Part2: The Association Of Toxoplasma Gondi IgG Antibody And Chronic Kidney Disease Biomarkers
Jun 16, 2022
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3. Results
3.1.Data Summary
A descriptive summary (survey-weighted percentages or means and standard errors)of the variables used in the analysis are presented in Table 1. The total number of sampled participants used in this analysis was 4692, of whom 47.7% were males and 52.3%were females. The mean age was 47.9 years, and the percentages of Mexican American, other Hispanic, non-Hispanic white, non-Hispanic black, and other races were 8.4, 4.8, 70.3,9.9, and 6.5, respectively. The percentage of T.gondi IgG-positive participants was 15.2%, while the percentage of participants with albuminuria was 6.9%(first) and 4.0%(second). The percentage of participants with persistent albuminuria was 3.5%, and the average eGFR was 87.8 mL/min/1.73 m². The percentage of CKD-positive participants was 10%，including 0.9% in stage 1,1.5% in stage2,7.1% in stage 3, and 0.5%in stage 4.The portion of participants with mild CKD was 2.4%, and those with moderate-to-severe CKD was 7.6%.
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3.2. Association of Toxoplasma and CKD Biomarkers and CKD Status
Table 2 presents the associations between T. gondii lgG status and CKD biomarkers without accounting for any covariates. The design-based t-test or Rho-Scott chi-square was used to assess the significance of these associations. The results indicated that there were statistically significant associations between the T.gondii status and each second albumin-to-creatinine ratio(p=0.0376), second albuminuria (p=0.0005), and persistent albuminuria (p<0.0001), CKDstatus (p=0.0001), and CKDstages (p=0.0004). The highest proportion of T.gondi-positive patients occurred in stage 4, while the mean of eGFR was lower among the T.gondi-positive participants, but it was not statistically significant(p=0.0913). However, the level of eGFR was significantly different among positive and negative participants (p=0.0014). The elevated level of eGFR (stages 4 and 5)had higher proportions of positive participants than the negative participants(Figure 1). Furthermore, there was a statistically significant association between the T.gondi status and CKD levels with higher proportions of mild and moderate-to-severe levels of CKD among the positive participants when compared to the negative participants (p <0.0001).
3.3.Age Factor
Figure 2 shows that the participants who were exposed to Toxoplasma were significantly older than the non-exposed participants (56 years vs.49 years, p-value<0.0001). Similarly, the participants with positive CKD were significantly older than the negative participants (67 years vs.48 years, p-value<0.0001). We investigated the possibility of the confounding effect of age on the association between T.gondi exposure and CKD status. The magnitude of the confounding effect of age was evaluated by calculating the CKDrisk ratio within each age stratum(younger, older) and comparing it with the unstratified risk ratio (Table3). The unstratified risk ratio of CKD(RRcrude=1.27,95% CI:1.09-1.49) indicated that participants with positive Toxoplasma had a significantly higher(27% higher) risk of having CKD than the negative participants. While the stratified risk ratio was higher among the older participants than the younger participants (1.11 vs. 0.76), the risk ratio was insignificant in both age groups, as indicated by the corresponding 95% confidence intervals. The unstratified and stratified effect estimates differ by 17%, indicating the magnitude of the confounding factor of age. The overall risk ratio was calculated by pooling the stratified risk ratios using the Mantel-Haenszel formula [34](RRMH=1.09,95% CI:0.93-1.28), which indicated an insignificant difference between the positive and negative T.gondi participants. These results clearly indicate that age is an important confounding factor that must be accounted for when studying the association between T. gondii exposure and CKD. To account for other potential covariates in addition to age, we made use of multivariable logistic regression models as detailed below.
The age standardization method recommended by NHANES was used to remove the confounding effect of age when comparing the prevalence of CKD by Toxoplasma in the USA population. The age distribution was standardized using the 2000 US census population. Figure 3 shows that the prevalence of CKD was higher among Toxoplasma positive than Toxoplasma negative individuals (10.45 vs. 8.99).
3.4. Association of Toxoplasma and CKD after Adjusting for Co-Variates
Several binomial logistic regression models were created to investigate the association between CKD status (negative vs. positive) and Toxoplasma exposure (negative vs. positive)while accounting for potential covariates. The first model describes the association between CKD status and Toxoplasma exposure adjusted for demographic covariates (age, gender, and race/ethnicity) and BMl. Table 4 summarizes the results of this first model. In this model, T.gondi was significantly associated with CKD(OR= 1.40,95% CI = 1.06-1.84, p-value=0.00447).Specifically, positive T.gondi significantly increased the odds of having CKD, holding all other variables unchanged. Age was also positively associated with CKD (OR=8.89,95% CI=6.31-12.51, p-value<0.0001), with the participants 45+ years old being 8.89 times more likely to have CKD than those who are<45 years old. Additionally, the model showed that female participants were more likely to have CKD(27% higher odds than males, p-value =0.0352), whereas there were no significant differences among the different race/ethnicity groups. BMI had slight positive association with CKD(OR=1.04, 95% CI =1.02-1.06, p-value= 0.0059).
In the second model, additional covariates were added, including alcohol use and diabetes status (Table 5). The effect of T.gondi was not significant in this model; however, age continued to have a strong association with CKD status(OR=8.59, 95%CI=5.48-13.47, p-value =0.0002). Unlike the first model, gender did not have any significant association with CKD status in the second model. Race/ethnicity did not show any significant association with CKD status, which is similar to the conclusion obtained from the first model. Furthermore, participants with no history of diabetes had 60%lower odds of having CKD(OR=0.40,95% CI=0.27-0.61, p-value=0.0075)than those with a history of diabetes. Alcohol use did not have a significant association with CKD status (p-value=0.7102). The model in Table 5 provides a somewhat better fit for the data than the model in Table4, as indicated by the better fit statistics(lower AIC and higher Pseudo R-Square for the model in Table 5).
A third model has been created for CKDstatus of the T.gondii lIgG antibody by dropping age from the second model but keeping all other covariates. This model showed a significant association between T.gondi exposure and CKD status(OR=1.56,95% CI=1.20-2.04, p-value=0.0168). However, the second model (the model in Table5) fit the data better than the third model(Table6), as indicated by the AIC and R-Square values for the two models.
Additional variables, including smoking and drug use, were added to the third model to investigate the association of Toxoplasma and CKD status in the presence of these conditions (Tables 7 and 8). The association of Toxoplasma was significant in the CKD status when the smoking risk was considered (p=0.0325); however, the addition of drug use variables reduced the sample size to 1551 and defused the effect of Toxoplasma and the other Variables.
To investigate the association between the progression in the CKD stages and Tox-oplasma while adjusting for potential covariates, we built the ordinal logistic regression model described in Table 9. The model showed a significant association between T.gondi exposure and CKDstages(OR=1.41,95% CI=1.07-1.86, p-value=0.0424), which indicates that positive T.gondi increases the odds of CKD progression. In this model, age, gender, and BMI were all significantly associated with CKD progression, but race/ethnicity was not.
Additional models were created to investigate the association of Toxoplasma and CKD using two levels of CKD(mild and moderate-severe). The association of Toxoplasma was not apparent when the age factor was included in the model (Table 10). Nevertheless, excluding age from the model (Table 1l)showed a significant association between levels of CKD and Toxoplasma (p =0.0371)
4. Discussion
4.1. Overview of Results and Implications
The Centers for Disease Control and Prevention (CDC) estimates that 15% of US adults have CKD, with 90% of these individuals unaware of their condition. Therefore, it is critical to adequately investigate the risk factors associated with CKD to monitor and adequately diagnose it at its early stages.
Exposure to T. gondii may cause acute or chronic damage to the kidney, triggering injury, which can affect the exposure over their life course. Prior studies have found a link between undergoing dialysis and an increased rate of T. gondii infection [35,36], but there has not been a thorough investigation of the association of Toxoplasma with CKD in the USA population. Thus, the purpose of this study was to investigate this association along with common risk factors of CKD.
The investigation began by examining the level of each individual CKD biomarker among Toxoplasma-positive and negative participants. We found that the levels of the second albumin-to-creatinine ratio, the second albuminuria, and persistent albuminuria were significantly higher among the Toxoplasma-positive participants when compared with the T.gondi-negative study participants (Table 2). These biomarkers are the common indicators of kidney injury and CKD progression [23]. Other studies have linked Toxoplasma infection to high levels of serum creatinine and blood urea nitrogen [30], and the results of this study confirmed that adverse levels of CKD biomarkers are associated with Toxoplasma infection. Despite the finding that the level of estimated GFR was not significantly lower among the Toxoplasma-positive compared to the negative participants, the proportions of elevated levels of eGFR were significantly higher among the positive participants compared to the negative participants (p = 0014, Table 2 and Figure 1).
In addition, the Toxoplasma-positive participants had a significantly higher proportion of CKD(p =0.0001, Table 2) when both eGFR and persistent albuminuria were used to assess CKD.
Moreover, progression from mild to moderate-to-severe stages of CKD was associated with Toxoplasma(p<0.0001, Table 2), and progression to each stage of CKD was associated with Toxoplasma infection in this study (p = 0.0004, Table 2).
In this study, the likelihood of having CKD was significantly higher with exposure to Toxoplasma infection than without exposure (RR=1.27 95% CI:1.09-1.49, Table3). The relative risk is greater than one, an indication that CKD is more likely to occur with T.gondii infection.
Prior CKD and Toxoplasma studies in the USA population suggest that the prevalence of CKD or Toxoplasma is associated with age[1,27]. In this study, the participants with positive Toxoplasma or CKD were significantly older than the negative participants (p<0.0001, Figure 2). This significant association between age and each CKD and Toxoplasma indicated that age might serve as a confounding factor in the association between CKD and Toxoplasma. This observation was supported by the age-stratified analysis. After age adjustments, the prevalence of CKD was higher among Toxoplasma-positive participants compared to the Toxoplasma-negative participants(10.45 vs.8.99, Figure 3).
The association between Toxoplasma and CKD was further investigated using multivariable models to account for the multiple potential risk factors associated with CKD. The most common risk factors suggested by previous studies were age, gender, race/ethnicity, and BMI. Our analysis confirmed the association between Toxoplasma and CKD after adjusting for these factors, where Toxoplasma infection was found to be associated with significantly increased odds of having CKD(OR=1.40,95% CI=1.06-1.84,p=0.0447, Table4). Additionally, age was significantly associated with CKD status, where older individuals were more likely to have CKD(OR=8.59,95% CI=5.48-13.47,p=0.0002, Table 4).
Prior studies suggested that behavioral factors such as alcohol use, smoking, drug use, and medical history (e.g., having diabetes)contribute to the risk of CKD. Thus, these factors were included in the predictive model to investigate the association of T.gondi and CKD. After adding these factors to the model, the number of samples decreased due to the missing values in these variables, and the association of T.gondi was not apparent. However, the association of Toxoplasma in this model was obvious when the strong effect of the age factor was excluded from the model (Tables 5-8).
In addition, the results of this study showed that positive Toxoplasma increased the odds of progression to more severe stages of CKD(OR=1.41,95%CI=1.07-1.86,p=0.0424, Table 9)after adjusting for the other common factors(age, gender, race/ethnicity, and BMI.
Additional classification of CKD was considered in investigating the Toxoplasma association by categorizing CKD into two levels: mild and moderate-to-severe CKD. The predictive model created with this CKD classification did not show a significant association of Toxoplasma; however, the association was obvious after excluding age from the model (OR=1.42,95% CI=1.07-1.90, p=0.0371, Table 11).
Our study confirms there is an association between T.gondi infection and CKD. This association could have two directions:(1) infection with CKD increases the risk of T.gondii infection, or (2)T.gondii infection increases the risk of CKD. Prior studies suggest that CKD reduces the immune response to T.gondi exposure or reactivates latent Toxoplasma in the body [29,31]. On the other hand, latent T.gondi infection could injure and damage kidney tissues [8].
The mechanism by which T. gondii exposure damages the renal system is unclear. Previous studies have demonstrated that Toxoplasma infection leads to an increase in the production of nitric oxide and reactive oxygen species(ROS) in cells, resulting in oxidative stress [37]. This oxidative stress is linked to renal failure and triggers an initial inflammatory response mediated by proinflammatory mediators TNF-alpha and IL-1b, and a transcriptional factor, NF-KB. The later stage of inflammation induces an increase in TGF-beta production, leading to the synthesis of extracellular matrix[38]. Thus, the chronic effect of oxidative stress on kidney tissues is mediated through inflammation, and subsequent tissue damage, leading to eventual organ dysfunction [38]. This process must be understood in the context of the markers of interest in this study albumin, creatinine, and eGFR. Kidney dysfunction is measured by the level of albumin and creatinine in the blood—a healthy kidney maintains the level of albumin in the blood and filters creatinine waste from the blood. Thus, CKD biomarkers investigated in this study, including albuminuria and eGFR, give an insight into the association between Toxoplasma Gondii and CKD. Studies by Gupta et al. have indicated that biomarkers of inflammation are inversely associated with measures of kidney function and positively with albuminuria, with inflammation being more elevated among those with lower eGFR and higher urine albumin to creatinine ratio [39].
4.2. Implications of Findings
It was critical to explore the association between T. gondii and CKD, as parasitic infection effects on renal diseases are understudied. This was especially critical since research is moving towards understanding the exposome, that is, the entirety of exposures that bring about adverse health outcomes. Examining the effects of T.gondi helps to close the gap in the literature on how T. gondii and similar apicomplexan parasites contribute to adverse renal health outcomes in the short term and over the life course. With this understanding, future studies will be better able to quantify the effects of T.gondii exposure in the context of other exposures, such as environmental and social.
4.3.Limitation
While this study focused on establishing the association between Toxoplasma infection and CKD, further studies are needed to investigate the direction of this association. Simply put, owing to the cross-sectional design of the study, temporality cannot be determined. Therefore, a longitudinal study would offer better insight into the direction of the exposure and outcomes. The used data included a limited set of biomarkers; additional biomarkers such as cystatin C,β-trace protein (BTP), and Neutrophil gelatinase-associated lipocalin (NGAL) should be considered in the future studies.
In addition, a larger sample size would allow for more analysis of subsamples within this study. Thus, caution should be taken when interpreting the results.
5. Conclusions
Positive T.gondii IgG antibody is associated with CKD and the progression of CKD stages. This association is more apparent among older people. Further investigations are needed to examine these findings in different geographical locations and among differentially exposed populations.
