Reinfection of liver graft by hepatitis C virus
after liver transplantation.
C Féray, … , H Bismuth, C Bréchot
J Clin Invest.
1992;
89(4)
:1361-1365.
https://doi.org/10.1172/JCI115723
.
We have investigated hepatitis C virus (HCV) viremia before and after orthotopic liver
transplantation (OLT). 38 patients were examined; 16 were HCV positive and 22
anti-HCV negative pre-OLT in a RIBA-2 test (Ortho Diagnostic Systems Inc., Westwood, MA).
HCV-RNA was detected using a modified nested polymerase chain reaction in 14/38 and
10/38 patients before and after OLT, respectively. 7 of these 14 subjects who were
HCV-RNA positive before OLT were also positive for serum hepatitis B surface antigen. After
OLT, six patients became HCV-RNA positive, likely as a result of transfusions, while four
developed a probable recurrence of HCV infection. Infection of the liver graft by the same
strain of HCV was indeed demonstrated by sequence analysis of a hypervariable domain
(in the envelope region) in two cases. This establishes the possibility of HCV recurrence
and shows the usefulness of polymerase chain reaction as the only assay currently capable
of identifying HCV infection after OLT.
Research Article
Find the latest version:
Rapid
Publication
Reinfection of Liver Graft by Hepatitis C Virus after Liver
Transplantation
C.Feray,*t D.
Samuel,*
V.Thiers,* M. Gigou,t F.Pichon,11A.Bismuth,'
M.Reynes,'P. Maisonneuve,** H.Bismuth,t and C.Brechot*#*Hybridotest, Institut Pasteur,Paris; *Unite dechirurgieHepatobiliaire,
OBanque
duSang,'IBacteriologie,
'Anatomo-pathologie, **Hopital P.Brousse, Villejuif;Banquedusang, Versailles,*tUnite'
d'hepatologie
and Institut Nationalde la Sante etde la RechercheMedical, U-75 Necker-Laennec, Paris, France
Abstract
We have
investigated
hepatitis
C virus
(HCV) viremia before
and
after
orthotopic
liver
transplantation (OLT).
38 patients
were
examined; 16
wereanti-HCV
positive
and 22 anti-HCV
negative
pre-OLT
in
aRIBA-2
test(Ortho Diagnostic
Systems
Inc.,
Westwood,
MA).
HCV-RNA
wasdetected
using
amodi-fied nested
polymerase
chain reaction in
14/38
and
10/38
pa-tients before and after
OLT,
respectively.
7
of these 14
subjects
who were
HCV-RNA
positive
before OLT
werealso
positive
for
serumhepatitis
Bsurface
antigen.
After OLT, six
patients
became HCV-RNA
positive, likely
as aresult
of
transfusions,
while four
developed
aprobable
recurrenceof HCV infection.
Infection of the liver
graft by the
samestrain
of HCV
wasindeed
demonstrated
by
sequenceanalysis
of
ahypervariable
domain
(in
the
envelope
region)
in
two cases.This establishes
the
possibility of HCV
recurrenceand showsthe
usefulness of
polymerase chain reaction
asthe
only
assaycurrently capable
of
identifying
HCV infection after OLT.
(J.
Clin. Invest. 1992.
89:1361-1365.)
Key words: polymerase chain reaction
*RNA
-viremia
*chronic
hepatitis
Introduction
Hepatitis
C
virus
(HCV)'
is
amajor etiological
agentof
non-A-non-Binfection (NANB) (1). Hepatitis
Cvirus-related chronicliver diseases
areincreasingly
anindication
fororthotopic
livertransplantation
(OLT). Indeed,
HCV tests arepositive
in40%of candidates
forOLT
in our center(D. Samuel, personal
com-munication).
It
has
recently been suggested by studies based
onserologi-cal
teststhat the
liver
graft might
be
infected by HCV
(2).
How-ever,
serological
assays aredifficult
tointerpret
inthe
context ofimmunosuppression.
Inaddition,
testsfor HCVantigens
are notavailable and
assaysonly detect antibodies
tostructural
and nonstructural
proteins (1). The distinction between
recur-Addressreprintrequests to:Cyril
Foray,
M.D.,Unite de chirurgieHe-patobiliare, HopitalP. Brousse,Villejuif,France.
Receivedfor publication 10April1991andinrevisedform 13
De-cember1991.
1.Abbreviations used in thispaper:HBSAg, Hepatitis B surfaceantigen; HBV,HepatitisBVirus; NANB, Non-A, Non-B; NPCR, nested poly-merasechain reaction; OLT, orthotopic liver transplantation; PCR, polymerase chain reaction.
rences and acquired HCV infections due, in particular, to
blood transfusions during OLT is thereforeamajorproblem. We have used the polymerase chain reaction (PCR) proce-dure(3)todetect HCV RNA(4) in theserumofpatients before and after OLT andto comparethecorresponding nucleotide sequences inahypervariable domain of thegenome.
Methods
Patients.38 patients who underwentliver transplantation between Oc-tober1986 and March 1989attheVillejuifliver transplantcenterwere studiedretrospectively. The patientsweredivided into three groups
accordingtotheir HCV and HBVserologiesat thetimeofOLT.
Group1comprised 16subjectswithapositivetestresultfor anti-HCVantibodies;7 werealsoHBsAg-positive.Theindications for OLT inthis groupwereHBsAg-positive fulminanthepatitis (n=2),
HBsAg-positive cirrhosis(n=5), NANBcirrhosis(n =8), and primary liver cancer(n= 1).
Group2comprised 12patientswho wereanti-HCV-negative and
HBsAg-positive.Theindicationsfor liver transplantation were cirrho-sis(n=8), primary livercancer(n=2), andfulminanthepatitis (n=2).
Group3 (controls) comprised 10 anti-HCV and HBsAg-negative
patients with primary biliarycirrhosis (n = 7) orprimary sclerosing
cholangitis (n=3).
Posttransplantimmunosuppressivetherapyconsisted ofa
combina-tion of prednisone, cyclosporin, and azathioprine. Each patient re-ceived blood products froman average of 103 (SD = 45)different
donors. All theHBsAg-positive patients(n=19) were treated withhigh
doses(10,000IU)of anti-HBs immunoglobulins(HBsIg) during OLT andthroughoutfollow-uptomaintain anti-HBslevels above 100 IU/
liter(5). Neitherthetransfusion products nor the liver donors were screenedforanti-HCVantibodiesatthetime of the study.
Procedures
Serum samples used for serology and PCR were obtained an average of 2mobeforeOLT (SD= 1.7)and 12 mo after OLT (SD=6.5).
Serological
tests
Anti-HCVantibodies.Thepresence of anti-HCV antibodies was tested
for usingarecombinant immunoblotassay RIBA-2 (Ortho Diagnostic
SystemsInc., Westwood, MA.).This test detects antibodies to epitopes located in capsid(c22)and nonstructural genes (c33, c100.3, and 51 1). HBVmarkers. Serum HBsAg, anti-HBs, anti-HBc, and HBeAg
titersweremeasuredby meansof radioimmunoassay using standard kits (Abbott Diagnostics, South Pasadena, CA). HBV DNA in serum wasdetected by a spot test assay as previously described (6).
Polymerase
chain reaction (PCR) for HCV RNA
EXTRACTION AND REVERSE TRANSCRIPTION OF HCV RNA RNAextraction. Sera(120
,l)
were incubated in a lysis buffer consistingof 100mMTris-HCl (pH8), 1%sodium dodecyl sulfate and 50
gg/ml
proteinase K, for2h at37'C. RNA was extracted with
phenol/chloro-form, precipitatedwith ethanol and lithium chloride (0.4 M), redis-solved in 30
,l
ofsteriledistilledwater,aliquoted and storedat-80°C. J.Clin. Invest.©TheAmericanSocietyfor Clinical Investigation,Inc. 0021-9738/92/04/1361/05 $2.00
cDNAsynthesis. 10 lofRNAsolution and 10pmol ofdownstream
primer wereincubated during5 min at65°Candrapidlycooled on ice. cDNAsynthesiswascarriedoutat37°C for45 minafter adjustment of
themixturetocontain 50 mmol/liter Tris-HCl pH 7.5,75mmol/liter potassiumchloride,6mmol/liter magnesium chloride, 1 mmol/liter
dNTPand200IUofMMLV reversetranscriptase (BethesdaResearch
Laboratories,Gaithersburg, MD),in a final volume of30 Ml.cDNA was stored at-20°C.
NESTED PCR (NPCR)
NPCR is a highly sensitive two-step procedure in which the products of afirstPCRusing"outer"primersarereamplifiedusing a second set of
"inner" primers located withinthepreviously amplified sequence (4). Amajor drawback oftheconventional nestingstrategyislinkedto the
opening of the reaction tube afterthefirstamplificationstep,givinga risk ofcontaminatingthe secondreaction with amplicons (7). To over-comethis problem,wehave developeda newprotocol forNPCR
with-outreopening the reaction tube betweenthe two stepsofthe procedure.
5Mlof cDNAwasfirst
deposited
ina700-,ulEppendorf tube,
then 45 Ml ofPCR mix(dNTP:
250,Meach,
KCI: 50mM, MgC12:
1.5mM,
gelatine: 0.01%, 8pmol ofeach outerprimer (Table I)and2.5 Uof
cloned Taqpolymerase (Cetus, Emeryville,CA) were added. The
reac-tion mixturewasthencoveredwith350 Mlof mineral oil.The second PCR mix(150
MI,
with100 pM eachinner primerbut no Taqpolymer-ase)wasinjectedontothesurface ofthemineraloil,formingadroplet within it. Thetubes were then subjectedto afirstamplification(25
cycles: 94°C for45 s,45°C for 45 s and72°C for 55s), and then
centrifugedfor 1 mininorder to mix the second PCRmixture withthe
first.The secondcycle ofamplification (94°C for45s,45°Cfor45 s
and72°C for30s)wasthuscarriedoutinafinal volumeof 200Mlwith
aninner/outer
primer
ratio of12:1.Theproducts ofthefirstPCRaretherefore diluted fourfold. Thesensitivity ofthis procedure is similarto thatof conventionalnested PCR.
Twodifferentsetsofprimerswereusedforeach cDNA(Table I).
Thefirstset(primers SRI, SR2, SF1,andSF2) islocated within the 5'
terminalsequence(5NC) ofthe HCVgenome(8) which is conservedin allHCVisolates.The second setof
primers
(82.0,82.1,82.2,and82.3), situated withinahypervariable region
oftheHCVgenomecoding
foranenvelope
protein "E2/NSI" (9),
waschosento testthevariability
of HCV sequences.ANALYSIS OF PCR PRODUCTS
20Ml ofeachnested PCR
product
wasanalyzed by
meansofelectropho-resison a 1.5%agarosegel.Bandswerevisualized
by
ethidium bromidestaining,transferredto nylon membranes (Gene-ScreenPlus; New En-gland Nuclear, Boston, MA) (10), and hybridized with32P-labeled
probes (TableI).
SPECIFICITYAND SENSITIVITYCONTROLS
Todetect carry-over, we performed HCV-PCR with the following con-trols:(a)serum-freelysis buffer for the detection of contamination at every stepofthe procedure,(b)three negative sera(blood donors) for 12 test sera, (c) the PCRmix alone. Measures for the prevention of carry overincluded drasticseparationof pre-PCR and post-PCR steps. Ithas been shown that most false results with PCR arise during
hand-ling ofamplificationproducts(11).A standard positivecontrol from an
HCV-infectedpatient with postransfusional hepatitis yielded positive results atdilutionsup to 10-8.
Sequencing ofthe
E2/NSI region
PCR products obtained with primers 82.0, 82.1, 82.2 and 82.3 were
purified usingthelow-melting-pointagarose procedure(12) and
se-quencedusingT7 DNA polymerase (Pharmacia, Uppsala, Sweden) andprimer82.1.
Histology
Livergraft biopsieswereobtained afterOLT at the timeof serum
col-lection.
Statistical
analysis
Data were analyzedusingFisher'sexact test.
Results
Before OLT.
HCV RNA was detected in 9 of the 16
patients
who
wereanti-HCV-positive (group 1),
5 of the 12 whowere anti-HCV-negative (group 2)
and none of the controls(group 3).
After
OLT. HCV RNA
wasdetected in
atotal of 10
pa-tients:
3, 4,
and 3 in groups
1,
2,
and
3,
respectively (Table
II
and
Fig. 1).
Four
patients
scored
positive
for HCV RNA both
before
and
after
OLT and
six
were
positive only after OLT
(Table II).
The
four patients with
a
positive amplification ofthe highly
conserved
sequence
(primers 5NC)
both
before
and
after OLT
were
then tested
with primers
encompassing the
hypervariable
domain,
located
in the
5'
part
of E2/NS1
sequence
(13).
Vari-Table
I.Oligonucleotide Sequences
Usedfor
HCV PrimersLengthof PCR 5' terminalsequence* products "Outer" SRl(-) 5'GCCATG
GGC
GTTAGT ATG AG3'(-259; -239)
259SR1(4-) 5'.GGTGC ACG GTCRACGAGACC3'(-21; -1) bp "Inner"
SF2(-*-)
5'GTGCAG CCT CCAGGACCCCCGA
3'(-236; -216)
212SR2(-) 5' ACGGGT GAG GTA GTAGACCC3'(-24; -44) bp
probe 5'.CCGGAA TTGCCAGGA CCGGGTCCTTTC TTG
3'(-76; -46)
Viralenvelope region (ES/NS 1)
82.0(-.) 5' CAC TGGGGA GTC CTAGCGGGC3'(1055-1076) 482
82.3(.*-) 5'GGG CTA GGA GTA AGC AAT A3'(1537-1518) bp
82.1(- ) 5'TCC ATG GTGGGG AAC TGG GC3'(1088-1108) 382
82.2(4-)
5'GGC TTTGGGGGGTAG TCCA3'(1470-1451)
bp
*Nucleotidesequencesarenumbered from
initiating
codon ATG. * ()sense§ () antisense
TableII. Histology, HCV-RNA and Anti-HCVBeforeandAfter OLT Before OLT
No. Etiology
Group 1
1 Fulm.Hep.*
2 Cirrhosis 3 Fulm. Hep. 4 Cirrhosis 5 Cirrhosis 6 Cirrhosis 7 Cirrhosis 8 Cirrhosis 9 Cirrhosis 10 Cirrhosis 11 Cirrhosis 12 Cirrhosis 13 Cirrhosis 14 Cirrhosis 15 Cirrhosis 16
PLCO
Group2 17 Cirrhosis 18 Cirrhosis 19 Cirrhosis 20 Cirrhosis 21 Cirrhosis 22 Cirrhosis 23 Fulm. Hep. 24 PLC 25 Cirrhosis 26 Fulm. Hep. 27 PLC 28 Cirrhosis Group3 29PBC"
30 PBC 31 PBC 32 PBC 33 PBC 34 PBC 35 PBC 36 PSC' 37 PSC 38 PSC Anti- HCV HCV RNA + +_ +_++
+ + + + + + + + + + HBV Anti-HBsAG DNA HCVHCV-RNA _ + _+_ _ + _+_ + + + + + _ +_ _ + + _ +_ _ + + _ +_ _ +_ _ +_ + + --+_ + + ---+ + +--__ + --+_ +-__ + _ + + _ + + _ + + _ + + _+_ - + + --+ _ + +___ _ + +___ _ + +___ _- + + _- + + _- + + HBsAG Histology - Normal - Normal - Normal +
CAHt
+ CAH - Normal - Normal - CAH - Rejection - CAH - Rejection - Normal - Normal - Normal - Normal - Normal - CAH - CAH - CAH - Normal - Normal - Cholestasis - Normal - Rejection - CAH - Normal - Cholestasis - Rejection - CAH - Normal - Normal - Normal - Normal - Normal - Normal - Normal - Normal - Normal*Chronic activehepatitis. § Primary livercancer.
"l
Primarybiliarycirrhosis 'Primary sclerosingcholangitis.ablesequencescould be amplified bothpre-and post-OLT in
twopatients (8, 13). Fig. 2andTable III show the resultsoW
tained in these two cases and in apatient (No. 29) with
ac-quired, probably posttransfusional hepatitis.In bothcases
pre-andpost-OLT nucleotidesequences werehighly conserved(95 and96%).Inaddition,the degreeofhomologywashigher
be-tween pairedpre- and post-OLT isolates than when thefive isolateswerecompared each otherorwithpublished American
(14)andJapanese isolates (15) (Fig. 2). These results demon-stratedthatthelivergraftwasindeed infectedby the pre-OLT strain inbothcases.
TableII comparestheresults of the anti-HCV and HCV
RNAassays.Two ofthe20patients whowereanti-HCV nega-tiveafterOLT,scoredpositive for HCV RNA. Conversely, 10
ofthe 18subjectswhowereanti-HCV positive after OLTwere HCVRNA-negative.
Liverhistologywasavailable for all patients. Chronic Hepa-titiswassignificantlymore frequentin patients with positive
HCV viremia (6/10 vs2/28; P< 0.01). Itis noteworthy that noneofthe 6patients with HCV viremia and chronic hepatitis
had evidence of associated HBV infection. Among the 28
HCV-RNA negative patients, 2 showed HBV-related chronic
After OLT Follow up (mo) 10 11 15 12 11 6 9 3 12 13 9 18 14 9 8 12 4 36 14 13 11 12 12 8 36 9 10 12 11 13 12 15 14 11 9 14 15 12
8
+ +
b a sc+
sc-|< 210 bp
A
Figure1. Detection of HCV RNA by nestedPCR(primers5' NC).
Patients 7,29,and 8 before (b) and after(a) OLT. SC+, positive standardcontrol. SC-, negative control. (A) Ethidium bromide staining. (B) Hybridization with 32P-labeled oligonucleotideprobe(30 min exposure).
hepatitis. These 2 subjects
werethe onlysubjects
amongthe 19HBsAg
positive individuals who showed evidence for
HBVre-currence as
assessed by positive
serumHBs Agand
HBV DNAassays.This
low
rateof
recurrenceis
explained by
alow
preva-lence of HBV replication before OLT (1/19)
togetherwith
long-term immunoprophylaxis with
HBsIg(16).
TableIII Nucleotide Sequence
Homologies
(%) in theHypervariable
Domain ofthe 5' Part ofE2/NSI
HCVJ1 (5) 5 63 66 55 52 55 63 HCV-Pt(4) 4 78 77 64 63 65 -No. 29 After OLT 3 76 78 65 65
-No. 13 AfterOLT 2' 70 67 -BeforeOLT 1' 68 69 -No.8 AfterOLT 2
-BeforeOLT 1
-1 2
1'
2'
3 4 5Sequences ofHCV RNAfrom 3patients in the present study are
shown, including2withHCVrecurrence (patients 8 and 13 in Table II) and 1 with posttransfusional hepatitis (patient 29 in Table II). Lanes Iand 2 correspond to pre-OLT and post-OLT HCV RNA se-quences inpatient8,whilelanes1'and 2'correspond to those of
patient 13. Lane3indicatesthesequence of HCV RNA in patient 29. Other sequencesfromthe literature are included for additional
comparison: HCVpt(4) and HCVJ1 (5) isolates (European patent
application90302866.0; Takeuchiet al., 1990). The
numbering
corresponds to HCVpt (European patent application 90302866.0).Discussion
1964
1 GGGGGA AGT GCC GCC CAC ACT ACC GGT TGA
2 ... ..C ..T
1' .. ¶TT CGT TCG ..C ..G CA. G..
2'.. ... ..T CGT TCT ..C ..G CA. G..
3.... ... ... ACA...G ..C ..C
4...
.G
. GTG TC. G..5.. G GTG CAA .G. ... GTC ... TC. AC.
1994
1 ATTEGTC TAG TAT TTA ACA CCA GGC GCC AAG 2 A. ... ... ..C ... ... ... ... ...
1'.C. CA. C . . .T. T CTC T. ..G .G.
2'.C. CA. C. C T. ... CTC T ..G ..G.
3CACTA. ... ..G TTG G. ACG ... ...
4T....T AGC CTC C.C G.... ... ... ...
5 C.CACG .CC CTC ..T .G. ..T ..G ..G TTC 2224
1 CAGAAC ATC CAG CTG ATC AAC ACC AAT GGC
22 ... ... .. .. .. ..
1'. T ... TGT .A..T. A. T.
,2'. .T .C TGT .A..T . T 3 ... ...
4 ... G..
5 A ..T
...CT ..G
....
... ... ... ..T G.A... ... ..C ... ... ... ..C ...
... ... ... ...
2254
1 AGTTGG CAC ATC AAT AGG ACG 2 ... ...
1' . ... .... .C
21'... ... ... ..C ... . ...
3. G . .. T
4 .T..C...T 5 ...T..T..C ..T
Figure 2. Nucleotide sequence of the
hypervariable
domain of 5' part ofE2/NSIof HCV RNA before and after OLT. Nucleotide sequences of thehypervariable domain oftheE2/NS1
region
before(lane 1;
lane1')
and after(lane 2;lane2)
OLT inpatients
8and13,
respec-tively. Nucleotidesequenceofthesame
region
ofE2/NS1
inpatient
29withacquired,
posttransfusional hepatitis (lane 3)
and fromtwo publishedreports.HCVpt(lane 4)and HCVJ1(lane 5)
isolates (Eu-ropean patentapplication 90302866.0;Takeuchietal.,1990).
Thenumbering correspondstoHCVpt (Europeanpatent
application
90302866.0).Dotsindicate identicalsequences.
Our
findings
provide direct evidence that
HCVinfection
canrecurin
liver transplant recipients.
Indeed,of
thetensubjectswho
wereHCV-RNA positive after
OLT, fourwerealsoposi-tivebefore
the
transplantation.
Wedocumented the
recurrenceof HCV
infection
intwopatients by the comparative
studyof
ahypervariable domain of the HCV
genomebefore
and afterOLT. The
nucleotide
homology
wasindeed
veryhigh,relative
tothat of
distinct
isolates.
The infection of the engrafted liver
by
the initial HCV strainprobably involved circulating virions.
However, it could
alsooccurvia
infected nonliver cells,
asprevi-ously suggested
for HBV(17, 18). Preliminary results indeed
suggestthatperipheral
bloodmononuclear
cellscanbeinfectedby
HCV(19,
andZignego,
A.L., manuscript submitted
forpublication).
The
actual
prevalence of HCV
recurrencein OLT
patients
cannotbederived fromourdata.Although the
ratewaslow,
it
should indeed be borne in mind
that
weonly analyzed
asingle
sample
obtained from eachpatients
atameanof
12moafter
OLT. Additional
intermittent,
transientorlateHCV
infectionscannotthusbe excluded.
It is also interesting
tonotethatsomeof these
patients bore
markers
of both HCV and HBVinfection
before OLT. It istempting
tospeculate
thatsuch coinfectionmight
attenuateHCV
viremia before OLTand, thus, the
risk of HCV reinfec-tion. In addition anti-HBsantibody
preparations,
usedtopre-vent HBV recurrences in these
subjects, might
containanti-HCV
antibodies
although
thebatcheswehavetested
havebeennegative
inaRIBA-IIassay.6 ofthe 38subjects
hadacquired
HCV infection after OLT. It is
noteworthy that
thisstudy
wasundertaken before the exclusion of anti-HCV
positive
blood and liver donors becamepossible. Although screening
ofbloodproducts
withserological
testsis
quite efficient (20),
thenum-berof donorsnecessarytotransfuse organ
recipients
islarge,
and thefrequency
oftransfusion-related infectionmight
well remainhigh.
1364 Feray, Samuel, Thiers,Gigou, Pichon, Bismuth,Reynes,Maisonneuve, Bismuth, and Brechot
patient No 7 RIBA2 + +
b a
29
Interestingly,
HCV viremia wasonly
associated
withchronic
hepatitis in patients who showed no evidence ofasso-ciated
HBV infection. Withregard
tothedevelopment ofhe-patic lesions,
therespective role of a direct cytopathiceffect
ofHCV and the
hostimmune response to viral and cellularanti-gens is
not known.However,
severechronic activehepatitis
hasbeen
reported
inpatients
infected with both HCV and HIV(21). Taken together
ourresults
indicate that HCV may beasignificant
causeof liver disease
following
OLT.Finally, although
anti-HCV antibodies weresought using
the RIBA-2 test, 2/20
ofthepatients
whowereanti-HCVnega-tive
after OLT
wereHCV
RNA-positive.
Thusserological
sta-tus
after
OLT was notabletodistinguish
recurrentfromac-quired, posttransfusional
HCV infection.Therefore,
detectionby
PCR ofHCV RNA iscurrently
theonly method forapprais-ing
HCV infection
following liver transplantation.
Acknowledgments
The authors thank D.Castaing,J. P.Benhamou, J. Bernuau, and F.
Saliba, for including patientsin theliver transplantprogram. C.
FRray
holds grantsfromtheFondationpour la RechercheMelicale
and theFondation Maurice Rapin. Thisworkwassupportedby Institut
Na-tionalde la
Sant6
etde la RechercheMedicale
U75, CNAM,ARC(6252),LNC andPasteur-Diagnostic.
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