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Serum Hepatitis B Virus DNA Detects Cryptic Hepatitis B Virus Infections in
Multitransfused Hemophilic Patients
By M.G. Rumi, M. Colombo, R. Romeo, G. Colucci, A. Gringeri, and P.M. Mannucci
The recognition of replicating hepatitis B virus (HBV) may
be important to both define the cause of and know how to
manage chronic liver disease in multitransfusedhemophilic
patients. Replicating HBV can be detected at the molecular
level by methods for HBV-specific DNA (HBV-DNA), which
are much more sensitive than the immunologic methods for
detecting hepatitis B surface antigen (HBsAg) and hepatitis
B e antigen (HBeAg). Unselected hemophilic patients (260;
6% with HBsAg, 4% with isolated anti-hepatitis B core
(anti-HBc), 52% with anti-HBs and anti-HBc, 26% with
isolated anti-HBs, and 12% with no HBV marker) were
investigated retrospectively with a dot spot hybridization
technique that detects serum HBV-DNA down to 0.5 pg
and by Southern blot analysis, which tests the specificity of
the HBV-DNA reactions. Eighteen patients (7%; five with
serum HBsAg and 13 HBsAg seronegative with antibodies
to HBV) had serum HBV-DNA. Serum HBV-DNA was
detected more frequently in HBsAg carriers than in seronegative patients (33% versus 6%. P c .01).and had no
relationship to serum alanine aminotransferase. Serum
HBV-DNA was more sensitive than the radioimmunoassay
for HBeAg was for detecting replicating HBV (7% versus
1.I %, P < .01],
These findings demonstrate that there is
cryptic HBV infection in a number of hemophiliacsand that
serum HBV-DNA may coexist with markers thought to
reflect immunity against HBV.
0 1990 by The American Society of Hematology.
H
These and other findings suggest that the role of HBV in
chronic hepatitis may be greater than previously thought.
With the advent of antiviral treatment of chronic hepatitis B,
detection of cryptic HBV infection may be important not
only for definition of the etiology but also for the management of chronic liver disease in hemophiliacs. Therefore, we
looked for HBV-DNA in the sera of 260 unselected hemophiliacs attending our clinic.
EPATITIS B virus (HBV) infection is an important
cause of morbidity in multitransfused hemophilic
patients, even in this era of clotting factor concentrates
treated with virucidal methods.’ Approximately 10% of
multitransfused hemophiliacs are chronic carriers of hepatitis B surface antigen (HBsAg) as a consequence of lifelong
exposure to infectious concentrate^.^^^ Chronic HBsAg carriers with elevated serum transaminases fall into two categories. In one category are patients with overt HBV replication,
as indicated by hepatitis B e antigen (HBeAg) in serum and
hepatitis B core antigen (HBcAg) in the l i ~ e r . ~This
.~
virologic profile, which, with few exceptions, is associated
with HBV-dependent liver disease, may be time-limited by
seroconversion to antibody to HBeAg (anti-HBe), which
leads to suppression of HBV replication.6 For the other
category, HBsAg carriers who have circulating anti-HBe, ie,
a marker of suppressed HBV replication, the etiology of
chronic liver disease is less certain. In these carriers, liver
disease has been attributed to hepatotoxic factors other than
HBV, such as the delta virus, the non-A, non-B viruses,
drugs, autoimmunity, or alcoh01.~However, recent studies
have clearly demonstrated that a subset of HBsAg carriers
harbor HBV with circulating anti-HBe. In these, cryptic
replication of HBV has been linked to preferential localization of HBcAg in the cytoplasm of infected hepatocytes and
with the probability of progression of liver disease to
c i r r h o ~ i s HBV-DNA
.~~~
sequences have also been found in
the serum and liver of HBsAg-seronegative patients with
chronic hepatitis by a molecular hybridization technique.”,’ ’
From the A . Bianchi Bonomi Hemophilia and Thrombosis
Center, Institute of Internal Medicine; and Institute of Human
Genetics. University of Milan, Italy.
Submitted March 9. 1989; accepted December 19, 1989.
Address reprint requests to M . Colombo, MD, Institute of
Internal Medicine, University of Milan. Via Pace, 9 Milano. Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
o 1990 by The American Society of Hematology.
0006-4971/90/7508-0001$3.00/0
1654
MATERIALS AND METHODS
Patients. We looked for HBV-DNA in serum samples from 260
unselected hemophilic patients attending the A. Bianchi Bonomi
Hemophilia and Thrombosis Center of the University of Milan.
They were all male, 2 to 71 years of age (mean, 28). Of the 260
patients, 209 had hemophilia A, 51 had hemophilia B. The clotting
defect was severe (factor VI11 or IX, less than 1%) in 176, moderate
(between 1% and 5%) in 34, and mild (higher than 5%) in the
remaining 50 patients. Table 1 shows other characteristics of these
patients. Of 260 patients, 244 had been infused lifelong with clotting
factor concentrates, either for prophylaxis or for treating bleeding
episodes; the remaining 16 patients were never treated with blood
products. Of the 244 treated patients, 227 had been given unmodified
concentrates until 1985. Since 1985, these patients were infused
exclusively with concentrates for which viral inactivation was
attempted by various heating procedures (dry-heating, hot vapor or
pasteurization). Serum samples for HBV-DNA assay were obtained
at least 2 weeks after infusion with clotting factor concentrates. The
remaining 17 patients had been treated exclusively with heated
concentrates. Fifty-five patients have had an average annual cumulative exposure to clotting factor concentrates greater than 30,000 IU
(intensively treated); 61, between 10,000 and 30,000 IU (moderately
treated); and 111, less than 10,000 IU (mildly treated). Table 2
shows the serum profiles of HBV markers for the whole group of
patients. Fifteen patients (6%) were chronic (greater than 1 year)
HBsAg carriers; 135 patients (52%) had both serum anti-HBc and
anti-HBs; 68 patients (26%) had isolated anti-HBs, 35 of them with
naturally occurring anti-HBs and 33 who developed anti-HBs after
vaccination against HBV (Hevac B, Pasteur, Sausfi, Paris, France;
or Engerix B, SKF, Prixeusart, Belgium); 11 patients (42%) had
isolated antibody to core antigen (anti-HBc); and 31 patients (12%)
had no HBV marker. Eighty-five patients (33%) were anti-human
immunodeficiencyvirus (HIV) seropositive. Of these, six had constitutional symptoms (fever, weight loss), and seven had full-blown
AIDS.
Serum HBV and HIV markers and liver function tests. The
Blood, Vol 75, No 8 (April 15), 1990: pp 1654-1658
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1666
SERUM HBV-DNA IN HEMCPHLIC PATIENTS
TrrmWn
No.
AoD.Mwnvn
lR-1
-
UnhOOtOdcmOnIrl3t8SUntil
1985 (IUI
230.000
10.000-30.000
< 10,Ooo
Subtotal
aJvhWtdlOCtCK#
Nobloodproduct
Totd
the EmRl site in pBR325. was purified by preparative e l e c t m p h
sis and radiolabeled by a random priming (Amersham Multiprime
D N A labeling system. Little Chalfont. England) to yield a specific
activity of 2 to 4 x Idcpm/rg. After high stringency washes in 2
and 0.2 x SSC and 0.1% SDS at 68%. nylon Qembranes were dried
and exposed to Kodak X-Omat AR film for I 2 to 60 hours at 8OOC.
The sensitivity of this technique was 0.5 pg. as a.ssesscd by the
calibration curve that was constructed with known amounts of
cloned HBV-DNA. Positive spots were graded from traces (:) to
4+. The samples graded as traces contained about 0.5 pg of
HBV-DNA and wen double retested. Grade 1 + corresponded to I
pg; grade 2+. 10 I O pg; grade 3 +. to 50 pg; and grade 4+. to 100 pg
of HBV-DNA (Fig la). Examples of HBV-DNA readings in
patients’ sera are shown in Fig I b. The specificity of the HBV-DNA
dot-blots was assessed by Southern blot hybridization. EcoRIdigested D N A samples (5 pg) were separated by electrophoresis on
0.8% and 1.5% agarose gel. Lambda Hind111 DNA fragments were
used as molecular weight markers. Digestion with restricted enzymes was carried out according to the instruction of the manufacturer. Electrophoresis (running bufer: 0.089 mol/L Tris. 0.089
mol/L boric acid. 0.002 mol/L EDTA) was carried out at 36 V.
overnight. After staining with ethidium bromide. the gel was
denaturated with I Y NaOH for 40 minutes. neutralized with I
mol/L Tris. pH 7.5. and processed for D N A transfer according to
66
61
111
227
17
16
260
23 (6-661
28 (10-611
22 (3-71)
26 (3-711
12 (2-621
23 (7-691
serum specimens were collected from patients during their regular
annual clinical visits and kept frozcn at -2OOC until used. At the
same time. liver function tests. including alanine aminotransferase
(ALT). aspartate aminotransferase (ASP. bilirubin. alkaline phosphatase (AP). gamma-glutamyltranspeptidase. and serum protein
electrophoresis. were performed. HBV markers, including HBsA&
anti-HBs. and anti-HBc. were tested for by radioimmunoassay
(RIA; Abbott Laboratories. North Chicago, IL). HBsAg reactive
sera were also tested for HBeAg. anti-H&. and antibody to delta
virus (anti-HDV) by RIA. Anti-HIV was measured by enzymelinked immunoassay (Organon Tecknica. Vcedijk Turnhout. Belgium), and positivity was confirmed by Western blot analysis.”
HBV-DNA assay. Serum HBV-DNA was detected in coded
serum samples as previously described” by a dot-spot hybridization
technique. Briefly. 20 pL of serum was applied directly to nylon
membranes that were denaturated with I N NaOH. neutralized
with I ml/L Tris. p H 7.5. and incubated with 200 &mL
proteinase K for 2 hours at 37%. Thereafter. nylon membranes were
soaked in 20 x SSC. dried and baked in a vacuum oven at 80% for 2
hours. Cloned HBV-DNA and normal human genomic DNA served
as positive and negative controls. respectively. for all hybridization
assays. To further assess the specificity of the hybridization reactions.xlected DNA blots were rehybridizedwith ”P-labeled pBR325
lacking the viral insert. Hybriditation. carried out at 42% in scaled
plastic bags. consisted of two steps: first. an overnight incubation in
the pre-hybridization solution (5 x SSC. 5 x Denhardt’s solution.
0.05 mol/L phosphate bufer. 50 &mL salmon sperm DNA. 0.1%
sodium dodecyl sulfate (SDS). 50% fonnamide); and second. overnight incubation in the hybridization solution (5 x S C . 5 x
Denhardt’s. 0.02 mol/L phosphate buffer. 100 r g / m L salmon sperm
DNA. 10% dextran sulphate. 50% formamide. 0.1% SDS) using
”P-labeled pHBV (I@cpm/cm: membrane) as probe. The probe
was kindly donated by Dr G.Ratti (Sclavo Institute. Siena. Italy).
pHBV. whichcontainsacopyofthe3.2kbgenomeof HBVclonedat
a
HB&g
HMO
Anti-HBe
Anti-HDV
Anti-HBs end anti-HBc
AmiHBs only
Natudlv Ocanjng
Port-Mceirution
Anti-HBc onC
No HBV marker
Tot&
15 (6)
3
12
4
136 1621
68 (261
35
33
11 (41
31 (121
260 (1001
2
3
4
5
6
7
1
2
3
4
5
6
7
A
B
b
c 0.
Flg 1.
~
1
.*
0
(a) Urm Aand B r h o w t t ~ ~ . m o c r d k g r w n o f w I ) V ~
spot hybddimkntoat of tho alibmion OUTV.. Spot. 1 and2 or.
tho mg.tiv0 controls containing “ P - h k h d pBR326 Wing tho
viral insort and “P-kkhd norm01 hunun genomic DNA. Spot. 3
through 7 aro tho por(tiv0 contrds containing sP-hkhd HBVDNA. Spot 3.0.6 pg of HBV-DNA: .pot 4.1 pg; .pot 6-10 pg: WOt
6,M) pg; spot 7.100 pg. Tho eom.ntiocUl grading (+an - to 4 + I
of HBV-DNA dot-blots in potionts’ UT. is g i v m on tho bottom lirw.
(bl Lima C and D show tho roauks in potionts‘ U
T
.
. Spot 1,
H B d g - and HB~Ag-poSitivOptim with 4 + HBV-DNA .pot 2.
HBsAg- and anti-HEo-pMitivo potimt with 1 HBV-DNA spot 3.
H B s A g ~ ~ g . t i vpotiont
o
with no HBV-DNA; spot 4. HBAg- and
HBoAg-poskivo potiont with 2 + HBV-DNA; #pot 6. H B d g nogotivo vaccinnn with t r m a m w m of HBV-DNA; .pot 6,
HBaAg-nogotivo potkm with no HBV-DNA; spot 7. HBOAg- and
H B ~ A g - p o ~ i Wtimt
t k ~ with 3+ HBV-DNA.
+
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RUM1 ET AL
1656
A
Southern.” The membrane was then dried. baked. hybridiml.
washed. and proc*rxd for autoradiography as previously described.
To assess the molecular status of HRV-DNA in our samples.
HRV-DNA isolated from circulating viral particles by phenolchloroform extraction. and ethanol precipitation was used as control.
Srorisrieal analysis. The prevalence of serum HRV-DNA in
relation to other markers of HRV infection. severity of coagulation
defect. intensity of previous treatments. ALT elevation. and presence
of HIV infection were analyzed by Fisher’s exact test. The prevalenceof serum HBV-DNA in relation to patient age was analyzed by
t he Student‘s I test.
B
C
D
E
Kb
-3.2
RESULTS
Eighteen patients (7%) tested repeatedly positive for
serum HRV-DNA (Table 3). Five were HBsAg-positive
(three with HReAg. two with anti-HRe). six had both
anti-HBs and anti-HBc. five had isolated anti-HBs (including two vaccinees). and two had isolated anti-Hk. HRVDVA was detected in none of the 31 patients who did not
have HBV markers. HRV-DNA was detected more frequently in HRsAg carriers than in HRsAg-negative patients
with antibodies to HRV ( 5 of 15.33%. versus 13 of 214.6’70.
P < .01). There was no relationship betwan serum HRVDNA and ALT. with the prevalence of HRV-DNA in
patients with elevated ALT similar to that of patients with
normal ALT (I5 of 170, 9%. versus 3 of 9. 33’70. not
statistically significant). HRV-DNA was more sensitive than
the RIA for HReAg for detecting patients with replicating
HBV ( I8 of 260.7‘7, versus 3 of 260. I . 1’70. P < .01). Serum
levelsof HRV-DNA were high (2+ t o 4 + ) in three HReAgpositive carriers. low ( I +) in two anti-Hbpositive carriers.
and t r a m ( 2 ) in 13 HRsAg seronegative patients with
antibodies to HRV. The presence of free HRV genome was
confirmed in all the HRV-DNA positive sera by Southern
blot analysis (Fig 2). The prevalence of HBV-DNA was not
influenced by patient age, severity of coagulation defect or
intensity of previous treatments. nor by elevated levels of
ALT or HIV infection (Table 3). Two patients with serum
HRV-DNA as the only marker of HRV infection. who had
been successfully vaccinated against hepatitis R when the
results for HRV-DNA were not yet available, developed
protective serum levels of anti-HRs I month after the fourth
dose of the vaccine (greater than 120 mlU/mL). In one
patient, HRV-DNA was detected during the course of
vaccination. During 2 years of follow-up. this patient re-
Fig 2. Autoradlogr” showing tho Southom hybddimkn
orulysls of HBV-DNA. b n o A is the positlw comrd consisting of
DNA i4at.d from circulating DNA panicles:b n e s B through D are
the patients’ sera containing trace amounts ( t 1 of HBV-DNA.
lanos B and C show serum HEV-DNA in two d n o n . Lene D
ahserum HBV-DNA in a patient who m s rerum HBsAgnegative and anti-HBs- and anti-HBc-podtiva. Lane E is tho
HBV-DNA-negative rerum usod as negative control.
mained persistently negative for HBsAg and anti-Hk. The
other patient was found to be HRV-DNA positive 2 years
after the booster dose of vaccine, coincident with a period of
heavy e x p u r e to plasma concentrates.
DISCUSSION
Serum HBV-DNA is a sensitive and specific marker of
HRV infection that is correlated quantitatively with the
replicating activity of the hepatitis virus in the liver.“
Consistent with previous data for non-hemophilic patients.
our study indicates that serum HRV-DNA is more sensitive
than the R I A for HBeAg for detecting patients with replicating HRV (7Rnversus 1.1%. P < .01). In fact. HBV-DNA was
found not only in the three HReAg-positive patients but also
in two HBsAg-positive patients with serum anti-HRe. in two
HBsAg-negative patients with anti-Hk. and in I I with
TOM.3. 8
.
” HBV-DNA md k.R . i n i w h i p With 8
.
” ALT ond
HBV M.r(tmh, 281) H.mophllkP n k n n
Pn*nts With WBV-DNA
~~
No.
Suun Mmkm
PMW3IltS
HBsAg
Anit-HBs and anti-HBc
Anti-HBs
Anti-HBc
No HBV “#cor
15
135
68
TOtd
260
Anti-HIV
Anti-HtV
+
-
11
31
85
175
OWd
No. 1%)
5 (33)
6 (4)
5 (71
2 (18)
018 (7)
9 (10)
9 (5)
N a n u I ALT
No. (961
E k m dU T
No. (961
0121 -
-
3/12 (251
5/94 (5)
5/45 (1 1)
219 (22)
0110
3/90(3)
151170 (9)
213 (67)
1/41 (2)
0113
012
4/19 (21)
2/71 (3)
-
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1657
SERUM HBV-DNA IN HEMOPHILIC PATIENTS
serum anti-HBs or anti-HBs and anti-HBc markers thought
to indicate immunity against HBV.16
In this study, trace amounts of HBV-DNA were detected
in 13 HBsAg-negative carriers. The interpretation of the
results of the dot-blot assay graded as trace amounts of
HBV-DNA was unequivocal, because the results were consistently reproducible, and dot-blot-positive serum samples
were shown to contain HBV-DNA by Southern blot analysis.
It was more difficult to interpret biologically these findings.
Trace amounts of HBV-DNA were unlikely to be passively
transferred from infusions with infected clotting factor
concentrates, since the HBV-DNA assays were carried out in
serum samples collected a t least 2 weeks after the last
infusion with concentrates. The serologic profile of HBVDNA associated with anti-HBc only, present in two HBsAgnegative carriers, might signal either infection with HBV
expressing subliminal serum levels of HBsAg or infection
with an HBV strain defective for HBsAg synthesis. The
existence of carriers with low serum levels of HBsAg is
substantiated by the circumstantial evidence that a small
proportion of blood donors with anti-HBc as the only marker
of HBV infection transmit hepatitis B to the recipients of
their blood donations." Recently, a D N A fragment that
hybridized with HBV-DNA was extracted from the liver of
an HBsAg-negative, anti-HBc-positive patient, demonstrating the existence of an HBV strain with a mutation within
the sequence coding for HBsAg synthesis."
The most intriguing finding of this study was the cooccurrence of serum HBV-DNA in six patients with antiHBs and anti-HBc and in five patients with isolated antiHBs, including two vaccinees. Studies of immunoprophylaxis
against HBV have indicated, with some exceptions, correlation of the presence of anti-HBs with immunity to subsequent
hepatitis B infections.16 The first such exception was the
finding in 1978 of Spero et al, who used an immunofluorescence technique for staining hepatic HBcAg in biopsy
samples, that HBsAg-negative hemophiliacs with serum
anti-HBs and anti-HBc can harbor replicating HBV.19 Another important exception was the report of well-documented
cases of hepatitis B in non-hemophilic patients with preexistent anti-HBs.20.21These findings suggest that exposure
to HBV may lead to convalescent anti-HBs with weak
reactivity against subsequent HBV infection. The cooccurrence of HBV-DNA and isolated anti-HBs that we
observed in five patients, including two vaccinees, might be
explained by infection with HBV-related variants that are
immunologically distinct from HBV and do not express core
A similar explanation was offered for the occurence of cryptic infection with HBV in successfully vaccinated newborns of chronically infected mothers and in adult
family contacts.24Taken together, these data confirm that in
high risk patients vaccination does not give 100% protection
against HBV. Whatever the significance of serum HBVD N A in HBsAg-negative patients with antibodies to HBV,
our data corroborate the evidence that HBV may have a
greater role in chronic hepatitis of hemophiliacs than previously thought. This biologic profile is not unique to hemophiliacs, since it can also be found in other settings. Recently,
HBV-DNA was detected in sera of 68 blood donors with
elevated ALT who tested negative for H B s A ~ .These
~~
findings emphasize the importance of serum HBV-DNA as a
test for detecting cryptic HBV infection. Assays for serum
HBV-DNA should, therefore, be incorporated in the routine
check-up of hemophilic patients, to better define the etiology
of the underlying liver disease. These assays should also be
employed as an adjunct to anti-HBc for detecting cryptic
HBV infection in vaccinees.
REFERENCES
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factor concentrates. Lancet 2:782, 1988
2. Mannucci PM, Capitanio A, Del Ninno E, Colombo M, Pareti
F, Ruggeri ZM: Asymptomatic liver disease in hemophiliacs. J Clin
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1990 75: 1654-1658
Serum hepatitis B virus DNA detects cryptic hepatitis B virus
infections in multitransfused hemophilic patients
MG Rumi, M Colombo, R Romeo, G Colucci, A Gringeri and PM Mannucci
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