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ORIGINAL CLINICAL SCIENCE
Trypanosoma cruzi persistence in the native heart is associated with high-grade myocarditis, but not with Chagas’ disease reactivation after heart transplantation Luiz A. Benvenuti, MD, PhD,a Alessandra Roggério, PhD,a Anna S. Nishiya, MS,b,c Silvia V. Campos, MD,a Alfredo. I. Fiorelli, MD, PhD,a and José E. Levi, PhDb From the aHeart Institute (InCor), University of São Paulo Medical School; the bFundação Pró-Sangue/Hemocentro of São Paulo, Molecular Biology Department; and cInfectious Disease Division (DIPA), Federal University of São Paulo, São Paulo, Brazil.
KEYWORDS: Chagas’ disease; Trypanosoma cruzi; heart transplantation; myocarditis; polymerase chain reaction
BACKGROUND: Chagas’ disease reactivation (CDR) after heart transplantation (HTx) is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. We investigated whether a detailed pathologic examination of the explanted heart at HTx with evaluation of myocarditis and parasitic persistence or load in the myocardium could be useful to identify patients at high risk of CDR. METHODS: The native hearts of 18 chagasic patients who presented CDR after HTx (CDRþ group) were compared with the native hearts of 16 chagasic patients who never presented CDR in a follow-up of at least 18 months after HTx (CDR– group). The intensity of myocarditis was evaluated semiquantitatively. Parasite persistence/load in the myocardium was investigated through immunohistochemistry for T cruzi antigens and by qualitative and quantitative real-time PCR for T cruzi DNA. RESULTS: The rate of high-grade myocarditis, parasite persistence, and the median of parasitic load and parasitic load/106 cells in the CDRþ group were 83.3%, 77.8%, 8.43 10–3, and 9.890, respectively, whereas in the CDR– group the values were 87.5%, 50%, 7.4910–3, and 17.800. There was no statistical difference between the groups. High-grade myocarditis was present in all 22 samples (100%) with parasite persistence and in 7 of 12 samples (58.3%) with no parasite persistence (p ¼ 0.003). CONCLUSIONS: Although associated with high-grade myocarditis, T cruzi parasite persistence in the myocardium of the native heart is not associated with the occurrence of CDR after HTx. J Heart Lung Transplant ]]]];]:]]]–]]] r 2014 International Society for Heart and Lung Transplantation. All rights reserved.
Human American trypanosomiasis (Chagas’ disease) is caused by infection with the hemoflagellate protozoan Trypanosoma cruzi. After a variable quiescent period, usually of many years, approximately 30% of infected Reprint requests: Luiz A. Benvenuti, MD, PhD, Heart Institute, University of São Paulo Medical School, Av. Dr. Enéas de Carvalho Aguiar 44, 05403–000 São Paulo-SP, Brazil. Telephone: þ55-11-26615080. Fax: þ55-11-2661-5279. E-mail address: [email protected]
people develop a chronic, inflammatory cardiomyopathy that may progress to ventricular systolic dysfunction.1,2 Heart transplantation (HTx) is a valid therapeutic option for end-stage heart failure due to Chagas’ disease.3,4 However, besides the usual morbidities related to HTx, patients can develop Chagas’ disease reactivation (CDR), a lifethreatening complication characterized by relapse of the infectious disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7
1053-2498/$ - see front matter r 2014 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2014.01.920
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CDR occurs in a significant proportion of HTx chagasic patients, usually in the first year of follow-up. Clinical studies have demonstrated that augmented immunosuppression increases the risk of CDR,5–7 probably due to a misbalance of the host-parasite relationship. In this study, we investigated retrospectively whether a detailed pathologic examination of the native, explanted heart at HTx, with evaluation of parasitic persistence/load in the myocardium, could be useful to identify patients at high risk of CDR.
Methods From March 1985 to December 2011, 112 patients with Chagas’ disease underwent HTx in our hospital. The disease was diagnosed from epidemiologic data and positive results on serologic tests. The immunosuppressive regimen included cyclosporine, azathioprine (changed to mycophenolate mofetil from 2001 to 2005), and steroids. As a routine in our HTx program, no patient received cytolytic induction therapy. Episodes of moderate, acute cellular rejection were treated with 3 days of methylprednisolone. Anti-thymocyte globulin was given for severe or persistent rejection or if associated with cardiac dysfunction. The diagnosis of CDR was based on direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues. The occurrence of CDR was monitored, and no prophylactic drug therapy was used. We selected for this study all patients who presented at least 1 episode of CDR up to 18 months after HTx (CDRþ group). Because the immunosuppressive regimen changed over time, we selected as controls those patients who underwent HTx at the same period as the CDRþ patients but who did not present CDR in a follow-up of at least 18 months after HTx (CDR– group). We analyzed retrospectively the pathologic report of the native heart explanted from each chagasic patient and selected for this study the largest paraffin-embedded sample of ventricular myocardium stored in our laboratory.
Histopathology One 4-μm-thick section of the paraffin-embedded sample was stained by hematoxylin and eosin (H&E). The slides were reviewed by 1 cardiac pathologist (L.A.B.) blinded to the patient group. Myocarditis was characterized by an inflammatory infiltrate adjacent to degenerating/necrotic myocytes according to the Dallas criteria8 and evaluated semiquantitatively. Myocarditis was defined as low-grade if there was mild, multifocal inflammatory infiltration with discrete foci of cardiomyocyte aggression and as high-grade if there was multifocal dense or diffuse inflammatory infiltration with multiple foci of cardiomyocyte aggression. We searched the slides for groups of amastigotes of T cruzi parasites in the cardiomyocytes (pseudocysts).
Immunohistochemistry for T cruzi antigens Immunohistochemistry (IHC) for T cruzi antigens was performed as previously reported,9 with minor modifications. Briefly, the sections underwent proteolytic digestion with 0.18 mg/ml trypsin T 7409 (Sigma-Aldrich, St Louis, MO) for 5 minutes at 371 C, incubated with rabbit polyclonal immune serum against T cruzi (Y strain) diluted 1:100,000 for 1 hour and MACH 4 (Biocare Medical, Concord, CA) for 30 minutes at room temperature. The reactions were developed with 3,3’-diaminobenzidine (Dako, Carpinteria, CA), and the slides were counterstained with hematoxylin. Myocardial sections previously known to contain pseudocysts of T cruzi parasites were used as positive controls.
Qualitative polymerase chain reaction for the detection of T cruzi DNA Qualitative polymerase chain reaction (PCR) was performed in sections of the paraffin blocks using the primers S34/S67, which amplify a 122-bp sequence localized within the minirepeat of the kinetoplast minicircles (kDNA).10 The DNA was extracted from three 8-μm sections of the paraffin blocks with QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions, and eluted in 200 μl. PCR was performed in a total volume of 25 μl containing PCR buffer 1, 1.5 mmol/liter MgCl2, 1.5 units Taq DNA polymerase (Invitrogen, São Paulo, Brazil), 200 μmol/liter deoxyribonucleotide triphosphate mixture, 1 μmol/liter each primer, and 1 μl extraction eluate. Reactions were denatured at 951C for 5 min and then underwent 45 cycles in a programmable thermal cycler (T3 Thermocycler; Biometra, Goettingen, Germany). Each cycle included 40 seconds at 951C, 1 minute at 571C, and 45 seconds at 721C, followed by a final incubation at 721C for 10 minutes. After thermal cycling, 10 μl each reaction mixture was analyzed using 2% agarose gel electrophoresis and visualized under ultraviolet light after ethidium bromide staining. Every sample was run at least in duplicate to obtain repetitive, concordant results. We used as positive control sections from a paraffinembedded endomyocardial biopsy specimen of a patient with CDR and T cruzi parasites in the myocardium. Negative controls included sections from paraffin blocks containing no tissue or myocardium of patients with idiopathic dilated or ischemic heart disease. We included in this study only samples in which the integrity of the extracted DNA could be checked through positive PCR amplification of T cruzi or human βglobin fragment.
Real-time PCR for T cruzi DNA Real-time PCR for T cruzi DNA was performed as described by Piron et al,11 using primers Cruzi 1, Cruzi 2, and probe Cruzi 3, labeled with 5’FAM (6-carboxyfluorescein) and 3’MGB (minor groove binder), targeting a 166-bp sequence from genomic DNA. To assess the dynamic range of the real-time PCR technique, 10-fold serial dilutions of DNA extracted from T cruzi culture (Y strain) were obtained corresponding to 2 104 to 2 10–4 parasites/μl. Real-time PCR was performed in a total volume of 25 μL containing 12.5 μL 1 TaqMan Universal Master Mix (Applied Biosystems, Branchburg, NJ), 750 nmol/liter each T cruzi primer, 250 nmol/liter T cruzi probe, and 5 μl DNA extraction eluate. Reactions were denatured at 951C for 10 minutes and then underwent 50 cycles (15 seconds at 941C and 1 minute at 601C) in a real-time PCR thermocycler (ICycler, BioRad, Hercules, CA). To verify the integrity of the extracted DNA and normalize the amount of T cruzi DNA, we also amplified the albumin gene, as previously reported.12 A sample was considered valid when the albumin gene was efficiently amplified and was considered positive for T cruzi DNA when the threshold cycle (Ct) was o 42.6. The Ct for a given sample is the first cycle of the PCR reaction where fluorescence is detected above baseline. Reactions were performed in duplicate, and the same positive and negative controls used in the qualitative PCR assay were included in each run. The parasitic load and parasitic load/106 cells were calculated for each sample as the mean of T cruzi copies and the mean of T cruzi copies/albumin copies times 2 106, respectively.
Benvenuti et al.
T cruzi Persistence in the Explanted Heart and CDR
Statistical analysis Continuous data from CDRþ and CDR– groups were compared using Student’s t-test or the Mann-Whitney rank sum test. Discrete data were compared using chi-square or Fisher exact test. A value of p r 0.05 was considered statistically significant.
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(Figure 2). The numbers of patients presenting with highgrade myocarditis and positive IHC for T cruzi antigens in CDRþ and CDR– groups are presented in Table 3. There was no statistical difference between the groups.
Qualitative PCR for T cruzi DNA
Results The CDRþ group consisted of 18 patients who were diagnosed as having CDR at a median of 4.5 months (range, 1–16 months) after HTx. The CDR– group consisted of 16 patients who were monitored for 18 months after HTx. The age and sex of the patients, use of mycophenolate mofetil, rate of endomyocardial biopsy specimens with moderate/severe rejection, and median and range of needing-treatment rejection episodes per month of follow-up for both groups are presented in Table 1. The CDRþ group patients presented a higher rate of endomyocardial biopsy specimens with moderate or severe acute cellular rejection and a higher number of needing-treatment rejection episodes per month compared with those in the CDR– group.
Macroscopic analysis of the hearts The native hearts were enlarged, presenting ventricular dilation and hypertrophy, and fulfilling the diagnostic criteria of dilated cardiomyopathy. The number of patients in the CDRþ and CDR– groups presenting chagasic rosary (small round whitish granules deposited sequentially along the coronary vessels),13 ventricular apical lesions (thinning and/or aneurysm), and ventricular thrombosis on the pathologic report, and with a permanent pacemaker are presented in Table 2. The apical portion of the ventricles could not be examined in 1 heart of the CDRþ group. There was no statistical difference between CDRþ and CDR– groups in the macroscopic analysis of the hearts.
Histopathology and immunohistochemistry for T cruzi antigens Myocarditis was present in 31 of 34 samples (91.2%) and classified as high-grade in most (Figure 1). IHC for T cruzi antigens was positive (Figure 2) in 6 of 34 samples (17.7%). Although amastigotes of T cruzi parasites were not detected in any sample at the first examination of H&E-stained sections, 4 of the 6 positive samples at IHC showed pseudocysts under reevaluation of the H&E-stained slides Table 1
PCR based on primers S34/S67 was positive in 13 of 18 samples (72.2%) of CDRþ patients and in 8 of 16 samples (50%) of CDR– patients (Figure 3). There was no statistical difference between the groups.
Real-time PCR for T cruzi DNA The albumin gene could not be amplified in 1 sample of the CDRþ group. In 2 samples of the CDRþ group and in 1 sample of CDR– group the Ct value was higher than 42.6, the upper limit of the dynamic range of the reaction. Therefore, these 4 cases were excluded from this part of the study. Real-time PCR was positive in 18 of 30 samples (60%), and when compared with the qualitative PCR for kDNA, agreement between the techniques occurred in 27 of 30 (90%). Real-time PCR was positive in 11 of 15 (73.3%) of the CDRþ group and in 7 of 15 samples (46.7%) of the CDR– group. The median and range of parasitic load and parasitic load/106 cells of the CDRþ and CDR– groups are presented in Table 3. There was no statistical difference between the groups.
Parasite persistence Parasite persistence, defined as any evidence of the presence of T cruzi parasites in the samples, was mainly detected through PCR techniques. All samples showing pseudocysts of T cruzi in H&E-stained sections or presenting positive IHC were positive in both qualitative and real-time PCR. With 1 exception, all samples positive for real-time PCR were also positive for qualitative PCR (kDNA); however, 2 positive samples for kDNA tested negative for real-time PCR. The number of patients with parasite persistence in CDRþ and CDR– groups is presented in Table 3. There was no statistical difference between the groups. High-grade myocarditis was present in all 22 samples (100%) with parasite persistence and in 7 of 12 samples (58.3%) with no parasite persistence (p ¼ 0.003).
Characteristics of Patients, Immunosuppressive Regimen, and Rejection Episodes
Variablea
CDRþ group
Age, years Male gender Usage of mycophenolate mofetil EMB specimens with moderate/severe rejection Rejection episodes needing treatment/month of follow-up, median
40.4 17/18 6/18 51/112 0.381
⫾ 15.1 (94.4) (33.3) (45.5) (0–1)
CDR– group 40.8 12/16 6/16 31/103 0.0556
CDR, Chagas’ disease reactivation; EMB, endomyocardial biopsy. a Continuous data are shown as mean ⫾ standard deviation or median (range), and categoric data as number (%).
⫾ 14.1 (75) (37.5) (30.1) (0–0.389)
p-value 0.942 0.164 0.916 0.029 o0.001
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CDR may occur after immunosuppression of chronic chagasic patients, being characterized by relapse of the acute phase of the disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7 Besides its occurrence after HTx, CDR has been reported after totalbody irradiation, hematologic malignances, chemotherapy, or infection with HIV.5,14 Two recent reviews reported CDR in 17 of 64 (26.5%) and in 17 of 44 (38.8%) chagasic HTx recipients monitored for a long period.5,7 Although the incidence of CDR after HTx is high, this complication
usually responds favorably to drug therapy with benznidazole or allopurinol, and the mortality rate is low.5–7 However, an early diagnosis of CDR and its differentiation from rejection or other infections is fundamental for effective therapy. Persistence of T cruzi parasites has been demonstrated in several organs of chronic chagasic patients, particularly the heart, gastrointestinal tract, and adrenal glands.15–22 Some studies were based on PCR techniques and demonstrated only the presence of T cruzi DNA, not necessarily live parasites,20–22 but the occurrence of CDR after immunosuppression is the most powerful evidence for the persistence of live parasites in chronic chagasic patients. In fact, amastigotes forms of T cruzi parasites were reported in previous studies involving detailed, careful examination of several histologic sections of different organs of chronic chagasic patients, particularly the heart, gastrointestinal tract, and adrenal glands.15–19 Therefore, we believe CDR is consequent to misbalance of the host-parasite relationship induced by immunosuppression, with proliferation and dissemination of T cruzi parasites from reservoirs in the body. In this study, we hypothesized T cruzi persistence in the body reservoirs would be reflected by parasitic persistence in the myocardium and investigated retrospectively if a detailed pathologic examination of the native, explanted
Figure 1 Semiquantitative evaluation of myocarditis shows (A) high-grade and (B) low-grade myocarditis.
Figure 2 Detection of Trypanosoma cruzi parasites at histology. Pseudocyst disclosed in (A) hematoxylin and eosinstained section (arrow) and (B) immunohistochemistry (arrow).
Table 2
Cardiac Macroscopic Pathology
Variable Pathologic finding Chagasic rosary Ventricular apical thinning/aneurysm Thrombus in ventricular cavities Permanent pacemaker
CDRþ group CDR– group No. (%) No. (%)
p-value
5/18 (27.8) 2/16 (12.5) 0.405 13/17 (76.5) 11/16 (68.8) 0.708 9/18 (50)
3/16 (18.8) 0.080
5/18 (27.8)
3/16 (18.8) 0.693
CDR, Chagas’ disease reactivation.
Discussion
Benvenuti et al. Table 3
T cruzi Persistence in the Explanted Heart and CDR
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Histopathology and Polymerase Chain Reaction Evaluation of Cardiac Samples
Pathologic findinga
CDRþ group
High-grade myocarditis IHCþ for T cruzi antigens Parasite persistence Parasitic load Parasitic load/106 cells
15/18 4/18 14/18 8.43 10–3 9.890
p-value
CDR– group (83.3) (22.2) (77.8) (4.02 10–4– 2.755) (0.886–48.800)
14/16 2/16 8/16 7.49 10–3 17.800
(87.5) (12.5) (50) (6.93 10–4– 1.590) (0.116 –556.000)
1.000 0.660 0.151 0.786 0.469
CDR, Chagas’ disease reactivation; IHC, immunohistochemistry; T cruzi, Trypanosoma cruzi. a Categoric data are shown as number (%) and continuous data as median (range).
heart at HTx with evaluation of parasitic persistence/load in the myocardium could be useful to identify patients at high risk of CDR. However, the CDRþ group in the follow-up showed similar pathologic findings compared with the CDR– group, mainly the rate of high-grade myocarditis, parasitic persistence, and parasitic load in the myocardium. Two possibilities may explain our findings: the evaluation of the parasitic persistence/load in the explanted heart does not reflect the parasitic status of other organs and/or the misbalance of host-parasite relationship which culminates with CDR is more complex, depending not only on the parasitic persistence/load in the body. A previous study from our group analyzed the clinical characteristics related to CDR and concluded a high number of rejection episodes, the development of neoplasms, and the use of mycophenolate mofetil constituted risk factors, probably mediated by excessive patient immunosuppression.5 Although the present study was not designed to evaluate the effect of immunosuppression or rejection episodes on the occurrence of CDR (patients of CDR– group were selected based on similar immunosuppressive regimen and presented a different follow-up period than the CDRþ patients), we had similar results and noticed that patients who presented CDR had a higher rate of endomyocardial biopsy specimens with moderate or severe acute cellular rejection and a higher number of episodes of rejection needing treatment per month compared with patients who did not present CDR. As a secondary finding of this study, we detected a high incidence of myocarditis (91.2%) and parasite persistence (64.7%) in the myocardium of patients with chronic chagasic cardiomyopathy. More important, the parasite persistence was associated with high-grade myocarditis, as demonstrated in previous studies,19–21 reinforcing the notion
that T cruzi parasites or their remnants are present in the myocardium in the chronic phase of disease and modulate local inflammation. Our study has some limitations. Although we included all patients who presented with CDR up to 18 months after HTx, the number of patients is small. The power of the statistical tests is poor, and therefore, the negative results obtained should be interpreted with caution. The study was retrospective, based on the analysis of 1 cardiac sample per patient, and the molecular methods were performed in formalin-fixed, paraffin-embedded tissue. In conclusion, we found no relation between the pathologic characteristics of the native, explanted heart of chagasic patients, particularly the persistence of T cruzi parasites in the myocardium, and the occurrence of CDR. On the other hand, parasite persistence was related to highgrade myocarditis in the native heart. The identification of chagasic, HTx patients susceptible to CDR should be pursued, and the possible relation of CDR to the parasitic load in biopsy specimens or blood obtained in the follow-up of the patients should be evaluated in future studies.
Disclosure statement This study was financially supported by grant 11/51580-1, São Paulo Research Foundation (FAPESP). Part of this work was presented at the International Academy of Pathology (IAP) 2012 XXIX International Congress, Cape Town, South Africa, September 3-October 5, 2012. None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
Figure 3 Qualitative polymerase chain reaction for Trypanosoma cruzi kinetoplast DNA. Chagas’ disease reactivation-positive group: lines 1 (–), 4 (þ), 5 (þ), 7 (þ), 11 (þ) and 12 (þ); Chagas’ disease reactivation-negative group: lines 2 (–), 3 (–), 6 (–), 8 (þ), 9 (þ) and 10 (þ); positive control: line 13; negative control: line 14.
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References 1. Higuchi ML, Benvenuti LA, Reis MM, Metzger M. Pathophysiology of the heart in Chagas’ disease: current status and new developments. Cardiovasc Res 2003;60:96-107. 2. Marin-Neto JA, Cunha-Neto E, Maciel BC, Simões MV. Pathogenesis of chronic Chagas heart disease. Circulation 2007;115: 1109-23. 3. Bocchi EA, Fiorelli A. The paradox of survival results after heart transplantation for cardiomyopathy caused by Trypanosoma cruzi. Ann Thorac Surg 2001;71:1833-8. 4. Fiorelli AI, Santos RH, Oliveira JL Jr, et al. Heart transplantation in 107 cases of Chagas’ disease. Transplant Proc 2011;43:220-4. 5. Campos SV, Strabelli TM, Amato Neto V, et al. Risk factors for Chagas’ disease reactivation after heart transplantation. J Heart Lung Transplant 2008;27:597-602. 6. Bacal F, Silva CP, Pires PV, et al. Transplantation for Chagas’ disease: an overview of immunosupression and reactivation in the last two decades. Clin Transplant 2010;24:E29-34. 7. Godoy HL, Guerra CM, Viegas RF, et al. Infections in heart transplant recipients in Brazil: the challenge of Chagas’ disease. J Heart Lung Transplant 2010;29:286-90. 8. Aretz HT. Myocarditis: the Dallas criteria. Hum Pathol 1987;18: 619-24. 9. Benvenuti LA, Roggério A, Coelho G, Fiorelli AI. Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients. J Heart Lung Transplant 2011;30:799-804. 10. Olivares-Villagómez D, McCurley TL, Vnencak-Jones CL, CorreaOliveira R, Colley DG, Carter CE. Polymerase chain reaction amplification of three different Trypanosoma cruzi DNA sequences from human chagasic cardiac tissue. Am J Trop Med Hyg 1998;59: 563-70. 11. Piron M, Fisa R, Casamitjana N, et al. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop 2007;103:195-200.
12. Gault E, Michel Y, Dehée A, Belabani C, Nicolas JC, Garbarg-Chenon A. Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol 2001;39:772-5. 13. Benvenuti LA, Gutierrez PS. Epicardial lesions in Chagas’ heart disease reflect an inflammatory process. Arq Bras Cardiol 2007;4:496-8. 14. Sartori AM, Ibrahim KY, Nunes Westphalen EV, et al. Manifestation of Chagas disease (American trypanosomiasis) in patients with HIV/ AIDS. Ann Trop Med Parasitol 2007;101:31-50. 15. Barbosa AA, Andrade ZA. Identificação do Trypanosoma cruzi nos tecidos extracardíacos de portadores de miocardite crônica chagásica. Rev Soc Bras Med Trop 1984;17:123-6. 16. Teixeira VP, Reis MA, Araújo MB, Silveira AS, Reis LR, Almeida HO. Comparação do parasitismo da veia central da supra-renal com o de outros tecidos em chagásicos crônicos. Rev Soc Bras Med Trop 1991;24:73-8. 17. Teixeira VP, Araújo MB, Reis MA, et al. Possible role of an adrenal parasite reservoir in the pathogenesis of chronic Trypanosoma cruzi myocarditis. Trans R Soc Trop Med Hyg 1993;87:552-4. 18. Côbo EC, Silveira TP, Micheletti AM, Crema E, Adad SJ. Research on Trypanosoma cruzi and analysis of inflammatory infiltrate in esophagus and colon from chronic chagasic patients with and without mega. J Trop Med 2012;2012:232646. 19. Higuchi ML, Brito T, Reis MM, et al. Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: light microscopy and immunohistochemical findings. Cardiovasc Pathol 1993;2:101-6. 20. Jones EM, Colley DG, Tostes S, Lopes ER, Vnencak-Jones CL, McCurley TL. Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy. Am J Trop Med Hyg 1993;48:348-57. 21. Benvenuti LA, Roggério A, Freitas HF, Mansur AJ, Fiorelli A, Higuchi ML. Chronic American trypanosomiasis: parasite persistence in endomyocardial biopsies is associated with high-grade myocarditis. Ann Trop Med Parasitol 2008;102:481-7. 22. Marcon GE, Albuquerque DM, Batista AM, et al. Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients. Mem Inst Oswaldo Cruz 2011;106:85-91.
ORIGINAL CLINICAL SCIENCE
Trypanosoma cruzi persistence in the native heart is associated with high-grade myocarditis, but not with Chagas’ disease reactivation after heart transplantation Luiz A. Benvenuti, MD, PhD,a Alessandra Roggério, PhD,a Anna S. Nishiya, MS,b,c Silvia V. Campos, MD,a Alfredo. I. Fiorelli, MD, PhD,a and José E. Levi, PhDb From the aHeart Institute (InCor), University of São Paulo Medical School; the bFundação Pró-Sangue/Hemocentro of São Paulo, Molecular Biology Department; and cInfectious Disease Division (DIPA), Federal University of São Paulo, São Paulo, Brazil.
KEYWORDS: Chagas’ disease; Trypanosoma cruzi; heart transplantation; myocarditis; polymerase chain reaction
BACKGROUND: Chagas’ disease reactivation (CDR) after heart transplantation (HTx) is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. We investigated whether a detailed pathologic examination of the explanted heart at HTx with evaluation of myocarditis and parasitic persistence or load in the myocardium could be useful to identify patients at high risk of CDR. METHODS: The native hearts of 18 chagasic patients who presented CDR after HTx (CDRþ group) were compared with the native hearts of 16 chagasic patients who never presented CDR in a follow-up of at least 18 months after HTx (CDR– group). The intensity of myocarditis was evaluated semiquantitatively. Parasite persistence/load in the myocardium was investigated through immunohistochemistry for T cruzi antigens and by qualitative and quantitative real-time PCR for T cruzi DNA. RESULTS: The rate of high-grade myocarditis, parasite persistence, and the median of parasitic load and parasitic load/106 cells in the CDRþ group were 83.3%, 77.8%, 8.43 10–3, and 9.890, respectively, whereas in the CDR– group the values were 87.5%, 50%, 7.4910–3, and 17.800. There was no statistical difference between the groups. High-grade myocarditis was present in all 22 samples (100%) with parasite persistence and in 7 of 12 samples (58.3%) with no parasite persistence (p ¼ 0.003). CONCLUSIONS: Although associated with high-grade myocarditis, T cruzi parasite persistence in the myocardium of the native heart is not associated with the occurrence of CDR after HTx. J Heart Lung Transplant ]]]];]:]]]–]]] r 2014 International Society for Heart and Lung Transplantation. All rights reserved.
Human American trypanosomiasis (Chagas’ disease) is caused by infection with the hemoflagellate protozoan Trypanosoma cruzi. After a variable quiescent period, usually of many years, approximately 30% of infected Reprint requests: Luiz A. Benvenuti, MD, PhD, Heart Institute, University of São Paulo Medical School, Av. Dr. Enéas de Carvalho Aguiar 44, 05403–000 São Paulo-SP, Brazil. Telephone: þ55-11-26615080. Fax: þ55-11-2661-5279. E-mail address: [email protected]
people develop a chronic, inflammatory cardiomyopathy that may progress to ventricular systolic dysfunction.1,2 Heart transplantation (HTx) is a valid therapeutic option for end-stage heart failure due to Chagas’ disease.3,4 However, besides the usual morbidities related to HTx, patients can develop Chagas’ disease reactivation (CDR), a lifethreatening complication characterized by relapse of the infectious disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7
1053-2498/$ - see front matter r 2014 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2014.01.920
2
The Journal of Heart and Lung Transplantation, Vol ], No ], Month ]]]]
CDR occurs in a significant proportion of HTx chagasic patients, usually in the first year of follow-up. Clinical studies have demonstrated that augmented immunosuppression increases the risk of CDR,5–7 probably due to a misbalance of the host-parasite relationship. In this study, we investigated retrospectively whether a detailed pathologic examination of the native, explanted heart at HTx, with evaluation of parasitic persistence/load in the myocardium, could be useful to identify patients at high risk of CDR.
Methods From March 1985 to December 2011, 112 patients with Chagas’ disease underwent HTx in our hospital. The disease was diagnosed from epidemiologic data and positive results on serologic tests. The immunosuppressive regimen included cyclosporine, azathioprine (changed to mycophenolate mofetil from 2001 to 2005), and steroids. As a routine in our HTx program, no patient received cytolytic induction therapy. Episodes of moderate, acute cellular rejection were treated with 3 days of methylprednisolone. Anti-thymocyte globulin was given for severe or persistent rejection or if associated with cardiac dysfunction. The diagnosis of CDR was based on direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues. The occurrence of CDR was monitored, and no prophylactic drug therapy was used. We selected for this study all patients who presented at least 1 episode of CDR up to 18 months after HTx (CDRþ group). Because the immunosuppressive regimen changed over time, we selected as controls those patients who underwent HTx at the same period as the CDRþ patients but who did not present CDR in a follow-up of at least 18 months after HTx (CDR– group). We analyzed retrospectively the pathologic report of the native heart explanted from each chagasic patient and selected for this study the largest paraffin-embedded sample of ventricular myocardium stored in our laboratory.
Histopathology One 4-μm-thick section of the paraffin-embedded sample was stained by hematoxylin and eosin (H&E). The slides were reviewed by 1 cardiac pathologist (L.A.B.) blinded to the patient group. Myocarditis was characterized by an inflammatory infiltrate adjacent to degenerating/necrotic myocytes according to the Dallas criteria8 and evaluated semiquantitatively. Myocarditis was defined as low-grade if there was mild, multifocal inflammatory infiltration with discrete foci of cardiomyocyte aggression and as high-grade if there was multifocal dense or diffuse inflammatory infiltration with multiple foci of cardiomyocyte aggression. We searched the slides for groups of amastigotes of T cruzi parasites in the cardiomyocytes (pseudocysts).
Immunohistochemistry for T cruzi antigens Immunohistochemistry (IHC) for T cruzi antigens was performed as previously reported,9 with minor modifications. Briefly, the sections underwent proteolytic digestion with 0.18 mg/ml trypsin T 7409 (Sigma-Aldrich, St Louis, MO) for 5 minutes at 371 C, incubated with rabbit polyclonal immune serum against T cruzi (Y strain) diluted 1:100,000 for 1 hour and MACH 4 (Biocare Medical, Concord, CA) for 30 minutes at room temperature. The reactions were developed with 3,3’-diaminobenzidine (Dako, Carpinteria, CA), and the slides were counterstained with hematoxylin. Myocardial sections previously known to contain pseudocysts of T cruzi parasites were used as positive controls.
Qualitative polymerase chain reaction for the detection of T cruzi DNA Qualitative polymerase chain reaction (PCR) was performed in sections of the paraffin blocks using the primers S34/S67, which amplify a 122-bp sequence localized within the minirepeat of the kinetoplast minicircles (kDNA).10 The DNA was extracted from three 8-μm sections of the paraffin blocks with QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions, and eluted in 200 μl. PCR was performed in a total volume of 25 μl containing PCR buffer 1, 1.5 mmol/liter MgCl2, 1.5 units Taq DNA polymerase (Invitrogen, São Paulo, Brazil), 200 μmol/liter deoxyribonucleotide triphosphate mixture, 1 μmol/liter each primer, and 1 μl extraction eluate. Reactions were denatured at 951C for 5 min and then underwent 45 cycles in a programmable thermal cycler (T3 Thermocycler; Biometra, Goettingen, Germany). Each cycle included 40 seconds at 951C, 1 minute at 571C, and 45 seconds at 721C, followed by a final incubation at 721C for 10 minutes. After thermal cycling, 10 μl each reaction mixture was analyzed using 2% agarose gel electrophoresis and visualized under ultraviolet light after ethidium bromide staining. Every sample was run at least in duplicate to obtain repetitive, concordant results. We used as positive control sections from a paraffinembedded endomyocardial biopsy specimen of a patient with CDR and T cruzi parasites in the myocardium. Negative controls included sections from paraffin blocks containing no tissue or myocardium of patients with idiopathic dilated or ischemic heart disease. We included in this study only samples in which the integrity of the extracted DNA could be checked through positive PCR amplification of T cruzi or human βglobin fragment.
Real-time PCR for T cruzi DNA Real-time PCR for T cruzi DNA was performed as described by Piron et al,11 using primers Cruzi 1, Cruzi 2, and probe Cruzi 3, labeled with 5’FAM (6-carboxyfluorescein) and 3’MGB (minor groove binder), targeting a 166-bp sequence from genomic DNA. To assess the dynamic range of the real-time PCR technique, 10-fold serial dilutions of DNA extracted from T cruzi culture (Y strain) were obtained corresponding to 2 104 to 2 10–4 parasites/μl. Real-time PCR was performed in a total volume of 25 μL containing 12.5 μL 1 TaqMan Universal Master Mix (Applied Biosystems, Branchburg, NJ), 750 nmol/liter each T cruzi primer, 250 nmol/liter T cruzi probe, and 5 μl DNA extraction eluate. Reactions were denatured at 951C for 10 minutes and then underwent 50 cycles (15 seconds at 941C and 1 minute at 601C) in a real-time PCR thermocycler (ICycler, BioRad, Hercules, CA). To verify the integrity of the extracted DNA and normalize the amount of T cruzi DNA, we also amplified the albumin gene, as previously reported.12 A sample was considered valid when the albumin gene was efficiently amplified and was considered positive for T cruzi DNA when the threshold cycle (Ct) was o 42.6. The Ct for a given sample is the first cycle of the PCR reaction where fluorescence is detected above baseline. Reactions were performed in duplicate, and the same positive and negative controls used in the qualitative PCR assay were included in each run. The parasitic load and parasitic load/106 cells were calculated for each sample as the mean of T cruzi copies and the mean of T cruzi copies/albumin copies times 2 106, respectively.
Benvenuti et al.
T cruzi Persistence in the Explanted Heart and CDR
Statistical analysis Continuous data from CDRþ and CDR– groups were compared using Student’s t-test or the Mann-Whitney rank sum test. Discrete data were compared using chi-square or Fisher exact test. A value of p r 0.05 was considered statistically significant.
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(Figure 2). The numbers of patients presenting with highgrade myocarditis and positive IHC for T cruzi antigens in CDRþ and CDR– groups are presented in Table 3. There was no statistical difference between the groups.
Qualitative PCR for T cruzi DNA
Results The CDRþ group consisted of 18 patients who were diagnosed as having CDR at a median of 4.5 months (range, 1–16 months) after HTx. The CDR– group consisted of 16 patients who were monitored for 18 months after HTx. The age and sex of the patients, use of mycophenolate mofetil, rate of endomyocardial biopsy specimens with moderate/severe rejection, and median and range of needing-treatment rejection episodes per month of follow-up for both groups are presented in Table 1. The CDRþ group patients presented a higher rate of endomyocardial biopsy specimens with moderate or severe acute cellular rejection and a higher number of needing-treatment rejection episodes per month compared with those in the CDR– group.
Macroscopic analysis of the hearts The native hearts were enlarged, presenting ventricular dilation and hypertrophy, and fulfilling the diagnostic criteria of dilated cardiomyopathy. The number of patients in the CDRþ and CDR– groups presenting chagasic rosary (small round whitish granules deposited sequentially along the coronary vessels),13 ventricular apical lesions (thinning and/or aneurysm), and ventricular thrombosis on the pathologic report, and with a permanent pacemaker are presented in Table 2. The apical portion of the ventricles could not be examined in 1 heart of the CDRþ group. There was no statistical difference between CDRþ and CDR– groups in the macroscopic analysis of the hearts.
Histopathology and immunohistochemistry for T cruzi antigens Myocarditis was present in 31 of 34 samples (91.2%) and classified as high-grade in most (Figure 1). IHC for T cruzi antigens was positive (Figure 2) in 6 of 34 samples (17.7%). Although amastigotes of T cruzi parasites were not detected in any sample at the first examination of H&E-stained sections, 4 of the 6 positive samples at IHC showed pseudocysts under reevaluation of the H&E-stained slides Table 1
PCR based on primers S34/S67 was positive in 13 of 18 samples (72.2%) of CDRþ patients and in 8 of 16 samples (50%) of CDR– patients (Figure 3). There was no statistical difference between the groups.
Real-time PCR for T cruzi DNA The albumin gene could not be amplified in 1 sample of the CDRþ group. In 2 samples of the CDRþ group and in 1 sample of CDR– group the Ct value was higher than 42.6, the upper limit of the dynamic range of the reaction. Therefore, these 4 cases were excluded from this part of the study. Real-time PCR was positive in 18 of 30 samples (60%), and when compared with the qualitative PCR for kDNA, agreement between the techniques occurred in 27 of 30 (90%). Real-time PCR was positive in 11 of 15 (73.3%) of the CDRþ group and in 7 of 15 samples (46.7%) of the CDR– group. The median and range of parasitic load and parasitic load/106 cells of the CDRþ and CDR– groups are presented in Table 3. There was no statistical difference between the groups.
Parasite persistence Parasite persistence, defined as any evidence of the presence of T cruzi parasites in the samples, was mainly detected through PCR techniques. All samples showing pseudocysts of T cruzi in H&E-stained sections or presenting positive IHC were positive in both qualitative and real-time PCR. With 1 exception, all samples positive for real-time PCR were also positive for qualitative PCR (kDNA); however, 2 positive samples for kDNA tested negative for real-time PCR. The number of patients with parasite persistence in CDRþ and CDR– groups is presented in Table 3. There was no statistical difference between the groups. High-grade myocarditis was present in all 22 samples (100%) with parasite persistence and in 7 of 12 samples (58.3%) with no parasite persistence (p ¼ 0.003).
Characteristics of Patients, Immunosuppressive Regimen, and Rejection Episodes
Variablea
CDRþ group
Age, years Male gender Usage of mycophenolate mofetil EMB specimens with moderate/severe rejection Rejection episodes needing treatment/month of follow-up, median
40.4 17/18 6/18 51/112 0.381
⫾ 15.1 (94.4) (33.3) (45.5) (0–1)
CDR– group 40.8 12/16 6/16 31/103 0.0556
CDR, Chagas’ disease reactivation; EMB, endomyocardial biopsy. a Continuous data are shown as mean ⫾ standard deviation or median (range), and categoric data as number (%).
⫾ 14.1 (75) (37.5) (30.1) (0–0.389)
p-value 0.942 0.164 0.916 0.029 o0.001
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CDR may occur after immunosuppression of chronic chagasic patients, being characterized by relapse of the acute phase of the disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7 Besides its occurrence after HTx, CDR has been reported after totalbody irradiation, hematologic malignances, chemotherapy, or infection with HIV.5,14 Two recent reviews reported CDR in 17 of 64 (26.5%) and in 17 of 44 (38.8%) chagasic HTx recipients monitored for a long period.5,7 Although the incidence of CDR after HTx is high, this complication
usually responds favorably to drug therapy with benznidazole or allopurinol, and the mortality rate is low.5–7 However, an early diagnosis of CDR and its differentiation from rejection or other infections is fundamental for effective therapy. Persistence of T cruzi parasites has been demonstrated in several organs of chronic chagasic patients, particularly the heart, gastrointestinal tract, and adrenal glands.15–22 Some studies were based on PCR techniques and demonstrated only the presence of T cruzi DNA, not necessarily live parasites,20–22 but the occurrence of CDR after immunosuppression is the most powerful evidence for the persistence of live parasites in chronic chagasic patients. In fact, amastigotes forms of T cruzi parasites were reported in previous studies involving detailed, careful examination of several histologic sections of different organs of chronic chagasic patients, particularly the heart, gastrointestinal tract, and adrenal glands.15–19 Therefore, we believe CDR is consequent to misbalance of the host-parasite relationship induced by immunosuppression, with proliferation and dissemination of T cruzi parasites from reservoirs in the body. In this study, we hypothesized T cruzi persistence in the body reservoirs would be reflected by parasitic persistence in the myocardium and investigated retrospectively if a detailed pathologic examination of the native, explanted
Figure 1 Semiquantitative evaluation of myocarditis shows (A) high-grade and (B) low-grade myocarditis.
Figure 2 Detection of Trypanosoma cruzi parasites at histology. Pseudocyst disclosed in (A) hematoxylin and eosinstained section (arrow) and (B) immunohistochemistry (arrow).
Table 2
Cardiac Macroscopic Pathology
Variable Pathologic finding Chagasic rosary Ventricular apical thinning/aneurysm Thrombus in ventricular cavities Permanent pacemaker
CDRþ group CDR– group No. (%) No. (%)
p-value
5/18 (27.8) 2/16 (12.5) 0.405 13/17 (76.5) 11/16 (68.8) 0.708 9/18 (50)
3/16 (18.8) 0.080
5/18 (27.8)
3/16 (18.8) 0.693
CDR, Chagas’ disease reactivation.
Discussion
Benvenuti et al. Table 3
T cruzi Persistence in the Explanted Heart and CDR
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Histopathology and Polymerase Chain Reaction Evaluation of Cardiac Samples
Pathologic findinga
CDRþ group
High-grade myocarditis IHCþ for T cruzi antigens Parasite persistence Parasitic load Parasitic load/106 cells
15/18 4/18 14/18 8.43 10–3 9.890
p-value
CDR– group (83.3) (22.2) (77.8) (4.02 10–4– 2.755) (0.886–48.800)
14/16 2/16 8/16 7.49 10–3 17.800
(87.5) (12.5) (50) (6.93 10–4– 1.590) (0.116 –556.000)
1.000 0.660 0.151 0.786 0.469
CDR, Chagas’ disease reactivation; IHC, immunohistochemistry; T cruzi, Trypanosoma cruzi. a Categoric data are shown as number (%) and continuous data as median (range).
heart at HTx with evaluation of parasitic persistence/load in the myocardium could be useful to identify patients at high risk of CDR. However, the CDRþ group in the follow-up showed similar pathologic findings compared with the CDR– group, mainly the rate of high-grade myocarditis, parasitic persistence, and parasitic load in the myocardium. Two possibilities may explain our findings: the evaluation of the parasitic persistence/load in the explanted heart does not reflect the parasitic status of other organs and/or the misbalance of host-parasite relationship which culminates with CDR is more complex, depending not only on the parasitic persistence/load in the body. A previous study from our group analyzed the clinical characteristics related to CDR and concluded a high number of rejection episodes, the development of neoplasms, and the use of mycophenolate mofetil constituted risk factors, probably mediated by excessive patient immunosuppression.5 Although the present study was not designed to evaluate the effect of immunosuppression or rejection episodes on the occurrence of CDR (patients of CDR– group were selected based on similar immunosuppressive regimen and presented a different follow-up period than the CDRþ patients), we had similar results and noticed that patients who presented CDR had a higher rate of endomyocardial biopsy specimens with moderate or severe acute cellular rejection and a higher number of episodes of rejection needing treatment per month compared with patients who did not present CDR. As a secondary finding of this study, we detected a high incidence of myocarditis (91.2%) and parasite persistence (64.7%) in the myocardium of patients with chronic chagasic cardiomyopathy. More important, the parasite persistence was associated with high-grade myocarditis, as demonstrated in previous studies,19–21 reinforcing the notion
that T cruzi parasites or their remnants are present in the myocardium in the chronic phase of disease and modulate local inflammation. Our study has some limitations. Although we included all patients who presented with CDR up to 18 months after HTx, the number of patients is small. The power of the statistical tests is poor, and therefore, the negative results obtained should be interpreted with caution. The study was retrospective, based on the analysis of 1 cardiac sample per patient, and the molecular methods were performed in formalin-fixed, paraffin-embedded tissue. In conclusion, we found no relation between the pathologic characteristics of the native, explanted heart of chagasic patients, particularly the persistence of T cruzi parasites in the myocardium, and the occurrence of CDR. On the other hand, parasite persistence was related to highgrade myocarditis in the native heart. The identification of chagasic, HTx patients susceptible to CDR should be pursued, and the possible relation of CDR to the parasitic load in biopsy specimens or blood obtained in the follow-up of the patients should be evaluated in future studies.
Disclosure statement This study was financially supported by grant 11/51580-1, São Paulo Research Foundation (FAPESP). Part of this work was presented at the International Academy of Pathology (IAP) 2012 XXIX International Congress, Cape Town, South Africa, September 3-October 5, 2012. None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
Figure 3 Qualitative polymerase chain reaction for Trypanosoma cruzi kinetoplast DNA. Chagas’ disease reactivation-positive group: lines 1 (–), 4 (þ), 5 (þ), 7 (þ), 11 (þ) and 12 (þ); Chagas’ disease reactivation-negative group: lines 2 (–), 3 (–), 6 (–), 8 (þ), 9 (þ) and 10 (þ); positive control: line 13; negative control: line 14.
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