Parvovirus B19 incidence, specific antibody response, and delayed hematopoietic recovery after allogeneic hematopoietic stem cell transplantation

Introduction

Parvovirus B19 (PVB19) is a well known small DNA virus from Erythrovirus genus which is in scope of pediatricians for decades being associated with erythropoiesis disturbances, arthropathies, myocarditis and other disabling clinical conditions [1]. PVB19 shows an affinity for the group P antigen of red blood cells, with lesser amounts in blood plasma [2]. The major hematotoxic effect of the virus is believed to occur at the pronormoblast stage, thus causing arrest of erythroid differentiation. PVB19 was occasionally found in aplastic anemias and pure red cell aplasia [3]. In this respect, most studies concerned resistant anemia cases in the patients subjected to renal transplantation where the PVB19 was not a rare finding [4].
Over past years, many cases of severe myocarditis and hepatitis were shown to be associated with parvovirus infection, as based on positive PVB19 antigen or DNA findings in affected tissues. Meanwhile, a latent persistence of PVB19 was quite common in skin, synovial tissues, myocardium and bone marrow [5]. The viral DNA was detectable in peripheral blood from 5% of healthy donors [6]. The authors suggest only a small risk for the donor-recipient viral transmission upon hematopoietic stem cell transplantation (HSCT).
Following allogeneic HSCT, a regular activation of herpesviruses and other latent pathogens is observed, due to acute myelosuppression and cellular immune deficiency [7]. Clinical significance of the PVB19 in immunocompromised patients is not yet properly evaluated. E.g., a prolonged study of the PVB19 viral load has been performed in 53 patients after allo-HSCT using quantitative PCR [8]. Specific viral DNA was detectable in blood serum from 30% of the HSCT recipients, either before, or after HSCT, at maximal viral load observed 2 months post-transplant. However, the patients with detectable PVB19 did not show specific clinical symptoms that could be ascribed to parvovirus infection.
Hence, the aim of our work was to compare the PVB19 DNA levels prior to allogeneic HSCT, and at 1-2 months post-transplant, as well as search for correlations with specific antibody levels, and rates of hematopoietic recovery within 60 days after allo-HSCT. Our preliminary data point to a prognostic significance of parvovirus DNA detection and increased antibody levels is possible predictors for delayed engraftment and febrile neutropenia.

Patients and Methods

A total of 54 pediatric and adolescent patients were involved into the study at the median age of 7.2 (0.6 to 19 years old), who had a malignant disease of hematopoiesis or  inherited disorders as initial diagnosis who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fifty-one patient of this group were observed for at least 2 months (60 days) after HSCT. Selection of the patients for allo-HSCT, choice of conditioning regimens, prophylaxis of acute graftversus-host disease was performed according to current EBMT recommendations. Most part of this group was represented by the patients with acute myeloblastic leukemia (AML, n=16; 30%), acute lymphoblastic leukemia (ALL, n=14; 26%); severe anemias (SAA) of different origin (13%; n=7). 33% of the patients were in first remission after previous treatment. Socio-demographic features their distribution for diagnosis and stage of the disease, main parameters of allo-HSCT, are shown in Tables 1 and 2.
Bone marrow was used as a source of hematopoietic stem cells in 83% of cases (45 of 54 patients), infusion of peripheral stem cells was applied in the rest of cases. Allo-HSCT from unrelated donors was performed in 45% of cases (24 of 54); grafting from matched related donors or haploidentical transplant was carried out in, resp., 20% (11/54) and 35%(19/54) patients. Reduced-intensity conditioning was used in nearly all cases (51 of 54 patients). Development of acute GvHD within early period after allo-HSCT was observed in 25 patients, of them, 9 exhibited grade 3-4 GvHD. Regular examination of the patients before and after HSCT was carried out according to a standard local clinical protocol. He study was approved by the Local Institutional Board at the St.Petersburg State I. Pavlov Medical University. The laboratory studies included routine blood counts and serum biochemistry, urinalysis etc. Quantitative determination of the PVB19 DNA as well as herpesviruses (CMV, EBV, HSV) and polyomaviruses (BK, JC) in blood plasma was performed before conditioning therapy which preceded allo-HSCT, as well as on day +30 (D+30) and day+60 (D+60) post-transplant. DNA extraction from the samples and quantitative evaluation of PVB19 DNA in the samples were performed by means of PCR with specific fluorescent probes using “Ampliprime” amplification kits» and «Amplisens® Parvovirus В19-FL» (Moscow, Russia). Moreover, quantitative determination of IgМ and IgG antibodies to PV B19 was performed by means of ELISA at 0, +30 and +60 days post-transplant using «Anti-Parvovirus B19 ELISA IgМ» and «Anti-Parvovirus B19 ELISA IgG» kits (EUROIMMUN, Germany). The diagnostic kits were used according to instruction. Statistical evaluation of the data was performed with a Winstat software package.

Results

Transplant-related changes of PVB19 DNA levels and anti-PVB19 antibodies

The pre-transplant contents of PVB19 DNA and IgG antibodies to the virus showed a broad-range scatter (Table 3). PVB19 was found in about 30% of this group. Meanwhile, 68% of the patients exhibited increased levels of IgG-anti- PVB19 antibodies (>10 IU/ml), thus reflecting high prevalence of adaptive immune response. Mean pre-transplant contents of PVB19 DNA did not correlate either with age of the patients, or with clinical disease status, physical state, or activation of other latent viruses.
The detectability and average levels of PVB19 DNA as well as concentrations of anti-PVB19 antibodies did not show any significant changes at 30 or 60 days after HSCT, as seen from Table 1. However, actual scatter of these data proved to be rather sufficient, thus suggesting some correlations between these laboratory indexes and clinical signs in the individual patients. In particular, positive (non-zero) viral loads have been registered in 28% before allo-HSCT, 29% and 30.4% on day+30 and day+60 after allo-HSCT, i.e., ca 70% of the patients showed negative results for PVB19 over the early period after allo-HSCT.

Association between the PVB19 presence and specific antibody response after allogeneic HSCT

Detection of PVB19 DNA, both before and after allo-HSCT was not accompanied by IgM antibody detection at any observation point, thus suggesting absence of acute infectious process caused by parvovirus infection.
Meanwhile, a significant positive correlation was revealed between the overall PVB19 viral load and serum levels of IgG antiviral antibodies (r=0.351; p=8x10-6, 153 assays in 54 clinical cases). In particular, a significant correlation was shown between initial viral load and anti-PVB19 levels at all three terms of the study (Table 3) being, however, maximal at the day+60 after allo-HSCT.

As seen from Fig. 1, a significant correlation exists between pre-transplant PVB19 load and expressed antibody response detected 60 days after allo-HSCT, i.e., the non-zero viral loads were associated with higher contents of specific antibodies, thus suggesting an association between the PVB19 persistence and production of virus-specific antibodies (Fig. 1).
A half-life time for endogenous IgG antibodies in humans is about 1 to 4 weeks [10]. These findings suggest an opportunity of specific antibody production at early terms after intensive cytostatic treatment, due to potential activity of surviving memory cells, e.g., plasmocytes which are able to function for months and even years after their maturation.

Parvovirus В19 activation and hematopoiesis recovery

In our clinical series, altered engraftment was, generally, more common at increased IgG PVB19 antibody levels when determined 60 days after HSCT (r=0.315; p=0.034; n=46).
Specifically, significant correlations were shown between initial parvovirus DNA detection, and delayed reconstitution of erythrocytes and platelets in peripheral blood (respectively, r=-0,281; p=0.02; r=-0,303, p=0.01). Moreover, a marked correlation was shown by the day +60 between decreased neutrophils and platelet counts, and increased anti-PVD19 antibody levels (Fig. 2-4). This association may suggest a relation between continued parvovirus persistence and slower hematopoiesis recovery at 30 to 60 days post-transplant.
In general, altered engraftment was also registered in cases with higher IgG antibodies against parvovirus 60 days after HSCT (r=0.315; p=0.034; n=46).

Parvovirus detection and febrile neutropenia

Positivity for PVB19 DNA by the day +30 after allo-HSCT was, in all cases (14/14), associated with febrile neutropenia (FN), as compared to 68% in patients with nondetectable PVB19 (23/34; р=0.015, see Table 4). Hence, active parvovirus infection could be a sufficient factor of common febrile reactions observed in early posttransplant neutropenia, thus supporting pathogenic significance of this infection, at least, in a subgroup of immunocompromised patiens. Development of these febrile responses could be either virus-induced, or combined with secondary bacterial infections caused by slow recovery of cellular immunity.

Discussion

The issue of optimal PVB19 diagnostics in heavily treated hematological patients is not yet clear, since the results of serological tests (serum IgM and IgG antibodies) did not correlate with detection of viral DNA in blood serum or bone marrow from the HSCT recipients [9]. In our study, a pilot group of the HSCT patients was studied for PVB19 DNA and specific serum antibodies before and at 30-60 days following transplantation. Generally, PVB19 was found at, generally, in about 30% of this group. Remarkably, these figures were at rather low levels, both pre-transplant and after HSCT and did not sufficiently increase post-translant. Mean initial concentrations of PVB19 DNA did not correlate either with age of the patients, or with clinical disease status, physical state, or activation of other latent viruses. IgM antibodies to PVB19 were not detectable in HSCT patients, thus suggesting absence of de novo infection. However, class IgG antibodies(>10 IU/ml) were, found in ca 70% of cases thus presuming previous contacts with the virus. Hence, the background levels of parvovirus exist both pre- and posttransplant.
A search for associations between PVB19 positivity and specific antibody production have shown a significant correlation between pre-transplant PVB19 load and expressed IgG antibody response detected 60 days after allo-HSCT, i.e., the non-zero viral loads were associated with higher contents of specific antibodies, thus suggesting an association between the PVB19 persistence and production of virus-specific antibodies.

Conclusions

1. Presence of parvovirus B19 in peripheral blood of children before allogeneic hematopoietic stem cell transplantation is followed by increased PV-specific antibodies of IgG class in blood serum at all terms after allo-HSCT.
2. Increased IgG levels of antibodies in blood of the patients after allo-HSCT is associated with relative neutropenia and thrombocytopenia at first 2 months after allo-HSCT.
3. Detection of parvovirus DNA at initial terms (before HSCT) and and 30-60 days later may be followed by development of early febrile neutropenias and slower recovery oferythrocyte and platelet counts in peripheral blood.

Conflict of interest

The authors report no conflicts of interest.

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