CLINICAL RESEARCH
Pre-Transplant Toxoplasma gondii Seropositivity Among Heart Transplant Recipients Is Associated With an Increased Risk of All-Cause and Cardiac Mortality
Satish Arora, MD*,*,
Pål A. Jenum, MD, PhD ,||,
Pål Aukrust, MD, PhD ,
Halvor Rollag, MD, PhD,¶,
Arne K. Andreassen, MD, PhD*,
Svein Simonsen, MD, PhD*,
Einar Gude, MD*,
Arnt E. Fiane, MD, PhD ,
Odd Geiran, MD, PhD,¶ and
Lars Gullestad, MD, PhD*
* Department of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway
Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway
Section of Clinical Immunology and Infectious Diseases, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway
Department of Thoracic and Cardiovascular Surgery, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway
|| Department of Microbiology, Central Laboratory, Hospital of Asker and Baerum, Oslo, Norway
¶ Faculty Division Rikshospitalet, University of Oslo, Oslo, Norway
Manuscript received April 10, 2007;
revised manuscript received June 22, 2007,
accepted July 30, 2007.
* Reprint requests and correspondence: Dr. Satish Arora, Department of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center, N-0027 Oslo, Norway. (Email: satish.arora{at}medisin.uio.no).
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Abstract
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Objectives: We evaluated the risk of mortality, development of cardiac allograft vasculopathy (CAV), and acute cellular rejection among Toxoplasma gondii (T. gondii) seropositive heart transplant (HTx) recipients and the 4 donor/recipient seropairing groups.
Background: Chronic T. gondii infection is known to trigger potentially adverse immunoregulatory changes, but the long-term implication for HTx recipients has not been assessed previously.
Methods: Frozen pre-HTx serum samples of 288 recipients and 246 donors were evaluated for T. gondii serostatus using Platelia immunoglobulin G immunoassay. Patients had undergone prospective serotesting using alternative assays, and results determined by the 2 methods were compared. Data regarding mortality, CAV, and acute cellular rejection were available for all patients.
Results: Overall, 211 recipients (73%) were seronegative and 77 (27%) were seropositive. In total, 82 recipients died, 76 developed CAV, and 82 had 1 or more episode of treated cellular rejection. Recipient seropositivity was associated with a significantly higher risk of all-cause (hazard ratio [HR] 1.9, 95% confidence interval [CI] 1.1 to 3.4; p = 0.02) and CAV mortality (HR 4.4, 95% CI 1.3 to 15.6; p = 0.02) and a higher risk of developing advanced CAV (HR 2.7, 95% CI 1.2 to 5.8; p = 0.01). Seropositivity did not influence the number of rejection episodes, and donor/recipient seropairing was not a risk factor for any end point.
Conclusions: T. gondii seropositivity among HTx recipients is associated with an increased risk of all-cause and CAV mortality and of development of advanced CAV. This may be mediated via immunoregulatory changes triggered by chronic T. gondii infection and needs to be explored further.
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Abbreviations and Acronyms
| | CAV = cardiac allograft vasculopathy | | CMV = cytomegalovirus | | EIA = enzyme immunoassay | | HTx = heart transplant/transplantation | | IFN = interferon | | IL = interleukin | | R = recipient | | T. gondii
= Toxoplasma gondii |
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Long-term survival after heart transplantation (HTx) is limited by the development of cardiac allograft vasculopathy (CAV), and chronic inflammation and immune activation are likely to play an important pathogenic role (1). Potential triggers for this response include persistent alloantigen-dependent reactions, but chronic low-grade infection may also be involved and there has been considerable interest regarding possible microbial triggers, such as cytomegalovirus (CMV) infection (2). Toxoplasma gondii (T. gondii) is a lifelong infection, and, although acute infection has been recognized as a clinical problem in HTx recipients, the literature is virtually devoid of data regarding the implication of chronic infection and associated immunoregulatory changes for prognosis in HTx recipients.
Primary T. gondii infection is largely asymptomatic in healthy individuals, and chronic parasite latency is maintained by an adaptive T-cell response particularly involving interleukin (IL)-12–driven interferon (IFN)- responses (3). Although a balanced immune response is required to control T. gondii reactivation, it is increasingly appreciated that an excessively vigorous response can lead to pathologic effects, including endothelial cell activation, and this may be particularly relevant in the setting of organ transplantation and immunosuppression (4). Therefore, we examined the association between recipient T. gondii seropositivity and mortality, development of CAV, and acute cellular rejection among HTx patients. We also determined whether donor and recipient T. gondii seropairing was related to the occurrence of these end points.
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Methods
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Patient serologic testing.
All HTx donors and recipients at our center have undergone routine prospective evaluation for T. gondii serostatus using commercially available enzyme immunoassays (EIAs). These analyses were performed with different methods and different lot numbers, and this could potentially result in diagnostic inaccuracy. Therefore, we retested serum samples of 288 recipients who underwent HTx between January 1994 and December 2005. The study protocol was approved by the institutional review board.
Serum samples had been obtained within 24 h before HTx (stored at –20°C) and were tested using an established immunoglobulin (Ig)G EIA (Platelia Toxo IgG TMB, Bio-Rad, Marnes-la-Coquette, France) with the same lot number. An EIA result of 6.0 IU/ml was used as a cutoff for the diagnosis of T. gondii seropositivity, according to the manufacturer's instructions and previously published data (5). Donor serum samples were available for 246 of these patients and were also retested using the same technique.
Prophylaxis and immunosuppressive regime.
All patients received prophylactic treatment for Pneumocystis jirovecii with trimethoprim-sulfamethoxazole for the first 6 months after HTx. No alternative prophylactic strategies were used. Triple immunosuppressive therapy was given according to local protocol, and no cytotoxic induction therapy was used.
Definition of end points.
mortality
Survival data was obtained on all patients using mortality information from the Norwegian Population Register. Cause of death was considered to be due to CAV based on 1 or more of the following: 1) clinical history, examination, and investigation findings consistent with the diagnosis of myocardial infarction/heart failure as cause of death in a patient known to have significant CAV; 2) sudden death occurring in a patient known to have significant CAV; or 3) autopsy findings confirming CAV as cause of death.
cardiac allograft vasculopathy
All patients had been evaluated annually by coronary angiography. The classification used by Costanzo et al. (6) was applied to grade the CAV as mild, moderate, or severe on the basis of left main stem involvement, primary vessel stenoses, and branch stenoses.
acute cellular rejection
Results of endomyocardial biopsies performed during the first year after HTx were obtained for all patients. Biopsies had been performed upon clinical indication and by protocol at the following intervals: weekly the first 2 months after HTx, biweekly in the third month, and then after 6 and 12 months.
Statistical analysis.
Analysis was performed with the SPSS statistical software (version 13.0, SPSS Inc., Chicago, Illinois), and a 2-sided p value of <0.05 was considered to be statistically significant. Student t test was used for normally distributed variables and the Mann-Whitney test for other variables. Categorical variables were compared using the chi-square test. Separate Kaplan-Meier analyses with log-rank test were performed for the end points mortality and CAV. When evaluating the latter end point, patients who had died without CAV development were considered to be censored cases. A secondary analysis was performed to assess the relationship between seropositivity and CAV death, and in this analysis deaths not due to CAV were considered to be censored. Variables with a p value of <0.05 upon univariate analysis were included in the final multivariate regression analysis with the forward stepwise method.
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Results
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Baseline characteristics of the 288 patients are shown in Table 1. Serum testing with Platelia Toxo IgG found 211 patients (73%) to be T. gondii seronegative and 77 patients (27%) to be T. gondii seropositive at the time of HTx. Of the 211 seronegative patients, 209 had also been diagnosed as seronegative by the initially used assays, indicating a 99% concordance rate. Of the 77 recipients identified as seropositive by Platelia Toxo IgG, 70 had been diagnosed as seropositive by initial assays (91% concordance). There was no significant differences in the immunosuppressive therapy between T. gondii seropositive and seronegative patients (Table 1).
Donor serum was available for 246 recipients, and 199 (81%) were T. gondii seronegative and 47 (19%) seropositive. The 4 donor (D) and recipient (R) T. gondii seropairing groups had the following frequencies: D–/R–: n = 145 (59%); D+/R–: n = 36 (15%); D–/R+: n = 54 (22%); and D+/R+: n = 11 (4%). Recipients in the D+/R– group were potentially at risk of seroconversion after HTx, but analysis of post-HTx samples (collected from recipients during the first year after HTx) revealed only 1 case of seroconversion. No patients developed acute toxoplasmosis infection during the follow-up period.
Recipient T. gondii seropositivity and mortality.
Median follow-up time for mortality was 5.5 (range 0 to 13.0) years, and 82 patients (28%) died during this period. There were 51 (24%) and 31 (40%) deaths among T. gondii seronegative and seropositive recipients, respectively (p = 0.018) (Fig. 1). As shown, the survival curves started to diverge at approximately 4 years after HTx, indicating a predominantly higher risk of long-term mortality among seropositive recipients. The increased mortality observed among T. gondii seropositive recipients was predominantly attributable to higher incidence of CAV mortality among this group (Table 2). Multivariate Cox regression analysis identified recipient T. gondii seropositivity as an independent predictor of mortality beyond 1 year after HTx, with an adjusted hazard ratio (HR) of 1.9 (95% confidence interval [CI] 1.1 to 3.4; p = 0.02) (Table 3). When considering only CAV mortality, recipient T. gondii seropositivity was an independent risk factor with an adjusted HR of 4.4 (95% CI 1.3 to 15.6; p = 0.02) (Table 4).
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Figure 1 Survival According to Recipient T. gondii Serostatus at Time of Heart Transplantation
Toxoplasma gondii (T. gondii) seropositive recipients had a significantly higher risk of all-cause mortality, and this was predominantly evident in the period beyond 4 years after transplantation.
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Donor T. gondii seropositivity and mortality.
Of 246 donors, 199 (81%) were T. gondii seronegative and 47 (19%) were seropositive. There were 60 (30%) and 13 (28%) deaths in these 2 groups, respectively (p = 0.74). When considering donor and recipient seropairing status, there were 37 (26%), 7 (19%), 23 (43%), and 6 (55%) deaths in D–/R–, D+/R–, D–/R+, and D+/R+ groups, respectively. When using D–/R– as the reference group, higher mortality was evident among both D–/R+ and D+/R+ groups (p = 0.04 and p = 0.01, respectively). However, when comparing D–/R+ with D+/R+, there was no significant difference in mortality (p = 0.10), indicating that donor T. gondii status did not influence mortality. Multivariate analysis with seropairing status and donor and recipient serostatus as 3 separate variables confirmed that only recipient T. gondii seropositivity was an independent predictor of mortality (p < 0.01).
T. gondii seropositivity and CAV.
Median follow-up time for CAV was 3.9 (range 0 to 13.0) years, and 76 patients (26%) developed CAV. Kaplan-Meier analysis indicated that T. gondii seropositive recipients were not at significantly higher risk of earlier CAV development (p = 0.10). During the study period, 20 recipients developed moderate CAV and 6 developed severe CAV. Among T. gondii seropositive recipients, 12 patients (16%) developed moderate/severe CAV compared with only 14 seronegative recipients (7%; p = 0.01) (Fig. 2). Multivariate analysis confirmed recipient T. gondii seropositivity as a risk factor for development of advanced CAV, with an adjusted HR of 2.7 (95% CI 1.2 to 5.8; p = 0.01). There was no significant difference regarding CAV incidence or progression among the 4 D/R seropairing groups. Similarly, statin therapy and CMV infection did not influence the risk of CAV development or progression (data not shown).
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Figure 2 Population Free From Moderate/Severe CAV According to Recipient T. gondii Serostatus
Toxoplasma gondii (T. gondii) seropositive recipients had a significantly higher risk of developing advanced CAV. Deaths without development of moderate/severe CAV and patients with only mild CAV are considered as censored. CAV = cardiac allograft vasculopathy.
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T. gondii seropositivity and acute cellular rejection.
During the first year after HTx, 19 T. gondii seropositive recipients (25%) had at least 1 episode of treated cellular rejection (grade 2R or 3R) compared with 63 seronegative recipients (30%; p = 0.39). Similarly, 4 seropositive recipients (5%) had more than 1 episode of treated cellular rejection compared with 13 seronegative recipients (6%; p = 0.76). There were 3 (4%) and 11 (5%) episodes of vascular rejection among seropositive and seronegative patients, respectively (p = 0.65). Similarly, there was no significant difference in the number of treated rejection episodes among the 4 D/R seropairing groups (data not shown).
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Discussion
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The present de novo study demonstrated that recipient T. gondii seropositivity is associated with a significantly increased risk of mortality, primarily reflecting a higher risk of CAV-related death. Recipient seropositivity is also a risk factor for developing more advanced CAV, as shown by serially performed angiographic examinations. In contrast, donor seropositivity and D/R seropairing status are not risk factors for mortality or CAV.
The relationship between recipient T. gondii seropositivity, reflecting chronic T. gondii infection, and all-cause and CAV mortality may have several explanations. First, chronic T. gondii infection is associated with an enhanced IL-12–driven IFN- response by T cells, and T-cell–mediated IFN- responses seem to play an important pathogenic role in CAV (7). It has also been shown that suppression of IFN- correlates well with prevention of CAV (8). Second, recent studies suggest that IL-17–producing T helper cells (TH17) may promote autoimmunity in various organs and are implicated in reduced graft survival (9), and, notably, T. gondii parasites have been shown to induce TH17-mediated responses at least partly involving IL-6 and transforming growth factor ß-related mechanisms (10). Finally, the vascular pathology that underlies CAV appears to be initiated by endothelial cell activation that in turn may promote a chronic immune response in the vessel wall, and with relevance to these data T. gondii has been shown to enhance endothelial cell activation through IFN-–related mechanisms (11). Although speculative, we suggest that our findings of a higher risk of total and in particular of CAV-related mortality in T. gondii seropositive recipients may be related to changes in the immunologic milieu in these patients, including an adaptive T-cell response with increased levels of IFN-, a key mediator in the pathogenesis of CAV. However, whether T. gondii infection is an important trigger of these immune responses in HTx will need to be explored further with in-depth studies.
The present study has also demonstrated that T. gondii seropositive recipients are at higher risk of developing advanced CAV. This is in concordance with the observed relationship regarding seropositivity and CAV mortality. Nevertheless, intravascular ultrasound would allow a more detailed assessment of CAV and should be used in future studies measuring the inflammatory response in T. gondii seropositive and seronegative recipients.
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Conclusions
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These findings suggest that T. gondii seropositivity among HTx recipients is associated with a significantly increased risk of all-cause and cardiac mortality as well as the development of advanced CAV. The observed higher risk of mortality and advanced CAV among seropositive recipients may be mediated via immunoregulatory changes triggered by chronic T. gondii infection and needs to be explored further.
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Footnotes
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Supported by an unrestricted grant from Inger and John Fredriksen to the Department of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway.
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References
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1. Hognestad A, Endresen K, Wergeland R, et al. Plasma C-reactive protein as a marker of cardiac allograft vasculopathy in heart transplant recipients J Am Coll Cardiol 2003;42:477-482.[Abstract/Free Full Text]2. Hussain T, Burch M, Fenton MJ, et al. Positive pretransplantation cytomegalovirus serology is a risk factor for cardiac allograft vasculopathy in children Circulation 2007;115:1798-1805.[Abstract/Free Full Text] 3. Scharton-Kersten TM, Wynn TA, Denkers EY, et al. In the absence of endogenous IFN-gamma, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection J Immunol 1996;157:4045-4054.[Abstract] 4. Koh KP, Wang Y, Yi T, et al. T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase J Clin Invest 2004;114:846-856.[CrossRef][Web of Science][Medline] 5. Jenum PA, Kapperud G, Stray-Pedersen B, Melby KK, Eskild A, Eng J. Prevalence of Toxoplasma gondii specific immunoglobulin G antibodies among pregnant women in Norway Epidemiol Infect 1998;120:87-92.[CrossRef][Medline] 6. Costanzo MR, Naftel DC, Pritzker MR, et al. Heart transplant coronary artery disease detected by coronary angiography: a multiinstitutional study of preoperative donor and recipient risk factorsCardiac Transplant Research Database. J Heart Lung Transplant 1998;17:744-753.[Web of Science][Medline] 7. van Loosdregt J, van Oosterhout MF, Bruggink AH, et al. The chemokine and chemokine receptor profile of infiltrating cells in the wall of arteries with cardiac allograft vasculopathy is indicative of a memory T-helper 1 response Circulation 2006;114:1599-1607.[Abstract/Free Full Text] 8. Yi T, Cuchara L, Wang Y, et al. Human allograft arterial injury is ameliorated by sirolimus and cyclosporine and correlates with suppression of interferon-gamma Transplantation 2006;81:559-566.[CrossRef][Web of Science][Medline] 9. Antonysamy MA, Fanslow WC, Fu F, et al. Evidence for a role of IL-17 in alloimmunity: a novel IL-17 antagonist promotes heart graft survival Transplant Proc 1999;31:93.[CrossRef][Web of Science][Medline] 10. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17–producing T cells Immunity 2006;24:179-189.[CrossRef][Web of Science][Medline] 11. Woodman JP, Dimier IH, Bout DT. Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replicationInhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. J Immunol 1991;147:2019-2023.[Abstract]
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Abstract
Methods