509
Congenital Enterovirus 71 Infection: A Case Study with Virology
and Immunohistochemistry
Kuan-Chih Chow,1 Chien-Chung Lee,2 Tze-Yi Lin,3
Wu-Chung Shen,4 Jen-Hsien Wang,5 Ching-Tien Peng,6
and Cheng-Chun Lee1
4
Departments of 1Medical Research, 2Obstetrics, 3Pathology,
Radiology, 5Internal Medicine, and 6Pediatrics, China Medical
College Hospital, Taichung, Taiwan
Previously reported enterovirus 71 (EV71) infections associated with hand-foot-mouth disease, aseptic meningitis, encephalitis, polio-like myelitis, and paralysis all have involved young
children. We report a 28-year-old woman who possibly contracted EV71 infection during
pregnancy. Obstetric ultrasonograms at 25 weeks of gestation revealed an abnormal placenta,
as well as hepatosplenomegaly, liver calcification, excessive ascites, and mild hydrocephalus
of the fetus. Presence of EV71 was determined by immunodot blotting, virus culture, and
partial sequencing of the amplified product of nested reverse transcription–polymerase chain
reaction. Postmortem immunohistochemistry further identified EV71 in the fetal midbrain
and liver. The findings indicate that intrauterine EV71 infection is possible during pregnancy.
Since the first isolation of enterovirus 71 (EV71) in 1969,
EV71 infection in young children has been associated with
hand, foot, and mouth disease, aseptic meningitis, encephalitis,
polio-like myelitis, and paralysis [1–8]. In the several reported
outbreaks (in Bulgaria in 1975 [2], Hungary in 1978 [9], Malaysia in 1997 [10], and Taiwan in 1998 [11, 12]), serious complications mostly involved the CNS. In each outbreak, 130
children died, most of them of cardiopulmonary complications
[2, 9–12]. Moreover, MRI [11] and postmortem examinations
showed that the pathological lesions were predominantly in the
brainstem and the spinal cord, rather than in the lung or heart
[2, 9–12].
From their observations concerning a case of hand, foot, and
mouth disease that developed into acute pulmonary edema,
Chang et al. [13] further speculated that the cause of death
could be neurogenic. It is interesting that no information on
intrauterine EV71 infection or the effect of transplacental EV71
infection on development of the fetus has been described in the
reports of outbreaks.
In the present case we used immunodot blotting (IDB) for
the immediate determination of intrauterine EV71 infection.
The identification of EV71 was confirmed by culture of the
virus in amniotic fluid, by the amplified product of nested reverse transcriptase PCR (RT-PCR) of the cord blood, and by
immunohistochemical staining of EV71 in the fetal midbrain
and liver.
Received 17 September 1999; revised 2 February 1999; electronically published 24 August 2000.
Financial support: China Medical College Hospital Foundation (DMR89-051) and National Science Council (NSC89-2320-B-039-025-M08).
Reprints or correspondence: Dr. Kuan-Chih Chow, Dept. of Medical
Research, China Medical College Hospital, #2 Yuh-Der Rd., Taichung,
Taiwan 404 ([email protected]).
Clinical Infectious Diseases 2000; 31:509–12
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3102-0019$03.00
Case Report
A 28-year-old woman (gravida 3, para 0) was referred to our
hospital because of severe abdominal cramping. Her pregnancy
had been complicated by an enlarged placenta (3 cm thicker
than the size normally observed) and mild hepatosplenomegaly
of the fetus, which were noted by obstetric ultrasonograms at
17 weeks of gestation. Amniocentesis was performed and the
amniotic fluid was subjected to a routine virus culture. At 25
weeks of gestation, obstetric ultrasonograms revealed a bulky
placenta with a thick margin. Fetal echosonograms showed
evidence of hepatosplenomegaly, liver calcification, excessive
ascites, mild hydrocephalus of the lateral ventricles, pleural effusion, and borderline dilatation of the left cardiac atrium.
Laboratory evaluation of the patient revealed a hemoglobin
value of 9.1 g/dL and a WBC count of 16,740 cells/mL (63.8%
neutrophils, 12.8% lymphocytes, and 0.4% monocytes). The
platelet count was 421 3 10 3 cells/mL. Blood chemistry values
were normal (calcium, 10.2 mg/dL; glucose, 142 mg/dL; alanine
aminotransferase, 27 U/L; and aspartate aminotransferase, 15
U/L). Tests for TORCH complex (toxoplasmosis, other disease
[congenital syphilis and viruses], rubella, cytomegalovirus infection, and herpes simplex virus infection), Epstein-Barr virus
infection, and parvovirus B19 infection were negative.
At 26 weeks of gestation, a 1300 g female stillborn fetus was
delivered. No apparent infarction nor purulent exudate was
observed in the placenta in addition to the visible thickened
margin and partial intervillous thrombosis. At postmortem examination, unilateral hydrocephalus was identified in the left
lateral ventricle of the fetus, along with the marked hepatosplenomegaly, fibrotic peritonitis, and meconium staining.
Methods
Presence of EV71 in the amniotic fluid, cord blood, and mother’s
plasma was primarily determined by IDB [11]. In brief, 50 mL of
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Chow et al.
a filter-prepared sample was siphoned through the well of a BioDot apparatus (Bio-Rad, Hercules, CA) that had been layered with
a nitrocellulose membrane (MSI, Westboro, MA). The membrane
was incubated with antibodies specific to EV71 (Chemicon, Temecula, CA) and then with biotinylated rabbit anti-mouse antibodies, together with alkaline phosphatase–conjugated streptavidin
(Dako, Kyoto, Japan). A positive reaction was identified by development in chromogen-containing NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (Boehringer Mannheim, Germany). The individual sample was run in duplicate.
Amniotic fluid and plasma from patients with thalassemia were
used as negative controls, and serial dilutions of EV71 strain TW/
2272/98 (stock at 106 TCID50/mL) were used as positive controls
(GenBank, GI:5524735).
A portion of the specimen was subjected to a routine virus culture
in A549 (ATCC, CCL-185), Vero (ATCC, CCL-81), and HEL299
(ATCC, CCL-137) cells. Cell culture with cytopathic effect was
examined by immunofluorescent microscopy with an enterovirus
screen set (Chemicon International, Temecula, CA), which included
pan-enterovirus, coxsackievirus B, echovirus, and poliovirus
blends. Monoclonal antibodies (3324) that were specific to EV71
were used to identify the presence of EV71. Amniotic fluid, cord
blood, mother’s plasma, and cell culture supernatant from patients
who were EV71-positive by IDB were further analyzed by a nested
RT-PCR method to confirm the presence of EV71 [14, 15].
Sequences of the first pair of primers were 50-ACCTTTGTACGCCTGTT-30 and 50-ATTGTCACCATAAGCAGCCA-30. Sequences of the second pair of primers were 50-AAGCACTTCTGTTTCCC-30 and 50-ATTCAGGGGCCGGAGGA-30. The
amplified product of 249 bp was resolved by a 2.5% agarose–
ethidium bromide gel. The specificity of the 249-bp amplified product of EV71 was confirmed by DNA sequencing (ABI Prism; Perkin-Elmer, Foster City, CA). Immunohistochemistry to detect
EV71 was performed on frozen sections of necropsy tissues, such
as midbrain, liver, spleen, umbilical cord, and heart, with use of
monoclonal antibodies (3324) and a labeled streptavidin-biotin
method (Dako, Carpenteria, CA) [16].
Vero cells, A549 cells, and liver biopsy specimens from patients
with hepatitis C infection were used as negative controls, and EV71
strain TW/2272/98–infected Vero cells were used as positive controls. The chromogenic reaction was visualized with peroxidaseconjugated streptavidin and aminoethyl carbazole (Sigma, St.
Louis). Slides were counterstained with Mayor’s hematoxylin, and
the positive staining was recognized under a microscope as crimson
granules.
Results
EV71 infection was preliminarily detected by IDB in both
mother’s plasma and the cord blood. The estimated intensity
of the EV71 signal (∼ 5 3 10 5 TCID50 /mL) in the cord blood
was about 3 magnitudes higher than that in the mother’s
plasma. Although results of RT-PCR confirmed the presence
of EV71 in the cord blood, no EV71 RNA was detected in the
mother’s plasma. In addition, we did not isolate EV71 from
the mother’s plasma or the cord blood. However, virus culture
of the amniotic fluid and the subsequent immunofluorescent
CID 2000;31 (August)
microscopy indicated that EV71 indeed was present at 17 weeks
of gestation (data not shown).
Results of RT-PCR of the amniotic fluid were identical to
those for the cord blood. Moreover, by a neutralization assay,
antibodies specific to EV71 were detected in the mother’s
plasma at week 26. In the frozen sections of necropsy-obtained
fetal tissues, the EV71 signal was identified in the neurons of
the midbrain and the hepatocytes of the liver (figure 1). The
EV71 signal was not detected in the spleen, umbilical cord, or
heart (data not shown). It is interesting that there was no
marked inflammation in tissues positive for EV71 signal.
Discussion
The findings presented above suggest that this patient was
infected with EV71 during pregnancy and that the virus then
passed through the placenta and infected the fetus. This conclusion is based on the detection of virus in the amniotic fluid,
the cord blood, and the postmortem tissues of the fetus by
IDB, RT-PCR, virus culture, and immunohistochemistry.
Previous reports have described several EV71 outbreaks with
clinical manifestations of aseptic meningitis, encephalitis, poliolike myelitis, and paralysis that suggested CNS involvement
[1–12]. Although infection-associated pathological lesions could
be detected in basically every part of the CNS by MRI, autopsy
results revealed that mortality-related damage was mainly located in the brainstem and the spinal cord [1, 2, 9, 10].
An elegant immunohistochemical study by Wong et al. [17]
further showed that EV71 infection was predominantly in the
neurons. In this study, we detected EV71 signal not only in
neurons of the midbrain but also in hepatocytes of the fetal
liver. Myelogenous cells of blood-forming units in the fetal liver,
however, were negative for EV71 signal. So were glial cells,
myocardial cells, endothelial cells, and cells in the spleen. It is
interesting that no obvious local inflammation was found in
tissues positive for EV71 signal. In addition, our observations
were not similar to those in cases of congenital infections with
echoviruses or coxsackieviruses. The pathological features of
echovirus or coxsackievirus infection are evident intravascular
coagulation, focal hemorrhage, and necrosis in the virus-infected organs, such as the heart, kidney, brain, spleen, and liver
[18–21].
From our experiences with an EV71 outbreak in 1998
[11–13], we learned that most patients who had CNS involvement in the midbrain and in the cervical spinal cord at the C45 levels developed pulmonary complications that appeared as
diffuse interstitial infiltration and consolidation on a chest radiograph. Some patients had tachycardia, a running pulse, or
(most of the time) no pulse. Sometimes, the heartbeat was not
detectable by electrocardiography. However, the infinitesimal
intra-aortic blood pressure suggested that the heart might be
at the point of going into spastic contraction.
These findings, considered together with the nuclei likely to
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Transplacental Enterovirus 71 Infection
511
Figure 1. Detection of enterovirus 71 (EV71) in fetal tissues by immunohistochemistry: top, EV71-positive neurons in the midbrain of a fetus
(stains, immunoperoxidase and methyl green); bottom, EV71-positive hepatocytes in the fetal liver (stains, immunoperoxidase and hematoxylin).
Original magnification, 3200.
be affected in the midbrain and in the spinal cord at the C4-5
levels, as well as the other clinical symptoms, clearly indicate
that the abrupt pulmonary complications might be neurogenic
[12, 13]. Neurogenic pulmonary edema could be lethal without
proper immediate care. Such a condition is similar to what has
been observed in a patient with poliomyelitis with C4-5 involvement [22]. Unlike poliomyelitis, though, EV71-associated
CNS lesions are mostly reversible, and the disease can resolve
completely within 2–4 weeks without residual symptoms.
From this case, it is clear that during pregnancy an intrauterine EV71 infection is possible in high-risk groups. Detection
of EV71 in hepatocytes of the fetal liver and in neurons of the
midbrain could provide partial explanations for hepatomegaly,
excessive ascites, pleural effusion, and hydrocephalus. The exact
role of each feature in causing fetal death, however, remains to
be determined.
Acknowledgments
We thank Ms. Li-Ching Lin for her excellent technical assistance
and are grateful to Dr. S. H. Chiou for critically reading the manuscript.
EV71 strain TW/2272/98 was kindly provided by Dr. Yuh-Chering
Huang, at Chang Gung Children’s Hospital.
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