Is Toxoplasma gondii infection protective against multiple sclerosis risk?

Author’s Accepted Manuscript Is Toxoplasma gondii Infection Protective Against Multiple Sclerosis Risk? Aslı Koskderelioglu, Ilhan Afsar, Bayram Pektas, Muhtesem Gedizlioglu www.elsevier.com/locate/msard

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S2211-0348(17)30074-3 http://dx.doi.org/10.1016/j.msard.2017.04.004 MSARD564

To appear in: Multiple Sclerosis and Related Disorders Received date: 15 February 2017 Revised date: 8 April 2017 Accepted date: 14 April 2017 Cite this article as: Aslı Koskderelioglu, Ilhan Afsar, Bayram Pektas and Muhtesem Gedizlioglu, Is Toxoplasma gondii Infection Protective Against Multiple Sclerosis Risk?, Multiple Sclerosis and Related Disorders, http://dx.doi.org/10.1016/j.msard.2017.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Is Toxoplasma gondii Infection Protective Against Multiple Sclerosis Risk?

Aslı Koskderelioglu1*, Ilhan Afsar2, Bayram Pektas2, Muhtesem Gedizlioglu1 1

Izmir Bozyaka Education and Research Hospital, Department of Neurology, Izmir, TURKEY

2

IKCU Ataturk Training and Research Hospital, Department of Medical Microbiology, Izmir, TURKEY

*

Correspondence to: Asli Koskderelioglu Izmir Bozyaka Education And Research Hospital, Neurology

Department, Saim Cikrikci cad No:59, Bozyaka, Karabaglar, 35170 Izmir, Turkey. Tel: +905334275100. [email protected]

ABSTRACT BACKGROUND Parasitic infections may play a protective role in neurodegenative diseases. OBJECTIVE To determine the association between Toxoplasma gondii (T. gondii) infection and Multiple Sclerosis (MS). METHODS One hundred fifteen patients with MS were included in the study. Sixty age and gender-matched healthy subjects were recruited as controls. Subjects were assessed for clinical and demographic parameters. The presence of specific IgG antibodies against T. gondii microorganism was searched by using an enzyme immunoassay test in the sera of the subjects. RESULTS T. gondii seropositivity was found to be lower in MS patients than in healthy controls (33.9% vs. 55%, p=0.007). Mean age and disease duration of the patients were 41.15±11.20 (18-74) and 1.90±1.44 (0-6) years, respectively.

MS patients with a high IgG titer had lower expanded disability status scale (EDSS) scores (p=0.001) and lower annualized relapse rates (ARR) (p=0.005). There was no significant association between T. gondii seropositivity and disease duration (p=0.598). Female MS patients tended to have higher T. gondii seropositivity than males although the difference did not reach statistical significance (p=0.192). We found a negative correlation between T. gondii seropositivity and both EDSS scores (r=-0.322, p<0.001) and ARR (r=-0.263, p=0.004). CONCLUSION We found a negative association between T. gondii infection and the presence of MS. Furthermore, parasite infected MS patients had experienced fewer relapses with lower disability scores supporting the hypothesis of immunomodulatory effects of parasitic infections in autoimmune diseases. Further studies are required to establish the protective role of parasitic infections in MS.

KEYWORDS: Toxoplasma gondii; Multiple sclerosis; Parasite; Hygiene

INTRODUCTION Multiple sclerosis (MS) is a chronic, heterogeneous, demyelinating disease of the central nervous system (1). The pathogenesis of MS involves an autoimmune process that is influenced by a complex interaction between genetic and environmental factors (2–4). MS is common in developed countries with higher sanitary standards consistent with decreased incidence of various infections. Hygiene hypothesis was first proposed by Strachan in 1998 (5). According to hygiene hypothesis, microorganisms such as parasitic worms play an important role in regulating immune system of the host. In modern life, the elimination of these parasites leads to autoimmune and allergic diseases by dysregulation of the immune system (6,7). Both epidemiological and experimental data provide substantial evidence to support autoimmune down-regulation secondary to parasitic infections in patients with MS. Regulatory T- and B-cells show complex effects extending beyond easily explainable responses to an infectious agent (8). Toxoplasma gondii (T. gondii) is an intracellular parasite that causes toxoplasmosis in both human and animals (9). It is a common parasitic infection that has infected almost 6 billion people worldwide (10,11). Despite wide geoepidemiological variance, the estimated global infection rates is around 30% (12).

T. gondii is an opportunistic infection frequently encountered in the central nervous system of immunosuppressed patients. In recent years, a relationship between T. gondii and many autoimmune diseases including Parkinson’s and Alzheimer’s diseases has been reported (13,14,15). Paradoxically, there are many studies reporting benefits of infection, including the remodulation of the immune system and protective effects against autoimmune diseases. However, the issue of any presumed association with demyelinating conditions such as MS is relatively unclear. Therefore, in this study, we aimed to investigate the prevalence of T. gondii infection in MS patients and to evaluate the possible association between anti-T. gondii IgG antibodies and MS clinical parameters, particularly disability. MATERIALS AND METHODS Study population We prospectively enrolled 115 consecutive patients with clinically definite relapsing-remitting MS, who were evaluated and followed up at outpatient clinic of Multiple Sclerosis, Department of Neurology, Izmir Bozyaka Education and Research Hospital in 2014. All patients were in remission, did not receive intravenous methyl prednisolone therapy during sera collection. We excluded MS patients presented with an acute demyelinating attack. They were receiving the same immunomodulatory/ immunosupressive treatment at least for 3 months. Of the total 115 MS patients, 12 were receiving glatiramer acetate, 78 IFN beta, 9 fingolimod, 2 natalizumab, 1 mitoxantrone, 1 cyclophosphamide and 12 none. The patient group did not receive any antiparasitic treatment, since there was no clinical sign or serological finding of an acute parasitic infection during study period. The control group consisted of 60 age and sex matched healthy subjects. The study protocol was approved by the local ethics committee, and all of the subjects gave written informed consent to participate. At the entry of the study, the outpatient medical records of all subjects were intensively reviewed, the EDSS scores, annualized relapse rates (ARR), disease duration, and demographics were recorded. Serum Samples Blood samples were taken from the participants by a nurse and then centrifuged to separate serum. The sera were stored at -20°C in the laboratory of the department of parasitology until the analyzes were carried out. The analyzes were completed by a trained microbiologist and a parasitologist.

Laboratory method The sera were studied with Immulite 2000 XPi (Siemens, Germany), a random access analyzer based on an amplified chemiluminescent immunoassay. This system can accomplish the quantitative measurement of IgG antibodies to T. gondii in serum, as an aid in the determination of serological status to T. gondii. Specimens with a result greater than or equal to 8 IU/mL are considered as positive and accepted indicative of a past infection. A result less than 6.5 IU/mL was considered to be non-reactive, thus negative. A result greater than or equal to 6.5 IU/mL and less than 8 IU/ml was considered as indeterminate. All samples were run against the T. gondii IgM assay as well. The results greater than or equal to 1.1 IU/mL were reported as positive. A result between 0.9 and 1.1 IU/mL was considered as indeterminate and negative if a result was less than 0.9 IU/mL. All samples that had indeterminate results were retested.

Statistical Analysis We used SPSS 16.0 software (SPSS Inc, Chicago, Illinois, USA) for data analysis. Continuous data were expressed as mean ± standard deviation (SD), whereas frequency data were expressed in percentages (%). The Chi-square and Student's t-test were used to test statistically significant differences for parametric data. Spearman rank correlation analysis was used to assess the relationship between toxoplasma seropositivity and clinical parameters. A p value less than 0.05 was considered to be significant. RESULTS We recruited 115 MS patients and 60 healthy controls in the study. No significant difference was found between healthy controls and MS patients with respect to age and gender (p=0.673 and p=0.632, respectively). The mean age of MS patients and controls were 41.16 ± 11.20 (median 40, IR 17) and 39.52± 7.45 (median 39, IR 9) years, respectively. Of the 115 MS patients, 77 (67%) were women. The demographic data is shown in Table-1. The mean disease duration was 9.38 ± 6.98 (0.25-45) years and the mean EDSS score was 1.90 ± 1.44 (0-6). The annualized relapse rate (ARR) was 0.54 ± 0.51(0.06-4). Of the total population, 41.1% of subjects were positive for anti- T. gondii IgG. Discriminatively, 39 of the MS patients (33.9%) and 33 of healthy controls (55%) had a positive test. Chi-square test revealed a significant difference between the two groups (p=0.007). The demographic and serological data of the participants is shown in Table-1. No significant association between seropositivity and gender was noted in both groups (p=0.085). All participants were negative for anti-Toxoplasma IgM.

The mean disease duration of anti-T. gondii Ig G positive MS patients was 9.39 ± 5.83 years; and for seronegative MS patients, 9.39 ± 7.54 years. There was no significant association between serological status and disease duration among MS patients (p = 0.598). The ARR and EDSS scores were significantly lower in MS patients who were seropositive for anti-T. gondii Ig G. There was a significant difference in ARR and EDSS scores between seropositive and seronegative MS patients (p=0.005 and p=0.001, respectively). The comparison between MS patients regarding seropositivity is summarized in Table-2. Although seropositivity rates were higher among female and older MS patients, no significant difference was noted for serological status based on age or gender (p=0.148, p=0.192, respectively). Of the thirty-nine seropositive MS patients, three of them were living in the rural area, and only eight (20.5%) had domestic animals. None of them reported a close contact with livestock animals. Chi-square tests showed no significant differences in seropositivity in respect to either place of residency or exposure to livestock / domestic animals or (p = 0.208 and p= 0.409, respectively). We found a negative correlation between anti- T. gondii Ig G seropositivity and relapse rate and disability scores. Seropositive MS patients tended to have lower relapse rates and EDSS scores (r=-0.263, p=0.004 and r=-0.322, p<0.001, respectively). There were no correlations between seropositivity and either disease duration or age. DISCUSSION Recent reports suggested a possible role of herpes viruses (herpes simplex, herpes zoster, HHV-6, CMV, EBV) in the pathogenesis of MS. However, the association with EBV infection is proposed to be more fundamental for MS than other viral agents. There is a strong evidence linking EBV and increased MS risk in the literature (16,17). Higher seroprevelance of EBV infection and higher antibody titers to Epstein-Barr nuclear antigen are well determined MS risk factors (17). It has been postulated that EBV infected B cells might have a direct role in inflammation and neurodegeneration in the central nervous system (18). Likewise, in genetically predisposed individuals, microbial agents are accounted for inducing autoimmunity. However, supporting the hygiene hypothesis, several studies considered some parasitic infections as protective factors against autoimmune diseases. Epidemiological data suggest that improvement in sanitary conditions and reduced exposure to pathogenic microorganisms might explain the increase in autoimmune and inflammatory diseases in highly industrialized countries (19,20). The ‘Old Friends’ mechanism, a variant of the hygiene hypothesis, states that mammals co-evolved with an array of organisms operating as inducers of immunoregulatory circuits (21,22). When the contact with these organisms (various microbiotas, commensals, chronic infections picked up at birth

and helminths) diminishes by industrialization, a defective immunoregulation occurs resulting in chronic inflammatory disorders, allergies and autoimmunity. Furthermore, Leibowitz and coworkers reported that MS prevalence showed a concordance with higher sanitation levels (23). Also, some epidemiological investigations showed an inverse correlation between the global distribution of MS and parasitic infections (24,25). There is limited data on the effects of T. gondii in the pathogenesis of autoimmune diseases. Krause et al. found a negative association of T. gondii IgG with type 1 diabetes mellitus patients (26). Previously SLE was associated with T. gondii IgG (27), several studies showed discordant association in consequence (28,29). Yet, it is still a debate whether past infection by T. gondii is associated with MS. Although Shapira et al. reported that T. gondii IgG was higher in autoimmune diseases, no association was found between T. gondii IgG seropositivity and MS in two different geographical regions (30). There was no significant difference in the seropositivity rates of T. gondii IgG between MS patients and controls. While the evidence for an involvement of parasitic agents in autoimmune disorders is relatively strong, there is less work about T. gondii infection and its possible pathogenetic role in MS. Although an interaction between MS susceptibility genes and T. gondii has previously been evaluated by Carter et al, no clinical studies have spesifically linked toxoplasmosis to MS so far (15). In Stascheit’ s recent study, it was for the first time that a correlation betwen seroprevalance of T. gondii IgG and MS has been assumed (31). Although infections are suspected for accelerating autoimmune diseases, early exposure to parasitic infections may have a protective role. Here, we searched for the existence of any effect of a particular parasitic infection, T. gondii on the MS course. This current study reflects the lower seroprevalence of an intracellular protozoan in MS patients compared to healthy subjects. In Turkey, there are very few seroepidemiological studies that show prevalence of T. gondii infection. Most of the studies encountered the seroprevalence of Toxoplasma in subjects with suspected toxoplasmosis or in certain regions. It was reported that the seroprevalence ranges from 13.9% to 85.3% in hospital patients, and from 23 to 43.7% in apparently healthy people (32). In the present study, we showed that the seropositivity rate of toxoplasmosis in MS patients was 33.9 % versus 55% in healthy controls. This rate was similar to the previous data of Stascheit et al. who reported a 32.9 % positivity rate in MS patients and 46.7 % in controls in Germany (31). Despite of higher seroprevalence of a suspected micoorganism in the etiology of autoimmune diseases, we have found lower seropositivity in the MS group. Our findings add further support to the suggested relationship between T. gondii and MS. On the other hand, the lower rate of toxoplasma seropositivity is also compatible

with the hygiene hypothesis involved in MS pathogenesis. In contrast, Pestehchian et al. showed similar seroprevalence rates between MS patients and their family members (33). It was reported that the risk for people of developing Toxoplasma infection is higher in rural enviroment (34). In contrast, in this study, the number of MS subjects living in urban areas was significantly higher than those living in rural areas. There were only three seropositive patients coming from rural areas. We could not determine any relationship between the seropositivity and the place of residency. Our study had several limitations. First, our study population was small. Second, our patient group consisted of relatively younger people. Young age might have affected the seropositivity rate; since age is a significant independent factor for seropositivity. Third, our study protocol did not involve a prospective design comparing initial and follow-up clinical or radiological outcomes. However, our study proposed to evaluate the possible role of a recent infection with T. gondii in MS patients. In this study, we observed a significant relationship between the seropositivity and relapse rates as well as disability scores. The results showed a relationship between T. gondii infection and MS course that supported the hypothesis of a protective effect. In contrast, Stascheit et al. found no association between toxoplasma seropositivity and clinical parameters (31). Correale et al. reported that MS subjects infected by helminths had a significant decrease in their relapses, lower disability scores and lower disease activity compared to MS subjects who were not infected (35). These findings raised a possibility of neuroprotective effects modulated by helminth infections in MS patients. We aimed to investigate whether past T. gondii infection could have a neuroprotective role in MS course. Our study is one of the promising studies that looks for the association of MS course and T. gondii infection. Further investigation is warrented to identify the pathophysiologic mechanism generated by T. gondii in MS course. CONCLUSION Toxoplasmosis is one of the parasitic infections that is related to several autoimmune diseases. This current study points out that seropositivity is lower in MS patients, and seropositive patients had a better clinical outcome. However, our results must be supported by further studies. In conclusion, T. gondii seropositivity may have a positive impact on MS disease course and progression.

CONFLICT OF INTEREST Authors have no conflict of interest to declare.

CONFLICT OF INTEREST, FINACIAL DISCLOSURE None.

ACKNOWLEDGEMENTS There were no funding sources for the study.

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Table 1: Demographic and serologic data in multiple sclerosis (MS) patients and controls. Variables

Group 1 (MS patients)

Group 2 (Controls)

n= 115

n= 60

Age (years)

41.16 ± 11.20 (40, IR 17)

39.52 ± 7.45 (39, IR 9)

0.673

Gender Male

38 (33 %)

22 (37 %)

0.632

77 (67 %)

38 (63 %)

Anti- T. gondii Ig M seropositivity

0

0

Anti- T. gondii Ig G seropositivity

39 (33.9 %)

33 (55 %)

Female

p-value

0.007

 p<0.05 is statistically significant. Median values, interquartile range (IR) and percentages are indicated in parenthesis.

Table 2: Demographic and clinical data among multiple sclerosis (MS) patients regarding seropositivity for AntiT. gondii Ig G. Median values, interquartile range (IR) and percentages are indicated in parenthesis. Variables

Anti- T. gondii Ig G seropositive MS patients

Anti- T. gondii Ig G seronegative MS patients

P value

N= 39

N= 76

Mean Age (years)

43.54 ± 12.40 (18-74)

39.93 ± 10.40 (19-74)

0.148

Gender (M/F)

16/23

22/54

0.192

Mean disease duration (years)

9.39 ± 5.83 (9.0, IR 12)

9.39 ± 7.54 (9.0, IR12)

0.598

Mean EDSS score (0-10)

1.23 ± 0.65 (1.0, IR 1)

2.24 ± 1.61 (2.0, IR 2)

0.001*

Mean ARR

0.37 ± 0.25 (0.35, IR 0.45)

0.63 ± 0.58 (0.45, IR 0.49)

0.005

Exposure to livestock/domestic

8 (20.5 %)

11 (14.5 %)

0.409

animals (positive subjects) Place of residency (rural area)

3 (7.7 %)

2 (2.6 %)

0.208

Abbreviations; EDSS: Expanded Disability Status Scale, ARR: Annualized Relapse Rate *

p<0.05 is statistically significant.

HIGHLIGHTS:  Toxoplasmosis is one of the most common parasitic infections worldwide.  Parasitic infections modulate the immune system of the host.  The role of Toxoplasma gondii infection in autoimmune diseases is a new area in research.  Up to date, the association between Toxoplasma gondii and multiple sclerosis has not been  entirely evaluated.