TOXOPLASMA GONDII INFECTION IN PATIENTS WITH MALIGNANT AND BENIGN BONE TUMOURS

. Toxoplasma gondii (T. gondii) is an intracellular parasite that infects humans, and seroprevalence of its infection varies from about 10 to 80 percent in different countries with a higher prevalence in warmer and humid regions. In this study, the rate of acute and chronic toxoplasmosis in patients with benign or malignant bone tumours was investigated. Fifty-three patients who suffered from various bone tumours, as well as sixty-five healthy controls with an unknown serological profile for anti- Toxoplasma antibodies, were enrolled in this cross-sectional study. Anti- Toxoplasma antibodies were detected in serum samples using enzyme-linked immunosorbent assay (ELISA) and blood samples of them were used for real-time PCR. Thirty-two (60.32%) and twenty-one (39.63%) of patients had malignant tumours and benign tumours, respectively. The results showed a higher and significant seropositivity rate of IgM antibodies in primary bone tumour patients compared to the control group and Toxoplasma DNA became positive in 18.86% of patients with primary bone tumours and 6.15% of controls. Surprisingly, the high presence of parasite DNA was detected in patients with malignant tumours. The seroprevalence of T. gondii IgM antibodies and DNA positivity among the cancer patients were significantly higher than healthy individuals. Also, chronic toxoplasmosis (it was shown with IgG positive) appears to be more common in people with benign cancers than malignancies. The study showed a relatively high seroprevalence of anti- T. gondii antibodies in patients with primary bone cancer. However, the considerable rate of positive blood samples for the presence of parasite’s DNA should not be ignored. A key to the effective management of diseases in immunosuppressed individuals is prompt and accurate diagnosis of toxoplasmosis. Moreover, it seems that PCR tests may be more reliable than serological methods and it could be considered as a precise method for diagnosis of acute toxoplasmosis.


Introduction
Toxoplasma gondii (T. gondii) is an obligatory intracellular parasite that infects humans and many animal species and approximately one-third of the world's population is at risk of infection with this protozoan [11,25]. Seroprevalence of T. gondii infection varies from about 10 to 80 per cent in different countries with a higher prevalence in warmer and humid regions [8,9,26]. Host immune system plays the most important role in pathological symptoms of toxoplasmosis, for example, immunocompetent individuals rarely show considerable signs, however, this infection in immunocompromised persons may lead to severe diseases such as encephalitis, pneumonia, retinochoroiditis and even death [1,31].
B lymphocytes produce various classes of antibodies in response to T. gondii infection which could be applied for serodiagnosis, like specific IgM which can be detected within 7-15 days in acute infection, however, class switching to IgG antibodies and production of a higher titer and avidity of this class of antibody is observed in chronic toxoplasmosis [12,28]. Therefore, one of the easiest diagnostic tests for the routine detection of toxoplasmosis is a screening of specific IgG and IgM antibodies in serum, however; the application of molecular techniques may be more sensitive and appropriative methods for diagnosis of acute toxoplasmosis in high-risk patients especially in cancerous people with low traceable antibodies in consequence of radio or chemotherapy [2,22,25].
Cancer is one of the major causes of mortality worldwide and is the second leading one in developing countries [18]. Primary bone tumours impose a burden of mortality and morbidity with a wide range of clinical manifestations including pain, peripheral inflammation and bone fractures on the patients, worldwide. Various histological types of primary bone tumours include benign (osteochondroma, giant cell tumour, exostosis) and malignant (osteosarcoma, Ewing's sarcoma, chondrosarcoma) tumours with nonspecific symptoms which make it difficult to be managed by clinicians [10,30].
Toxoplasmosis can cause opportunistic lifethreatening infection in cancer patients [4]. On the other hand, several studies hypothesized that T. gondii infection is responsible for the progression of malignant diseases due to inhibition of apoptosis and motility of macrophages [6]. Therefore, epidemiological studies are required to estimate the rate of infection in high-risk individuals, especially in immunocompromised patients with malignancies undergoing chemotherapy [1,27]. A casecontrol study of 900 different cancer patients and 900 controls was conducted in China for evaluating the epidemiology of T. gondii infection and the results showed a high significant prevalence of anti T. gondii IgG in cancer patients but because of rare incidence of different bone tumours, this type of tumour hadn't been mentioned [7], so in this study, we aimed to evaluate the serum levels of IgG and IgM using enzyme-linked immunosorbent assay (ELISA) and determine parasite-specific DNA by quantitative real-time poly merase chain reaction (qPCR) in aforementioned patients.

Materials and methods
Fifty-three patients who suffered from various bone tumours, as well as sixty-five healthy controls with an unknown serological profile for anti-Toxoplasma antibodies, were enrolled in this cross-sec-2021, Т. 11, № 6 Toxoplasma gondii infection in bone tumours patients tional study with local ethical approval and informed consent, from March to August 2019. The blood samples were collected from patients who were diagnosed for primary bone cancer and subjected to surgery at the Shafa Orthopedic Hospital. The patient group was divided into patients with benign tumour subgroups including osteochondroma, giant cell tumour (GGT), exostosis, and other types and patients with malignant tumour subgroups including osteosarcoma, Ewing's sarcoma, chondrosarcoma, and other sarcomas. Approximately five ml of blood was obtained from each participant and divided into two tubes. The tube with no anticoagulant was used for serum isolation to be applied in serological tests and the second tube containing EDTA was used for isolation of buffy coat leukocytes and subsequently DNA purification. All samples were stored at −20°C until use. The serum titre of anti-T. gondii antibodies (IgG and IgM) were measured using commercial enzymelinked immunosorbent assay (ELISA) kits according to the manufacturer's setting (Pishtazteb, Iran) and the absorbance was read by ELISA reader (Biohit, BP800, Finland) at 450 nm versus 630 nm. All experiments were performed in duplicate and equivocal results were repeated [2,29]. Genomic DNA was extracted from the isolated buffy coat samples using DNA extraction mini kit (YTA Co., Iran) according to the manufacturer's instruction. Specific primers for repeated element (RE) gene of T. gondii (F 5'-AGG GAC AGA AGT CGA AGG GG-3' and R 5'-GCA GCC AAG CCG GAA ACA TC-3') was designed using NCBI blast software [3]. The DNA samples were tested for the presence of the 164-bp fragment of RE gene of T. gondii by realtime PCR machine (Rotor-Gene Q, Qiagen, USA), in 20 μl volumes, as previously described [3]. Briefly, 1 μg of DNA template was added to SYBR Green PCR Master Mix (YTA Co., Iran). Afterwards, 0.8 μl of each forward and reverse primers with a concentration of 1 pmol/μl was added and distilled water was used for volume adjustment. Quantitative PCR (qPCR) was performed at 95°C for 10 min, followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The PCR experiments were repeated three times.
Statistics. Data were recorded and analysed with the SPSS v.18 software (SPSS Inc., Chicago, ILL, USA) using Mann-Whitney and Chi-square tests. The p-value < 0.05 was considered as significant.

Results
The demographic data of the participants are presented in Table 1. Thirty-two (60.32%) and twentyone (39.63%) of patients had malignant tumours and benign tumours, respectively. The results showed a higher and significant seropositivity rate of IgM antibodies in primary bone tumour patients compared to the control group ( Table 2). All samples were tested by real-time PCR for Toxoplasma DNA which became positive in 18.86% of patients with primary bone tumours and 6.15% of controls. Surprisingly, the high presence of parasite DNA was detected in patients with malignant tumours ( Table 2). We founded a higher IgG and IgM titer against T. gondii in patients with benign versus malignant bone tumours but PCR results in malignant patients had a higher percentage than benign patients and control group.
A p-value less than 0.05 was considered significant statistically and the seroprevalence of T. gondii IgM antibody and DNA positivity among the cancer patients were significantly higher than healthy individuals (p = < 0.001 and p = 0.005 respectively). Also, chronic toxoplasmosis (it was shown with IgG positive) appears to be more common in people with benign cancers than malignancies.

Discussion
One of the accepted hypothesis is a higher incidence of opportunistic infections such as toxoplasmosis in cancer patients as a group of immunocompromised individuals [20], for example, the seroprevalence rate of T. gondii infection in Iran  was 45-51% in cancer patients but 39% in healthy individuals [1,8]. However, the geographical factors, eating habits and livestock farming practices can be effective in the prevalence of this infection. Khabaz et al. showed that anti-T. gondii IgG was detected in 63.6% of patients with neoplasia and 58% of healthy controls, although, all of the participants (case and control groups) were negative for anti-T. gondii IgM antibodies [19]. T. gondii seroprevalence was 60.3% in immunocompromised patients and 33.3% in healthy individuals in other provinces during 2014-2015 [17]. Otherwise, the higher rate of T. gondii infection in immunocompromised individuals, such as cancer patients could be due to many reasons such as a difference in genetic susceptibility and the source of Toxoplasma infection [1,32]. So, it might be concluded that a high incidence of acute toxoplasmosis in bone cancer patients could be due to impaired genetic system or decreased immunity to encounter this protozoan. The high rate of PCR results obtained for T. gondii DNA in cancer patients (18.86% against 6.15% in a healthy individual) could be a witness of this hypothesis and the lower immunity in malignant patients may be explained with more incidence of this infection in patients with malignant tumours versus benign types (21.87% and 14.28%, respectively). On the other hand, this parasite could remain silent in tissue cysts that are commonly formed in different organs such as the central nervous system and the situations such as the immune suppression in cancer patients or the therapeutic process can cause reactivation of parasite and latent toxoplasmosis [24]. So, this can account as another reason for a high percentage of positive PCR and IgM results in cancer.
In the current study, the findings emphasize raising the possible role of cancer on T. gondii infection susceptibility, because the seroprevalence of anti-Toxoplasma antibodies in patients was higher (47.16% versus 38.46%). The other hypothesis is the effect of persistent infection on the promotion of cancer due to rising mutation rates as a result of long-term host defence responses in inflammation situations [15]. Also, intercellular pathogens like T. gondii may disrupt cell barriers against oncogenic agents and might cause mutations after accumulating over time [14].
Interestingly, we observed that two PCR positive patients in the malignant group were negative for anti-Toxoplasma IgM and IgG antibodies in ELISA which might be due to the recent infection or impaired immune response following immunosuppressive therapy. It was mentioned that serological tests could be sometimes inadequate for detecting active infection in immunosuppressed individuals, because the antibody titre may not rise enough to be detected [13,23]. Overall, these patients are incapable of developing high titres of antibodies against T. gondii. Moreover, anti-neoplastic drug therapy could impair specific anti-Toxoplasma antibody production [16,21].
Hence, serological methods alone have low reliability in both patients and healthy; it was strongly recommended that serologic tests should be combined with other diagnostic methods like gene amplification for accurate clinical diagnosis of active toxoplasmosis. Molecular methods are efficient techniques that allow specific amplification of DNA. The real-time qPCR could be successfully used to T. gondii diagnosis and is capable to detect low concentrations of target DNA [3,22,32]. Besides, to increase the specificity and sensitivity of immunological assays in immunocompromised patients, new antigenic targets should be designed the parasite to have a potential immunogenic antigen in immunodiagnostic tools of toxoplasmosis in patients with cancer [5].

Conclusion
The study showed a relatively high seroprevalence of anti-T. gondii antibodies in patients with primary bone cancer. However, the considerable rate of positive blood samples for the presence of parasite's DNA should not be ignored. A key to the effective management of diseases in immunosuppressed individuals is prompt and accurate diagnosis of toxoplasmosis. Moreover, it seems that PCR tests may be more reliable than serological methods and it could be considered as a precise method for diagnosis of acute toxoplasmosis.