Kidney International, Vol. 42 (1992), pp. 1012—1016

Polymerase chain reaction detection of cytomegalovirus genome in renal biopsies SUSAN KADEREIT, SUSAN MICHELSON, BEATRICE MOUGENOT, PHILIPPE THIBAULT,

PIERRE J. VERROUST, F1&NçoISE MIGNON, RONALD C0LIM0N', and PIERRE M. RoNco Unite de Virologie Médicale, Institut Pasteur; and Laboratoire d'Histologie et Cytologie Pathologiques, Département d' Urologie and Département de Nephrologie, Hôpital Tenon, Paris, France

Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality in renal transplant recipients, and may be associated with reduced graft survival U, 21. Restriction enzyme analysis of CMV isolates [3—5) has clearly established that the donor kidney may be the source of CMV contamination, especially in CMV seronegative recipients who are at high risk for serious CMV disease [6]. This study, based on gene amplification by the polymerase chain reaction (PCR) [7], was

son) [15] and an enzyme immunoassay (ELISA) set up in our laboratory. Antigen was prepared from MRC-5 cells infected by CMV and showing >90% cytopathic effects. A control prepa-

ration was made from uninfected cells treated in parallel. Ninety-six-well NUNC plates were coated with 2.5 g antigen! well/0.l ml of PBS, saturated with 1% BSA and successively

incubated with sera diluted 1/100 in PBS containing 0.1%

Tween 20 (PBS-T), peroxidase-labeled sheep anti-human IgG (Diagnostics Pasteur) and substrate (OPD). Each incubation kidneys from 30 patients. PCR has been successfully applied to step was followed by washes in PBS-T. Results are expressed the identification of CMV genome in urine, blood and saliva as delta OD (average OD of two wells coated with infected cell [8—13]. Since for ethical reasons we were reluctant to perform antigen-average OD of two wells coated with uninfected cell donor kidney biopsies in this initial study, we selected samples antigen). A delta OD of 0.2 was considered negative. from patients undergoing a kidney biopsy justified by renal disease symptoms or a nephrectomy for cancer. The results show that kidneys from 8 of 20 seropositive patients contained Detection of CMV genome by PCR amplification CMV genetic material by Southern blot analysis. UnexpectProcessing of samples. Renal biopsy fragments were ground edly, the latter could also be detected in 3 of 10 seronegative mechanically and incubated for 48 hours at 37°C in 500 p1 of patients. lysis buffer (0.1 M Tris, pH 7.4, 0.01 M EDTA, 0.5% SDS) containing 200 g/ml of Proteinase K (Merck). Samples were Methods then extracted twice with a phenol (vol/vol 50%)-chloroform Patients (48%)-isoamyl alcohol (2%) mixture. DNA was precipitated CMV genome was searched for in kidney biopsies taken from with ethanol and resuspended in 20 .d of distilled water. Amplification procedure. The primers used for specific am27 consecutive patients referred for symptoms of renal disease (proteinuria, hematuria, and/or renal insufficiency) and in three plification of CMV DNA (A, 5'-GGA TCC GCA TGG CAT additional specimens from the "normal" pole of kidneys with TAC CGT ATG T-3' and D, 5'-GAA TTC AGT GGA TAA CCT cancer. None of the patients showed clinical signs of CMV GCG GCG A-3') were synthesized (Laboratoire de Chimie infection; only one with endocarditis had persistent fever. Organique, Institut Pasteur, France) based on sequence data of Biopsies were processed as previously described for light Weston and Barrell [16]. They frame a 406 bp region within the microscopy and immunofluorescence studies [14]. Histological HindIII-X fragment of the Ad- 169 CMV strain which region was found to be highly conserved in 60 fresh human isolates of CMV diagnoses are given in Table 1. examined (unpublished results and Fig. 1). Each DNA speciSerological studies men was amplified for 30 cycles using a Hybaid apparatus (Cera The serologic status of the patients was established using the Labo) in two independent double-blind experiments. Briefly, 3 CMV latex agglutination assay (CMV Scan®, Becton Dickin- .d of DNA solution were first diluted to a final volume of 20 p1

undertaken to determine the incidence of CMV genome in

in distilled water and boiled for 30 minutes. The reaction

mixture (100 Ld) consisted of 15 p1 of each deoxyribonucleotide 1Present address is: Centre Hospitalier Regional, Service de Microbiologic, Hôpital de Pontchaillou, 35000 Rennes, France.

(stock solution: 10 mM); 6 p1 of 0.6 M Tris, pH 8.8, 0.17 M

Received for publication July 11, 1991 and in revised form April 27, 1992 Accepted for publication April 30, 1992

/iM EDTA, 1.7 mg!ml bovine serum albumin; 10 p1 dimethylsulfoxide; 1 p1 of each primer (stock solution: 1 mM); and 3 p1 (1.5 U) of Taql polymerase (Perkin-Elmer). After five minutes of annealing at 55°C, elongation was carried out at 70°C for five

© 1992 by the International Society of Nephrology

ammonium sulfate, 67 mivi MgCl2, 0.1 M /3-mercaptoethanol, 50

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Kadereit et a!: CMV genome detected in biopsies

Table 1. Characteristics of pa tients and CMV status Serology

Patient number

Age

Sex

Lesionsa

Scan

ELISA

67 48

M F F M

Cancer Amyl. Memb.GN En.Ex.GN (Endoc.) Granul.TIN Fibrosis MPGN (Cryo.) FSG FSGN (IgA)

+ + + + + + + + + + + + + + + + +

+ + +

1

2 3 4 5 6 7 8 9 10

64 60 70 63 66 54 57 23 34 35 63 48 54 78 42 49 66 68

11

12 13 14 15

16 17 18 19 20 21

18

22 24

67 72 23

25 26 27

17 51 16

28 29 30

54 22 18

23

M

M F M

F

Minimal

M M M M M M M M

FSG En.Ex.GN (IgA) Amyl.

Ex.Nec.GN FSG (Diab.) Ex.Nec.GN Goodpasture Fibrosis Memb.GN Tub.Necr. Post-inf.GN Cancer Ex.Nec.GN Uncl.GN Mes.GN (Lupus) Memb.GN

F M F M M M M

F

M M M M M

Min.change Cancer Ex.GN (IgA) En.GN (IgA)



+



+ + + + ND



+ + — — — — —

+





ND





+





+





+ + + ND — —





— —





— —



-



— —





-



-





— — —

+ + +

— —









+ + + + + + +

+

+



+

+ + +





Hybridization of PCR productsb Southern Dot dig

ND

+

-

+ + +

a Abbreviations are: Amyl., amyloidosis; Cryo., cryoglobulinemia; Diab., diabetes; En. Ex., endo- and extra-capillary; Endoc., endocarditis;

Ex. Nec., extra-capillary and necrotizing; FSG, focal and segmental glomerulosclerosis; GN, glomerulonephritis; Granul. TIN, granulomatous tubulointerstitial nephritis; Memb., membranous; Mes., mesangiocapillary; Mm., minimal; MPGN, membranoproliferative; Post-inf., postinfectious; Tub. Necr., tubular necrosis; Unci., unclassified; N.D., not done because of serum shortage. b Following 30 cycles of amplification, PCR products were hybridized with an insert from plasmid pPR containing a 208bp sequence framed by the primers. Dot-blot hybridization was detected using this probe labeled with digoxygenin (Dot dig).

I

II

I ! ON HI

ethidium bromide staining. Blots were made of the same gels

K H IV!f0l according to the Suothern technique [17].

I tI'II°IIII

pPR*

Specificity of amplification was controlled using cellular

s

B

.

406bp0

Fig. 1. Hind III restriction map of Ad-169 CMV strain according to Fleckenstein et a! [22] and Oram et a! [23]. The 406 bp region that we amplified is delineated by a BamHI (B) and an EcoRI (E) restriction site. These restriction sites are conserved among 60 wild CMV isolates. The probe pPR (*), delimited by two Sau3AI (S) sites, was cloned in a pGEM vector.

minutes. Samples were subsequently cycled as follows: denaturation, 10 seconds at 94°C; annealing, one minute at 55°C and elongation, one minute at 70°C. Amplification products were transferred to nitrocellulose by dot-blotting and hybridized with a specific probe. They were also analyzed by electrophoresis in 2% agarose gels followed by

DNA as well as DNA purified from other human Herpes viruses

(Herpes Simplex virus types 1 and 2, Varicella-Zoster virus, Epstein-Barr virus, Human herpes virus 6) and Colburn Simian virus. Controls also consisted of DNA samples prepared from 30 mice kidneys which were amplified in parallel with the human renal biopsies. Each reaction contained a positive viral DNA and a negative water control. All amplifications were performed twice independently. Hybridization of filters. Hybridization was performed with the gel purified plasmid pPR, a Sau3Al subfragment of the HindIII-X fragment. The probe was labeled with either alpha 32PdCTP (Southern blots) by nick translation or with digoxygenin tagged-deoxyuridine triphosphate (dig-dUTP) by random priming (pPR-dig) using a kit purchased from BoehringerMannheim (dot blots). Filters were hybridized overnight at 68°C and washed under stringent conditions in sequential baths of 2 x SSC, 0.1% SDS, then 1 x SSC, 0.1% SDS, and finally 0.1 x

SSC, 0.1% SDS, as previously described [18]. Dot blots were

developed according to the manufacturer's instruction, and results were scored "+" or "—" after drying of the blots.

1014

I

Kadereit et a!: CMV genome detected in biopsies

V —

0' t fl 0

tO 0' L C') V fli

p. HAWSU WUSI

plo— —

t.

03

B —

A COI',

+

+

Fig. 2. Southern blot hybridization of PCR products with a speqflc CMV probe. After amplification in parallel of mouse kidney DNA and human renal biopsy DNA, PCR products were separated by electrophoresis and transferred to nitrocellulose according to the Southern technique 117]. Blots were hybridized with a subfragment of the Hind III-X fragment. Numbers for human biopsies correspond to the numbers in Table 1. Arrowheads indicate hybridization with the specific 406 bp amplification product. A negative (—) and a positive (+) control were

HnWSU L91J91

amplified in parallel.

Results and discussion

Detection of CMV genome in seropositive patients

although a small number of samples (patients 3, 18 and 20) gave discordant results with the two tests. By Southern blot (Fig. 2), 8 of the 20 kidney specimens yielded a 406 bp band character-

Twenty patients of this series had anti-CMV antibodies by istic of the PCR amplification product of the CMV genome. the latex agglutination assay and/or EIA (Table 1). They in- Only 3 of them showed clear positivity by dot blot with a eluded 14 males and 6 females aged from 23 to 78 years (mean nonradioactive labeled probe. Although this technique can be age 59). The sensitivity of the latex agglutination was similar to more widely used in hospital-associated laboratories, its interthat of the enzyme immunoassay developed in our laboratory, est was limited in our hand by its lower sensitivity. It may also

Kadereit et a!: CMV genome detected in biopsies

1015

lead to false-positive results, as in patient 9 in whom the

Pathogenesis and Prevention of Human Infection (vol 20), edited

positive dot blot could not be confirmed by a detectable band on

by PLOTKIN SA, MICHELSON 5, PAGANO J, RAPP F, New York, Alan R. Liss, Inc., 1984, pp. 87—99 2. COLIMON R, MICHELSON S: Human cytomegalovirus: Pathology, diagnosis, treatment, in Advances in Nephrology, Necker Hospital, Year Book Medical Publishers, Inc., 1990, pp. 302—325 3. WERTHEIM P, BUURMAN C, GEELEN J, VAN DER NOORDAA J:

the Southern blot. These results suggest that the CMV genome may persist in the kidney after serologically-proven viral infection, and most likely explain why kidneys from seropositive individuals may be a source of contamination upon grafting. Detection of CMV genome in seronegative patients

This group was composed of nine males and one female ranging in age from 16 to 72 years (mean age 36; Table 1). All were negative by both serological tests. CMV DNA could be found in 3 of 10 patients (Fig. 2), who were positive by both dot and Southern blots, while one was only positive by dot blot. All samples of mouse kidney amplified in parallel were negative.

These results confirm the high sensitivity of PCR methods.

Transmission of cytomegalovirus by renal allograft demonstrated by restriction enzyme analysis. Lancet 1:980—981, 1983 4. GRUNDY JE, SUPER M, SwENY P, MOORHEAD J, Liji SF, BERRY NJ, FERNANDO ON, GRIFFITHS PD: Symptomatic cytomegalovirus

infection in seropositive kidney recipients: Reinfection with donor virus rather than reactivation of recipient virus. Lancet 11:132—135, 1988 5. KANESAKI T, BABA K, TANAKA K, ISHIBASHI M, YABUUCHI H:

Characterization of cytomegalovirus isolates recovered during repeated infection in renal transplant recipients. J Med Virol 28:140— 143, 1989 6. Ho M, SUWANSIRIKUL 5, DOwLING JN, YOUNGBLOOD LA, ARM-

They are in keeping with previous observations made in transSTRONG JA: The transplanted kidney as a source of CMV infection. planted patients. First, in renal transplant recipients, PCR assay NEnglJMed 293:1109—1112, 1975 was shown to be the most sensitive test to detect CMV in urines 7. Snu RK, SCHARF 5, FALOONA F, MULLIS KB, HORN GT, ERLICH HA, ARNHEIM N: Enzymatic amplification of p-globin genomic when compared with enzyme-linked immunosorbent assay, sequences and restriction site analysis for diagnosis of sickle cell DNA hybridization and direct virus isolation [121. This finding anemia. Science 230:1350—1354, 1985 was recently confirmed by LOning et al [19] who demonstrated 8. DEMMLER GJ, BUFFONE GJ, SCHIMBOR CM, MAY RA: Detection CMV DNA by PCR despite negativity of in situ hybridization of cytomegalovirus in urine from newborns by using polymerase and immunohistochemistry. Second, in bone marrow grafted chain reaction DNA amplification. Jlnfect Dis 158:1177—1184, 1988 patients, a prospective longitudinal study indicated that CMV 9. SHIBATA D, MARTIN WJ, APPLEMAN MD, CAUSEY DM, LEEDOM JM, ARNI-IFIM N: Detection of cytomegalovirus DNA in peripheral DNA detection by PCR in blood was more sensitive than virus blood of patients infected with human immunodeficiency virus. J culture and CMV antibody status for the diagnosis of CMV Infect Dis 158:1185—1191, 1988 infection [10]. Moreover, epidemiological studies have shown 10. CASSOL SA, PooN M-C, PAL R, NAYLOR MJ, CULVER-JAMES J, that seronegative subjects may be positive for CMV genome by BOWEN TJ, RUSSELL JA, KRAWETZ SA, PON RT, HOAR DI: PCR [20]. Our data further suggest that kidneys from seronegPrimer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection ative donors may also be contaminating, although further study in marrow transplant recipients. J Clin Invest 83:1109—1115, 1989 is required to establish correlation between PCR positivity and 11. JIWA NM, VAN GEMERT GW, RAAP AK, VAN DE RIJKE FM, infectious potential.

It is interesting to note that in the two groups of patients taken as a whole, serologic and PCR data did not correlate with any type of nephropathy. In conclusion, the present study shows the feasibility of PCR

MULDER A, LENS PF, SALIMANS MMM, ZwAAN FE, VAN DORP W, VAN DER PLOEG M: Rapid detection of human cytomegalovirus

DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction. Transplantation 48:72—76, 1989

assay for the specific detection of CMV genome in kidney 12. OLIVE DM, SIMSEK M, AL MUFTI 5: Polymerase chain reaction assay for detection of human cytomegalovirus. J Cliii Microbiol biopsies. This test may be a powerful tool to study in more 27:1238—1242, 1989 detail the transmission of CMV infection in kidney recipients 13. H5IA K, SPECTOR DH, LAWRIE J, SPECTOR SA: Enzymatic ampliand to analyze the relationship between CMV infection and fication of human cytomegalovirus sequences by polymerase chain graft rejection. Furthermore, it indicates that, because of the reaction. J Clin Microbiol 27:1802—1809, 1989 high incidence (37%) of CMV genome in kidney biopsies, one must be very cautious when interpreting the possible relationship between its detection and the pathogenesis of certain types of glomerulonephritis recently suggested by Muller et al for IgA nephropathy [21].

14. MELCION C, MOUGENOT B, BAUDOUIN B, RONCO P, MouLONGUET-DOLERIS L, VANHILLE Pu, BEAUFILS M, MORELMAROGER L, VERROUST P, RICHET G: Renal failure in myeloma:

Relationship with isoelectric point of immunoglobulin light chains. Clin Nephro! 22:138—143, 1984 15. MCHUGH TM, CASAVANT CH, WILBER JC, STITES DP: Compari-

son of six methods for the detection of antibody to cytomegalovi-

Acknowledgments We thank Drs. P. Bindi, C. Michel and B. Viron for referring several patients. This work was supported by grants N° CRE 893010 from the "Institut National de La Sante et de La Recherche Médicale" (INSERM) and N° 998593 from the CNAMTS.

rus. J Cliii Microbiol 22:1014—1019, 1985 16. WESTON K, BARRELL BG: Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus. J Mol Biol 192:177—208, 1986

17. SOUTHERN EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mo! Biol 98:503—5 17, 1975

Reprint requests to P. Ronco, INSERM U.64, HOpital Tenon,

Pavilion Castaigne, 4 rue de la Chine, 75970 Paris Cedex 20, France.

18. COLIMON R, SOMOGYI T, BERTRAND C, MICHELSON S: Demon-

stration of colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned

References I. BETTS RF: The relationship of epidemiology and treatment factors to infection and allograft survival in renal transplantation, in CMV:

subgenomic fragments. J Gen Virology 66:2183—2198, 1985 19. LONING T, STIL0 K, RIVIERE A, HELMCHEN U: Cytomegalovirus

detection in kidney transplants: Results obtained from the polymerase chain reaction. Clin Nephroi 37:78—83, 1992

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21.

TYMS AS: Persistence of cytomegalovirus in mononuclear cells in peripheral blood from blood donors. Br Med J 299:897—898, 1989 MULLER GA, KUHN W, MULLER CA, RISLER T, BOHLE A, MARKOVIC-LIPKOVSKI J: Detection of human cytomegalovirusDNA in IgA nephropathy. Nephron 57:383—384, 1991

B, MULLER I, COLLINS J: Cloning of the complete cytomegalovirus genome in cosmids. Gene 18:39—46, 1982

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Polymerase chain reaction detection of cytomegalovirus genome in renal biopsies.

Kidney International, Vol. 42 (1992), pp. 1012—1016 Polymerase chain reaction detection of cytomegalovirus genome in renal biopsies SUSAN KADEREIT, S...
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