Using polymerase chain reaction in early diagnosis of re-activated Trypanosoma cruzi infection after heart transplantation

Using Polymerase Chain Reaction in Early Diagnosis of Re-activated Trypanosoma cruzi Infection After Heart Transplantation Cecilia Maldonado,a Susana Albano,a Lorena Vettorazzi,a Oscar Salomone, MD,b Juan C. Zlocowski, MD,c Claudio Abiega,c Marcos Amuchastegui, MD,b Roque Co ´ rdoba, MD,b and Teresita Alvarellos, MDa Background: Heart transplantation is an effective treatment for patients with end-stage Chagas’ heart disease. Re-activation of Chagas’ disease in transplant recipients is frequent, triggered by immunosuppression level. Therefore, highly sensitive methods for early diagnosis of Chagas’ disease relapse are necessary to initiate appropriate therapy. We analyzed the use of polymerase chain reaction (PCR) in the clinical follow-up of heart transplant recipients. Methods: We prospectively evaluated 4 heart transplant recipients at the Hospital Privado, Co ´ rdoba, Argentina, who had terminal Chagas’ disease. The parameters analyzed were presence of parasites in the blood (blood culture, Strout) and in endomyocardial biopsy (EMB) samples, and PCR was performed with oligonucleotides directed to a nuclear repetitive sequence of Trypanosoma cruzi. We evaluated these parameters weekly from the day of transplantation until results were negative and then during regular follow-up visits. Results: In 2 patients, we detected T cruzi using PCR in peripheral blood 30 days before clinical evidence of re-activation. In the 3rd case, PCR results in peripheral blood were positive from the day before transplantation, followed by positive results in EMB and sub-cutaneous chagomas biopsy specimens. Only 1 patient had positive Strout results for parasites in skin lesions, and none showed amastigotes in the biopsy specimens. After clinical diagnosis, all patients received 5 mg/kg/day benzimidazole for 6 months, with acceptable tolerance and good clinical outcome. All patients had negative peripheral blood PRC results after 30 days of treatment. One patient had intermittent positive PCR results during follow-up, with no evidence of clinical re-activation. Conclusion: Polymerase chain reaction detection of T Cruzi in heart transplant recipients is a more sensitive and specific procedure in diagnosing Chagas’ disease re-activation. J Heart Lung Transplant 2004;23: 1345-8. Copyright © 2004 by the International Society for Heart and Lung Transplantation.

American trypanosomiasis or Chagas’ disease is a major cause of morbidity and mortality in Latin America. Trypanosoma cruzi, an obligate cytoplasmic parasite in mammals, is the agent responsible for Chagas’ disease.1,2 Chagas’ disease is endemic in 18 Latin American countries, in which the estimated prevalence of human infection ranges from 16 to 20 million. In addition, another 70 to 90 million people are considered at risk.3 Formerly considered typical of rural areas, Chagas’ disease is now ubiquitous because of changes in the

From the aMolecular Diagnosis Laboratory, Fundacio ´ n para el Progreso de la Medicina, Co ´ rdoba, bHeart Transplant Program, Hospital Privado Centro Me´dico de Co ´ rdoba, Co ´ rdoba, and cInfectious Diseases and Microbiology, Co ´ rdoba, Argentina. Submitted June 30, 2003; revised September 8, 2003; accepted September 10, 2003. Reprint requests: Teresita Alvarellos, Fundacio ´ n para el Progreso de la Medicina, 9 de Julio 941, X5000 EMS Co ´ rdoba, Argentina. Telephone: 54-351-421-0669/0777. Fax: 54-351-425-7678. E-mail: [email protected] Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter. doi:10.1016/ j.healun.2003.09.027

social patterns of many countries, mainly as a result of rural-to-urban migration. Chagas’ disease also occurs in non-endemic areas, where it may be acquired through blood transfusions. Furthermore, a serologic study of T cruzi infection performed in 205 Latin American immigrants to the United States showed a prevalence of 4.5%. This leads to the estimate that at least 400,000 to 500,000 infected people currently live in the United States.4 In Argentina, the incidence of Chagas’ disease in endemic areas ranges from 5% to 30%.4 The infection can be acquired through vectorial transmission, blood transfusion, breast-feeding, laboratory accidents, and congenital means.5 Two phases can be distinguished in the natural history of Chagas’ disease. The acute phase generally is asymptomatic and only 5% of infected individuals have symptoms. This evolves into an indeterminate asymptomatic phase, and after 10 to 15 years, 10% to 25% of infected people have organspecific lesions, mainly gastrointestinal tract and myocardial.5,6 The symptomatic acute phase is characterized by increased parasite burden, occasional fever, liver and spleen enlargement, and myocarditis.6,7 This is 1345

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followed by a decrease in parasitemia concentration and clinical symptoms. The chronic phase of the disease presents small concentrations of parasite. Because of the lack of specificity of signs and symptoms in any phase, the diagnosis of Chagas’ disease is based mainly on serologic testing (complement fixation, immunofluorescence, and hemagglutination) and detection of parasitemia.7–9 The serologic diagnosis of Chagas’ disease has several difficulties in that these methods have great sensitivity but poor specificity because of antigenic cross-reactivity.10 In transplant recipients, these tests present an additional problem because of decreased antibody production caused by immunosuppressive therapy. In addition, current parasitologic methods have low sensitivity and are useful only in the presence of large concentrations of parasitemia.11 Therefore, several molecular biology laboratories are trying to create new methods to detect T Cruzi DNA using polymerase chain reaction (PCR), which can be used in any phase of the disease independent of the immune status of the host.11–12 The main objective of this study was to evaluate the usefulness of PCR for early diagnosis of T cruzi reactivation, because the specificity and sensitivity of serologic and parasitologic methods are not adequate to detect small concentrations of parasitemia in patients with immunosuppression. METHODS We studied 54 heart recipients and their donors. All belonged to the Heart Transplant Program of the Hospital Privado in Co ´ rdoba. The patients underwent conventional clinical examination and serologic testing for Chagas’ disease. Four of the 54 heart transplant recipients (mean age, 53 ⫾ 8 years) were serologically positive for Chagas’ disease. The period of evaluation spanned from the day of transplantation until 40 months after transplantation. Patients received immunosuppression therapy according to hospital protocols with cyclosporine, azathioprine, and prednisone. We performed serology using indirect hemagglutination (HemAve Chagas kit, Polychaco, Argentina, qualitative positive at ⬎1/16), indirect immunofluorescence assay (IFI Fluor Parasitest Kit, Argentina, positive at ⬎1/30), and enzyme-linked immunosorbent assay (ChagateK ELISA BioMerieux, Argentina, cut off at 0.20 OD). Patients were considered serologically positive for Chagas’ disease when the serum was reactive in both tests. We detected parasitemia using the Strout test. This method uses blood serum obtained from spontaneous clot retraction of approximately 5 ml blood without anti-coagulant. The serum is first centrifuged at 160g to separate the red blood cells and centrifuged a second time at 350g to 380g to obtain the sediment

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used to search for the parasite. The estimated sensitivity is 95% for acute cases.11 We evaluated patients clinically and with peripheral blood PRC weekly during the 1st month and monthly thereafter at regular outpatient visits. We performed PCR only when indicated by endomyocardial, gastric, and skin biopsy samples. DNA Extraction We collected and immediately processed 3 ml peripheral blood, containing 5 ␮l 0.2 mol/liter EDTA. We extracted DNA according to a previously described protocol.12,13 Briefly, 1 ml buffy coat was lysed with 5 volumes of lysis buffer (0.3 mol/liter sucrose, 10 mmol/ liter Tris-HCl, pH 7.5, 5 mmol/liter MgCl, and 1% Triton x-100), digested with 10% sodium dodecyl sulfate proteinase K (300 ␮g/ml) and incubated overnight at 37°C. The DNA was then extracted with phenol-chloroform and precipitated with isopropanol. After centrifugation, the pellet was rinsed with 70% ethanol, air dried, and resuspended in 50 ␮l nuclease-free water. We used 10 ␮l of this material as substrate for PCR. PCR Amplification We performed PCR using TCZ1 (5⬘-CGAGCTCTTGCCCACACGGGTGCT-3⬘) and TCZ2 (5⬘-CCTCCAAGCAGCGGATAGTTCAGG-3⬘) oligonucleotides, which amplify 188 base pairs of a repetitive nuclear sequence (⫽100,000 copies per parasite).12,13 Thermal cycling conditions were performed for a total of 33 cycles for 1 minute each at 95°C, 65°C, and 72°C, respectively (9600 thermal cycler, Perkin Elmer; Norwalk, CT). To assess performance of the assay, we included negative and positive controls. Negative controls consisted of DNA samples from patients with negative serology results, and positive controls consisted of DNA samples obtained from a T Cruzi culture (trypomastigotes). Other controls included distilled water as templates to check for contamination of the PCR reagents and to check for cross-contamination. These samples were processed in parallel with the specimens and tested in duplicate. To visualize PCR products, we used electrophoresis through a 2% agarose gel with ethidium bromide in 1⫻ tris-borate/EDTA buffer. PCR Assay Performance Tests of a serial 1-to-10 dilution of T Cruzi DNA obtained from culture allowed us to assess the analytic sensitivity of the T Cruzi PCR assay. Serial dilutions containing 24 ⫻ 105 parasites/ml down to 1 parasite/ml were prepared in saline solution and added to 1 ml buffy coat from an uninfected individual. We extracted DNA as described previously12,13 and performed PCR testing. We detected DNA at up to 1 parasite/ml. To study specificity and possible false-positive reactions, we evaluated samples from 10 transplant recipients

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Table 1. Results From 4 Patients Serologically Positive for Chagas Disease Patient # 1

Clinical re-activation Hematogenous chagoma

Parasitemia Strout (⫺)

Histology Without amastigotes

2

Myocarditis

Strout (⫺)

Without amastigotes

3

Hematogenous chagoma Myocarditis

Strout (⫹)

Without amastigotes

4

Hematogenous chagoma

ND

ND

PCR PB (⫹) EMC Bx (⫹) Skin lesion (⫹) PB (⫹) EMC Bx (⫹) PB (⫹) EMC Bx (⫹) Gastric Bx (⫹) Skin Bx (⫹) PB (⫹) Skin Bx (⫹)

Clinical response Good

Negative PCR results 1 month

Good

2 months

Good

1 month

Good

1 month

Bx, biopsy; EMC, endomyocardial; ND, not determined; PB, peripheral blood; PCR, polymerase chain reaction.

with negative serology results for Chagas’ disease; from 10 anonymous blood donors; and from 5 patients infected with cytomegalovirus, Toxoplasma gondii, and Herpes simplex virus types 1 and 2. None of these samples showed amplification with TCZ1 or TCZ2 primer set (data not shown). We used this assay during follow-up of the patients. RESULTS We evaluated 4 patients from the Heart Transplant Program of the Hospital Privado de Cordoba, Argentina. Pre-transplant diagnosis was end-stage Chagas’ heart disease diagnosed by clinical exam and by at least 2 different positive serologic tests. The immunosuppression protocol was similar in all patients, based on cyclosporine, azathioprine, and prednisone therapy. The parameters evaluated after transplantation were parasites in blood (blood culture, Strout test) and in EMB samples and parasites found using PCR. (Table 1 summarizes results). Patient 1 was a 50-year-old man with positive T Cruzi PCR results in peripheral blood at 6 days after transplantation. He had sub-cutaneous nodules as evidence of clinical Chagas’ disease re-activation at Day 52. Polymerase chain reaction results were positive in peripheral blood and in skin biopsy specimen, whereas Strout test and histology exam results were negative. The patient received 5 mg/kg/day benzimidazole for 6 months with good clinical outcome. The PCR results became negative after 42 days of therapy. Patient 2 was a 62-year-old man with positive T Cruzi PCR results in peripheral blood at Day 12 after transplantation. At Day 132, heart failure was diagnosed clinically, with positive PRC results in EMB specimens and in peripheral blood. Strout and EMB specimen results were negative for the presence of parasites. The patient underwent therapy with 5 mg/kg/day benzimidazole for 6 months with good clinical outcome. Positive and negative PCR results alternated during follow-up,

with no evidence of clinical re-activation. Finally, PCR results were negative at 1 year after the end of therapy. Patient 3, a 57-year-old woman, had positive T Cruzi PCR results in peripheral blood the day before transplantation. At Day 5, she experienced heart failure and PCR results became positive in peripheral blood, in EMB specimens, and in subcutaneous chagomas biopsy specimens—in addition to positive Strout test results. This patient received benzimidazole at 5 mg/kg/day with acceptable tolerance and PCR results were negative in peripheral blood and in EMB specimen after 30 days of treatment. Patient 4 was a 46-year-old woman who had positive T Cruzi PCR results in peripheral blood and in subcutaneous nodules, considered signs of clinical re-activation of Chagas’ disease, at 15 days after transplantation. Strout test and histologic exam were not performed. At this time, the patient received 5 mg/kg/day benzimidazole for 6 months with good clinical outcome and negative PCR results after 30 days of treatment. DISCUSSION The frequency of solid-organ transplantation has increased during past decades, and new developments in immunosuppressive therapy clearly have improved patient outcome. However, the incidence and spectrum of opportunistic infections also have increased. Because serologic testing has low sensitivity in diagnosing these patients, new techniques are necessary for early detection of infection relapse.3 Chagas’ disease is an endemic cause of heart failure in South American.14 –18 Frequently asymptomatic infection flares and recurs after immunosuppression.16 –18 Even though the incidence of re-activation in infected patients who undergo immunosuppression therapy is unknown, the experience in heart transplant recipients shows the frequent presence of fever, subcutaneous nodules, and myocarditis.17 Serologic tests that indicate previous exposure to specific pathogens are useful in

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the pre-transplant period but are used rarely afterward. Transplant recipients who receive immunosuppressive therapy do not develop enough antibodies to establish serologic diagnosis of infection.17–19 Thus, tests aimed at detecting a pathogen’s nucleic acids, such as PCR, should be used.20 –22 In this study, we analyzed the use of PCR in the clinical follow-up of heart transplant recipients who had Chagas’ disease. Polymerase chain reaction results were positive 30 days before clinical onset in 3 patients; in 1 patient, PCR was the only method that provided a positive diagnosis. Nucleic acid detection of pathogen in blood, fluids, and tissues is a sensitive and specific method for diagnosing infection re-activated in immunosuppressed patients.19 We consider PCR reliable for monitoring patients at risk of Chagas’ disease re-activation after transplantation. Further investigation of larger populations are necessary to establish whether quantitative concentrations of T Cruzi in the blood predict clinical flares of Chagas’ disease. REFERENCES 1. Kirchhoff L. American trypanosomiasis: a tropical disease now in the United States (Chagas’ disease). N Eng J Med 1993;329:639 –44. 2. Moncayo A. Chagas’ Disease. Tropical Disease Research: 11th Program Report. Geneva, Switzerland: World Health Organization, 1993, p. 62. 3. Marin Neto J. Etiology and pathogenesis of Chagas’ disease. 2000 Up to Date. www.uptodate.com. 4. De Arteaga J, Massari P, Galli B, Garzo ´ n Maceda F, Zlocowsky J. Renal transplantation, and Chagas’ disease. Transplant Proc 1992;24:1900 –1. 5. Storino R, Jo ¨ rg M. Vı´as de infeccio ´ n y aspectos clı´nicos. In: Storino R, Milei J, eds. Enfermedad de Chagas. Doyma Argentina: Ed Mosby, 1995, pp. 1185–208. 6. Lugones H, Ledesma O, Storino R. Chagas agudo. In: Storino R, Milei J, eds. Enfermedad de Chagas. Doyma Argentina: Ed Mosby, 1994, pp. 209 –34. 7. Zhang L, Tarleton L. Parasite persistence correlates with disease severity and localization in chronic Chagas’ disease. J Infect Dis 1999;180:480 –6. 8. Mendes RP, Hoshino-Shimizu S, Da Silva A, et al. Serological diagnosis of Chagas’ disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi. J Clin Microbiol 1997;35:1829 –34.

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9. Solana ME, Katzin AM, Umezawa ES, et al. High specificity of Trypanosoma cruzi epimastigote ribonucleoprotein as antigen in serodiagnosis of Chagas’ disease. J Clin Microbiol 1995;33:1456 –60. 10. Oelemann W, Teixeira M, Da Costa G, et al. Evaluation of three commercial enzyme-linked immunosorbent assays for diagnosis of Chagas’ disease. J Clin Microbiol 1998; 36:2423–7. 11. Freilij H, Storino R. Diagno ´ stico de laboratorio. In: Storino R, Milei J, eds. Enfermedad de Chagas. Doyma, Argentina: Ed Mosby, 1994, pp. 343–58. 12. Moser D, Kirchoff L, Donelson J. Detection of Trypanosoma cruzi by polymerase chain reaction gene amplification. J Clin Microbiol 1989;27:1477–82. 13. Kirchhoff L, Votava J, Ochs D, Moser D. Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi. J Clin Microbiol 1996;34:1171. 14. Avila H, Pereyra JB, Thiemann O, et al. Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis. J Clin Microbiol 1993;31:2421. 15. Bocchi E, Bellotti G, Mocelin A, et al. Heart transplantation for chronic Chagas heart disease. Ann Thorac Surg 1996;61:1727–33. 16. Blanche C, Aleksic I, Takkenberg J. Heart transplantation for Chagas’ cardiomyopathy. Ann Thorac Surg 1995;60: 1406 –8. 17. Bocchi E, Bellotti D, Uip J, et al. Long-term follow up after heart transplantation in Chagas’ disease. Transplant Proc 1993;25:1329 –30. 18. Salomone O, Juri D, Omelianuk M, et al. Prevalence of circulating Trypanosoma cruzi detected by polymerase chain reaction in patients with Chagas⬘ cardiomyopathy. Am J Cardiol 2000;85:1274 –6. 19. Reilij H, Storino R. Chagas e inmunodepresio ´ n. In: Storino R, Milei J, eds. Enfermedad de Chagas. Doyma, Argentina: Ed Mosby, 1994, pp. 293–301. 20. Fishman Jay A, Rubin R. Infection in organ–transplant recipients. N Engl J Med 1998;338:1741–51. 21. Schijman AG, Vigliano C, Burgos J, et al. Early diagnosis of recurrence of Trypanosoma cruzi infection with polymerase chain reaction after heart transplantation of chronic Chagas heart disease patient. J Heart Lung Transplant 2000;11:1114 –7. 22. Chiari Egler. Chagas disease diagnosis using polymerase chain reaction, hemoculture and serologic methods. Mem Inst Oswaldo Cruz 1999;94:(suppl 1):299 –300.