Baba. Department of Pediatrics, Nishi-Kobe Medical Center, 5-7-1,. Koji-dai, Nishi-ku, Kobe, .... cytes, giant proerythroblasts, or evidence of hemophagocytosis.
Ann Hematol (1999) 78 : 83–86
Q Springer-Verlag 1999
CASE REPORT M. Osaki 7 K. Matsubara 7 T. Iwasaki 7 T. Kurata H. Nigami 7 H. Harigaya 7 K. Baba
Severe aplastic anemia associated with human parvovirus B19 infection in a patient without underlying disease
Received: August 28, 1998 / Accepted: November 18, 1998
Abstract Human parvovirus B19 (B19 virus) infection is known to induce aplastic crisis in patients with hemolytic anemia. In healthy subjects, B19 infection may sometimes cause mild pancytopenia, but these changes are transient and recovery is spontaneous. We report the first case of aplastic anemia in a previously healthy boy without any underlying diseases, following asymptomatic infection with the B19 virus. Laboratory examination initially showed thrombocytopenia, mild leukopenia in the peripheral blood, and severe hypoplastic bone marrow. Since pancytopenia developed and worsened progressively, immunosuppressive therapy was given, resulting in a complete remission. Despite the lack of an infectious prodrome, serological and histological analysis revealed an underlying infection with the B19 virus. Thus, B19 virus infection must be considered one of the causes of aplastic anemia in patients without underlying hemolytic anemia and an apparent episode of the viral infection.
mia or hereditary spherocytosis. Chronic bone marrow failure may also develop with a persistent B19 infection in immunocompromised individuals [8]. On the other hand, B19 virus infection in immunocompetent hosts has been shown to induce subclinical transient anemia of a mild degree, associated with or without slight thrombocytopenia and leukopenia [1]. In experimental infections with normal volunteers, reticulocytes and platelet counts in the peripheral blood decreased 10–14 days after intranasal inoculation, and these hematological abnormalities recovered spontaneously within a few weeks [1]. Here, we report an otherwise healthy boy who developed aplastic anemia due to B19 virus infection without any apparent manifestations of erythema infectiosum.
Method Serological detection of anti-B19 virus antibodies
Key words Aplastic anemia 7 Human parvovirus B19 7 Asymptomatic infection 7 Healthy subject
Introduction Human parvovirus B19 (B19 virus) is known as a cause of erythema infectiosum in children and an influenzalike condition with polyarthropathy in adults [4]. One of the most serious complications of B19 virus infection is a transient aplastic crisis of hematopoiesis in patients with chronic hemolytic anemia such as sickle cell aneM. Osaki 7 K. Matsubara (Y) 7 H. Nigami 7 H. Harigaya 7 K. Baba Department of Pediatrics, Nishi-Kobe Medical Center, 5-7-1, Koji-dai, Nishi-ku, Kobe, 651-2273, Japan Tel.: 081-78-997-2200, Fax: 081-78- 993-3728) T. Iwasaki 7 T. Kurata Department of Pathology, National Institute of Infectious Diseases, Tokyo/Japan
Anti-B19 virus IgG and IgM antibodies were examined using an enzyme immunoassay method with a commercially available kit (IDEIA, Dako, Denmark). When the index value of the OD450/ cut-off OD450 ratio was 1.2 or more, the result was interpreted as positive. Detection of the B19 virus antigen and genome The presence and localization of the B19 virus were examined on paraffin sections of bone marrow using an immunohistochemical method for the viral capsid antigen (VCA) and in situ hybridization for the genome. The antiserum for detection of the VCA was prepared by immunization of the recombinant VP1 protein of the B19 virus expressed in Escherichia coli in rabbits. For histochemical staining, the deparaffinized sections were incubated with the antiserum at 4 7C overnight and reacted with biotinylated antirabbit IgG and sequentially with avidin-biotin complex solution (Vector Lab, Burlinghame, Calif.). Peroxidase activity was developed in a diaminobenzine solution with hydrogen peroxide. Counterstaining was performed using the Giemsa method and Berlin blue staining. Nonisotopic in situ hybridization was performed as follows: After digestion of deparaffinized sections with proteinase K, the slides were prehybridized with 20 ml of the hybridization buffer
84 (50% deionized formamide, 3! standard saline citrate (SSC), 5! Denhardt’s solution and 50 mM HEPES at pH 7) and hybridized with 5 ng of a biotinylated cloned DNA fragment of the B19 virus (kindly supplied by J. P. Clewley, Virus Reference Laboratory, Central Public Health Lab, London) [5] at 42 7C overnight. Hybridized DNA was detected by a streptavidin-biotin-alkaline phosphatase method using BCIP and NBT as substrates.
Case report A 14-year-old boy had been healthy until 1 week prior to admission, when dizziness and nasal bleeding developed. He had no apparent symptoms, signs of preceding infection, or history of recent medication. Physical examination on admission revealed no petechiae, exanthema, arthralgia, or hepatosplenomegaly. Blood analysis revealed Hb of 13.1 g/dl, a RBC of 4.39!10 12/l, reticulocytes 3.4!10 9/l, platelets 38!10 9/l, and a WBC of 3.0!10 9/l (neutrophils 26.5%, lymphocytes 64.5%, monocytes 7%, atypical lymphocytes 1.5%). Blood chemistries were within normal ranges. Bone marrow aspiration showed a marked decrease of nucleated cell cellularity (20.3!10 9/l) with a myeloid component of 16.2% and an erythroid component of 7.0%. No megakaryocytes, giant proerythroblasts, or evidence of hemophagocytosis were observed (Fig. 1a, b). Platelet-associated IgG and haptoglobin were within the normal range. The autoantibody against neutrophils was not detected in the serum. Direct and indirect Coombs’ tests revealed no abnormal findings. There was no evi-
Fig. 1a–d Bone marrow findings on admission. a A hypoplastic hematopoiesis was accompanied by the absence of megakaryocytes (hematoxylineosin, !100). b Neither giant proerythroblasts nor eosinophilic intranuclear inclusions of erythroblasts were observed (hematoxylin-eosin, !200). c Brown signals for capsid antigen (arrows) were detected in the remaining erythroid progenitor cells (counterstained with methylgreen and Berlin blue for iron, !400). d The blue signal for the genome (arrow) of the B19 virus was detected (counterstained with hematoxylin !400)
dence of iron deficiency, and osmotic fragility and sucrose gradient lysis tests yielded normal results. The patient was initially observed without any medical treatment for more than 4 weeks. However, pancytopenia developed and worsened progressively, so repetitive platelet transfusion became necessary. Six weeks after admission, his blood analysis results were RBC 3.29!10 12/l, reticulocytes 2.9!10 9/l, platelets 17!10 9/l, and WBC 1.6!10 9/l with 12.5% neutrophils. A repeat bone marrow aspiration again showed decreased cellularity, meeting the diagnostic criteria for severe aplastic anemia. Subsequently, the patient was treated with combination therapy consisting of an intravenous infusion of horse anti-lymphocyte globulin (Lymphoglobulin, Institute Pasteur Merix, Lyon, France; 15 mg/kg on days 1–5), methylprednisolone (2 mg/kg on days 1–7 i.v., 1 mg/kg on days 8–14 p.o., slowly tapered off and stopped by day 30), cyclosporin A (2–5 mg/kg p.o. for 6 months, with dose adjustment to maintain a serum trough concentration of 100–200 ng/ml), and danazol (5 mg/kg p.o. for 6 months) [15]. No adverse effects were observed except for transient Coombs’ positive hemolysis, which recovered without specific treatment. The hematopoiesis had not recovered 1 month after the start of the therapy (Fig. 2a), but by 3 months after the initiation of treatment he had achieved a complete recovery (Hb 1 11.5 g/dl, platelets 1 150!10 9/l, absolute neutrophil count 1 1.5!10 9/l). Bone marrow aspiration also showed recovery of hematopoiesis (Fig. 2c). To clarify the causative infectious agent associated with aplastic anemia, we performed serological analysis. Anti-B19 virusspecific IgG and IgM antibodies in the serum were found to be positive on admission (cut-off indices 2.0 and 5.3, respectively).
85 Fig. 2a–d Bone marrow findings following immunosuppressive therapy. a Bone marrow tissue obtained 1 month after therapy remained hypoplastic, with no megakaryocytes (hematoxylin-eosin, !100). b The cells positive (arrows) for the B19 virus genome increased in number (in situ hybridization, !200). c Bone marrow in remission (3 months after treatment) showed megakaryocytes and enlarged cells with large nuclei and nucleoli (hematoxylin-eosin, !200). d No genome-positive cells were detected (in situ hybridization, !100)
Other recent infections were excluded by serological tests for hepatitis A, B, and C, rubella, measles, cytomegalovirus, and Epstein-Barr virus. Although the genome of the B19 virus was not detected in the serum by polymerase chain reaction, the VCA and genome of the B19 virus were detected in the erythroid progenitor cells by immunohistochemistry (Fig. 1c) and by in situ hybridization (Fig. 1d). One month after the start of immunosuppressive therapy, the number of cells positive for the B19 virus genome increased (Fig. 2b). However, the VCA and B19 virus genomes disappeared from the bone marrow 3 months after the treatment (Fig. 2d), when the anti-B19 virus-specific IgM titer became negative while the anti-B19 virus-specific IgG titer remained positive. Based on these findings, we attributed the boy’s aplastic anemia to asymptomatic B19 infection. He has maintained a complete remission for more than 1 year since the start of medical treatment.
Discussion We have described a 14-year-old boy with no obvious underlying disease who developed severe aplastic anemia following B19 virus infection. Although the patient had neither fever, exanthema, nor arthralgia, serological and histological studies confirmed an underlying B19 virus infection. Since an outbreak study showed that a substantial population of B19 virus-infected indi-
viduals does not manifest any symptoms [6], we believed this patient to have an infection of B19 virus that was asymptomatic except for the hematological disturbance. Despite great advances in clinical and experimental hematology, definitive assignment of an etiological agent for aplastic anemia is usually difficult, and most cases are attributed to an idiopathic origin [9]. In some patients with aplastic anemia, viral infections such as Epstein-Barr virus, cytomegalovirus, and rubella precede aplastic anemia. However, little is known about the causal relationship between viral infections and aplastic anemia. As for B19 infection as a cause of aplastic anemia in immunocompetent hosts, only one case of a 20-year-old woman without underlying disease has been reported [7]. However, in this patient, infection with the B19 virus was determined only by serological testing. As far as we know, our case is the first in which severe aplastic anemia developed in association with histologically and serologically proven B19 virus infection. In a recent study, Langans et al. postulated that B19 was a possible cause of fulminant liver failure and subsequent aplastic anemia [10]. In their study, B19 virus DNA was detected at a significantly higher rate in
86
liver tissue from the affected patients. More recently, some cases of aplastic anemia associated with mild liver dysfunction induced by B19 virus were also reported [12]. However, it is clear that the present case is different from post-hepatitis aplastic anemia because of the lack of liver dysfunction. One may argue that this patient suffered from “idiopathic” aplastic anemia and concomitant B19 infection, because B19 infection is a common event. It is, however, quite difficult to answer this argument properly on the basis of only one case. The patient in our study initially developed marked thrombocytopenia, followed by anemia and granulocytopenia. Experimental infections with the B19 virus in normal volunteers demonstrated that not only erythroid production, but also myeloid and platelet production were affected [1]. Colony-forming assays showed erythroid colonies were involved earlier than myeloid colonies [13]. The difference in onset of clinical manifestations of hematopoiesis in the present case may merely reflect the difference in half-life between each hematopoietic progenitor cell after the simultaneous growth failure. The role of B19 virus infection in the pathogenesis of aplastic anemia remains unclear. The B19 virus is well documented as infecting the erythroid progenitor cells and is associated with aplastic crisis in patients with chronic hemolysis [3, 11]. Although in vitro and in vivo analysis showed that the B19 virus infects mainly the erythroid progenitor cells [4, 16], other progenitor cells may also be affected by inefficient viral replication [9]. The lack of absolute reticulopenia in the peripheral blood and the presence of normoblasts in the bone marrow, as well as the absence of giant proerythroblasts, in this case are quite different from the findings in chronic bone marrow failure induced by B19 virus infection in immunocompromised hosts [8]. It is clear that the patient was immunocompetent, because he had no past history of frequent infections, and immunological screening analysis (data not shown) revealed no remarkable findings. Moreover, he did indeed produce antibodies recognizing B19 virus. The most plausible explanation for the pathogenesis of aplastic anemia is that some immune-mediated mechanisms induce a disturbance in the bone marrow and lead to decreased hematopoiesis. The recovery of hematopoietic function after immunosuppressive therapy is the strongest argument for this hypothesis. Many authors attributed the pathogenesis of aplastic anemia associated with Epstein-Barr virus or hepatitis virus to the autoimmune process [2, 14]. The precise mechanisms involved in the development of B19 virus-associated aplastic anemia remain to be clarified.
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