A case-control study of the association of toxoplasmosis with antirheumatic therapy in rheumatoid arthritis

Rabie G. Abdullah 1, Souzan H. Eassa 2, Fouad K. Mohammad 3
Author affiliations:
  1. Rabie G. Abdullah, Department of Pharmacology, College of Pharmacy, University of Duhok, Duhok, Kurdistan Region, Iraq; E-mail: rabie.gabriel@uod.ac
  2. Souzan H. Eassa, Department of Basic Sciences, College of Medicine, University of Duhok, Duhok, Kurdistan Region, Iraq; Molecular and Microbiology Division, School of Medicine, University of Kurdistan Hewlêr, Erbil, Iraq; E-mail: souzan.hussain@ukh.edu.krd
  3. Fouad K. Mohammad, Department of Physiology, Biochemistry, and Pharmacology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq; College of Nursing, American University of Kurdistan, Duhok, Kurdistan Region, Iraq; E-mail: fouadmohammad@yahoo.com
Correspondence: Fouad K. Mohammad, E-mail: fouadmohammad@yahoo.com
 

ABSTRACT

 

Background & Objective: Toxoplasmosis is a globally prevalent opportunistic zoonotic infection. Rheumatoid arthritis (RA) is an autoimmune disease which is manifested as joint inflammation, pain, stiffness, and swelling. The patients may contract toxoplasmosis, especially when receiving antirheumatic immunosuppressive therapy. However conflicting results have been reported on toxoplasmosis in RA patients. We aimed to evaluate Toxoplasma gondii positivity and associated risk factors in patients with RA in Duhok province, Iraq, and to assess the role of antirheumatic therapy in toxoplasmosis and the possibility of its reactivation.

Methodology: This case-control study was carried out in Duhok, Kurdistan Region, Iraq, from February 2022 to June 2023. A total of 88 RA patients, with and without concurrent antirheumatic therapy, were recruited. The diagnosis of RA was according to the 2010 American College of Rheumatology/European League. RA-patients were categorized into three groups: Group 1 - no-therapy (n = 14); Group 2 - conventional therapy (n = 49) and Group 3 - biologics in combination with conventional medications (n = 25). Healthy subjects (n = 61) were also selected as controls. Plasma samples obtained from the participants were screened for T. gondii infection using Human anti-Toxoplasma gondii antibody IgG. IgG positive samples were tested utilizing human anti-Toxoplasma gondii antibody IgM ELISA Kit and the avidity T. gondii IgG ELISA kit was used to determine the state of infection.

Results: Of the 88 RA patients, with or without antirheumatic therapy, 76 (86.36%) were females and 12 (13.64%) were males. Patients on therapy (n = 74) received several conventional antirheumatic drugs of which methotrexate was the most frequently used one (86.36%). Among biological therapies, etanercept, a TNF-alpha inhibitor, was mostly used (13.51%).

The toxoplasmosis was significantly less in the older age group (> 50 y) compared to individuals under 30 y. Gender had no significant effect. Toxoplasma IgG positivity was found to be significantly higher in the three groups of RA patients in comparison with healthy controls. Further assessment of anti-Toxoplasma IgM positivity and Toxoplasma IgG avidity results to identify the state of T. gondii infection in anti-Toxoplasma IgG positive subjects revealed that the frequency of reactivation of toxoplasmosis in RA patient groups was not significantly different from that of the control counterparts.

Conclusion: The study showed higher seroprevalence of anti-Toxoplasma antibodies in patients with RA irrespective of therapy; therefore, we could consider toxoplasmosis interaction with RA in the pathogenesis of both diseases. This seroprevalence was higher in younger age groups, but with no significant role of gender, duration of disease, duration of therapy and its type. No significant difference was noted in rates of reactivation of latent T. gondii infection among RA patients and healthy controls.

Abbreviations: CI - Confidence interval; O - Odds ratio; RA - Rheumatoid arthritis; SD - Standard deviation; T. gondii - Toxoplasma gondii; TNF - Tumor necrosis factor.

Keywords: Autoimmune disease; Rheumatoid arthritis; Antirheumatic drugs; Toxoplasma gondii; Toxoplasma seroprevalence; Toxoplasma IgG avidity.

Citation: Abdullah RG, Eassa SH, Mohammad FK. A case-control study of the association of toxoplasmosis with antirheumatic therapy in rheumatoid arthritis. Anaesth, pain intensive care 2024;28(4):674−680; DOI: 10.35975/apic.v28i4.2510
Received: May 14, 2024; Reviewed: June 28, 2024; Accepted: June 28, 2024

 

1. INTRODUCTION

 

Toxoplasmosis is a globally prevalent, opportunistic protozoan, zoonotic infection. It is caused by the obligate intracellular protozoan parasite known as Toxoplasma gondii (T. gondii).1 All warm-blooded animals are susceptible to infection by the parasite that causes chronic infection in about one-third of the human population.1,2 Human infection usually occurs through contact with contaminated cat feces or consuming inadequately cooked meat derived from an infected animal.1,2
Rheumatoid arthritis (RA) is an autoimmune disease, which is manifested as joint inflammation, resulting in signs and symptoms that include pain, stiffness, swelling, and reduced functionality, with substantial morbidity and diminished quality of life of the affected individuals.3 The age-standardized global prevalence rate of RA was estimated to be 208.8 cases per 100,000 population, with a higher preponderance in females (82.76%) compared to males (17.24%).4 In Iraq, the prevalence was estimated to be 104.5 per 100,000 population,4 with higher rates in females compared to males.5,6 The latter study was a preliminary one we conduced to examine plasma cholinesterase activity in RA patients with toxoplasmosis.6
The primary focus of dealing with RA, in addition to an early diagnosis, is to effectively manage pain and inflammatory responses, improve mobility, and minimize the risk of joint degeneration and deformity.3,7 Major therapeutic options against RA include conventional antirheumatic drugs and biologic agents.3,7,8 However, the administration of immunosuppressive medications, including both conventional and biologic therapies, has been associated with an elevated vulnerability to opportunistic infections like tuberculosis, histoplasmosis, and toxoplasmosis.9,10 Within this context, the association between toxoplasmosis and RA is complex and largely unclear from the pathophysiological aspects of the disease.10,11
Severe forms of toxoplasmosis may occur in RA patients who receive immunosuppressive therapy, especially the anti-tumor necrosis factor (anti-TNF) medications.12,13 However, in spite of reports of toxoplasmosis and/or RA in Iraqi patients a few studies addressed the role of antirheumatic immunosuppressive therapy, though with conflicting results,5,6,14-16 regarding the possible association of toxoplasmosis with RA.11 This research aimed to further expand our previous study6 and evaluate T. gondii positivity and associated risk factors in patients with RA in Duhok province, Iraq, and to assess the role of antirheumatic therapy in toxoplasmosis and the possibility of its reactivation.

 

2. METHODOLOGY

 

This case-control study was carried out in Duhok, Kurdistan Region, Iraq, from February 2022 to June 2023. The aims and significance of the study were explained to all participants and signed informed consents were obtained from them. The study received approval from the Committee of Higher Studies, College of Pharmacy, University of Duhok, Iraq (No. 320, 4/8/2021) and the final approval from the Research Ethics Committee, Duhok Directorate General of Health, and registered with the reference number 15092021-9-14 R1, August 7, 2023

2.1. Inclusion and exclusion criteria
We recruited 88 RA patients, with and without concurrent antirheumatic therapy, from the Duhok Center of Rheumatic Diseases & Medical Rehabilitation, Duhok, Iraq. The patients had been diagnosed by rheumatologists based on the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria.17 Patients with moderate to severe comorbidities were excluded from the study. Pregnant women and patients with past surgical procedures on the joints were also excluded from the study.

2.2. Patients groups
RA patients were categorized into three groups: RA patients who did not receive therapy up to the time of the interview (RA Group 1, n = 14), and those who received conventional therapy including methotrexate, corticosteroids, and other disease-modifying antirheumatic drugs (RA Group 2, n = 49), and the last group comprised patients who were given biologics (such as infliximab, adalimumab, etanercept) mostly in combination with conventional medications (RA Group 3, n = 25). Healthy subjects (n = 61) were selected as controls from the staff of the rheumatic center, and from blood donors who attended Duhok blood bank.

2.3. Blood sampling / T. gondii seroprevalence assays
About 5 mL of venous blood samples were obtained from the patients and controls and they were collected into heparinized vacutainer tubes. Plasma was separated by centrifuging and stored at –20°C pending analysis within one month. All samples were screened for T. gondii infection using Human anti-Toxoplasma gondii antibody IgG ELISA Kit (Cat. No ED0537Hu, BT LAB Co., China). IgG positive samples were tested utilizing Human anti-Toxoplasma gondii antibody IgM ELISA Kit (Cat. No ED0538Hu, BT LAB Co., China) and the avidity T. gondii IgG ELISA kit (NovaLisa®, Dietzenbach, Germany) was used to determine the state of infection (whether the infection was current, past, or a reactivation one) according to the manufacturer’s instructions.

2.4. Statistical analysis
Continuous data are shown as mean and standard deviation (SD), and categorical data are presented as numbers and percentages. The Odds ratio (OR) with 95% confidence interval (CI) were estimated by binary logistic regression with backward selection of variables at P < 0.2.18 Fisher's exact probability test was performed to compare the state of infection among groups. The statistical package Minitab software (Version 21.4.1, Minitab Inc., PA, USA), as well as the statistical software PAST4.1319 were used for statistical analyses. A P < 0.05 was considered statistically significant.

 

3. RESULTS

 

This study included 88 RA patients with or without antirheumatic therapy. Seventy-six (86.36%) patients were females and 12 (13.64%) were males. Their mean age was 44.51 ± 13.404 y, and their mean body mass index (BMI) was 30.463 ± 7.554 kg/m2 (Table 1).

 

Table 1: Demography of rheumatoid arthritis (RA) patients and healthy controls
Variables RA patients
(n = 88)		 Healthy controls
(n = 61)		 p-value
Gender
·        Female 76 (86.36) 22 (36.07) 0.0001
·        Male 12 (13.64) 39 (63.93)
Age (y) 44.51 ± 13.404 37.21 ± 9.91 0.0001
BMI (kg/m2) 30.463 ± 7.554 27.88 ± 4.538 0.015
Data presented as mean ± SD or n (%)
 

Table 2: Medications used by 74 rheumatoid arthritis (RA) patients. [n (%)]
Medications RA patients received therapy  
A. Conventional agents
  ·    Methotrexate 56 (75.68)
  ·    Prednisolone 8 (10.81)
  ·    Hydroxychloroquine 3 (4.05)
  ·    Azathioprine 1 (1.35)
  ·    Deflazacort 1 (1.35)
  ·    Sulfasalazine 1 (1.35)
B. Biological agents
  ·    Etanercept 10 (13.51)
  ·    Infliximab 6 (8.11)
  ·    Tocilizumab 5 (6.76)
  ·    Adalimumab 2 (2.70)
  ·    Rituximab 2 (2.70)
 

Patients on antirheumatic therapy (n = 74) received several conventional antirheumatic drugs of which methotrexate was the most frequently utilized one (n = 56, 86.36%). Among biological therapies, etanercept, a TNF-alpha inhibitor, was mostly used (n = 10, 13.51%) (Table 2).

The frequency of occurrence of toxoplasmosis was significantly lesser in the older age group (> 50 y) when compared to individuals under 30 y of age (OR, 0.11; 95% CI, 0.03–0.49; P = 0.004) (Table 3). Within this context, gender had no statistically significant effect.

Toxoplasma IgG positivity was found to be significantly higher in RA patients (n = 14) without therapy (OR, 14.01; 95% CI, 2.82–69.55; P = 0.001), RA patients (n = 49) who received conventional therapy (OR, 5.71; 95% CI, 1.67–19.52; P = 0.006), and those (n = 25) with combination biologic and conventional therapy (OR, 3.90; 95% CI, 1.10–13.86; P = 0.035) in comparison with healthy controls (Table 3).

Upon further assessment of anti-Toxoplasma IgM seroprevalence and Toxoplasma IgG avidity results, we were able to identify the state of T. gondii infection in anti-Toxoplasma IgG-positive subjects (Table 4). As total numbers of T. gondii-infected patients varied from 8 to 17 among the tested groups, current and past infections also varied accordingly from 0 to 3 and 3 to 8,

 

Table 3: Association of toxoplasmosis with demographics in rheumatoid arthritis (RA) patients (n = 88), and non-RA healthy controls (n = 61)
Variables Frequency Toxoplasma IgG positive n (%) Toxoplasma IgG negative n (%) Odds ratio
(95% C.I.)		 P-value
Age group
< 30 y 29 15 (51.7) 14 (48.3) 1.00 (reference) -
30-50 y 85 27 (31.8) 58 (68.2) 0.37 (0.13, 1.12) 0.078
> 50 y 35 9 (25.7) 26 (74.3) 0.11 (0.03, 0.49) 0.004
Gender
Female 98 37 (37.8) 61 (62.2) 1.00 (reference) -
Male 51 14 (27.5) 37 (72.5) 0.62 (0.20, 1.90) 0.406
Participants
Control 61 16 (26.2) 45 (73.8) 1.00 (reference) -
RA group 1 14 8 (57.1) 6 (42.9) 14.01 (2.82, 69.55) 0.001
RA group 2 49 17 (34.7) 32 (65.3) 5.71 (1.67, 19.52) 0.006
RA group 3 25 10 (40.0) 15 (60.0) 3.90 (1.10, 13.86) 0.035
RA Group 1 = RA patients without therapy, RA Group 2 = RA patients received conventional therapy, RA Group 3 = RA patients received combination therapy (biologic agent + conventional drugs)
 

Table 4: State of T gondii infection in anti-Toxoplasma IgG positive subjects
Groups Current infection
n (%)		 Past infection
n (%)		 Reactivation
n (%)		 Undefined
n (%)		 Total
Control 2 (12.50) 5 (31.25) 5 (31.25) 4 (25.00) 16
RA Group 1 3 (37.50) 3 (37.50) 0 (0.00) 2 (25.00) 8
RA Group 2 3 (17.65) 8 (47.06) 3 (17.65) 3 (17.65) 17
RA Group 3 0 (0.00) 5 (50.00) 2 (20.00) 3 (30.00) 10
No statistically significant differences among groups.
RA Group 1 = RA patients without therapy, RA Group 2 = RA patients received conventional therapy, RA Group 3 = RA patients received combination therapy (biologic agent + conventional drugs)
 

respectively (Table 4). Most importantly and within the context of the state of infection, the results revealed that the frequency of reactivation of toxoplasmosis in RA patient groups was not significantly different among themselves and from that of the control counterparts. To this end, as shown in Table 4, the percentages of reactivation, which were of low values, ranged from 0% to only 3% in RA patients when compared to that of the control group (5%).

 

4. DISCUSSION

 

The present study aimed to assess the seroprevalence of T. gondii infection among individuals diagnosed with RA in Duhok, Iraq. The high percentage of RA in female Iraqi patients from Duhok was in agreement with those reported locally in Iraq or globally.4-6,15 However, other studies did not report gender effects on the occurrence of RA.11,16,20 The types of antirheumatic therapy recorded in the present study are in accordance with recent RA findings in Duhok, however, with varying adherence rates to medications.15 The main reason to start antirheumatic treatments is to stop the progress of RA and improve the quality of life of the patients.3-5,7,8
A unique finding of the present study showed that the rates of toxoplasmosis in patients with RA who received no treatment, conventional therapy were considerably higher than that of the controls (26.2%). To this end, gender seemed not to significantly affect this frequency in RA patients. Besides, regarding toxoplasmosis frequency, no statistically significant differences were found among the subgroups of RA patients, and there was no evidence of association between the RA duration, or the duration of therapy with the seroprevalence of anti-T. gondii IgG (data not shown in the results). Therefore, the impact of duration of RA therapy on the occurrence of toxoplasmosis needs additional in-depth exploration.

Our results are in accordance with those of Kuba et al.16 who demonstrated significantly high prevalence of toxoplasmosis among Iraqi RA patients receiving methotrexate (33.3%) and RA group without treatment (36%) when compared to control group (12%). Similarly, earlier studies from Egypt and Turkey, found a statistically greater frequency of toxoplasmosis in patients with RA versus healthy controls, without significant dissimilarity among patients receiving conventional antirheumatic medications and those treated with a biologic agent.9,21
Several studies conducted on RA patients also concluded greater frequency of T. gondii seropositivity in comparison to healthy participants without referring to therapy received or its impact on toxoplasmosis.11,22,23 In addition, others assessed the influence of RA duration or the age of the patients on the prevalence of toxoplasmosis without significant outcome.11,20,24
While some studies revealed no significant association of age with the prevalence of toxoplasmosis among RA patients;11,23,25 in contrast we report significantly lesser frequency in the older age group (> 50 y). Our results are in agreement to those of others.26,27 However, the latter age effect on the seropositivity of toxoplasmosis has been reported earlier without any correlation with the pathogenesis of RA.24 The present younger age-related finding of toxoplasmosis could be attributed to increased frequency of cat ownership (the primary source of toxoplasmosis1,2) in young individuals in our local community.28 Nevertheless, autoimmunity status of RA patients, being immunocompromised, might be a factor in the prevalence of toxoplasmosis among them.10,12,13
Various studies have postulated various hypotheses to explain the high prevalence of toxoplasmosis in patients with chronic autoimmunity conditions like RA. The protozoan T. gondii may trigger a strong and long-lasting cellular response mediated by T helper cells, a response which is characterized by the production of many pro-inflammatory cytokines that help in defending the host’s cells against the parasite, but at the same time, induce significant pathological and immunological alterations, ultimately leading to an autoimmune condition.1,10,29 It is also possible that immunosuppressive therapy for RA, such as TNF-alpha antagonists, cause dormant toxoplasmosis to be reactivated in patients and increase their susceptibility to opportunistic infections.25 T. gondii protozoon upregulates the expression of interleukin-17 in infected people; this cytokine has a role in the development of several autoimmune disorders, hence establishing a notable connection between toxoplasmosis and autoimmune illnesses.30 The increased vulnerability of individuals with RA toward opportunistic infections may also be elucidated by the pathobiology of the illness itself. The premature degeneration of the immune system in immunological-mediated disorders like RA may contribute to the impairment of crucial immune processes, resulting in reduced protection against infectious parasites.30
The present study employed human anti-T. gondii antibody IgM ELISA test and avidity T. gondii IgG ELISA test in Toxoplasma IgG positive samples to identify the state of infection, particularly, the reactivation one. The results showed non-significant difference in the rate of Toxoplasma reactivation among RA groups themselves and when they were compared to healthy controls. This notion was supported by low percentages of reactivation, which were 0% to 3% in RA patients versus the 5% of the control group. A similar study also reported no significant differences in Toxoplasma seropositivity among RA treatment and non-treatment groups.16
Nevertheless, the presence of anti-Toxoplasma IgM antibodies in serum of T. gondii IgG positive individuals may indicate a recent infection, but not necessarily an activation of latent toxoplasmosis. The application of T. gondii IgG avidity test, as we did in the present study, is a crucial attempt to differentiate recent toxoplasmosis from reactivation of latent infection.14,31 However, limited number of patients in each RA group in the present study, may have contributed to this non-significant reactivation outcome. Furthermore, we could not delineate false negative expression of anti-Toxoplasma IgG among the RA groups, since it was reported that such a phenomenon can find its way in patients, especially the elderly, with dysregulation of immunity.11-13,32,33
 

5. LIMITATIONS

 

In the present study, the sample size of RA patients without therapy was small (n = 14). However, this is anticipated due to the need for intense and prompt therapeutic action as existing joint erosions cannot be reversed, given that the welfare of the patients is contingent upon the effectiveness of treatment, along with other variables that impact the course of the disease6-7
This may explain the lack of such a group (RA patients with no therapy) in the majority of similar studies mentioned previously with few exceptions.16,20 False negative results for anti-Toxoplasma IgG can also occur, though uncommon, in people with hypogammaglobulinemia or severe immunosuppression, especially in elderly.

Another obscure point that needs additional exploration is the relationship of toxoplasmosis to the duration of therapy in RA patients.

 

6. CONCLUSION

 

Our study showed higher seroprevalence of anti-Toxoplasma antibodies in patients with rheumatoid arthritis irrespective of therapy when compared to healthy individuals. Therefore, we should consider toxoplasmosis interaction with rheumatoid arthritis in the pathogenesis of both diseases.

This seropositivity was noted to be higher in younger age groups, but with no significant role of gender, duration of disease, duration of therapy and its type. No significant difference was noted in rates of reactivation of latent T. gondii infection among rheumatoid arthritis patients and healthy controls.

Additional research should be conducted to elucidate the significance of T. gondii infection in patients with rheumatoid arthritis and its relevance to antirheumatic therapy as well as the duration of such therapy. Despites these findings, the risk of reactivation of latent toxoplasmosis should not be ruled out, especially in patients receiving immunosuppressive medications. Hence, extra caution should be taken when prescribing these therapeutic agents in toxoplasma positive rheumatoid arthritis patients.

7. Data availability
The numerical data of this study is available with the first author.

8. Conflict of interests
The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.

9. Acknowledgments
This report represents a portion of a dissertation to be submitted by the first author to the University of Duhok, Iraq in partial fulfillment of the requirements for the Ph.D. degree in Clinical Pharmacology. The authors thank the College of Pharmacy, University of Duhok for providing facilities and supplies to conduct this study.

10. Authors’ contribution
RGA: Dealt with the participants, obtained blood samples and executed laboratory assays; conducted literature search, statistical analyses and shared in drafting the manuscript.

SHE & FKM: Conceptualized and supervised the study, shared in literature search, statistical analyses and drafting the manuscript. All authors have read the manuscript and approve it for publication.

 

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