorrhagia while in IgM negative, they presented as fatigue 29.2%, bony aches 16.9, arthralgia 15.4% & poly arthritis 10.8%.IgM positive cases showed 20.8% big joint affection and 16.7% both small and big joint affection while IgM negative cases showed 4.6% big joint affection and 26.2% both small and big joint affection.Heart rate was significantly higher in IgM-positive cases.Affected organs were mainly joints, blood, and thyroid in IgM-positive cases meanwhile; in IgG-positive cases, the affected organs were mainly joints and blood, thyroid, lymph nodes, and glands (Table 2).
The positivity of IgG was significantly related to the female sex and also showed a higher level of systolic and diastolic blood pressure than IgG negative cases, toxoplasmosis IgG positive cases showed a higher rate of RA while negative cases showed a higher percentage of SLE & ITP.The common presenting symptoms among positive cases were 25.9% poly arthritis, 18.5% fatigue, 18.5% ecchymosis & purpura, and 11.1% recurrent abortion, while in IgG negative, they presented as fatigue 33%, bony aches 17, arthralgia 15% & ecchymosis & Purpura 21%.IgGpositive cases showed 33.3% of big and small joint affection while IgG-negative cases showed 52% small joint affection and 20% of both small and big joint affection.Heart rate was significantly higher in IgGpositive cases.Affected organs were mainly joints and blood (80%) in IgG-positive cases meanwhile; in IgG-negative the percentage was 95% (Table 2).
Clinical signs and symptoms were generally found to be less severe in autoimmune patients with higher parasite prevalence in the intestine and positive toxoplasmosis IgM and IgG than in people with lower parasite prevalence.According to this, parasite infection may serve as a protective factor and exacerbate the clinical signs of autoimmune illnesses (Table 2).
Positive cases of intestinal parasites were significantly related to the females and also showed higher levels of systolic blood pressure than negative cases.Also, the percentage of positive cases was 32.2% ITP but it was 55% SLE in negative cases.Ecchymosis and purpura were of higher rate at 34.5% in intestinal positive cases but fatigue and arthralgia were of higher rate among negative cases.Intestinal positive cases were mainly of no symptoms or small joint affection but in negative cases, they were mainly small joint or both small and big joint affection.The percentage of affected organs intestinal-positive cases was 51.7% blood meanwhile; in intestinalnegative cases, the percentage of joint affection was 85.1% (Table 2).Higher eosinophils, RBCs, hemoglobin, hematocrit, LDH, ferritin, and ESR values were detected in IgM-positive patients, whereas decreased MCV, platelets, and ALT values were found.IgMpositive subjects had higher intestinal parasite rates on stool analysis, as well as higher RBC and albuminuria rates on urine analysis (Table 3).
IgG-positive patients had greater rates of RBCs and albuminuria on urine examination, as well as higher rates of eosinophils, LDH, ferritin, CRP, and ESR, but had lower levels of ALT.They also had higher rates of intestinal parasite affection (Table 3).
Positive cases of intestinal parasites exhibited greater levels of WBCs, eosinophils, hemoglobin, and hematocrit and indicated lower levels of CRP, ALT, and AST, whereas negative cases showed higher rates of RBCs in urine and albuminuria as well as higher rates of splenomegaly and hepatosplenomegaly (Table 3).

SD = Standard deviation
Intestinal parasites were present in 100% of ITP patients while presented in 52.6% of AIHA patients, and 47.6% in RA patients.According to the type of parasite, Enterobius vermicularis was present mainly in ITP patients followed by AIHA patients, amoebiasis was present mainly in RA followed by SLE & AIHA patients, Giardia lamblia was present in SLE & ITP patients and finally Ascaris lumbricoides was existed in ITP patients only.RA & AIHA patients were 100% toxoplasmosis IgM positive, while toxoplasmosis IgG was mainly presented in SLE (80.4%) and in AIHA patients (73.7%) (Table 4).

Table 4: Intestinal Parasites and toxoplasmosis infection in relation to the type of autoimmune
disease among the studied groups.
ITP & RA cases were 100%, female patients.SLE & AIHA patients had significantly higher weight.The age of the patients also varied greatly, being greater in RA & AIHA patients.In RA patients, both systolic and diastolic blood pressure were greater.Patients with RA and SLE had higher body temperatures, and those with AIHA had tachycardia as well (Table 5).SLE and ITP patients had the lowest white blood cell counts, respectively.In SLE cases, followed by RA cases, eosinophils likewise had the lowest value.In AIHA cases, the lowest levels of RBCs, Hb, and HCT were found, whereas ITP cases had the highest levels of platelets.
The LDH value was highest in AIHA cases and lowest in ITP cases.RA patients had the highest levels of CRP and ESR, respectively.Splenomegaly was discovered by abdominal ultrasound in 82.1% of ITB cases, 66.7% of SLE cases, and 47.4% of AIHA patients (Table 6).

DISCUSSION
Immunologists and parasitologists are now and urgently studying the consequences of parasitic infections on the immune system.Many parasitic worms have a tendency to suppress the host's immune response, which permits them to ameliorate some conditions while aggravating others (Bashir et al., 2002).
In order to demonstrate the prevalence and effects of intestinal parasitic infections on patients with autoimmune diseases, the current study was carried out.
The present study documented a high prevalence of intestinal parasites and high seroprevalence of toxoplasmosis IgM and IgG in the studied cases of autoimmune disorders.Also, the positivity of toxoplasmosis IgM among cases with autoimmune disease was significantly related to the female sex.Toxoplasmosis IgM-positive cases were detected in SLE, ITP, and HT.Also, the positivity of IgG was significantly related to the female sex and showed a higher percentage in RA patients.
According to Osada and Kanazawa (Osada and Kanazawa, 2010), helminths mostly induce T-helper Type 2 (Th2) cells while reducing T-helper Type 1 (Th1) cells.This theory would only clarify how parasitic worms regulate autoimmune disorders brought on by Th1 cells (Rook, 2008).Some parasite illnesses reportedly have an anti-inflammatory response.Previous research has demonstrated that helminths like Necator americans and Trichuris suis can reduce the symptoms of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (Varyani et al., 2017).
Due to the Th2-based immune response they produce; parasitic worms commonly impair the immune system's ability to respond to a vaccine effectively.This makes the immune system less susceptible to antigens than usual.This is a severe problem in underdeveloped nations where parasitic worms and the need for vaccinations are common (Kamal and Khalifa, 2006).
Numerous parasites have the ability to regulate their own immune responses by driving the production of regulatory genes including the suppressor of cytokine signaling (SOCS) gene and immunosuppressive cytokines like IL-4 and IL-10.In another way, they can also hinder the activity of sensitive T-cells that are stimulated by autoimmune conditions including diabetes, arthritis, encephalomyelitis, and multiple sclerosis (MS) (Dhingra et al., 2013).
It was revealed that helminths, particularly nematodes and flatworms, viruses, and bacteria can alter the effect of autoimmune illnesses due to their immunosuppressive activity, which is in line with certain recent findings (Saunders et al., 2007;Sfriso et al., 2010;He et al., 2010;Akdis et al., 2011).
In the current study, the positivity of toxoplasmosis IgM among cases with autoimmune disease was significantly related to the female sex.Also, positive cases were clinically presented with recurrent abortion, fatigue, ecchymosis, purpura, and menorrhagia.Affected organs were mainly joint, blood, and thyroid in IgM-positive cases.Regarding the positivity of IgG, it was significantly related to the female sex and also showed higher levels of systolic and diastolic blood pressure.In toxoplasmosis IgG-positive cases, the common presenting symptoms were polyarthritis, fatigue, ecchymosis, purpura, and recurrent abortion.
A similar study revealed that 49.2% of the patients had cerebral toxoplasmosis and 50.9% of the cases were attributed to reactivation.The highest prevalence associated with autoimmune disease was rheumatoid arthritis (28%) (Durieux et al., 2022).

Population-based
case-control research for Toxoplasma gondii and multiple sclerosis was conducted, and it documented a seroprevalence of anti-T.gondii antibodies in 29.5% and 66.7% of the cases.It was hypothesized that Toxoplasma gondii and MS had a negative correlation, indicating that this parasite may have a protective function (Nicoletti et al., 2020).
By studying the prevalence of CNS toxoplasmosis in individuals with autoimmune disorders, immunocompetent people, and immunocompromised patients, it was discovered that patients with autoimmune diseases have clinical indices of brain toxoplasmosis more frequently than immunocompromised patients (Graham et al., 2021).
Studying the seroprevalence of Toxoplasma gondii infection in patients with arthritis in Eastern China in 2017 as a result showed that the prevalence of anti-Toxoplasma gondii IgG was significantly higher in patients with arthritis (18.8%) compared to healthy controls.Patients with rheumatoid arthritis had the greatest infection seropositivity, which was subsequently followed by reactive arthritis, osteoarthritis, infectious arthritis, and gouty arthritis (Tian et al., 2017).
Furthermore, it was demonstrated that T. gondii infection improves experimental autoimmune encephalomyelitis because it modifies the immune response to enhance brain expression suppressor of cytokine signaling (SOCS3) and IL-27.This finding was in line with a prior study (Ham et al., 2021).
According to the type of parasites, all ITP patients had intestinal parasites, followed by AIHA patients and RA patients.ITP patients were more likely to have Enterobius vermicularis than AIHA patients, RA patients were more likely to have amoebiasis than SLE or AIHA patients, ITP patients were more likely to have giardiasis than AIHA patients, and ITP patients were more likely to have Ascaris lumbricoides than AIHA patients.Toxoplasmosis IgM was detected in all RA and AIHA patients.Although toxoplasmosis IgG was more common in SLE and AIHA patients.
Helminth infection consistently strengthens the immune system, reducing the impact of rheumatoid arthritis.As a result, parasitic helminths can be employed to treat rheumatoid arthritis.Additionally, it is advised that more research be done to make use of these helminth species' immunological qualities and that helminth organisms be considered symbiotic organisms rather than parasites (Ike et al., 2020).
Similarly, employed mouse models were used to examine the impact of a parasite-derived 68-mer peptide on type 1 diabetes and multiple sclerosis, two autoimmune diseases.It was discovered that this parasite with peptide origins can improve various autoimmune conditions (Lund et al., 2016).Demonstrated malarial infection in mice decreased pathogenic leukocyte infiltration but had no effect on the autoimmune inflammation of glomeruli, corroborating this finding (Bolland et al., 2018).
Interestingly, one research suggested that cDC2s generated by CCL17 play a crucial role in systemic lupus erythematosus's end-stage lupus nephritis and are inhibited by a parasite infection.Malaria parasite infection did not alter the lupus-causing autoimmune processes, but it did prevent end-stage tissue damage in the kidney and draining lymph nodes (Amo et al., 2021).
A prevalence study was carried out in agreement with the current study to detect the frequency of intestinal parasites found in autoimmune illnesses.Cryptosporidium (48.0%) was the parasite with the highest prevalence, and it was followed by C. cayetanensis (32%), G. lamblia (24%), B. hominis (20%), and E. histolytica (8%).There were 4% for each of Schistosoma mansoni, Microsporidia, S. stercoralis, and A. lumbricoides (Hussein et al., 2019).
In a corresponding study for the relationship between intestinal parasites and inflammatory bowel disease (IBD), it was found that Blastocystis species, Entamoeba histolytica and Cryptosporidium species were the most common intestinal parasites in IBD patients compared to healthy controls (Kamal et al., 2022).

Conclusion
Numerous autoimmune disorders patients, including those with SLE, ITP, RA, and AIHA, typically had high prevalence rates of toxoplasmosis and intestinal parasites, which changed the clinical course and investigatory indicators of these illnesses.Therefore, more research may be indicated to determine the primary mechanisms by which those parasites might alter the course of several autoimmune illnesses.

Table 1 :
Prevalence of intestinal parasites and toxoplasmosis in autoimmune disease patients.

Table 2 :
Socio-demographic and clinical data in relation to parasitic infection among the studied cases (Total n=154).

Table 3 :
Laboratory investigations in relation to parasitic infection among the studied cases.

Table 5 :
Socio-demographic and clinical data in relation to the type of autoimmune disease among cases.
SD = Standard deviation

Table 6 :
Lab investigations in relation to the type of autoimmune disease among the patients.