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Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients
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Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients Luiz A. Benvenuti, MD, Alessandra Roggério, PhD, Guilherme Coelho, MD, and Alfredo I. Fiorelli, MD From the Heart Institute (InCor), University of São Paulo Medical School, São Paulo, SP, Brazil.
KEYWORDS: Chagas’ disease; Trypanosoma cruzi; heart transplantation; endomyocardial biopsy; polymerase chain reaction
BACKGROUND: Chagas’ disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. METHODS: We performed retrospectively qualitative polymerase chain reaction for T cruzi DNA using 2 sets of primers targeting nuclear DNA (nDNA) or kinetoplast DNA (kDNA) in 61 EMB specimens of 11 chagasic heart transplant recipients who presented with CDR. Thirty-five EMB specimens were obtained up to 6 months before (pre-CDR group) and 26 up to 2 years after the diagnosis of CDR. The control group consisted of 6 chagasic heart transplant recipients with 18 EMB specimens who never experienced CDR. RESULTS: Amplification of kDNA occurred in 8 of 35 (22.9%) EMB specimens of the pre-CDR group, in 5 of 18 (27.8%) of the control group, and in 17 of 26 (65.4%) EMB specimens obtained after the successful treatment of CDR. Amplification of nDNA occurred in 3 of 35 (8.6%) EMB specimens of the pre-CDR group, 0 of 18 (0%) of the control group, and 6 of 26 (23.1%) EMB specimens obtained after the successful treatment of CDR. CONCLUSIONS: Amplification of kDNA in EMB specimens is not specific for the diagnosis of CDR, occurring also in patients with no evidence of CDR (control group). However, amplification of nDNA occurred in a few EMB specimens obtained before CDR, but in none of the control group specimens. Qualitative PCR for T cruzi DNA in EMB specimens should not be used as a criterion for cure of CDR because it can persist positive despite favorable clinical evolution of the patients. J Heart Lung Transplant 2011;30:799 – 804 © 2011 International Society for Heart and Lung Transplantation. All rights reserved.
American trypanosomiasis (Chagas’ disease) is caused by infection with the hemoflagellate protozoan Trypanosoma cruzi. After a variable quiescent period, usually of many years, a chronic, inflammatory cardiomyopathy develops in approximately 30% of infected people that may evolve with progressive cardiac systolic dysfunction.1,2 Reprint requests: Luiz A. Benvenuti, MD, Heart Institute, University of São Paulo Medical School, Av. Dr. Enéas de Carvalho Aguiar 44, 05403-000 São Paulo-SP, Brazil. Telephone: ⫹55-11-3069-5080. Fax: ⫹55-11-3069-5279. E-mail address: [email protected]
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, chagasic patients can develop Chagas’ disease reactivation (CDR), a life-threatening complication characterized by relapse of the infectious disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7 CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. For this purpose, careful search for parasites in hematoxylin and eosin (HE)–
1053-2498/$ -see front matter © 2011 International Society for Heart and Lung Transplantation. All rights reserved. doi:10.1016/j.healun.2011.02.012
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stained sections and immunohistochemistry (IHC) for T cruzi antigens are mandatory. In this study, we investigated retrospectively the usefulness of qualitative polymerase chain reaction (PCR) for T cruzi DNA in EMB specimens of chagasic heart transplant recipients, particularly for the early detection of CDR.
Methods Patients and sample selection From January 2001 to June 2009, 11 chagasic HTx recipients monitored at our institution were diagnosed as having the first episode of CDR through direct identification of T cruzi parasites in EMB specimens (6 patients), skin biopsy specimens (3 patients), blood (1 patient), or cerebrospinal fluid (1 patient). We analyzed retrospectively all biopsy specimens that were diagnosed with CDR (6 EMB, 3 skin) plus 37 EMB specimens obtained up to 6 months before (pre-CDR group) and 28 EMB specimens obtained up to 2 years after that diagnosis. In addition, 20 EMB specimens obtained up to 1 year after HTx of 6 chagasic patients who never experienced CDR constituted the control group.
Histopathology The previous slides of each EMB specimen were reviewed by one pathologist (L.A.B.), and additional HE-stained sections and IHC were performed if necessary. We carefully searched the slides for T cruzi parasites and T cruzi antigens.
IHC for T cruzi antigens IHC for T cruzi antigens was performed as previously reported, with minor modifications.8 Briefly, the sections underwent proteo-
lytic digestion with 0.18 mg/ml trypsin T 7409 (Sigma, St Louis, MO) for 5 minutes at 37°C, incubated with rabbit polyclonal immune serum against T cruzi (Y strain) diluted 1:400,000 for 18 hours at 4°C (overnight), and with MACH 4 (Biocare Medical, Concord, CA) for 30 minutes at room temperature. The reactions were developed with 3,3=-diaminobenzidine (Sigma), and the slides were counterstained with HE. Myocardial sections previously known to contain groups of complete T cruzi parasites (pseudocysts) were used as positive controls.
PCR-based assay for the detection of T cruzi DNA PCR was performed in sections of the paraffin-embedded EMB specimens using 2 sets of primers: TCZI/TCZII, which amplifies a 188-bp repetitive nuclear sequence of T cruzi DNA (nDNA) and S34/S67, which amplifies a 122 bp sequence localized within the mini-repeat of the kinetoplast mini-circles (kDNA). PCR was performed as previously reported, with minor modifications.9,10 Briefly, sections of the paraffin blocks were extracted with proteinase K (Invitrogen, Camarillo, CA) and underwent 35 cycles in a programmable thermal cycler (PTC-200, BioRad, Hercules, CA). PCR was performed in a total volume of 50 l containing PCR buffer 1x, 1.5 mmol/liter of MgCl2, 1.5 units of Taq DNA polymerase (Invitrogen, Brazil), 200 mol/liter of deoxyribonucleotide triphosphate mixture, 1 mmol/liter of each primer, and 1 l of extraction solution. After thermal cycling, 10 l of each reaction mixture was analyzed using gel electrophoresis and visualization after ethidium bromide staining under ultraviolet light. Every sample was run at least twice to obtain 2 consecutive, concordant results. To be called positive, a sample had to yield 1 consecutive positive result. Negative controls included sections from paraffin blocks containing no tissue. This study only included EMB specimens in which the integrity of the DNA extracted from the paraffin-embedded specimens could be checked through positive PCR amplification of a human -globin fragment.
Figure 1 Histopathology of Chagas’ disease reactivation in an endomyocardial biopsy specimen. Lymphocytic myocarditis with groups of Trypanosoma cruzi parasites (arrows) in the cardiomyocytes (hematoxylin and eosin stain). (Inset) Positive immunohistochemistry for T cruzi antigens is shown.
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Table 1 Number of Positive Patients or Positive Endomyocardial Biopsy Specimen Positive PCR by primer
Group Control, No. (%) Patients EMB specimen Pre-CDR, No. (%) Patients EMB specimen Post-CDR, No. (%) Patients EMB specimen
TCZI/TCZII (nuclear DNA)
S34/S67 (kDNA)
801
The 6 patients in the control group were a median age of 41 years (range, 15–52 years) at HTx, and 5 (83%) were male. There was no statistical difference between the patients with CDR and the control group in age (Mann-Whitney rank sum test) or gender (Fisher’s exact test).
EMB specimens of pre-CDR and control groups 0/6 (0) 0/18 (0)
3/6 (50) 5/18 (27.8)
2/11 (18.2) 3/35 (8.6)
4/11 (36.4) 8/35 (22.9)
3/8 (37.5) 6/26 (23.1)
6/8 (75) 17/26 (65.4)
CDR, Chagas’ disease reactivation; EMB, endomyocardial biopsy; PCR, polymerase chain reaction.
Statistical analysis Data from pre-CDR and control groups were compared using the Mann-Whitney rank sum test or Fisher’s exact test. A value of p ⱕ 0.05 was considered statistically significant.
Results The 11 patients that presented with CDR were a median age of 41 years (range, 17– 61 years) at HTx, and 9 (82%) were men. They were diagnosed as having CDR a median of 87 days (range, 40 –381 day)s after HTx. Parasites were clearly identified in HE-stained sections and/or IHC in all specimens that were diagnosed with CDR (Figure 1). All of those specimens (6 EMB, 3 skin) tested positive for PCR based on both sets of primers. The patients were treated with benznidazole (5–10 mg/kg daily) for 60 days, and all but 1 made a full recovery. A patient with CDR diagnosed through direct identification of T cruzi parasites in the cerebrospinal fluid died of chagasic meningoencephalitis 39 days after that diagnosis.
The study excluded 2 EMB specimens each of the preCDR and control groups that tested negative in the PCR assay for human -globin fragment. The 35 EMB specimens of the pre-CDR group were obtained a median of 30 days (range, 6 –336 days) after HTx and a median of 64 days (range, 10 –164 days) before the diagnosis of CDR. The 18 EMB specimens of the control group were obtained at a median of 35 days (range, 7–309 days) after HTx. There was no statistical difference between the pre-CDR and control groups regarding the time elapsed between HTx and EMB specimen collection (MannWhitney rank sum test). IHC for T cruzi antigens was negative in all EMB specimens of the pre-CDR and control groups. The numbers of patients or EMB specimens that tested positive by PCR based on each set of primers are presented in Table 1. PCR based on primers TCZI/TCZII was positive in 3 EMB of 2 patients in the pre-CDR group, up to 164 days before that diagnosis, but in none of the EMB specimens of the control group (Figure 2). Although PCR based on primers S34/S67 was positive in several EMB specimens of pre-CDR group, it was also positive in EMB specimens of the control group (Figure 3). All EMB specimens that amplified nDNA also amplified kDNA. There was no statistical difference between pre-CDR and control groups in the number of positive patients or the number of positive EMB specimens (Fisher’s exact test).
EMB obtained after the diagnosis of CDR The study excluded 2 EMB specimens obtained after the diagnosis of CDR that tested negative in the PCR assay for human -globin fragment. We analyzed 26 EMB specimens from 8 patients obtained a median of 69 days (range, 10 –
Figure 2 Polymerase chain reaction detection of Trypanosoma cruzi DNA in endomyocardial biopsy (EMB) specimens of chagasic heart transplant recipients using primers TCZI/TCZII. Amplicons were generated from EMB specimens that diagnosed Chagas’ disease reactivation (CDR; line 12) and in EMB specimens of pre-CDR group (lines 8 and 10). No EMB specimens of the control group generated amplicons (lines 1 to 7).
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Figure 3 Polymerase chain reaction detection of Trypanosoma cruzi DNA in endomyocardial biopsy (EMB) specimens of chagasic heart transplant recipients using primers S34/S67. Amplicons were generated from EMB specimens that diagnosed Chagas’ disease reactivation (CDR; line 12), in EMB specimens the pre-CDR group (lines 8 to 11), and in EMB specimens of the control group (lines 2, 5 and 6).
488 days) after the diagnosis of CDR. A new episode of CDR occurred in 1 patient, with direct identification of T cruzi parasites in HE-stained sections of 2 sequential EMB specimens collected 203 and 231 days after the initial CDR diagnosis. The patient was a 23-year-old woman who was diagnosed as having CDR 381 days after HTx and had multiple positive pre-CDR EMB specimens in the PCR assay for T cruzi DNA. PCR based on primers TCZI/TCZII was positive in 6 of 26 (23.1%) EMB specimens from 3 of 8 patients (37.5%), up to 151 days after the diagnosis of CDR. PCR based on primers S34/S67 was positive in 17 of 26 EMB specimens (65.4%) of 6 of 8 patients (75%) at up to 488 days after the diagnosis of CDR (Figure 4). All EMB specimens that amplified nDNA also amplified kDNA. Timelines of the PCR results for each patient are presented in Figure 5.
Discussion Being a systemic chronic infection, Chagas’ disease was initially considered a contraindication to HTx. In fact, at the beginning of our HTx program, we observed a high incidence of neoplasm and CDR, life-threatening complications due to immunosupression.3,6 Currently, after an adjustment of the immunosuppressant regimen, chagasic HTx recipients present a good clinical evolution, and according to
some authors, even a better survival rate than patients who undergo HTx for other reasons.4 CDR is equivalent to the acute phase of Chagas’ disease, being characterized by direct detection of T cruzi parasites in organic fluids (usually blood) and/or tissues (myocardium or skin). CDR is clearly linked to immunosuppression: besides its occurrence in the follow-up of HTx, it has been reported in chronic chagasic patients after total-body irradiation, hematologic malignances, chemotherapy, or infection with HIV.5,11 Two recent reviews reported CDR occurred in 17 of 64 (26.5%) or 17 of 44 (38.8%) chagasic HTx recipients followed-up for a long period.5,7 If correctly treated, patients with CDR present good clinical evolution: only 1 patient from that above mentioned series died of CDR.5 Therefore, an early diagnosis and the differentiation of CDR from rejection or other infections is crucial for the correct drug therapy, which is based on oral benznidazole or allopurinol. CDR that affects the myocardium induces lymphocytic myocarditis and produces a histologic picture quite similar to acute cellular rejection. The only reliable criterion to distinguish both entities in EMB specimens is to find any evidence for the presence of T cruzi parasites. Therefore, in addition to the routine search for groups of parasites (pseudocysts) in HE-stained sections and T cruzi antigens by IHC, some authors advocate the use of qualitative PCR for T cruzi DNA as a very sensitive method to detect a
Figure 4 Polymerase chain reaction detection of Trypanosoma cruzi DNA using primers S34/S67 in endomyocardial biopsy (EMB) specimens obtained after Chagas’ disease reactivation (CDR). Amplicons were generated from EMB specimens obtained 37 (line 2), 124 (line 6), and 488 days after CDR (line 1). Line 7, positive control; line 8, blank; line 9, negative control.
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803
Finally, in our study, the PCR was positive for T cruzi DNA in some EMB specimens obtained even a long time after CDR was successfully treated. This finding indicates qualitative PCR should not be used as a criterion for cure of CDR and suggests T cruzi DNA, but not necessarily intact or live parasites, can persist in the myocardium of chagasic HTx recipients, as previously reported in chronic chagasic cardiomyopathy.10,17,18
Disclosure statement This study was financially supported by Fundação de Amparo a Pesquisa do Estado de São Paulo—FAPESP (Grant No. 09/ 50181– 6). 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 5 Multiple scatter plot graph shows the interval in days between the episode of Chagas’ disease reactivation (CDR) and each endomyocardial biopsy (EMB) specimen according to the positive (filled symbols) or negative (open symbols) polymerase chain reaction (PCR) status in each occasion. Negative numbers correspond to the interval in days of EMB performed before the CDR episode. Each patient is depicted in contiguous horizontal lines. , positive PCR using primers TCZI/TCZII; Œ, negative PCR using primers TCZI/TCZII; ’, positive PCR using primers S34/S67; , negative PCR using primers S34/S67.
parasitic presence in the EMB specimen. According to these authors, the PCR technique would be able to make an early diagnosis of CDR, up to few months before the direct detection of T cruzi parasites by other techniques.12–14 The usefulness of qualitative PCR techniques to diagnose CDR should be critically assessed, however. Owing to parasitic persistence, PCR for T cruzi DNA in blood is positive in most chronic chagasic patients.15,16 Furthermore, only 1 of those studies had a control group (ie, chagasic HTx recipients with no CDR). Diez et al14 found that 1 of 5 patients (20%) without CDR generated positive PCR results. In the present study, we investigated retrospectively the results of qualitative PCR for T cruzi DNA in EMB specimens of chagasic HTx recipients and evaluated particularly its usefulness for the early detection of CDR. The last goal was pursued comparing the PCR results of EMB obtained previously to the diagnosis of CDR (pre-CDR group) with the PCR results of EMB obtained from a control group of chagasic HTx recipients who never presented CDR. We concluded that amplification of kDNA is not specific for the diagnosis of CDR, which occurs in both pre-CDR and control groups. However, although there was not statistical difference between pre-CDR and control groups, the fact that nDNA was only amplified from EMB specimens of patients of the pre-CDR group suggests that it could be an earlier marker of CDR. Future studies should explore this possibility. Furthermore, the negative results of PCR for T cruzi DNA in EMB specimens are useful to exclude CDR in samples doubtful for that diagnosis.
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, Bellotti G, Mocelin AO, et al. Heart transplantation for chronic Chagas’ heart disease. Ann Thorac Surg 1996;61:1727-33. 4. 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. 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 immunosuppression and reactivation in the last two decades. Clin Transplant 2010;24:E29-E34. 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. Palomino SA, Aiello VD, Higuchi ML. Systematic mapping of hearts from chronic chagasic patients: the association between the occurrence of histopathological lesions and Trypanosoma cruzi antigens. Ann Trop Med Parasitol 2000;94:571-9. 9. Benvenuti LA, Roggério A, Sambiase NV, Fiorelli A, Higuchi ML. Polymerase chain reaction in endomyocardial biopsies for monitoring reactivation of Chagas’ disease in heart transplantation: a case report and review of the literature. Cardiovasc Pathol 2005;14:265-8. 10. 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. 11. Sartori AM, Lopes MH, Benvenuti LA, et al. Reactivation of Chagas’ disease in a human immunodeficiency virus-infected patient leading to severe heart disease with a late positive direct microscopic examination of the blood. Am J Trop Med Hyg 1998;59:784-6. 12. Schijman AG, Vigliano C, Burgos J, et al. Early diagnosis of recurrence of Trypanosoma cruzi infection by polymerase chain reaction after heart transplantation of a chronic Chagas’ heart disease patient. J Heart Lung Transplant 2000;19:1114-7. 13. Maldonado C, Albano S, Vettorazzi L, et al. Using polymerase chain reaction in early diagnosis of re-activated Trypanosoma cruzi infection after heart transplantation. J Heart Lung Transplant 2004;23:1345-8.
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14. Diez M, Favarolo L, Bertolotti A, et al. Usefulness of PCR strategies for early diagnosis of Chagas’ disease reactivation and treatment follow-up in heart transplantation. Am J Transplant 2007;7:1633-40. 15. Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 1994;51:771-7. 16. Ribeiro-dos-Santos G, Nishiya AS, Sabino EC, Chamone DF, Sáez-Alquézar A. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples. Ann Trop Med Parasitol 1999;93:689-94.
17. 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. 18. 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.
Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients Luiz A. Benvenuti, MD, Alessandra Roggério, PhD, Guilherme Coelho, MD, and Alfredo I. Fiorelli, MD From the Heart Institute (InCor), University of São Paulo Medical School, São Paulo, SP, Brazil.
KEYWORDS: Chagas’ disease; Trypanosoma cruzi; heart transplantation; endomyocardial biopsy; polymerase chain reaction
BACKGROUND: Chagas’ disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. METHODS: We performed retrospectively qualitative polymerase chain reaction for T cruzi DNA using 2 sets of primers targeting nuclear DNA (nDNA) or kinetoplast DNA (kDNA) in 61 EMB specimens of 11 chagasic heart transplant recipients who presented with CDR. Thirty-five EMB specimens were obtained up to 6 months before (pre-CDR group) and 26 up to 2 years after the diagnosis of CDR. The control group consisted of 6 chagasic heart transplant recipients with 18 EMB specimens who never experienced CDR. RESULTS: Amplification of kDNA occurred in 8 of 35 (22.9%) EMB specimens of the pre-CDR group, in 5 of 18 (27.8%) of the control group, and in 17 of 26 (65.4%) EMB specimens obtained after the successful treatment of CDR. Amplification of nDNA occurred in 3 of 35 (8.6%) EMB specimens of the pre-CDR group, 0 of 18 (0%) of the control group, and 6 of 26 (23.1%) EMB specimens obtained after the successful treatment of CDR. CONCLUSIONS: Amplification of kDNA in EMB specimens is not specific for the diagnosis of CDR, occurring also in patients with no evidence of CDR (control group). However, amplification of nDNA occurred in a few EMB specimens obtained before CDR, but in none of the control group specimens. Qualitative PCR for T cruzi DNA in EMB specimens should not be used as a criterion for cure of CDR because it can persist positive despite favorable clinical evolution of the patients. J Heart Lung Transplant 2011;30:799 – 804 © 2011 International Society for Heart and Lung Transplantation. All rights reserved.
American trypanosomiasis (Chagas’ disease) is caused by infection with the hemoflagellate protozoan Trypanosoma cruzi. After a variable quiescent period, usually of many years, a chronic, inflammatory cardiomyopathy develops in approximately 30% of infected people that may evolve with progressive cardiac systolic dysfunction.1,2 Reprint requests: Luiz A. Benvenuti, MD, Heart Institute, University of São Paulo Medical School, Av. Dr. Enéas de Carvalho Aguiar 44, 05403-000 São Paulo-SP, Brazil. Telephone: ⫹55-11-3069-5080. Fax: ⫹55-11-3069-5279. E-mail address: [email protected]
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, chagasic patients can develop Chagas’ disease reactivation (CDR), a life-threatening complication characterized by relapse of the infectious disease, with direct detection of T cruzi parasites in blood, cerebrospinal fluid, or tissues.5–7 CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. For this purpose, careful search for parasites in hematoxylin and eosin (HE)–
1053-2498/$ -see front matter © 2011 International Society for Heart and Lung Transplantation. All rights reserved. doi:10.1016/j.healun.2011.02.012
800
The Journal of Heart and Lung Transplantation, Vol 30, No 7, July 2011
stained sections and immunohistochemistry (IHC) for T cruzi antigens are mandatory. In this study, we investigated retrospectively the usefulness of qualitative polymerase chain reaction (PCR) for T cruzi DNA in EMB specimens of chagasic heart transplant recipients, particularly for the early detection of CDR.
Methods Patients and sample selection From January 2001 to June 2009, 11 chagasic HTx recipients monitored at our institution were diagnosed as having the first episode of CDR through direct identification of T cruzi parasites in EMB specimens (6 patients), skin biopsy specimens (3 patients), blood (1 patient), or cerebrospinal fluid (1 patient). We analyzed retrospectively all biopsy specimens that were diagnosed with CDR (6 EMB, 3 skin) plus 37 EMB specimens obtained up to 6 months before (pre-CDR group) and 28 EMB specimens obtained up to 2 years after that diagnosis. In addition, 20 EMB specimens obtained up to 1 year after HTx of 6 chagasic patients who never experienced CDR constituted the control group.
Histopathology The previous slides of each EMB specimen were reviewed by one pathologist (L.A.B.), and additional HE-stained sections and IHC were performed if necessary. We carefully searched the slides for T cruzi parasites and T cruzi antigens.
IHC for T cruzi antigens IHC for T cruzi antigens was performed as previously reported, with minor modifications.8 Briefly, the sections underwent proteo-
lytic digestion with 0.18 mg/ml trypsin T 7409 (Sigma, St Louis, MO) for 5 minutes at 37°C, incubated with rabbit polyclonal immune serum against T cruzi (Y strain) diluted 1:400,000 for 18 hours at 4°C (overnight), and with MACH 4 (Biocare Medical, Concord, CA) for 30 minutes at room temperature. The reactions were developed with 3,3=-diaminobenzidine (Sigma), and the slides were counterstained with HE. Myocardial sections previously known to contain groups of complete T cruzi parasites (pseudocysts) were used as positive controls.
PCR-based assay for the detection of T cruzi DNA PCR was performed in sections of the paraffin-embedded EMB specimens using 2 sets of primers: TCZI/TCZII, which amplifies a 188-bp repetitive nuclear sequence of T cruzi DNA (nDNA) and S34/S67, which amplifies a 122 bp sequence localized within the mini-repeat of the kinetoplast mini-circles (kDNA). PCR was performed as previously reported, with minor modifications.9,10 Briefly, sections of the paraffin blocks were extracted with proteinase K (Invitrogen, Camarillo, CA) and underwent 35 cycles in a programmable thermal cycler (PTC-200, BioRad, Hercules, CA). PCR was performed in a total volume of 50 l containing PCR buffer 1x, 1.5 mmol/liter of MgCl2, 1.5 units of Taq DNA polymerase (Invitrogen, Brazil), 200 mol/liter of deoxyribonucleotide triphosphate mixture, 1 mmol/liter of each primer, and 1 l of extraction solution. After thermal cycling, 10 l of each reaction mixture was analyzed using gel electrophoresis and visualization after ethidium bromide staining under ultraviolet light. Every sample was run at least twice to obtain 2 consecutive, concordant results. To be called positive, a sample had to yield 1 consecutive positive result. Negative controls included sections from paraffin blocks containing no tissue. This study only included EMB specimens in which the integrity of the DNA extracted from the paraffin-embedded specimens could be checked through positive PCR amplification of a human -globin fragment.
Figure 1 Histopathology of Chagas’ disease reactivation in an endomyocardial biopsy specimen. Lymphocytic myocarditis with groups of Trypanosoma cruzi parasites (arrows) in the cardiomyocytes (hematoxylin and eosin stain). (Inset) Positive immunohistochemistry for T cruzi antigens is shown.
Benvenuti et al.
Chagas’ Disease Reactivation After Heart Transplantation
Table 1 Number of Positive Patients or Positive Endomyocardial Biopsy Specimen Positive PCR by primer
Group Control, No. (%) Patients EMB specimen Pre-CDR, No. (%) Patients EMB specimen Post-CDR, No. (%) Patients EMB specimen
TCZI/TCZII (nuclear DNA)
S34/S67 (kDNA)
801
The 6 patients in the control group were a median age of 41 years (range, 15–52 years) at HTx, and 5 (83%) were male. There was no statistical difference between the patients with CDR and the control group in age (Mann-Whitney rank sum test) or gender (Fisher’s exact test).
EMB specimens of pre-CDR and control groups 0/6 (0) 0/18 (0)
3/6 (50) 5/18 (27.8)
2/11 (18.2) 3/35 (8.6)
4/11 (36.4) 8/35 (22.9)
3/8 (37.5) 6/26 (23.1)
6/8 (75) 17/26 (65.4)
CDR, Chagas’ disease reactivation; EMB, endomyocardial biopsy; PCR, polymerase chain reaction.
Statistical analysis Data from pre-CDR and control groups were compared using the Mann-Whitney rank sum test or Fisher’s exact test. A value of p ⱕ 0.05 was considered statistically significant.
Results The 11 patients that presented with CDR were a median age of 41 years (range, 17– 61 years) at HTx, and 9 (82%) were men. They were diagnosed as having CDR a median of 87 days (range, 40 –381 day)s after HTx. Parasites were clearly identified in HE-stained sections and/or IHC in all specimens that were diagnosed with CDR (Figure 1). All of those specimens (6 EMB, 3 skin) tested positive for PCR based on both sets of primers. The patients were treated with benznidazole (5–10 mg/kg daily) for 60 days, and all but 1 made a full recovery. A patient with CDR diagnosed through direct identification of T cruzi parasites in the cerebrospinal fluid died of chagasic meningoencephalitis 39 days after that diagnosis.
The study excluded 2 EMB specimens each of the preCDR and control groups that tested negative in the PCR assay for human -globin fragment. The 35 EMB specimens of the pre-CDR group were obtained a median of 30 days (range, 6 –336 days) after HTx and a median of 64 days (range, 10 –164 days) before the diagnosis of CDR. The 18 EMB specimens of the control group were obtained at a median of 35 days (range, 7–309 days) after HTx. There was no statistical difference between the pre-CDR and control groups regarding the time elapsed between HTx and EMB specimen collection (MannWhitney rank sum test). IHC for T cruzi antigens was negative in all EMB specimens of the pre-CDR and control groups. The numbers of patients or EMB specimens that tested positive by PCR based on each set of primers are presented in Table 1. PCR based on primers TCZI/TCZII was positive in 3 EMB of 2 patients in the pre-CDR group, up to 164 days before that diagnosis, but in none of the EMB specimens of the control group (Figure 2). Although PCR based on primers S34/S67 was positive in several EMB specimens of pre-CDR group, it was also positive in EMB specimens of the control group (Figure 3). All EMB specimens that amplified nDNA also amplified kDNA. There was no statistical difference between pre-CDR and control groups in the number of positive patients or the number of positive EMB specimens (Fisher’s exact test).
EMB obtained after the diagnosis of CDR The study excluded 2 EMB specimens obtained after the diagnosis of CDR that tested negative in the PCR assay for human -globin fragment. We analyzed 26 EMB specimens from 8 patients obtained a median of 69 days (range, 10 –
Figure 2 Polymerase chain reaction detection of Trypanosoma cruzi DNA in endomyocardial biopsy (EMB) specimens of chagasic heart transplant recipients using primers TCZI/TCZII. Amplicons were generated from EMB specimens that diagnosed Chagas’ disease reactivation (CDR; line 12) and in EMB specimens of pre-CDR group (lines 8 and 10). No EMB specimens of the control group generated amplicons (lines 1 to 7).
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Figure 3 Polymerase chain reaction detection of Trypanosoma cruzi DNA in endomyocardial biopsy (EMB) specimens of chagasic heart transplant recipients using primers S34/S67. Amplicons were generated from EMB specimens that diagnosed Chagas’ disease reactivation (CDR; line 12), in EMB specimens the pre-CDR group (lines 8 to 11), and in EMB specimens of the control group (lines 2, 5 and 6).
488 days) after the diagnosis of CDR. A new episode of CDR occurred in 1 patient, with direct identification of T cruzi parasites in HE-stained sections of 2 sequential EMB specimens collected 203 and 231 days after the initial CDR diagnosis. The patient was a 23-year-old woman who was diagnosed as having CDR 381 days after HTx and had multiple positive pre-CDR EMB specimens in the PCR assay for T cruzi DNA. PCR based on primers TCZI/TCZII was positive in 6 of 26 (23.1%) EMB specimens from 3 of 8 patients (37.5%), up to 151 days after the diagnosis of CDR. PCR based on primers S34/S67 was positive in 17 of 26 EMB specimens (65.4%) of 6 of 8 patients (75%) at up to 488 days after the diagnosis of CDR (Figure 4). All EMB specimens that amplified nDNA also amplified kDNA. Timelines of the PCR results for each patient are presented in Figure 5.
Discussion Being a systemic chronic infection, Chagas’ disease was initially considered a contraindication to HTx. In fact, at the beginning of our HTx program, we observed a high incidence of neoplasm and CDR, life-threatening complications due to immunosupression.3,6 Currently, after an adjustment of the immunosuppressant regimen, chagasic HTx recipients present a good clinical evolution, and according to
some authors, even a better survival rate than patients who undergo HTx for other reasons.4 CDR is equivalent to the acute phase of Chagas’ disease, being characterized by direct detection of T cruzi parasites in organic fluids (usually blood) and/or tissues (myocardium or skin). CDR is clearly linked to immunosuppression: besides its occurrence in the follow-up of HTx, it has been reported in chronic chagasic patients after total-body irradiation, hematologic malignances, chemotherapy, or infection with HIV.5,11 Two recent reviews reported CDR occurred in 17 of 64 (26.5%) or 17 of 44 (38.8%) chagasic HTx recipients followed-up for a long period.5,7 If correctly treated, patients with CDR present good clinical evolution: only 1 patient from that above mentioned series died of CDR.5 Therefore, an early diagnosis and the differentiation of CDR from rejection or other infections is crucial for the correct drug therapy, which is based on oral benznidazole or allopurinol. CDR that affects the myocardium induces lymphocytic myocarditis and produces a histologic picture quite similar to acute cellular rejection. The only reliable criterion to distinguish both entities in EMB specimens is to find any evidence for the presence of T cruzi parasites. Therefore, in addition to the routine search for groups of parasites (pseudocysts) in HE-stained sections and T cruzi antigens by IHC, some authors advocate the use of qualitative PCR for T cruzi DNA as a very sensitive method to detect a
Figure 4 Polymerase chain reaction detection of Trypanosoma cruzi DNA using primers S34/S67 in endomyocardial biopsy (EMB) specimens obtained after Chagas’ disease reactivation (CDR). Amplicons were generated from EMB specimens obtained 37 (line 2), 124 (line 6), and 488 days after CDR (line 1). Line 7, positive control; line 8, blank; line 9, negative control.
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Finally, in our study, the PCR was positive for T cruzi DNA in some EMB specimens obtained even a long time after CDR was successfully treated. This finding indicates qualitative PCR should not be used as a criterion for cure of CDR and suggests T cruzi DNA, but not necessarily intact or live parasites, can persist in the myocardium of chagasic HTx recipients, as previously reported in chronic chagasic cardiomyopathy.10,17,18
Disclosure statement This study was financially supported by Fundação de Amparo a Pesquisa do Estado de São Paulo—FAPESP (Grant No. 09/ 50181– 6). 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 5 Multiple scatter plot graph shows the interval in days between the episode of Chagas’ disease reactivation (CDR) and each endomyocardial biopsy (EMB) specimen according to the positive (filled symbols) or negative (open symbols) polymerase chain reaction (PCR) status in each occasion. Negative numbers correspond to the interval in days of EMB performed before the CDR episode. Each patient is depicted in contiguous horizontal lines. , positive PCR using primers TCZI/TCZII; Œ, negative PCR using primers TCZI/TCZII; ’, positive PCR using primers S34/S67; , negative PCR using primers S34/S67.
parasitic presence in the EMB specimen. According to these authors, the PCR technique would be able to make an early diagnosis of CDR, up to few months before the direct detection of T cruzi parasites by other techniques.12–14 The usefulness of qualitative PCR techniques to diagnose CDR should be critically assessed, however. Owing to parasitic persistence, PCR for T cruzi DNA in blood is positive in most chronic chagasic patients.15,16 Furthermore, only 1 of those studies had a control group (ie, chagasic HTx recipients with no CDR). Diez et al14 found that 1 of 5 patients (20%) without CDR generated positive PCR results. In the present study, we investigated retrospectively the results of qualitative PCR for T cruzi DNA in EMB specimens of chagasic HTx recipients and evaluated particularly its usefulness for the early detection of CDR. The last goal was pursued comparing the PCR results of EMB obtained previously to the diagnosis of CDR (pre-CDR group) with the PCR results of EMB obtained from a control group of chagasic HTx recipients who never presented CDR. We concluded that amplification of kDNA is not specific for the diagnosis of CDR, which occurs in both pre-CDR and control groups. However, although there was not statistical difference between pre-CDR and control groups, the fact that nDNA was only amplified from EMB specimens of patients of the pre-CDR group suggests that it could be an earlier marker of CDR. Future studies should explore this possibility. Furthermore, the negative results of PCR for T cruzi DNA in EMB specimens are useful to exclude CDR in samples doubtful for that diagnosis.
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14. Diez M, Favarolo L, Bertolotti A, et al. Usefulness of PCR strategies for early diagnosis of Chagas’ disease reactivation and treatment follow-up in heart transplantation. Am J Transplant 2007;7:1633-40. 15. Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 1994;51:771-7. 16. Ribeiro-dos-Santos G, Nishiya AS, Sabino EC, Chamone DF, Sáez-Alquézar A. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples. Ann Trop Med Parasitol 1999;93:689-94.
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