# Toxoplasma gondii Infection in Diabetes Mellitus Patients in China: Seroprevalence, Risk Factors, and Case-Control Studies

Authors: Yong-Xin Li; Hai Xin; Xiang-Yan Zhang; Cui-Ying Wei; Yu-He Duan; Hao-Fu Wang; Hai-Tao Niu
Journal: BioMed Research International
 (2018)

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## Abstract

The association betweenToxoplasma gondii (T. gondii) infection and diabetes mellitus remains controversial. With the improvement of living standards, the prevalence rate of diabetes is steadily increasing in China. Thus, it is necessary to explore the possible association between toxoplasmosis and diabetes mellitus in China. Hence, case-control studies were conducted to explore theT. gondii seroprevalence and identify the risk factors and possible transmission routes ofT. gondii infection in different types of diabetes, including type 1 diabetes (T1DM), type 2 diabetes (T2DM), and gestational diabetes (GDM) patients in China. Four hundred serum samples for each type of diabetes mellitus, matched with 400 control subjects for each group, were collected and examined for anti-T. gondii IgG and IgM antibodies using commercially available enzyme immunoassay kits. The totalT. gondiiseroprevalence in T1DM, T2DM, and GDM patients was 16.50%, 23.50%, and 21.25%, respectively. Each type of diabetes mellitus patients had a significantly higherT. gondii seroprevalence than the control subjects. Multivariate regression identified three variables as risk factors forT. gondiiinfection in diabetes patients, including keeping cats at home and consumption of raw oysters for T1DM patients and consumption of raw/undercooked meat and raw oysters for T2DM patients, which may help to guide future research and control policies in diabetes mellitus patients.


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## Body

## 1. Introduction

Toxoplasma gondii, an obligate intracellular opportunistic parasite, can infect nearly all types of warm-blood animals, including humans [1]. Notably, nearly one-third of the human population worldwide have been estimated to be infected with this parasite, and over 7% of Chinese have chronicT. gondiiinfection [1–3]. In general, mostT. gondii infections do not cause significant clinical symptoms [4]. But in some cases, infected persons may present clinical symptoms of toxoplasmosis such as lymphangoncus, cerebral, and eye diseases [4–6]. In some extreme cases,T. gondii infection can be reactivated and lead to a life-threatening disease with involvement of the central nervous system in immunocompromised patients [4, 5].T. gondiican reach many organs of the host after infection [7], including the pancreas [8].Diabetes mellitus is a common chronic metabolic disease and more than 300 million persons worldwide are projected to be affected by this disease in 2030 [9]. With the improvement of living standards, the prevalence rate of diabetes has steadily increased in China. The sensitivity and susceptibility to various infections can be higher in diabetes mellitus patients [10]. In some cases, the Apicomplexan parasite,T. gondii, has been proposed as a possible cause of diabetes, and current information is nearly predicated on this issue [11–13]. Meanwhile, chronic toxoplasmosis has been considered as a potential risk factor for type 2 diabetes (T2DM) identified by a meta-analysis of studies on the association between chronic toxoplasmosis and diabetes mellitus [13]. However, type 1 diabetes (T1DM) patients in Colombia were found to have significantly lowerT. gondii seroprevalence [14]. In USA, no association was found betweenT. gondiiinfection and diabetes mellitus in a prospective cohort of elderly Latinos [15]. Since data of previous studies on the association betweenT. gondii infection and diabetes mellitus remain controversial, we conducted matched case-control studies to determine whetherT. gondii seropositivity is associated with different clinical types of diabetes mellitus, including T1DM, T2DM, and gestational diabetes (GDM), and explore the risk factors forT. gondiiinfection in diabetes patients for the first time in China.


## 2. Materials and Methods

### 2.1. Study Sites

The study was conducted in The Affiliated Hospital of Qingdao University, Qingdao (35°35′–37°09′N, 119°30′–121°00′E), Shandong province, eastern China. The Affiliated Hospital of Qingdao University is a large comprehensive hospital in Shandong province that occupies an important position in the national medical system. The patients at this hospital mainly come from five provinces (Shandong, Jiangsu, Liaoning, Jilin, and Heilongjiang).


### 2.2. Study Design and Sample Collection

Through case-control studies, we studiedT. gondii seroprevalence and identified the risk factors and possible transmission routes ofT. gondii infection in diabetes mellitus patients and control subjects in China between September 2014 and January 2017. A total of 1200 diabetes mellitus patients who visited the Affiliated Hospital of Qingdao University were included in the study. Three types of diabetes mellitus patients (T1DM, T2DM, and GDM) were invited to participate in this study. The number of patients of each type was 400 (Tables 1–3).Table 1
Seroprevalence ofT. gondii infection by sociodemographic factors in type 1 diabetes (T1DM) patients and controls in eastern China.
Characteristic T1DM (N=400) Controls (N=400) T1DMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 62 15 24.19 0.187 62 10 16.12 0.591 0.263 31-40 70 12 17.14  85 12 14.12  0.604 41-50 100 13 13.00  90 8 8.89  0.367 51-60 92 18 19.57  86 8 9.30  0.053 >60 76 8 10.53  67 8 11.94  0.789 Region Shandong 170 34 20.00 0.237 170 23 13.53 0.493 0.110 Jilin 141 21 14.89  141 13 9.22  0.144 Heilongjiang 89 11 12.36  89 10 11.24  0.816 Gender Male 207 31 14.98 0.395 221 23 10.41 0.447 0.155 Female 193 35 18.13  179 23 12.85  0.160 Area of residence Urban 202 30 14.85 0.370 211 16 7.85 0.031 0.019 Rural 198 36 18.18  189 27 14.29  0.299 Employment Unemployed 156 28 17.95 0.261 137 21 15.33 0.147 0.549 Employed part-time 139 26 18.71  161 13 8.07  0.006 Employed full-time 105 12 11.43  102 12 11.76  0.940 Obesity and overweight Yes 156 21 13.46 0.191 194 24 12.37 0.596 0.762 No 244 45 18.44  206 22 10.68  0.021 Keeping cats at home Yes 50 16 32.00 0.002 58 10 17.24 0.138 0.074 No 350 50 14.29  342 36 10.53  0.134 Consumption of raw/undercooked meat Yes 26 8 30.77 0.043 60 10 16.67 0.174 0.140 No 374 58 15.51  340 36 10.59  0.052 Consumption of oyster Raw 94 32 34.04 <0.001 134 16 11.94 0.845 <0.001 Boiled 306 34 11.11  266 30 11.28  0.950 Consumption of fish Raw 147 34 23.13 0.007 167 19 11.38 0.948 0.006 Boiled 253 32 12.65  233 27 11.59  0.721 Consumption of raw vegetables and fruits Yes 204 37 18.14 0.368 228 27 11.84 0.805 0.066 No 196 29 14.80  172 19 11.05  0.287 Exposure to soil Yes 232 40 17.24 0.639 247 32 12.96 0.246 0.190 No 168 26 15.48  153 14 9.15  0.182 Gardening or agricultural activities Yes 257 42 16.34 0.909 257 33 12.84 0.260 0.261 No 143 24 16.78  143 13 9.09  0.053 Washing hands before meals Yes 262 44 16.79 0.827 266 28 10.53 0.390 0.036 No 138 22 15.94  134 18 13.43  0.559 Source of drinking water Spring/well 85 19 22.35 0.101 68 13 19.12 0.031 0.625 Tap 315 47 14.92  332 33 9.94  0.054 Total 400 66 16.50  400 46 11.50  0.042Table 2
Seroprevalence ofT. gondii infection by sociodemographic factors in type 2 diabetes (T2DM) patients and controls in eastern China.
Characteristic T2DM (N=400) Controls (N=400) T2DMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 37 9 24.32 0.956 37 4 10.81 0.793 0.127 31-40 83 19 22.89  87 11 12.64  0.080 41-50 96 23 23.96  99 13 13.13  0.052 51-60 93 24 25.81  97 12 12.37  0.018 >60 91 19 20.88  90 7 7.78  0.012 Region Shandong 156 45 28.85 0.074 156 25 16.03 0.025 0.007 Jilin 142 32 22.54  142 17 11.97  0.019 Heilongjiang 102 17 16.67  102 5 4.90  0.007 Gender Male 202 50 24.75 0.551 211 27 12.80 0.492 0.002 Female 198 44 22.22  189 20 10.58  0.002 Area of residence Urban 200 50 25.00 0.479 229 18 7.86 0.005 <0.001 Rural 200 44 22.00  171 29 16.96  0.223 Employment Unemployed 163 43 26.38 0.118 169 24 14.20 0.326 0.006 Employed part-time 133 23 17.29  148 13 8.78  0.033 Employed full-time 104 28 26.92  83 10 12.05  0.012 Obesity and overweight Yes 152 30 19.74 0.152 190 20 10.53 0.470 0.017 No 248 64 25.81  210 27 12.86  <0.001 Keeping cats at home Yes 52 19 36.54 0.017 56 9 16.07 0.279 0.015 No 348 75 21.55  344 38 11.05  <0.001 Consumption of raw/undercooked meat Yes 28 14 50.00 <0.001 161 5 3.11 <0.001 <0.001 No 372 80 21.51  339 42 12.39  0.001 Consumption of oyster Raw 91 41 45.05 <0.001 143 15 10.49 0.559 <0.001 Boiled 309 53 17.15  257 32 12.45  0.119 Consumption of fish Raw 150 50 33.33 <0.001 167 22 13.17 0.454 <0.001 Boiled 250 44 17.60  233 25 10.73  0.031 Consumption of raw vegetables and fruits Yes 253 66 26.09 0.109 286 32 11.19 0.581 <0.001 No 147 28 19.05  114 15 13.16  0.203 Exposure to soil Yes 228 54 23.68 0.920 248 34 13.71 0.120 0.005 No 172 40 23.26  152 13 8.55  <0.001 Gardening or agricultural activities Yes 248 56 22.58 0.580 254 35 13.78 0.096 0.011 No 152 38 25.00  146 12 8.22  <0.001 Washing hands before meals Yes 270 63 23.33 0.910 285 30 10.53 0.232 <0.001 No 130 31 23.85  115 17 14.78  0.075 Source of drinking water Spring/well 90 20 22.22 0.745 64 13 20.31 0.020 0.776 Tap 310 74 23.87  336 34 10.12  <0.001 Total 400 94 23.50  400 47 11.75  <0.001Table 3
Seroprevalence ofT. gondii infection by sociodemographic factors in gestational diabetes (GDM) patients and controls in eastern China.
Characteristic GDM (N=400) Controls (N=400) GDMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 185 35 18.92 0.043 201 26 12.94 0.017 0.107 31-40 184 38 20.65  71 19 26.76  0.294 >40 31 12 38.71  28 3 10.71  0.014 Region Shandong 239 59 24.69 0.079 239 32 13.39 0.451 0.002 Jilin 65 8 12.31  65 8 12.31  1.00 Heilongjiang 95 18 18.95  95 8 8.42  0.035 Area of residence Urban 202 45 22.28 0.612 245 25 10.20 0.165 0.001 Rural 198 40 20.20  155 23 14.84  0.192 Employment Unemployed 126 24 19.05 0.081 98 15 15.31 0.470 0.464 Employed part-time 143 39 27.27  197 20 10.15  <0.001 Employed full-time 131 22 16.79  105 13 12.38  0.343 Obesity and overweight Yes 79 15 18.99 0.582 117 9 7.69 0.086 0.018 No 321 70 21.81  283 39 13.78  0.011 Keeping cats at home Yes 40 12 30.00 0.135 47 4 8.51 0.433 0.010 No 360 73 20.28  353 44 12.46  0.005 Consumption of raw/undercooked meat Yes 33 12 36.36 0.027 77 5 6.49 0.098 <0.001 No 367 73 19.89  323 43 13.31  0.021 Consumption of oyster Raw 84 30 35.71 <0.001 153 18 11.76 0.909 <0.001 Boiled 316 55 17.41  247 30 12.15  0.084 Consumption of fish Raw 137 42 30.66 <0.001 158 17 10.76 0.086 <0.001 Boiled 263 43 16.35  242 41 16.94  0.858 Consumption of raw vegetables and fruits Yes 282 64 22.70 0.275 299 37 12.37 0.692 0.001 No 118 21 17.80  101 11 10.89  0.149 Exposure to soil Yes 233 47 20.17 0.534 254 34 13.39 0.261 0.045 No 167 38 22.75  146 14 9.59  0.002 Gardening or agricultural activities Yes 257 52 20.23 0.505 263 35 13.31 0.265 0.034 No 143 33 23.08  137 13 9.49  0.002 Washing hands before meals Yes 266 54 20.30 0.513 302 38 12.58 0.529 0.013 No 134 31 23.13  98 10 10.20  0.011 Source of drinking water Spring/well 78 14 17.95 0.840 66 15 22.73 0.003 0.476 Tap 322 61 18.94  334 33 9.88  <0.001 Total 400 85 21.25  400 48 12.00  <0.001A total of 1200 control subjects, matched with diabetes mellitus patients by age, gender, and residence, were included in the study. Serum samples were obtained from persons who participated in health screening at the Affiliated Hospital of Qingdao University.Approximately 5 mL of venous blood samples was drawn from participants who gave their consent to participate in this study. Blood samples were left overnight at room temperature to allow clotting and centrifuged at 3000 rpm for 10 minutes. The sera were collected in Eppendorf tubes and stored at 4°C for 24-72 hours and then kept at -20°C until testing.


### 2.3. Sociodemographic, Clinical, and Behavioral Data Collection

Sociodemographic data including age, gender, area of residence, and employment were obtained from all participants. Clinical data including the type of diabetes and behavioral data including obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, fish, oysters, raw vegetables and fruits, gardening or agricultural activities, exposure to soil, source of drinking water, and washing hands before meals were collected from the participants. These variables were selected based on published literature [15, 16]. Data was obtained from the patients/guardians, medical examination records, and informants. Patients were invited to provide veridical information and were informed that their data were used in a confidential manner.


### 2.4. Serological Assay

Sera were analyzed for the presence of IgG and IgM antibodies againstT. gondiiusing commercially available enzyme immunoassay kits (Demeditec Diagnostics GmbH, Germany) according to the manufacturer’s instructions. Clinical specificity and sensitivity of IgG kit were 99% and 98%, respectively. Clinical specificity and sensitivity of IgM kit were 99% and 100%, respectively [16, 17]. Positive and negative serum controls were included in every plate. To avoid bias of results, the serology test was performed double-blinded. Samples from diabetes mellitus patients and control group were randomly mixed, and the person performing the test did not know the source of samples in advance [16, 17].


### 2.5. Statistical Analysis

Results were analyzed with SPSS 18.0 software package. For the univariate analysis, Chi-square test was used to compare the categorical variables. The Mantel-Haenszel test was used to probe any differences between the patient and control groups. Multivariate regression models were used to adjust for potential confounders. Variables were included in the multivariate analysis if they had ap value ≤0.25 in the univariate analysis [17, 18]. Odds ratios (ORs) and the corresponding 95% confidence interval (CI) were calculated, in order to identify the independent risk factors forT. gondii infection. Results with ap value <0.05 were considered as statistically significant.


### 2.6. Ethics Approval and Consent to Participate

The study protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. The aim of the study was explained to the patients and they provided written consent for their participation in the study. Control sera were collected from volunteers.


## 2.1. Study Sites

The study was conducted in The Affiliated Hospital of Qingdao University, Qingdao (35°35′–37°09′N, 119°30′–121°00′E), Shandong province, eastern China. The Affiliated Hospital of Qingdao University is a large comprehensive hospital in Shandong province that occupies an important position in the national medical system. The patients at this hospital mainly come from five provinces (Shandong, Jiangsu, Liaoning, Jilin, and Heilongjiang).


## 2.2. Study Design and Sample Collection

Through case-control studies, we studiedT. gondii seroprevalence and identified the risk factors and possible transmission routes ofT. gondii infection in diabetes mellitus patients and control subjects in China between September 2014 and January 2017. A total of 1200 diabetes mellitus patients who visited the Affiliated Hospital of Qingdao University were included in the study. Three types of diabetes mellitus patients (T1DM, T2DM, and GDM) were invited to participate in this study. The number of patients of each type was 400 (Tables 1–3).Table 1
Seroprevalence ofT. gondii infection by sociodemographic factors in type 1 diabetes (T1DM) patients and controls in eastern China.
Characteristic T1DM (N=400) Controls (N=400) T1DMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 62 15 24.19 0.187 62 10 16.12 0.591 0.263 31-40 70 12 17.14  85 12 14.12  0.604 41-50 100 13 13.00  90 8 8.89  0.367 51-60 92 18 19.57  86 8 9.30  0.053 >60 76 8 10.53  67 8 11.94  0.789 Region Shandong 170 34 20.00 0.237 170 23 13.53 0.493 0.110 Jilin 141 21 14.89  141 13 9.22  0.144 Heilongjiang 89 11 12.36  89 10 11.24  0.816 Gender Male 207 31 14.98 0.395 221 23 10.41 0.447 0.155 Female 193 35 18.13  179 23 12.85  0.160 Area of residence Urban 202 30 14.85 0.370 211 16 7.85 0.031 0.019 Rural 198 36 18.18  189 27 14.29  0.299 Employment Unemployed 156 28 17.95 0.261 137 21 15.33 0.147 0.549 Employed part-time 139 26 18.71  161 13 8.07  0.006 Employed full-time 105 12 11.43  102 12 11.76  0.940 Obesity and overweight Yes 156 21 13.46 0.191 194 24 12.37 0.596 0.762 No 244 45 18.44  206 22 10.68  0.021 Keeping cats at home Yes 50 16 32.00 0.002 58 10 17.24 0.138 0.074 No 350 50 14.29  342 36 10.53  0.134 Consumption of raw/undercooked meat Yes 26 8 30.77 0.043 60 10 16.67 0.174 0.140 No 374 58 15.51  340 36 10.59  0.052 Consumption of oyster Raw 94 32 34.04 <0.001 134 16 11.94 0.845 <0.001 Boiled 306 34 11.11  266 30 11.28  0.950 Consumption of fish Raw 147 34 23.13 0.007 167 19 11.38 0.948 0.006 Boiled 253 32 12.65  233 27 11.59  0.721 Consumption of raw vegetables and fruits Yes 204 37 18.14 0.368 228 27 11.84 0.805 0.066 No 196 29 14.80  172 19 11.05  0.287 Exposure to soil Yes 232 40 17.24 0.639 247 32 12.96 0.246 0.190 No 168 26 15.48  153 14 9.15  0.182 Gardening or agricultural activities Yes 257 42 16.34 0.909 257 33 12.84 0.260 0.261 No 143 24 16.78  143 13 9.09  0.053 Washing hands before meals Yes 262 44 16.79 0.827 266 28 10.53 0.390 0.036 No 138 22 15.94  134 18 13.43  0.559 Source of drinking water Spring/well 85 19 22.35 0.101 68 13 19.12 0.031 0.625 Tap 315 47 14.92  332 33 9.94  0.054 Total 400 66 16.50  400 46 11.50  0.042Table 2
Seroprevalence ofT. gondii infection by sociodemographic factors in type 2 diabetes (T2DM) patients and controls in eastern China.
Characteristic T2DM (N=400) Controls (N=400) T2DMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 37 9 24.32 0.956 37 4 10.81 0.793 0.127 31-40 83 19 22.89  87 11 12.64  0.080 41-50 96 23 23.96  99 13 13.13  0.052 51-60 93 24 25.81  97 12 12.37  0.018 >60 91 19 20.88  90 7 7.78  0.012 Region Shandong 156 45 28.85 0.074 156 25 16.03 0.025 0.007 Jilin 142 32 22.54  142 17 11.97  0.019 Heilongjiang 102 17 16.67  102 5 4.90  0.007 Gender Male 202 50 24.75 0.551 211 27 12.80 0.492 0.002 Female 198 44 22.22  189 20 10.58  0.002 Area of residence Urban 200 50 25.00 0.479 229 18 7.86 0.005 <0.001 Rural 200 44 22.00  171 29 16.96  0.223 Employment Unemployed 163 43 26.38 0.118 169 24 14.20 0.326 0.006 Employed part-time 133 23 17.29  148 13 8.78  0.033 Employed full-time 104 28 26.92  83 10 12.05  0.012 Obesity and overweight Yes 152 30 19.74 0.152 190 20 10.53 0.470 0.017 No 248 64 25.81  210 27 12.86  <0.001 Keeping cats at home Yes 52 19 36.54 0.017 56 9 16.07 0.279 0.015 No 348 75 21.55  344 38 11.05  <0.001 Consumption of raw/undercooked meat Yes 28 14 50.00 <0.001 161 5 3.11 <0.001 <0.001 No 372 80 21.51  339 42 12.39  0.001 Consumption of oyster Raw 91 41 45.05 <0.001 143 15 10.49 0.559 <0.001 Boiled 309 53 17.15  257 32 12.45  0.119 Consumption of fish Raw 150 50 33.33 <0.001 167 22 13.17 0.454 <0.001 Boiled 250 44 17.60  233 25 10.73  0.031 Consumption of raw vegetables and fruits Yes 253 66 26.09 0.109 286 32 11.19 0.581 <0.001 No 147 28 19.05  114 15 13.16  0.203 Exposure to soil Yes 228 54 23.68 0.920 248 34 13.71 0.120 0.005 No 172 40 23.26  152 13 8.55  <0.001 Gardening or agricultural activities Yes 248 56 22.58 0.580 254 35 13.78 0.096 0.011 No 152 38 25.00  146 12 8.22  <0.001 Washing hands before meals Yes 270 63 23.33 0.910 285 30 10.53 0.232 <0.001 No 130 31 23.85  115 17 14.78  0.075 Source of drinking water Spring/well 90 20 22.22 0.745 64 13 20.31 0.020 0.776 Tap 310 74 23.87  336 34 10.12  <0.001 Total 400 94 23.50  400 47 11.75  <0.001Table 3
Seroprevalence ofT. gondii infection by sociodemographic factors in gestational diabetes (GDM) patients and controls in eastern China.
Characteristic GDM (N=400) Controls (N=400) GDMvs. Prevalence ofT. gondii infection Prevalence ofT. gondii infection Controls No. tested No. positive % P-value No. tested No. positive % P-value P-value Age ≤ 30 185 35 18.92 0.043 201 26 12.94 0.017 0.107 31-40 184 38 20.65  71 19 26.76  0.294 >40 31 12 38.71  28 3 10.71  0.014 Region Shandong 239 59 24.69 0.079 239 32 13.39 0.451 0.002 Jilin 65 8 12.31  65 8 12.31  1.00 Heilongjiang 95 18 18.95  95 8 8.42  0.035 Area of residence Urban 202 45 22.28 0.612 245 25 10.20 0.165 0.001 Rural 198 40 20.20  155 23 14.84  0.192 Employment Unemployed 126 24 19.05 0.081 98 15 15.31 0.470 0.464 Employed part-time 143 39 27.27  197 20 10.15  <0.001 Employed full-time 131 22 16.79  105 13 12.38  0.343 Obesity and overweight Yes 79 15 18.99 0.582 117 9 7.69 0.086 0.018 No 321 70 21.81  283 39 13.78  0.011 Keeping cats at home Yes 40 12 30.00 0.135 47 4 8.51 0.433 0.010 No 360 73 20.28  353 44 12.46  0.005 Consumption of raw/undercooked meat Yes 33 12 36.36 0.027 77 5 6.49 0.098 <0.001 No 367 73 19.89  323 43 13.31  0.021 Consumption of oyster Raw 84 30 35.71 <0.001 153 18 11.76 0.909 <0.001 Boiled 316 55 17.41  247 30 12.15  0.084 Consumption of fish Raw 137 42 30.66 <0.001 158 17 10.76 0.086 <0.001 Boiled 263 43 16.35  242 41 16.94  0.858 Consumption of raw vegetables and fruits Yes 282 64 22.70 0.275 299 37 12.37 0.692 0.001 No 118 21 17.80  101 11 10.89  0.149 Exposure to soil Yes 233 47 20.17 0.534 254 34 13.39 0.261 0.045 No 167 38 22.75  146 14 9.59  0.002 Gardening or agricultural activities Yes 257 52 20.23 0.505 263 35 13.31 0.265 0.034 No 143 33 23.08  137 13 9.49  0.002 Washing hands before meals Yes 266 54 20.30 0.513 302 38 12.58 0.529 0.013 No 134 31 23.13  98 10 10.20  0.011 Source of drinking water Spring/well 78 14 17.95 0.840 66 15 22.73 0.003 0.476 Tap 322 61 18.94  334 33 9.88  <0.001 Total 400 85 21.25  400 48 12.00  <0.001A total of 1200 control subjects, matched with diabetes mellitus patients by age, gender, and residence, were included in the study. Serum samples were obtained from persons who participated in health screening at the Affiliated Hospital of Qingdao University.Approximately 5 mL of venous blood samples was drawn from participants who gave their consent to participate in this study. Blood samples were left overnight at room temperature to allow clotting and centrifuged at 3000 rpm for 10 minutes. The sera were collected in Eppendorf tubes and stored at 4°C for 24-72 hours and then kept at -20°C until testing.


## 2.3. Sociodemographic, Clinical, and Behavioral Data Collection

Sociodemographic data including age, gender, area of residence, and employment were obtained from all participants. Clinical data including the type of diabetes and behavioral data including obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, fish, oysters, raw vegetables and fruits, gardening or agricultural activities, exposure to soil, source of drinking water, and washing hands before meals were collected from the participants. These variables were selected based on published literature [15, 16]. Data was obtained from the patients/guardians, medical examination records, and informants. Patients were invited to provide veridical information and were informed that their data were used in a confidential manner.


## 2.4. Serological Assay

Sera were analyzed for the presence of IgG and IgM antibodies againstT. gondiiusing commercially available enzyme immunoassay kits (Demeditec Diagnostics GmbH, Germany) according to the manufacturer’s instructions. Clinical specificity and sensitivity of IgG kit were 99% and 98%, respectively. Clinical specificity and sensitivity of IgM kit were 99% and 100%, respectively [16, 17]. Positive and negative serum controls were included in every plate. To avoid bias of results, the serology test was performed double-blinded. Samples from diabetes mellitus patients and control group were randomly mixed, and the person performing the test did not know the source of samples in advance [16, 17].


## 2.5. Statistical Analysis

Results were analyzed with SPSS 18.0 software package. For the univariate analysis, Chi-square test was used to compare the categorical variables. The Mantel-Haenszel test was used to probe any differences between the patient and control groups. Multivariate regression models were used to adjust for potential confounders. Variables were included in the multivariate analysis if they had ap value ≤0.25 in the univariate analysis [17, 18]. Odds ratios (ORs) and the corresponding 95% confidence interval (CI) were calculated, in order to identify the independent risk factors forT. gondii infection. Results with ap value <0.05 were considered as statistically significant.


## 2.6. Ethics Approval and Consent to Participate

The study protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. The aim of the study was explained to the patients and they provided written consent for their participation in the study. Control sera were collected from volunteers.


## 3. Results

### 3.1. Epidemiology of T1DM Patients with T. gondii Infection

T1DM patients (16.50%) had a significantly higherT. gondiiseroprevalence than the control subjects (11.50%) (p=0.042). Of these, 53 T1DM patients (13.25%) were found to be positive forT. gondii IgG antibodies, as compared to 40 controls (10.00%), and the difference was not statistically significant (p=0.152).T. gondii IgM antibodies were detected in 15 of the 400 T1DM patients and in seven of the 400 controls (3.75%versus 1.75%, respectively,p=0.084). The details of T1DM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 1. The highest seroprevalence ofT. gondii infection was detected in T1DM patients in the age range of ≤30 years (24.19%). T1DM patients living in Shandong province (20.00%) had a higherT. gondii seroprevalence than those living in Jilin (14.89%) and Heilongjiang (12.36%) provinces, but the difference was not significant (p=0.237). There were no significant differences between female (18.13%) and male (14.98%) T1DM patients (p = 0.395). The seroprevalence ofT. gondii infection among the T1DM patients who lived in rural areas (18.18%) was slightly higher than those who lived in urban areas (14.85%), but the difference was not statistically significant (p=0.370). Moreover,T. gondii infection seroprevalence was not significantly different among T1DM patients with different types of employment (p=0.261).


### 3.2. Epidemiology of T2DM Patients with T. gondii Infection

T2DM patients (23.50%) had a significantly higherT. gondiiseroprevalence than the control subjects (11.75%) (p<0.001). A total of 77 T2DM patients (19.25%) were found to be positive forT. gondii IgG antibodies, as compared to 37 controls (9.25%), and the difference was statistically significant (p<0.001).T. gondii IgM antibodies were detected in 19 of the 400 T2DM patients and in 11 of the 400 controls (4.75%versus 2.75%, respectively,p=0.137). The details of T2DM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 2. The highest seroprevalence ofT. gondii infection was detected in T1DM patients in the age range of 51-60 years (25.81%). T2DM patients living in Shandong province (28.85%) had a higherT. gondiiseroprevalence than those living in Jilin (22.54%) and Heilongjiang (16.67%) provinces, but the difference was not significant (p=0.074). There were no significant differences between male (24.75%) and female (22.22%) T2DM patients (p = 0.551). The seroprevalence ofT. gondiiinfection among the T2DM patients who lived in urban areas (25.00%) was slightly higher than those who lived in rural areas (22.00%), but the difference was not statistically significant (p=0.479). Moreover,T. gondii infection seroprevalence was not significantly different among T2DM patients with different types of employment (p=0.118).


### 3.3. Epidemiology of GDM Patients with T. gondii Infection

GDM patients (21.25%) had a significantly higherT. gondiiseroprevalence than the control subjects (12.00%) (p=0.042). A total of 70 GDM patients (17.50%) were found to be positive forT. gondii IgG antibodies, as compared to 37 controls (9.25%), and the difference was statistically significant (p<0.001).T. gondii IgM antibodies were detected in 18 of the 400 GDM patients and in 11 of the 400 controls (4.50%versus 2.75%, respectively,p=0.186). The details of GDM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 3. The highest seroprevalence ofT. gondii infection was detected in GDM patients in the age range of >40 years (38.71%), and significant difference was found among different age groups (p=0.043). GDM patients living in Shandong province (24.69%) had a higherT. gondiiseroprevalence than those living in Jilin (12.31%) and Heilongjiang (18.95%) provinces, but the difference was not significant (p=0.079). The seroprevalence ofT. gondiiinfection among the GDM patients who lived in rural areas (20.20%) was slightly lower than those who lived in urban areas (22.28%), but the difference was not statistically significant (p=0.612). Moreover,T. gondii seroprevalence was not significantly different among GDM patients with different types of employment (p=0.081).


### 3.4. Risk Factors Associated with T. gondii Infection

Univariate analysis showed that some lifestyle variables of T1DM patients had apvalue ≤0.25, including obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, oysters, and fish, and source of drinking water. Six lifestyle variables of T2DM patients had apvalue ≤0.25 through univariate analysis. They are obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, fish, oysters, and raw vegetables and fruits. In GDM patients, keeping cats at home and consumption of raw/undercooked meat, fish, and oysters had apvalue ≤0.25 by univariate analysis. In the multivariate analysis, keeping cats at home (OR=2.885; 95% CI: 1.37-6.07;p = 0.005) and consumption of oysters (OR=13.19; 95% CI: 2.91-59.82;p = 0.001) were associated with significantly increased odds ofT. gondii infection in T1DM patients (Table 4). Consumption of raw/undercooked meat (OR=2.663; 95% CI: 1.08-6.56;p= 0.033) and oysters (OR=4.785; 95% CI: 1.98-11.45;p<0.001) was associated with significantly increased odds ofT. gondiiinfection in T2DM patients (Table 4). There was no evidence of a significant association betweenT. gondii status and the selected variables in GDM patients (Table 4).Table 4
Multivariate analysis of selected characteristics of diabetic patients and their association withToxoplasma gondii infection.
Type of diabetic patients Characteristica Adjusted Odds ratiob 95% Confidence interval P value T1DM Obesity and overweightKeeping cats at home 0.5842.885 0.32-1.081.37-6.07 0.0860.005 Consumption of raw/undercooked meat Consumption of oyster 2.17713.19 0.81-5.882.91-59.82 0.1250.001 Consumption of fish 0.295 0.06-1.36 0.117 Source of drinking water 1.713 0.78-3.77 0.181 T2DM Obesity and overweightKeeping cats at homeConsumption of raw/undercooked meat 0.6731.5842.663 0.39-1.150.79-3.171.08-6.56 0.1470.1930.033 Consumption of oyster 4.758 1.98-11.45 <0.001 Consumption of fish 0.747 0.32-1.72 0.493 Consumption of raw vegetables and fruits 1.296 0.72-2.33 0.385 GDM Keeping cats at homeConsumption of raw/undercooked meat 1.3761.906 0.63-2.990.83-4.36 0.4200.127 Consumption of oyster 1.764 0.78-3.99 0.173 Consumption of fish 1.516 0.72-3.18 0.271 T h e   a variables with a p<0.25 in the univariate analysis were included. A d j u s t e d   b by age and the other characteristics included in this table.


## 3.1. Epidemiology of T1DM Patients with T. gondii Infection

T1DM patients (16.50%) had a significantly higherT. gondiiseroprevalence than the control subjects (11.50%) (p=0.042). Of these, 53 T1DM patients (13.25%) were found to be positive forT. gondii IgG antibodies, as compared to 40 controls (10.00%), and the difference was not statistically significant (p=0.152).T. gondii IgM antibodies were detected in 15 of the 400 T1DM patients and in seven of the 400 controls (3.75%versus 1.75%, respectively,p=0.084). The details of T1DM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 1. The highest seroprevalence ofT. gondii infection was detected in T1DM patients in the age range of ≤30 years (24.19%). T1DM patients living in Shandong province (20.00%) had a higherT. gondii seroprevalence than those living in Jilin (14.89%) and Heilongjiang (12.36%) provinces, but the difference was not significant (p=0.237). There were no significant differences between female (18.13%) and male (14.98%) T1DM patients (p = 0.395). The seroprevalence ofT. gondii infection among the T1DM patients who lived in rural areas (18.18%) was slightly higher than those who lived in urban areas (14.85%), but the difference was not statistically significant (p=0.370). Moreover,T. gondii infection seroprevalence was not significantly different among T1DM patients with different types of employment (p=0.261).


## 3.2. Epidemiology of T2DM Patients with T. gondii Infection

T2DM patients (23.50%) had a significantly higherT. gondiiseroprevalence than the control subjects (11.75%) (p<0.001). A total of 77 T2DM patients (19.25%) were found to be positive forT. gondii IgG antibodies, as compared to 37 controls (9.25%), and the difference was statistically significant (p<0.001).T. gondii IgM antibodies were detected in 19 of the 400 T2DM patients and in 11 of the 400 controls (4.75%versus 2.75%, respectively,p=0.137). The details of T2DM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 2. The highest seroprevalence ofT. gondii infection was detected in T1DM patients in the age range of 51-60 years (25.81%). T2DM patients living in Shandong province (28.85%) had a higherT. gondiiseroprevalence than those living in Jilin (22.54%) and Heilongjiang (16.67%) provinces, but the difference was not significant (p=0.074). There were no significant differences between male (24.75%) and female (22.22%) T2DM patients (p = 0.551). The seroprevalence ofT. gondiiinfection among the T2DM patients who lived in urban areas (25.00%) was slightly higher than those who lived in rural areas (22.00%), but the difference was not statistically significant (p=0.479). Moreover,T. gondii infection seroprevalence was not significantly different among T2DM patients with different types of employment (p=0.118).


## 3.3. Epidemiology of GDM Patients with T. gondii Infection

GDM patients (21.25%) had a significantly higherT. gondiiseroprevalence than the control subjects (12.00%) (p=0.042). A total of 70 GDM patients (17.50%) were found to be positive forT. gondii IgG antibodies, as compared to 37 controls (9.25%), and the difference was statistically significant (p<0.001).T. gondii IgM antibodies were detected in 18 of the 400 GDM patients and in 11 of the 400 controls (4.50%versus 2.75%, respectively,p=0.186). The details of GDM patients and control subjects, including age distribution, gender, employment, and area of residence, are shown in Table 3. The highest seroprevalence ofT. gondii infection was detected in GDM patients in the age range of >40 years (38.71%), and significant difference was found among different age groups (p=0.043). GDM patients living in Shandong province (24.69%) had a higherT. gondiiseroprevalence than those living in Jilin (12.31%) and Heilongjiang (18.95%) provinces, but the difference was not significant (p=0.079). The seroprevalence ofT. gondiiinfection among the GDM patients who lived in rural areas (20.20%) was slightly lower than those who lived in urban areas (22.28%), but the difference was not statistically significant (p=0.612). Moreover,T. gondii seroprevalence was not significantly different among GDM patients with different types of employment (p=0.081).


## 3.4. Risk Factors Associated with T. gondii Infection

Univariate analysis showed that some lifestyle variables of T1DM patients had apvalue ≤0.25, including obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, oysters, and fish, and source of drinking water. Six lifestyle variables of T2DM patients had apvalue ≤0.25 through univariate analysis. They are obesity and overweight, keeping cats at home, consumption of raw/undercooked meat, fish, oysters, and raw vegetables and fruits. In GDM patients, keeping cats at home and consumption of raw/undercooked meat, fish, and oysters had apvalue ≤0.25 by univariate analysis. In the multivariate analysis, keeping cats at home (OR=2.885; 95% CI: 1.37-6.07;p = 0.005) and consumption of oysters (OR=13.19; 95% CI: 2.91-59.82;p = 0.001) were associated with significantly increased odds ofT. gondii infection in T1DM patients (Table 4). Consumption of raw/undercooked meat (OR=2.663; 95% CI: 1.08-6.56;p= 0.033) and oysters (OR=4.785; 95% CI: 1.98-11.45;p<0.001) was associated with significantly increased odds ofT. gondiiinfection in T2DM patients (Table 4). There was no evidence of a significant association betweenT. gondii status and the selected variables in GDM patients (Table 4).Table 4
Multivariate analysis of selected characteristics of diabetic patients and their association withToxoplasma gondii infection.
Type of diabetic patients Characteristica Adjusted Odds ratiob 95% Confidence interval P value T1DM Obesity and overweightKeeping cats at home 0.5842.885 0.32-1.081.37-6.07 0.0860.005 Consumption of raw/undercooked meat Consumption of oyster 2.17713.19 0.81-5.882.91-59.82 0.1250.001 Consumption of fish 0.295 0.06-1.36 0.117 Source of drinking water 1.713 0.78-3.77 0.181 T2DM Obesity and overweightKeeping cats at homeConsumption of raw/undercooked meat 0.6731.5842.663 0.39-1.150.79-3.171.08-6.56 0.1470.1930.033 Consumption of oyster 4.758 1.98-11.45 <0.001 Consumption of fish 0.747 0.32-1.72 0.493 Consumption of raw vegetables and fruits 1.296 0.72-2.33 0.385 GDM Keeping cats at homeConsumption of raw/undercooked meat 1.3761.906 0.63-2.990.83-4.36 0.4200.127 Consumption of oyster 1.764 0.78-3.99 0.173 Consumption of fish 1.516 0.72-3.18 0.271 T h e   a variables with a p<0.25 in the univariate analysis were included. A d j u s t e d   b by age and the other characteristics included in this table.


## 4. Discussion

The association betweenT. gondii infection and diabetes mellitus remains controversial, with few studies reporting conflicting results [19, 20]. Thus, the present study was conducted to determine whetherT. gondii infection is associated with different types of diabetes mellitus in eastern China. The results showed that diabetes mellitus patients had higher frequencies of antibodies againstT. gondii as compared to control subjects. Thus, our findings based on serological methods supported an association between diabetes mellitus andT. gondii infection.Type 1 diabetes mellitus (T1DM) is an autoimmune disease with complex interactions between genetic and environmental factors [14]. The enteroviruses and other infectious agents were found to be associated with T1DM [21]. In the present study, T1DM patients had a significantly higherT. gondii seroprevalence than the controls (p=0.042), suggesting that T1DM patients are more likely to be infected withT. gondii. However, further targeted studies should be conducted to explore and confirm the association between T1DM andT. gondii infection.Type 2 diabetes mellitus (T2DM), a major global health problem, is a complex metabolic disease [11]. The incidence of T2DM has notably increased in recent years, in both developed and developing countries [22]. Various infections, includingT. gondii, may easily appear in T2DM patients because they are immunocompromised [23]. In the present study, T2DM patients had a significantly higherT. gondiiseroprevalence than the control subjects (p<0.001). These evidences indicated a potential association betweenT. gondiiinfection and T2DM implying thatT. gondiiinfection may increase susceptibility to T2DM, while T2DM patients are more vulnerable to opportunistic infections such asT. gondii. Importantly, the clinician should pay more attention toT. gondii infection when they diagnose and treat T2DM patients given the high prevalence ofT. gondii infection in T2DM patients, andT. gondii infection may aggravate the T2DM status.T. gondii infection during pregnancy may cause serious consequences such as miscarriage, microcephaly, hydrocephalus, and severe neurological disorders in the fetus [5]. In addition, in immunodeficient individuals, released bradyzoites from tissue cysts switching back into rapidly multiplying tachyzoites could cause reactivation of latent infection and dissemination throughout the body [24]. The immune system in diabetes mellitus patients is affected, and GDM patients are more susceptible toT. gondii infection. In the present study, GDM patients had a significantly higherT. gondii seroprevalence than the control subjects (p<0.001). Thus, serological screening of GDM patients is needed, followed by proper treatment of theT. gondii infection [25]. Moreover, information about toxoplasmosis and its transmission routes should be given to GDM patients as part of prenatal care.The first epidemiological investigation onT. gondii infection in humans in China was conducted in Guangxi Zhuang Autonomous Region in 1978 [26]. Now, toxoplasmosis has become a notifiable disease in China. However, there are no national guidelines for the prevention of toxoplasmosis in China. Humans acquire the infection through three major routes: consumption of undercooked meat containingT. gondii tissue cysts, ingesting oocysts-contaminated water, soil, vegetables, and fruits, and transmission from mother to fetus during pregnancy [1, 27]. As expected, we found that keeping cats at home and consumption of raw/undercooked meat were associated with significantly increased odds ofT. gondii infection in diabetic patients. These two risk factors have been identified in many studies in China [16, 28–30]. Interestingly, fresh oyster consumption was also a potential risk factor forT. gondii infection in T1DM and T2DM patients, which was similar to a study reported from the United States [31].T. gondii oocysts can be washed into the sea via rainwash and runoff [32, 33], and shellfish including oysters, clams, and mussels can ingest the oocysts directly from seawater [1, 32–37]. In China,T. gondiioocysts have been detected in oysters [38] and consumption of fresh oysters is common in recent years, which may explain the higherT. gondiiseroprevalence in the diabetes mellitus patients who consume raw oysters than those who do not consume raw oysters. Thus, knowledge of these risk factors will help in prevention efforts.Some limitations of the present study should be kept in mind. First, our study participants might not represent the general clinically healthy individuals, pregnant women, and diabetes mellitus population due to the potential limitation of enrollment methods. Therefore, potential selection bias should be considered when interpreting our results. Second, serology could not clearly indicate the infection status as current infection or past infection; potential bias caused by such misclassification could not be eliminated. Moreover, molecular identification, taxonomy, genetic variation, and diagnosis ofT. gondii should be considered in further studies. Third, more effective statistical analysis methods should be used to confirm the association between diabetes mellitus andT. gondii infection. Therefore, our results need to be proved in further studies.


## 5. Conclusion

This study provided serological evidence of an association betweenT. gondii infection and three types of diabetes mellitus (T1DM, T2DM, and GDM). Moreover, keeping cats at home and consumption of raw/undercooked meat and raw oysters were risk factors forT. gondii positivity using multivariate regression, which may help to guide future research and control policies. Further studies should be conducted to elucidate the role ofT. gondii in diabetes mellitus.



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*Source: 4723739-2018-12-18.xml*