EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Pure red cell aplasia (PRCA) is a rare disease that presents with anemia secondary to erythropoiesis failure. It is characterized by normocytic, normoc-hromic anemia associated with reticulocytopenia in the peripheral blood and a sparse presence or an absence of erythroblasts in the bone marrow.¹ Primary infection is asymptomatic in most cases. Acute infection causes fifth disease in individuals with normal immune system, transient aplastic crisis in patients with chronic hemolysis, and PRCA in immunocompromised hosts.² The target cell is the erythroid progenitor in the bone marrow. During active infection, the virus binds to the globoside (antigen P) receptor on erythroid precursors, infects the cell, replicates, and lyses the cell.³ This reveals the presence of giant pronormoblasts, which may aid the diagnostic process leading to discovery of PRCA on bone marrow examination.⁴ Passive immunization with intravenous immunoglobulin (IVIG) containing large amounts of anti-parvovirus B19 (HPV-B19) immunoglobulin G (IgG) is the treatment of choice for HPV-B19-associated PRCA.

Here, we present 2 renal transplant recipients with HPV-associated PRCA who were seen at our center. We summarize their clinical features and provide a review of the relevant literature. The patients provided written informed consent.

A 34-year-old female patient diagnosed with focal segmental glomerulosclerosis had received a kidney transplant 6 years previous. While she on a regimen of tacrolimus (2 × 4 mg), mycophenolate sodium (MPA; 2 × 360 mg), and Deltacortril (prednisolone, 5 mg) in posttransplant year 2, her hemoglobin decreased to 3 g/dL. Her anemia persisted, so a bone marrow biopsy was performed, and 3 units of erythrocyte suspension were given every month. Myelodysplastic syndrome was considered. When the patient presented to our center, biochemical parameters and laboratory test results were within reference range except hemoglobin (leukocytes 7500 cells/mL, hemoglobin 7.4 g/dL, mean corpuscular volume 86.5 fL, platelets 362?000 cells/mL, and reticulocytes 0.94%). The test results for direct Coombs and for HPV-B19 IgG and IgM levels were negative. After repeated bone marrow aspirations, we observed dyshematopoiesis, mega-loblastic erythropoiesis, and large-nucleated erythroblasts, as well as normocellular bone marrow with a reduction in erythroid series (Figure 1, A and B). Polymerase chain reaction (PCR) for HPV-B19 DNA detected 4?500?000 IU/mL. TheIVIG treatment was given in response to the diagnosis of HPV-B19-associated PRCA. Meanwhile, the patient was referred to her nephrologist to reduce her immunosuppressive therapy. On day 15, hemoglobin increased to 11.6 g/dL. In months 2 and 3 of treatment, hemoglobin increased to 19 g/dL, and phlebotomy was performed twice. At month 5 of treatment, hemoglobin started to decrease again. The PCR results showed 4?350?000 IU/mL, so IVIG was administered for the second time. The hemoglobin level started to increase in week 2 of treatment and was completely normal by month 2. In month 5, hemoglobin levels started to decrease again. The HPV-B19 PCR results showed 2?262?000 IU/mL, so IVIG was given for the third time. Anemia resolved in week 4. Then, in month 2 of IVIG treatment, hemoglobin decreased again. The HPV-B19 PCR results showed 5?270?000 IU/mL. After the third relapse, the patient was again given IVIG, and the immunosuppressive therapy was changed (MPA discontinued, dose of tacrolimus reduced, and everolimus added), after which the hemoglobin level increased to 11.4 g/dL. The treatment course and hemoglobin-parvovirus serology results of the patient are shown in Table 1 and Figure 2.

Case 2
A 51 year-old male patient had received a kidney transplant 8 months previous in response to diagnosis of chronic renal failure secondary to hypertension. Anemia developed 2 months after the transplant, and multiple erythrocyte transfusions were performed. He was on a regimen of tacrolimus (2 × 3.5 mg), MPA (2 × 360 mg), and Deltacortril (1 × 5 mg). Darbepoetin was also started, but no response was observed. Profound anemia was discovered, so the patient was referred to the hematology department for tests. The biochemical parameters and the test results were all within reference ranges except hemoglobin (leukocytes 4100 cells/mL, hemoglobin 5.6 g/dL, mean corpuscular volume 82 fL, platelets 343?000 cells/mL, and reticulocytes 0.2%). Direct Coombs test results were negative. It was discovered that MPA was discontinued in response to lymphopenia. Repeated tests for HPV-B19 IgM and IgG were also negative. Bone marrow aspiration showed normocellular aspirate with erythroid hypoplasia and giant erythroblasts containing darkly stained nucleoli/inclusion structures, as well as megaloblastic changes (Figure 1, C and D). This was suspected to be HPV-B19 infection. The PCR results for HPV-B19 PCR in serum showed >15?000?000 IU/mL. The immunosuppressive dose was reduced, and IVIG was given. On day 10 of the treatment, the transfusion dependency disappeared and hemoglobin level increased to 12.1 g/dL. On day 75, another HPV-B19 PCR test was performed for control purposes and showed >15?000?000 IU/mL. The highest level of hemoglobin was detected in month 3 (15.4 g/dL). However, anemia recurred in month 5, and the HPV-B19 PCR results remained at >15?000?000 IU/mL. In response to relapse, IVIG was given to the patient again, and hemoglobin level increased to 10.1 g/dL at week 1 of treatment. In week 2, hemoglobin level was 9.1 g/dL and PCR was >15?000?000 IU/mL. In week 3, hemoglobin decreased to 6.7 g/dL. The dose of tacrolimus was reduced, and everolimus was added to the treatment. A third round of IVIG was given. The treatment course and hemoglobin-parvovirus serology of the patient are shown in Table 2 and Table 2.

Kidney transplant patients are highly susceptible to infections such as HPV-B19 because kidney transplant recipients are typically treated with immunosup-pressive therapy. Common clinical manifestations of HPV-B19 infection in immunocompromised patients are fever, arthralgia, and rash. However, most patients have severe anemia due to the absence of reticulocyte response and no erythropoietin response.⁵ As a result of an impaired immune response, production of anti-HPV-B19 antibodies may be inadequate, and typical signs of clinical syndrome may be absent. Thus, the diagnosis of parvovirus infections in renal transplant patients remains challenging, and parvovirus PCR is required to determine the diagnosis.⁶

Both patients in this study presented with profound anemia and had no other signs of infection. When the patients were referred to the hematology department, they received an extended course of transfusion support; a PCR test was not performed because HPV-B19 serologies were negative. Giant pronormoblasts seen in patients’ bone marrow aspirations were highly diagnostic, and positive HPV-B19 PCR results also supported our diagnosis. In the differential diagnosis of a case with anemia and reticulocytopenia, other conditions such as nutritional deficiencies, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, immune deviations, and drug effects should also be considered. Clinical and laboratory diagnoses were considered in the differential diagnosis and excluded in our cases.

In the literature, HPV-B19-associated PRCA is seen more frequently in the early period after transplant. One of our 2 patients was in the early period after the transplant, and the second patient was in the late period.

According to current guidelines, standard therapy against HPV-B19 infection consists of IVIG in a total dose of 2 g/kg in 5 divided doses.⁷ This management course is usually successful in acute symptomatic patients. It is believed that a reduction in immunosuppression contributes to resolution of the infection. There is no clear evidence to support a best practice for adjustment of immunosuppressive medication. In a previously published study, 34% of the patients relapsed within 4.3 months and frequently required IVIG courses.⁸ Therefore, the disadvantages of this treatment are disease relapse and high financial cost. We applied the IVIG protocol of 400 mg/kg/d for 5 days with the reduction of immunosuppressive therapy in our patients. The course of IVIG was tolerated well, and no side effects were observed. In both of our patients, transfusion independence was achieved by week 2, and the hemoglobin level returned to normal levels in approximately 4 weeks. In both cases, the first relapse was in month 5, consistent with the literature. The first patient relapsed 3 times, and the second patient relapsed 2 times. In our first patient, repetitive IVIG applications produced a response, but the gradual shortening of response times was remarkable. In our second patient, partial response was obtained after repeated IVIG application, and relapse was observed in a short time.

It is difficult to treat patients who relapse after IVIG or who are resistant to IVIG. For a definitive treatment to be established, a reduction/modification of the etiologic cause of immunosuppression or an effective antiviral agent against HPV-B19 is required.

Reduction of immunosuppressive therapy, esp-ecially calcineurin inhibitors, is a fundamental aspect of HPV-B19 infection management, but there remains a concern of an increased risk of graft rejection. It has been shown that solid-organ transplant recipients can benefit from the antiviral properties of inhibitors of the mechanistic target of rapamycin (mTORi). In memory T cells, these drugs can increase efficacy, functionality, and ability to inhibit viral cell growth. Multiple studies indicate that mTORi treatment is useful to prevent infection by cytomegalovirus or BK virus or may play a role in treatment of Epstein-Barr virus posttransplant lymphoproliferative disease and herpes virus-related Kaposi sarcoma.⁹ A previously published case report showed successful results with the conversion from calcineurin inhibitors to a mTORi in a case of HPV-B19-associated PRCA with relapses.¹⁰ We also reduced the dose of tacrolimus and added everolimus in our patients who developed relapse after IVIG. In addition, there have been several cases reported in the literature of tacrolimus-mediated PRCA that showed rapid recovery after the replacement of tacrolimus with cyclosporine A.^11,12

Antiviral drugs are not available for the treatment of HPV-B19 infection. Successful use of foscarnet in relapsed or unresponsive patients after IVIG has been published recently.¹³

In conclusion, HPV-B19 infection should be considered in the differential diagnosis of anemia with reticulocytopenia in patients receiving posttransplant immunosuppressive therapy. In such immunocompromised patients, standard HPV-B19 serological tests may not detect HPV-B19 infection. Therefore, HPV-B19 PCR testing should be performed, especially in serologically negative patients. Reduction of immunosuppressive therapy and administration of IVIG is an effective option with an early treatment response. In addition, because relapses are possible, patients should be followed in this respect. However, there remains a need for an established, definitive treatment for relapsed/refractory patients.

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