Risk of Dementia in Patients with Toxoplasmosis- A Nationwide, Population-based Based Cohort Study in Taiwan

Background Toxoplasma gondii (T. gondii) has infected about 25–30% of individuals worldwide, and it was dicult to detect its latent status. We aimed to evaluate the association between toxoplasmosis, the risk of dementia, and the effects of antibiotics in Taiwan. This nationwide, population-based, retrospective cohort study was conducted by using the two million Longitudinal Health Insurance Database retrieved from Taiwan’s National Health Insurance Research Database. The Fine and Gray’s competing risk analysis was used for the development of dementia in the toxoplasmosis cohort relative to the non-toxoplasmosis cohort. A sensitivity analysis was also conducted. The effects of the antibiotics (sulfadiazine or clindamycin) on the risk of dementia were also analyzed.


Introduction
Toxoplasmosis, caused by Toxoplasma gondii (T. gondii), which has infected about 25-30% individuals worldwide [1]. Individuals can become infected due to the ingestion of tissue cysts, infected meat, or food contaminated with sporulated oocysts [1]. After ingestion, the bradyzoites and sporozoites released from the cysts and oocysts would change the form to tachyzoites [2], which could spread into the blood stream and lymphatic system and cause distant organ invasion. The tachyzoites could then induce acute in ammation in the organs which might cause myocarditis, hepatitis, pneumonitis or retinochoroiditis [2]. They can also cross the blood-brain barrier and invade the brain cells during the rst week of infection [1]. In addition, it would become a chronic infection by the latent toxoplasmic cysts and remain in the tissues or central nervous system (CNS) [1]. Elevated risk of cerebral toxoplasmosis was noted in the elderly due to the possibility of the increase of immunosuppression [3]. In Taiwan, Administration appoints several senior external specialists in psychiatry, neurology, infection medicine, and other related medical specialties for randomly reviewing the records of ambulatory care visits and inpatient claims to verify the accuracy of the diagnoses [33]. Thus, it is therefore suitable to use the NHIRD to examine the longitudinal association between the toxoplasmosis and the potential risk of developing dementia afterward.

Ethical approval
This study was approved by the Institutional Review Board of the Tri-Service General Hospital (TSGH IRB No. 2-107-05-026). Because the patient identi ers were encrypted before their data were used for research purposes to protect con dentiality, the requirement for written or verbal consent from patients for data linkage was waived.

Study population
This was a retrospective cohort study. From the LHID of two million individuals, a sub-database, randomly strati ed retrieved from the NHIRD, we identi ed individuals as being ≧50 years old with a diagnoses as toxoplasmosis, that were selected between January 1, 2000, and December 31, 2015, according to ICD-9-CM code of 130.x. In this 15-year follow-up study, patients diagnosed with dementia or toxoplasmosis before 2000 or before the rst visit for toxoplasmosis, and all patients aged < 50 were excluded. The date of the toxoplasmosis diagnosed was de ned as the index date. Figure 1 depicts the ow chart of this study for the comparison of patients with toxoplasmosis and the controls. In addition, Figure S1 depicts the ow chart of this study for the comparison of patients with toxoplasmosis with and without antibiotics treatment.

Covariates
The covariates included sex, age group (50-64, ≥ 65 years), geographical area of residence (north, center, south, and east of Taiwan), urbanization level of residence (levels 1 to 4), levels of hospitals as medical centers, regional and local hospitals, and monthly income (in New Taiwan Dollars [NT$]; < 18,000, 18,000-34,999, ≥ 35,000). The urbanization level of residence was de ned according to the population, along with various indicators of the level of political, economic, cultural, and metropolitan development. Level 1 was de ned as a population of > 1,250,000, and a speci c designation as political, economic, cultural, and metropolitan development. Level 2 was de ned as a population between 500,000 and 1,249,999, and as playing an important role in the political system, economy, and culture. Urbanization levels 3 and 4 were de ned as a population between 149,999 and 499,999, and < 149,999, respectively.
The comorbidities in this study were diabetes mellitus, hypertension, hyperlipidemia, coronary artery disease, human immunode ciency virus infections/ acquired immune de ciency syndrome, and other immune de ciency diseases.
All the ICD codes of the comorbidities are as listed in Table S1.
Data on the usage of antibiotics, sulfadiazine and clindamycin, were collected. The data of the de ned daily dose (DDD) were obtained from the WHO Collaborating Centre for Drug Statistics Methodology (https://www.whocc. no/), and the duration of the usage of antibiotics was calculated by dividing the cumulative doses by the DDD of the antibiotics. While we analyzed the effects on the risk of dementia between the two subgroups with or without the antibiotics treatment, the sample was divided by the covariates with the references of previous studies using the NHIRD, regarding the treatment effects of medications medication [34][35][36]. The yearly times of the visits for psychiatry, neurology, and infection medicine clinics were also recorded.

Study Outcomes
All the participants were followed from the index date until the onset of dementia, withdrawal from the NIHRD, or the end of 2015. The patients with dementia were grouped as Alzheimer dementia (AD), vascular dementia (VaD). At least three visits in one consecutive year in the NHIRD records would be regarded as a diagnosis of dementia. All the ICD codes of dementia are as listed in Table S1.

Statistical Analysis
All analyses were performed using the SPSS software version 22 (SPSS Inc., Chicago, Illinois, USA). χ2 and t tests were used to evaluate the distribution of the categorical and continuous variables, respectively. The Fisher exact test for categorical variables was used to statistically examine the differences between the two cohorts. The Fine and Gray's survival analysis was used to determine the risk of dementia, and the results are presented as a hazard ratio (HR) with a 95% con dence interval (CI). A sensitivity analysis by excluding the diagnosis of dementia within the rst year, and the rst ve years, was conducted to avoid protopathic bias. The difference in the risk of dementia, between the genital warts subjects and control groups was estimated using the Kaplan-Meier method with the log-rank test. A 2-tailed P value < 0.05 was considered to indicate the statistical signi cance.

Results
Sample characteristics Table 1 shows that a total of 800 patients that were enrolled, including 200 subjects with toxoplasmosis and 600 controls without toxoplasmosis, which were 1:3 matched for age, sex, and index year. There were no differences in sex and age. The toxoplasmosis cohort tended to have a higher percentage in the comorbidities of DM, but a slightly lower percentage of HIV/AIDS and other immunode ciency diseases, in comparison to the non-toxoplasmosis controls. The patients with toxoplasmosis tended to have the monthly insurance premiums of NT$18,000-34,999, live in the middle, eastern, and the outlying islands, residing in areas of urbanization levels 2, 3, and 4, and seek medical care from the medical center and regional hospital. In addition, patients with toxoplasmosis have visited more clinics of infection disease and neurology, than the control group. Kaplan-Meier model for the cumulative incidence of dementia Of the toxoplasmosis patients, 21 in 200 (457.84 per 10 5 person-years) developed dementia, when compared to 56 in 600 (323.42 per 10 5 person-years) in the control group, and the difference was statistically signi cant in the Kaplan-Meier survival analysis (log-rank, p =0.030, Figure 2).
Of the toxoplasmosis patients, 20 in 191 with antibiotics treatment (455.72 per 10 5 person-years) developed dementia when compared to 1 in 9 without antibiotics treatment (507.67 per 10 5 person-years) in the control group, and the difference was statistically signi cant in the Kaplan-Meier survival analysis (log-rank, p =0.099, Figure S2). Types and sensitivity analysis of dementia after toxoplasmosis Table 3 reveals that the toxoplasmosis patients were associated with overall dementia, AD, and other degenerative dementia, with an adjusted HR as 2.878 (p<0.001), 6.675 (p<0.001), and 3.162 (p<0.001), respectively. Toxoplasmosis was noted as being associated with VaD. Table 3 also depicts that, after the exclusion of diagnosis within the rst year or rst ve years, toxoplasmosis was only associated with the other degenerative dementia.

HR analysis of dementia in the patients with toxoplasmosis
The effects of antiprotozoal medications for toxoplasmosis and the risk of toxoplasmosis Antiprotozoal medications usage for toxoplasmosis was associated with a lower risk than the comparison group, both sulfadiazine and clindamycin, either monotherapy or combination treatment, were associated with a lower risk of dementia (Table 4).

Discussion
In this retrospective cohort study, there are several noteworthy ndings. First, the patients with toxoplasmosis had a nearly 2.8-fold increased risk of the development of dementia, that is, after the sensitivity analysis by excluding the diagnosis of dementia the rst year and the rst ve years since toxoplasmosis was diagnosed, the patients with toxoplasmosis still had a two-fold increased risk for developing dementia. Second, the sensitivity analysis revealed that, by excluding the AD diagnosis in the rst year and rst ve years after toxoplasmosis, the association became insigni cant, but VaD and other degenerative dementia were still associated with toxoplasmosis. However, other types of degenerative dementia were found to be proportionately higher than AD and VaD, and most of the community studies revealed that Alzheimer-type dementia is the most common cause of dementia (40-60% in all dementias), followed by vascular dementia (20-30% in all dementias), and mixed or other dementias (7-15%) in Taiwan [37][38][39]. One possible explanation for this disparity is that some subjects were classi ed as other degenerative type of dementia, similar to the ndings of previous studies [32,35]. Thirdly, the usage of medications such as sulfadiazine and clindamycin, either monotherapy or combination treatment, were associated with a lower risk of dementia. To the best of our knowledge, this is the rst nationwide, population-based study on the topic about the association between the toxoplasmosis and the risk of dementia, and the effects of antibiotics usage in the reducing risk after toxoplasmosis infections.
In previous studies, the brain is the main target organ in T. gondii infection and may cause life-threatening encephalitis in immunosuppressed patients [40]. In healthy individuals, the tachyzoites of the parasite could be cleaned by cellular immune response in the proliferative stage of the systemic infection [41]. In the infected mice brain model, the interferon-gamma (IFN-γ) produced by lymphocyte, microglial cell and blood-derived macrophage could mediate the cell-immune response to the proliferating tachyzoites [42]. The IFN-γ could also activate the astrocytes which inhibit the tachyzoites replication by nitric oxide (NO) production [42]. The microglial are also the resident innate immune cells in the CNS which are the main cause of the in ammatory process. Uncontrolled activation of microglial cells may cause neurotoxicity due to the releasing of in ammatory cytokines, NO or superoxide (SOD). In addition to the acute toxoplasmosis caused by tachyzoites, there were also bradyzoites of the parasite which could produce a tissue cyst and slowly replicate in the brain or muscles and become latent toxoplasmosis [41]. Although the tachyzoites could induce more obvious in ammatory cytokine production than bradyzoites [42], the dormant parasite could resume the pathogenic activity and kill the host with immune de ciency. The latent toxoplasmosis revealed the asymptomatic in a normal condition. However, in comparison with the acute toxoplasomosis, the latent toxoplasmosis might cause a slow and cumulative effect that decreased the psychomotor performance [43]. The early animal model study had already discovered the pathological change in the cyst-containing region of the brain in mice including the granulomatous change of the perivascular areas and necrotic tissue deposition with vascular sclerosis [44]. Torres et al., (2018) had designed another mice model and argued that Möhle et al., didn't evaluate the advanced signs of AD which could be caused by cerebral amyloid angiopathy (CAA) driven by T. gondii, and there is the Aβ immunoreactivity co-localized with the T. gondii cysts as early as day 15 post infection and widespread Aβ immunoreactivity They were detected in other areas of the brain where they didn't co-localize with cysts at days 60 to 90 post infection [45]. Moreover, Torres et al., pointed out that Aβ immunoreactivity may lead to N-methyl-D-aspartate receptor (NMDAR) loss. In the CNS system, the glutamate plays the role of neuron excitation and could be endocytosed or released at the synapse through NMDAR on neural cells. Therefore, the NMDAR plays an important role of the synaptic connection which controls the function of learning and memory. The NMDAR dysfunction is highly associated with AD [46]. There was also strong evidence that the NMDAR antagonist could avoid the neuron dysfunction by Aβ immunoreactivity [47]. However, some studies had found that the countries with high seropositivity of T. plasma didn't have a higher AD prevalence. For example, in the 1970s, the seroprevalance of France was 70% [1], but only 3% prevalence of AD in people more than 60 years old were noted in 2012 [48]. Möhle et al., (2016) had declared a mice model study and discovered that there were reduced Aβ plaques in T. plasma infected mice compared to the non-infected mice [49]. The association between toxoplasmosis and AD, as well as the underlying mechanisms, is as yet to be clari ed.
Our study has several strengths: First, we used Taiwan's NHIRD, which is a valuable resource to cover a nationwide population, to address this issue. Second, several previous studies have demonstrated the accuracy and validity of several diagnoses of neuropsychiatric disorders in the NHIRD, such as Tourette syndrome [50], stroke [29,[51][52][53], sleep apnea [54], and major depressive disorder [55]. Besides, as aforementioned, the in-hospital licensed medical Therefore, this study was conducted in a large, nationwide, and reliable database for the association between toxoplasmosis and psychiatric morbidities in an Asian country.
The present study has several limitations that warrant consideration. Firstly, similar to the previous studies, not all data were recorded in the NHIRD, and we were unable to evaluate the family history, neurological severity, types, laboratory parameters, the availability of rehabilitation, and the additional examination ndings (e.g., neuroimaging). Therefore, the lack of data about the clinical and radiological course and treatment of the disease was a limitation. Secondly, other factors, such as genetic, psychosocial, and environmental, were not included in the dataset. However, since the present study had covered all of Taiwan's hospitals and > 99% of the Taiwanese population during a 15year period, which thereby increased the likelihood of our data being valid and representative. Thirdly, the prevalence of the Toxoplasma infection in Taiwan was about 10% in 2006 (https://nidss.cdc.gov.tw) but only focused on pregnant women, and it couldn't represent the general prevalence of the total population. Therefore, we couldn't correlate the current prevalence of dementia with the prevalence of toxoplasmosis in the past.

Conclusion
To the best of our knowledge, we have provided the rst evidence that toxoplasmosis is associated with AD and other dementia in Taiwan, but the association between toxoplasmosis and VaD is not signi cant. The antibiotics for toxoplasmosis treatment had the effect to prevent the potential risk of dementia in the future. Clinicians should pay more attention on the risk of dementia in the patients with toxoplasmosis.

Declarations
Acknowledgements: This study was supported by the Tri-Service General Hospital Research Foundation under the grants from the Medical Affairs Bureau, the Ministry of Defense of Taiwan (MAB-107-084 and MND-MAB-110-087), the Tri-Service General  Hospital Research Foundation (TSGH-C108-003, TSGH-C108-151, TSGH-B-109-010, TSGH-E-110240, and TSGH-B-110-012), and the Taoyuan Armed Forces General Hospital (TYAFGH-A-110020). The sponsor had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also appreciate the provision of the National Health Insurance Research Database by the Taiwan's Health and Welfare Data Science Center and Ministry of Health and Welfare (HWDC, MOW).

Data Availability:
The data on the study population that were obtained from the NHIRD (http://nhird.nhri.org.tw/en/index.html) are maintained in the NHIRD (http://nhird.nhri.org.tw/). The NHRI is a nonpro t foundation established by the government. Only citizens of the Taiwan who ful ll the requirements of conducting research projects are eligible to apply for the NHIRD. The use of the NHIRD is limited to research purposes only. Applicants must follow the Computer-Processed Personal Data Protection Law (http://www.winklerpartners.com/?p=987) and the related regulations of the National Health Insurance Administration and NHRI, and an agreement must be signed by the applicant and their supervisor upon application submission. All applications are reviewed for approval of data release.
Con icts of Interest: none    Table 3.; CI = con dence interval  Toxoplasmosis tracking to dementia

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