Silingardi, Enrico MD*; Santunione, Anna Laura MD*; Rivasi, Francesco MD†; Gasser, Bernard MD‡; Zago, Silvia MD§; Garagnani, Lorella PhD†
Parvovirus B19 (B19) is a nonenveloped, single stranded DNA virus.1 It exclusively infects man and shows a marked trophism for the precursors of erythroid cells.2
B19 infections show a seasonal trend, having greater frequency in the winter and the beginning of spring. Because the major part of the infections are found among young children of school age, the most exposed adults are the parents of children in this age group and people working in the scholastic community.2
B19 is usually transmitted through the respiratory tract, but other possible modalities of transmission are organ and bone marrow transplants as well as blood transfusions and hematic derivatives.2 Clinical manifestations are present in only 25% to 50% of the infected subjects and include, above all, erythema infectiosum (also known as fifth disease), characterized by a cutaneous rash and, in particular in adults, by arthralgia. In immunocompromised patients, the infection can cause aplastic crises, whereas immunocompetent patients recover spontaneously, leaving them with a permanent immunity.2
Transplacental transmission from mother to fetus has been documented.1 It can take place in any phase of pregnancy,2 but is more frequent in the first 2 trimesters and can have variable consequences. In fact, it can be asymptomatic both for the pregnant woman and for the fetus but, in some cases, it can provoke severe fetal complications and, in rare cases, intrauterine death (IUFD) or miscarriage,2 with possible consequences on a medico-forensic level.
We present 5 cases of maternal-fetal infection from B19 followed by IUFD. All the patients had been exposed to infection from B19 but the absence of symptoms of infection had convinced physicians not to activate programs monitoring fetal conditions. In all the cases examined, the investigation pointed out pathologic signs and laboratory characteristics of B19 infection, in 4 cases as a direct infection of the fetus and in 1 case as a placental involvement.
From 2001 to 2005, we reviewed 5 cases of maternal-fetal infection from B19 followed by IUFD observed. Three of these cases took place in the second trimester of pregnancy and the other 2 cases in the third trimester. In 4 cases, the IUFD took place suddenly in the absence of prodromic clinical manifestations; in 1 case, the patient was hospitalized following an ultrasound diagnosis of fetal hydrops and IUFD took place 5 days after admission.
All the fetuses, whose degree of development corresponded to their gestational age, were autopsied and appeared to be in an optimal state of conservation. Histopathological examinations were carried out on the encephalon, heart, lungs, liver, spleen, kidneys, thymus, bone marrow, and placenta, using HE methods, Azan-Mallory for collagen fibers, Weigert for elastic fibers, and Perls for hemosiderin. The diagnosis was made by the histopathological identification of the typical nuclear inclusions in cells of erythroid series (erythroblasts) or placental involvement. Diagnostic confirmation was obtained through research on B19 DNA, using PCR analysis carried out on histologic sections of the lung, liver, spleen and placenta.
A 38-year-old patient in her 21st week, in good health, who had a normal pregnancy, was admitted following a casual ultrasound finding of fetal hydrops and notable placental thickening. The same ultrasound had documented increase in peak velocity of the middle cerebral artery due to a condition of anemia and encephalic hypoxia; the amniotic fluid was within the norm. IUFD took place 5 days after admittance.
The fetus was male, weighed 440 g, 25.5 cm in length. Autopsy showed cutaneous purpura, diffuse hydrops and ascites, increased liver, and placental volume. Malformations or structural anomalies were absent. Histology showed erythroid hypoplasia of the bone marrow, numerous intravascular erythroblasts with eosinophilic nuclear inclusions from B19 in the liver and the kidneys, endocardial fibroelastosis (Fig. 1A), and thymic hypoplasia with lymphocytic depletion.
Furthermore, there was hepatic hemosiderosis and intense edema of the chorionic villi.
A 29-year-old patient, in good health who had a normal pregnancy. IUFD in the absence of symptoms in the 24th week.
The fetus was male, weighed 640 g, 32.5 cm in length. Autopsy showed cutaneous purpura, diffuse hydrops, bilateral pleural effusion and ascites, and increase in liver and spleen volume. Malformations or structural anomalies were absent. Histology showed intravascular erythroblasts with eosinophilic nuclear inclusions from B19 in various organs (Figs. 1B, C), evident endocardial fibroelastosis extending to the 4 cardiac cavities, moderately branched into myocardial interstice, thymic hypoplasia with lymphocytic depletion, and, in addition, hepatic hemosiderosis and edema of the chorionic villi.
A 35-year-old patient in good health who had had a normal pregnancy. IUFD in the absence of symptoms in the 28th week.
The fetus was female, weighed 1200 g, 38.5 cm in length. Autopsy showed diffuse hydrops, very pronounced in the cranial and thoracoabdominal region, bilateral hydrothorax and ascites, and modest increase in liver and spleen volume.
Histology showed eosinophilic nuclear inclusions from B19 in numerous intravascular erythroblasts in liver and spleen. In addition, there was hepatic hemosiderosis and pronounced placental hydrops.
A 28-year-old patient in good health who had had a normal pregnancy. IUFD in the absence of symptoms at the 21st week.
The fetus was male, weighed 495 g, 25 cm in length. Autopsy showed diffuse hydrops, hydropericardium, hydrothorax and a moderate ascites, pronounced dilatation of cardiac ventricular chambers and volumetric increase of the spleen.
Histology showed erythroid hypoplasia of the bone marrow, intravascular erythroblasts with eosinophilic nuclear inclusions from B19 at the splenic level, endocardial fibroelastosis particularly evident in the right ventricle, thymic hypoplasia with lymphocytic depletion; in addition, hepatic hemosiderosis and fibrosis, intense edema, and erythroblastosis of chorionic villi (Fig. 1D).
A 28-year-old patient in good health who had had a normal pregnancy. At the 31st week, ultrasound showed regular fetal growth and slightly increased amniotic fluid. IUFD at the 39th week.
The fetus was male, weighed 3050 g, 54 cm in length. Autopsy showed cutaneous purpura, moderate subcutaneous edema, ascites, hydroceles, and subpleural petechiae.
Histology showed edema and infiltration of the polynucleated cells in the chorionic membranes, appearance of necrosis, calcification of the villi, and absence of erythroblastosis.
Analysis was carried out to determine the presence of B19 DNA in cases of IUFD using paraffin-embedded tissue: fetal lung, liver, spleen tissue, and placenta.
Three 5 μm sections from each block were cut with a microtome. To prevent carryover of contaminating DNA, a fresh blade was used for each sample and the microtome overlay was covered with a piece of adhesive tape changed for every sample. The cut sections were deparaffinized by washing twice with xylene and twice with 100% ethanol. After evaporation of the ethanol, 200 μL of digestion buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween 20 and 200 μg of proteinase K per mL) was added and the mixture was incubated for 3 hours at 56°C. Proteinase K was inactivated by incubating the samples at 95°C for 10 minutes.
Polymerase Chain Reaction
For the PCR, 2 different primer sets were used: one set (primer set A) generating a DNA fragment located on the genoma part coding for nonstructural proteins and another set (primer set B) in the structural protein part of the genome.
Primer set A:
1. 5′-ACTGGTGGTGCTCTTTACTG-3′ (bp 497–516)
2. 5′-TAACCCCTCTACACACACTG-3′ (bp 744–725)
Primer set B:
1. 5′-CAAAAGCATGTGGAGTGAGG-3′ (bp 3187–3206)
2. 5′-CCTTATAATGGTGCTCTGGG-3′ (bp 3290–3271)
Primer sets A and B generate DNA fragments of 248 bp and 104 bp, respectively.
The total reaction volume in PCR was 50 μL and the reaction mixture contained 200 μM each of dATP, dCTP, dGTP, and dTTP, 50 mM KCl, 10 mM Tris (pH: 8.3), 2 mM MgCl2, 1 μg of each primer for both strands and 1 U of AmpliTaq Gold DNA Polymerase (Applera Italia). Thirty-two incubation cycles were carried out. The PCR for both primer sets was conducted with an initial 10 minutes denaturation step at 94°C coupled with a repeating cycle of 1 minute at 94°C, 2 minutes at 55°C (reannealing) and 2 minutes at 72°C (primer extension).
Detection of PCR Products
For the analysis of the amplified products of the PCR performed on each primer set, 10 μL of reaction solution were analyzed on 3% agarose gel containing 1 μg of ethidium bromide per mL.
The PCRs were positive in overall the tissues examined (Fig. 2).
B19 infection during pregnancy can have very different clinical pictures and results, ranging from the total absence of maternal and fetal symptoms to transitory hydrops of the fetus, to IUFD and miscarriage which constitute the most serious events3,4 and can raise important medico-forensic questions.
Fifty percent of pregnant women are immunologically susceptible to contracting B19 infection, but the incidence of maternal infection varies from 16.7% to 21% of the total number of women exposed.3–5 About 25% of these patients do not present clinical symptoms.6 The risk of vertical maternal-fetal transmission through the placenta is about 30% of the maternal infections2 and is considered to be more probable in the first 2 trimesters, becoming less frequent as the gestational age progresses. This depends on the fact that the antigen of blood group P (P-ag), the principal antigen necessary for maternal-fetal transmission, is present to a lesser degree at the placental level as the gestational age increases.7 The total incidence of important effects on the fetus (hydrops, IUFD) is between 5% and 10% of the patients infected.4,8 Nevertheless, there are a number of studies in which no case of hydrops was reported in pregnant patients exposed to the infection.3 The incidence of IUFD from B19 in North America and Europe has been estimated to be less than 1 of 1000 pregnancies per year.9 A large study carried out by the Society of Perinatal Obstetricians pointed out that IUFD affects 30% of fetuses with hydrops who are not transfused, and only 6% of those fetuses which are transfused.10
Between the exposition of a pregnant woman to the infection and the manifestation of the clinical fetal picture, there is, on the average, an interval of 6 weeks, at times even a few months, and infection of the fetus can take place even in the absence of clinical and laboratory signs of maternal infection.2 In fact, the infection can be asymptomatic, and seroconversion can take place even months after contagion.11 All this does not make it easy to predict fetal infection and IUFD.
Fetal infection can cause anemia since the virus determines cytotoxic apoptosis and the lysis of erythroid precursors and, in this way, it inhibits erythropoiesis.9 The presence of the virus in the erythroid precursors is revealed by the typical nuclear inclusions.12 The nuclei of the erythroblasts present an intensely margined and colored chromatin; the center of the nuclei is more luminous and appears homogeneous and vitreous.12
Anemia can lead to cardiac insufficiency and hydrops, which can be of varying degrees. Other anatomopathologic aspects12 correlated to fetal infection from B19, such as hepatosplenomegaly, hemosiderosis, and hepatic fibrosis from an excess of erythrolysis, have been described. The placenta can show an increase in weight, a pallid and edematous appearance due to hydrops, and inflammatory infiltrates of the villi which can lead to necrosis and calcification. The heart can be normal or symmetrically enlarged, with endocardial fibroelastosis; the thymus can be abnormally small due to lymphocytic depletion.12
The characteristics and the extent of the fetal damage vary according to the trimester of pregnancy in which the contagion takes place. In the first trimester, miscarriage has been reported in 3% of cases but the causal connection with B19 infection remains difficult to demonstrate.13 Isolated reports of cases of embryopathy have not been confirmed.14
Clinical manifestations of fetal damage are more frequent in the second trimester.2 This probably depends on the fact that, in this phase of the pregnancy, the concentration of P-ag in the placental trophoblast is still elevated.9 Furthermore, very intense hematopoiesis is prevalently located in the liver, the average life of the erythrocytes is shortened (45–70 days) and fetal growth requires a rapid increment of 3 to 4 times the erythrocytic mass.15 In this way, the fetus is more vulnerable to a condition of erythrocytic damage induced by the virus. Histopathological examinations reveal the typical nuclear inclusions in intravascular erythroblasts.12
Our investigation documented 3 cases of IUFD which took place in the second trimester (cases 1, 2, 4) in which the findings of major importance corresponded with a typical anatomopathologic picture, confirming the specific pathogenesis. In addition, we observed an endocardial fibroelastosis of varying degrees in these 3 cases which was not always accompanied by an increase in cardiac dimension.
IUFD can also take place, but with a lesser frequency, in a more advanced phase of gestation. In the major part of cases which take place during the third trimester, hydrops is absent,16 and, in many cases, clinical and laboratory signs of a recent maternal infection are not found.9,11 According to the prevailing opinion, this comes from the reduced concentration of placental P-ag and the normalization of the average erythrocytic life.2 In this period, the persistence of the maternal infection can, nevertheless, determine inflammatory and degenerative phenomena in the placenta,17 whose dysfunction may be a cause of IUFD, even without fetal infection.2 In these cases, histopathological examinations of the fetal organs do not show particular alterations or the typical viral inclusions.18
A similar typology finds substantial correspondence in case 5 of our collection, in which only flogosis of the chorionic membranes was found, with the absence of signs of fetal infection.
Our case 3 took place in the beginning phase of the third trimester and presented signs of fetal infection superimposable with cases 1, 2, 4, without endocardial fibroelastosis.
IUFD, above all when it takes place suddenly and unexpectedly (an eventuality of which our case histories constitute a significant example) can raise medico-forensic problems connected to the diagnosis of the cause of death and to eventual obstetric liability.
In fact, the cases of IUFD from B19 have appeared recently in the field of forensic medicine. This is due to increased knowledge of the pathology which today permits a rapid diagnostic approach, valid monitoring strategies ad relatively simple therapies, not without risks but effective in many cases. The parents informed that the pathology can be diagnosed and cured in cases at risk, sometimes do not accept the unfavorable results calmly and may take legal action.
Naturally, the involvement of forensic pathology in the study and evaluation of these cases is possible in those countries in which, by law, the question of medical responsibility for the cases involving a death is managed by the Judicial Authorities, who charge the forensic pathologist to carry out necessary verifications. Thus, it can happen that the case is directly entrusted to the forensic pathologist, as happened in one of our cases; in other cases, if the legal initiative is undertaken at a later date, the forensic pathologist can be entrusted with this after the autopsy has been carried out by the hospital pathologist, which is what happened in the other 4 cases in our series.
We believe that the forensic pathologist should orient his research in 2 directions. As for the first point, for medico-forensic ends, the exact diagnostic definition of the cause of death through a complete anatomopathologic investigation is important; this, however, can be very difficult especially if the fetus is macerated.19
The diagnosis, liable to suspicion on the basis of the autoptic findings, namely hydrops, serous effusion and placental thickening, is initially confirmed by the documentation of typical nuclear inclusions in the intravascular erythroblasts. Definitive confirmation can come from a search for B19 DNA in the fetal tissues and in the placenta using PCR, which is the most sensitive method for diagnosing fetal infection.9 The above-mentioned anatomopathologic and laboratory findings are convergent and conclusive signs for diagnosing IUFD from B19.
In cases of IUFD of the third trimester, if there are no signs of fetal infection, the diagnosis involves the identification of inflammatory-degenerative placental lesions and PCR.
The second point concerns the obstetrical liability in the management of clinical cases.
Every patient who is pregnant should be made aware of the risks coming from exposition to infection from B19. If a patient who is pregnant is exposed to the infection, the first thing is to diagnose the infection which is done using a serologic study of the IgM-IgG relationship.9 If the IgGs are positive and the IgMs are negative, the patient is in condition of immunity which excludes any risk. If the test is negative for both the IgGs and the IgMs, it has to be repeated 2 to 4 weeks later to evaluate an eventual positivity. If the IgM test, or both tests, are positive, the patient has an active infection. It then becomes necessary to inform the patient about the risks to which the fetus is potentially exposed, and begin monitoring to diagnose fetal infection early.6
In these cases, besides the daily counting of the fetal movements (starting from the end of the second trimester), ultrasound monitoring is advised, to be continued weekly for a varying amount of time according to the gestational age up to a maximum of 12 weeks, the time within which the major part of fetal complications manifested themselves.6 The most common ultrasound signs are hydrops, effusion towards the serous cavities, placental edema and polyhydramnios.6 Doppler ultrasound can also allow us to recognize anemia, under the form of an increase in the peak velocity of the systolic flow of the medial cerebral artery.20 Anemia can also be identified with greater precision using an invasive method (cordocentesis).6
Early diagnosis is very important because the incidence of IUFD can be reduced considerably by the timeliness of the diagnosis and the therapy.
Therapy includes the intravenous administration of immunoglobulins at a high concentration which can give positive results.21 The greatest benefits can be obtained by a blood transfusion which corrects the anemia and significantly reduces mortality.22 Without therapy, IUFD takes place in 30% of the total number of cases of hydrops and in all cases of severe hydrops,23 whereas intrauterine hematic transfusion can increase the survival rate to over 80%.10,23 However, transfusion can add risks in 2% to 5% of cases.6 Possible complications of transfusion include hematomas, lacerations and hemorrhage of the funiculus, infections, breaking of the membranes, increase in uterine activity and miscarriage which, in turn, are possible causes of obstetric liability.
If the pregnancy is in a more advanced stage (at least 32–34 weeks), the diagnosis of hydrops and fetal anemia can lead to considering the eventuality of an early delivery.6
In conclusion, accurate research on the anatomopathologic and laboratory signs of B19 infection furnishes fundamental elements for diagnostic judgment. On the one hand, it can explain the nature of the pathology which caused the IUFD and, on the other hand, it constitutes the basis for judging the conduct of the obstetricians.
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