Serological and molecular detection of Toxoplasma gondii infection in patients with hematological malignancies

DOI: https://doi.org/10.21203/rs.3.rs-1774386/v1

Abstract

Introduction:

Toxoplasma gondii causes life-threatening disease in immunocompromised patients such as those with hematological malignancy. Serological diagnosis of toxoplasmosis in these patients is challenged by the impaired antibody response; meanwhile, molecular testing is necessary to demonstrate reactivation. We aimed to study rates and risk factors of toxoplasmosis in hematological malignancy patients using serological and molecular methods.

Methodology:

The study was conducted on 40 adult hematological malignancy patients and 40 age and sex-matched healthy individuals. Data on plausible risk factors of toxoplasmosis were collected. Serologic testing for anti-Toxoplasma antibodies was performed using enzyme-linked immunosorbent assays. Blood samples were examined by polymerase chain reaction (PCR) for detection of T. gondii AF146527 (a 529-bp repeat element).

Results

T. gondii seropositive rate was significantly higher among patients (75%) compared to healthy individuals (42.5%). However, seropositive patients displayed lower anti-Toxoplasma IgG concentrations compared to healthy individuals (p < 0.05). Positive PCR results were obtained in 13 patients (32.5%) and one healthy individual (2.5%) with a significant difference. Among patients, serology and PCR showed slight agreement. Seropositive patients were more likely to be PCR positive (36.7%) compared to seropositive healthy individuals (5.9%) (p < 0.05). No statistically significant association was found between toxoplasmosis and demographic variables, cat contact, undercooked meat consumption, type of malignancy, blood transfusion, and chemotherapy.

Conclusions

Patients with hematological malignancy are at high risk of T. gondii infection and reactivation. The combination of serology and PCR would be more accurate for a definite diagnosis. Follow-up is necessary to prevent the development of life-threatening toxoplasmosis in these patients.

Introduction

Patients with hematological malignancy (HM), including those with acute myelogenous leukemia, and those who have undergone hematopoietic stem cell transplantation or treated with aggressive immunosuppressive regimens are at high risk of opportunistic infections. Prompt correct diagnosis is essential for effective management of these infections [1, 2].

Toxoplasma gondii is a widespread obligately intracellular protozoan parasite causing opportunistic infection in immunocompromised patients. Humans acquire T. gondii infection by ingestion of cysts in undercooked meat or oocysts in contaminated food or water. Infection may be also transmitted congenitally or through organ transplantation [3]. After initial dissemination, the parasite can evade the immune system and persists throughout the host life within dormant cysts, predominantly in the brain, retina, and muscles [4]. Infection is generally asymptomatic and has little clinical relevance in immunocompetent subjects [5]. However, in immunocompromised patients, reactivation of latent cysts may occur if the immune system fails to maintain an efficient T helper-1 immune response. This can lead to cerebral or disseminated disease [6]. The incidence of reactivated toxoplasmosis is closely related to T. gondii seroprevalence in the population. The duration and degree of immune suppression are strong predictors of reactivation [7]. Primary T. gondii infection in immunocompromised patients is also life-threatening due to the involvement of multiple organs [8].

Definitive diagnosis of toxoplasmosis can be made by isolation of the parasite from blood or body fluids, and by histological examination of affected tissues using specific stains [7]. As these methods are difficult and time-consuming, the classical diagnosis of toxoplasmosis usually relies on the detection of specific antibody responses [9]. However, serological methods may have poor efficiency in immunocompromised patients due to delayed or impaired antibody production [3, 10]. Polymerase chain reaction (PCR) assays for the detection of T. gondii DNA using primers specific to different regions of the parasite genome have been in routine use for more than 30 years [11, 12]. The ability of molecular methods to detect low amounts of parasites is valuable, as Toxoplasma can circulate inconstantly and at low concentrations with the potential for reactivation [13]. Thus, the use of PCR assays for detection of T. gondii DNA in blood or body fluids is strongly recommended as an additional diagnostic method in immunocompromised patients [14].

The present study aimed to evaluate rates of T. gondii infection in patients with HM compared to healthy individuals using serological and molecular methods and to shed light on possible risk factors of toxoplasmosis in these patients.

Methodology

Study design and setting

A case-control observational study was carried out on 40 adult patients with different types of HM and 40 age and sex-matched healthy subjects in Alexandria, Egypt. This sample size was calculated using Epi info 7 (Centers for Disease Control and Prevention; Atlanta, GA, USA). Based on Toxoplasma prevalence rate of 65% in the Egyptian population [15] and with a risk ratio of 1.5 in cancer patients [16, 17], the sample size was considered adequate at 95% confidence interval with a statistical power of 90%.

All participants were recruited from the Hematology and Parasitology Departments, Medical Research Institute (Alexandria University- Egypt) during the period from February 2019 to October 2019.

The study protocol was approved by the Ethical Committee of the Institute. All patients and healthy subjects gave consent to participate and were offered information about the procedure and objectives of the study. Informed consent was obtained from each participant before inclusion in the study.

Data and samples collection

All participants were interviewed using a preformed structured questionnaire to collect demographic characters and data on the lifestyle habits that pertain to exposure to T. gondii such as contact with cats and consumption of undercooked meat. The medical history of patients including the type of malignancy, blood transfusion, and the treatment received was also recorded.

A single venous blood sample (about 5ml) was drawn from each participant and divided into two tubes; a plain tube and a tube with an anticoagulant. Samples were immediately transported to the laboratory of the Parasitology Department, Medical Research Institute, Alexandria University. The sample in the plain tube was centrifuged at 3000 rpm for 5 minutes to obtain sera. Clear, non-hemolyzed sera were divided into two clean labeled Eppendorf tubes and stored at -20o C until used for serology. The anti-coagulated blood sample was stored at -80o C until used for DNA extraction and molecular testing.

Serological testing

The serum samples were tested for anti-T. gondii IgM and quantitative determination of anti-Toxoplasma IgG concentration using commercially available ELISA assays (Biocheck Inc., Foster City, California, 94404 USA). The tests were performed according to the manufacturer’s instructions. For qualitative determination of IgM, the optical density (OD) of each analyzed sample was divided by the OD of the kit calibrator to obtain the IgM index. Samples with IgM index ≥ 1 are considered positive. The IgG cut-off calibrator provided with the kit has a concentration of 32 IU/ml. For the quantitative determination of the Toxoplasma IgG antibody levels, the OD of the kit calibrators was plotted on the Y axis versus their corresponding concentrations on the X axis. The Toxoplasma IgG antibodies levels in patients' sera were read off the graph using their individual OD values.

Molecular detection

DNA was extracted from the anticoagulated blood samples using QIAamp DNeasy Mini kit (Qiagen cat. no. U.S.69504 and 69506) according to the manufacturer’s directions. Amplification of Toxoplasma AF146527 (a 529-bp repeat element) was performed in a thermal cycler (Beco, Germany) using Red Taq master mix (Bioline, UK) and the primers TOXO 4: 5'CGCTGCAGGGAGGAAGACGAAAGTTG3' and TOXO 5: 5′CGCTGCAGACACAGTGCATCTGGATT-3′ [18]. The amplification steps included initial denaturation step at 94°C for 5min, 35 cycles of denaturation at 94°C for 45s, annealing at 55°C for 30s, and extension at 72°C for 45s, and a final extension step at 72°C for 10 min. The final PCR products were separated in 1% agarose gel electrophoresis at 100 v for 30 min. The bands of the expected size (529 bp) were visualized against a 100 bp DNA ladder on a UV transilluminator. Each PCR round included a negative control of nuclease-free water and a positive control of DNA extracted from T. gondii RH strain maintained in the Parasitology Department, Medical Research Institute, Alexandria University.

Statistical analysis

The obtained data were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 20 (Chicago, IL, USA). Categorical variables were compared using Pearson’s Chi-squared test. The t-test was used to analyze the difference between the means of quantitative data of two groups. The agreement between serology and PCR was analyzed by Cohen’s Kappa agreement test. A p-value of less than 0.05 was considered statistically significant.

Results

Characteristics of the study population

The mean age of patients was 40 ± 15.88 years (range: 17–76 years) and the mean age of the healthy individuals was 38.6 ± 15.69 years (range: 18 to 76). In both groups, 65% of participants were aged ≤ 40 years. Males constituted 47.5% of patients and 40% of healthy individuals. Among the 40 HM patients, 19 (47.5%) had acute myeloid leukemia (AML) and 12 (30%) had acute lymphoid leukemia (ALL). Less common forms of HM among participating patients were chronic lymphoid leukemia (CLL) (5 patients), chronic myeloid leukemia (CML) (3 patients), and hairy cell leukemia (one patient). Collectively, 75% of the patients were on cytotoxic chemotherapy for a duration ranging from 2 weeks to 10 months. and 25% had not yet received treatment. The main symptoms among HM patients were fever, unexplained fatigue, weight loss, and anemia-related symptoms. None of the patients had neurological or pulmonary symptoms.

Toxoplasma gondii infection in HM patients and healthy individuals

Serological testing revealed that 30 HM patients (75%) were IgG seropositive, of whom three were IgM positive, suggesting potential acute infection. Among healthy participants, 17 were IgG seropositive, of whom one had IgM antibodies. The overall T. gondii seropositive rate was significantly higher among HM patients compared to the healthy participants (X2 = 8.717, P = 0.003). PCR gave positive results in 13 out of the 40 HM patients (32.5%) and one of the 40 healthy participants (2.5%) with a highly statistically significant difference (P = 0.0004) (Table 1). Examples of PCR positive and negative samples are shown in Fig. 1.

Among patients with different types of HM, T. gondii seropositive rate ranged from 58.3–100% and the PCR positive rate ranged from 21.1–100%. Neither serology nor PCR positive results showed a significant association with the type of HM (Table 2).

Seropositive HM patients displayed significantly lower anti-Toxoplasma IgG concentrations (mean ± SD = 290.69 ± 147.8 IU/ml) compared to seropositive healthy individuals (mean ± SD = 550.6 ± 281.95) (p = 0.0001). Seropositive patients were more likely to be PCR positive compared to seropositive healthy individuals ((36.7% versus 5.9%, p < 0.05) (Table 3).

Serology versus PCR for detection of Toxoplasma gondii infection

No significant association could be observed between PCR results and detection of anti-Toxoplasma antibodies (Table 4). Detection of specific IgG antibodies showed a slight agreement with PCR for detection of T. gondii infection in HM patients (k = 0.011). Among the healthy individuals, the single PCR positive individual was IgG positive-IgM negative (data not shown).

Factors associated with Toxoplasma gondii infection among the examined patients

There was no significant difference in T. gondii positivity rate in relation to patients' age or gender. Contact with cats and consumption of undercooked meat did not affect seropositivity rates. Higher rate of positive PCR result was observed among patients with a history of cat contact compared to those with a negative history, but this did not reach the level of statistical significance (P = 0.053). Regarding clinical history, it was found that the chronic form of malignancy, receiving chemotherapy, and blood transfusion were not significantly associated with the T. gondii positive rates (Table 5).

Discussion

T. gondii is recognized as an opportunistic parasite in immunocompromised patients [3, 10]. Results of the present study revealed that T. gondii seropositive rate ranged from 58–100% in patients with different forms of HM. The overall seropositive rate was significantly higher in HM patients compared to healthy individuals. In Egypt, seroprevalence rates as high as 90% and as low as 20% were previously reported among patients with other forms of malignancy [1921]. Similar studies conducted worldwide reported concordant results. In Iran, Gharavi et al. (2017) detected T. gondii IgG antibodies in 56.4% of leukemia patients and 42.4% of the control group [22]. A meta-analysis incorporating nineteen studies conducted in China concluded that the overall T. gondii seroprevalence was significantly higher in the population with cancer compared to those without (20.59% vs 6.31%) [23]. The variations in Toxoplasma seroprevalence rates among different countries may be attributed to different social and cultural habits, distinct environmental and geographical factors, and different livestock rearing practices [7].

Weakened host immunity is a major sequalae in all types of HM [2427]. Treatment regimens can further exacerbate immunosuppression in these patients [2]. In the case of immunodeficiency, patients with latent toxoplasmosis are at risk for reactivation whereas recently infected patients are at risk for acute disseminated toxoplasmosis, in which case the diagnosis of infection is an emergency [8, 28]. A positive IgG test merely indicates that the host has been infected at some time in the past. Although serologic detection of IgM and high IgG titers suggests an acute infection, and could, therefore, be valuable in emergency diagnosis, the application is limited in immunocompromised patients due to reduced antibody production [7, 29]. This was confirmed in the present study by the significantly lower IgG concentration observed in seropositive patients with HM compared to controls. Therefore, direct detection of the parasite or its DNA in clinical samples is essential [30].

In the present study, PCR analysis revealed detection of Toxoplasma DNA in 32.5% of patients compared to 2.5% of controls with a statistically significant difference. The PCR positivity was not significantly related to the type of malignancy. Generally, detection of T. gondii in clinical samples confirms the presence of parasites which can be due to primary or reactivated infection [31]. Although the presence of Toxoplasma DNA in blood indicates parasitemia, its clinical significance in immunocompromised patients is unclear. T. gondii DNA was detected by PCR in blood samples of 80% of patients with cerebral toxoplasmosis but it may be also detected in the blood of immunocompromised patients who had no localizing signs and symptoms [3234]. The detection of T. gondii may precede the onset of disease reactivation, indicating that molecular monitoring of T. gondii in peripheral blood may contribute to early diagnosis [35]. Noteworthy, molecular methods can detect parasites that have been released from tissue cysts into the blood stream, but it cannot differentiate the DNA of dead and viable parasites. The parasite may be rapidly destroyed by the immune system but the DNA remains in the blood for up to 13 weeks [36, 37 ]. Accordingly, diagnosis of Toxoplasma-related illness in immunocompromised patients should be based on molecular, clinical, and radiologic findings [38].

PCR amplification of Toxoplasma AF146527 is extremely sensitive and specific for molecular detection of T. gondii as this 529bp fragment is repeated 200- to 300-fold in the genome [18, 31]. In the present study, most seropositive controls (16 out of 17 subjects) were PCR negative. On the other hand,19 out of the 30 ELISA-positive HM patients were negative by PCR. Failure to detect Toxoplasma DNA in a considerable number of seropositive individuals was reported in different population groups [3941]. This can be explained by the low amount of DNA in blood samples. In chronic T gondii infection, parasites appear rarely in the blood at intervals during the asymptomatic phases of the disease and may be detected by PCR unless efficiently eliminated by the immune system [42]. Bavand et al., (2019) studied HIV patients in Iran and reported that Toxoplasma DNA was detected in only five out of 69 T. gondii IgG-positive patients. A non-severely depressed immunological status can contribute to the clearance of blood parasitaemia [43]. It has to be noted that the absence of DNA does not exclude the presence of active disease [7, 38, 44]. Mild local reactivation of latent cysts may not be associated with positive PCR results in peripheral blood samples. Molecular diagnosis is more useful in cases associated with severe localized infection or dissemination [42].

Intriguingly, the present study showed that Toxoplasma DNA was detected in two patients for whom the results of serological tests were negative. A possible explanation is that blood samples were drawn very early after infection and before the production of antibodies. Another explanation is that the immune system was unable to produce enough immunoglobulins to be detected by the serological ELISA assay due to immunodeficiency. That is why parasite detection by molecular methods is strongly recommended when serology is negative in immunocompromised patients. Collectively, PCR and serological examination showed slight agreement. Therefore, the combination of both techniques would be more accurate for a definite diagnosis of toxoplasmosis.

In the present study, the seropositivity of T. gondii showed a non-statistically significant association with the age and gender of patients. Similar results were reported in previous studies [17, 21, 45].

Contact with infected cats and consumption of undercooked meat are the main sources of T. gondii infection in man [46]. However, both were not significantly associated with T. gondii infection in HM patients. These findings correlate with the findings of an earlier report among different population groups in Egypt [47]. Oocysts are not usually found on cat fur but are often buried in soil along with cat feces. Sanitation and hygienic habits would play a significant role in limiting the transmission of oocysts to humans [48]. Transmission through undercooked meat is largely determined by the prevalence of the parasite in the animals as well as local methods of meat cooking. [49].

Although blood transfusion is a potential transmission route for Toxoplasma infection [50, 51]. it was not linked to T. gondii transmission in HM in the present study. Toxoplasma parasitemia was previously reported in 10% of blood donors in Egypt which suggests that the rate of Toxoplasma transmission is low [15].

One of the limitations of the study is that the more advanced molecular methods such as real time PCR and sequencing of the PCR product were not performed. However, the conventional PCR protocol used proved to be highly sensitive due to the high copy number of the amplified gene fragment [18]. Moreover, the sequence of the used primers was subjected to a BLAST (Basic Local Alignment Search Tool) analysis to ensure specificity of the assay to T. gondii. In each PCR run, we included a positive and a negative control samples to validate the results.

Conclusion

patients with different types of HM are at high risk of Toxoplasma infection and reactivation. Although serology helps in estimating the risk for reactivation, its application is limited in immunocompromised patients due to the reduced antibody levels. Toxoplasma DNA may be detected in seronegative HM patients. This may be attributed to early acute infection with delayed or impaired antibody production. No statistically significant association could be observed between other plausible risk factors and T. gondii infection among these patients. HM appears to be the only predisposing factor for Toxoplasma infection in the studied patients. Further studies should investigate the effects of disease duration, length, and type of anti-cancer therapy and the severity of immune suppression on the outcome of T. gondii infection in HM patients.

Declarations

Ethics approval: The study protocol was approved by the Ethical Committee of the Medical Research Institute, Alexandria University, Egypt. All patients and healthy subjects gave consent to participate and were offered information about the procedure and objectives of the study. Informed consent was obtained from each participant before inclusion in the study. All methods were carried out in accordance with relevant guidelines and regulations. 

Consent to participate: All patients and healthy subjects included in the study gave consent for participation in the study. Informed consent was obtained from each participant before inclusion in the study.

Consent for publication: Not applicable

Availability of data: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors have no relevant financial or non-financial interests to disclose.

Funding: No funds or other support were received during the preparation of this manuscript.

Authors' contributions: All authors contributed to the study conception and design. Data collection was performed by Taha Shawa, Maha El-Gammal, and Heba Ibrahim. Data analysis and interpretation was performed by Hend El-taweel and Heba Ibrahim. The first draft of the manuscript was written by Taha Shawa, and Heba Ibrahim. All authors revised and approved the final manuscript.

Acknowledgements: The authors appreciate and would like to thank the Hematology Department and patients for their support in collecting blood samples. They also appreciate Mrs Doaa Gaber Shalaby, Hematology Department, Medical Research Institute, Alexandria University, for their support and help in sample collection. The authors also appreciate and would like to thank the all members of the staff of Parasitology Department.

References

  1. Scerra S, Coignard-Biehler H, Lanternier F, Suarez F, Charlier-Woerther C, Bougnoux ME, Gilquin J, Lecuit M, Hermine O, Lortholary O (2013) Disseminated toxoplasmosis in non-allografted patients with hematologic malignancies: report of two cases and literature review. European journal of clinical microbiology & infectious diseases, 32(10), 1259–1268.
  2. Christopeit M, Schmidt-Hieber M, Sprute R, Buchheidt D, Hentrich M, Karthaus M, Penack O, Ruhnke M, Weissinger F, Cornely OA (2021) Prophylaxis, diagnosis and therapy of infections in patients undergoing high-dose chemotherapy and autologous haematopoietic stem cell transplantation. 2020 update of the recommendations of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO). Annals of hematology, 100(2), 321–336.
  3. Hill DE, Dubey JP (2018) Toxoplasma gondii. In Foodborne parasites (pp. 119–138). Springer.
  4. Skariah S, McIntyre MK, Mordue DG (2010) Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion. Parasitology research, 107(2), 253–260.
  5. Remington JS (1974) Toxoplasmosis in the adult. Bulletin of the New York Academy of Medicine, 50, 211–227.
  6. Vidal JE (2019) HIV-related cerebral toxoplasmosis revisited: current concepts and controversies of an old disease. Journal of the International Association of Providers of AIDS Care (JIAPAC), 18, 2325958219867315.
  7. Robert-Gangneux F, Dardé M-L (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. Clinical microbiology reviews, 25(2), 264–296.
  8. Signorini L, Gulletta M, Coppini D, Donzelli C, Stellini R, Manca N, Carosi G, Matteelli A (2007) Fatal disseminated toxoplasmosis during primary HIV infection. Current HIV research, 5(2), 273–274.
  9. Pappas G, Roussos N, Falagas ME (2009) Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. International journal for parasitology, 39(12), 1385–1394.
  10. Weiss LM, Dubey JP (2009) Toxoplasmosis: A history of clinical observations. International journal for parasitology, 39(8), 895–901.
  11. Su C, Zhang X, Dubey JP (2006) Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. International journal for parasitology, 36(7), 841–848.
  12. Su C, Shwab EK, Zhou P, Zhu XQ, Dubey JP (2010) Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology, 137(1), 1–11.
  13. Romand S, Chosson M, Franck J, Wallon M, Kieffer F, Kaiser K, Dumon H, Peyron F, Thulliez P, Picot S (2004) Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii. American journal of obstetrics and gynecology, 190(3), 797–802.
  14. Robert-Gangneux F, Sterkers Y, Yera H, Accoceberry I, Menotti J, Cassaing S, Brenier-Pinchart M-P, Hennequin C, Delhaes L, Bonhomme J (2015) Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study. Journal of clinical microbiology, 53(5), 1677–1684.
  15. El-Geddawi OA, El-Sayad MH, Sadek NA, Hussien NA, Ahmed MA (2016) Detection of T. gondii infection in blood donors in Alexandria, Egypt, using serological and molecular strategies. Parasitologists United Journal, 9(1), 24.
  16. Wang Z-D, Liu H-H, Ma Z-X, Ma H-Y, Li Z-Y, Yang Z-B, Zhu X-Q, Xu B, Wei F, Liu Q (2017) Toxoplasma gondii infection in immunocompromised patients: a systematic review and meta-analysis. Frontiers in microbiology, 8, 389.
  17. Kalantari N, Rezanejad J, Tamadoni A, Ghaffari S, Alipour J, Bayani M (2018) Association between Toxoplasma gondii exposure and paediatrics haematological malignancies: a case–control study. Epidemiology & Infection, 146(15), 1896–1902.
  18. Homan WL, Vercammen M, De Braekeleer J, Verschueren H (2000) Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. International journal for parasitology, 30, 69–75.
  19. El Shazly AM, Afify EM, Morsy TA (1996) Antibodies against toxoplasma in children and adults with malignancy. Journal of the Egyptian Society of Parasitology, 26(3), 781–787.
  20. Mostafa NES, Hamed EFA, Rashed HES, Mohamed SY, Abdelgawad MS, Elasbali AM (2018) The relationship between toxoplasmosis and different types of human tumors. The Journal of Infection in Developing Countries, 12(02), 137–141.
  21. Malek RA, Wassef R, Rizk E, Sabry H, Tadros N, Boghdady A (2018) Toxoplasmosis an overlooked disease: seroprevalence in cancer patients. Asian Pacific journal of cancer prevention: APJCP, 19(7), 1987.
  22. Gharavi MJ, Roozbehani M, Mandeh Z (2017) Detection of anti-Toxoplasma gondii antibodies in chronic myeloid leukemia and acute myeloid leukemia patients. Veterinary world, 10(9), 1063.
  23. Jiang C, Li Z, Chen P, Chen L (2015) The seroprevalence of Toxoplasma gondii in Chinese population with cancer: a systematic review and meta-analysis. Medicine, 94(50).
  24. Chandran R, Hakki M, Spurgeon S (2012) Infections in leukemia. Sepsis-an ongoing and significant challenge.
  25. Dearden C (2008) Disease-specific complications of chronic lymphocytic leukemia. ASH Education Program Book, 2008(1), 450–456.
  26. Zhu Y-D, Xu W, Miao K-R, Cao X, Fan L, Liu Q, Yao L, Wu Y-J, Hong M, Li J-Y (2009) Abnormality of serum immunoglobulin in peripheral blood of patients with chronic lymphocytic leukemia. Zhongguo shi yan xue ye xue za zhi, 17(5), 1159–1162.
  27. Görgün G, Holderried TAW, Zahrieh D, Neuberg D, Gribben JG (2005) Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. The Journal of clinical investigation, 115(7), 1797–1805.
  28. Costa JM, Pautas C, Ernault P, Foulet F, Cordonnier C, Bretagne S (2000) Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. Journal of clinical microbiology, 38(8), 2929–2932.
  29. Ben-Bassat I, Many A, Modan M, Peretz C, Ramot B (1979) Serum immunoglobulins in chronic lymphocytic leukemia. The American journal of the medical sciences, 278(1), 4–9.
  30. Babady NE (2016) Laboratory diagnosis of infections in cancer patients: challenges and opportunities. Journal of clinical microbiology, 54(11), 2635–2646.
  31. Liu Q, Wang Z-D, Huang S-Y, Zhu X-Q (2015) Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasites & vectors, 8(1), 1–14.
  32. Colombo FA, Vidal JE, Oliveira ACPd, Hernandez AV, Bonasser-Filho F, Nogueira RS, Focaccia R, Pereira-Chioccola VL (2005) Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: importance of molecular and immunological methods using peripheral blood samples. Journal of clinical microbiology, 43(10), 5044–5047.
  33. Adurthi S, Sahoo TP, Chakka K, Radhika B, Appaji L, Bapsy PP, Ramesh C, Jayshree RS (2008) Acute toxoplasmosis in nonstem cell transplant patients with haematological malignancies: a study from a regional cancer institute in South India. Hematological oncology, 26(4), 229–233.
  34. Gashout A, Amro A, Erhuma M, Al-Dwibe H, Elmaihub E, Babba H, Nattah N, Abudher A (2016) Molecular diagnosis of Toxoplasma gondii infection in Libya. BMC infectious diseases, 16(1), 1–8.
  35. Matsuo Y, Takeishi S, Miyamoto T, Nonami A, Kikushige Y, Kunisaki Y, Kamezaki K, Tu L, Hisaeda H, Takenaka K (2007) Toxoplasmosis encephalitis following severe graft-vs.‐host disease after allogeneic hematopoietic stem cell transplantation: 17 year experience in Fukuoka BMT group. European journal of haematology, 79(4), 317–321.
  36. Garweg J, Boehnke M, Koerner F (1996) Restricted applicability of the polymerase chain reaction for the diagnosis of ocular toxoplasmosis. German journal of ophthalmology, 5(2), 104–108.
  37. Guy EC, Joynson DHM (1995) Potential of the polymerase chain reaction in the diagnosis of active Toxoplasma infection by detection of parasite in blood. Journal of Infectious Diseases, 172(1), 319–322.
  38. Martino R, Maertens J, Bretagne S, Rovira M, Deconinck E, Ullmann AJ, Held T, Cordonnier C, European Group for B, Marrow Transplantation Infectious Diseases Working P (2000) Toxoplasmosis after hematopoietic stem cell transplantation. Clinical infectious diseases, 31(5), 1188–1194.
  39. Gallego C, Saavedra-Matiz C, Gómez-Marín JE (2006) Direct genotyping of animal and human isolates of Toxoplasma gondii from Colombia (South America). Acta tropica, 97(2), 161–167.
  40. Messaritakis I, Detsika M, Koliou M, Sifakis S, Antoniou M (2008) Prevalent genotypes of Toxoplasma gondii in pregnant women and patients from Crete and Cyprus. The American journal of tropical medicine and hygiene, 79(2), 205–209.
  41. Nakashima F, Pardo VS, Miola MP, Murata FHA, Paduan N, Longo SM, Brandão de Mattos CC, Pereira-Chioccola VL, Ricci Jr O, De Mattos LC (2020) Serum IgG Anti-Toxoplasma gondii Antibody Concentrations Do Not Correlate Nested PCR Results in Blood Donors. Frontiers in cellular and infection microbiology, 9, 461.
  42. Khalifa Ke-S, Roth A, Roth B, Arasteh KN, Janitschke K (1994) Value of PCR for evaluating occurrence of parasitemia in immunocompromised patients with cerebral and extracerebral toxoplasmosis. Journal of Clinical Microbiology, 32(11), 2813–2819.
  43. Bavand A, Aghakhani A, Mohraz M, Banifazl M, Karami A, Golkar M, Babaie J, Saleh P, Mamishi S, Ramezani A (2019) Prevalence of Toxoplasma gondii antibodies and DNA in Iranian HIV patients. Iranian journal of pathology, 14(1), 68.
  44. Fricker-Hidalgo H, Bulabois C-E, Brenier-Pinchart M-P, Hamidfar R, Garban F, Brion J-P, Timsit J-F, Cahn J-Y, Pelloux H (2009) Diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of DNA detection and serological techniques. Clinical Infectious Diseases, 48(2), e9-e15.
  45. Ali MI, Abd El Wahab WM, Hamdy DA, Hassan A (2019) Toxoplasma gondii in cancer patients receiving chemotherapy: seroprevalence and interferon gamma level. Journal of Parasitic Diseases, 43(3), 464–471.
  46. Djurković-Djaković O, Dupouy-Camet J, Van der Giessen J, Dubey JP (2019) Toxoplasmosis: overview from a one health perspective. Food and Waterborne Parasitology, 15, e00054.
  47. Taman A, Alhusseiny S (2020) Exposure to toxoplasmosis among the Egyptian population: A systematic review. Parasitologists United Journal, 13(1), 1–10.
  48. Dubey JP (1995) Duration of immunity to shedding of Toxoplasma gondii oocysts by cats. The Journal of parasitology, 410–415.
  49. Belluco S, Patuzzi I, Ricci A (2018) Bovine meat versus pork in Toxoplasma gondii transmission in Italy: a quantitative risk assessment model. International journal of food microbiology, 269, 1–11.
  50. Alvarado-Esquivel C, Sánchez-Anguiano LF, Hernández-Tinoco J, Ramos-Nevarez A, Estrada-Martínez S, Cerrillo-Soto SM, Medina-Heredia GE, Guido-Arreola CA, Soto-Quintero AA, Beristain-Garcia I (2018) Association between Toxoplasma gondii infection and history of blood transfusion: a case-control seroprevalence study. Journal of International Medical Research, 46(4), 1626–1633.
  51. Montoya, JG, Liesenfeld, O (2004) Toxoplasmosis. Lancet, 363(9425), 1965–1976.

Tables

Table (1): Serological and molecular detection of T. gondii in patients with hematological malignancy compared to healthy individuals

 

 

Test

Patients

n=40

Healthy individuals

 n=40

 

 

P-value

No.

No

Anti-Toxoplasma IgG only

27

67.5

16

40.0

0.013 ⃰

Anti-Toxoplasma IgG+ IgM

3

7.5

1

2.5

 

0.304

Total seropositivity

30

75.0

17

42.5

0.003 ⃰

PCR 

13

32.5

1

2.5

0.0004 ⃰

P values were calculated using the Chi-square test,  ⃰ : Statistically significant at p ≤ 0.05

Table (2) T. gondii among the examined patients according to the type of hematological malignancy

Type of hematological malignancy

No.

examined

Serology 

 

       PCR 

 

No.

%

P

 

 

0.4

No

%

P

 

 

0.263

AML

19

15

78.9

4

21.1

ALL

12

7

58.3

6

50.0

CLL

5

5

100.0

1

20.0

CML

3

2

66.7

1

33.3

HCL

1

1

100.0

1

100.0

AML: Acute myeloid leukemia, ALL: acute lymphoid leukemia, CLL:  chronic lymphoid leukemia, CML: chronic myeloid leukemia, HCL:  hairy cell leukemia. 

 p: p-value for Chi-square test 

Table (3) Anti-Toxoplasma IgG concentration and PCR results among seropositive hematological malignancy patients and healthy individuals 

Test

Seropositive patients

(n=30)

Seropositive healthy individuals   (n=17)

P

IgG concentration

 

 

0.0001 *1

(IU/ml)          Mean ± SD

290.69 ±       147.81

550.62 ± 281.95

PCR positive

No. (%)

11 (36.7)

1 (5.9)

0.007*2

*: Statistically significant at P ≤ 0.05, p-value for t-test, p-value for Chi-square test

Table (4) Association between IgG and IgM ELISA and PCR results for detection of T. gondii infection among patients with hematological malignancy

Serology

PCR

p

+ve 

-ve

No.

%

No.

%

0.3139

IgG +ve-/ IgM-ve

9

33.3

18

66.7

IgG +ve/ IgM +ve

2

66.7

1

33.3

IgG -ve/ IgM-ve

2

20.0

8

80.0

Total

13

100.0

27

100.0

 

p: p-value for Chi-square test 

Table (5) T. gondii infection diagnosed by serology and PCR among patients with hematological malignancy according to plausible risk factors 

Characteristic variable

No. examined

Serology+ ve

 p-value           

PCR +ve

p-value

No.

%

No.

%

Age in years

≤40

26

19

73.7

 0.702

9

34.6

0.697

>40

14

11

78.6

4

28.6

Gender

Male

19

14

73.7

 0.855

6

31.6

0.906

Female

21

16

76.2

7

33.3

Contact with cats

Yes

22

18

81.8

0.271

10

45.5

0.053

No

18

12

66.7

3

16.7

Consumption of under cooked meat

Yes

32

24

75

  1

11

34.4

0.613

No

8

6

75

2

25

Receiving

chemotherapy 

Yes

30

23

76.7

 0.673

8

26.7

0.172

No

10

7

70

5

50

Blood transfusion

Yes

22

18

81.8

 0.271

8

34.6

0.564

No

18

12

66.7

5

27.8

Malignancy condition

Acute

31

22

71

 

0.274

10

32.3

0.952

Chronic

9

8

88.9

3

33.3

p values were calculated by Chi-square test