Infectious Disease Pathology
Case 2 -
Parvovirus B19 Infection with Severe Anemia, Fetal Hydrops and Cardiomegaly Leading to Intrauterine Fetal Demise
A. Brian West, Yale University, New Haven, CT
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A 24 year-old primagravida presented to her obstetrician at 36 weeks gestation because she had felt no fetal movements for 5 days. She had been a regular attender at prenatal visits, the last at 32 weeks, and her pregnancy up to this had been uneventful. Intrauterine demise was confirmed by ultrasound examination, and the fetus was submitted for autopsy. The patient was a male fetus of 3,560 g weight, with hydrops fetalis and Grade III maceration. The external and internal features were otherwise normal, with the exception of the heart which showed cardiomegaly (35 g; normal range for body weight, 23.1 +/- 3.1 g), and the liver which was severely autolysed. The brain was heavy for gestational age at 390 g, and there were recent foci of subarachnoid hemorrhage. The placenta weighed 480 g and was complete with unremarkable membranes and umbilical cord.
Case 2 - Slide 1
Case 2 - Figure 8
Immunoreactive nucleated red cells in placental villus capillaries
Case 2 - Figure 9
B19-infected cells in area of hematopoiesis in fetal spleen
Case 2 - Figure 10
B19 in erythropoietic cells in hepatic parenchyma, with lack of staining in portal tract
Case 2 - Figure 11
Giant proerythroblasts in bone marrow biopsy from a renal transplant patient with chronic anemia. Naked nucleus in panel on bottom right.
Case 2 - Figure 12
Giant proerythroblasts in bone marrow biopsy (top left) and aspirate from an AIDS patient with chronic anemia. Naked nucleus on bottom right.
Case 2 - Figure 13
B19 immunostaining of giant proerythroblasts in the biopsy illustrated in Fig k.
The question is why this previously apparently healthy fetus died in utero at about 35 weeks
Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The gross findings in the fetus included hydrops fetalis, biventricular cardiomegaly and grade 3
maceration. The placenta was heavy for gestational age (480 g) and of normal gross appearance, except
for the presence of mild pallor.
Microscopic examination revealed severe autolysis of the liver and spleen with extramedullary
hematopoiesis, and a few foci of subarachnoid hemorrhage. The main finding in the placenta was prominent
erythroblastosis with abundant nucleated red blood cells in the fetal vessels. On closer examination of
the nucleated erythroid cells in placenta, liver and spleen, many were noted to have chromatin
concentrated at the nuclear membrane, while the center of the nuclei was occupied by a glassy amphophilic
substance, features strongly suggestive of parvovirus B19 infection. Immunohistochemistry for CMV,
Herpes and Adenovirus was negative, but there was positive staining for B19 in the abnormal nucleated red
The differential diagnosis of hydrops fetalis includes immune and non-immune causes, though immune
hydrops is now rarely seen because of the use of anti-D (RhoGAM). Non-immune categories include cardiac
and pulmonary conditions, genetic defects and severe anemia of many etiologies. The presence of
erythroblastosis here points towards severe anemia, and therefore to increased red cell destruction or
failure of erythropoiesis.
The differential diagnosis of intrauterine infection is large but the presence of inclusions in the
nucleated red blood cells and the absence of an inflammatory reaction suggests a viral infection.
Immunostains for Herpes, CMV and Adenovirus were performed because of the appearance of some cells in the
severely autolysed liver which looked as if they might have inclusions, and it was not clear what type of
cells they were.
Parvovirus B19 infection with severe anemia, fetal hydrops and cardiomegaly leading to intrauterine
This case illustrates the importance of looking carefully for inclusions when viral infections are in
the differential diagnosis, and the utility of specific immunostaining in establishing and confirming
diagnoses. Immunohistochemistry can be particularly valuable when there is severe autolysis of organs
such as the liver and spleen (a common problem with still births), provided that appropriate negative
controls are performed.
Morphology of the inclusions: The morphology of the viral inclusions in B19 infections is
particularly interesting. As illustrated in this case, in fetal infections nucleated red blood cells are
affected predominantly. Chromatin is pressed to the nuclear membrane while the central part of the
nucleus contains amorphous amphophilic material that on EM can be shown to be a mass of viral capsids.
Cytoplasmic inclusions are not seen. The light microscopic features are not specific, however, and can
be mimicked by degenerative change, but are highly suggestive of B19. The diagnosis can be confirmed by
immunohistochemistry or by in situ hybridization.
In children and adults hematopoiesis is concentrated mainly in the bone marrow and nucleated red blood
cells are not present in the circulation, in contrast to the situation described above. Here B19 may
also be seen as inclusions, but typically in giant pro-erythroblasts in a setting of erythroid
hypoplasia. These cells are 25 – 45 µm in diameter and contain a large nucleus with uncondensed
chromatin and a prominent inclusion or nucleolus, and usually a scant rim of foamy cytoplasm (Florea
2007). Bare nuclei, devoid of cytoplasm, are also seen (Koduri 1998). The presence of viral antigen in
both nucleus and cytoplasm can be demonstrated by immunohistochemistry and in situ hybridization (Liu
1997, Crook 2000).
Clinical diagnosis of B19 infections: Although the morphologic features described above are
suggestive of B19 infection, they are not specific. Tests useful in making a firm diagnosis include IgM
and IgG anti-B19, and methods for detecting B19 in peripheral blood and quantifying B19 viral load
(Soliman 2009). IgM is indicative of recent infection, but both IgM and IgG may be negative in very
early infection. IgG alone is merely indicative of past infection.
Review of the Literature/Treatment Options:
Parvovirus B19 is a small (~24 nm) single-stranded DNA virus that is only known to affect humans.
It causes its most significant clinical effects by inhibiting erythropoiesis, although in the majority of
cases of childhood or adulthood infection the scenario is that of a mild exanthem, often accompanied by
arthropathy. The virus is spread in respiratory secretions, and attaches to red blood cells and their
precursors through a specific receptor, globoside (also known as blood group P antigen), that is
expressed predominantly on erythroid cells. Globoside is also expressed on fetal cardiac myocytes and
endothelial cells (Broliden 2006), but the infection appears to be non-permissive because of the absence
of an essential co-receptor, α5β1−integrin. Once internalized the virus takes advantage
of the proliferation machinery of erythroblasts and uses it to reproduce, forming nuclear and later
cytoplasmic inclusions and causing toxic injury to the host erythroid cells and the induction of
apoptosis. This can result in several different clinical syndromes.
Discovery of the virus: B19 was discovered in 1974 during screening of blood samples for Hepatitis B
(Cossart 1975). Small capsids of an unknown virus were detected in well 19 of plate B, and because of
its size and EM appearance it was identified as a member of the parvoviridae, a group of small viruses of
high host specificity many of which are well known to veterinarians. No disease was associated with B19
for several years following its discovery, but increasing sero-positivity with age suggested that it was
a childhood infection. In 1981 it was noted that transient aplastic crises in children with sickle cell
disease were associated with the presence of virus or of anti-viral antibodies in serum, suggesting a
role for the virus (Pattison 1981). Then, in 1984, Anderson and colleagues showed that the virus was the
causative agent in an epidemic of erythema infectiosum among school children in England (Anderson 1984).
Epidemiology: B19 has a world-wide distribution. Seroprevalence increases with age, ranging from
~15% among pre-school children to ~85% among the elderly. Infection is more common in winter
and spring, and there are epidemics every 3 – 4 years. Transmission in usually via respiratory spread,
but also occurs from transfusion of blood and blood products, materno-fetal passage, and through bone
marrow and organ transplantation. The virus is robust and resistant to heat and detergent inactivation
because of its lack of a lipid envelope, making it difficult to remove from blood products.
In European studies, approximately 25 - 45% of women of child-bearing age are thought to be
susceptible to B19 infection, vertical transmission of the virus to the fetus occurs in 33 – 51% of
infections, and the risk of an adverse fetal outcome is estimated at 10% (de Jong 2011). In the
Netherlands, B19 infections account for 2 – 3 fetal deaths per 10,000 live births annually.
Erythema Infectiosum: Erythema infectiosum is also known as 'Fifth Disease' (i.e. the fifth most
common viral exanthem in children following measles, scarlet fever, rubella, and fourth disease), and
'Slapped Cheek Syndrome'. During the first 10 days following infection patients may be asymptomatic or
have mild flu-like symptoms. Viremia peaks at about day 10, and virus is secreted via the respiratory
tract. At about day 20 a mild maculo-papular rash may develop on the cheeks, but leaving a ring of
circumoral pallor (hence the 'slapped cheek' appearance), followed by involvement of the upper body.
Mild symmetrical peripheral arthralgia may affect adults. The rash lasts usually for a few days, and
arthralgias for a few weeks, but without sequelae.
In immune competent individuals IgM antibodies develop from about day 8, peaking at about day 25, and
are quickly followed and replaced by IgG antibodies. The rash and facial erythema only develop after
formation of IgG, and are a result of immune complex deposition. Clinical signs of the infection resolve
over a few weeks, and thereafter there is solid immunity to reinfection. If one was to monitor the
reticulocyte and erythrocyte counts in peripheral blood during the course of infection one would see
transient erythroid hypoplasia and reticulocytopenia, the counts reaching their nadir when viral load is
highest, and rapidly returning to baseline values as immunity develops and the virus is cleared from the
Transient Aplastic Crisis: Transient aplastic crisis occurs when a naïve patient with an underlying
condition resulting in shortened red cell life span, such as sickle cell disease, thalassemia, and
hereditary spherocytosis, is infected with B19. Here the synergistic effects of viral inhibition of
erythropoiesis and the short-lived erythrocytes can lead to an aplastic crisis with acute anemia that can
lead to cardiac failure and can even be fatal. The process is transient because as soon as the immune
system kicks in and anti-B19 antibodies are synthesized, the virus is cleared, erythropoiesis resumes,
and there is reversion of peripheral blood counts to the baseline levels.
Persistent B19 Infection: Under normal circumstances B19 is cleared by the humoral limb of the
acquired immune system through the synthesis of IgM and IgG specific anti-B19 antibodies. Persons with
compromise of humoral immunity lack the capacity to clear the virus and a persistent infection ensues
with the development of red cell aplasia and chronic anemia. Infected individuals do not present with
the rash and facial erythema of fifth disease or arthropathy because these are immune complex mediated
symptoms. Managing these patients can be difficult, but treatment with transfusions may be necessary to
reverse the anemia, and pooled immunoglobulin infusion (which contains IgG anti-B19 since about 60% of
adults have immunity to B19) can reduce the viral load. Interestingly, this may result in immune complex
formation and the classic signs of fifth disease. Conditions in which persistent B19 can occur include a
wide spectrum of immunodeficiency states including congenital forms of immunodeficiency, cytotoxic
chemotherapy, HIV infection and AIDS, and post-transplant immunosuppression. In the latter setting
treatment is often a delicate balance between having enough immunosuppression to prevent rejection while
having sufficient immunocompetence to control the virus and avoid red cell aplasia and anemia.
Hydrops fetalis: When a maternal infection is acquired during pregnancy there is a risk of vertical
transmission of the virus to the fetus. This is most likely to occur when infection arises during the
second and early third trimester, and may be facilitated by the presence of the receptor for B19,
globoside, on syncytiotrophoblast (Jordan 1998). As in children and adults, in the fetus cells of
erythroid lineage are infected, and erythropoiesis is inhibited. This leads to anemia which may be
severe and life-threatening for the fetus, and, as in other forms of severe intra-uterine anemia, can
lead to hydrops fetalis and cardiac insufficiency.
In the fetal vessels in the placenta nucleated red cells are abnormally numerous, and on close
examination show characteristic morphological features: chromatin is pushed to the periphery of the
nuclei while the central part of the nucleus has a glassy amphophilic appearance due to virus
accumulation. Since fetal hematopoiesis takes place largely in the liver and spleen, viral accumulation
in erythroid precursors is most easily found there.
Management of B19 infections: Children or adults with subclinical infection, erythema infectiosum or
arthropathy require no treatment other than pain relief for the arthropathy. The infection is
self-limiting (including the arthropathy, which heals without sequelae) and results in solid immunity.
Patients with transient aplastic crisis may require red cell transfusions, but once erythropoiesis
recommences, no further treatment is required. Immunity is established here also, and aplastic crises
due to B19 do not recur. Patients with immunodeficient states develop chronic B19 infection that may
result in chronic anemia. Pooled immunoglobulin infusions are usually effective in controlling viremia,
but eliminating the infection may be a challenge and require prolonged treatment. In transplant patients
there may be a delicate balance between immunosuppression to prevent rejection and immunocompetence to
control viremia and clear infection.
In women who develop acute B19 infection during pregnancy, weekly ultrasound monitoring has been
recommended (Heegaard 2002, Schild 1999). If hydrops fetalis is diagnosed during pregnancy clinical
work-up for the differential diagnosis should be undertaken. Cord blood can be sampled to evaluate for
anemia, antibodies, viral load and other factors, and intra-uterine transfusions may be given if anemia
is significant (Chauvet 2011).
Parvovirus B19 infection is almost restricted to cells of the erythroid series because of the limited
distribution of its receptor, globoside, and co-receptor, α5β1−integrin. Consequently,
the chief pathologic effects of infection are mediated through red cell deficiencies. While most cases
of parvovirus B19 infection are of minor clinical consequence, infections in patients with underlying
defective erythropoiesis, increased red cell destruction and impaired immunity may cause severe acute or
chronic anemia and their clinical consequences. Moreover, B19 can cause acute infection during
pregnancy, and through vertical transmission to the fetus induce severe fetal anemia, hydrops, cardiac
insufficiency and death.
It is important to recognize the strikingly different cytopathic features of B19 infection in
erythropoietic cells in the fetus and in the bone marrow of children and adults with impaired immunity,
both of which can be conveniently highlighted by immunohistochemistry.
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