Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by the loss of dopamine-producing neurons in the substantia nigra and the accumulation of intraneuronal α-synuclein in Lewy bodies. This pathology results in various neurological symptoms, including resting tremor, bradykinesia, and rigidity (Alexoudi et al. Reference Alexoudi, Alexoudi and Gatzonis2018; Maiti et al. Reference Maiti, Manna and Dunbar2017; Poewe et al. Reference Poewe, Seppi, Tanner, Halliday, Brundin, Volkmann, Schrag and Lang2017). PD prevalence is increasing in low- and middle-income countries, where it affects 1.51% of the world’s population (Ou et al. Reference Ou, Pan, Tang, Duan, Yu, Nong and Wang2021; Zhu et al.). Over the past two decades, the global incidence of PD has shown a remarkable annual percentage increase of 16.32% between 2004 and 2023 (Zhu et al.). Therefore, identifying the underlying causes of this disease can assist us in preventing this alarming increase.
Neuron degeneration in PD likely arises from a combination of genetic and environmental factors (Koprich et al. Reference Koprich, Johnston, Huot, Reyes, Espinosa and Brotchie2011; Shulman et al. Reference Shulman, De Jager and Feany2011), although the exact causes remain unknown. Evidence suggests inflammation and mitochondrial dysfunction may contribute to dopaminergic neuron death (Johnson et al. Reference Johnson, Stecher, Labrie, Brundin and Brundin2019; Kannarkat et al. Reference Kannarkat, Boss and Tansey2013). Notably, infections have been implicated in persistent microglia inflammation and potentially in PD development (Navarro-López et al. Reference Navarro-López, Çelikok and Şengör2021; Zorina et al. Reference Zorina, Jurja, Mehedinti, Stoica, Chita, Floris and Axelerad2023). Several studies have shown associations between PD and viral and bacterial infections such as HSV-1 (Bu et al. Reference Bu, Wang, Xiang, Shen, Wang, Liu, Jiao, Wang, Cao, Yi, Liu, Deng, Yao, Xu, Zhou and Wang2015; Hemling et al. Reference Hemling, Röyttä, Rinne, Pöllänen, Broberg, Tapio, Vahlberg and Hukkanen2003), EBV (Bu et al. Reference Bu, Wang, Xiang, Shen, Wang, Liu, Jiao, Wang, Cao, Yi, Liu, Deng, Yao, Xu, Zhou and Wang2015; Espay & Henderson, Reference Espay and Henderson2011), influenza (Cocoros et al. Reference Cocoros, Svensson, Szépligeti, Vestergaard, Szentkúti, Thomsen, Borghammer, Sørensen and Henderson2021), Helicobacter pylori (Shen et al. Reference Shen, Yang, Wu, Zhang and Jiang2017), and HCV (Wijarnpreecha et al. Reference Wijarnpreecha, Chesdachai, Jaruvongvanich and Ungprasert2018). However, definitive links between infectious agents and PD have yet to be established (Li et al. Reference Li, Huang, Ren and Yang2022). Parasitic infections play a major role in neurological disorders. Numerous studies have indicated that Toxoplasma gondii may play a role in PD (Bisetegn et al. Reference Bisetegn, Debash, Ebrahim, Mahmood, Gedefie, Tilahun, Alemayehu, Mohammed and Feleke2023; Firouzeh et al. Reference Firouzeh, Ziaali, Sheibani, Doustimotlagh, Afgar, Zamanpour, Keshavarz, Shojaee, Shafiei, Esmaeilpour and Babaei2021; Nohtani et al. Reference Nohtani, Asgari, Mikaeili, Ostovan, Mirzaeipour, Bahreini and Rashidi2022; Virus et al. Reference Virus, Ehrhorn, Lui and Davis2021). Despite limited direct evidence linking other parasites to PD progression, certain parasites share characteristics with T. gondii that might be relevant to PD progression. One such parasite is the Toxocara species.
Toxocariasis, a neglected zoonotic parasitic infection prevalent in low-income areas (Chen et al. Reference Chen, Liu, Liu, Zheng, Hong, Sugiyama, Zhu and Elsheikha2018; Ma et al. Reference Rostami, Wang, Hofmann, Hotez and Gasser2020), is caused by Toxocara cati and T. canis in cats and dogs, respectively (Quintero-Cusguen et al. Reference Quintero-Cusguen, Gutiérrez-Álvarez and Patiño2021). This infection’s global prevalence stands at 19% (Rostami et al. Reference Rostami, Riahi, Holland, Taghipour, Khalili-Fomeshi, Fakhri, Omrani, Hotez and Gasser2019). Humans, as accidental hosts, acquire the parasite’s embryonated eggs through contaminated water, food, or soil (Choi et al. Reference Choi, Lim, Choi, Lee, Paik, Kim, Choi and Huh2012; Morimatsu et al. Reference Morimatsu, Akao, Akiyoshi, Kawazu, Okabe and Aizawa2006; Raissi et al. Reference Raissi, Sohrabi, Bayat, Etemadi, Raiesi, Jalali, Karami, Abdollahi, Hoseiny, Shayanfard, Alizadeh, Gadalla and Ibrahim2021). Toxocara larvae can migrate to various organs, including the brain. Larval migration in human toxocariasis can lead to severe clinical consequences, including internal organ damage (de Almeida Carvalho and Rocha Reference de Almeida Carvalho and Rocha2014), vision loss (Fata et al. Reference Fata, Hosseini, Woo, Zibaei, Berenji, Farash and Moghaddas2021; Zibaei et al. Reference Zibaei, Mahdavi, Firoozeh, Hasani and Bahadory2022), and neurological disorders (Chatzikonstantinou et al. Reference Chatzikonstantinou, Polymeropoulos, Stavrati, Konstantinidis and Kazis2022; Faure et al. Reference Faure, Goulenok, Lariven, Dossier, Henry-Feugeas, Argy and Papo2021; Ma et al. Reference Holland, Wang, Hofmann, Fan, Maizels, Hotez and Gasser2018). Specifically, neurotoxocariasis has been associated with meningoencephalitis, dementia, schizophrenia, epilepsy, and Alzheimer’s disease (Fan Reference Fan2020; Fan et al. 2015a; Gale and Hedges Reference Gale and Hedges2020; Luna et al. Reference Luna, Cicero, Rateau, Quattrocchi, Marin, Bruno, Dalmay, Druet-Cabanac, Nicoletti and Preux2018; Nicoletti Reference Nicoletti2020; Taghipour et al. Reference Taghipour, Habibpour, Mirzapour and Rostami2021b; Taghipour et al. Reference Taghipour, Rostami, Esfandyari, Aghapour, Nicoletti and Gasser2020). Despite the range of neurological disorders linked to toxocariasis, its connection with PD has received limited attention. A case-control study found a higher seroprevalence of Toxocara among PD patients (6%) compared to controls (0%), with no statistical significance (Çelik et al. Reference Çelik, Kaplan, Ataş, Öztuna and Berilgen2013). Notably, animal studies have suggested altered neurotransmitter levels, including serotonin, GABA, monoamines, and dopamine, in Toxocara-infected subjects (Abdel Ghafar et al. Reference Abdel Ghafar, Elkowrany, Salem, Menaisy, Fadel and Awara1996; Othman et al. Reference Othman, Abdel-Aleem, Saied, Mayah and Elatrash2010). Therefore, the potential for neuroinflammation and neural damage caused by Toxocara larvae in the brain could theoretically contribute to PD pathological processes.
Given the rising global prevalence of PD and the potential role infections may play in its pathogenesis, understanding the possible contribution of Toxocara infection to PD could provide valuable insights into its etiology. Investigating the links between this common parasitic infection and neurodegenerative disorders may lead to novel treatments and preventive strategies. Therefore, we conducted this study to evaluate this association in northern Iran.
Methods
Study site
Between June 5, 2022, and Julu 20, 2023, a case-control study was conducted at Rouhani Hospital, a referral hospital in Babol, Mazandaran province, northern Iran. This region experiences a hot, humid summer (20–35°C) and a mild, humid winter (13–20°C), with an annual precipitation exceeding 800 mm and a relative humidity over 70%. These conditions contribute to a high prevalence of parasitic diseases like Toxocariasis. Mazandaran province has a seroprevalence of over 23% (Aghamolaie et al. Reference Aghamolaie, Seyyedtabaei, Behniafar, Foroutan, Saber, Hanifehpur, Mehravar and Rostami2019; Fallah et al. Reference Fallah, Davoodi, Najafi-Vosough, Sardari, Kordi, Faizi, Azarghoon, Afrabandpey, Zare and Shojaei2021), which is two-fold higher than Iran’s mean seroprevalence (9.3%, range of 6.3–13.1%) (Eslahi et al. Reference Eslahi, Badri, Khorshidi, Majidiani, Hooshmand, Hosseini, Taghipour, Foroutan, Pestehchian and Firoozeh2020).
Study population and design
All participants provided written informed consent, and the research was approved by the Research Ethics Committee of Babol University of Medical Science, Babol, Iran (IR.MUBABOL.HRI.REC.1401.081). Patients presenting PD symptoms referred to the Ayatollah Rouhani Hospital’s neurology department and clinic were included in the study. PD diagnosis relies on the clinical symptoms outlined by the Movement Disorder Society (MDS), which include tremor, hypokinesia, rigidity, and postural instability (Goetz et al. Reference Goetz, Tilley, Shaftman, Stebbins, Fahn, Martinez-Martin, Poewe, Sampaio, Stern, Dodel, Dubois, Holloway, Jankovic, Kulisevsky, Lang, Lees, Leurgans, LeWitt, Nyenhuis, Olanow, Rascol, Schrag, Teresi, van Hilten and LaPelle2008). An expert neurologist confirmed the patient’s clinical condition, encompassing the duration, primary clinical manifestations, and severity of the disease. UPDRS-MDS criteria were used to assess movement disorders according to the MDS movement disorder association. A modified Yahr & Hoehn classification system was utilized to categorize the disease’s severity into three groups: 1–2 (mild), 3–5–2 (moderate), and more than 3 (severe). Brain imaging was performed to eliminate conditions such as brain tumors and cerebrovascular diseases. Patients with kidney and liver failure were excluded, as were those with neurologic disorders induced by neuroleptic drugs or toxins. Additionally, patients who did not provide consent or had incomplete information were excluded from the study. Healthy control subjects, matched in sex and age, were referred to the Rouhani Hospital’s General Health Outpatient Clinic. A neurologist examined all subjects, finding no evidence of PD or cognitive disorders.
Covariates
Based on an examination of existing literature, covariates linked with reduced cognitive abilities were selected: age (years), gender (male or female), education (below high school, high school graduate, or some college or higher), place of residence (urban or rural), diabetes (yes/no), prevalent coronary heart disease (CHD) (yes or no), systolic blood pressure (mm Hg), alcohol consumption (never or any use), and family history of PD. Additionally, covariates related to Toxocara infection included contact with dogs, cats, and soil (yes/no).
Sample collection and laboratory analysis
All participants underwent venipuncture, and their blood samples were immediately centrifuged for 5 minutes at 3500 rpm to separate serum. The sera samples were then aliquoted and transported on ice to the Laboratory of the Infectious Diseases and Tropical Medicine Research Center, Health Research Institute at Babol University of Medical Sciences, where they were stored at -20°C until analysis. Testing of the blood samples was conducted by expert technicians who were blinded to the individuals’ health conditions. Analysis of anti-Toxocara IgG serum antibodies was performed using enzyme-linked immunosorbent assays (ELISAs) from NovaTec Immunodiagnostics, Dietzenbach, Germany, which boasted a diagnostic specificity and sensitivity of over 95%. Test results were reported as international units (IU), with values less than 9.0 IU/mL, 9.0–11.0 IU/mL, and more than 11.0 IU/mL classified as ‘test-negative’, ‘suspicious’, and ‘test-positive’ for anti-Toxocara IgG serum antibodies, respectively.
Statistical analysis
Stata statistical software (v.16 Stata Corp., College Station, TX, USA) was used for all analyses. To summarize participant characteristics, we used mean and standard deviations (SD) for continuous variables and proportions for categorical variables. Pearson’s χ2 and Fisher’s exact tests were used to examine between-group differences. The seroprevalence of Toxocara infections in cases and healthy controls was also presented as percentages with 95% confidence intervals (CI). Using approximate Bayesian logistic regression with penalized likelihood (PL) estimation via data augmentation, we examined the association between Toxocara seropositivity and PD and calculated the odds ratios (ORs) and 95% CI (Discacciati et al. Reference Discacciati, Orsini and Greenland2015). Using the penlogit command, we added specific prior-data records to a data set automatically. Using these records, we generated a penalty function for the log-likelihood of a logistic model, which equals (up to an additive constant) a set of independent log prior distributions on the parameters of the model (Discacciati et al. Reference Discacciati, Orsini and Greenland2015). Using directed acyclic graphs (DAGs), variables were adjusted based on a minimal sufficient adjustment set for potential confounders, including age, gender, and parent or relative history of PD (Knüppel, Reference Knüppel2010). Statistical significance was defined as a P value of less than 0.05.
Results
The study comprised 91 patients with PD (35.2% female) and 90 healthy individuals (37.8% female) as controls. The mean ages for PD patients and healthy controls were 68.7 ± 10.1 and 68.4 ± 10.6 years, respectively. Among the PD patients, 20 individuals (22%) were younger than 60. Among them, 35 people (38.5%) were from urban areas and 56 people (61.5%) from rural areas. Forty-three individuals (47.3%) had no formal education. Thirty-one (34.1%) of the case group members had high blood pressure, 16 (17.6%) had diabetes, 3 (3.3%) were alcoholics, and 15 (16.5%) had a history of head trauma. Table 1 displays baseline characteristics of cases and controls.
Demographic and Clinical characteristics of patients with Parkinson’s disease and healthy controls
A total of 60 out of 181 participants (33.15%, 95% CI: 26.3–40.5%) were seropositive for anti-Toxocara IgG based on the ELISA test. Among the 91 PD patients, 30 (32.9%, 95% CI: 23.4–43.6%) tested positive for anti-Toxocara IgG. Comparatively, 30 out of 90 control participants (33.3%, 95% CI: 23.7–44.0%) were also seropositive. To further investigate the potential predictive value of Toxocara infection on PD, we performed univariate and multivariate logistic regression analyses. As shown in Table 2, with the control group serving as the reference, the ORs for univariate and multivariate analyses were 0.98 (95% CI: 0.52–1.82) and 0.95 (95% CI: 0.49–1.83), respectively. These findings indicate no statistically significant association between Toxocara infection and PD.
Univariable and multivariable analyses to assess whether there is an association between Parkinson’s disease (PD) and seropositivity to Toxocara spp. in elderly people
To assess the potential association between Toxocara infection and the severity of PD, we analyzed the data based on the modified Yahr & Hoehn classification system. Of the 91 PD patients, 22 were classified as mild, 30 as moderate, and 39 as severe. After adjusting for confounding variables in a multivariate analysis (Table 2), we observed a trend suggesting that individuals with Toxocara infection had a 63% lower chance of experiencing severe PD compared to those without the infection. However, this association did not reach statistical significance.
Discussion
Our case-control study investigated the potential associations between Toxocara infection/exposure and PD risk in the elderly, and the severity of the disease in an endemic area in northern Iran. Results revealed a similar prevalence of Toxocara infection/exposure among PD patients (33%) and controls (33.3%). In multivariate logistic regression analyses, there was no statistically significant association between Toxocara infection/exposure and PD. Additionally, Toxocara infection seropositivity was lower among patients with mild PD than among those with moderate to severe PD, but there was no significant association between seropositivity and severity. Previously, only one study examined the association between PD and toxocariasis. According to Celik et al., although the seroprevalence of T. canis was higher in patients with IPD (6.0%) than in controls (0%), no statistical differences were found (Çelik et al. Reference Çelik, Kaplan, Ataş, Öztuna and Berilgen2013).
Toxocara larvae can invade the brains of humans, and while case descriptions of cerebral toxocariasis are historically rare, improved diagnosis and greater awareness have contributed to increased detection. Despite this, cerebral or neurological toxocariasis (NT) remains a poorly understood phenomenon. Furthermore, our understanding of cognitive deficits due to toxocariasis in human populations remains particularly deficient. Recent data describe an enhanced expression of biomarkers associated with brain injury, such as GFAP, AβPP, TGF-β1, NF-L, S100B, tTG, and p-tau, in mice receiving even low doses of Toxocara ova (Fan et al. Reference Fan, Holland, Loxton and Barghouth2015b). The lack of a significant association between Toxocara infection and PD in this study raises questions about the underlying mechanisms involved in PD pathogenesis. Several viruses and bacteria have been linked to increased PD risk (Bopeththa and Ralapanawa Reference Bopeththa and Ralapanawa2017; Bu et al. Reference Bu, Wang, Xiang, Shen, Wang, Liu, Jiao, Wang, Cao, Yi, Liu, Deng, Yao, Xu, Zhou and Wang2015; Cocoros et al. Reference Cocoros, Svensson, Szépligeti, Vestergaard, Szentkúti, Thomsen, Borghammer, Sørensen and Henderson2021; Dourmashkin et al. Reference Dourmashkin, Dunn, Castano and McCall2012; Espay and Henderson Reference Espay and Henderson2011; Harris et al. Reference Harris, Tsui, Marion, Shen and Teschke2012; He et al. Reference He, Yuan, Zhang and Han2015; Hemling et al. Reference Hemling, Röyttä, Rinne, Pöllänen, Broberg, Tapio, Vahlberg and Hukkanen2003; Laurence et al. Reference Laurence, Benito-León and Calon2019; Sasco and Paffenbarger Reference Sasco and Paffenbarger1985; Shen et al. Reference Shen, Yang, Wu, Zhang and Jiang2017; Vlajinac et al. Reference Vlajinac, Dzoljic, Maksimovic, Marinkovic, Sipetic and Kostic2013; Wijarnpreecha et al. Reference Wijarnpreecha, Chesdachai, Jaruvongvanich and Ungprasert2018). There is, however, a different aspect to the relationship between parasitic infections such as T. gondii and Toxocara. Toxoplasma infection has been associated with many different aspects, including an increase in PD progression (Firouzeh et al. Reference Firouzeh, Ziaali, Sheibani, Doustimotlagh, Afgar, Zamanpour, Keshavarz, Shojaee, Shafiei, Esmaeilpour and Babaei2021) or a reduction in PD complications (Nohtani et al. Reference Nohtani, Asgari, Mikaeili, Ostovan, Mirzaeipour, Bahreini and Rashidi2022). According to Bayani et al., in a meta-analysis (Bayani et al. Reference Bayani, Riahi, Bazrafshan, Ray Gamble and Rostami2019), there was no statistically significant association between PD and toxoplasmosis. Similarly to Toxoplasma, Toxocara infection has been shown to have different effects on dopamine levels in vivo. Compared to uninfected mice, Othman et al. demonstrated significantly reduced levels of dopamine, serotonin, monoamines, and GABA in a murine model (Othman et al. Reference Othman, Abdel-Aleem, Saied, Mayah and Elatrash2010). However, Fan has shown that outbred ICR mice infected with Toxocara have elevated levels of dopamine (Fan Reference Fan2020). The study hypothesized that mice infected with Toxocara would exhibit increased expression of tyrosine hydroxylase, an enzyme that plays a crucial role in dopamine production. Toxocara infection/exposure may also contribute to the development of Schizophrenia, a disease characterized by an increased production of dopamine (Taghipour et al. Reference Taghipour, Habibpour, Mirzapour and Rostami2021a). It is important to further explore the possibility of dopamine-related PD occurring in Toxocara infection in light of the difference in dopamine expression between the two animal studies.
When the T-helper 2 (Th2) response is triggered by helminth infections such as Toxocara spp, it differs from that triggered by microbial pathogens. Th2 increases cytokine levels in the body such as IL-4, IL-5, and IL-13 (Maizels Reference Maizels2013). Moreover, Toxocara induces downregulated cytokines, including TGF-β and IL-10 (Allen and Maizels Reference Allen and Maizels2011). These cytokines are associated with suppression of the Th1 immune reaction along with decreased IL-17, IFN-γ, and TNF-α levels which are critical for PD progression (Di Lazzaro et al. Reference Di Lazzaro, Picca, Boldrini, Bove, Marzetti, Petracca, Piano, Bentivoglio and Calabresi2024). Pro-inflammatory cytokines can be neuroprotective, but a sustained or excessive release can damage neurons (Othman et al. Reference Othman, Abdel-Aleem, Saied, Mayah and Elatrash2010). It has been shown that in Toxocara-infected mice’s brains, nitric oxide and pro-inflammatory cytokines are significantly increased (Othman et al. Reference Othman, Abdel-Aleem, Saied, Mayah and Elatrash2010). Early in the course of PD, pro-inflammatory cytokines are expressed more frequently, and later in the course, a Th2- and Th17-mediated response is found (Di Lazzaro et al. Reference Di Lazzaro, Picca, Boldrini, Bove, Marzetti, Petracca, Piano, Bentivoglio and Calabresi2024). IL-5, IL-10, and IL-17 levels were lower in patients with a more recent onset of disease and higher in patients with a more extensive disease (Di Lazzaro et al. Reference Di Lazzaro, Picca, Boldrini, Bove, Marzetti, Petracca, Piano, Bentivoglio and Calabresi2024). Based on these facts, more studies are needed to investigate the immune response and inflammatory effects of Toxocara spp on PD, especially at different stages of PD progression and infection with Toxocara. Nevertheless, it is important to remember that not all PD patients exhibit consistent signs of an inflammatory cytokine imbalance. Furthermore, chronic inflammation is not necessarily associated with PD (Acioglu et al. Reference Acioglu, Heary and Elkabes2022; Zheng et al. Reference Zheng, Zhang, Zhang, Gao, Wang, Liu, Xue, Yao and Lu2022). In conclusion, there is no way to prove that every instance of PD is associated with elevated inflammatory processes and concomitant chronic infection.
Several limitations should be considered when interpreting the findings of this study. The relatively small sample size and the single-center design may limit the generalizability of the results. Additionally, the cross-sectional nature of the study prevents the establishment of causality between Toxocara infection and PD. Future research endeavors could involve larger, multicenter studies with longitudinal designs to validate these findings and explore potential causal relationships. Moreover, investigating the mechanistic pathways underlying the observed associations and considering other potential confounding variables could provide a more comprehensive understanding of the relationship between Toxocara infection and PD.
In conclusion, this study did not find a statistically significant association between Toxocara infection and Parkinson’s disease. However, the trend suggesting a potential protective effect against severe PD with increasing Toxocara infection warrants further investigation. These findings contribute to the ongoing discourse on the role of infectious agents in neurodegenerative diseases and highlight the need for additional research to elucidate the mechanisms underlying these complex relationships. Ultimately, understanding the interplay between infections and neurodegenerative disorders may offer novel insights into disease prevention and treatment strategies.
Acknowledgements
The authors are very thankful to the staff of the Rouhani Hospital, Babol, Iran. The authors would also like to thank all the participants in this study.
Author contribution
Conceptualization: A.A.K. and A.R.; methodology and laboratory analysis: A.A.K, A.A., D.A., and M.S.; statistical analysis: M.S. and S.S.; writing—original draft preparation: A.A.K, A.F., and A.R.; writing—review and editing: A.R., A.F. and S.S.; supervision and funding acquisition: A.A.K and A.R. All authors have read and agreed to the final version of the manuscript.
Data availability statement
All relevant data are within the manuscript and further data that support the findings of this study are available from the corresponding author upon reasonable request.
Financial support
The authors received no specific funding for this work.
Competing interest
The authors declare no conflict of interest.
