Toxoplasma gondii seropositivity and cognitive functioning in older adults: The national health and nutrition examination survey 2011 to 2014

DOI: https://doi.org/10.21203/rs.3.rs-2133394/v1

Abstract

Objective

To examine the relationship between Toxoplasma gondii infection and cognitive functioning in older adults.

Method

A total of 2,956 older adults aged 60 and above from the National Health and Nutrition Examination Survey from 2011 to 2014 were included. Serum toxoplasma gondii antibody was analyzed in the lab. A value > 33 IU/mL was categorized as positive for toxoplasma gondii infection; <27 IU/mL was categorized as negative. Cognitive tests included the Consortium to Establish a Registry for Alzheimer’s Disease Word Learning subtest (CERAD-WL) immediate and delayed memory, the Animal Fluency test (AFT), and the Digit Symbol Substitution Test (DSST). Multivariate linear regression models were constructed to examine the association between toxoplasma gondii seropositivity and standardized cognitive test-specific and global cognition z scores. All models were adjusted for age, race/ethnicity, education, depressive symptoms, smoking status, body mass index, prevalent coronary heart disease, stroke, and systolic blood pressure.

Results

Most of the 2,956 participants (mean age 70.0) were female (51.0%), non-Hispanic White (48.3%), and completed some college or above (48.3%). A total of 703 participants were positive for toxoplasma gondii infection (23.8%). Adjusted linear regression showed that compared with participants with negative toxoplasma gondii infection, those with positive toxoplasma gondii infection had lower CERAD-WL immediate memory (beta [β] -0.16, 95% confidence interval [CI] -0.25,-0.07), CERAD-WL delayed memory (β -0.15, 95% CI -0.24,-0.06), AFT (β -0.15, 95% CI -0.24,-0.06), DSST (β -0.34, 95% CI -0.43,-0.26), and global cognition (β -0.24, 95% CI -0.32,-0.16) z scores after controlling for the covariates.

Conclusions

Toxoplasma gondii seropositivity is independently associated with worse immediate and delayed verbal learning, language proficiency, executive functioning, processing speed, sustained attention, working memory, as well as global cognition in older adults. Public health measures are needed to prevent toxoplasma gondii infection, which may help preserve cognitive functioning in older adults.

1. Introduction

Alzheimer’s disease and related dementia (ADRD) is a serious public health threat worldwide. In 2016, a total of 43.8 million people had dementia in the world. With a growing number of people with ADRD, families, communities, and healthcare systems around the world are heavily burdened (1). Although dementia is currently not curable, identifying modifiable risk factors associated with ADRD can help reduce the burden of the disease. Cognitive test performance in older adults is an important indicator of their cognitive functioning (2). By examining their correlations to cognitive tests, risk factors for cognitive decline can be identified and intervened.

Toxoplasma gondii is among the most prevalent human zoonoses (3) and affects about 30% of the global population (4). Toxoplasma gondii infection can happen prior to or after a person is born, with postnatal infection being more common. By consuming oocysts found in cat feces-contaminated soil or water (5, 6) or eating tissue cysts in undercooked meat (7, 8), humans can be infected with toxoplasma gondii after birth. While most people with a healthy immune system are asymptomatic following acute infection with toxoplasma gondii, some may experience unspecific symptoms lasting from several weeks to months, including fever, malaise, and lymphadenopathy (9).

Previous studies have found that toxoplasma gondii infection is associated with neurocognitive changes in humans (10–13). However, their direction of findings and effect sizes are inconsistent. In addition, this area is understudied, given the prevalence of toxoplasma gondii infection in humans. In a systematic review and meta-analysis of the association of toxoplasma gondii seropositivity and cognitive function in healthy people, only thirteen studies were included, most of which had small sample sizes (4). In addition, two included studies (14, 15) utilized the National Health and Nutrition Examination Survey (NHANES) 1988–1994 cycle data and thus could not reflect the current epidemic of toxoplasma gondii infection. The findings of this study showed that seropositivity to toxoplasma gondii was modestly but significantly associated with poorer processing speed, working memory, verbal short-term memory, and executive functioning. However, studies using large, the latest, and nationally representative population-based data are needed to better elucidate the cognitive effects of toxoplasma gondii seropositivity.

In this study, taking advantage of the NHANES, we aimed to examine the relationship between toxoplasma gondii seropositivity and cognitive functioning in a nationally representative sample of U.S. older adults. The findings of this study will provide implications for understanding the cognitive effects of toxoplasma gondii infection and developing tailored public interventions to protect cognitive functioning and prevent dementia in the growing number of older adults in the US.

2. Method

2.1 The parent study design and study population

The NHANES is an ongoing, cross-sectional survey of civilian, non-institutionalized adults and children in the United States conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention. About 5,000 nationally representative participants across the country are surveyed bi-annually (16). Their sociodemographic, health, and nutritional status are evaluated using in-person interviews and physical exams. The interviews are conducted at participants’ homes; health exams are conducted in specially equipped mobile exam centers. Health exams include laboratory testing of urine and blood specimens and medical, dental, and physiological assessments. Participants' serum toxoplasma gondii antibody levels and cognitive functioning were measured in the NHANES 2011–2014 cycles. In this study, two survey cycles (2011–2012 and 2013–2014) were merged to increase sample size and power. Between 2011 and 2014, a total of 19,151 individuals participated in the NHANES. They were recruited from a selection of census blocks or clusters of census block area segments. The detailed sampling method has been published elsewhere (17).

Of the 19,151 individuals, we excluded individuals aged < 60 (n = 15,679) or had missing data on serum Toxoplasma gondii IgG (n = 511). Participants with equivocal serum Toxoplasma gondii IgG (≥ 27 and < 33 IU/mL) were further excluded (n = 5). Finally, a total of 2,956 participants aged 60 and above were included in the analysis. The characteristics of the excluded participants due to missing data (n = 516) were in the Appendix. Compared with the included participants, people who were excluded were more likely to be of other ethnicities than Non-Hispanic Whites, overweight/obese, completed lower education, had higher systolic blood pressure and had lower Digit Symbol Substitution Test (DSST) and Animal Fluency test (AFT) scores.

2.2 Ethical considerations

The National Center for Health Statistics Research Ethics Review Board approved the parent study. The University of Houston-Downtown Committee for the Protection of Human Subjects granted this study an exemption because only publicly accessible and de-identified data were used.

2.3 Independent variable: Toxoplasma gondii seropositivity

An enzyme immunoassay (EIA) that measures IgG against toxoplasma gondii was used to measure the presence of toxoplasma gondii (16). Toxoplasma gondii IgG was measured with two enzyme immunoassay kits (18). After all the antibody testing was finished, data was collated. Strict quality control was implemented for every plate. Values between 27 and 33 IU/mL were deemed as “equivocal”; values ≥ 33 IU/mL were deemed as “positive”; values < 27 IU/mL were deemed as “negative” (16). For our analysis, we categorized the participants into “negative” or “positive” for toxoplasma gondii infection.

2.4 Dependent variable: Cognitive functioning

Three cognitive tests were used to assess participants’ various domains of cognitive functioning, including the Consortium to Establish a Registry for Alzheimer’s Disease Word Learning subtest (CERAD-WL), the AFT, and the DSST. The detailed method has been published elsewhere (19).

1) The CERAD-WL assessed participants' capacity for both immediate (immediate memory) and delayed (delayed memory) verbal learning (20). It consisted of a delayed recall after three consecutive learning trials. For each learning trial, participants must read aloud ten randomly selected words that are displayed on a computer screen in huge, bolded characters, one at a time. Following the presentation of the words, participants were encouraged to retain and recall as many words as they could. In each of the three trials, the order of the ten words was changed. There was a ten-point maximum for each trial. A participant's immediate memory score, which varied from zero to 30, was the sum of their three trial scores. After the AFT and the DSST, participants took the delayed recall test, which asked them to recall as many words from the same ten-word list as they could. The delayed memory score, which varied from zero to ten, depended on how many accurate words a subject could recall.

2) Participants' language proficiency and executive function were assessed by the AFT (21). Each animal a participant named received one point, and they had one minute to name as many animals as they could. Participants were first prompted to identify three pieces of clothing as a warm-up.

3) The DSST measured the participants' working memory, sustained attention, and processing speed (22). A paper form with a top-mounted key that included nine numbers and paired symbols was used to conduct the exam. Participants got two minutes to copy the symptoms to the matching symbols in the 133 boxes that were placed next to the numbers.

The score was based on the total number of correct matches (23). Before participants started the formal test, a sample practice test was provided. The possible score range of the DSST was between zero and 133 (24).

2.5 Covariates

Covariates of this study included age (years), sex (male or female), race/ethnicity (Mexican Americans, other Hispanics, non-Hispanic White, or non-Hispanic Black), education (below high school, high school graduate, or some college or above), depressive symptoms, smoking status (never, former, or current smokers), BMI (< 18.5 kg/m2, 18.5–24.9 kg/m2, 25-29.9 kg/m2, or ≥ 30 kg/m2), prevalent coronary heart disease (CHD) (yes or no), stroke (yes or no), and systolic blood pressure (mmHg). The information was self-report or collected from health exams. The Patient Health Questionnaire (PHQ-9) was used to measure depressive symptoms (25). It is a nine-item screening tool on the frequency of depressive symptoms over the past two weeks and has a total score ranging from 0–27. A higher score indicates more severe depression symptoms. The PHQ-9 total score was used to indicate depressive symptoms.

2.6 Statistical analysis

Means (standard deviation [SD]) were used to describe the characteristics of the study population for continuous data that followed a normally distributed distribution. Medians (interquartile range) were used for non-normally distributed continuous data. Data for categorical variables were summarized using frequency (percentages). Independent T-tests were used to compare group differences for continuous variables between the negative and positive groups. Chi-square tests were used to compare group differences for categorical variables between the negative and positive groups.

The CERAD-WL immediate memory, CERAD-WL delayed memory, AFT, and DSST were standardized with mean zero and variance one to compute cognitive test-specific z-scores. The cognitive test-specific z scores of the four tests were then averaged to calculate the global cognition z scores. Linear regression models were constructed between toxoplasma gondii seropositivity (negative or positive) and each of the four cognitive test-specific and global cognition z scores. All models were adjusted for the covariates mentioned above. We considered a 95% confidence interval not including one as statistically significant. All analyses were performed using SPSS 25.0.

3. Results

The sociodemographic and health information of the study population, stratified by toxoplasma gondii seropositivity, was presented in Table 1. Of the 2,956 participants, 1,403 were from the 2011-2012 cycle, 1,553 from the 2013-2014 cycle. A total of 703 participants were positive for toxoplasma gondii infection (23.8%). They had a mean age of 70.0 (SD 7.0). Most of the 2,952 participants (mean age of 70.0 [SD 7.0]) were female (51.0%), non-Hispanic White (48.3%), completed some college or above (48.3%), were never smokers (49.8%), and had a BMI ≥30 (35.6%) and an average of 8.7 (SD 10.7) hours of physical activity every week. Their mean total cholesterol and systolic blood pressure were 190.2 mg/dL and 133.5 mmHg. Their mean delayed memory, immediate memory, AFT, and DSST score were 5.8 (SD 2.4), 18.5 (SD 5.0), 16.4 (SD 5.6), and 45.8 (SD 17.5), respectively. Compared with participants with negative toxoplasma gondii seropositivity, participants with positive toxoplasma gondii seropositivity were older, less educated, and more likely to be male, current smokers, and had more physical activities. They were also more likely to have different ethnicities and lower CERAD W-L immediate recall, CERAD W-L delayed recall, AFT, and DSST scores.

            Table 2 showed the mean and 95% CI of the cognitive test-specific z-scores by toxoplasma gondii infection status. The mean of CERAD W-L immediate call, CERALD W-L delayed recall, AFT and DSST was 0.04 (95% CI -1.94, 2.01), 0.03 (95% CI -1.92, 1.99), 0.03 (95% CI -1.95, 2.01) and 0.08 (95% CI -1.88, 2.03), respectively, among participants with negative toxoplasma gondii infection. Among participants with seropositive toxoplasma gondii infection, the mean of CERAD W-L immediate call, CERALD W-L delayed recall, AFT and DSST was -0.12 (95% CI -2.01, 1.77), -0.12 (95% CI -2.07, 1.83), -0.11 (95% CI -1.99, 1.76) and -0.26 (95% CI -2.15, 1.63), respectively. For the global cognition z-scores, the mean was 0.06 (95% CI -1.92, 2.03) for participants with negative toxoplasma gondii infection and -0.18 (95% CI -2.06, 1.70) for those with positive toxoplasma gondii infection.

Adjusted linear regression (Table 3) showed that compared with participants with negative toxoplasma gondii infection, those with positive toxoplasma gondii infection had lower CERAD-WL immediate memory (beta [β] -0.16, 95% CI -0.25,-0.07), CERAD-WL delayed memory (β -0.15, 95% CI -0.24,-0.06), AFT (β -0.15, 95% CI -0.24, -0.06), and DSST (β -0.34, 95% CI -0.43,-0.26) z scores controlling for age, race/ethnicity, education, depressive symptoms, smoking status, BMI, prevalent CHD, stroke, and systolic blood pressure. For the global cognition z score by averaging the four cognitive test-specific z-scores, the negative association remains (β -0.24, 95% CI -0.32,-0.16).

4. Discussions

In this sample of 2,956 nationally representative older adults in the US, positive toxoplasma gondii infection is independently associated with worse immediate and delayed verbal learning, language proficiency, executive functioning, processing speed, sustained attention, working memory, as well as global cognition. This relationship is independent of age, race/ethnicity, education, depressive symptoms, smoking status, BMI, prevalent CHD, stroke, and systolic blood pressure. Although our findings should be validated using longitudinal studies, they suggest that serum toxoplasma gondii seropositivity may be used as a biomarker of cognitive decline and preventing toxoplasma gondii infection a target of public health intervention to protect cognitive functioning in older adults. This is very important given the prevalence of toxoplasma gondii infection and the increasing population aging in the US.

A limited number of studies have examined the associations of toxoplasma gondii seropositivity with cognitive outcomes in humans. Two studies utilized the NHANES 1988–1994 cycle data and thus could not reflect the current epidemic of toxoplasma gondii infection (14, 15). In addition, one of them only included school-aged children (15). To the best of our knowledge, only two relevant studies exclusively targeted older adults (26, 27). In one study of 84 older adults aged 65 years and above in Germany, toxoplasmosis-positive participants showed impaired working memory, attention, and word fluency, but not processing speed measured by DSST, compared with those who were toxoplasmosis negative (26). It is important to note that the sample size of that study is very small. Then, in another study including older adults in the US, while no statistically significant association was found between toxoplasma gondii IgG levels and memory performance or attention, toxoplasma gondii IgG levels were inversely associated with global cognition measured by Mini-Mental State Examination (MMSE) (27). However, in that study, researchers did not adjust BMI, exercise, or depressive symptoms. In another longitudinal study targeting adults aged > 30 years with eleven years’ follow-up, researchers found no associations of toxoplasma gondii infections with verbal fluency and verbal learning assessed by CERAD (3). However, their study population was middle-aged, which was different from our participants. Overall, the findings of existing cross-sectional or longitudinal studies on this topic are inconsistent. Most of these studies were based on relatively small sample sizes, had methodological limitations, or targeted a different age group. However, in our study, we took advantage of a nationally representative, relatively large, and latest sample and calculated global cognition, adding stronger evidence on the negative relationship between toxoplasma gondii infection and cognitive functioning.

The possible mechanisms that account for the association between toxoplasmosis gondii infection and worse cognitive functioning are complicated. T gondii infection has been shown to increase dopamine release in vitro and animal trials (28–31). Excess dopamine turnover has been associated with worse cognitive decline (32–34). Dysregulated dopamine may influence neuronal plasticity in the hippocampus in humans, a brain region important for memory and spatial orientation (35, 36). In addition, as a defense mechanism against toxoplasma gondii infection, the host may rapidly catabolize tryptophan and produce more kynurenine and quinolinic acid (37). It is reported that higher levels of dopamine, kynurenine, and quinolinic acid were associated with increased neurotoxic effects and impulsive behavior incidence (38). Furthermore, toxoplasma gondii infection was associated with the dysbiosis of gut microbiota in mice, which may increase gut-blood-barrier permeability and induce mental disturbances and behavioral changes (39–41). Future studies are expected to explore the underlying mechanism.

The major strength of this study is the relatively large, nationally representative sample of older adults in the US. Stringent quality control and assurance measures were implemented throughout the NHANES study, including the rigorous assessment of toxoplasma gondii IgG and the adoption of validated cognitive tests to assess multiple cognitive functioning domains, therefore guaranteeing the quality of data used in this study. Moreover, a comprehensive list of sociodemographic, lifestyle, mental, and physical health covariates was adjusted, minimizing residual confounding. Thus, the findings of our study are generalizable to U.S. older adults. Importantly, the cognitive effects of toxoplasma gondii infection in humans are understudied in the literature. Thus, our study fills in a research gap. Last but not least, the findings of lower DSST score associated with toxoplasma gondii infection is important as previous studies have shown that lower DSST scores were independently associated with a higher risk of dementia (42, 43).

The major limitation of this study is the cross-sectional design which prevented us from examining whether participants had long-term exposure to toxoplasma gondii or a recent exposure where the IgG immune response to toxoplasma gondii had just started (4). Reverse causation is also possible. Additionally, research has revealed that specific genes affect susceptibility and immune response to toxoplasma gondii infection (44). However, our study did not assess any genetic factors. Finally, with three cognitive tests, we may not assess all domains of participants’ cognitive functioning. In addition, the excluded people (n = 516) were different from the included participants (n = 2,956) in many aspects; thus, selection bias is possible (45).

Future students are expected to 1) use more advanced methods for identifying specific strains and stages of toxoplasma gondii infection (46), 2) explore the pathophysiological mechanisms on the cognitive effects of toxoplasma gondii infection, 3) include non-western populations, and 4) utilize longitudinal designs to assess the temporal relationship between toxoplasma gondii infection and cognitive functioning. These studies may enable the identification of new biomarkers for cognitive decline and enlighten the development of toxoplasma gondii medications and vaccinations to protect people from the adverse effects of toxoplasma gondii infection.

In conclusion, toxoplasma gondii seropositivity is prevalent in U.S. older adults and is independently associated with worse immediate and delayed verbal learning, language proficiency, executive functioning, processing speed, sustained attention, working memory, as well as global cognition in this population. Future studies are expected to examine the longitudinal relationship and pathophysiological mechanism between toxoplasma gondii infection and cognitive functioning. Public health measures are needed to prevent toxoplasma gondii infection, which may help preserve cognitive functioning in older adults.

Declarations

5. Funding

The study was funded by the Organized Research and Creative Activities (ORCA) Program at the University of Houston-Downtown.

 

6. Conflict of Interest

The authors have no conflict of interest to declare.

 

7. Data Availability Statement

The data that support the findings of this study are openly available on the NHANES website and can be accessed at https://wwwn.cdc.gov/nchs/nhanes/Default.aspx

 

8. Acknowledgment

ZQ and HL performed data analysis; MZ, SG, XL, DY, and TX drafted the original manuscript. All the authors significantly provided feedback to the manuscript.

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Tables

Table 1. Characteristics of the participants by toxoplasma gondii seropositivity

Variables

Negative
 (n=2,253)

Positive
 (n=703)

Total
 (n=2,956)

P Value

Age, years

69.7(6.9)

70.9(7.0)

70.0(7.0)

<0.001

Sex, n (%)

 

 

 

<0.001

Male

1,057(46.9%)

390(55.5%)

1,447(49.0%)

 

Female

1,196(53.1%)

313(44.5%)

1,509(51.0%)

 

Race/ethnicity, n (%)

 

 

 

<0.001

  Mexican Americans

231(10.3%)

43(6.1%)

274(9.3%)

 

  Other Hispanics

161(7.1%)

131(18.6%)

292(9.9%)

 

  Non-Hispanic Whites

1,111(49.3%)

317(45.1%)

1,428(48.3%)

 

  Non-Hispanic Blacks

479(21.3%)

165(23.5%)

644(21.8%)

 

  Other

271(12.0%)

47(6.7%)

318(10.8%)

 

Education, n (%)

 

 

 

<0.001

  Below high school

594(26.3%)

256(36.4%)

850(28.7%)

 

  High school graduate

510(22.6%)

165(23.5%)

421(14.2)

 

  Some college or above

 1,147(50.9%)

281(39.9%)

1,428(48.3%)

 

Depressive symptoms

3.5(4.8)

3.3(4.5)

3.5(4.8)

0.334 

Smoking, n (%)

 

 

 

0.043

  Never

1,130(50.2%)

342(48.6%)

1,472(49.8%)

 

  Former

846(37.5%)

249(35.4%)

1,095(37.0%)

 

  Current

274(12.2%)

111(15.8%)

385(13.0%)

 

Body mass index, n (%)

 

 

 

0.444

  <18.5 kg/m2

35(1.6%)

9(1.3%）

44(1.5%)

 

  18.5-24.9 kg/m2

592(26.3%)

166(23.6%)

758(25.6%)

 

  25.0-29.9 kg/m2

784(34.8%)

262(37.3%)

1,046(35.4%)

 

  ≥30 kg/m2

800(35.5%)

251(35.7%)

1,051(35.6%)

 

Physical activity, hours/week

8.2(10.3)

10.6(12.1)

8.7(10.7)

0.025

Total cholesterol, mg/dL

190.7(42.5)

188.4(43.4)

190.2(42.7)

0.209

Systolic blood pressure, mmHg

133.2(19.8)

134.5(42.5)

133.5(27.0)

0.271

CERAD W-L immediate recall

18.7(5.0)

17.9(4.8)

18.5(5.0)

<0.01

CERAD W-L delayed recall

5.9(2.4)

5.5(2.4)

5.8(2.4)

<0.01

Animal Fluency Test

16.6(5.6)

15.8(5.3)

16.4(5.6)

<0.01

Digit Symbol Substitution Test

47.2(17.4)

41.2(16.8)

45.8(17.5)

<0.001

 

Table 2. Cognitive z-scores and 95% confidence intervals by toxoplasma gondii seropositivity 

 

Negative

(n=2,253)

Positive

(n=703)

CERAD W-L immediate recall

0.04 (-1.94, 2.01)

-0.12 (-2.01, 1.77)

CERAD W-L delayed recall

0.03 (-1.92, 1.99)

-0.12 (-2.07, 1.83)

Animal fluency test

0.03 (-1.95, 2.01)

-0.11 (-1.99, 1.76)

Digit symbol substitution test

0.08 (-1.88, 2.03)

-0.26 (-2.15, 1.63)

Global cognition

0.06 (-1.92, 2.03)

-0.18 (-2.06, 1.70)

 

Table 3. The independent associations of toxoplasma gondii seropositivity (Reference: Negative) with cognitive specific test and global cognition z-scores

 

 

Beta

95% CI

CERAD W-L immediate recall

-0.16

(-0.25，-0.07)

CERAD W-L delayed recall

-0.15

(-0.24，-0.06)

Animal fluency test

-0.15

(-0.24，-0.06)

Digit symbol substitution test

-0.34

(-0.43，-0.25)

Global cognition

-0.24

(-0.32，-0.16)