Epstein-Barr Virus Infection-Associated Renal Disease:Diagnostic Use of Molecular Hybridization Technology inPatients with Negative Serology1
Tibor Nadasdy, Chang-Soo Park, Stephen C. Peiper, James E. Wenzl, James Oates, and
Fred G. SiIva2
1. Nadasdy. C-S. Park, F.G. Silva, Department of Pa-thology, University of Oklahoma, Oklahoma City, OK
S.C. Peiper. Department of Pathology. James GrahamBrown Cancer Center, University of Louisville, Louis-ville. KY
J.E. Wenzl, Department of Pediatrics, University of 0kb-homa, Oklahoma City. OK
J. Oates, Presbyterian Hospital, Albuquerque, NM
(J. Am. Soc. Nephrol. 1992; 2:1734-1742)
ABSTRACTThere are only a few reports of renal disease associ-ated with Epstein-Barr virus (EBV) infection. The diag-nosis of EBV infection in these previously reported
patients was based primarily on positive serology.Two patients with renal disease who, despite repeat-edly negative serologies, were shown by molecularhybridization techniques-in situ hybridization (ISH)and polymerase chain reaction (PCR)-to have EBVinfection are reported here. Site-specific molecularprobes directed against specific, tandemly re-peated EBV genomic regions were used. A synthetic23-mer terminally biotin-labeled oligonucleotideprobe selected from the EBV No/I region was usedfor ISH. For PCR, oligonucleotide primers were de-
signed from sequences of the highly conserved, longinternal direct repeat region of EBV to specificallyamplify a I 10-base-pair segment. The first patient,
a 3-yr-old girl with a 1-yr history of fatigue, fever,splenomegaly, and lymphadenopathy developedhematuria. A renal biopsy revealed widespread gb-
merular mesangiolysis admixed with segmental mes-
angial sclerosis; no immune deposits were noted byelectron microscopy or immunofluorescence. ISH onparaffin sections of the resected spleen and lymph
I Received October 14. 1991. Accepted February 21, 1992.2 Correspondence to Dr. F.G. Silva, Department of Pathology, University of
Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City. OK 73190.
nodes was positive for EBV. The second patient, a 28-yr-old male renal allograft recipient, received a dou-ble dose of OKT3. Seven weeks after transplantation,a renal biopsy revealed a lymphoproliferative dis-order. Paraffin sections of the nephrectomy speci-men were positive for EBV by both ISH and PCR. It wasconcluded that (1) EBV cannot be excluded on thebasis of multiple negative serologies in some pa-tients, and (2) ISH and PCR may lead to the detectionof viral genomic information in renal and nonrenal
tissues.
Key Words: Epstein-Barr virus, in situ hybridization, polymerase
ciation of Epstein-Barr virus (EBV) infection andrenal disease. Nephnitis associated with infectiousmononucleosis was first described by Pfeiffer (1) in
1 889; this, however, probably represented an acutepoststreptococcal gbomerubonephnitis. Poststrepto-coccal on nonpoststreptococcal acute postinfectious
glomerubonephritis after infectious mononucleosishas also been described by others (2,3). Tennant (4)did not note any morphologic signs of renal disease
in a patient with infectious mononucleosis and din-ically acute gbomerubonephnitis. Hematuria In pa-tients with infectious mononucleosis is not infre-
quent, but cases documented with renal biopsy arerare (5). Occasional occurrences of immunogbobulin
A (IgA) nephropathy (3), steroid-dependent minimal-change nephrotic syndrome (6), and hemolyticuremic syndrome (7) in patients with infectiousmononucleosis have been also reported. A recent
study from France suggested the robe of EBV in thepathogenesis of primary IgA nephropathy (9). Taub
(8) published a case with gbomerular scarring and avasculitic lesion. However, the most frequently re-
ported renal disease in infectious mononucleosis ap-
pears to be interstitial nephnitis (2,3, 1 0- 1 2). In ad-dition, in the early and mid- 1 980s, posttransplant
lymphoproliferative disorders (PTLD) have been nec-ognlzed to be caused by EBV, which frequently in-volves the graft itself (1 3- 1 8). Recent studies confirm
this data (19-33), and some of them suggest that
Nadasdy et al
Journal of the American Society of Nephrology I 735
OKT3 (CD3) antibody treatment may facilitate the
development of PTLD (21,24,26,28,34).EBV infection is usually diagnosed by serologic
examination for the presence of anti-EBV antibodies.
Primary EBV infection is defined senobogicabby bycirculating antibodies to the viral capsid antigen (1gMand IgG) and to the EBV early antigen (IgG). The EBVearly antigen consists of two components, diffuseand restricted, as defined by immunofluonescencestaining characteristics. The 1gM response to theviral capsid antigen lasts for only a few months,
whereas the IgG response is thought to be lifelong.Antibodies to EBV nuclear antigen (EBNA) usually
develop several weeks or months after the infection.We report two patients with EBV infection-asso-
dated renal disorders (one with a primary gbomerulan
disease and one with a PTLD) in whom EBV wasdemonstrated by In situ hybridization (ISH) andpolymerase chain reaction (PCR) in routinely prod-
The clinical symptoms of the 3-yr-old girl startedin December 1988 with a mononucleosis-like syn-drome (generalized lymphadenomegaly, hepatosple-nomegaly, and Coombs negative hemolytic anemia).An evaluation of her anemia included a bone marrow
biopsy, which showed lymphoid and enythnold hyper-plasia. EBV serology (IgG, 1gM against viral capsidantigen, early antigen, EBNA) was negative. In June
1 989, a lymph node biopsy was performed and re-active hyperplasia was noted. Her differential bloodcounts over time showed the following: polymorpho-
nuclears beukocytes, 1 2 to 43% ; bands, 0 to 1 %:lymphocytes, 3 1 to 6 1 %; atypical lymphocytes, 2 to23%; monocytes, 3 to 15%; eosinophils, 1 to 16%. AT arid B cell panel, performed on October 1 1 . 1989,was normal. The serum lmmunogbobullns were also
all within normal ranges for hen age. Her sedimen-tation rate was usually elevated, up to 105 mm/h.Her red blood cell morphology was interpreted asnormal in the peripheral blood smears examined. Henplatelet count varied between 1 46 x 1 0� and 730 x
1 09/L, but it was within the normal range duringmost checkups. EBV serology (IgG, 1gM against viralcapsid antigen, early antigen, EBNA) continued to be
repeatedly negative. The patient was also tested forantibodies to cytomegabovirus, human immunodefi-dlency virus, toxoplasmosis, mycoplasma. leptospi-
rosis, tubaremia, diphtheria, tuberculosis, hepatitis Bsurface antigen, as well as for the presence of hepa-
titis B core 1gM antibody and hepatitis A 1gM anti-body-all with negative results. Her antinuclear an-
tibody tests were always negative. In November1989,
she developed macroscopic hematunia with red blood
cell casts in the urine. Proteinunia was 0 to 1 + bydipstick. She was admitted to Chibdren’s Hospital ofOklahoma on November 23, 1989, in acute oligunicrenal failure, which appeared to be secondary to uricacid nephropathy (serum uric acid, 1 6. 1 mg/dL). Her
serum potassium rose to 7.8 mg/dL, BUN was 88 mg/dL, and serum creatinine was 1 .5 mg/dL. She re-ceived 3 consecutive days of hemodiabysis, which
resulted in a decrease in her serum uric acid level to
8.8 mg/dL and reestablishment of normal renal func-tion. A renal biopsy was performed on December 12,1989. In January 1990, the patient underwent asplenectomy because of the persistent splenomegalywith hemolytic anemia. Splenic penihilar and mes-
entenic lymph nodes were also removed for exami-nation. Histology showed sinusoidal congestion and
lymphold hyperplasia in the spleen and parafollicu-lan hyperplasia in the lymph nodes. After the sple-nectomy, the hemolytic anemia subsided. The he-matunia also resolved, and the renal function hasremained satisfactory until the present time. How-
ever, hen general condition did not improve and themalaise and fatigue, as well as the severe generalized
lymphadenopathy, persisted. The patient’s wholeblood was tested for various adenoviruses and her-
pesvinuses (including human herpesvirus 6) by PCRin the laboratory of Dr. Robert Fox (Scripps Clinic,La Jolla, CA). The results were positive for EBV, and
only for EBV. Subsequent workup at the NationalInstitutes of Health (NIH) revealed a peculiar lmmu-nodeficiency with the appearance of an increasingpercentage of CD3 + CD4- CD8- T lymphocytes with-out cbonal rearrangement in her peripheral blood (Dr.
Steve Strauss, NIH, Bethesda, MD, personal commu-
nication).
Patient 2
The patient, a 28-year-old male with end-stagerenal disease from presumed chronic gbomenuboneph-ntis (not documented by renal biopsy), received a
cadavenic renal albognaft on November 1 1 , 1 989. Thealbognaft was biopsied pretransplant and was normal.He did well postoperatively except for a transient
pancytopenia, which was considered secondary to
azathiopnine and acycbovir (which he received as pro-phylaxis to prevent CMV infection), and the acycbovir
was discontinued. One month after transplantation,his serum creatinine level began to rise to 6 mg/dLand a fine needle aspirate showed acute cellular re-
jection. Ultrasound and renogram were consistentwith rejection. He was given 5 days of methylpred-nisobone but failed to respond, and his serum creati-
nine level rose to 6.3 mg/dL. He was then treatedwith OKT3, and his serum creatinine diminished to3 mg/dL. He again received a double dose of OKT3,
but in spite of that, his serum creatinine rose to 3.8
mg/dL. On January 3, 1 990, a renal transplant bi-
opsy was performed and showed a lymphoprobifera-tive disorder. The diagnosis was confirmed by a bone
marrow biopsy. At that time, the differential peniph-eral blood count showed 24% polymorphonuclear leu-
kocytes, 4 1 % lymphocytes, 6% monocytes, and 29%atypical lymphocytes. EBV serology (1gM and IgG an-tibody response against viral capsid antigen, early
antigen, EBNA) was repeatedly negative. Because ofthe PTLD involving the renal transplant, the graftwas resected on February 9, 1990, and the patientreturned to the hemodlalysis program. After the ces-sation of the immunosuppressive therapy, the lym-
phoproliferative disorder spontaneously regressed.The patient is currently on hemodialysis without anysigns of PTLD. The other kidney of the donor wastransplanted into a different recipient and is func-tioning well.
METHODS
The renal biopsy specimen of patient 1 was fixedin 1 0% buffered formalln and embedded in methac-
rylate. All other tissue specimens for light microscopy
were formabin fixed and paraffin embedded. Directimmunofluorescence on frozen sections was per-formed in both renal biopsies with antibodies to hu-man IgG, 1gM, IgA, kappa and lambda light chains,
C3, Cbq, fibninogen, and albumin. For ultrastructuralexaminations, a small piece of both renal biopsyspecimens were fixed in 3% glutaraldehyde, postfixedin osmium tetroxide, contrasted with lead citrate and
uranyl acetate, and embedded in Epon.ISH was performed on paraffin sections of the re-
sected spleen and the splenic hilar lymph nodes of
patient 1 and on the removed renal albograft of pa-tient 2. A terminally biotin-labeled ollgonucbeotide
probe (kindly provided by Dr. David Brigati, Depart-ment of Pathology, University of Oklahoma HealthSciences Center, Oklahoma City), composed of 23
consecutive nucbeotides selected from the EBV NotI
region, was used (35). The NotI region is repeated intandem an average of 12.6 times in the EBV genome.This synthetic sequence has a 9 1 % homology withanother EBV genomic tandem repeat, the PstI region,which is reiterated about 25 times in the EBV ge-
nome. The NotI/PstI probe and the hybridization pro-cedure are described in detail elsewhere (35). TheUltraProbe Kit (Biomeda, Foster City, CA) was usedas a detection system and Included streptavidin-con-
jugated alkaline phosphatase and Fast Red TRchromogen. ISH was performed with the Code-OnAutomated Molecular Pathology System (Fisher Sd-entific, Pittsburgh, PA) in the laboratory of Dr. Bni-gati, according to the procedure described by Mon-tone et al. (35).
PCR was performed on the same tissue blocks asISH. Templates from EBV genomes were amplified
by PCR, as described by Peiper et at. (36). Briefly,
oligonucleotide primers were designed from Se-quences of the highly conserved, long internal directrepeat region of EBV to specifically amplify a 1 10-base-pair segment. PCR amplification reactions con-taming extracts from 8-��m sections of paraffin-em-bedded tissues were subjected to 30 cycles of ampli-ficatlon, each consisting of 1 -mm denaturation at
94#{176}C,2 mm at 46#{176}Cfor the annealing of the primersto the template, and 3 mm at 72#{176}Cfor DNA synthesisby taq pobymerase. Nucleic acids present in the am-
plification reactions were precipitated with ethanoland separated by size on agarose gels. The nucleicacids were then transferred to hybridization mem-branes by Southern blotting. Amplified EBV se-quences were detected by hybridizing the blot with alabeled oligonudleotide probe designed from se-
quences specific to the targeted 1 1 0-base-pair seg-ment. DNA from a Burkitt’s bymphoma cell line (Raji)
and a placenta were the positive and negative con-
trols, respectively. Samples were considered positivefor EBV templates when an amplification product of1 1 0 base pairs that annealed to the specific oligonu-
cbeotide probe was detected. A whole blood sample of
the patient was also tested by PCR for various ade-noviruses and herpesviruses, including human her-
pesvirus 6 in the laboratory of Dr. Robert Fox (LaJolla, California). His PCR method for the detectionof EBV In whole blood, including the careful controls,is detailed elsewhere (37).
RESULTS
Patient I
The percutaneous renal biopsy contained renal con-tex and medulla with 23 gbomeruli. Histologically, themajority of the gbomeruli showed segmental or global,mild to severe mesangiab expansion with sclerosisand moderate mesangiab hypercellularity (Figure 1A).In 1 0 gbomenubi, segmental mesanglolysis with micro-aneurysm formation adjacent to sclerotic segmentswas noted. In five gbomeruli, mesangiolysis domi-nated (Figure 1 B). Central mesangiab sclerosis in thegbomenular lobules with penipherab microaneurysmsalso occurred in three glomeruli. One gbomerulus withsclerosis contained a cellular crescent. The intersti-
tium was not widened, and no interstitial cellularInfiltrate was detected. Some tubules contained red
blood cell casts. No prominent vascular changes wereobserved. Immunofluorescence was negative withthe antisera employed. Three gbomerubi were exam-med ultrastructunally. Electron microscopy demon-strated widened and moderately hypercellular gb-merulan mesangial areas with dissolution of the mes-
angial matrix and occasional microaneurysmformation (Figure 2). A mild, focal, irregular gbomer-
ular subendotheliab accumulation of electron-lucentfluffy material was noted. A tubule contained dys-
- . - . � - .i-� Nadasdyetal
morphic red blood cells admixed with electron-dense
bundles of fibrillar material corresponding to fibninstrands. A few fibrin strands were also observed ina Bowman’s capsule. No discrete electron-dense im-
mune-type deposits or viral particles were identified.The diagnosis of a diffuse mesanglal gbomerubopathy
with mesangiab sclerosis and mesangiolysis was
made.Both the lymph node and the spleen showed reac-
tlve hyperplastic changes (Figure 3). Scattered p051-tive cells for EBV were demonstrated by ISH in the
parafollicubar areas of the splenic hibar lymph node
and in the white pulp of the spleen. The number ofpositive cells, similarly to the positive control sec-
tions, varied between 0 and 5 (usually 1 and 2) perhigh-power field. The staining was nucleolar or nu-clear, but some cells also appeared to show cyto-plasmic reaction (Figure 4). PCR in the tissue sped-
Figure 1 . Glomerular pathology of patient I . (A) A gbomer-ulus showing pronounced mesangial sclerosis with moder-ate hypercellularity. (B) Widespread glomerular mesangi-olysis with coalescence of the capillary loops. Jones; mag-nifications: panel A, x600; panel B, xl,000.
Figure 3. Paracortical hyperplasia with polymorphic cells inthe splenic hilar lymph node from patient I . Hematoxylinand eosin; magnification, x200.
Figure 2. A large dilated glomerular capillary loop with Figure 4. Two cells in the spleen of patient I labeled withdissolving mesangium. No electron-dense deposits are the biotinylated oligonucleotide probe for the EBV Noflseen. Uranyl acetate, lead citrate; magnification, x2000. region (arrows). Magnification, x400.
Journal of the American Society of Nephrology 1737
1234567
Figure 5. PCR on tissue samples from patients I and 2. Lanes:1, placenta: 2, buffer: 3, RaJi cell: 4, transplanted kidneyfrom patient 2: 6 and 7, spleen and lymph node, respec-lively, from patient I.
Figure 6. The resected renal transplant of patient 2 showswidespread tumor-like infiltrate consistent with PTLD. Notethe many plasmacytoid cells and the cell showing pyknosisand karyorrhexis (arrow). Hematoxylin and eosin: magnifi-cation, x200.
EBV Infection-Associated Renal Disease
I 738 Volume 2 . Number 12 ‘ 1992
mens (lymph node, spbeen) were negative (Figure 5).However, PCR performed on whole blood samples inthe laboratory of Dr. Robert Fox was positive for EBV
and negative for a variety of other herpesviruses(including human henpesvinus 6) and adenoviruses(Dr. Fox, personal communication).
Patient 2
The percutaneous renal biopsy specimen showed
renal medulla and cortex with four normal-appearinggbomeruli. The intenstitlum contained a heavy,patchy, lymphoplasmacytic infiltrate with displace-ment and compression of the renal tubules. In theareas with a slight, diffuse infiltrate, mainly lympho-cytes were seen; however, in the heavily infiltrated
areas, plasma cells, plasmacytoid lymphocytes, andimmunoblasts predominated. Altogether, the Infil-tnate had a polymorphic appearance. Immunofluores-cence showed cytoplasmic positivities for both kappa
and lambda bight chains in the interstitial plasma-
cytic-lymphocytic cells and slight granular gbomeru-bar mesanglal staining with the antibody against C3.No reactions were noted with the other applied anti-
bodies. Electron microscopy demonstrated a numberof interstitial inflammatory cells-mainly large lym-phocytes and plasma cells. The gbomeruli appearedessentially normal by ultrastructural examination;no discrete immune-type , electron-dense depositswere identified, and no viral on viral-bike particles
were seen. The diagnosis of PTLD was made and waslater confirmed by a bone marrow biopsy. which
showed a polymorphous polyclonal B cell probifera-tion.
The resected renal allograft showed similar mon-phobogic findings to those described in the renal bi-
opsy specimen. The polymorphic interstitial infib-trate was widespread with displacement of the renaltubules (Figure 6). Scattered infiltrating cells hadeosinophilic cytoplasm and pyknotic nuclei with oc-caslonal karyornhexis. A slight-to-moderate cellularintimal proliferation in the arteries was also detected.ISH revealed 2 to 25 (average, 1 2) positive cells per
high-power field in the heavily Infiltrated areas. Thepositive cells appeared to be mainly lymphocytes,plasmacytoid lymphocytes, and cells showing pyk-nosis and karyorrhexis. Some of the cells showedonly nucleolar or nuclear positivities, but cytoplasmicstaining was also frequently seen, particularly incells with pyknotic nuclei, and occasionally karyor-rhexis (Figure 7). Tubular epithelial cells and gbomer-
uli did not stain with the NotI/PstI probe. PCR per-
formed on the nesected kidney was also positive forEBV genomic material (Figure 5).
DISCUSSION
Results from our two patients indicate that renaldisease can be associated with EBV infection. Thepathogenetic role of EBV in PTLD is well known;
however, the relationship of EBV infection with gb-merular diseases is bess well established. We believethat the gbomerular disease of our first patient is
related to her chronic, devastating EBV infection. In
addition to the clinical signs and symptoms, the di-agnosis of EBV infection is usually made by thedetection of the patient’s antibody response to a va-niety of different viral antigens of EBV. Of particularinterest in our two patients is that neither of them
-: . Nadasdy et aI
Journal of the American Society of Nephrology I739
Figure 7. ISH with the EBV probe on a section of the removedrenal transplant. Note the many positive cells with strongnuclear, nucleolar (arrows), and also cytoplasmic staining(arrowheads). Magnification, x400.
developed detectable antibody responses to EBVantigens by the routinely used serologic methods,despite their characteristic clinical symptoms. Mole-cuban hybridization methods (ISH, PCR), however,revealed the presence of EBV in both patients.
There are occasional reports on deficient immuneresponses in immunologically compromised individ-
uals (17,25,26,38-43), such as patients with X-linked bymphoproliferative disorder (39.4 1), renaltransplant patients developing PTLD after cycbospor-Inc and OKT3 treatment (25,26), patients with ataxiatelanglectasia (17,38,40), patients with chronic EBVInfection (42), and bone marrow transplant redipi-
ents (33). It is speculated that the presence of bow onundetectable bevels of antibodies to EBV signifies a Tcell deficiency (40). This might be particularly truein transplant patients, because the immunosuppres-sive regimen, including cycbosponine and OKT3, pni-many suppresses T cell responses. There is both in
vitro (34) and in vivo (2 1 ,24-26) evidence that OKT3treatment may facilitate the development of PTLD.
No signs of immunodeficlency were detected beforeand at the time of the renal biopsy in the first patIent.Recent examinations, however, revealed that the pa-
tient developed a peduliar Immune defect character-ized by the appearance of an increasing percentageof CD3+ CD4- CD8- T lymphocytes without cbonalrearrangement (Dr. Steve Strauss, NIH, Bethesda,
MD, personal communication). This resembles thelpr and gid single gene models In the mice (44). TheclInical syndrome In the mice with each of these twononabbebic genes is very sImilar and includes the ap-
pearance of nonmalignant CD3+ CD4- CD8- T lym-phocytes with systemic lymphoprollferative dIseaseand autoantibodies. This type of immune disordercould explain the patIent’s negative serology for EBV
but not the renal disease. The lpr/gbd mIce developImmune complex glomerubonephnitls, whereas In ourcase, no gbomerular immune complexes were noted.
We are unsure whether the gbomerulopathy of pa-
tient 1 is specific for chronic EBV infectIon. Mesan-giab sclerosis is not specific to any particular glomer-ular disease; rather, It is generally considered to be asign of chronic gbomerubar injury. Mesangiolysis may
heal by mesanglal expansion and later sclerosis (45).Morita and Chung (44) gave a detailed revIew on mes-
angiolysis and suggested that it may be the conse-quence of a number of injuries such as toxic effects,gbomerular Ischemia, hypertension, thrombotic ml-
ntis, and diabetic glomerubosclerosis. Mesangiolysiswas also recently described in graft versus host re-
action after bone marrow transplantation (46), Tak-atsukl’s syndrome (47), Takayasu’s arteritis (48), and
sickle cell disease (49). Huang and Wiegenstein (50)
published a case of mesangiolytic gbomenubonephritlsassociated with systemic echovirus infection. Of in-terest is that their patient was an infant with corn-bined immunodeficiency who subsequently devel-
oped a devastating disease that bed to death. Theseauthors did not note if their patient was tested forEBV or other viral pathogens. Mesangiolysis followed
by mesangiab sclerosis is usually seen in thromboticmicroangiopathies (5 1 ). The light and electron micro-scopic changes in the renal bIopsy of patIent 1 could
be consistent with a late stage of the gbomenular typeof thrombotic microangiopathy; the hemolytic ane-mia of the patIent, however, was not of the microan-
giopathlc type clinically. We are uncertain whetherthe gbomerular pathology with the concomitant ap-pearance of marked mesangial sclerosis and mesan-giobysis, noted in patient 1 , Is the direct consequence
of EBV infection on the action of various viral-asso-dated mediators (e.g. , interferons) produced duringthe devastating illness. Further studies are needed todirectly resolve this issue.
The history of our second patient is very similar tothat of the patient of Denning et at. (25); this patientwas also on cycbosponine and OKT3 treatment and
developed PTLD despite entirely negative serobogiesfor EBV. ISH In the renal transplant revealed thepresence of the viral sequences in both Denning et
at. (25) and our second patient. The lack of immuneresponse against EBV antigens in their and ourtransplant patients could be explained by the highdoses of OKT3 and cycbosponine. The defective im-mune response to EBV in PTLD appears to be theconsequence of deficient Immunosurveillance ratherthan the down-regulation of immunogenic EBV-en-
coded antigens (52). After transplant nephrectomyand the cessation of immunosuppressIve therapy,the PTLD regressed in both the patient of Denninget at. (25) and our second patient, which is not un-
EBV Infection-Associated Renal Disease
I 740 Volume 2 . Number 12 ‘ 1992
usual, particubarby If the dIsease is polydbonab(17,19,20,23,30).
ISH yielded strongly positive reactions in the tissue
specImens of both of our patients, particularly in the
renal transplant of patIent 2. In the batter case, most
of the cells demonstrating signs of necrobiosis (some
Infiltrating interstitial cells with pyknotic nuclei, eo-sinophilic cytoplasm, and occasionally, karyor-rhexis), were strongly reacting with the NotI/PstI
probe and displayed not only nuclear but also cyto-
plasmic positivitles. Some indistinct cytoplasmic
staining was also seen in the spleen and lymph nodeof patient 1 . These cells, particularly those with nec-robiotlc features, probably represent cells In the lytic
cycle of EBV Infection with a high number of viralDNA copies in both the nucleus and cytoplasm, as
has been noted before with this nucleotide probe(1 7,35). This is supported by the experimental cvi-
dence that the NotI/PstI tandem repeat Is among themost transcribed regions In the EBV genome during
the extreme early and bate stages of chemically in-
duced, experimental, lytlc EBV infections (53).
Strong nuclear as well as cytoplasmic stainIng of byticcells for EBV sequences was also demonstrated re-cently by Bashir et at. (54). The difference In thenumber of EBV-positive cells between our two cases
Is not surprising; it may vary from case to case, andnot Infrequently, only a few cells show positive sig-
nals (55-57). In our positive control, which was aspleen from the same patient reported in the publi-
cation of Montone et at. (35), the number of positivecells did not exceed the number of positive cells in
the sections of patIent 1 . PCR In the renal transplant
tissue was obviously positive, but the paraffin blocksof the spleen and the lymph node from patient 1 werenegative with the same primers and probe. This is in
contradiction to the positive ISH on the tissue sec-tions and the positive PCR performed on whole blood.
PCR, although a very sensitive method, may occa-sionally show false-positive or false-negative results(58). In patient 1 , either the ISH was false positive or
the PCR on the tissue was false negative. The clinical
symptoms, the positive ISH, and the positive PCR onthe whole blood suggest that probably the PCR on the
tissue specimen was false negatIve. An explanationfor that could be that we may be dealing with an
aberrant or mutant EBV strain and the primers were
different in the two PCR procedures. We attemptedto perform ISH on the resin-embedded and frozentissue of the renal biopsy specimen from patient 1.
The resin-embedded sections were technically un-
suitable for performing ISH, and the results on the
frozen sections were uninterpretabbe because of thesmall piece of tissue, the poorly preserved structure,
crush artifacts, and some background staining.In summary, results from these two patients con-
firm the usefulness of molecular hybridization meth-
ods in the diagnosis of human viral disease in tworenal conditions. We were able to demonstrate thepresence of EBV genomic sequences by both ISH andPCR In the tissues of two patients with repeatedly
negative EBV serobogles. Thus, the absence of de-tectable antibody response does not necessarily cx-
dude EBV Infection, even In Individuals with anapparently normal immune system.
ACKNOWLEDGMENTS
We are grateful to Dr. David Brigati (University of Oklahoma Health
Sciences Center) for providing the NotI/PstI probe and for his helpful
suggestions. We acknowledge Ms. Elaine Broussard and Ron Child-
ers for performing the PCR, Dr. Michael F. Hensley (St. Francis
Hospital, Tulsa, OK) for providing clinical data, and Ms. Cheryl
Ronck for her editorial assistance. This study was supported by
contract #3896 from the Oklahoma Center for the Advancement of
Science and Technology.
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