Clin Exp Immunol 1993; 94:306-312

Factors influencing the occurrence of active cytomegalovirus (CMV)infections after organ transplantation

G. J. BOLAND, R. J. HENE*, C. VERVERS, M. A. M. DE HAAN & G. C. DE GASTDepartment of Immuno-Haematology and *Nephrology, University Hospital Utrecht, Utrecht, The Netherlands

(Acceptedfor publication 3 August 1993)

SUMMARY

In this study, several factors influencing the occurrence of active CMV infection after organtransplantation (Tx) are analysed. For this purpose, 105 heart, kidney and lung transplant recipientswho were CMV-positive or had a CMV-positive donor, were closely monitored for active CMVinfection by antigenaemia, cultures, CMV serology and lymphocyte proliferation (LP) to CMV.Univariate and multivariate regression analysis were performed. As pretransplant risk factors theHLA-type and numbers of HLA mismatches between recipients and their donors, and the CMVserology of the recipient and donor were analysed. A new finding was that recipients of donorspositive for HLA-B7 were especially at risk for developing active CMV infection (P= 0 03) and CMVdisease (P= 0-03). This was not due to increased rejection treatment in these patients. Post-transplantrisk factors for development ofactiveCMV infection were absence ofdetectable cellular immunity toCMV (lymphocyte proliferation) after Tx (P< 001) and rejection treatment with OKT3 or ATG(P= 0-05). High levels ofIgG anti-CMV did not prevent occurrence ofactive CMV infection or CMVdisease in the CMV+ recipients.

Keywords cytomegalovirus transplantation organ transplantation risk factors

INTRODUCTION antigenaemia, viraemia and CMV excretion [13-15]. Immunityto CMV was measured by specific lymphocyte proliferation andActive CMV infections occur frequently after organ trans- aniCVgGndgM ntbie.Rslswraayedn

planatin [-41nd ay ead o te dvelomen ofCMV anti-CMV IgG and IgM antibodies. Results were analysed inplantation [1-4] and may lead to the development of CMV reaintrjciotetm tadHL tyefdnraddisease, which is associated with lower graft survival [4]. Risk . .factors for the development of active CMV infection are recipient.primary CMV infection in non-immune recipients [5,6], but alsoreinfection with donor CMV strain in immune recipients is PATIENTS AND METHODSassociated with a higher incidence of active CMV infection andCMV disease [7]. Patients

Several interactions ofCMV with the immune system have From May 1988 until February 1991, 33 heart, 106 kidney andbeen reported. In CMV-positive patients, active CMV infec- three lung transplantations were monitored for CMV infection.tions occur frequently after severe immunosuppressive therapy For analysis of factors influencing occurrence of active CMVwith anti-T cell antibodies [8]. Active CMV infection itself is infection and CMV disease, patients were divided into severalalso immunosuppressive in vitro [9] and in vivo [10], especially groups (Table 1). The results of the heart, lung and kidneyfor cell-mediated immune responses. Allogeneic reactions have transplants are shown together; in case of differences betweenbeen shown to induce active CMV infection in mice [11], and the transplants this is mentioned in the text.primary CMV infection was related to more rejection afterkidney transplantation, despite HLA matching [12]. To analyse Immunosuppressive treatmentfurther the relation of CMV with the immune system of the Maintenance immunosuppression consisted of cyclosporinerecipients, we evaluated the results obtained from the monitor- and steroids for most kidney recipients. Six of the 106 kidneying of 33 heart, 106 kidney and three lung transplant recipients. transplant recipients were treated with azathioprine and ster-Monitoring consisted of detection of active CMV infection by oids during CMV monitoring because cyclosporine was contra-

indicated. The heart and lung transplant recipients receivedCorrespondence: Greet J. Boland, Department Immuno-Haemato- triple therapy with cyclosporine, steroids and azathioprine.

logy, Bloodtransfusionlab. F03722, University Hospital Utrecht, Rejections were treated with high dose steroids (dexamethasoneP.O. Box 85 500, 3508 GA Utrecht, The Netherlands. 200 mg or methylprednisolone 1000 mg); severe and persistent

306

Risk factorsfor CMV infection 307

Table 1. Patient groups in relation to CMV infection

Active CMV infectiondetected by:

Group* CMV infection n M/F HTx/NTx/LTx LDt Ant Excr Both IgM

I Latent 26 18/8 7/19/0 3 0 0 0 4Actinf 40 29/11 9/29/2 3 15 5 20 6Disease 10 4/6 2/8/0 1 4 0 6 5

Total 76 51/25 18/56/2 7 19 5 26 15

II No inf 15 9/6 1/14/0 2 0 0 0 0Actinf 6 4/2 3/3/0 0 0 0 6 5Disease 8 6/2 2/6/0 0 4 0 4 8

III No inf 37 29/8 9/27/1 5 - - - 0

Total 66 48/18 15/50/1 7 4 0 10 13

* Group I, CMV-positive patients; Group II, CMV-negative patients with a CMV-positive donor; Group III, CMV-negative recipients with CMV-negative donors.

t LD, Living (related) kidney donor.t No inf, latent, no active infection detected by antigenaemia or cultures; act inf,

asymptomatic antigenaemia and/or cultures; ant, antigenaemia only; excr, CMV excretiononly detected positive; both, both antigenaemia and CMV excretion positive; IgM,IgM-positive.

rejections with OKT3. Also, ATG was given to some patients. which, in a comparative study, correlated with a four-foldRejection treatment with ATG was given to patients who were increase in IgG titres.transplanted in 1988 and in some cases of very persistentrejections when OKT3 had already been given. Lymphocyte proliferation

Lymphocyte proliferation was measured by incubation of 105Methods usedfor CMV monitoring peripheral blood mononuclear cells (PBMC), isolated by FicollCMV monitoring was performed at least every 2 weeks for 3 density centrifugation, with several antigens and mitogens.months or longer. In total, 1320 blood samples were tested for Isolated PBMC were washed and resuspended in RPMI 1640resp. antigenaemia and viraemia. The median number of (GIBCO, Uxbridge, UK) supplemented with glutamine (2 mM),samples tested was eight (range 4-20) per patient. On average penicillin (100 U/ml), streptomycin (100 ug/ml) and 15% pooled9-3 samples per patient were tested. Approximately 1300 urine human AB serum. CMV antigen was prepared from CMV-and saliva samples were cultured to detect CMV excretion. infected fetal fibroblasts (CMV-FF) by repeated freezing andCMV-negative recipients with CMV-negative donors were only thawing. Uninfected cells (FF) served as a control. PBMC (105)monitored with CMV serology. were incubated with 104CMV-FF and FF for 6 days. On the last

day, the cultures were pulsed with 3H-thymidine. After harvest-ing the cells, incorporation of3H-thymidine was measured using

CMV-antigen test and cultures a liquid scintillation counter. As a control, other mitogenicThe methods used to detect activeCMV infection by the antigen (phytohaemagglutinin (PHA) and concanavalin A (Con A)) andtest and cultures have been described before [13-15]. Detection antigenic (candida antigen, 104 herpes simpex virus I-infectedofCMV antigen in granulocytes by the antigen test is defined as FF and 104 varicella zoster virus-infected FF) stimulations wereantigenaemia. Positive buffycoat cultures are defined as virae- also measured. Lymphocyte proliferation tests with a stimula-mia, positive cultures of urine or saliva as CMV excretion. tion index of at least 3 (CMV-infected FF/uninfected FF) and

with a net pulse of at least 1000 ct/min (CMV-infected FF minusCMV serology uninfected FF) were considered positive.Anti-CMV IgG and IgM levels were measured using a commer-cially available ELISA (Sorin Biomedica, Saluggia, Italy), Definition of active CMV infection and CMV diseaseperformed and interpreted according to the manufacturer's Active CMV infection was defined as antigenaemia and/orinstructions. In short, quantitative results for IgG levels were CMV excretion. CMV disease was diagnosed when active CMVobtained by diluting the sera 1:400 and comparing the extinc- infection was present and the patients were at the same timetion measured with references. Results are expressed as relative suffering from two or more of the following symptoms: feverELISA units (REU). IgG was considered to be negative below (38 5°C for at least 2 days and not due to other causes),5 units; a significant rise in serum IgG was detected when values leucocytopenia or thrombocytopenia, elevated hepatic enzymeincreased within 2 weeks by at least 30 units and remained high, levels, kidney dysfunction, retinitis, pneumonia.

308 G. J. Boland et al.

HLA typing to CMV and rejection treatment, were analysed to determineHLA typing was performed on PBMC from patients and living their influence on the development of active CMV infection and(related) donors; from cadaveric donors spleen cells were used. CMV disease.HLA-A, -B and -C antigens were typed using the standard NIH First, a univariate analysis was performed for several of thelymphocytotoxicity assay, and HLA-DR antigens were typed risk factors. The results are shown in Table 2. The factors thatusing highly selected alloantisera by two-colour fluorescence proved to be significant, or surprisingly showed not to be ofassay, described by van Rood et al. [16]. significant influence, are specified below.

For the CMV-positive recipients, and the CMV-negativeStatistical analysis recipients separately, a multivariate analysis was performed forUnivariate and multivariate regression analysis of the factors the factors that proved of significant influence in the univariatethat influenced CMV infection and CMV disease were per- analysis. These results are shown below.formed. This was done for the CMV-positive and -negativerecipients separately. Pretransplant risk factors

We used the statistical program SPSS/PC+, version 4.01, Donor CMV serology. The frequency of active CMVfor the univariate logistic regression in both patient groups. A infection in CMV+ recipients was similar in the patients withmultivariate analysis of the significant factors for active CMV CMV-positive donors (21/30) as in those with negative donorsinfection and CMV disease was then performed for the CMV- (25/36, NS, x2, results not shown); from 10 donors CMVnegative recipients, and for the CMV-positive recipients with serology was unknown. CMV disease was found in 20% (6/30)CMV disease as dependent variable. In the CMV-positive where the donor was seropositive, compared with 8% (3/36)recipients, the severity of active CMV infection (positive where the donor was CMV-, but these differences were notantigenaemia, positive cultures, or both) was also taken into statistically significant.account. This multivariate analysis was performed using the HLA type. To determine whether CMV infection was relatedstatistical software program BMDP version 1990, as a polyto- to HLA specificities, incidence of active CMV infection andmous logistic regression with 'the severity of infection' as CMV disease was analysed in relation to HLA-A, -B, -C andordinal dependent variable. Confidence intervals (CI) were -DR typing.calculated if possible. In the CMV-negative recipients, in some The incidence of active CMV infection in the CMV+instances the patient group was too small to calculate the CI. recipients was higher in patients who were mismatched withFor further analysis, in some instances the X2-test was used as their donors for HLA-B7. The frequency of active CMVindicated. infection and disease was especially high in recipients from

HLA-B7-positive donors compared with recipients from HLA-B7-negative donors. The relation CMV infection and patient

RESULTS and/or donor HLA-B7 is shown in Table 3. Rejection treatmentDetection of active CMV infection with OKT3 or ATG, however, was given significantly lessIn 50/76 CMV+ recipients (Table 1, group I) an active CMV (P< 001, X2) in CMV-positive recipients from an HLA-B7-infection was detected. In the seronegative patients with sero- positive donor compared with those with an HLA-B7-negativepositive donors (group II) primary CMV infection occurred in donor. This last finding was also significant (P < 0-01, x2) when5/6 heart (83%) and 9/23 kidney recipients (39%), with corrected for the number of HLA antigens tested.antigenaemia in all primary infections. In the patients at risk of primary infection (Table 3b), 6/14

High antigenaemia (> 15 positive cells/105) was associated patients who had primary CMV infection were HLA-B7-with symptomatic infection; in 7/87 (8%) asymptomatic and positive, whereas none of the 15 patients in whom no primary8/18 (44%) symptomatic CMV infections, high antigenaemia CMV infection occurred were HLA-B7-positive (P=002).was present (P < 0-01, x2). Viraemia was present in 7/87 (80/;,) of Moreover, all three HLA-B7-positive patients with an HLA-B7-the asymptomatic infections and in 4/18 (22%) symptomatic positive donor developed CMV disease.infections (NS). An increased risk for developing active CMV infection in the

Positive IgM anti-CMV was detected in all but one of the CMV-positive recipients from HLA-B7-positive donors was notpatients with primary CMV infection. In CMV-positive reci- related to donor CMV serology; both in recipients from CMV-pients, IgM occurred more often in patients with CMV disease positive, HLA-B7-positive donors and in recipients from CMV-than in asymptomatic infections or when CMV remained latent negative, HLA-B7-positive donors, active CMV infection was

(5/10 compared with 10/66, P=0-02, X2). In four patients IgM frequently present. Also, lymphocyte proliferation (LP) towas found positive without antigenaemia-detection or positive CMV after Tx was comparable in CMV-positive recipients ofcultures. In these patients, we may have missed the antigenaemia HLA-B7-positive donors (10/21 with negative LP to CMV) andand/or cultures, or IgM was false positive. HLA-B7-negative donors (26/55 with negative LP). Neither

Whether or not IgM became positive was not dependent on were any differences observed between heart and kidneythe serum IgG levels in the CMV-positive recipients (results not transplant recipients.shown). Also, positive IgM occurred in patients at risk for Another significant relation between active CMV infectionreinfection (n = 6) as well as in CMV reactivations (n = 9). or disease was found with HLA-DRw6 (Table 2). This relation,

however, was not significant in the total patient group, only inAnalysis of risk factors for development ofCMV disease the CMV-negative recipients ofCMV-positive donors. Further-Pretransplant determined factors, e.g. CMV serology of the more, in the CMV-positive recipients of donors who werepatient and donor and HLA* type and compatibility of donor positive for HLA-DR2, CMV disease occurred slightly moreand recipient, as well as post-transplant factors, e.g. immunity often (Table 2).

Risk factorsfor CMV infection 309

Table 2. P values of the univariate analysis of risk factors

Total group CMV-negative rec. CMV-positive rec.

Act. inf. Disease Act. inf. Disease Act. inf. Disease

Tx-type 00004 NS NS NS 00002 NSLP 0 01 0001 - - 00004 0-002OKT3/ATG NS 0-004 NS NS NS 0007Steroids NS NS NS NS NS NSOKT3 or steroids NS 0-03 NS NS NS NSAge NS NS NS NS NS NS

Ganciclovir 0-03 <0-0001 0-03 0-0005 NS <0-000

HLA-B7Patient NS NS 0-02 0-02 NS NSDonor 0-03 003 0-02 0003 0-05 NSEither NS 0-05 0-02 0-02 NS NS

HLA-DR2Patient NS NS NS NS NS NSDonor NS NS NS NS NS 0-03

HLA-DRw6Patient NS NS 0-01 0-05 NS NSDonor NS NS 0-002 0.01 NS NS

HLA-DR7Patient NS NS NS NS NS NSDonor NS NS NS NS NS NSMismatches HLA-A NS NS NS NS NS NSMismatches HLA-B NS NS NS NS NS NSMismatches HLA-DR NS NS NS NS 0-006 NS

Total HLAMismatches 0-02 NS NS NS 0-02 NS

Number ofmismatches (Tables 2 and 4). Patients with active patients with negative LP to CMV (14/21) than in patients withCMV infection and CMV disease had a higher number of positive LP to CMV (6/19, P=003,x2).mismatches than those without infection (P=0 02). However, From all CMV-positive patients with negative LP to CMVthis did not reach statistical significance when the heart and after Tx, 86% developed an active CMV infection and 28%kidney recipients were analysed separately. The heart and lung CMV disease. From all CMV-positive recipients with positivetransplant recipients were less well matched with their donors LP toCMV after Tx, active CMV infection was detected in 48%,compared with the kidney recipients (P<0-01, X2). Anti- and none of these had CMV disease.rejection treatment, however, was not given more frequently to In all CMV-negative recipients LP to CMV was negativepatients who were less well matched with their donors. 2 weeks after Tx.

Rejection treatment. Only rejection treatment given withinPost-transplant risk factors 4 months after Tx was analysed (Tables 2 and 4) In the CMV-

Lymphocyte proliferation to CMVafter Tx. LP to CMV was positive patients, patients with CMV disease received signifi-measured in all patients 2 or 3 weeks after Tx, and before active cantly more often anti-T cell antibodies compared with theCMV infection occurred. patients in whom no active CMV infection developed. Rejection

In the CMV-positive recipients in whom CMV disease treatment with anti-T cell antibodies preceded active CMVdeveloped, none of the 10 patients had LP to CMV after Tx, infection in 13 CMV-positive patients, of whom three becamewhereas in the patients in whom no active CMV infection symptomatic. In another five CMV-positive patients anti-T celloccurred, 21/26 patients had LP to CMV after Tx (P <0-01, x2 antibody treatment was started during antigenaemia, and inTable 4). This was also significant for the heart (P=0 02) and three of these patients CMV disease developed. Four patientskidney (P= 0 01l) recipients separately. In the patients in whom were treated with anti-T cell antibodies, and no active CMVan asymptomatic CMV infection developed, 21 patients had infection occurred. Time-dependent analysis of the relationnegative and 19 positive LP to CMV. Within this group, between time of occurrence of active CMV infection and theantigenaemia as well as cultures were positive more often in time of rejection treatment did not reach statistical significance.

310 G. J. Boland et al.

Table 3. Relation between HLA-B7 and active CMV infection risk factor was donor HLA-DR2: P=0 03, CI 1 2-3-2. AlsoOKT3 or ATG treatment influenced the occurrence of CMVdisease in the CMV-positive recipients: P= 0-04, CI 1-34).

active CMV infection In the CMV-negative recipients, lymphocyte proliferationHLA-B7 Active CMV was not analysed, since this was negative for all patients. In thisrecipient/donor n infection* (%) OKT3 (%)t CMV disease(%) patient group, the multivariate analysis gave the following

significant relations with primaryCMV infection: patient HLA-a CMV-positive recipients B7 P=0-001 (CI 4 4-7 8), patient HLA-DR6 P=0 05 (CI 1-0-

47 18 (38) 17 (94) 5 (28) 45*0). The relation with CMV disease was strongest with donor+/- 8 3(44) 3(100) 0 HLA-B7: P=0003, followed by donor HLA-DR6 (P=001),

11 7(64) 2(29) 3(43) HLA-B7 patient (P= 001) and donor HLA-DR6 (P= 0007).+/+ 10 7 (70) 1 (14) 2 (29)

b CMV-negative recipients with CMV-positive donors Graftfailure within 6 months-/- 23 8 (35) 3 (38) 4 (50) In 7/29 CMV-negative kidney transplant recipients at risk for+1- 3 3(100) 1(33) 1(33) primary CMV infection, because they had a CMV-positive-/+ 0 0 0 0 donor, the kidney was removed. Five of these seven patients+/+ 3 3 (100) 3 (100) 3 (100) indeed had primary CMV infection, all of them symptomatic.

All primary infections in the heart transplant recipients were

Active CMV infection with antigenaemia and positive cultures, or asymptomatic or mild, and these patients all survived..,. . . ' ~~Graft failure was furthermore seen in one of the 26 CMV-antigenaemia and symptoms (in case ganciclovir was given before

positive cultures were detected) was significantly more often detected in positive patients who had no CMV infection, and in 6/10 CMV-CMV-positive recipients from HLA-B7-positive donors compared with positive patients who had CMV disease. Six ofthese were kidneyHLA-B7-negative donors (P=0 04) and in the total patient group recipients and one had received a heart transplant. Thus in the(P< 001). CMV-positive recipients as well as in the CMV-negative

f OKT3 treatment for rejection was given significantly less often in recipients, CMV disease was related to graft failure (P= 0 01,CMV-positive recipients from HLA-B7-positive donors compared with X2). Also, graft failure was more often seen in patients receivingHLA-B7-negative donors (P<0-01). more rejection treatment (P<0-01, x2). No relation between

graft failure and donor HLA-B7 was observed.LP to CMV was negative in 18/23 CMV-positive patients

treated for rejection with OKT3 or ATG and in 18 of the DISCUSSIONpatients who were not treated (P< 0-01, x2). High dose steroidswere given more frequently to patients in whom CMV disease In this study the relation of active CMV infection to immunitydeveloped (70%) compared with the patients in whom no active of the recipient to CMV has been analysed. All patients at riskCMV infection was detected (46%) or with asymptomatic for active CMV infection were closely monitored by antigenae-infection (55%). However, this did not reach statistical signifi- mia, viraemia, CMV excretion and anti-CMV IgG and IgMcance (Table 4). antibody titres. These methods are very suitable for monitoring

Humoral immunity to CMV after Tx. In the CMV-positive after transplantation, as described before [13-15]. Active CMVpatients without an active CMV infection, IgG levels 2 weeks infection and CMV disease occurred in CMV-negative, non-after Tx were on average 71 + 80 REU. IgG levels 2 weeks after immune, recipients, as well as in CMV-positive, immune,Tx in the CMV-positive patients in whom active CMV infection recipients. An increased incidence or severity in patients at riskwas detected later on, but in whom no disease developed, were for reinfection (CMV-positive recipients with positive donors),52+ 40 REU, and in the CMV-positive patients who had CMV as reported by others [7], could not be confirmed by us.disease 43 + 19 REU. These differences are not statistically Like Roenhorst et al. [17], we also found an increased risk ofsignificant. In all CMV-negative recipients IgG anti-CMV was CMV infection when donor and/or patient had the HLA-DRw6negative shortly after Tx. phenotype. However, this increased risk was only apparent in

the patients at risk of primary CMV infection or primary CMVResults of the multivariate risk analysis disease.To get more insight into the relation between the risk factors, a Reports of increased incidence of active CMV infection inmultivariate regression analysis was done. Seventy-four of the HLA-DR7 [18,19] positive recipients could not be confirmed inCMV-positive recipients were included in the multivariate our study. However, we found a relation between CMVanalysis with ordinal dependent variable the severity of CMV infection and another HLA specificity, HLA-B7. Active CMVinfection. In these patients, absence of LP correlated best with infection andCMV disease were present more often in recipientsactive CMV infection: P<0.0001 (CI 0-06-0 40), followed by who were mismatched with their donors for HLA-B7 (onlydonor HLA-B7 (P=0-02; CI 1 2-9 1) and the transplant type recipient or only donor HLA-B7-positive), or when both the(P= 0-03, CI 0 1-0-9). After this, the number ofmismatches was recipient and the donor were HLA-B7-positive. This was seen inno longer significant, indicating its relationship with the trans- primary CMV infection as well as in recurrent infection. Mostplant type (since kidney recipients were matched with their remarkable was that active CMV infections in recipients ofdonors). HLA-B7-positive donors occurred without previous rejection

Symptomatic CMV infections occurred in 1 1/74 patients. treatment. Since HLA-B7 is an important restriction element forHere, also, the strongest correlation wasfound with the absence several cytotoxic T cell clones [20-22] and in view of theof cellular immunity (P=0-001, CI 0-01-0 58). The following existence of several HLA-B7 subtypes [23], an explanation can

Riskfactors for CMV infection 311

Table 4. Number of HLA mismatches, lymphocyte proliferation to CMV 2weeks after Tx and rejection treatment

Number of HLA- Rejection treatmentmismatches LP to CMV pos

Groups* n (mean + s.d.) 2 weeks post-Tx Steroids OKT3/ATG

I Latent 26 1 7+1 7 21/26(81)t 12 4Act inf 40 2-3+1-6 19/40 (48) 22 12Disease 10 2-7+0-8 0/10 (O)t 7 6

II Noinf 15 15+1 1 0/15(0) 9 7Act inf 6 2-2+2-1 0/6 (0) 4 2Disease 8 2 1+15 0/8 (0) 4 5

* Group I, CMV-positive recipients; group II, CMV-negative recipientswith a CMV-positive donor.

t Lymphocyte proliferation (LP) to CMV was less in the patients in group Iwith CMV disease compared with group I who remained latently infected(P < 0-01). Numbers in parentheses are percentages.

be that the HLA-B7 subtype specificity may be important incellular immunity to CMV. The HLA-B7 phenotype may beespecially important as a restriction element for the recognitionof CMV peptides in the context of HLA, by cytotoxic Tlymphocytes. This could explain the phenomenon that inpatients who were mismatched with their donors for HLA-B7,CMV infection is more severe and rejection therapy has minorinfluence on this. When patients as well as their donors areHLA-B7-positive, the HLA-B7 subtype may be important.

An alternative explanation may be, in view of the existenceof cytotoxic T cell clones against HLA-B7 in vivo and theexistence of HLA-B7 subtypes [23], that allogeneic reactionsagainst donor HLA-B7 (subtype) in the graft enhanced activeCMV infection. Enhancement of CMV reactivation by allo-geneic reaction has been shown in mice [11]. In our study,patients who were less well matched with their donors had ahigher incidence of active CMV infection. However, hearttransplant recipients were the majority of patients with highnumbers ofHLA mismatches. Therefore, increased incidence ofactive CMV infection in mismatched recipients may reflectdifferences between heart and kidney recipients, and beside(mis)matching, other factors may also be involved, like anti-genic dose and immune suppression. However, in view of therelation we found between HLA-B7 and the number ofmismatches with the increased incidence of active CMV infec-tions, it is certainly worthwhile to investigate this further.Looking at the results in a time-dependent fashion, we found noevidence that the reverse, enhancement of rejection by activeCMV infection, occurred [12].

HLA type of donor and recipient were not the only factorsthat influenced occurrence of active CMV infection. Regardingpost-transplant immunity to CMV, cellular immunity proved tobe more important than humoral immunity to CMV in theCMV+ recipients for prevention ofactive CMV infection. CMVdisease occurred only during negative LP to CMV, and in noneof the patients with positive LP to CMV was active CMVinfection detected. The importance of cellular immunity for theprevention of active CMV infection and CMV disease has alsobeen noticed in pregnancy [24]. Presence of cellular immunity inthe mother during pregnancy prevented transmission and CMV

disease in the fetus. In our study in transplant recipients, not inall patients with negative LP to CMV after transplantation didactive CMV infection occur. This indicated that other factorsalso, for instance natural killer (NK) cell function, susceptibilityfor CMV virulence of the CMV strain and HLA type, may beimportant [25-27].

Whereas rejection treatment with high dose steroids had nosignificant influence on the occurrence of CMV infection,rejection treatment with anti-T cell antibodies increased the riskof active CMV infection, and also had a pronounced effect onLP. Increased incidence of active CMV infection and CMVdisease in patients treated with anti-T cell antibodies wasprobably due to decreased cellular immunity during and aftertreatment; in most patients, treatment preceded active infection.In patients in whom antigenaemia was detected and who weresubsequently treated for rejection, the incidence ofCMV diseasewas high. Negative lymphocyte proliferation to CMV was alsomeasured in some patients not treated for rejection with anti-lymphocyte antibodies.

High-levels of IgG after transplantation were not correlatedwith lower incidence of active CMV infection. We measuredIgG levels against a mixture ofcellular and viral CMV antigens.Levels ofCMV neutralizing antibodies or levels of IgG againstspecific CMV proteins may be more relevant for protectionagainst infection [28-90].

In conclusion, we found an increased incidence of activeCMV infection in patients with negative lymphocyte prolifera-tion to CMV 2 weeks after transplantation, in patients treatedfor rejection and in patients receiving an organ from an HLA-B7-positive donor.

ACKNOWLEDGMENTSThis study was supported by grant NHS 87087 from the Dutch HeartFoundation. With special thanks to G. Jambroes, R. A. M. G.Donckerwolcke and R. J. Hen6 for providing patients for this study.

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