(HBsAg) but this marker does not indicate the presence of infectious particles in the blood. In individuals, infected with H B V , H B s A g is found predominantly in ...
Rev. Inst. Med. trop. São Paulo 31(6): 377-383, novembro-dezembro,
1989
POTENTIAL INFECTIVITY OF BLOOD FROM HBsAg ASYMPTOMATIC CARRIERS DUE TO THE PRESENCE OF HBV-DNA AND COMPARISON WITH OTHER MARKERS OF HBV INFECTION
M . V a l e r i a T E D E S C H I (1), C l á u d i a F i g u e i r e d o P A D I L L A ( 1 ) , I n á F e r r a z d e C A M A R G O (2) & C l a r a F . T a c h i b a n a Y O S H I D A (1).
S U M M A R Y
Serum
s a m p l e s f r o m 356 H B s A g
positive
titrated by reverse passive hemagglutination, H B V - D N A , H B s A g and IgM
asymptomatic were
carriers, which
a n a l y s e d for
were
the presence
a n t i - H B c . T h e s a m p l e s were divided in three
of
classes,
a c c o r d i n g to the titers of H B s A g a n d I g M a n t i - H B c a n d the d i s t r i b u t i o n of H B V - D N A and H B s A g
among
these classes was studied. In
the
6 5 % of s a m p l e s h a v e o n e or b o t h m a r k e r s a g a i n s t o n l y
high
titer c l a s s of
19% in the low
F r o m t h e t o t a l o f 3 5 6 s a m p l e s , 121 g a v e p o s i t i v e r e s u l t s f o r I g M F r o m these, 38.9% of H B V - D N A
a n d 4 7 . 9 % of H B e A g
A
higher frequency
of a g r e e m e n t b e t w e e n
t i t e r s of H B s A g ;
however,
were observed, whereas
c l a s s of H B s A g a n d little or n o I g M even in H B s A g positive borderline
K E Y
WORDS:
was detected
in the
anti-HBc, showing potential blood
in
found.
all these m a r k e r s w a s found
H B V - D N A
class.
a n t i - H B c (33.9%).
s a m p l e s w i t h a b s e n c e of I g M a n t i - H B c , 18.3% a n d 16.6% w e r e r e s p e c t i v e l y
c l a s s of h i g h
HBsAg,
titer
in
low
the titer
infectivity
samples.
HBV-DNA; HBsAg; IgM anti-HBc Asymptomatic
carriers.
I N T R O D U C T I O N
The Hepatitis
B
v i r u s ( H B V ) is o n e of
the
tion being found
in some areas
2
1 2
2 9
,
mainly
viruses that cause hepatitis in m a n , a n d the clini-
in W e s t A m a z o n where its p r e s e n c e is often asso-
cal picture is extremely variable, c a u s i n g acute
c i a t e d to the D e l t a v i r u s
and chronic forms with different prognosis
3
1 3
.
and is
usually
strong epidemiological and biochemical eviden-
a c h i e v e d b y a s s a y of the H B V s u r f a c e
antigen
ce for
( H B s A g ) but this m a r k e r
degrees of s e v e r i t y , i n c l u d i n g
cirrhosis and
association with hepatocellular
The
a
carcino-
d i a g n o s i s of H B V infection
does not indicate
p r e s e n c e of i n f e c t i o u s p a r t i c l e s i n t h e b l o o d .
ma.
the In
i n d i v i d u a l s , infected w i t h H B V , H B s A g is found T h e infection
is e n d e m i c
in m a n y
parts
of
predominantly
i n t h e f o r m of 22 n m
non-infec-
it p o s e s a s i g n i f i -
t i o u s p a r t i c l e s a n d a l s o a s p a r t of s u r f a c e a n t i g e n
c a n t p r o b l e m , w i t h h i g h f r e q u e n c i e s of t h e infec-
present in complete H B V particles . T h e s e com-
the world a n d in our country
8
(1) D e p a r t a m e n t o d e V i r o l o g i a , F u n d a ç ã o O s w a l d o C r u z , R i o d e J a n e i r o , B r a z i l . (2) B i o - M a n g u i n h o s , F u n d a ç ã o O s w a l d o C r u z , R i o d e J a n e i r o , B r a z i l . A d d r e s s f o r c o r r e s p o n d e n c e : D r a . M . V a l e r i a T e d e s c h i , F u n d a ç ã o O s w a l d o C r u z , D e p a r t a m e n t o d e V i r o l o g i a , A v . B r a s i l 4365. C E P 21040 R i o d e J a n e i r o , R J , B r a z i l .
plete particles also h a v e a n inner core w h i c h con
d i l u t e d i n 50 m M
tains the Hepatitis B core antigen ( H B c A g )
coated in polystirene
H B e A g in a cryptic form
1
2 4 ,
3 1
and
H B e A g in the s e r u m a s a soluble protein is asso
a n d is therefore
utilized
as a marker
virion
2 5
microplates. After
over
night incubation, the plates were w a s h e d 4 times
. T h e p r e s e n c e of
c i a t e d w i t h t h e p r o d u c t i o n of c o m p l e t e
c a r b o n a t e b u f f e r p H 9.6 w e r e
,
of infec
i n B S A ( 0 . 5 % ) i n P B S , a n d 100 p l e s (2 x
10
3
in N H S - P B S )
of d i l u t e d
sam
were incubated
for
l h / 3 7 C . T h e p l a t e s w e r e w a s h e d a g a i n , 100
fú
o f p u r i f i e d H B c A g ( 5 0 n g / m l ) d i l u t e d i n 1% N H S /
tivity.
P B S w a s a d d e d a n d the reaction w a s i n c u b a t e d circular
f o r 1 h o u r a t 37 C . A f t e r w a s h i n g t h e p l a t e s , 100
and a virus specific D N A polymerase can
fjá o f r a b b i t a n t i - H B c c o n j u g a t e d t o p e r o x i d a s e
Inside the core, a double-stranded D N A
be f o u n d
1 5
'
1 6
-
2 0
d i l u t e d i n 10%) N H S / 1 0 % N R S / P B S w e r e
.
added
a n d i n c u b a t i o n p r o c e e d e d for l h / 3 7 C . A f t e r w a Different t e c h n i q u e s are a v a i l a b l e for the de
s h i n g , 1 0 0 fú o f s u b s t r a t e ( 1 . 2 m l o f l O X s u b s t r a t e a n d 0.25 m l
of
low sensitivity, like electron microscopy, H B c A g
5 m g T M B / m l D M S O ) were a d d e d a n d after
30
d e t e c t i o n a n d D N A p o l y m e r a s e a s s a y or i n d i r e c t
minutes at room temperature, the reaction was
tection of c o m p l e t e v i r u s b u t t h e y are either
of
buffer
+
10.8 m l d i s t i l l e d w a t e r
stopped with
l i k e the d e t e c t i o n of H B e A g .
100 /xl o f 4 N S u l f u r i c A c i d .
(The
c o m p o n e n t s of 1 0 X s u b s t r a t e buffer are S o d i u m R e c e n t l y , the u s e of m o l e c u l a r h y b r i d i z a t i o n analysis
allowed
HBV-DNA
the
serological detection
of
by nucleic acid hybridization, provi
d i n g a direct a n d h i g h l y s e n s i t i v e m a r k e r of v i r a l 4
5
replication ' '
6
2 8
A c e t a t e 34.0 g , C i t r i c A c i d 0.73 g , i n 250 m l d i s t i l l e d w a t e r a n d 0.3 m l H
0
2
of
30%). T h e reac
t i o n is r e a d a t 450 n m . T h e cut-off v a l u e is c a l c u lated
using the m e a n negative control
+
0.1
m e a n positive control. N e g a t i v e controls are co
.
lourless (extinction I n t h i s w o r k , w e s o u g h t to e s t a b l i s h t h e p o tential infectivity
2
of H B s A g p o s i t i v e s e r a
from
a r o u n d 0.05) a n d
positive
controls give a colour that is clearly discernible from the negative control.
a s y m p t o m a t i c carriers as determined by the pre s e n c e of H B V - D N A . T h e c o r r e l a t i o n
of
I g M a n t i - H B c and H B s A g levels with
H B V - D N A
HBeAg,
Preparation of samples and molecular
hybri
dization
positivity was studied. T h e serum samples were treated M A T E R I A L S AND
M E T H O D S
b e d b y S C O T T O et al. same volume
H B s A g
positive
serum samples from
356
2 3
. T o 50
as descri
of s a m p l e , the
of 2 M N a C l w a s a d d e d p l u s
v o l u m e s of 1M N a O H . incubation
de J a n e i r o were
were spotted by filtration through a B R L H y b r i dot
onto nitrocellulose
SSC
Detection of serological H B V m a r k e r s
temperature,
two
minutes
b l o o d d o n o r s of different b l o o d b a n k s f r o m R i o analysed.
at room
F o l l o w i n g a 10
( I X SSC:
the s a m p l e s
filters prewet
with
0.015 M N a c i t r a t e , 0.15 M
6 X
NaCl,
p H 7.0). N e u t r a l i z a t i o n w a s c a r r i e d o u t b y a d d i H B s A g
detection
confirmed by
— These
Elisa — F I O C R U Z
by R - P H A (Bio-Manguinhos —
samples were
t i o n o f 2 0 0 jxl o f 0.5 M T r i s p H 7.4, 3 M N a C l , e v e r y
7
five s a m p l e s . T h e positive a n d n e g a t i v e controls
and
titrated
FIOCRUZ)
3
0
a d d e d to
.
the filters were treated
in the
same
way. HBeAg
detection
— H B s A g
positive
sam
p l e s w e r e t e s t e d for t h e p r e s e n c e of H B e A g u s i n g
T h e filters w e r e p r e h y b r i d i z e d for l h i n a h y
E l i s a K i t s from O r g a n o n as well as reagents pro
bridization m i x containing 50% deionized forma-
duced and standardized in our
laboratory
1 9
.
mide, 5 X
I g M a n t i - H B c detection — A l l s a m p l e s were
Denhardt
s o l u t i o n , 50 m M
sodium
p h o s p a t e b u f f e r p H 7.5, 5 X S S C a n d 100
/mg/ml
tested with reagents standardized in the Centers
denatured s a l m o n s p e r m D N A , a n d then the ra-
for D i s e a s e s C o n t r o l , A t l a n t a , U S A , u s i n g H B c A g
d i o a c t i v e l y l a b e l l e d p r o b e w a s a d d e d to the h y
o b t a i n e d f r o m c l o n e d E . c o l i B r i e f l y , 1 2 5 fil
b r i d i z a t i o n s o l u t i o n a t a c o n c e n t r a t i o n o f 1-2 x
of
goat a n t i - h u m a n I g M (/x-chain specific, S i g m a ) ,
10
6
cpm/ml. Hybridization was carried out
at
37°C for
15-18 h s , f o l l o w e d
by
two
20
minutes
washes in 2 X S S C ,
0.1% S D S at 65°C a n d
20
in 0.1X S S C ,
minutes
washes
65°C. the filters
two
0.1% S D S at
were e x p o s e d to
Sakura
X-ray
t a g e of H B V - D N A , H B e A g a n d I g M a n t i - H B c i n creases a s the c o n c e n t r a t i o n of H B s A g
increases
(Table I).
f i l m s for 24 h s . The
i n t e r m e d i a t e t i t e r s ( 1 / 2 5 6 t o 1 / 1 0 2 4 ) a n d 101 s a m p l e s w i t h h i g h t t i t e r s ( o v e r 1/2048). T h e p e r c e n
probe u s e d in this work w a s the p l a s m i d
p C P I O w h i c h w a s a gift f r o m D r . P i e r r e T i o l l a i s from the Institut Pasteur. T h i s p l a s m i d consists of t h e
vector
p B R 322, c o n t a i n i n g
of the H B V g e n o m e . I n
two
copies
some experiments
the
whole plasmid was used as a probe, whereas others the H B V - D N A
was purified
by
in
electroe-
lution from agarose gels. T h e probe w a s labelled by
nick translation
v i t y o f 1-2 x 1 0 d A T P
7
2 2
with
3
2
P to a s p e c i f i c acti3 2
c p m / / i , g of D N A . T h e
was supplied by
Dr. José
P-alpha-
Carlos
Maia,
f r o m the U n i v e r s i t y of S ã o P a u l o .
R E S U L T S
The sed
distribution
of 356 s a m p l e s w a s a n a l y -
a c c o r d i n g to the h e m a g g l u t i n a t i o n titers
HBsAg. than
T h e titers varied from
1/4096, of w h i c h
28.4% were
with R - P H A titer greater t h a n The
1/16 t o high
by
two
titered,
1/2048.
r e s u l t s of the h y b r i d i z a t i o n
evaluated
of
greater
independent
tests
were
observers
and
d o u b t f u l c a s e s w e r e r e p e a t e d . 94 o u t o f 356 s e r u m samples
were
positive
when
hybridized
to
p C P I O , a s e x e m p l i f i e d i n F i g u r e 1. A s i t h a s b e e n reported that false positive cases m a y arise, poss i b l y a s a r e s u l t of b a c t e r i a l c o n t a m i n a t i o n ring storage
1 0
, all positive s a m p l e s were
du-
hybri-
d i z e d to p B R 3 2 2 , the v e c t o r i n w h i c h the H B V DNA
w a s c l o n e d . I n d e e d , o u t of 94, t e n s a m p l e s
w e r e f o u n d to h y b r i d i z e a l s o to p B R 3 2 2 a n d w e r e therefore
h y b r i d i z e d to
insert. T o pBR322
the purified
ascertain the
purity
D N A was also spotted
of onto
H B V - D N A the
insert,
the
g i v i n g a n e g a t i v e r e s u l t . F i v e of t h e t e n
filter,
pBR322
p o s i t i v e s a m p l e s w e r e s h o w n to b e p o s i t i v e for t h e c l o n e d H B V - D N A , b e i n g t h e r e f o r e ded
also
inclu-
a m o n g the H B V positive samples, w h i c h we-
re a t o t a l of 90. The and
serological markers H B V - D N A ,
H B e A g
I g M a n t i - H B c detected in the present
ples were
correlated w i t h the
H B s A g , semi-quantitatively R - P H A
t i t e r , d i v i d e d i n t h r e e g r o u p s : 106
R
PHA* titer
number H B V - D N A ( /r) of s a m p l e s c
HBeAg c
( /c
)
IgM anti-HBc ( A ) C
u n d e r 1/128
106
14 (12.3)
10 ( 9.4)
28 (26.4)
1/256 to 1/1024
149
18 (12.1)
21 (14.1)
52 (34.9)
o v e r 1/2048
101
58 (57.4)
66 (65.3)
41 (40.6)
Total
356
90 (25.0)
97 (27.2)
121 (33.9)
sam-
concentration
represented by
TABLE 1 Correlation between H B V serological markers and hemaggluti n a t i o n t i t e r of H B s A g .
of the
sam-
p l e s w i t h l o w t i t e r ( u n d e r 1/128), 149 s a m p l e s w i t h
* R - P H A = reverse passive h e m a g g l u t i n a t i o n
A clear result c a n be observed w h e n the per
s a m p l e s 6 5 % h a v e o n e or b o t h of t h e s e m a r k e r s
c e n t a g e of H B V - D N A a n d / o r H B e A g m a r k e r s is
against only
c o r r e l a t e d w i t h the titer of H B s A g . I n h i g h titer
2).
19% in low
titer s a m p l e s (Figure
HBsAg
HBsAg
HBsAg
R-PHA *UNDER 1/128 LOW T I T E R N= 106
R - P H A 1/256 TO 1/1024 INTERMEDIATE T I T E R N= 149
R - P H A OVER 1/1024 HIGH T I T E R 101
ED HBV-DNA £3 HBeAg *R-PHA
R E V E R S E PASSIVE HEMAGGLUTINATION
T I T E R OF HBsAg
F i g . 2 — P e r c e n t a g e of H B V D N A a n d or H B e A g m a r k e r s of H B V i n f e c t i o n i n r e l a t i o n to H B s A g h e m a g g l u t i n a t i o n t i t e r .
TABLE 2 C o r r e l a t i o n of a n t i H B c I g M a n t i b o d i e s w i t h H B V D N A a n d H B e A g s e r o l o g i c a l m a r k e r s of H B V infection.
w i n g t h a t e x c e p t for s t r o n g l y p o s i t i v e s a m p l e s , the p r e s e n c e of I g M a n t i - H B c s e e m s to b e i n d e p e n d e n t of the p r e s e n c e of e i t h e r H B V - D N A
I g M anti H B c
n u m b e r of samples
NEG
235 90 27 4 356
P / N < 10 P N 11 50 P / N > 50
c
H B V D N A ( '< )
H B e A g {
