Prophylaxis of cytomegalovirus in renal transplant recipients

AS MACDONALD, DL NICOL, P BELITSKY, S L EE. Prophylaxis of cytomegalovirus in renal transplant recipients. Can J Infect Dis 1993:4(Suppl C):51C-57C. The incidence and outcome of cytomegalovirus (CMV) infection and d isease is compared in renal transplant recipients in relation lo the use of prophylaxis wiU1 h igh Ulre anli-CMV immunoglobulin. Sevenly-iliree CMV-negalive recipients (R-) who received kidneys from CMV-posilive donors (D+) were given prophylactic CMV hyperimmune globulin inlravenously al three-week intervals lo six monilis . They a lso received Lhree months of oral low dose acyclovir as did lhe rema ining 288 palienls who did nol receive hyperimmune globu lin. There was a low incidence of CMV disease wh ich did nol di1Ter between groups (D+R-. 10%: D+R+. 5 .5%. DR+. 7%: DR-. 0.8%). The major risk factor was U1e use of OKT3 lo treat. rejection. CMV disease was seen in 22% of ilii group (ll of 50) . compared wiili on ly 2% (seven of3ll) of iliose nol requiring OKT3. There was only one CMV-relaled dealh. bul palienls wiU1 CMV disease had a reduced graft survival rate (62% versus 90%). CMV hyperimmune globuli n added lo acyclovir appears lo reduce ilie incidence of CMV disease in high risk renal recipients (D+R-) in Lhe lower risk groups.

. .C YfOMEGALOVlRUS (CMV) CONTINUES TO BE AN IMPORTANT cause of morbidity and graft loss in renal transplant recipients.Most commonly, quiescent virus is reactivated in previously infected patients under the influence of immunosuppression or is transmitted in the graft from a seropositive donor to a seronegative recipient.This latter group is most at risk because de novo infections in immunocompromised hosts are usually more severe and more lilcely to be lethal.Transplant patients developing CMV-induced disease are also more likely to suffer Joss of their grafts.
A variety of strategies has been employed to minimize the risks of CMV in transplant programs.A common approach is to avoid grafting from CMV seropositive donors to CMV seronegative recipients.Although this eliminates the group at most risk, it does not address the problem of reactivation disease.It also decreases the opportunity for seronegative patients to receive a graft.
Based on the observation that prophylactic treatment of transplant recipients with acyclovir to prevent herpesvirus infections is accompanied by a reduction in CMV disease.we have elected to give U1is agent to all our kidney recipients.In addition, the high risk group, ie.seronegative recipients of grafts from seropositive donors, also received intravenous immunoglobulin with a high titre of antibodies against CMV.
This report describes the results of this strategy on the incidence and severity of CMV infection and disease in patients receiving kidney transplants.

PATIENTS AND METHODS
Patient population: All 355 patients who received 361 renal transplants between January 1987 and July 1991 were included.The patients were divided into four groups based on the CMV status of donor and recipient before transplantation, ie, D+/R-(n=73).D+/R+ (n=9ll.D-/R+ (n=74) and D-/R-(n=123).Other donor and recipient characteristics were also compared (Table 1).
Anti-CMV hyperimmune globulin (CMV-HIG) (Cytomegalovirus-Hyperimmunoglobulin IV, Immuno.Vienna) was administered intravenously to all D+ j R-patients.CMV-HIG is a virus-inactivated, lyophilized concentrate of pooled human plasma obtained from donors reactive for CMV, with a standardized reconstituted concentration of anti-CMV antibodies of 50 U/mL.according to the reference preparation of the Paul Ehrlich Institute.Using a standardized complement fixation antibody test, the preparation was found to contain a CMV antibody titre of 1:128.Patients were given 1 mL/kg preoperatively and then at three-week intervals for six months.The six-month lime was arbitrary.but was felt to encompass the major at-risk period and overlaps the three months of acyclovir.
All patients.regardless of CMV status.received low close oral acyclovir 600 mg/ clay in divided doses for three months unadjusted for level of renal function.

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CMV serology: Pretransplant donor and recipient CMV status was determined using a passive latex agglutination assay (CMV Scan, Becton Dickinson).Post-transplant anli-CMV tilres from D+/R-patients were routinely determined using a standardized complement fixation antibody test on samples obtained immediately before each CMV-H!G treatment and again at one year.Other patients had similar post-transplant CMV serology performed only if clinical disease was suspected.The serology did not distinguish between immunoglobulin (Ig) G and lgM.Seroconversion was therefore defined as seropositive reactivity six to 12 months posttransplantation in recipients who were previously CMV seronegative, to exclude contamination with passively administered antibody.CMV disease diagnosis and treatment: All patients who developed a fever or in whom there was a clinical suspicion of CMV disease were investigated by serial CMV serology.This consisted of a complement fixation antibody test and serial cultures for CMV in Uiine, blood bufty coat specimen and sputum, using the shell vial culture technique (1).If kidney biopsy material was available, it was stained for CMV inclusions.
Clinical CMV disease was treated by supportive measures, antibiotic therapy against secondary inJections and reduction of immunosuppression.Since January 1990, patients with disease have been treated with intravenous ganciclovir at a dosage of 10 mg/kg/day for 14 days, adjusting U1e dose according to renal function.Definitions: CMV infection was defined as any one of: seroconversion of previously CMV-negative patient: a fourfold or greater rise in the serum antibody litre in a previously CMV-positive patient; culture of the virus from blood or other body fluids: or the presence of CMV inclusion bodies in histological sections of biopsy specimens.
CMV disease was defined as any combination of features defined under CMV infection plus clinically significant signs and/or symptoms of tissue invasion.Patients were classified according to severity of their CMV disease as follows: •  All but three patients received prednisone and CsA.CsA was initially administered by continuous intravenous infusion at a dosage of 3 to 4 mg/kg/ day for five days and subsequently orally at 10 mg/kg/day in two divided doses.Dosages were adjusted to maintain target radioimmunoassay whole-blood trough levels (Incstar) of approximately 300 mg/mL for the first 180 days and 100 lo 200 mg/mL thereafter.depending on efficacy and nephrotoxicity.Methyl prednisone 500 mg wa given intravenously at transplantation followed by oral prednisone ( 1 mg/kg/ day) which was tapered lo be discontinued at about 105 days.All patients received azathioprine 2 mg/kg intravenously during surgery.then 1.5 mg/kg/day orally until it was discontinued after 30 days.Patients who h ad received a transplant previously or who received a 'marginal ' kidney (donor age less than five years or more than 60 years.or extended ischemia) received a polyclonal equine antilymphocyte globu lin (ALG) (Lymphoglobulin.Merieux.Paris.France) 0.5 mL/kg/day intravenously loa maximum of 35 mL/day commencing immediately following transplantation.ALG was continued until serum creatinine fell below 300 J.lmol/L, al which time intravenous CsA was commenced as above.In addition , 57 recipients of first cadaver kidney transplants also received ALG as above before CsA as part of a randomized study comparing ALG with CsA for induction immunosuppression (2).In total.127 patients received ALG , with a mean duration of therapy of three days .Rejection episodes were all confirmed histologically by fine needle aspiration biopsy and/or minicore biopsy.Initial antirejection therapy consisted of a rapidly tapering high dose oral prednisone pulse (500 mg.250 mg, 125 mg.75 mg.50 mg.40 mg.30 mg, 20 mg on successive days) added to the maintenance prednisone regimen.In the absence of a clinical and biochemical re pon c by clay 5 of anlirejeclion treatment, biopsy was repealed to determine the cause of unr sponsiveness.Histologically confirmed steroid-resistant rejection was treated with OKT3 monoclonal antibody (Orthoclone, Orlho).5 mg/ day intravenously for 10 to 14 days.cllll;ng which time th CsA dose was reduced by 50o/o.During the first three days of OKT3 therapy, patients received methyl prednisone intravenously (500 mg. 100 mg and 100 mg) to minimize early side effects.

RESULTS
The four groups of patients had similar donor and recipient characteristics and immunosuppression as outlined in Table 1.

Relationship of CMV status to infection and disease:
The incidence of post-transplant CMV infection and disease according to pretransplant donor /recipient CMV serology is shown in Figure 1.The D+/R-gro up had a greater number of patients with a rise in CMV complement fixation antibody titre post transplant.both asymptomatic and associated with a transient unexplained fever.Of the patients who demonstrated e raconversional or before six months.all continued lo have positive CMV t.itres at 12 months.Statistical significance between the groups cou ld not be tested because routine serology was only performed in the D+/Rgroup.Among these patients, 38 of 73 (52%) seroconverted.
Only one of 123 D-/R-patients (0.8%) developed clinical CMV di ease.A significantly higher incidence (P<0.05) was seen in the three other groups.each of which had a comparable incidence of clinical CMV disease (D+/R-.lOo/o: D+/R+, 5.5%: and D-/R+, 7%).Disease severity and associated graft loss were likewise similar in these latter three groups (Figure 2).Of the 238 graft recipients in whom either the donor or recipi- Non OKT 3 Treated ents had positive serology and who would therefore be considered at risk, only 17 (7%) developed clinical CMV disease: 13 moderate (5.4%): three severe (1.2%); and one lethal (0.4%).The only CMV-related death was in a D+ /R-patient who developed CMV sepsis, despite clearing of the virus with gancic lovir therapy.Of four patients with severe disease, three had pneumonitis requiring ventilatory support, and the fourth had a bowel perforation requiring laparotomy and resection.
Of the remaining 13 patient with moderate disease.five developed pneumonitis; five had renal dysfunction or subsequent rejection: two had anemia, leukopenia or thrombocytopenia for wh ich another cause could not be found: and one had hepatitis with fever and a transient elevation in liver enzymes associated with a rise in CMV titres.Fewer than 10% of patients experienced initial nonfunction of the kidney, equally distributed among the four groups.There was no correlation between risk of CMV disease and initial function.Eleven of 50 patients requiring OKT3 rescue treatment for steroid-resistant rejections developed clinical CMV disease, compared with only seven of 311 patients who did not require OKT3 (P<0.001) (Figure 3).Of Lhe patients who received OKT3, the prior use of ALG , regardless of duration, did not increase the risk of CMV disease, which occurred in four of 24.Other viral diseases: No patient developed active herpes simplex or zoster infections, but four patients developed lymphoproliferative disorders probably related to Epstein-Barr virus infection .Al l four patients had received OKT3 for steroid-resistant rejection, with three receiving prior ALG (of whom two died).

Complications of prophylaxis:
No patient developed complications attributable to the use of either acyclovir or CMV-HIG.Patient and allograft survival: Post-transplant CMV disease was associated with significanUy reduced graft survival at one year (62% versus 90%, P<O.Ol), but patient survival was similar to that without CMV disease (97% versus 93%) (Figure 4).Pretransplant CMV status of donor and recipient, however, did not adversely affect either graft or patient survival (Figures 5 ,6).D+/R-patients actually had better one-and threeyear graft survival than the others (94% versus 87% and 86% versus 74%, P<0.05) .

DISCUSSION
CMV disease can be transmitted to recipients by the transplanted organ, especially if there is no prior immunity as measured by serum antibody.even in situations where antibody is present.Occasionally, this may b due to virus strain differences.Most commonly, if U1e patient is seropositive, the occurrence of disease after cu > •;;:  It is true, however, that reactivation disease occurs less commonly and is less severe in seropositive recipients than in seronegative recipients given an organ from a seropositive donor.Here seroconversion occurs in 50 to 85% and disease in 40 to 60% of recipients (3-9).Because of the high incidence of disease in lhe transplant population, a variety of approaches to prophylaxis has been tried.The use ofCMV-HIG as a single agent has been reported for a variety of organ transplants.These preparations are made from pools of sera from preselected donors with high titr s of CMV antibody which cover the spectrum of CMV strains.In a prospective randomized trial by Snydman et al (7) , the use of CMV-HIG without acyclovir did not alter the overall incidence or onset of CMV infection, but resulted in a threefold reduction in symptomatic infections.In the present study, with a much larger number of patients at risk, CMV seroconversion rates were similar (52% versus 68%) but the incidence of CMV disease in D+/R-kidney b•ansplants was less (lOo/o versus 20%).as was graft loss from CMV (3% versus The combined use of CMV-HIG and low dose acyclovir would therefore appear to inhibit the development of CMV disease in those at risk for primary infection, although it does not prevent seroconversion.Presenlly the mechanism of action of CMV-HIG in preventing CMV disease is unclear.Conceivably, CMV-HIG may form immune complexes with CMV, which in turn may augment some or all of the humoral or cell-mediated immune responses provoked by CMV activation as suggested previously (10,11).
Oral acyclovir has also been noted to reduce the incidence of CMV disease following transplantation (12).The authors have used low dose acyclovir, 600  mg/day , initially for herpes simplex virus prophylaxis (13).for which it has been highly effective.Conceivably.the low overall incidence (5%) of clinical CMV dis a e in the series under discussion may a lso be attributed to its use.Balfour at al (12). in a randomized , placebo controlled trial , demonstrated a reduction in CMV disease in renal transplant patients from 29% in controls to 7. 5% in those receiving high dose oral acyclovir.Th authors noted that the doses of acyclovir used in their study, 1800 to 3200 mg/day, resulted in p lasma concentrations lower than required for inhibition of CMV in vitro.
The present study suggests an additive affect of acyclovir and CMV-HIG in preventing CMV disease in USE D+/R-renal transplant recipients.The 10% incidence of CMV disease noted in the group who received both acyclovir and CMV-1-IIG was lower than the 20% reported in the studies using either agent alone (7,12).CMV-HIG has been observed to have a synergistic effect against CMV with ganciclovi.r(14).In trealing CMV pneumonitis in bone marrow recipients.a much higher response rate was noted when the two were used to-geUler than when either agent was used alone (15,16).
The intensity of immunosuppression contributes to U1e development of CMV disease alU1ough it is less frequent with CsA-based therapy (5.17).CsA may be less suppressive of CMV-specific cytotoxic T cell and natural killer cell responses.In addilion.lower doses of corticosteroids are used with CsA-based immunosuppression.and acute rejeclion episodes requiring additional immunosuppression are less common.Serious CMV disease is observed with much greater frequency in patients receiving anlilymphocytic serum preparations (6).In our series.the use of OKT3 in setting of rejection was a major risk factor for CMV disease.The incidence of 22% in patients receiving OKT3 for steroid-resistant rejection compares with rates of 20 to 59% reported in recent studies (18,19).The fact that prior treatment with ALG did not further increase the risk of CMV disease in our patients was surprising.but in keeping with a recent study by Chiu et al (20).It is not clear whether ALG or OKT3 are equally culpable in predisposing to CMV disease when used alone.Both ALG and OKT3 have been associated with a similar incidence of CMV disease when used for induction of inlmunosuppression (21).In contrast.when used as rescue therapy for refractory acute myocardial rejection, OKT3 has resulted in significantly more CMV disease and associated morbidity than ALG (22).Expe1imental evidence also suggests OKT3 may be a specific risk factor for CMV disease.Carrano et al (23) demonstrated that anti-anti-idiotypic antibodies, generated in some renal transplant recipients after receiving OKT3 for rejection, were specifically associated with the appearance of CMV disease.A subsequent study (24) demonstrated structural similarity between the T cell receptor /CD-3 complex and antibodies generated against CMV virion epitopes and may account in part for the association between the use of OKT3 for rescue therapy and CMV disease.

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Certainly.additional prophylactic measures appear indicated in OKT3-treated patients.Ganciclovir, a more effective agent than either acyclovir or CMV-HIG in treating established CMV disease (9).would appear to be an attractive option.Recent reports (25,26) have shown beneficial effects when used prophylactically in bone marrow transplantation, albeit at the cost of side effects such as leukopenia and thrombocytopenia (15. 16,25,26).
Acute rejection and graft loss are known to be more frequent in association with CMV disease (3,6,8.27).perhaps related to regulation of major histocompatibility complex class II anligens by viral-released gamma interferon (27).This increased graft loss was confirmed in our study, in which patients with CMV disease had a one-year graft survival of only 62% versus 90% for U1ose without CMV disease.With only one CMV-related death, however, patient survival was not reduced.Overall, graft and patient survival were comparable at one and three years between groups, with better graft survival in D+/R-recipients.This is consistent with recent data from the Collaborative Transplant Study (28).but contrasts with previously published studies (6,29) in which graft survival was significantly reduced in D+/Rkidney transplant recipients.A higher rate of clinical CMV disease in unprotected patients may explain the lower graft survival in the latter studies.although surprisingly.Opelz (28) found U1at the survival figures were only marginally improved by the use of prophylaxis against CMV infection.
In summary, the routine use of CMV-HIG in combination with low dose oral acyclovir allows CMV-negalive recipients to be safely transplanted with kidneys from CMV-positive donors.The incidence of early morbidity and graft loss due to CMV infection was similar to that in recipients who were CMV-posi.tivebefore grafting.
In addition, llie use of a low does acyclovir regimen (600 mg/day) appears effective in reducing the incidence of CMV disease in lower risk CMV-positive recipients.Despite the use ofCMV-HIG and/or acyclovir, the use of OKT3 for steroid-resistant rejection was a major risk factor for CMV disease with increased morbidity and graft loss.Additional prophylactic measures seem necessary in this group.Short term ganciclovir appears to only delay the appearance of disease, at least in cardiac recipients (30).but different drug regimens may overcome this.

Figure 1 )
Figure 1) Patterns of post-transplant cytomegalovirus (CMV) infection according to pretransplant donor and recipient CMV serology.Seroconversion is considered the L east serious manifestation.and clinical disease the most serious.D Donor: R Recipient

Figure 2 )
Figure 2) Severity of clinical cytomegalovirus (CMV) disease according to pretransplant donor and recipient CMV serology.D Donor: R Recipient

Figure 4 )
Figure 4) Comparison of graft and patient survival in patients with cytomegalovirus (CMV) disease to concurrent recipients without CMV disease.GS Graft survival: PS Patient survival: Tx Transplant