detection of parvovirus b19 infection in first and second trimester fetal loss
TRANSCRIPT
DETECTION OF PARVOVIRUS B19 INFECTION IN FIRST AND SECOND TRIMESTER FETAL LOSS
Ronald R. de Krijger, MD, PhD Surgery and Anatomy, Erasmus University and University Hospital, Rotterdam, The Netherlands
Anne-Marie W. van Elsacker-Niele, MD Department of Virology, State University and University Hospital, Leiden, The Netherlands
Adri Mulder-Stapel Departments of Plastic and Reconstructive Surgery and Anatomy, Erasmus University and University Hospital, Rotterdam, The Netherlands
Marcel M . M . Salimans, PhD University Hospital, Leiden, The Netherlands
Enno Dreef Leiden, The Netherlands
Departments of Plastic and Reconstructive
Department of Virology, State University and
Department of Pathology, State University and University Hospital,
Harro T. Weiland, MD, PhD University Hospital, Leiden, The Netherlands
Johannes H. J. M. van Krieken, MD, PhD University and University Hospital, Leiden, The Netherlands
Christ1 Vermeij-Keers, MD, PhD Department of Plastic and Reconstructive Surgery and Anatomy, Erasmus University and University Hospital, Rotterdam, The Netherlands
Department of Virology, State University and
Departments of Pathology, State
Fetal and placental tissues and maternal serafrom a series of273 cases offirst and second trimester fetal loss were collected to detect thefiequenq of parvouims B19 infection. I n addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and i m m u n e
Received 28 May 1996; accepted 4 March 1997. This study was supported by grants from the “Praeventiefonds,” project numbers 0028151 7 (A.M.W.
The authors would like to thank Mr. L. Boshart for expert technical and photographic assistance. Present address of Anne-Marie W. van Elsacker-Niele: Public Health Laboratory, Department of
Address correspondence to Dr. Chr. Vermeij-Keers, Department of Anatomy, Erasmus University
van E.-N., A.M.-%, H.T.W.) and 0028-1518 (R.R. de K., C.V.-R).
Medical Microbiology, Leeuwarden, The Netherlands.
Rotterdam, P.O. Box 1738,3000 DR Rotterdam, The Netherlands.
Pediatric Pathology €9 Laboratmy Medicine, 18:23-34, I998 Coprright 0 I998 Taylor &Francis 1077-1042/98$12.00+ .OO 23
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histochemist? (IHC) were used for the detection of paroovirus BI 9 infection. Sera were tested for BI Pspecific immunoglobulin M (IgM) and/or IgG using a n enzyme-linked immunosorbent assay technique. Based on serology, I 4 9 cases not related to B19 infection 7uere excluded from further analysis. Two of the remaining 124 cases (0.7% o f all 273 cases) had parvovirus R l PspeciJic IgM and IgG at the time of abortion, indicating a recent maternal parvovirus BI 9 infection. I n our histolopal examination, I0 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either i n fetal tissues {n = 2) or in placental tissue {n = 8). However, this vacuolization was considered a fixation art fact and not identical to paruovirus B19-speeific nuclear inclusions described i n previous reports. Only I of these 10 cases had parvovirus B l 9DNA detectable in placental tissue by PCR analysis. Neither i n this case nor i n any ofthe other cases tested was paruovirus B19 DNA or protein detectable 4 ISH or IHC, respectively. I n none of 4 1 cases i n which fetal tissues were available were congenital anomalies found. I n conclusion, the frequency of maternal panlovirus BI 9 infection in this series offetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from paruovirus BI 9 infection and the usage of nuclear histology for the detection offetalparvovirus B19 infection is considered a nonspeafic parameter that requires confirmation by PCR.
Keywords first trimester fetal loss, histology, parvovirus B19, polymerase chain reaction, serology
Human parvovirus B19, first described in 1975 [ l ] , has a tropism for a limited number of cell types, including the human erythroid progenitor cell, endothelial cells, and cardiac myocytes [2]. This tropism is mediated by the P-antigen, which functions as the cellular receptor for parvovirus B19 [3,4]. Parvovirus B19 causes erythema infectiosum, mainly in children [5], and may cause aplastic crises in patients with chronic hemolytic anaemia and persist- ent red cell aplasia in immunodeficient patients [6, 71.
In the human fetus, the tropism for erythroid progenitor cells may cause fetal anemia. Combined with myocarditis and endothelial cell damage, this may lead to fetal death through nonimmune hydrops [S, 91. The time between maternal infection and fetal complications may be up to 12 weeks [lo-121. Most cases of B19-related fetal death have been reported in the period between 20 and 28 weeks of gestation. A single study has dealt with the relationship between parvovirus B19 infection and first trimester fetal loss but evaluated a relatively small series of abortions [ 131.
Unlike animal parvoviruses, human parvovirus B19 seems to have a low teratogenic potential, with few reports of congenital anomalies in B19-in- fected fetuses [ 14-1 61. These case reports may represent parvovirus B19-in- duced anomalies or coincidental findings. Further analysis is needed to detect a potential developmental effect of parvovirus B19 on the human fetus. For the detection of parvovirus B19 infection in mother and fetus a series of methods have been employed: serologic testing for parvovirus B19 antibodies in maternal or cord blood, histology for the detection of typical nuclear inclusions associated with parvovirus B19 infection [ 17-19], electron micros- copy [ 201, amplification of viral DNA from sera or tissues by the polymerase
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chain reaction (PCR) [21, 221, in situ hybridization [23-251, and imniuno- histochemistry by an antibody to a B19 capsid protein [26]. Yet, few studies have used these techniques to study cases of first trimester fetal loss.
In this study, our first aim was to determine the frequency of parvovirus B19 infection in a series of 273 cases'of first and second trimester fetal loss. We have used serology on maternal sera, histology, polymerase chain reac- tion, in situ hybridization, and immunohistochemistry for the detection of fetal parvovirus B19 infection. Second, we have analyzed all fetal tissues available for the presence of congenital anomalies, both in parvovirus B19- infected cases and in noninfected cases.
MATERIALS AND METHODS
Serology
A total of 402 serum samples from 273 cases of first and second trimester fetal loss were tested for parvovirus B19-specific immunoglobulin G (IgG) and IgM using an enzyme-linked immunosorbent assay (ELISA) method, as described before [22]. The collection of sera for research purposes was approved by the ethical committee of the Leiden State University. In short, parvovirus B19 VP2 antigen produced in a baculovirus expression system was used as the antigen. For the detection of parvovirus B19-specific IgG, mi- crotiter plates (Polysorb F96-3, Nunc, Roskilde, Denmark) were coated with VP2 antigen. Patient serum was added and incubated for 1 h at 37°C. After washing, a peroxidase-labeled rabbit anti-human IgG (F(ab),, Dako, Glos- trup, Denmark) was added at a 1:2000 dilution. After 1 h at 37°C the plates were washed and then incubated with ephenylenediamine.ZHC1 (OPD, Abbott, USA) for 30 min in the dark at room temperature. The reaction was stopped with 4 N H,SO,. The optical density ( A ) was measured at 492 nm. For the detection of parvovirus B19-specific IgM, microtiter plates, coated with rabbit anti-human IgM, were incubated overnight at 4°C with diluted patient serum ( 1 : l O O in phosphate-buffered saline, PBS). After washing, biotin-labeled B19 capsids in dilution buffer were added. The plates were incubated for 2 h at 37"C, washed, and incubated for 1 h at 37°C with horseradish peroxidase-labeled avidin ( 1 :25,000, Dako) . Final washing and staining were identical to those in the IgG procedure.
For both IgG and IgM ELISAs control samples were included: blank, negative control serum, cutoff serum, and a strongly positive serum. The cutoff value was determined by the mean A value of 50 indirect fluorescent antibody (1FA)-negative sera increased by twice the standard deviation. An ELISA test result was acceptable if the A value of the cutoff serum was below 0.500 and at least twice the A valuc of the blank and the A value of the strongly
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positive serum was between 1.500 and 2.500. A serum sample was considered equivocal when its ( A value minus the Avalue of the blank) was between once and twice the (A value of the cutoff serum minus the A value of the blank) and positive if it was at least twice this value.
Fetal Tissue
Placental tissues were obtained from spontaneous abortions and from cases of intrauterine fetal death. A total of 273 cases between 6 and 29 weeks of gestation (defined as the time elapsed since the first day of the last normal menstruation) were obtained over a period of 4 years. In 41 cases fetal tissues were recovered, of which 16 were complete autopsies. A further 6 cases concerned fragmented embryos or fetuses, including lung, heart, thymus, thyroid, intestine, spleen, pancreas, adrenal, kidney, testis, skeletal parts, and/or eye, that were amenable to gross analysis and constituted the major part of the embryo or fetus. The remaining 19 cases were small embryos, present in tissue blocks, that could not be examined externally and showed various degrees of maceration. Gross anomalies were analyzed before tissues were fixed in 4% paraformaldehyde in PBS, pH 7.4, and embedded in paraffin.
Histology
Hematoxylin and eosin-stained 5-pm sections of all blocks with placental tissue from 124 cases, selected according to serological data, were analyzed by three independent investigators for the presence of nuclear inclusions in erythroid progenitor cells, which are known to be specific for parvovirus B19 infections [ 17-19]. In the 41 cases in which fetal tissues were available serial sections (20-200 sections per case) were studied for congenital anomalies and nuclear inclusions.
Polymerase Chain Reaction
PCR was performed after histological examination of the tissue sections. Placental tissue was used for PCR in all 124 cases and fetal tissues in the 41 available cases. Five-micrometer microtome sections were cut. To avoid contamination, a disposable knife was used for each tissue sample and sections of paraffin blocks without tissue were taken between every two cases.
Sections were deparaffinized by washings with xylene, 100% ethanol, and acetone. After evaporation of the acetone, 50 pL of lysis buffer-10 mM Tris/HCl, pH 8.0, 1.5 mM MgC12, 50 mM NaCl, 0.2 mg/mL bovine serum
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albumin (BSA) , 0.5% Tween-20, 0.4 mg/mL proteinase K-was added for 1 h at 37°C. Proteinase Kwas inactivated by heatingfor 10 rnin at 100°C and 10 pL of lysate was used for PCR.
Parvovirus B19 PCR was essentially performed as the three-step cycle PCR described by Salimans [27]. The specificity of the amplification product was confirmed by dot spot hybridization using a digoxygenin-labeled oligonu- cleotide (B19 nucleotides 581-600, sequence: 5'-GCAGTGTGGCTTCTA- AGCTT-3'). Controls were used in each PCR experiment: a B19-positive and B19-negative control serum (tested in a series of dilutions as a sensitivity control), a B19-positive and B19-negative tissue sample, lysis buffer, and water.
In Situ Hybridization
Three-micrometer sections of placental tissues were mounted on coated slides and air dried overnight at 37°C. Subsequently, they were deparaffinized by three 10-min washings in xylene and two 5-min washings in methanol. Slides were incubated for 30 min with 1% hydrogen peroxide in methanol, washed in water, and air dried. Then they were pretreated with 0.5% pepsin in 0.2 M HCl for 30 min at 37"C, washed in 70% and 100% ethanol to inactivate pepsin, and air dried. The biotinylated parvo-probe (2 ng/pL, sequence GCA GTG TGG CTT CTA AGC 'IT) was denatured at 80°C for 10 min and the hybridization was carried out overnight at 42°C in a moist chamber. Stringent washing consisted of two 10-min changes of 2x(SSC), pH 7.0, at room temperature followed by the same washing steps at 50°C. Visualization of the probe was performed as follows: slides were preincubated with 4xSSC, pH 7.0, and 5% nonfat dry milk for 15 min; incubated with avidin-D-HRPO (1:100, Vector Labs, Burlingame, CA, USA) for 30 min; washed twice in 4xSSC; incubated with biotinylated goat anti-avidin-D (1:200, Vector Labs); washed twice in 4xSSC; and finally incubated with avidin-D- HRPO (1:100, Vector Labs). Subsequently, slides were washed twice with 4xSSC and rinsed with 0.1 M buffered sodium acetate. Then slides were incubated for 10 rnin with 0.05% hydrogen peroxide in 3-amino-9ethylcarba- zole, rinsed twice in water, and counterstained with Mayer hematoxylin for 3 min. Finally, they were washed in tap water for 10 min, air dried, and mounted with Kaiser's glycerin (Merck, Darmstadt, Germany). Negative controls were performed as already described, with the omission of the parvo-probe.
lmmunohistochemistry
Three-micrometer sections of placental tissues were mounted on coated slides, deparaffinized, and incubated for 25 rnin with 1 % hydrogen peroxide
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in methanol. After washing, sections were preincubated for 10 min in 10% normal goat serum and subsequently overnight with a monoclonal antibody against parvovirus B19-specific protein-NCL-parvo, dilution 1:4000 in PBS/BSA (1%) (Novocastra, Newcastle upon Tyne, UK). The rest of the avidin-biotin complex (ABC) procedure was carried out according to manu- facturers’ prescriptions using a biotin-conjugated rabbit anti-mouse second antibody (1:200, Dako) and astreptavidin-peroxidase complex (1:100, Dako). Sections were developed for 5 min in sodium acetate solution, to which 7 mL of 3-amino-9-ethyl-carbazol in dimethylformamide (0.25 g/L) (Sigma, St. Louis, MO, USA) and 75 pL of 30% H202 were added. Finally, sections were counterstained with hematoxylin and mounted in Kaiser’s glycerin (Merck). Negative control experiments were performed by omitting the primary an tiserum.
RESULTS
Serology
ln 149 of 273 cases (55%) of first or second trimester fetal loss and intrauterine fetal death, either specific IgG was already present in the mater- nal blood sample drawn before conception or specific IgG was not found in the maternal blood sample, drawn within 1 month after the termination of pregnancy. These cases are therefore considered not to be related to par- vovirus B19 infection and were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM, immediately following the abortion, indicating a recent parvovirus B19 infec- tion in the mother.
Histology
Ten of 124 cases (8.0%) showed nuclear vacuolization in fetal erythroid progenitor cells, which were present either in the placenta ( n = 8) or in feral tissues ( n = 2). The vacuolization most likely represented a fixation artifact and was not considered to be an unequivocal hallmark of parvovirus B19 infection. These 10 cases included the case with both B19-specific IgM and a positive PCR reaction (Figure I ) . None of the remaining nine cases showed a positive PCRreaction upon repeated testing. Neither did these cases present parvovirus B19-specific IgM upon serologic testing. The other case with specific IgM in maternal blood did not show nuclear inclusions.
The 41 cases in which fetal tissues were available did not present gross or microscopic evidence (in serial sections) of congenital abnormalities. None of the cases showed evidence of hydrops fetalis.
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Figure 1. Details of placental tissue of' cases with nuclear vacuolization. The upper left panel ( ~ 4 0 0 ) represents the parvovirus B19 PCR-positive case. The other three panels (~1700) represent cases negative by serological screening and PCR. Nucleated red blood cells with nuclear inclusions are indicated by arrows.
Polymerase Chain Reaction
A single case of 124 cases (0.8%), which also had parvovirus B19-specific IgM antibodies at the time of abortion, had parvovirus B19 DNA in placental tissue. No fetal tissue was available from this case. Parvovirus B19 DNA could repeatedly be demonstrated by PCR (Figure 2). The positive and negative
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Figure 2. Dot-blot hybridization of PCR amplificates. Lane lC, positive tissue control; lane l D , negative tissue control; lane 1F (= lane 3C), positive serum control; lane 1G (= lane 3D), negative serum control; lane 2A, positive experimental sample; lane 2B (= lane ZH), positive tissue control; lane 2C (= lane SA), negative tissue control; other spots represent negative experimental samples or other negative controls (water, lysis buffer).
serum and tissue control samples resulted in a consistent positive and negative PCR reaction, respectively. N o positive PCR was obtained with the other serologically B19-positive case (specific IgM antibodies), nor with any other case, including the control sections of paraffin blocks without tissue.
In Situ Hybridization and Immunohistochemistry
None of the placental tissues ( n = 18, including two cases of serologi- cally proved recent maternal infection, all cases with nuclear vacuoliza- tion, and seven other randomly chosen samples) that were studied by either of these techniques showed the presence of parvovirus B19 DNA or protein, respectively. The positive control samples showed the ex- pected reactivity (Figure 3) .
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Figure 3. (A) In situ hybridization for panrovirus B19 DNA on positive control tissue with multiple parvovirus B19-infected cells showing a nuclear staining pattern. (B) No staining was observed in any of the experimental cases. (C) Immunohistochemistry for parvovirus B19 DNAon positive control tissue with multiple parvovirus B19-infected cells, showing a predominantly cytoplasmic staining pattern. (D) No staining was observed in any of the experimental cases.
DISCUSSION
In this study we have examined the frequency of pavovirus B19 infections in cases of first and second trimester fetal loss. This frequency appears to be low in a series of 273 cases, randomly obtained over 4 years. In 2 of all 273
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cases (0.7%) the maternal serum sample showed evidence of recent infection with parvovirus B19 (specific B19 IgM present). In one of these cases a positive PCR reaction of placental tissue was obtained and nuclear inclusions in fetal erythrocytes were found, suggesting transmission to the embryo. However, no fetal tissues were available to prove this transmission. No conclusions could be drawn with regard to the effect of parvovirus B19 infections on the development of congenital anomalies. Another study that analyzed par- vovirus B19 infections in early spontaneous abortions found 5 of 80 cases (6%) with positive maternal serology (IgM and IgG), 2 of which were also positive in the PCR reaction of placental tissues (chorionic vesicles), suggesting that indeed the frequency of parvovirus B19 infection is low [13]. The majority of cases in the study by Rogers et al. were collected between January and March, during which period parvovirus B19 epidem- ics in the general population are known to occur. This may have resulted in an increase in parvovirus B19 cases among aborters as well as non- aborters. The abortions and fetal deaths we studied were not clustered in any period of the year.
We have used several techniques for the detection of parvovirus B19 infection in pregnancy. Serology can be used only as an indicator of maternal infection. Other techniques are needed to prove parvovirus B19 infection of the fetus. Until the advent of' molecular biology, characteristic nuclear inclu- sions were used as the criterion for parvovirus B19 infection. Many studies have reported such inclusions in PCR-verified cases. In our study we found 10 cases with nuclear vacuolization that, although similar to the inclusions in PCR-proven cases, were considered fixation artifacts or degenerative phe- nomena. We therefore consider nuclear inclusions, unless in experienced hands, a nonspecific parameter that may also occur in non-parvovirus-associ- ated conditions. Indeed, Rogers et al. [13] describe five cases with nuclear inclusions, without positive maternal serology and tissue PCR. Thus, all cases with suggestive nuclear abnormalities should be analyzed by PCR to confirm the presence of parvovirus B19. With regard to PCR in placental tissues, it must be mentioned that the placenta may be contaminated by viremic maternal blood; a positive PCR of placental tissue may thus be of exclusive maternal origin. Therefore, preferably fetal tissues should be used for par- vovirus B19 PCR.
Alternatively, parvovirus B19 infections may be confirmed by in situ hybridization and/or immunohistochemistry, using a monoclonal antibody to a parvovirus-specific protein, enabling the morphological detection of infected cells [23-25, 281. In this study none of the cases were positive with either ISH or IHC. The number of parvovirus B19-infected cells and/or the amount of viral DNA or antigen in the tissue sections may hamper detection with these techniques, which generally have a lower sensitivity than PCR.
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In conclusion, we have found 2 of 273 cases (0.7%) of maternal serocon- version (specific IgM present), indicating a low incidence of maternal par- vovirus B19 infection in cases of early spontaneous abortions. Placental tissue from one of these cases showed evidence of parvovirus B19 infection by a positive PCR reaction. ISH and IHC remained negative in this and all other cases studied. Nine other cases with nuclear vacuolization did not show evidence of parvovirus B19 infection by any of the techniques used. We therefore recommend for all cases with positive maternal serology and nuclear abnormalities to perform PCR specific for parvovirus B19 DNA on fetal tissues.
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