Viral and Toxoplasma gondii infections inchildren after liver transplantation

A Salt, G Sutehall, M Sargaison, C Woodward, N D Barnes, R Y Calne, T G Wreghitt

AbstractThe incidence and morbidity of viral andToxoplasma gondii infections were

studied in 40 children who underwentliver transplantation between December1983 and February 1988. The incidenceof primary and reactivated cytomegalo-virus (CMV) infection was 19% and 47%,respectively; primary infection causedclinical disease in all five cases affectedand was fatal in one. Primary Epstein-Barr virus (EBV) infection occurred in 10(26%) recipients but caused only milddisease. No reactivatedEBV infection wasrecorded and no lymphoproliferative dis-orders associated with EBV were foundafter amaximum offour years' follow up.Adenovirus infection occurred in seven

(18%) patients; this was associated in onecase with fatal pneumonia and fulminanthepatitis, but otherwise with only mildrespiratory disease. Primary T gondiiinfection was detected in one patient whoremained asymptomatic. Other virusescausing infection included herpes sim-plex, varicella zoster, and respiratorysyncytial virus.

Surveillance for these infections andthe long term sequelae should be includedin the follow up of all children whoundergo transplantation.

Public HealthLaboratory,Addenbrooke'sHospital, CambridgeDepartment ofPaediatricsA SaltN D BarnesDepartment ofSurgeryR Y CalneDepartment ofPathologyM SargaisonDepartment ofClinical MicrobiologyT G WreghittPublic HealthLaboratory, LeedsC WoodwardCorrespondence to:Dr T G Wreghitt, ClinicalMicrobiology and PublicHealth Laboratory,Addenbrooke's Hospital,Cambridge, England.Accepted for publication31 August 1989

Viral infections are a major cause of morbidityand mortality after organ transplantation,when cytomegalovirus (CMV), Epstein-Barrvirus (EBV), and adenovirus infections are

particularly common."2 CMV has been studiedextensively, and its potential for transfer withthe donor organ3 and for reactivation withimmunosuppression4 is well recognised in ren-

al,5 bone marrow, heart67 and liver transplantrecipients.8 EBV and adenovirus, although nottransmitted with the donor organ, have similarpotential and may cause serious disease afterorgan transplantation.9 There is also increasingevidence associating EBV infection with thesubsequent development of lympho-proliferative disorders. '°" Toxoplasma gondiican also be transmitted with the donor organ,

and fatal disseminated disease has beenreported after heart, renal, and liver transplan-tation in adults.'2-'4The impact of these infections in children

after liver transplantation has not been welldocumented, but the Pittsburgh group haverecently reported their experience." The

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Cambridge/King's College Hospital livertransplantation programme now includes morethan 70 children under 16 years. Here we

review our experience with CMV, EBV,adenovirus, other viral infections and Tgondii,and compare our findings with those in our

adult liver transplant recipients and with thoseof the children reported from Pittsburgh.

Patients and methodsBetween December 1983 and February 1988,63 children received liver transplants. Therewere sufficient serum samples available foranalysis from 40 (14 boys and 26 girls) whosurvived for four weeks or more after trans-plantation. At the time of transplantation theirages ranged from seven months to 17 years witha mean age of 6-7 years. Seventeen childrenwere aged 3 years or less. Twenty eight of 40patients had biliary atresia/hypoplasia, theremainder had a variety of conditions, includ-ing cx-1-antitrypsin deficiency and othermetabolic disorders. Five children received a

second transplant during this period. For two,who underwent a further transplantationwithin two weeks of the first, only the secondpostoperative period was included.

In all children intravenous cyclosporine wasstarted 48 to 72 hours after transplantation, in adose of2 mg/kg/day increasing to 4 mg/kg/day.This was changed to oral cyclosporine, 10 mg/kg/day, in the second week when the T-tubewas clamped. Methylprednisolone, 10 mg/kg,was given intraoperatively and prednisolone(2 mg/kg/day initially, reducing to 1 mg/kg/day by two weeks), and azathioprine (1 5 mg/kg/day) were started immediately after theoperation. Episodes of acute rejection were

treated with intravenous methylprednisolone,1-0 g/1-76 m2/day for three days. Immuno-suppression was reduced or temporarily with-drawn during episodes of severe infection.

Samples for serological analysis were takenbefore transplantation, then weekly through-out the hospital admission, and on subsequentfollow up visits. Serum samples were alsoavailable from 25 donors.

Serological tests were performed to detectantibodies to CMV, EBV, adenovirus, herpessimplex virus (HSV), varicella zoster virus(VZV) and Tgondii.The complement fixation test (CFT) was

performed as described by Bradstreet andTaylor,'6 with three 5000 haemolytic doses ofguinea pig complement to detect antibody to

CMV, HSV, VZV and adenovirus. Sera before

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Salt, Sutehall, Sargaison, Woodward, Barnes, Calne, Wreghitt

and after transplantation from each patientwere titrated in parallel.To detect CMV antibody in those sera with a

CMV complement fixation titre of less than 8,and in certain others which showed anomalousCMV complement fixation titres, the more

sensitive competitive enzyme linked immuno-sorbent assay (ELISA), as described byWreghitt et al,'7 was performed. Serum sam-

ples from patients who showed a > four-foldrise in CMV complement fixation titre, or whoseroconverted, were tested for CMV specificIgM by means of a p capture ELISA.'8Serum samples before and after transplanta-

tion were titrated for EBV anti-viral capsidantigen IgG by the indirect immunofluores-cence method of Henle and Henle'9 with theHRI-K subline of the P3J line of Burkitt'slymphoma cells.20 Serum samples were alsotested for EBV nuclear antigen (EBNA) anti-body in an anti-complement immunofluores-cence test2' and anti-EBV early antigen (EA)antibody in an immunofluorescence test22 usingP3HR-1 virus superinfection.2'

Antibodies to T gondii were assayed in thelatex agglutination test (Toxoreagent, EikenChemical Company of Japan), as described byBalfour et al.24Some sera were also tested for the presence of

antibodies to T gondii by the dye test and theindirect haemagglutination test. T gondiispecific IgM was detected by ELISA, as des-cribed by Payne et al.25Throat swabs, nasopharyngeal aspirates, and

urine samples were obtained for viral culturewhen clinically indicated.

INFECTIONPrimary infection was defined as seroconver-

sion from initially undetectable to detectableantibody titres to a specific viral antigen. Thisdiagnosis was supported where appropriate bydetection of specific IgM, and in many cases,

by virus isolation.Reactivated infection was diagnosed by a

four-fold (16-fold in the case ofEBV) or greaterrise in antibody titre.The clinical course of all patients was re-

viewed to correlate clinical symptoms and signsof infection with the laboratory data. Symp-tomatic viral infection was defined as a contin-uous or intermittent fever of > 38°C for at leastseven days, with no evidence of other microbialinfections or rejection episodes, in associationwith other clinical and laboratory evidence ofviral infection.

ResultsTwenty seven children had serologicalevidence of viral infection, with a total of 34infective episodes. Six children had more thanone viral infection (table 1).

CYTOMEGALOVIRUS (CMV)Of the 29 seronegative recipients, five (19%)developed primary CMV infection; all fivedeveloped CMV specific IgM (> 10 units).CMV was isolated from urine culture in threepatients.

Table 1 Patients with multiple infections

Case No CMV EBV Adenovirus HSV VZV

3 - + +5 + + - - -

6 - + + + -

14 - + + - -

24 + - - + -

31 + +

The donor CMV antibody status was notknown for all cases, particularly those whoreceived a transplant before 1986. Since thenthe importance of the donor organ in transmis-sion ofCMV has been recognised. Three of thefive children who developed primary CMVinfection received organs from CMV positivedonors. The donor CMV antibody status wasnot known for the other two. Only one childwho was CMV antibody negative, and wasknown to have received a CMV positive organfailed to acquire primary infection.

All children with primary CMV infectionhad clinically important disease. The mainclinical features of the infection are summar-ised in table 2. The three patients whodeveloped infection within one month of livertransplantation had severe disease. One childdied with pneumonitis and encephalitis 47 daysafter transplantation. Another, who hadreceived methylprednisolone for an episode ofacute rejection, subsequently developed CMVhepatitis; liver function was abnormal for sixmonths but has remained normal since. Thethird child developed moderately severepneumonitis and duodenal ulceration. Each ofthese children was treated with Cymevene(ganciclovir) soon after the onset of symptoms.

Reactivated CMV infection arose in eight(47 O%) of the 17 children who were seropositivebefore transplantation. In six this occurredwithin four weeks of transplantation during thetime of maximal immunosuppression, in one ateight weeks, and in the last at seven monthsduring a terminal episode of septicaemia. Onlytwo children were symptomatic. The symp-toms were mild and included fever of one tothree weeks' duration, anorexia and diarrhoea,coinciding with the rise in CMV complementfixation antibody titre, development of CMVspecific IgM (< 10 units), and isolation ofCMV from the urine.

EPSTEIN-BARR VIRUS (EBV)Of the 39 children from whom serum wasavailable, 22 were seronegative before trans-plantation. Of these, 12 showed a > 16-fold risein antibody to EBV viral capsid antigen. In twopatients who showed a minimum 16-fold rise in

Table 2 Primary CMV infection: clinicalfeatures

Case NoSymptoms 5 16 22 25 27

Onset (days after transplantation) 52 76 23 28 26Fever (> 38C) (No of days) 7 21 21 35 32Hepatitis - + - - +Pneumonitis - - + +Gastrointestinal haemorrhage - - + +Central nervous system + - - +Leucopenia + - + - -Thrombocytopenia +Death - - - +

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Paediatric viral and Tgondii infections

VCA IgG antibody, however, this could not beconfirmed in tests for viral capsid antigen IgM,EBNA, and EA tests. Ten (45%O) children,therefore, had confirmed primary infection.Their average age was 2-9 years (range 0-11years) and eight (73%) were under 3 years.

Six children had symptoms which coincidedwith seroconversion. The most commonfeature was prolonged fever of two to eightweeks' duration, with mild, non-specific con-stitutional upset. Concurrent mild deteriora-tion in liver function was noted in most cases.None of the children displayed the typicalfeatures of a mononucleosis syndrome withpharyngitis and lymphadenopathy, and noatypical lymphocytes were found. Infectionwas noted between three weeks and 13 monthsafter transplantation. Serological evidence ofEBV infection was found in three cases simul-taneously or within a few weeks of changes inCMV and adenovirus antibody titres, whichincreased the difficulty of ascribing symptomsspecifically to EBV infection.

Reactivated EBV infection was not found inany of the 17 children who had antibodies toEBV before transplantation. Follow up ofthesepatients (to September 1989) has shown noevidence oflymphoproliferative disorders. Thelongest period of follow up for patients withEBV infection in this series was four years andthe shortest nine months.

ADENOVIRUSAdenovirus infection was diagnosed on thebasis of a > four-fold rise in adenoviruscomplement fixation antibody titre togetherwith culture when available. Eight (19%) chil-dren showed evidence of infection; three hadserological evidence of previous infection.Infection arose late postoperatively, betweenthree and 12 months in all but two patients.Adenovirus was isolated and typed in only twocases because most patients were no longer inhospital at the time of infection.One child, from whom adenovirus type 7 was

isolated from a nasopharyngeal aspirate takenon the fourth day after surgery, was retrans-planted on day 14. She subsequently developeda high fever and interstitial pneumonia anddied of respiratory failure nine days after hersecond transplantation. Adenovirus type 7 wasisolated from lung, liver, spleen and kidney atnecropsy.Four other children had symptoms

associated with adenovirus infection. All hadfever of about one week's duration, coryza,pharyngitis and conjunctivitis. One child alsohad a prominent mobilliform rash.

HERPES SIMPLEX VIRUSThere were no primary infections with HSV inthis series, but three of the 10 children whowere seropositive preoperatively had sub-sequent reactivated HSV infection. Mucosalulceration was present in all cases. Two ofthesehad associated fever in the early period aftersurgery at four and eight weeks, respectively,and were treated with intravenous Zovirax(acyclovir). A significant (> four-fold) rise inHSV antibody titre was shown in all cases and

Table 3 T gondii antibody titres in one patient withprimary infection

Antibody titres

Transplantation LA test Dye test HA test IgM

Before < 8 < 8 < 32 -

After (day 21) 2048 8000 1024 198 units

LA = Latex agglutinationHA = Haemagglutination

HSV was isolated in two. In a fourth child HSVwas isolated without a noticeable change inantibody titre or symptoms.

VARICELLA ZOSTER VIRUSOne child developed chicken pox after contactwith a brother with the disease. She had twobrief grand mal convulsions, but no VZVantibody was found in the cerebrospinal fluid.The immunosuppression was interrupted; shewas treated with zoster hyperimmune globulinand acyclovir and recovered without sequelae.

RESPIRATORY SYNCYTIAL VIRUSRespiratory syncytial virus infection was foundin one child who developed moderately severebronchiolitis eight weeks after transplantation,10 days after recovering from EBV infection.

T GONDII INFECTIONTgondii infection was diagnosed serologicallyin only one patient who showed a > four-foldrise in latex agglutination T gondii antibodytitre (table 3). This child, who was T gondiiantibody negative before transplantation,received an organ from an antibody positivedonor. The rise in titre occurred three weekslater, one week after the withdrawal ofimmunosuppressive treatment because of a lefthepatic lobe abscess. This was after treatmentfor acute rejection with methylprednisolone.Treatment with pyrimethamine was given forsix weeks. There were, however, no clinicalsymptoms or signs which could be ascribed tothis infection.

DiscussionThe aim of this study was to assess the impactof viral and T gondii infections on ourpaediatric liver transplant recipients and todescribe some of the patterns of clinical diseaseassociated with these infections. Our findingswill be compared with those of other series, inparticular, those of Breinig et al.'5CMV is the most common viral cause of

morbidity in immunosuppressed patients,including all organ transplant recipients. Ahigh incidence of CMV infection has beenreported in adult series for kidney (50-96%),3 4liver (50-77%),' 26 and heart (48 %)12 transplan-tation. In our study CMV was responsible formost of the severe morbidity experienced andfor one of the two fatal viral infections.

Thirty per cent ofour study group developedCMV infection, and primary infection was seenin 19% (five of 26) of CMV antibody negativechildren. This is comparable with the findingsofBreinig et al"5 and also with those ofour adultliver transplant series (table 4). ReactivatedCMV infection was found in eight of 17 (47%o)

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Table 4 CMV infection: comparison between Cambridgeand Pittsburgh liver transplant recipients

Children Adults

Cambridge Pittsburgh Cambridge

Total No of patients 43 43 32Total No (0)

with CMV infection 13 (30) 13 (30) 15 (50)Total No (°h)

seronegative 26 (60) 35 (81) 12 (38)No (0o) seronegative

with CMV infection 5 (19) 6 (17) 3 (25)Total No (Oo)

seropositive 17 (40) 8 (19) 20 (62)No (0 ) seropositive

with CMV infection 8 (47) 7 (88) 12 (60)

of our antibody positive recipients, which iscomparable with the findings in our adultgroup (table 4). Children who had primaryCMV infection experienced the most seriousdisease, with one fatality. Only mild symptomswere experienced by the two symptomaticpatients who had reactivated infection.

In contrast, the Pittsburgh group found thatprimary CMV infection occurred less frequen-tly in their paediatric recipients than in adults.They also found a significantly higherincidence of reactivated CMV infection in bothchildren and adults.The incidence of CMV antibody increases

with age from 5-20% at 12 months,27 28 to 400oat 20 years, and 70-80% by 60 years.29 Thelikelihood of a child receiving an organ from aCMV seropositive donor is therefore less thanthat for an adult. This may change in the futurewith the increasing use in children of liverreduction operations using older donors: we donot have complete serological data for all don-ors in this series. The proportion of positivedonors, however, was 26% (five of 19) of thosetested and that of recipients 40%o (17 of 43).There is therefore a considerable risk ofCMVantibody negative children receiving organsfromCMV antibody positive donors and henceof serious donor acquired CMV disease inchildren. Every effort should be made to screendonors for CMV antibody and to avoid CMVmismatch. This has proved difficult in the pastbecause of the scarcity of child donors and theurgency of transplantation. The recentavailability of the CMV scan latex agglutina-tion test, however, (Becton-Dickinson), whichis reliable, rapid, and simple to perform, hasmeant that antibody testing can easily be donebefore donors and recipients are matched.30EBV infection has been increasingly recogn-

ised as a cause of morbidity after organ trans-plantation and also of long term sequelae in theform of lymphoproliferative disorders.'01 Ten(260%) of39 recipients in our series experiencedprimary EBV infection, which is less than theproportion reported by Breinig et al (24/42,57%)."5 Although the proportion of childrenwho were EBV antibody positive was similar inboth groups (43% v 50%), reactivated EBVinfection was not seen in our series butoccurred in 48% of children in the Pittsburghgroup."'A variety of lymphoproliferative disorders,

from transient non-malignant states to B celllymphoma, have been associated with EBVinfection and also with cyclosporine treat-

ment.3' 32 Ho et al also established that the riskis increased with primary infection.'0 Breinig etal described two children with lym-phoproliferative disorders, developing twomonths and two years after primary EBVinfection, one ofwhom developed an intestinallymphoma.'5 In our group, despite close followup of the children at risk following EBVinfection, no lymphoproliferative disorderswere noted during nine months to four years offollow up.

Eighty per cent (eight of 10) of the childrenin our series who experienced primary infec-tion were less than 3 years old. Hence this agegroup has a potentially increased risk of longterm sequelae resulting from primary EBVinfection.Adenovirus infection causes a wide spectrum

of illness in children.33 It has also beenrecognised as a cause of fulminant hepatitisand disseminated life threatening diseasein immunocompromised hosts, includingbone morrow, renal,3' and liver transplantrecipients.'°.We found adenovirus infection in eight of 43

(I9%) recipients, which is comparable with theincidence reported by Koneru et al of22/262 or8-4% in a large study ofadenovirus infection inpaediatric liver transplant recipients.9 Theirstudy relied on viral culture and isolationmethods alone; we used chiefly serologicalevidence, with viral isolation and thereforetyping in only two cases. Adenovirus types 1and 2 accounted for 60% of isolates in Kon-eru's study and were responsible only for mildillness. All five patients who experiencedadenovirus type 5 infection had severe fulmin-ant hepatitis, all following treatment for rejec-tion, and two patients died. Adenovirus type 7was isolated only in one case and was associatedwith only mild disease. In our study the onechild who had adenovirus type 7 infection hadsevere disseminated disease leading to fatalrespiratory and hepatic failure.Evidence of EBV infection in three cases,

and CMV in one, was found simultaneously orwithin four weeks of adenovirus infection,suggesting an association between these infec-tions in immunosuppressed patients. Such anassociation after organ transplantation has beendocumented.9 15

No specific treatment is available for adeno-virus infection, although some therapeuticmeasures have been reported.3536 We recentlyfound in vitro evidence of a response to ganci-clovir.36 Reduction of immunosuppressivetreatment is essential and, to date, the onlyeffective means of reducing morbidity.

Toxoplasma gondii has not been reported as acause ofmorbidity in children who have under-gone transplantation. In Cambridge thegreatest incidence of T gondii infection hasbeen found in heart transplant recipients.7 12Fifty seven per cent of those who were mismat-ched developed primary donor acquired infec-tion, two ofwhom died ofdisseminated disease.One of two cases in the adult liver transplantseries was also fatal.8 The child in our serieswho was infected with T gondii was asymp-tomatic. The mild course of the infection may

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be explained by the concomitant reduction ofimmunosuppressive treatment and pyrimeth-amine.

In conclusion, this study confirms thatCMVinfection is the principal cause of serious viraldisease in children after liver transplantation.EBV, adenovirus, and T gondii infection alsohave the potential for producing serious dis-ease. Surveillance for these infections and forlong term sequelae should be included in thefollow up of all children after transplantation.It is important to screen donors forCMV and Tgondii infection and to avoid donor/recipientmismatch whenever possible.

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