Seroprevalence of anti-Toxocara antibody among multiple sclerosis patients: a case–control study

Abstract

Although previous studies have shown an association between parasitic infections and multiple sclerosis, the possible role of Toxocara infection on the etiology of multiple sclerosis has been overlooked. The present study aimed to investigate the seroprevalence of anti-Toxocara IgG antibodies among patients with multiple sclerosis compared to healthy controls. Seventy patients with prior diagnosis of multiple sclerosis were selected as cases and 70 healthy matched individuals as controls. The presence of serum anti-Toxocara IgG antibody was investigated by ELISA technique. The Chi square test was used to test statistically significant differences for parametric data. A total of 140 serum samples were collected and analyzed. In the case and control groups, 20 (28.6%) and 8 (11.4%) participants had positive serum anti-Toxocara IgG antibodies, respectively, indicating a statistically significant difference (OR 3.1; 95% CI 1.26–7.63; p value = 0.02). The seroprevalence rate was also higher among individuals with a history of contact with dogs (OR 2.7; 95% CI 1.17–6.37; p value = 0.03).The results declare that a protective role of Toxocara canis against the development of multiple sclerosis is unlikely.

Introduction

Multiple sclerosis (MS) is a chronic demyelinating disease with an autoimmune origin that is characterized by multifocal and temporally scattered central nervous system (CNS) damage that mainly affects young adults. Autoimmune diseases like MS are currently considered to be caused by a combination of both genetic vulnerability and environmental factors (Kaminska et al. 2017). Several infectious agents have been proposed as potential environmental or causal factors in MS, for instance, human cytomegalovirus (CMV), Epstein–Barr virus (EBV), human herpes virus 6, and Chlamydia pneumonia (Giovannoni et al. 2006; Ascherio and Munger 2007). Epidemiological data provide evidence that parasite infections may also be associated with a risk of autoimmunity and initiate diseases such as MS (Sondergaard and Theorell 2004; Kuk et al. 2006; Zibaei and Ghorbani 2014).

Human toxocariasis is a zoonotic parasitic disease caused by the larvae of two species of Toxocara roundworms, Toxocara canis, from dogs, and less commonly Toxocara cati, from cats. Human infection mostly occurs by exposure to contaminated soil containing infective larvae or rarely, by consumption of raw meat or giblets of paratenic animals, such as chickens, lambs, or cows (Azizi et al. 2007; Woodhall and Fiore 2014). The larvae hatch in the small intestine and migrate through vessels to the muscles, liver, and lung and sometimes to the eye and brain. Infection of humans with embryonated eggs of T. canis mainly remains asymptomatic, but sometimes results in covert toxocariasis, visceral larva migrans syndrome, or ophthalmologic and neurologic symptoms. Neurological manifestations of T. canis larvae are rare, but toxocariasis still remains an important differential diagnosis of several neurological disorders. Central nervous system involvement includes dementia, meningo-encephalitis, myelitis, cerebral vasculitis, epilepsy, and optic neuritis (Finsterer and Auer 2007; Nicoletti et al. 2008).

Recent studies have shown that the prevalence of MS in Iran has increased considerably, especially during the last decade. Based on the most recent study, the prevalence of MS in Shahrekord is estimated to be 60 per 100,000 person (95% CI 55.1–65.3) (Azami et al. 2019). To the best of our knowledge, the role of toxocariasis in triggering autoimmune diseases, MS, in particular, has not received as much attention as that of other microbial pathogens. Cicero and his colleagues recently found no association between MS and T. canis seropositivity in a population-based study (Cicero et al. 2019). Although some studies have discussed the possible role of Toxocara infection as a protective or harmful factor in autoimmune diseases such as ankylosing spondylitis, systemic lupus erythematosus and MS (Kuk et al. 2006; Kaya et al. 2009; Zibaei and Ghorbani 2014; Levy et al. 2015), the question still remains whether an autoimmune phenomenon is evoked in Toxocara infections. The main objective of this study was to investigate the seropositivity of anti-Toxocara IgG antibodies in a cohort of MS patients and healthy controls.

Materials and methods

Study population

In this case–control study, seventy consecutive patients with a definite diagnosis of MS according to McDonald’s criteria (Polman et al. 2011) were chosen as the case group and seventy healthy individuals among the patients’ family members who had no previous history of neurologic disorders were chosen as controls. Informed consent was obtained from each participant. This study was carried out in Hazrat-e-Rasoul clinic, Shahrekord, Iran during 2018. The patients, who were all in the remission period, visited this clinic as part of their routine follow-up and had not received intravenous corticosteroids in the past 3 months. For demographic data (age, sex, education, occupation, history of contact with dogs, and place of residence) a questionnaire was given to each patient. In case the participant was illiterate, a blind evaluator would read and answer the questions. Five milliliters of blood samples of all participants were collected in tubes containing k2-EDTA as anticoagulant. The tubes were transported to the research laboratory by using the cold chain process. After centrifuging, plasma was separated from the samples and stored at − 20 °C for further examination.

Serological tests

Toxocara antibodies (IgG) were detected by an Enzyme-Linked Immunosorbent Assay (ELISA) kit (IBL, International Gmbh, Hamburg, Germany) according to the manufacturer’s guidance. In brief, after 30 min incubation at 37 °C, diluted sera samples (1:100) were added to antigen-coated wells; then horseradish peroxidase (HRP) labeled Protein A Conjugate was added at a 1:1000 dilution (30 min at 37 °C), followed by the tetramethylbenzidine (TMB) substrate. Absorbance readings were made at 450 nm; a cut-off absorbance value was defined as the mean absorbance reading for three negative control sera plus two standard deviations. A positive absorbance value would be defined as more than 10% above the mean cut-off point. Absorbance rates between 10% below and above the cut-off point would be defined as borderline, meaning they were not classified as a negative or positive result, thus excluded. Finally, if the absorbance rate was less than 10% of the cut-off point it would be considered as negative for the presence of anti-Toxocara antibody. The specificity and sensitivity of the mentioned kit was more than 95% (Buehrer et al. 2015).

Statistical analysis

Statistical analysis was conducted using SPSS software (version 23.0). Continuous data were expressed as mean ± standard deviation (SD), whereas frequency data were expressed in percentages (%). The Chi square test was used to test statistically significant differences for parametric data. Age was stratified into four subgroups (≤ 14, 15–29, 30–44, and ≥ 45) and was analyzed as a categorical variable. A p value of less than 0.05 was considered statistically significant.

Ethical considerations

Informed consent was obtained from all participants or parents/legally authorized representative of participants prior to data collection and serum sampling. All procedures performed in this study were in accordance with the ethical standards of Iran National Committee for Ethics in Biomedical Research (Ethics Code: IR.SKUMS.REC.1394.32) and with the 1964 Helsinki declaration and its later amendments.

Results

The study was conducted on a total of 140 subjects. At the time of enrolment, MS patients had a mean age of 41.2 ± 9.5 years, and 52 (74.3%) of them were female. Controls had a mean age of 38.8 ± 7.6, and 45 (64.3%) were female. Demographic characteristics of the study population are represented in Table 1. As shown, there was no significant difference between recruited patients and controls with respect to sex, age, education, place of residence, occupation, and history of contact with dogs (p value = 0.27, 0.06, 0.06, 0.61, 0.1, and 0.31, respectively).

Table 1.

Demographic characteristics of MS patients and controls

	MS patients (n = 70)	Controls (n = 70)	p value
Gender
Male	18 (25.7%)	25 (35.7%)	0.27
Female	52 (74.3%)	45 (64.3%)
Age (years)
≤ 14	2 (2.9%)	4 (5.7%)	0.06
15–29	21 (30%)	28 (40%)
30–44	28 (40%)	31 (44.3%)
≥ 45	19 (27.1%)	7 (10%)
Education
Illiterate	3 (4.3%)	7 (10%)	0.06
Primary school	8 (11.4%)	18 (25.7%)
High school	31 (44.3%)	25 (35.7%)
University degree	28 (40%)	20 (28.6%)
Place of residence
Urban	38 (54.3%)	42 (60%)	0.61
Rural	32 (45.7%)	28 (40%)
Occupation
Rancher	6 (8.6%)	10 (14.3%)	0.10
Employee	6 (8.6%)	12 (17.1%)
Homemaker	31 (44.3%)	17 (24.3%)
Farmer	24 (34.3%)	29 (41.4%)
Student	3 (4.2%)	2 (2.9%)
History of contact with dogs
Yes	28 (40%)	35 (50%)	0.31
No	42 (60%)	35 (50%)

The overall anti-Toxocara seroprevalence rate was 20% across all participants. The presence of anti-Toxocara canis antibody was found in 20 MS patients giving a seroprevalence rate of 28.6% and 8 controls resulting in a seroprevalence rate of 11.4%, thus, reflecting a significant difference between MS patients and their controls in terms of anti-Toxocara IgG antibody (OR 3.1; 95% CI 1.26–7.63; p value = 0.02).

When the analysis was performed across all participants (n = 140), no significant relationship was found between seropositivity and age, gender, education or occupation (p value = 0.86, 0.82, 0.71 and 0.49 respectively). However, the seropositivity rate among urban and rural residents was 27.5% and 10%, respectively, which was statistically significant (OR 3.41; 95% CI 1.29–9.06; p value = 0.01). There was also a significant relationship between individuals’ history of contact with dogs and positive serologic status (OR 2.7; 95% CI 1.17–6.37; p value = 0.03). As shown in Table 2, based on a subgroup analysis among seropositive patients, female gender was significantly associated with developing multiple sclerosis (p value = 0.07).

Table 2.

Distribution of variables among seropositive individuals

Variables	Total (n = 28)
	n (%)	p value
Sex
Male	9 (32)	0.07
Female	19 (68)
Place of residence
Urban	6 (21.5)	0.64
Rural	22 (78.5)
Age (years)
≤ 14	2 (7)	0.73
15–29	10 (36)
30–44	11 (39)
≥ 45	5 (18)
Occupation
Rancher	5 (18)	0.78
Employee	4 (14)
Homemaker	9 (32)
Farmer	8 (29)
Student	2 (7)
Education
Illiterate	2 (7)	0.87
Primary school	6 (21.5)
High school	13 (46.5)
University degree	7 (25)
History of contact with dogs
Yes	18 (64)	> 0.99
No	10 (36)

Similar to the entire population, place of residence and history of contact with dogs were significantly associated with anti-T. canis seropositivity among MS patients (p value = 0.04 and 0.01, respectively) (Table 3).

Table 3.

Demographic data among MS patients based on seropositivity (n = 70)

Variables	Seropositive MS patients (n = 20)	Seronegative MS patients (n = 50)	p value
	n (%)	n (%)
Sex
Male	4	14	0.56
Female	16	36
Place of residence
Urban	15	23	0.04
Rural	5	27
Age (years)
< 14	1	1	0.56
15–29	8	13
30–44	7	21
> 45	4	15
Occupation
Rancher	4	2	0.28
Employee	2	4
Homemaker	7	24
Farmer	6	18
Student	1	2
Education
Illiterate	1	2	0.31
Primary school	4	4
High school	10	21
University degree	5	23
History of contact with dogs
Yes	13	15	0.01
No	7	35

Discussion

Limited studies have investigated the possible role of Toxocara infection on the etiology of multiple sclerosis. This study showed that subjects with positive anti-Toxocara IgG antibodies are approximately three times more likely to develop MS throughout their life as compared to those who are seronegative. In a study by Kuk et al., the seroprevalence of Toxocara antibodies was assessed in patients with multiple sclerosis and ankylosing spondylitis. Based on the results of this study, those with Toxocara seropositivity were more than five times more likely to develop MS as compared to those with Toxocara seronegativity (OR 5.94). However, there was no significant difference in Toxocara seropositivity between the case and control population (p = 0.08) (Kuk et al. 2006). In a similar study, Zibaei and Ghorbani investigated the association between Toxocariasis and MS. They found that seropositivity was significantly higher in MS patients in comparison to the control group, suggesting an increase in the risk of developing MS after infection with Toxocara (Zibaei and Ghorbani 2014). In a later study in 2017, Posova and his colleagues examined the presence of anti-Toxocara antibodies in CIS and MS patients and found a very low number of positive patients, supporting the concept of a protective rather than a harmful effect of helminth infections (Posová et al. 2017). However, in the most recent population-based study, Cicero and his colleagues found no association between MS and T. canis seropositivity (Cicero et al. 2019).

Epidemiological studies have shown that Toxocara infection rate varies widely within and between countries. Our study showed a prevalence rate of 20% among all participants. A meta-analysis in Iran found a 15.8% (95% CI 9–22.5) seroprevalence rate of Toxocariasis (Abdi et al. 2012). Several other studies have evaluated the seroprevalence of Toxocariasis among the general population in Iran, with one study reporting the infection rate as high as 25% (Fallah et al. 2007; Sharif et al. 2010; Baghani et al. 2018; Khoshnood et al. 2018; Sarkari et al. 2018; Shokouhi and Abdi 2018; Aghamolaie et al. 2019). According to a systematic review carried out in Brazil, Toxocara seroprevalence rates ranged from 4.2 to 65.4% (Fialho and Correa 2016). Studies from the United States have also reported varying infection rates ranging from 5 to 13.9% (Berrett et al. 2017; Farmer et al. 2017; Liu et al. 2018).

As it is already known, dogs and cats are important in the life cycle of Toxocara and owning them is a risk factor for Toxocara infection. In the present study, a higher seropositivity rate was observed among participants with a history of exposure to dogs although not statistically significant. There was no statistically significant difference among patients and controls in regards to history of contact with dogs. Previous studies investigating the possible effect of exposure to dogs and cats on MS have demonstrated conflicting results (Gustavsen et al. 2014; Siejka et al. 2016; de Jong et al. 2019).

Although our study failed to show a relationship between seropositivity and age across the entire cohort, the prevalence of anti-Toxocara IgG antibodies was significantly different across rural and urban residents, with a higher rate observed in urban areas. These findings are in line with a previous study by Zibaei and Ghorbani, indicating that the seroprevalence rate was not affected by age. The higher seropositivity rate among urban residents in this study is an interesting finding as it can be possibly explained by the growing trend of pet ownership in the cities of Iran. Correale and his colleagues have conducted multiple studies on the association between helminth infections and MS (Correale and Farez 2007; Correale et al. 2008; Correale and Farez 2011, 2012; Correale and Equiza 2014). They recently demonstrated that parasite-infected MS patients showed a favorable clinical response compared to uninfected MS individuals; with an increase in clinical and radiological activity after administration of antihelminthic drugs (Correale and Farez 2007, 2011).

There have also been incompatible results with studies designed to investigate the influence of other parasitic infections such as Toxoplasmosis on MS, with some showing a protective role while others suggesting an etiologic role for infections (Stascheit et al. 2015; Oruç et al. 2016; Tavalla and Sabzevari 2017; Saberi et al. 2018). Studies affirming a protective role for helminth infections against MS have been mostly conducted in the Western world; therefore, conducting larger multinational studies with the enrollment of subjects with different geographical distribution is warranted.

Unlike the methods used for the immunodiagnosis of bacterial, viral or protozoal (Toxoplasmosis) infections, it is not possible with toxocariasis to assess the age of the presence of specific IgG using the levels of specific IgM (Fillaux and Magnaval 2013). Hence, we were not able to discriminate between acute and chronic infections in our study, making it unclear as to whether the infection had occurred prior to diagnosis of MS or not. Thus, it might not be accurate to conclude that infection with T. canis can act as a potential trigger and risk factor for developing MS. Another limitation of this study is that analysis was not performed based on the treatment patients were receiving. Studies have shown that immunomodulatory or immunosuppressive therapies can have an impact on the infective risk of patients (Buonomo et al. 2018).

Our results rationally declare that a protective role of T. canis against the development of MS is unlikely. While no definitive conclusions may be drawn from such early, exploratory studies, these investigations provide an opportunity for novel observations to perhaps contribute to important implications. Clearly, more research in this area is required to evaluate the effect of helminth infection on the pathogenesis of multiple sclerosis. Most importantly, although the results are intriguing, none of these studies can exclude the possibility that the changes observed are merely a consequence of MS, rather than its cause.

Funding

None.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.