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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 2, 1996, pp. 147-151
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1),
Mar/Apr 1996
Human Parvovirus B19 Infection and Hydrops Fetalis in
Rio de Janeiro, Brazil
Rita CN Cubel /**/^+ , Aparecida GP Garcia*, Claudia S Pegado^*,
Hilda I Ramos*, Maria EF Fonseca***, Jonhatan P Clewley****,
Bernard J Cohen****, Jussara P Nascimento
Departamento de Virologia, Instituto Oswaldo Cruz, Av. Brasil
4365, 21045-900 Rio de Janeiro, RJ, Brasil ^ *Servico de
Anatomia Patologica, Instituto Fernandes Figueira, Av. Rui
Barbosa 716, 22250-020 Rio de Janeiro, RJ, Brasil **Departamento
de Microbiologia e Parasitologia, CCM/UFF, Rua Prof. Ernani Melo
101, 24210-130 Niteroi, RJ, Brasil ***Setor de Microscopia
Eletronica, Instituto de Microbiologia, UFRJ, Ilha do Fund o,
21944-790 Rio de Janeiro, RJ, Brasil ****Virus Reference
Division, Central Public Health, Laboratory, 61 Colindale Av.,
NW9 5HT, London, UK
Code Number: OC96028
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[TABLES AT END OF TEXT]
Formalin-fixed paraffin embedded lung and liver tissue from
23 cases of non immune hydrops fetalis and five control cases,
in which hydrops were due to syphilis (3) and genetic causes
(2), were examined for the presence of human parvovirus B19 by
DNA hybridisation. Using in situ hybridisation with a
biotynilated probe one positive case was detected. Using 32P-
labelled probes in a dot blot assay format, five further
positives were obtained. These were all confirmed as positive
by a nested polymerase chain reaction assay. Electron microscopy
revealed virus in all these five positive cases. The six B19 DNA
positive cases of hydrops fetalis were from 1974, 1980, 1982,
1987 and 1988, four of which occurred during the second half of
the year, confirming the seasonality of the disease.
Human parvovirus B19 was first discovered in blood donors by
Cossart et al. (1975) and has been identified as the causative
agent of erythema infectiosum, an acute exanthem of childhood
(Anderson et al. 1983). In adults, particularly women, the rash
illness is less frequent, but there may be involvement of joints
leading to acute arthritis (Reid et al. 1985). In addition, B19
is also a major cause of aplastic crisis in patients suffering
from hemolytic anemias (Pattison et al. 1981).
Infection with the virus was first shown to be associated with
fetal death in 1984 (Brown et al. 1984). Since then, several
other reports have described B19 infection in pregnancy (Knott
et al. 1984, Bond et al. 1986, Anderson et al. 1988). Fetal
infection may cause hydrops fetalis and stillbirth, but normal
delivery usually occurs (Kinney et al. 1988). Estimates of the
risk of fetal death from intrauterine infection range from 1.66%
to 9% (CDC 1989, PHLS 1990, Guidozi et al.1994, Gratacos et al.
1995). More recent reports suggest that the risk of fetal loss
in pregnancy may be higher with asymptomatic B19 infections
(Smoleniec et al. 1994, Gratacos et al. 1995). The incidence of
congenital malformations after maternal infection is, however,
no higher than the expected rate in the general population
(Kinney et al. 1988). Thus, parvovirus B19 is considered to be
embryocidal rather than teratogenic. The infection is diagnosed
by the detection of IgM antibodies in both maternal and fetal
blood (Knott et al. 1984) but, in some cases, maternal anti-B19
IgM can no longer be detected when hydrops fetalis develops (Bond
et al. 1986, Anderson et al. 1988). In most of the reported
cases, DNA detection in fetal tissues is the method of choice for
diagnosis (Clewley et al. 1987, Salimans et al. 1989). Electron
microscopy has also been used (Caul et al. 1988, Knisely et al.
1988, Naides & Weiner 1989, Field et al. 1991).
A serological survey of the Rio de Janeiro population showed
that 30 to 60% of women of child-bearing age have antibodies
(Nascimento et al. 1990) and thus at least 40% of women are
still susceptible to infection during pregnancy. A previous
study of hydrops fetalis done by the Pathology Department of
the Instituto Fernandes Figueira (IFF/FIOCRUZ) during 1954 to
1992 showed that infectious diseases predominate over immunologic
causes in the aetiology of hydrops fetalis (Garcia et al. 1995).
We report here the results of B19 detection in fetal tissues from
non-immune hydrops fetalis (NIHF). In all but one, where
syphilis has also been diagnosed, no other infectious agent was
found.
MATERIALS AND METHODS
Specimens - Formalin-fixed paraffin embedded tissues.
From a collection of 86 cases of NIHF analyzed during 38 years
(1954 to 1992) at the Pathology Department in IFF/FIOCRUZ, 28
fetuses were selected because histology suggested intrauterine
viral infection. For 23 of these 28 fetuses the aetiology of the
NIHF was not known (Garcia et al. 1995). In five additional
fetuses included as controls the hydrops was diagnosed as a
result of syphilis (three) or genetic causes (two). Lung and
liver tissues from all 28 cases were used for screening for B19
DNA. Other tissues, when available, were also examined from the
six cases found positive for B19 DNA in lung or liver.
In situ hybridisation (ISH) - ISH was performed as
described previously (Nascimento et al. 1991). The only
modification was that a pBR322 plasmid containing citomegalovirus
DNA was used as a negative control probe for each tissue.
Dot blot hybridisation (DBH) - A 10% homogenate of
fetal tissue was prepared and DNA extracted as described by
Clewley et al. (1987) except that the extracted DNA was applied
to nitrocellulose filters and the blots were hybridised with a
32P- labelled B19 probe. The probe was made from the 5.2 Kb Eco
RI fragment excised from the B19/pGem-1 plasmid (Mori et al.
1989). Diluted plasma from a viremic blood donor (Cruz et al.
1989) and normal human serum were used as positive and negative
controls.
Nested polymerase chain reaction (PCR) - B19 DNA was
recovered from 10% fetal tissue suspensions according to the
method described by Boom et al. (1990). PCR amplifications were
carried out as described by Clewley (1993). Two sucessive sets
of amplification were performed using primers derived from the
B19 parvovirus non-structural coding sequence. Primers pair H
(1417-1424) and C (2160-2141) were used for the first round of
reaction, primers pair F (1498-1520) and I (2029-2065) for the
second round. The numbers indicate nucleotide positions relative
to the clone sequenced by Shade et al. (1986), Genbank accession
number, M13178. Ten ul of PCR products were analyzed by
electrophoresis on a 4% Nusieve-agarose (3:1) gel. Deionized
H^2O extracted alongside the specimens was used in other to check
for possible contamination. Positive and negative B19 DNA human
sera were also used as controls. Amplifications were carried out
in a suite of physically separated PCR rooms.
Electron microscopy - Direct electron microscopy (DEM)
of the 10% fetal tissue suspensions were done as described before
(Field et al. 1991). The tissue suspensions were negative
stained with 2% PTA pH 7.2 and observed in a Philips 301 EM.
RESULTS
Fifty-six lung and liver tissues from 28 fetuses were tested
by ISH. Only one fetus was found to be positive for B19 DNA in
both lung and liver tissues. From this case other tissues
(placenta, heart, kidney, thymus and adrenal) were available.
These were all found to be negative for B19 DNA, when tested by
ISH. When DNA was extracted from these tissues and tested by
DBH, using nitrocellulose membranes and a 32P-labelled probe,
another two (kidney and adrenal) were found to be positive for
B19 DNA. Electron microscopy of ultrathin sections was not able
to demonstrate virus particles in any of these tissues (Table I).
There was no material remaining for direct electron microscopy
(DEM).
DNA was extracted from the lung and liver tissue from the other
27 cases and hybridized against a 32P-labelled probe. Five
additional fetuses were found to be positive for B19 DNA for one
of each of their tissues tested. A nested PCR was performed to
confirm the five positive results found by DBH. Analysis of
nested PCR products by agarose gel electrophoresis showed a DNA
band of the expected size (591 bp), in at least one of the
tissues for each of the samples tested. DEM was also performed
on these tissues and parvovirus-like particles were observed in
all of them. In one of these five positive cases (1757) the NIHF
had been first diagnosed as syphilis.
The B19 positive fetuses were found during the years 1974,
1980, 1982, 1987 (one case each year) and 1988 (two cases) as
shown in Table II. Of these, only two were not found during the
second half of the year.
DISCUSSION
In the present study using DNA hybridisation we were able to
detect six fetuses infected with human parvovirus B19.
In a previous study of a collection of fetuses in England, we
found that ISH is as sensitive as DBH with 32P-labelled probe for
formalin-fixed tissues (Nascimento et al. 1991). In this study
of a collection of Brazilian tissues, DBH was much more efficient
than ISH for detecting B19 DNA. One explanation for this could
be that the fetuses were accumulated during a 21 year period
compared to the five year period of the English collection. The
quality of paraffin and the formalin may differ between the
collections. For example, the formalin used in the preservation
of the tissues in Brazil is not buffered. This may contribute
either to degradation of nucleic acids or prevention of probe
access to the B19 DNA in the tissue. This would not interfere
with DBH since DNA is concentrated by extraction from the tissue
and treated with alkali before binding to the filter and
subsequent hybridisation (Clewley 1985).
The difference between the detection of B19 DNA by DBH in
different tissues from the same fetus could be explained by
postmortem changes associated with the inevitable time lapse
between fetal death and formalin fixation of the tissues (Lohr
& Neremberg 1990). The site of B19 replication is erythroid
progenitors which accumulate in fetal liver between 12 to 30
weeks of gestation (Clewley et al. 1987, Knisely et al. 1988).
Since liver is subject to greater autolysis than lung it is
likely that differences in B19 DNA detection in these tissues is
due to postmortem changes.
Recent reports have used sensitive PCR assay to detect B19 DNA
in clinical specimens (Clewley 1989, Salimans et al. 1989),
especially when studying B19 infection during pregnancy
(Torok et al. 1992, Cassinotti et al. 1993). We used nested
PCR to confirm the five positive cases found by DBH. Although
a single-step PCR should be sufficient for detecting B19 DNA, a
nested PCR may be necessary for maximum sensitivity, particularly
when investigating fetal tissues in which partially degraded
nucleic acids may be recovered (Clewley 1993).
Although a previous study showed that hybridisation is more
sensitive than electron microscopy for testing formalin-fixed
samples (Field et al. 1991), B19 parvovirus-like particles could
be observed in this work, by DEM, in all five fetuses positive
for B19 DNA by DBH.
A study carried out by the Pathology Department in IFF/FIOCRUZ
showed that of 86 cases of NIHF, 31 were diagnosed as syphilis
by identification of Treponema pallidum. One of
these cases (1757) was used as a negative control in our study.
B19 DNA was found in this fetus by DBH and PCR. Since congenital
syphilis is also considered a cause of hydrops fetalis (Bulova
et al. 1972) this may be a case of dual infection.
Four of the parvovirus B19 positive cases were found during the
second half of different years. This would be consistent with
the seasonality described in Rio de Janeiro for exanthematic
disease (Schatzmayr 1985). Two of the positive cases occurred
during November and December 1988, coinciding with the finding
by chance of a B19 infected blood donor (Cruz et al. 1989). This
supports the suggestion that 1988 was an epidemic year for B19
infection in Rio de Janeiro.
Human parvovirus B19 should be considered as a virus to be
monitored during pregnancy, since it is clinically very similar
to rubella (Shirley et al. 1987) and it has become evident as a
cause of fetal infection in countries were rubella is controlled
by vaccination (Cohen 1993). It would be particularly important
to survey for B19 infection in some Brazilian states, for
instance, S o Paulo and Parana which have started rubella
vaccination programmes.
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This paper is part of the PhD thesis of the first author to be
submitted at Instituto de Microbiologia, UFRJ. ^
+Corresponding author. Fax: 55-21-230.7638
Received 19 May 1995
Accepted 1 September 1995
TABLE I
Positive B19 hydrops cases
Month Gestacional Autopsy findings Tissue B19 Detection
Year age ISH DBH EM PCR
-----------------------------------------------------------------
October 33 wks macerated, lung - - + +
1974 hydropic, liver - + - +
(1447)^a,b fibro-elastosis,
spleen and lung anomaly
March 28 wks macerated, lung - + + +
1980 hydropic, liver - - + +
(1757)^a syphilis lesional cord - nd + nd
complex
placenta - nd + nd
November 22 wks macerated, lung - + + +
1982 hydropic, liver - - + +
(2023)^a fibro-elastosis cord - nd + nd
placenta - nd + nd
spleen - nd + nd
adrenal - nd + nd
May 27 wks macerated, lung - + + -
1987 hydropic, liver - - + +
(2387)^a hepatomegaly,
esplenomegaly
November 31 wks stillbirth, lung - + + +
1988 hydropic, liver - - + +
(2595)^a hepatomegaly, cord - nd + nd
esplenomegaly placenta - nd + nd
spleen - nd + nd
kidney - nd + nd
brain - nd + nd
adrenal - nd + nd
December 30 wks macerated, lung + + - nd
1988 hydropic, liver + + - nd
(2601)^a,b fibro-elastosis, placenta - - nd nd
hepatomegaly, kidney - + nd nd
esplenomegaly adrenal - + nd nd
heart - - nd nd
thymus - - nd nd
a: registration number; b: mother with sickle cell disease; nd:
not done
TABLE II
Cases of NIHF from 1968 to 1989 tested for the presence of human
parvovirus B19
Year of death No. positive/No. tested
----------------------------------------
1968/69 0/02
1974 1/02
1976/79 0/02
1980 1/01
1981 0/01
1982 1/03
1983 0/02
1987 1/03
1988 2/02
1989 0/05
Total 6/23
Five fetuses showing NIHF used as negative controls were selected
in the years of 1980, 1986 and 1987.
Copyright 1996 Fundacao Oswaldo Cruz
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