_J Clin Pathol 1995;48:351-357

Histological diagnosis of cytomegalovirushepatitis in liver allografts

F Colina, N T Juca, E Moreno, C Ballestin, J Farinia, M Nevado, C Lumbreras,R Gomez-Sanz

Department ofPathological Anatomy,Hospital Universitario12 de Octubre, Crta,Andalucia 5400, 28041Madrid, SpainF ColinaN T JuciC BallestinJ FarifiaM Nevado

Abdominal OrganTransplant ServiceE MorenoR G6mez-Sanz

Microbiology ServiceC Lumbreras

Correspondence to:Dr F Colina.Accepted for publication1 July 1994

AbstractAims-To determine the incidence ofhistologically documented cytomegalo-virus (CMV) hepatitis following orthotopicliver transplantation (OLT) and to assessthe effectiveness of immunohistochem-istry and in situ hybridisation (ISH) indetecting CMV. To describe the histo-logical pattern most frequently associatedwith CMV hepatitis in order to select thebiopsy group in which these modern tech-niques are most effective.Methods-A prospective histological studywas carried out on 853 biopsy specimens,obtained from 191 liver allografts (160patients). Specimens were stained withhaematoxylin and eosin and immuno-histochemically (avidin-biotin complex)using monoclonal antibodies directedagainst early and late CMV antigens. Aretrospective selection was made of 23specimens with viral inclusion bodies incytomegalic cells (group A) to characterisethe most frequently associated histologicalpattern, and of 34 other specimens with-out viral inclusion bodies (group B) butwith the same microscopic features asgroup A. Re-cuts from both specimengroups were studied using immuno-histochemistry and ISH with a CMV spe-cific complementary DNA probe.Results-CMV infection was confirmed in35 specimens (29 by immunohistochem-istry, 23 by presence ofinclusion bodies inhaematoxylin and eosin stained sections,16 by ISH) from 27 patients (incidence16.9%). CMV hepatitis was diagnosedwithin 46 + 19 (range 21-114) days post-transplant. Twenty one (91.3%) of the 23biopsy specimens with inclusion bodies(group A) displayed heterogeneous in-flammatory foci disseminated throughoutthe hepatic lobule. Nineteen specimens(82-6%) were positive by immunohisto-chemistry and 14 (60-9%) by ISH. In eight(23.5%) of the 34 group B specimens CMVinfection was confirmed by immuno-histochemistry (n=6) or ISH (n=2). An-other 12 (35.3%) ofthe group B specimensnegative on staining with haematoxylinand eosin, immunohistochemistry andISH came from allografts in which pre-vious or subsequent biopsy specimenswere CMV positive.Conclusions-Demonstration of cyto-megalic inclusion bodies in haematoxylinand eosin sections is sufficient for a diag-nosis of CMV hepatitis. The routine use

of immunohistochemistry in all allograftbiopsy specimens in more sensitive thandemonstration of inclusion bodies bystaining with haematoxylin and eosin butmay yield false negative results because ofthe focal distribution ofpositive cells. ISHwas less sensitive than staining withhaematoxylin and eosin and/or immuno-histochemistry. A histological picture of"disseminated focal hepatitis" withoutviral inclusion bodies selects a group ofallograft biopsy specimens in which im-munohistochemistry and/or ISH may im-prove detection of CMV.(JT Clin Pathol 1995;48:351-357)

Keywords: Cytomegalovirus, liver transplant, hepatitis.

Cytomegalovirus (CMV) is the opportunisticagent which causes greater morbidity than all ofthe viruses which infect immunocompromisedtransplant recipients.'` CMV infection maylead to allograft dysfunction in orthotopic livertransplants (OLT), and the incidence ofCMVallograft hepatitis has been reported at between4 and 34 6%.-12 Several factors" are re-sponsible for this wide range ofincidences. Theimmunosuppressive regimen,5 differences inthe proportion ofCMV seronegative recipientsof a liver from CMV seropositive donors,6 andthe use of antiviral prophylaxis are three of themost important factors.11

Allograft biopsy may be necessary for es-tablishing a diagnosis of CMV hepatitis'0 andthe presence of CMV in liver tissue can bedocumented by several histopathological tech-niques.'3 The demonstration of cytomegalicinclusion bodies in routine haematoxylin-eosin stained sections has been regarded as thegold standard for assessing the sensitivity ofimmunohistochemistry with anti-CMV anti-bodies, of in situ hybridisation (ISH) with aCMV specific complementary DNA probe,'4and of the rapid shell vial culture.6 13 Im-munohistochemistry and/or ISH can be usedroutinely, either separately or together, in liverallograft biopsy specimens. These two tech-niques may demonstrate the presence of thevirus in some cases in which its characteristicviral inclusion bodies are not detected by stain-ing with haematoxylin and eosin.'3-16 However,the routine use of these techniques in all liverallograft biopsy specimens is expensive andtime-consuming.One aim of this study was to evaluate com-

paratively the sensitivity and effectiveness ofeach of the three histopathological methods(staining with haematoxylin and eosin,

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Table 1 Incidence and number of days post-OLT to diagnosis of CMV hepatitis distributed by positive histopathology

No. ofMethod No. ofpatients Days post-OLT (range) specimens (s) Days post-OLT (range)Haematoxylin and eosin 20 44+11 (21-61) 23 (0 66) 43+10 (21-61)Immunohistochemistry 24 47+19 (23-114)* 29 (0 83) 59 +62 (23-373)Insituhybridisation 14 47+15 (25-88) 16 (046) 50+15 (25-88)Total positive 27 46+ 19 (21-114) 35 59+57 (21-373)

* If one patient is excluded (outlier from day 114), the x + a for IHC is 44 + 13 (range 23-73) (n = 23); s =sensitivity (number ofpositives/total number of positive biopsy specimens).

immunohistochemistry, ISH) for diagnosingCMV hepatitis in a series of liver allograftbiopsy specimens. In CMV hepatitis viralinclusion bodies are normally accompaniedby other microscopic lesions in immuno-competent and immunosuppressed patients.910Another aim was to describe what could bedefined as a histological pattern highly sug-gestive of CMV infection even in the absenceof the fundamental morphological lesion (viralinclusion bodies). Concentrating the use ofthese complementary techniques in biopsyspecimens showing this histological patterncould increase the number of CMV hepatitisdiagnoses less expensively than their routineuse in all liver allograft biopsy specimens.

MethodsOver a three year period, 244 consecutive OLTswere performed on a group of 198 patients.This study does not include those patients andgrafts surviving for less than 15 days or thosefor which follow up biopsy specimens were not

available to avoid diluting the true incidenceof CMV hepatitis in recipients survivingthrough the period ofgreatest risk. Patients whohad received prophylactic antiviral treatmentwere also excluded. Immunosuppressive ther-apy after transplantation was as follows: methyl-prednisolone, 200 mg/dl decreased graduallyto 30 mg/dl for one month; cyclosporin, atdoses adjusted to maintain its trough levelsat 200-300 mg/ml in blood, and azathioprine(2 mg/kg/day). Initial rejection episodes weretreated with three consecutive intravenousdoses of methylprednisolone (1 g). If no histo-logical response was obtained, OKT3 (OrthoPharmaceuticals, Raritan, New Jersey, USA)was administered intravenously every day for14 days.Liver biopsies were routinely performed im-

mediately after revascularisation of the liverallograft and two weeks after transplantation. Ifliver allograft dysfunction occurred, additionalbiopsy specimens were taken to establish adiagnosis. Liver biopsy was also carried out inthose cases in which no biochemical response

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Figure 1 Immunohistochemistry. A: Three positive cels associated with a histological picture of rejection and no lobularinflammatory infiltrate. Black arrow shows a positive, morphologically normal cell (x 40). B: Positive nuclear andcytoplasmic inclusions (open arrow). Note the high degree of the immunostaining in the "normal" nucleus in thisuncounterstained slide (x 40).

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was observed in liver function tests after anti-rejection treatment and occasionally as a con-

trol after normalisation of liver function tests.The histopathological material available for thisstudy came from 160 patients in whom a totalof 191 OLTs were performed, with a mean

histological follow up of 416+324 days (lastbiopsy), yielding a total of 1022 liver biopsyspecimens. The prospective study was carriedout on 853 percutaneous Menghini needleparaffin wax embedded liver biopsy specimensafter excluding 169 specimens obtained im-mediately after revascularisation. At least threehaematoxylin and eosin stained sections withmore than three portal tracts/biopsy were ex-

amined. Immunohistochemical studies were

performed with monoclonal anti-CMV anti-bodies (clone CCH2 which recognises earlyand late antigens (Dako-CMV, code M757;Gostrup, Denmark)) in a single section. Im-munohistochemical staining was carried outusing ABC standard techniques.1718 A sectionof lung with CMV pneumonitis served as a

positive control.Two groups were selected from among the

853 biopsy specimens taken: group A, 23 speci-mens previously displaying viral inclusion bod-ies were re-cut. Immunohistochemistry andISH with a CMV specific complementaryDNAprobe (Enzo-Pathogene, 32872; Syosset, NewYork, USA) were applied to this group, as

reported elsewhere. 9 Positive controls were

also used. The following histological featureswere tabulated to characterise the histologicalpattern most frequently associated with viralinclusion bodies9 02024: presence of dis-seminated inflammatory foci, types of foci clas-sified according to their inflammatory cell

population, diffuse sinusoidal lymphocytic in-filtration, presence of hepatocytic aniso-karyosis, hyperchromatism and anisocytosis,density ofthe inclusion bodies (number/haema-toxylin and eosin section), and type of cellinfected. The percentages of biopsy specimensfor which immunohistochemistry and ISHtested positive were evaluated. Group B: re-

cuts of the 34 biopsy specimens displaying theabovementioned histological patterns in theabsence of viral inclusion bodies were alsostudied using immunohistochemistry and ISH.

Conventional culture and rapid shell vialculture25 for CMV detection were carried outon biological products (blood, urine, exudates)of 82 patients (122 OLTs) (once a week untilday 60 and thereafter once a month or when-ever warranted by the patient's clinical con-

dition).

Statistical analysis-Results are presented as

mean + SD time of diagnosis. Univariate ana-

lysis was performed by the X2 test for categoricalvariables and Fisher's exact test when data weresparse. Comparative analysis of mean timesof first positive biopsy specimens by differenthistological techniques was performed by theKapan-Meier test (table 1); p< 05 was con-

sidered significant.

ResultsCMV was detected by one or more of the threemethods in 35 liver allograft biopsy specimensfrom 27 (16-9%) patients (table 1). The mean

time for the first diagnosis ofCMV hepatitis was46 + 19 days following surgery (range 21-114).Statistical comparison of the mean post-trans-

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Colina, Jucd, Moreno, Ballestin, Faninia, Nevado, et al

Table 2 Histopathological characterisation of CMV hepatitis

Group A Group BHistopathological features (n= 23) (%) (n=34) (%)Location of inclusions bodies

hepatocyte 23 (100)nuclear 3 ( 13-0)cytoplasmic 0 ( 0 0)both 20 ( 87-0)

biliary epithelium 4 ( 17-4)endothelial or sinusoidal cell 6 ( 26-1)

Disseminated focal hepatitis 21 ( 91-3) 34 (100)type of inflammatory cells in foci *mixed 17 ( 73 9) 17 ( 50-0)neutrophilic 8 ( 34 8) 12 ( 35 3)lipogranuloma 2 ( 8-7) 1 ( 2-9)granuloma 1 ( 4 3) 4 ( 11-8)pure lymphocytic 0 ( 0 0) 6 ( 17-6)

Other featuresdiffuse lymphocytic infiltration 1 ( 4 3) 3 ( 9*7)

anisocytosis 12 ( 52 2) 20 ( 58-8)anisokaryosis 9 ( 39-1) 13 ( 38-2)hyperchromatism 14 ( 60 9) 15 ( 44-1)mitosis 10 ( 43 5) 11 ( 32 4)

* Several biopsy specimens showed more than one type of inflammatory foci in their disseminated focal hepatitis pattem.

Table 3 Distribution ofpositive biopsy specimens by positive cell densityHaematoxylin and eosin Immunohistochemistry In situ hybridisationn(%) n(%) n(%)

Total number of positive biopsy specimens 23 (100%) 29 (100%) 17 (100%)<5 cells/biopsy 12 ( 52-2) 16 ( 55 2) 11 ( 64 7)5-10 cells/biopsy 7 ( 304) 3 ( 10-3) 4 ( 23 5)>10 cells/biopsy 4 ( 17-4) 10 ( 34 5) 2 ( 11-8)

plant time (Kaplan-Meier test) for the initialdiagnosis of CMV hepatitis by each of thetechniques did not reveal significant differences(table 1). Recurrence of CMV hepatitis wasobserved (by immunohistochemistry) in onepatient at day 373 post-transplant (333 daysafter a previous biopsy had confirmed CMVhepatitis, but the next three biopsy specimenswere negative for CMV). Another patient wasonly diagnosed as CMV positive by im-munohistochemistry in a biopsy specimentaken on day 114 post-transplant. Excludingthis outlier, the initial time of diagnosis byimmunohistochemistry was 44 + 13 days (range23-73), which was not statistically significantwith respect to the mean time for diagnosingthe total series (table 1).Of the 35 biopsy specimens with CMV doc-

umented by one of the three methods, 29were positive by immunohistochemistry (sen-sitivity = 0-83) and 23 (group A) demonstratedviral inclusion bodies in the haematoxylin andeosin stained sections (sensitivity= 0 66) (table1). It should be noted that in the prospectivestudy of 853 biopsy specimens, immuno-histochemistry was positive in four specimensin which staining with haematoxylin and eosindid not detect viral inclusion bodies and whichdid not have the inflammatory lesion describedin groups A or B. Of the 23 group A biopsyspecimens, immunohistochemistry was positivein 19 (82-6%). The inclusion bodies displayedin these new sections appeared to stain im-munohistochemically but morphologically nor-mal cells also stained occasionally (fig 1). Infour ofthe specimens the new sections to whichimmunohistochemistry was applied displayedneither inclusion bodies nor morphologicallynormal positive cells and were regarded as

false negative results. Immunohistochemistrywas positive in two patients without liver dys-function, whose allografts had been biopsiedto check for resolution of cellular rejection, butno positive culture developed in blood, throatand bronchoalveolar exudates, or urine samplesobtained at the same time as the graft biopsy.Rejection induced cellular infiltration dis-appeared after additional immunosuppressivetreatment and only the finding of CMV an-tigens in liver tissue by immunohistochemistrycould be considered pathological. ISH waspositive in 14 (60 9%) of the 23 group Aand in two of the group B specimens. Thistechnique also demonstrated, although veryinfrequently, positive cells without mor-phological lesions (cytomegalic and/or in-clusion) in both groups (fig 2).The histological characteristics of the 23

group A specimens are detailed below (table2). Viral inclusion bodies: hepatocytes werebeing most frequently affected with nuclear(100%) and/or cytoplasmic inclusion bodies(87%). The density of inclusion bodies perbiopsy varied widely (52% of the specimenspresented with less than five cells with viralinclusion bodies per histological section) (table3). Demonstration of a single inclusion bodyoccurred regularly (five specimens). Numerousinflammatory foci scattered throughout theliver tissue (termed disseminated focal hep-atitis) (fig 3) were present in 91% of the groupA specimens (table 2). The inflammatory fociwere sometimes located around cells carryingviral inclusion bodies but often no such prox-imity to these cytomegalic cells was evident.Cells with inclusion bodies not surroundedby inflammatory infiltrate were also observed.Three types of foci were characterised (fig 4):

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pure polymorphonuclear leucocytes, mixedpolymorphonuclear leucocytes and round cells

(macrophages, lymphocytes, plasma cells), andgranulomatous foci. In 34-8% of cases co-

incident foci of these three types could beobserved heterogeneously distributed in thesame section with no defined preferential zonallocation in the hepatic lobule. Anisocytosis,anisokaryosis, hyperchromatism, and mitosisof the hepatocytes were frequent histologicalchanges accompanying the inflammatory histo-logical pattern. Diffuse sinusoidal lymphocyticinfiltration with a mononucleosis-like patternwas observed in only one specimen in thisgroup (table 2).

In group B specimens disseminated focalhepatitis was detected a mean of 69 + 63 dayspost-transplant, which was not significantlydifferent to that for the 35 CMV positive speci-mens (59 + 57 days) (table 1). Nor were therestatistically significant differences (X2 test) be-tween the incidence of the histological featuresobserved in groups A and B (table 2). Eight(23-5%) of the 34 biopsy specimens with dis-seminated focal hepatitis without viral inclusionbodies were positive by immunohistochemistry(n=6) or by ISH (n= 2). Another 12 (35.3%)specimens from this group were taken fromallografts which were CMV positive in previousor subsequent biopsy specimens.

DiscussionCMV hepatitis was defined as invasive CMVinfection of the liver with histological evidenceof a viral cytopathic effect.'2 It is a common

cause of complications among immuno-

suppressed OLT recipients.5 Liver biopsy is animportant tool for diagnosing the causes ofliver allograft dysfunction and can differentiateCMV hepatitis from cellular rejection. Othermicroscopic diagnoses are possible as very littleoverlap exists between the inflammatory re-

sponse provoked by CMV and other sim-ultaneous processes.'0 26 The incidence ofCMVhepatitis in the series studied here was 16-8%.The period ofmaximum risk was within 46 + 19days post-transplant. Recurrence ofCMV hep-atitis was rare (one case), as was late onset (onecase: day 114). The presence of viral inclusionbodies in liver tissue correlated with activedisease in most cases, as has been reported inother series.6 Histological study has also beenreported to offer greater sensitivity than rapidshell vial culture,'3 however, Paya et al6 detectedCMV hepatitis on culture of biopsy specimens(viral inclusion bodies present) but these same

specimens were negative on histology. Thisapparent anomaly could be explained by thefocal and scarce distribution of infected cellsin some of the biopsied allografts (table 3).

Ifused on all liver allograft biopsy specimens,immunohistochemistry is more sensitive thanhistological demonstration of inclusion'3 16 (29v 23 in this study). Unlike other studies,'527in this series immunohistochemistry did notprovide early warning ofCMV infection, whichcan be attributed in part to the focal and scarce

distribution of positive cells on immuno-histochemistry. The possibility of easily study-ing more re-cut sections by staining withhaematoxylin and eosin than just one or twoby immunohistochemistry and ISH could alsoexplain the false negative results produced with

4'4-.1-...

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the latter two methods. In some series,28 as wasthe case in ours, ISH has found to be lesssensitive,'419 although its use may increase thenumber of diagnoses. One possible explanationof the greater sensitivity of immunohisto-chemistry compared with ISH is that the formercan detect more cells harbouring viral products(antigens), which could be more numerousthan cells holding viral DNA.'213 ISH, on theother hand, demonstrates viralDNA and there-fore identifies cells with active replication.'422Intracellular or extracellular influences (suchas oestrogens) can modulate or alter the CMVreplication cycle and thus explain why not allinfected cells host full viral replication and/orharbour inclusion bodies.2930The indiscriminate application of all of these

diagnostic methods to liver allograft biopsyspecimens is time-consuming and expensive.The disseminated focal hepatitis pattern, suchas the one evaluated in this study, could beuseful as a basis for selecting biopsy specimensfor subsequent examination using immuno-histochemistry and/or ISH and serve as a guide-line for histopathology laboratories. As was thecase in this study, Paya et al'3 have reportedbiopsy specimens with a disseminated focalnecrosis pattern accompanied by CMV vir-

emia, although they could not always confirmthe presence of CMV in the tissue using im-munohistochemistry. The presence of so-calleddisseminated focal hepatitis on histology shouldspark a search for CMV. An algorithm is pro-posed for determining when such techniquescan be effectively used (fig 5). The time post-transplant during which patients are at in-creased risk of CMV infection (46 + 19 post-transplant days) is another parameter whichshould be borne in mind when deciding on theadditional use of ISH and/or immunohisto-chemistry on histological sections.Demonstration ofinfected liver allograft cells

using immunohistochemistry in the absence ofallograft dysfunction or a histological in-flammatory lesion and negative on culture,raised questions as the validity of certain ac-cepted methods of diagnosing CMV hepatitis.The great sensitivity of immunohisto-chemistry` and other new techniques such asPCR'2 for detecting antigens or genomes oflatent viruses (permanent infection withoutreplication or disease) makes it advisable thatthe clinician be informed as to whether or notCMV positive results on the above methodscoincide with microscopic lesions on stainingwith haematoxylin and eosin.

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Histlogical diagnosis of cytomegalovinus hepatitis in liver allografts

CMV cultures and serology?CMV infection risk period?Other opportunistic viruses?Other histological diagnoses?

Figure S Proposed algorithm for diagnosing CMV hepatitishaematoxylin and eosin.

This work was supported by a research grant (94/0242) fromthe Fondo de Investigaciones Sanitarias, Ministry of Health,Madrid, Spain. N T Juca was sponsored by the ConselhoNacional de Desenvolvimento Cientifico e Tecnol6gico de Bra-sil. Preliminary results were presented in 1993 at the XVINational Congress of the Sociedad Espafiola de AnatomiaPatol6gica, Tenerife, Spain.

1 Ho M. Human cytomegalovirus infections in im-munosuppressed patients. In: Cytomegalovirus. Biology andinfection. New York: Plenum Press, 1991:249-300.

2 Ho M. Cytomegalovirus infection and indirect sequelae inimmunocompromised transplant patients. Transplant Proc1991;23 (Suppl 1):2-7.

3 Rubin RH. The indirect effects of cytomegalovirus infectionon the outcome of organ transplantation. JrAMA 1989;261:3607-9.

4 Rubin RH. Impact of cytomegalovirus infection on organtranplant recipients. Rev Infect Dis 1990;12 (Suppl 7):754-66.

5 Stratta RJ, Shaeffer MS, Markin RS, Wood RP, LangnasAN, Reed EC, et al. Cytomegalovirus infection and diseaseafter liver transplantation. An overview. Dig Dis Sci 1992;37:673-88.

6 Paya CV, Hermans PE, Wiesner RH, Ludwig J, SmithTF, Rakela J, et al. Cytomegalovirus hepatitis in livertransplantation: Prospective analysis of 93 consecutiveorthotopic liver transplantations. Y Infect Dis 1989;160:752-8.

7 Rakela J, Wiesner RH, Taswell HF, Hermans PE, SmithTF, Perkins JD, et al. Incidence of cytomegalovirus in-fection and its relationship to donor-recipient serologicstatus in liver transplantation. Transplant Proc 1987;19:2399-402.

8 Singh N, Dummer JS, Kusne S, Breinig MK, ArmstrongJA, Makowka L, et al. Infections with cytomegalovirusand other herpes viruses in 121 liver transplant recipients:transmission by donated organ and the effect of OKT3antibodies. Y Infect Dis 1988;158:124-31.

9 Snover DC, Hutton S, Balfour HH Jr, Bloomer JR. Cyto-megalovirus infection of the liver in transplant recipients.Y Clin Gastroenterol 1987;9:659-65.

10 Bronsther 0, Makowka L, Jaffe R, Demetris AJ, BreinigMK, Ho M, et al. Occurrence of cytomegalovirus hepatitisin liver transplant patients. J Med Virol 1988;24:423-34.

11 Paya CV, Wiesner RH, Hermans PE, Larson-Keller nI,Ilstrup DM, Krom RAF, et al. Risk factors for cyto-megalovirus and severe bacterial infections following livertransplantation: A prospective multivariate time-de-pendent analysis. Y Hepatol 1993;18:185-95.

12 Stratta RJ, Shaefer MS, Markin RS, Wood RP, KennedyEM, Langnas AN, et al. Clinical patterns of cyto-megalovirus disease after liver transplantation. Arch Surg1989;124: 1443-50.

13 Paya CV, Holley KE, Wiesner RH, Balasubramaniam K,Smith TF, Espy MJ, et al. Early diagnosis of cyto-megalovirus hepatitis in liver transplant recipients: Roleof immunostaining, DNA hybridization and culture ofhepatic tissue. Hepatology 1990;12:119-26.

14 Naoumov NV, Alexander GJ, O'Grady JG, Sutherland S,Aldis P, Portmann BC, et al. Rapid diagnosis of cyto-megalovirus infection by in-situ hybridisation in livergrafts. Lancet 1988;i:1361-4.

in liver grafts. IHC, immunohistochemistry; H and E,

15 Theise ND, Conn M, Thung SN. Localization of cyto-megalovirus antigens in liver allografts over time. HumPathol 1993;24: 103-8.

16 Niedobitek G, Finn T, Herbst H, Gerdes J, Grillner L,Landqvist M, et al. Detection of cytomegalovirus by insitu hybridisation and immunohistochemistry using newmonoclonal antibody CCH2: a comparison of methods.J Clin Pathol 1988;41:1005-9.

17 Hsu SM, Raine L, Fanger H. Use of avidin-biotin-per-oxidase complex (ABC) in immunoperoxidase techniques:A comparison between ABC and unlabeled antibody(PAP) procedures. JHistochem Cytochem 1981;29:577-80.

18 Zweygberg-Wirgart B, Landqvist M, Hokeberg I, ErikssonBM, Oldin-Stenkvist E, Grillner L. Early detection ofcytomegalovirus in cell culture by a new monoclonalantibody CCH2.J Virol Methods 1990;27:211-19.

19 Masih AS, Linder J, Shaw BW Jr, Wood RP, Donovan JP,White R, et al. Rapid indentification of cytomegalovirusin liver allograft biopsies by in situ hybridisation. Am JSurgPathol 1988;12:362-7.

20 Reller LB. Granulomatous hepatitis associated with acutecytomegalovirus infection. Lancet 1973;i:20-2.

21 Bonkowsky HL, Lee RV, Klatskin G. Acute granulomatoushepatitis: occurrence in cytomegalovirus mononucleosis.JAMA 1975;233:1284-8.

22 Ten Napel CHH, Houthoff HJ, Teh TH. Cytomegalovirushepatitis in normal and immune compromised hosts. Liver1984;4: 184-94.

23 Colina F, Mollejo M, Moreno E, Alberti N, Garcia I,G6mez-Sanz R, et al. Effectiveness of histopathologicaldiagnoses in dysfunction of hepatic transplantation. Re-view of 146 histopathological studies from 53 transplants.Arch Pathol Lab Med 199 1;l1S:998-1005.

24 Snover DC. Biopsy diagnosis of liver disease. Baltimore: Wil-liams and Wilkins, 1992:127-54.

25 Paya CV, Smith TF, Ludwig J, Hermans PE. Rapid shellvial culture and tissue histology compared with serologyfor the rapid diagnosis of cytomegalovirus infection inliver transplantation. Mayo Clin Proc 1989;64:670-5.

26 Colina F. The role of histopathology in hepatic trans-plantation. Semin Diagn Pathol 1992;9:200-9.

27 Rabah R, Jaffe R. Early detection of cytomegalovirus in theallograft liver biopsy: A comparison of methods. PediatrPathol 1987;7:549-56.

28 Espy MJ, Paya CV, Holley KE, Ludwig J, Hermans PF,Wiesner RH, et al. Diagnosis of cytomegalovirus hepatitisby histopathology and in situ hybridisation in liver trans-plantation. Diagn Microbiol Infect Dis 1991;14:293-6.

29 Lafemina RL, Hayward GS. Differences in cell-type-specificblocks to immediate early gene expression and DNAreplication of human, simian, and murine cyto-megalovirus. J Gen Virol 1988;69:355-74.

30 Koment RW. Restriction to human cytomegalovirus rep-lication in vitro removed by physiological levels of cortisol.JMed Virol 1989;27:44-7.

31 Toorkey CB, Carrigan DR. Immunohistochemical detectionof an immediate early antigen of human cytomegalovirusin normal tissues. J Infect Dis 1989;160:741-51.

32 Delgado R, Lumbreras C, Alba C, Pedraza MA, Otero JR,G6mez R, et al. Low predictive value of polymerase chainreaction for diagnosis of cytomegalovirus disease in livertransplant recipients. J Clin Microbiol 1992;30: 1876-8.

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