Introduction
Toxoplasma gondii is an apicomplexan parasite that infects approximately one-third of the world population [Reference Montoya and Liesenfeld1]. Humans are infected with T. gondii mainly by ingesting cysts from unsanitary food, ingesting food contaminated with cat-derived T. gondii oocysts, as well as transmission from mother to the foetus [2].
Efficient immunity can limit the relapse of T. gondii infection in the multiplying tachyzoite stage, and hence, acute infection is usually asymptomatic in immunocompetent individuals. However, for the immunocompromised host, the cysts can infect various organs, such as the liver, spleen and nervous system, which, in turn, cause severe symptoms as well as death [Reference Nimir3, Reference Ferreira and Borges4]. Currently, T. gondii infection in patients with malignancy is of great concerns, and thus, the correlations between T. gondii infection and malignancy have been evaluated [Reference Cong5–Reference Alvaradoesquivel7].
Malignant lymphoma (ML) is a common malignancy in children, with more than 13 000 new cases and 1800 disease-related deaths in China in 2015 [Reference Chen8]. The genetic, physical and chemical factors are responsible for the development of lymphoma [Reference Hoppe9, Reference Ferlay10]. Also, a potential correlation between T. gondii infection and lymphoma has been reported [Reference Herold11–Reference Hashiguchi and Onozawa13]; however, a study conducted by Stamatovic et al. did not show any such association [Reference Stamatovic14], thereby rendering it controversial. A few reports have focused on T. gondii infection among children with ML in Eastern China, but little is known about the potential risk factors in this group. Thus, the present study was conducted to explore the seropositivity and risk factors associated with T. gondii infection in children with ML.
Methods
Subjects
Children with ML were followed up and agreed to participate in this study from July 2012 to October 2018. A total of 314 children with primary ML, who presented to the Affiliated Hospital of Qingdao University, were recruited. In addition, 314 healthy children, age-, gender- and residence-matched to the ML patients, were recruited as controls. None of the participants received intravenous immunoglobulin therapy and/or immunotherapy before enrolment. Written informed consent was obtained from all participants/guardians. The study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (No. 201311683).
Sample and data collection
Approximately 2 ml of venous blood was withdrawn from the participants. Blood samples were left at room temperature for 2 h to allow clotting, followed by centrifugation at 3000 rpm for 10 min. The sera were collected in and stored at −80 °C until further analysis.
Socio-demographic and clinical data
A structured questionnaire was employed to obtain information about the socio-demographic data, including age; gender; residence area; any history of contact with cats, dogs and swine; consumption of raw/undercooked meat, raw vegetables, fruits and oysters; the source of drinking water; and the parents' occupation [Reference Zhou15]. Clinical data collected from the medical examination records encompass the infection status of the mother during pregnancy, history of blood transfusion, chemotherapy and the histological type of ML. Participants/guardians did not know the infection status before the data were collected.
Serological assay
T. gondii antibodies (including IgG and IgM) in sera were tested using the commercially available enzyme immunoassay kits (ELISA) (Demeditec Diagnostics GmbH, Germany) according to the manufacturer's instructions. Sera from the ML patients and healthy children were randomly mixed. Positive and negative controls were included in every assay [Reference Zhou15].
Statistical analysis
The results were analysed using the statistical software SPSS 19.0. For the single variable analysis, χ 2chi-square test or Fisher's exact test was used to assess the association between T. gondii seroprevalence and various variables. The risk factors associated with T. gondii infection were defined by a multivariable backward stepwise logistic regression analysis. Adjusted odds ratio (OR) with 95% confidence interval (CI) were calculated to identify the effect size of risk factors. A P-value < 0.05 was considered statistically significant in the multivariate analysis.
Results
Socio-demographic and risk factors of ML children with T. gondii infection
The overall seroprevalence of T. gondii antibodies in ML patients and healthy controls was 62/314 (19.8%) and 31/314 (9.9%) (P = 0.001), respectively. A significant difference (P = 0.001) was detected while comparing the seroprevalence of T. gondii IgG antibodies between children with ML and healthy children, i.e. 60 ML children (19.1%) vs. 31 (9.9%) control subjects. Interestingly, we found 13 (4.1%) ML patients and six (1.91%) healthy children positive for IgM antibodies (P = 0.103). The baseline data, including socio-demographic and clinical treatment, are shown in Table 1. In ML patients, the seroprevalence of T. gondii was higher in 11–14-year-old patients (13/50, 26%) than in those ⩽2-year-old (7/47, 14.89%), although a not statistically significant difference was detected (P = 0.18).
Table 1. Seroprevalence of T. gondii infection in children with lymphoma and control subjects in eastern China
a Fisher's exact test was used.
Multivariable analysis revealed that contact with cat (OR 2.5; 95% CI 1.4–4.5; P = 0.002), consumption of oysters (OR 1.9; 95% CI 1.1–3.6; P = 0.035) and history of chemotherapy (OR 2.2; 95% CI 0.88–4.2; P = 0.031) were significantly associated with T. gondii infection in ML patients, whereas in healthy controls, contact with cat (OR 2.5; 95% CI 1.2–5.4; P = 0.017) and consumption of oysters (OR 2.4; 95% CI 1.0–5.6; P = 0.042) were the risk factors for the infection (Table 2). Other variables did not show an association with T. gondii infection in the present study.
Table 2. Multivariable analysis of children with lymphoma and healthy controls and the association with T. gondii infection
a Backwards stepwise multivariable analysis.
b Adjusted by age.
Seropositivity of T. gondii in children with ML
Table 3 shows the seroprevalence of different histological type of ML. The maximal seroprevalence of T. gondii antibodies was detected in children with NK/T-cell lymphoma (38.46%), followed by B-small lymphocytic lymphoma (30.77%), marginal zone B-lymphoma (25.58%) and diffuse large B-cell lymphoma (21.05%). Compared to the control subjects, patients with NK/T-cell lymphoma, B-small lymphocytic lymphoma, marginal zone B-lymphoma and Hodgkin's lymphoma have significantly higher seroprevalence (all P < 0.05).
Table 3. Clinical diagnosis and seroprevalence of T. gondii in children with lymphoma in eastern China
As compared with 9.9% seroprevalence of T. gondii antibodies in controls (31/314).
Discussion
The genetic, physical and chemical factors are known to be responsible for the development of lymphoma [Reference Hoppe9, Reference Ferlay10]. Although some reports have revealed a possible association between T. gondii infection and lymphoma [Reference Herold11–Reference Hashiguchi and Onozawa13], the infection status of T. gondii in children with lymphoma remains unclear. Therefore, we tested the T. gondii antibodies, i.e. IgG and IgM in 314 children with lymphoma and 314 healthy controls to explore the seroprevalence and risk factors associated with T. gondii infection in children with ML.
In this study, we found a higher seroprevalence of T. gondii IgG antibodies in children with lymphoma as compared to the control subjects (19.1% vs. 9.9%, P = 0.001), suggesting that the exposure to T. gondii is common in children with lymphoma. However, the seroprevalence of T. gondii IgM antibodies was not significantly different between children with ML and the controls. Reportedly, T. gondii IgG antibodies present later than IgM antibodies in the blood, suggesting a recent infection of T. gondii [Reference Liesenfeld16]. IgM can persist for several years, and in the presence of a positive IgM result, caution must be exercised since a chronic T. gondii infection can be erroneously classified as an acute or false positive; in such cases, IgG avidity tests are crucial [Reference Reshika17]. In the current study, two lymphoma patients with IgM antibodies solely were diagnosed with toxoplasmosis according to the clinical features and IgG avidity tests. This result was similar to a study conducted by Zhou et al. [Reference Zhou15]. Thus, it is necessary to notify the doctors to focus on the significance of T. gondii IgM seropositivity in children with malignancy, and patients with solely T. gondii IgM antibodies should be tested for IgG avidity to avoid misdiagnosis.
Several studies have demonstrated that the positive T. gondii antibodies increased with age in healthy children, but in young patients with malignancy, T. gondii is likely to occur [Reference Cong5, Reference Zhou15, Reference Marchioro18]. This could be attributed to the fact that younger patients with malignancy may be immunocompromised and are inclined towards the T. gondii infection [Reference Cong5, Reference Zhou6]. However, the reasons that younger ML patients were susceptible to T. gondii infection have not been well explained, thereby necessitating further investigation.
In the present study, the multivariate logistic analysis showed that contact with cat and consumption of oocysts are associated with the T. gondii infection. Interestingly, felines are the only definitive hosts for T. gondii, and oocytes can be transmitted via cat faeces and cause toxoplasmosis if the oocyst passed by this method was ingested by humans [Reference Montoya and Liesenfeld1]. Some studies also demonstrated that contact with cats was a risk factor for T. gondii infection in patients with malignancy [Reference Cong5, Reference Zhou6]. Moreover, oocysts shed by felines can be washed into the sea by rain, and T. gondii oocysts can be maintained in the sea for many years [Reference Dubey19]. If these oocysts were ingested by oysters, it might be a potential risk factor for the transmission of T. gondii. Qingdao is a coastal city, and oysters are a popular snack for people, and hence, the consumption of oysters might increase the possibility of T. gondii infection in patients with malignancy [Reference Zhou6]. Therefore, publicizing the information of this risk factor could be conducive to prevent T. gondii infection. In addition, recent studies in Shandong province showed that contact with cats and consumption of undercooked oysters were the risk factors for T. gondii in oral cancer patients [Reference Zhou6], and contact with cats and consumption of undercooked meat could increase the risk of T. gondii infection in patients with diabetes mellitus [Reference Li20]. These similar results indicate the necessity to conduct an epidemiological investigation to identify the risk factors for T. gondii infection in different diseases.
Previous studies demonstrated that blood transfusion was a risk factor for T. gondii infection in patients with malignancy [Reference Zhou6, Reference Zhou15, Reference Alvarado-Esquivel21]. In the current study, we found among many clinical variables, the seroprevalence of T. gondii was only associated with the history of chemotherapy. Chemotherapy is not only an effective treatment for lymphoma but also suppresses the patients' immune system, which would render them vulnerable to T. gondii. Moreover, some studies also demonstrated that chemotherapy augments the infection risk of T. gondii [Reference Ortiz-Munoz22, Reference Scerra23]. Thus, T. gondii infection should be monitored in children with lymphoma; if toxoplasmosis was confirmed, microbiotic antibiotic, such as sulfamethoxazole, should be administered to prevent toxoplasmosis [Reference Zhou15].
Among the various histological types of lymphoma, the seroprevalence of T. gondii in aggressive lymphomas, such as NK/T-cell lymphoma, B-small lymphocytic lymphoma, marginal zone B-lymphoma and Hodgkin's lymphoma, was significantly higher than that in the controls. However, patients with indolent lymphoma present a lower seroprevalence of T. gondii as compared to the controls, which was consistent with the current findings [Reference Scerra23]. Some reports showed a potential association between T. gondii infection and non-Hodgkin's lymphoma [Reference Herold11], intraocular B-cell lymphoma [Reference Shen12] and B-cell lymphoproliferative disorders [Reference Scerra23]. Also, T. gondii can dysregulate the immune response pathway and reduce lipid synthesis by downregulating the activity of butyrylcholinesterase [Reference He24, Reference Da Silva25]. In addition, the infection of T. gondii RH strain contributes to the high levels of T helper cell type 1 (Th1) cytokine and a robust inflammatory response and breaks the balance between apoptosis and anti-apoptosis [Reference Mordue26, Reference He27], thereby leading to an imbalance in the hosts' gene expression, which would result in carcinogenesis [Reference Gharavi28].
Nonetheless, aggressive chemotherapy and immunosuppressive therapeutics used for treating the patients suffering from aggressive lymphoma cause deficiency in cell-mediated immunity; therefore, these patients were at risk to T. gondii infections and inclined to manifest toxoplasmosis [Reference Zhou15]. This phenomenon could partially explain the high seroprevalence of the infection in aggressive lymphoma patients. However, further studies are needed to explore the causes of the difference in the seroprevalence in various histological types of lymphoma.
Nevertheless, the present study has some limitations. First, the limited data did not represent the whole of China. Second, the treatment data of the patients were lacking, and hence, the influence of immunosuppressive management for antibody seroprevalence was uncertain. Third, the donors' sera were not tested, and thus, the influence of the donor-derived antibody was unclear.
Conclusion
The present study revealed that T. gondii infection is prevalent in children with lymphoma, and contact with cats, consumption of raw oysters and history of chemotherapy were independently associated with the risk of infection in this patient group. Thus, the clinicians should be careful with this pathogen infection in patients with lymphoma and efforts should be directed towards evaluating the effect of T. gondii in lymphoma patients.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0950268819001869.
Financial support
This study was supported by the Shandong Provincial Natural Science Foundation, China (ZR2016HQ18). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
The authors declare that there are no conflicts of interest.
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