Combined human cytomegalovirus and hepatitis C virus infections increase the risk of allograft vascular disease in heart transplant recipients

Combined Human Cytomegalovirus and Hepatitis C Virus Infections Increase the Risk of Allograft Vascular Disease in Heart Transplant Recipients B.Dal Bello, C. Klersy, P. Grossi, A. Gavazzi, G. Ippoliti, M. Rinaldi, M. Vigano´, and E. Arbustini

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ARDIAC allografts may undergo chronic rejection which is characterized by the development of an accelerated graft vessel pathology termed allograft vascular disease (AVD).1 Progression of the disease is usually much faster than is the case with spontaneous atherosclerosis, from which AVD differs in several morphologic features.2– 4 Despite improvements in early survival, AVD is the major cause of late death in recipients of cardiac allografts.5 Several immunologic,6 – 8 infectious,9,10 and metabolic10 –13 factors have been proposed as able to influence the occurrence of AVD: they may be related to donors (age, sex, body weight, and cigarette smoking),14 –16 to recipients before transplantation (age, sex, native heart disease, body weight, serologic human cytomegalovirus [HCMV] status, and lipid profile),11,12,17,18 and to the transplant itself (total graft ischemic time,14 number and severity of acute rejection episodes,6 – 8 HLA mismatches,7 number and nature of systemic infections,9,10 posttransplant lipid profile,12,13 and plasma levels of cyclosporine [CyA]19). Nevertheless, the multifactorial etiopathogenesis is still incompletely understood. Infections, particularly viral, have been shown in several reports to be associated with an increased risk of AVD9 –11,20 –24: HCMV systemic infection seems to be a major determinant of the risk,20 –24 and recently, hepatitis C virus (HCV) infection has been shown to affect about 11% of patients after transplantation25 and to cause vasculitis.26 We performed a multivariate statistical analysis aimed at investigating which, if any, immunologic, metabolic, infectious factors, particularly viral, may be associated with an increased risk of AVD. MATERIALS AND METHODS Clinical Series The series consists of 267 allografts from the 258 patients for whom coronary artery status (coronary angiography and/or pathologic study) was known. Of these 267 grafts, 252 were orthotopic, 5 heterotopic, and 10 were retransplants. There were 25 females and 233 males; mean age at transplantation was 49 6 10 years (range 9 to 66). Native heart disease was idiopathic dilated cardiomyopathy in 130 patients, ischemic “cardiomyopathy” in 104, and other heart diseases in 24 (19 cases of valvular heart disease, 2 cases hypertrophic cardiomyopathy, 1 case of cardiac amyloidosis, 1 case of restrictive cardiomyopathy, and 1 case granulomatous myocarditis).

Coronary tree anatomy status was derived from angiography in 203 of the 267 grafts, from pathologic examination at autopsy in 71, and at retransplantation in 13; for 20 of these grafts both angiography and postmortem or retransplantation histopathologic examination were available.

Immunosuppression Patients were immunosuppressed with a CyA-based regimen. Azathioprine and prednisone were also used as part of a triple-drug regimen with a policy of reducing the use of steroids in the long term. FK 506 was substituted for CyA27 in 11 of these patients.

Acute Rejection Acute rejection was diagnosed in accordance with the ISHLT grading system28; endomyocardial biospies (EMB) seen before publication of the above grading were reviewed and reclassified accordingly. For the present statistical analysis, acute rejection was evaluated in terms of the number of positive EMBs per patient. Biopsy monitoring, endomyocardial sampling, and sample processing were performed as reported in detail elsewhere.29 AVD was diagnosed on clinical data and/or coronary angiography and/or pathologic examination of the coronary trees.

Infections Before transplantation, all candidates were screened according to a standard infection-surveillance protocol.30 During the follow-up, HCMV infections were monitored by early and conventional virus isolation from peripheral blood leukocytes (PBL; viremia) and by direct antigen detection and quantification in PBL (antigenemia). Heparinized blood samples were collected weekly during the first 3 months after transplantation and at least twice a week if antigenemia and viremia levels were increasing. After 3 months, samples were collected monthly or when clinically indicated. During antiFrom the Department of Pathology (B.D.B., E.A.), Biometric Unit-Scientific Direction (C.K.), and the Departments of Infectious Diseases (P.G.), Cardiology (A.G.), Internal Medicine (G.I.), and Cardiac Surgery (M.R., M.V.), IRCCS-Policlinico San Matteo, Pavia, Italy. Supported by grant RF030RFM92/01 from the Health Ministry to IRCCS Policlinico San Matteo, Pavia-Ricerche Finalizzate 1994. Address reprint requests to Dr E. Arbustini, Istituto di Anatomia Patologica, Viale Forlanini 16, 27100 Pavia, Italy.

0041-1345/98/$19.00 PII S0041-1345(98)00583-1

© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

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Transplantation Proceedings, 30, 2086–2090 (1998)

ALLOGRAFT VASCULAR DISEASE viral treatment, heparinized blood samples were collected twice a week. HCMV infections in EMB were diagnosed on the bases of standardized criteria detailed in prior reports.31 Anti-HCV antibodies were detected by a second or third-generation enzyme immunoassay, HCV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR).32

Data for Statistical Analysis

Pretransplant and donor-recipient matching data. Recipient: age at surgery, sex, native heart disease, blood group (ABO, Rh). Donor: age, sex, blood group (ABO, Rh), cause of death. Donor/recipient: concordance of the blood groups (ABO and Rh), number of mismatches HLA-A, HLA-B, HLA-DR, HLA-A plus B, HLA-A plus -B plus -DR (HLA-A and -B mismatches were available for 250 grafts, HLA-DR for 98 grafts). Type of transplant (orthotopic, heterotopic, or retransplantation); time of ischemia between explantation from the donor and implantation in the recipient. Posttransplant data. (1) Pathologic: acute rejection: number of positive EMBs in the first 3 months of follow-up (254 grafts with at least one EMB), number of positive EMBs between the 3rd and the 12th month of follow-up (248 grafts with survival .3 months), number of positive EMBs after 12 months of follow-up (219 grafts with survival .12 months), total number of positive EMBs (calculated in 254 grafts), number of episodes of acute rejection additionally immunosuppressed, namely those $2 of the ISHLT grading (data available for 254 grafts); Quilty effect, both A and B types, in EMBs (information available for 254 grafts); type A ischemic damage of myocytes in early posttransplant EMBs (data available for 254 grafts); vasculitis in EMBs (data available for 254 grafts). (2) Clinical: systo-diastolic hypertension (systolic pressure .150 mm Hg; diastolic pressure .90 mm Hg) (data on 182 grafts with repeated consecutive controls); cholesterol plasma levels $220 mg/dL (data on 194 grafts with repeated consecutive controls); triglyceride plasma levels $200 mg/dL (data on 194 grafts with repeated consecutive controls). (3) Infections: bacterial, protozoal, or fungal, Toxoplasma gondii infections; viral infections: number of episodes of symptomatic HCMV infections, number of episodes of asymptomatic HCMV infections, total viral infection episodes (HCMV, HCV, Epstein-Barr virus [EBV], herpes simplex virus [HSV], varicella zoster virus [VZV]). (4) AVD presence or absence of AVD: as evaluated with coronary angiography (203 grafts) and with pathologic examination (at autopsy or at retransplantation; 84 grafts). Statistical Analysis Statistical analysis consists of descriptive statistics, calculation of AVD incidence rate, and of Poisson model multivariate analysis.

Incidence rate of AVD. After stratification for potential risk factors, incidence rates were calculated for the whole population. Rates are expressed as the number of AVDpositive patients per 1000 person-months; 95% CI for rates are reported as well. Given the low number of retrans-

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planted patients, these were considered as new subjects for the purposes of statistical analysis. Multivariate analysis. Factors to be included in the multivariate model were selected with the chi-square test for unequal rates. The subsequent multivariate analysis was based on a Poisson model. Variables significant at 0.1 at univariate analysis were included in the multivariate model. Backward stepwise selection identified the independent predictors of the AVD incidence rate (P value to enter and P value to stay were fixed at .15 and .2, respectively). In the final model, the relative risk of AVD incidence is reported, together with its 95% CI. The presence of influential observations was checked (and excluded) for Poisson model by means of delta-beta plots.33 STATA 5.0 (Stata Corp 1995, College Station, Tex) was used for calculations. RESULTS

AVD was diagnosed in 70 of the 267 grafts (26.2%). Diagnosis was done with angiography in 49 (later confirmed by pathologic study in 11), with pathologic study in 18, with clinical data in 3. These three patients died of anterolateral and anteroseptal acute myocardial infarctions (n 5 2) and of low output syndrome with inferior Q waves (n 5 1) at 57, 70, and 100 months posttransplantation, respectively. Analysis of AVD-Associated Risk Factors

The AVD incidence rate was estimated at 5.36 per 1000 person-months (95% CI 5 4.24 – 6.77). Variables considered as potential risk factors, as well as associated rates of AVD, 95% CI, and P value from the chi-square test unequal trends are listed in Table 1. Viral infections (association of HCMV and HCV), nonidiopathic dilated cardiomyopathy (DCM) native heart disease, number of mismatches for HLA-B, systo-diastolic hypertension, and the number of EMBs positive for acute rejection (seven or more) proved to be univariate risk factors and were included in the Poisson multivariate model. In the multivariate analysis, the association of HCMV and HCV infections multiplied the incidence rate of AVD by 3.9, with a 95% CI range from 1.2 to 12.4; systo-diastolic hypertension persistent after transplantation multiplied the incidence ratio by 2.2; two HLA-B mismatches by 2; seven or more EMBs positive for acute rejection by 2.2; and finally non-DCM native heart disease by 1.8 (Table 2). DISCUSSION

In our heart transplant population, we found a positive correlation between increased risk of AVD and viral infections (association of HCMV and HCV) as well as HLA-B mismatches, acute rejection positive EMBs, posttransplantation hypertension, and non-DCM native heart disease. The highest risk in our series was for viral infections, which included HCMV infections. HCMV infection-related risk was reported by Gao et al,9 Loebe et al,24 Stovin et al,23 Braunlin et al,22 Everett et al,21 and Koskinen et al.10

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BELLO, KLERSY, GROSSI ET AL Table 1. Rates of AVD: Factors Associated With AVD at Univariatei Analysis (Chi-Square Test for Unequal Rates) Variables

Viral infections 0 –1 2* Systo-diastolic hypertension no yes Diagnosis DCM Other HLA B Mismatches 0 –1 2 No. of EMBs with acute rejection 0–6 $7

Events

Person-Mo

Rates

95% CI

P Value

66 4

12873.0 195.0

5.127 20.512

4.028 – 6.526 5.590 –52.513

.004

41 20

9936.4 2464.0

4.126 8.117

3.038 –5.604 5.237–12.581

.011

27 43

6761.7 6306.2

3.993 6.819

2.738 –5.823 5.057–9.194

.027

14 53

3966.8 8725.7

3.529 6.074

2.090 –5.959 4.640 –7.951

.067

42 28

9387.8 3675.0

4.447 7.619

3.225– 6.047 5.261–11.035

.027

*Association of HCMV and CMV.

Conversely, Le Bidois et al,34 Gulizia et al,35 and Sharples et al36 did not confirm the association, but none of the above studies considered viral infections other than HCMV. In our series, HCMV infections by themselves did not increase the risk of AVD. Vice versa, when viral infections were evaluated combining both HCMV and HCV infections, the risk was multiplied by 3.8. None of the other viral infections (EBV, VZV, HSV) proved to be associated with an increased risk of AVD. Both HCMV and HCV viruses can affect the vascular wall: HCMV characteristically localizes and replicates in endothelial cells,37 while HCV causes vasculitis.38 There is definite evidence that HCMV selectively affects endothelial cells which are the site of latency, replication, and diffusion of the infection itself.39 In a prior study,37 we documented the occurrence of HCMV endothelialitis in transplanted patients with systemic major infection. The same observation applies not only to hearts but also to transplanted

lungs: HCMV-positive cytopathic cells may be seen in the lumen of the vessels.40 Circulating large cytopathic cells have been shown to be CD34- and factor VIII Ragpositive.39 A recent study reports that, in a series of 88 heart transplant recipients, 2.3% were anti-HCV positive prior to transplantation, 11.1% of the recipients with a follow-up $1 year seroconverted to anti-HCV, and 6.8% of the antiHCV-negative recipients were HCV-RNA positive. The combination of HCMV and HCV in the same patient may have a “malignant” effect and causes both endothelial cell damage and vascular wall inflammation.25 As far as other risk factors for AVD are concerned, our data on hypertension, HLA-B mismatches, number of acute rejection-positive EMBs, and non-DCM native heart disease merit a short comment. For systo-diastolic hypertension, results similar to ours were found by Radovancevic et al41 and Schutz et al,42 but not by Le Bidois et al,34 Gao et al,43 Eich et al,44 and Braunlin et al.22 This association is

Table 2. Rates of AVD: Multivariate Analysis (Poisson Model) Variables

Incidence Rate Ratio

Diagnosis Other DCM HLA B mismatches 2 0 –1 Systo-diastolic hypertension Yes No No. EMBs with acute rejection ($7) Yes No HCV and HCMV infections Yes No

1.758

1.042–2.967

.035

2.006

1.037–3.878

.039

2.210

1.281–3.813

.004

1.821

1.081–3.069

.024

3.861

1.202–12.406

.023

Note: Log likelihood 5 2153.525; x2 (5) 5 23.369; P value (model) 5 .0003.

95% CI

P Value

ALLOGRAFT VASCULAR DISEASE

not surprising, given that hypertension is a major risk factor for native coronary atherosclerosis. Any preexisting hypertension can be further worsened by the hypertensive effects of immunosuppressive drugs which may contribute to the severity of the vascular disease. However, hypertension represents a potentially reversible risk factor given that it can be controlled by appropriate drug strategies. Again, the risk of AVD was doubled in our series by a high number of HLA-B mismatches. Survival, rather than AVD itself, has been reported to be associated with a higher number of HLA mismatches.45 Given that AVD is a major factor affecting survival, the association between HLA mismatches and AVD also seems likely. Data on the association between AVD and HLA mismatches have been reported for HLA class II (DR) and HLA-B5 and B8.23 Conversely, one study46 reported that a high number of HLA mismatches is protective from AVD-related death but not from AVD itself. Other studies provided negative results.43,44 Our series, as are most other series,6,11 is small. The Opelz and Wujciak45 study clearly demonstrated that, in this type of analysis, a high number of observations is essential. In any case, current evidence supporting the hypothesis of an association between HLA mismatches and AVD is not conclusive. This topic deserves further investigation. AVD risk was also increased in patients with a high number of acute rejection-positive EMBs. These data, along with the forementioned association between risk and HLA diversity, confirm that AVD is influenced by immunologic factors. Most published data reported concordant results,6,8,34 while discordant data have been only occasionally reported.47 Although the debate is still open, most evidence supports the hypothesis that the risk of AVD increases in patients with numerous and/or severe acute rejection episodes. Non-DCM native heart disease seems to be a further determinant of increased risk of AVD; similar data have been reported in other series.12,16,18,36,48 Although older age was not a risk factor, our ischemic heart disease patients were older (52 6 8.2 years) than our nonischemic patients (46.7 6 11.8 years) (P 5 .000). They obviously have an atherogenic habitus which may persist after transplantation, and is not necessarily controlled by treatment or by modified lifestyle. Rather, it may worsen on account of the hyperlipidemic effect of immunosuppressive therapy. In the overall context of AVD risk factors, comparison with the literature is often difficult, since the statistical methods used vary substantially. Specifically, we considered time modeling through Poisson regression; therefore, parallelism with other series is meaningful only when time to AVD is taken into account in the assessment of the risk factor-AVD association. For a minority of factors, an almost universal agreement seems to be reached; for the remaining factors, the picture is confusing, with the results of each transplant center and/or study demonstrating a high degree of variability. This variability needs rationalization in the form of both a list of official risk factors and of

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standardized guidelines for angiographic or intracoronary ultrasound (ICUS) scheduling protocols. A major limit of our study lies in the method with which we assessed the presence of AVD in living patients: ICUS is proving to be much more sensitive than coronary angiography (80% versus 15%) in disease detection.11 However, this superiority in sensitivity applies to the detection of early AVD rather than to its severity. In the Rickenbacher et al.11 study, only 15 of the 67 (22.3%) ICUS-positive cases had angiographic luminal narrowing $30%. Accordingly, until ICUS is routine in allograft coronary artery study, the in vivo diagnosis of AVD in most heart transplant centers will continue with coronary angiography. CONCLUSIONS

Among factors increasing the risk of AVD in heart transplant recipients, viral infections, particularly HCMV combined with HCV, seem to be the major risk factor, multiplying the risk by 3.8. REFERENCES 1. Billingham ME: J Heart Transplant 11:S38, 1992 2. Billingham ME: Transplant Proc 27:2013, 1995 3. Arbustini E, Roberts WC: Am J Cardiol 78:814, 1996 4. Arbustini E, Dal Bello B, Morbini P, et al: Am J Cardiol 78:795, 1996 5. Hosenpud JD, Novick RJ, Bennett LE, et al: J Heart Lung Transplant 15:655, 1996 6. Hosenpud JD, Everett JP, Morris TE, et al: Circulation 92:205, 1995 7. Stempfle HU, Mudra H, Strom C, et al: Transplant Proc 27:1977, 1995 8. Hornik P, Smith J, Pomerance A, et al: Circulation 96 (suppl II): II148, 1997 9. Gao SZ, Hunt SA, Schroeder JS, et al: J Am Coll Cardiol 28:673, 1996 10. Koskinen PK, Nieminen MS, Krogerus LA, et al: J Heart Lung Transplant 12:724, 1993 11. Rickenbacher PR, Kemna MS, Pinto FJ, et al: Transplantation 61:46, 1996 12. Johnson MR: J Heart Lung Transplant 11:S124, 1992 13. de Lorgeril M, Boissonat P, Mamelle N, et al. Circulation 89:2590, 1994 14. Rickenbacher PR, Pinto FJ, Lewis NP, et al: Am J Cardiol 76:340, 1995 15. Ott GY, Herschberger RE, Ratkovec RR, et al: Ann Thorac Surg 57:76, 1994 16. Gao SZ, Hunt SA, Alderman EL, et al: J Am Coll Cardiol 29:623, 1997 17. Mehra MR, Stapleton DD, Ventura HO, et al: Circulation 90:I178, 1994 18. Wallwork J: Clin Transplant 8:341, 1994 19. Miller L, Kobashigawa J, Valantine H, et al: J Heart Lung Transplant 14:S227, 1995 20. McGiffin DC, Savunen T, Kirklin JK, et al: J Thorac Cardiovasc Surg 109:1081, 1995 21. Everett JP, Hershberger RE, Norman DJ, et al: J Heart Lung Transplant 11:S133, 1992 22. Braunlin EA, Hunter DW, Canter CE, et al: Circulation 84:III303, 1991

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BELLO, KLERSY, GROSSI ET AL 36. Sharples LD, Caine N, Mullins P, et al: Transplantation 52:244, 1991 37. Arbustini E, Grasso M, Diegoli M, et al: Am J Clin Pathol 98:205, 1992 38. McMurray RW, Elbourne K: Semin Arthritis Rheum 26:689, 1997 39. Percivalle E, Revello MG, Vago L, et al: J Clin Invest 92:663, 1993 40. Arbustini E, Morbini P, Grasso M, et al: Transplantation 61:418, 1996 41. Radovancevic B, Poindexter S, Birovlijev S, et al: Eur J Cardiothorac Surg 4:309, 1990 42. Schutz A, Kemkes BM, Kugler C, et al: Eur J Cardiothorac Surg 4:300, 1990 43. Gao SZ, Schroeder JS, Hunt SA, et al: J Heart Lung Transplant 12:1029, 1993 44. Eich D, Thompson JA, Ko D, et al: J Heart Lung Transplant 10:45, 1991 45. Opelz G, Wujciak T: N Engl J Med 330:816, 1994 46. Kerman RH, Kimball P, Scheinen S, et al: Transplantation 57:884, 1994 47. Cocanougher B, Ballantyne CM, Pollack MS, et al: Transplant Proc 25:233, 1993 48. Stovin PGI, Sharples LD, Schofield PM, et al: J Heart Lung Transplant 12:110, 1993