Background

Toxoplasma gondii is a ubiquitous, apicomplexan parasite of warm-blooded animals (Tenter et al. 2000). Toxoplasmosis is probably one of the most common parasitic infections of humans. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immuno-compromised (Jones et al. 2001 and Dubey 2005).

Toxoplasma infection is ranked among the most commonly occurring opportunistic infections in AIDS patients and may well be a greater direct cause of morbidity and mortality than other more common opportunistic infections (Dubey 2005).

Serological diagnosis of toxoplasmosis represents the most widely used approach for defining the stage of infection, ELISA showing higher sensitivity and specificity (Remington et al. 2004 and Sensini 2006).

Diabetes mellitus is a widespread metabolic complex disorder causing various dysfunctions in most of the body systems including cellular and humoral immunity. Diabetic patients were included in a classification of the immuno-compromised patients (Olefsky 1985). Insulin-dependent diabetes mellitus (IDDM) or type I diabetes is a debilitating chronic disease that impairs production and secretion of the key hormone insulin and alters blood sugar metabolism. Insulin is synthesized and secreted by pancreatic islet cells of Langerhans (Orci et al. 1988).

Toxoplasmosis in diabetic patients may occur due to recently acquired acute infection or they may suffer from dissemination of reactivated latent infection (Montoya et al. 2001). However, studies of toxoplasmosis in diabetic patients are scanty (Cavallazzi 1985 and Yamamoto et al. 2003).

As regards autoimmunity, initial studies suggested that certain infections might precipitate the onset of autoimmune pathology (Benoist and Mathis 2001). With the improvement of living standards, the prevalence rate of diabetes has steadily increased. The sensitivity and susceptibility to various infections can be higher in diabetes mellitus patients (Joshi et al. 1999). In some cases, Toxoplasma gondii has been proposed as a possible cause of diabetes, and current information is nearly predicated on this issue (Shin et al. 2009 and Shirbazou et al. 2013).

Circulating auto-reactive antibodies such as anti-nuclear antibodies (ANA) play a central role in the diagnosis and classification of autoimmune disorders. Auto-reactive antibodies can appear long before the onset of clinical disease, and detection of these antibodies in serum has been shown to have strong predictive value (Youinou et al. 2010).

The aim of the present work was to study the seroprevalence of Toxoplasma gondii among a group of patients with type I diabetes mellitus and to consider the possible relationship between diabetes mellitus, Toxoplasma gondii infection, and auto-reactive antibodies using ELISA technique.

Materials and methods

Participants

A case-control study was carried out in the diabetes clinic at the Research Institute of Ophthalmology (RIO), Giza City, Egypt, over a period of 7 months. All studied patients gave informed written consent before the involvement in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics and Research Committee of RIO.

Sample size calculation

Using the WHO manual for sample size determination in health studies, the sample size was determined by using the proportion difference approach with the assumption of 95% confidence level, 80% power, control to case ratio 1:1, the odds ratio to be detected ≥ 2, and the 30% control group will be exposed (Chadha 2006). The minimal sample size required 52 cases and 52 controls with a total of 104. To account for possible non-response and for more precision, 15% was added to give a total sample of 120 subjects (60 cases and 60 controls).

Study design

The case group comprised 60 patients with type I diabetes mellitus (T1DM) in the age group of 15–60 years who were diagnosed with T1DM based on WHO criteria (WHO 2011) and who were receiving care at the diabetes clinic of RIO. The diabetes clinic was held twice a week and the average number of diabetic (type I) patients attending per day was two patients. The control group involved an equal number of non-diabetic subjects chosen from other outpatient clinics of RIO hospital by selecting for every case a matched age and sex control. T1DM was excluded from controls by history taking and measuring their random blood sugar. The response rates were 80% and 85% among cases and controls respectively.

All subjects in the present study were subjected to the interview questionnaire through complete history taking and clinical examination according to structural questionnaire comprised the following items:

  • Name, age, gender, occupation, residence (urban/rural)

  • Symptoms and signs of toxoplasmosis: medical history of fever, malaise, swelling in the neck, skin rash, diminution of vision, and history of bad outcome of pregnancy

  • Risk factors for contracting toxoplasmosis: consumption of undercooked meat (ham, sausages, luncheon), consumption of unwashed vegetables and fruits, contact with cats, and contact with soil (gardening or agricultural)

  • Past history of medical disease: diabetes mellitus, DM complications such as renal disease and eye manifestations, intake of immunosuppressive drugs, and history of blood transfusion

Blood samples were collected from all subjects included in the study, and the separated sera were stored at − 20 °C in duplicate vials until used. All serum samples were tested for:

  1. 1.

    Detection of anti-Toxoplasma antibodies (IgG and IgM) by the indirect enzyme-linked immunosorbent assay (ELISA) using the Calbiotech Toxoplasma IgG ELISA kit (catalog no: TX022G)

  2. 2.

    Detection of anti-nuclear antibodies (ANA) by the indirect enzyme-linked immunosorbent assay (ELISA) using the Calbiotech ANA screen ELISA kit (catalog no: AN033G)

Statistical analysis

The obtained data were entered, revised, and analyzed using the Statistical Package for the Social Sciences software (SPSS), version 18. The results were presented as frequencies and percentages. Two-tailed Fisher’s exact test or chi-squared test (χ2) were used as appropriate for comparison of categorical variables between groups. p value < 0.05 was considered as statistically significant following Bonferroni correction for multiple testing (Daniel 2005).

Results

Sociodemographic data of patients and controls are shown in Table 1, which revealed that 38 (63.3%) of the patient group were living in urban areas versus 55 (91.7%) of the control group. Patients consuming undercooked meat were significantly lower (p = 0.001) than those of the control group (36.7% vs. 66.7%, respectively), while 85% vs. 8.3% were in contact with cats and 90% vs. 1.7% were in contact with soil for patient and control groups respectively with highly statistically significant difference (p < 0.0001); 5% of cases vs. 3.3% of control were consuming unwashed vegetables.

Table 1 Socio-demographic data of patients and controls
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Distribution of the patients studied and their correlation with T. gondii seroprevalence was shown in Table 2; anti-T. gondii IgG antibodies were found in 27 (45%) of the cases and in 14 (23.3%) of the control group. The seroprevalence of T. gondii infection was significantly higher in cases than in controls (OR = 2.69; 95% CI 1.23–5.89; p = 0.01). Anti-T. gondii IgM antibodies were found in three (11.1%) of 27 anti-T. gondii IgG seropositive cases group; none of the 14 anti-T. gondii IgG seropositive control had anti-T. gondii IgM antibodies. There was no statistically significant difference in the frequency of IgM seropositivity among cases and controls (p = 0.244). Results also showed that only one patient of group I and control group had positive ANA; thus, no possible association could be detected between the studied groups.

Table 2 Distribution of patients and control groups according to anti-Toxoplasma IgG, IgM, and ANA prevalence
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Table 3 revealed statistical significant differences regarding the consumption of undercooked meat, contact with cats and contact with soil, and Toxoplasma seroprevalence. There was no variation, neither with sex nor with age groups.

Table 3 Relation between anti-Toxoplasma IgG prevalence and sociodemographic data among patients and control groups
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Table 4 showed the clinical findings in the study group I in relation to seropositivity where 12 (44.4%) patients with diabetic retinopathy had positive anti-T. IgG. Also, it was found that 2 (7.4%) patients with nephropathy had positive anti-T. IgG, 4 (14.9%) patients with neuropathy, and 1 (3.7%) patient with liver disease were seropositive. No statistically significant differences could be detected between the clinical findings among patients with type I DM group and Toxoplasma seropositivity.

Table 4 Relation between anti-Toxoplasma IgG prevalence and clinical findings among patients (group 1)
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Discussion

The aim of this study was to determine the seroprevalence of Toxoplasma gondii among a group of patients with type I diabetes mellitus and to detect the relationship between Toxoplasma gondii infection and auto-reactive antibodies.

Overall, the current hypothesis suggests that toxoplasmosis increases susceptibility to acquiring diabetes and, on the other hand, diabetic patients are more vulnerable to opportunistic infections such as toxoplasmosis (Zhu et al. 2006; Gokce et al. 2008; Shirbazou et al. 2013; Saki et al. 2016).

In our study, the overall seroprevalence of toxoplasmosis in diabetic patients was 45.0% which was in agreement with 46% reported as by Hemida et al. (2017) while it was lower than 57.2% as reported by Gokce et al. (2008), higher than 16.5% as reported by Li et al. (2018) in China, and much higher than 5.4% as reported by Krause et al. (2009). It has been found that seroprevalence of T. gondii infection was significantly higher in cases than in controls (OR = 2.69; 95% CI 1.23–5.89; p = 0.01) which was incompatible with the results reported by Gokce et al. (2008) (OR = 5.066; 95% CI 2.579–9.952; p = < 0.001), Hemida et al. (2017) (p = 0.03), and Li et al. (2018). However, Siyadatpanah et al. (2013) found no statistically significant difference in the prevalence of toxoplasmosis between diabetics and non-diabetic individuals.

Concerning the association between age and seropositivity in the study population, there was a non-significant higher prevalence of seropositivity(IgG) among age group above 30 years old (81.5% and 85.7%) for the cases and control group respectively. The reason might be the increased possibility of an individual coming into contact with one of the transmission routes (Ajioka and Soldati 2007; Spalding et al. 2005). Similarly, Shin et al. (2009) reported that the prevalence of toxoplasmosis tended to be increased with age, and also, this increase was not statistically significant. On the other hand, Hemida et al. (2017) found a non-significant negative relation between seropositivity and increase in age.

With respect to gender, the seropositivity for anti-Toxoplasma IgG among females was higher than that among males, but the difference was not significant. Similar results were obtained in the survey performed at Al-Hussien University Hospital, Cairo, Egypt (Hemida et al. 2017), on Cheju Island, Korea (Yang et al. 2000), and Seoul National University Children’s Hospital, Korea (Kook et al. 1999). In contrast, a report has been made that male patients had a higher infection rate than female patients at Kangnam St. Mary’s Hospital, Korea (Choi et al. 1989).

All patients were subjected to fulfill a questionnaire sheet to verify the possible risk factors of Toxoplasma infection and to evaluate the environmental and socioeconomic pattern of both cases and control groups. In our study, there was no statistical difference in seroprevalence between rural and urban areas (p value = 0.86) that agreed with what has been found with Hemida et al. (2017), although it was demonstrated in other studies that living in rural areas had a higher risk of toxoplasmosis than those in urban areas (Garcia et al. 1999).

According to the present data, there was a statistically significant difference regarding the consumption of undercooked meat, contact with cats and contact with soil, and Toxoplasma seroprevalence. The participants who frequently do gardening were highly infected (85.2%) compared with the ones who did not spend time in contact with soil (14.8%). This could be explained due to the fact that the buried sporulated oocysts of cats might be contaminating the soil and sand and the oocysts remain infectious for several months and can last beyond 1 year (Frenkel and Dubey 1972).

Kapperud et al. (1996), Bobić et al. (1998), and Cook et al. (2000) demonstrated a significant association between eating raw or unwashed vegetables and fruits and Toxoplasma gondii seroconversion. Spalding et al. (2005) and Jones et al. (2005) reported that this mode of infection is possibly responsible for Toxoplasma infection in vegetarians and herbivores. Kapperud et al. (1996), Baril et al. (1999), and Kravetz and Federman (2005) emphasized the relevance of cats in toxoplasmosis as the most likely method for transmission. Epidemiological studies and several outbreaks have identified the handling and consumption of raw or undercooked meat as a source of toxoplasmosis (Cook et al. 2000; Ross et al. 2001; Carme et al. 2002 and Dubey and Jones 2008).

The present study revealed that no patients gave history of fever with lymphadenopathy, while 26 patients (43.3%) had a history of retinopathy. Previous studies reported that Toxoplasma infection is often clinically silent (Thulliez 1992; Gorgievski-Hrisoho et al. 1996; Remington et al. 2001 and Nimri et al. 2004; Kravetz and Federman 2005). However, Montoya (2002) demonstrated that fever, lymphadenopathy, and retinochoroiditis were suggestive presentations of toxoplasmosis.

To clarify the characteristics of T. gondii-seropositive diabetic patients, the patients’ medical records were checked. The major coincidental diseases in seropositive patients were retinopathy, nephropathy, neuropathy, liver disease, and cancer. Shin et al. (2009) reported that major co-morbidities of seropositive patients included malignant neoplasms, diabetes mellitus, arthritis, chronic hepatitis B, chronic renal diseases, schizophrenia, and acute lymphadenitis.

In the present study, no association could be detected between Toxoplasma gondii infection and auto-reactive antibodies.

Conclusion

In the present study, the overall seroprevalence of toxoplasmosis in diabetic patients was 45.0%. A significant association was detected between consumption of undercooked meat, contact with cats and contact with soil, and Toxoplasma seroprevalence (possible risk factors of Toxoplasma infection). The major coincidental diseases in seropositive patients were retinopathy, nephropathy, neuropathy, liver disease, and cancer. No association could be detected between Toxoplasma gondii infection and auto-reactive antibodies. The absence of specific antibodies does not exclude active disease in immune-suppressed patients whom toxoplasmosis is suspected. So, additional diagnostics methods including PCR amplification of T. gondii are strongly recommended. A prospective exposure-outcome study is suggested to investigate the effect of Toxoplasma infection in individuals with auto-reactive anti-islet antibodies in children and adolescents with a family history of diabetes mellitus type 1 on disease outcome.