Human Parvoviruses B19, PARV4 and Bocavirus in Pediatric Patients with Allogeneic Hematopoietic SCT

ORIGINAL ARTICLE Human parvoviruses B19, PARV4 and bocavirus in pediatric patients with allogeneic hematopoietic SCT

J Rahiala1,2, M Koskenvuo1,3, P Norja4, M Meriluoto4, M Toppinen4, A Lahtinen4,EVa¨isa¨nen4, M Waris5, T Vuorinen5, U Saarinen-Pihkala1, M Lappalainen6, T Allander7, O Ruuskanen3, K Hedman4,6,MSo¨ derlund-Venermo4 and K Vettenranta8

Among the immunocompetent, infections with parvovirus B19 (B19V) and human bocavirus (HBoV) 1 range clinically from asymptomatic to severe, while following allogeneic hematopoietic SCT (HSCT) B19V can cause a persistent severe illness. The epidemiology and clinical impact of HBoV1 and the other emerging parvovirus 4 (PARV4) among immunocompromised patients have not been established. To determine the occurrence and clinical spectrum of B19V, PARV4 and HBoV1 infections, we performed a longitudinal molecular surveillance among 53 allogeneic HSCT recipients for pre- and post-HSCT DNAemias of these parvoviruses. Quantitative real-time PCR showed B19V DNA in sera of 16 (30%) patients, at mean levels of 4.6 Â 103, 9.9 Â 107, 1.1 Â 1010 and 1.6 Â 102 B19V DNA copies/mL pre-HSCT (9/53), and at 1 (6/53), 2 (4/53) and 3 months (1/25) post HSCT, respectively. However, no clinical manifestation correlated with the presence of B19V viremia. All B19V sequences were of genotype 1. None of the sera investigated contained PARV4 or HBoV1 DNAs. Our data demonstrate B19V viremia to be frequent among pediatric allogeneic HSCT recipients, yet without apparent clinical correlates. PARV4 or HBoV1 viremias were not seen in these immunocompromised patients.

Bone Marrow Transplantation (2013) 48, 1308–1312; doi:10.1038/bmt.2013.63; published online 20 May 2013 Keywords: parvovirus B19V; parvovirus 4; bocavirus; childhood; hematopoietic SCT

INTRODUCTION impact also in non-transplanted patients with hematologic Infections not only by opportunistic pathogens but also by malignancy.15 Although specific antiviral therapy is not available, community-acquired respiratory viruses can cause severe illness in i.v. Ig results in clinical improvement in most cases. However, immunocompromised hosts following allogeneic hematopoietic relapses occur in up to 25% of immunocompromised patients.1 SCT (HSCT). Virus detection is of key importance in early diagnosis HBoV1 is increasingly recognized as a cause of respiratory among these patients.1–3 infection worldwide; it has been estimated to be among the four Only a few years ago parvovirus B19 (B19V) was the only known most prevalent viruses along with respiratory syncytial, rhino- and member of Parvoviridae causing illness in humans.4 Since then the adenoviruses in children hospitalized for respiratory disease.16–21 emerging parvoviruses, parvovirus 4 (PARV4) and human HBoV1 genomes have also been observed in stool among 0–13% bocavirus 1 (HBoV1), have also been shown to infect humans.5,6 of patients with or without gastroenteritis22 and in 0–44% of The epidemiology and clinical impact of HBoV1 and the other respiratory samples from individuals with non-infectious ill- emerging PARV4 among immunocompromised patients have not nesses.23,24 Serodiagnosis or HBoV1 DNA detection in serum been fully established. therefore provides more reliable diagnosis of acute HBoV1 Three genotypes of B19V exist, 1–3, of which genotype 1 is the infection.21,25–28 The role of HBoV1 among the immuno- most prevalent.7,8 Approximately half of 15-year-old adolescents suppressed is poorly understood. Tozer et al.29 observed that and up to 80% of adult blood donors are B19V IgG seropositive.9 It among 229 whole blood samples collected over 18 months from causes a self-limiting aplasia of erythroid progenitor cells, followed immunocompromised children (n ¼ 31) 2.6% were HBoV1 DNA by erythema infectiosum (fifth disease) and/or arthralgia. positive. One patient, however, accounted for three (50%) of the Allogeneic HSCT patients are at a particular risk for B19V findings. HBoV1 infections have also been reported among pediatric infection, which may lead to severe, persistent and usually leukemia and immunocompromised patients.30–32 One disclosed a nonspecific illness.10–13 A few studies address the incidence and child with disseminated bocavirus infection shortly after allogeneic clinical spectrum of B19V infections among HSCT patients; HSCT.33 The clinical manifestations were fever, rhinorrhea, cough, however, large prospective studies among the pediatric diarrhea and hypoxia, and the virus occurred in high quantities in population are lacking. B19V infections can be severe after plasma, nasopharyngeal aspirates and stool. Fecal shedding was allogeneic HSCT and in addition to aplastic crises14 cause present for several weeks after clinical resolution. No data are pneumonia10 and multiorgan failure.11 B19V can have a clinical available on antiviral therapy of HBoV1 infection.

1Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Children’s Hospital, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland; 2Department of Pediatrics, Porvoo Hospital, Porvoo, Finland; 3Department of Pediatrics, University of Turku, Turku, Finland; 4Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland; 5Department of Virology, University of Turku, Turku, Finland; 6Department of Virology and Immunology, Helsinki University Central Hospital Laboratory Division (HUSLAB), Helsinki, Finland; 7Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden and 8Division of Hematology- Oncology, Department of Pediatrics, University of Tampere, Tampere, Finland. Correspondence: Dr J Rahiala, Department of Pediatrics, Porvoo Hospital, Sairaalantie 1, PO Box 500, Porvoo FIN-06151, Finland. E-mail: jaana.rahiala@hus.fi Received 23 November 2012; revised 28 March 2013; accepted 3 April 2013; published online 20 May 2013 Pediatric patients with HSCT J Rahiala et al 1309 PARV4 is a human parvovirus first detected in the plasma of an PCR procedures 6 individual with an acute HIV infection-like illness. Three Because of the high sensitivity of PCR, strict precautions, including the use genotypes of PARV4 have been identified, two of them in of separate rooms, aerosol-resistant tips and disposable racks, were Europe and one in Africa.6,34–36 The viral DNA has been shown to employed to avoid false-positive results.48 In each PCR run, water and persist in lymphoid and some other tissues.37–41 The virus is most plasmids containing the respective viral genomes served as negative and frequently detected in persons with a history of i.v. drug use or as positive controls, respectively. For all PCRs 5 mL of the sample DNA extract co-infections with other parenterally transmitted viruses, such as was applied per 20-mL reaction. 37,42–45 For an initial qualitative PCR for B19V genotypes 1–3, a 248-base-pair HIV or hepatitis C and B. Viremia seems to accompany fragment of the B19 VP1 gene was amplified and the results confirmed by acute PARV4 infection, and the viral DNA has been demonstrated Southern hybridization.49 To further solidify the results and to quantify the also in blood products, indicating a risk for parenteral DNA, all positive samples were re-examined by a commercial quantitative 34,39,42–43 transmission. The seroprevalence in Northern Europe PCR (qPCR) detecting all the three genotypes of B19V50 (Artus Parvo B19 reaches 78% among HIV-positive i.v. drug users, but is negligible LC PCR Kit; Qiagen). The criterion for a positive B19V PCR result was among non-users.42,44 In all, the epidemiology and clinical positivity by both the methods. 46,47 For HBoV1 a real-time quantitative PCR assay targeting the NP1 gene implications of PARV4 are still under evaluation. 20,51 The aim of this study was to determine the occurrence and of HBoV1 was applied as described with a Stratagene Mx3005P clinical associations of B19V, PARV4 and HBoV1 viremias among instrument (Agilent Technologies, Santa Clara, CA, USA). For standardization of quantification, a plasmid pST2 (GenBank accession pediatric patients immunocompromised due to allogeneic HSCT. DQ000496) containing the HBoV1 NP1 gene was used in serial dilutions covering a range of 7 logs. For PARV4-DNA detection a multiplex quantitative PCR was applied, MATERIALS AND METHODS which identifies and quantifies all three PARV4 genotypes currently Patients known.52 This quantitative PCR has been thoroughly evaluated with both The retrospective study included all 53 pediatric (of 195) patients (Table 1) plasmids and patient samples containing the three genotypes. The with a hematologic malignancy, who underwent allogeneic HSCT and had analytical sensitivity, determined with PARV4 plasmids serially diluted in serum samples collected at least pre-, 1 and 2 months post HSCT at the purified human DNA or buffer alone, was 20 copies per reaction for each of Division of Hematology-Oncology and Stem Cell Transplantation, the three genotypes. Children’s Hospital, University of Helsinki, Finland, between 1997 and 2006. The time from the primary diagnosis to HSCT ranged from 0.3 to 6.5 Statistical analysis years, with a mean of 1.7 years. The patient files were studied and Data were analyzed by using Statistical Package for Social Science (SPSS demographics and details of infectious symptoms reviewed. version 19). The Mann–Whitney U-test was used to compare the nonparametric data. The Fisher’s exact and w2 tests were used to compare Samples the frequency of qualitative variables. P-values o0.05 were considered The study material consisted of a total of 184 sera collected from the 53 statistically significant. patients and stored at À 70 1C until analysis. The time points of interest were pre-HSCT and 1, 2 and 3 months post HSCT until discharge or death. Ethical considerations The study was approved by the Ethics Committee of the Medical Faculty of Extraction of viral DNA the Turku University. Also, the Health Care Supervision Centre granted their permission for the analysis. DNA was extracted from 100 mL of serum by using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RESULTS The sera were collected from the 53 pediatric patients receiving Table 1. Characteristics of the study population allogeneic HSCT within a 10-year period (years 1997–2006). The pre-transplant sample and samples taken at 1 and 2 months post No. of patients 53 HSCT were collected from all the patients, while only 25 samples Type of malignancy ALL 37 taken at 3 months were available. The mean point of discharge AML 10 was 78 days (median 64, range 30–188) post HSCT. Among the CML 4 deceased, the time from HSCT to death (n ¼ 23, 43%), ranged from JMML 1 0.2 to 5.6 years (mean 1.4). Two patients died within the first MDS 1 3 months. Sex (male/female) 31/22 One or more serum samples were B19V PCR positive in 30% (16/ 53) of the patients (Table 2), all of genotype 1. In four patients two Age (years) at diagnosis, median (range) 6.0 (0.2–15.9) samples were positive, once with negative sample in between. Altogether 11% (20/184) serum samples were B19V PCR positive; Age (years) at HCST, median (range) 8.2 (0.9–19.0) at pre-HSCT 17% (9/53), and at one 11% (6/53), two 8% (4/53), and Donor three 4% (1/25) months post HSCT. The viral loads ranged from 1 8 3 MUD 36 2.9 Â 10 to 1.9 Â 10 /mL (mean 4.6 Â 10 /mL) at pre-HSCT, Matched family donor 10 6.3 Â 101–5.9 Â 108/mL (mean 9.9 Â 107/mL) at 1, 9.0 Â 101– Cord blood 4 4.4 Â 1010/mL (mean 1.1 Â 1010/mL) at 2 and 1.6 Â 102/mL at Haploidentical 3 3 months post HSCT, respectively. During 1997–2001 an HSCT was performed on 28 patients in Virological samples the study group, of whom 46% (13/28) had at least one B19V- Pre-HSCT 53 positive serum sample, but after 2001 only 12% (3/25) of samples 1 month post HSCT 53 were B19V positive (odds ratio 6.4, P ¼ 0.008; Fisher’s exact test). In 2 months post HSCT 53 4 3 months post HSCT 25 total, only three patients had B19V DNA levels exceeding 10 /mL and one exceeding 1010/mL; these patients were clinically Abbreviations: HSCT ¼ hematopoietic SCT; JMML ¼ juvenile monomyelo- indistinguishable. cytic leukemia; MDS ¼ myelodysplastic syndrome; MUD ¼ matched unre- In all, no clinical manifestation showed a significant correlation lated donor. with the presence of B19V viremia (Table 2). In particular, the

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 1308 – 1312 Pediatric patients with HSCT J Rahiala et al 1310 the recipients remained B19V PCR negative for 6–12 months Table 2. Medical records of B19V PCR-positive or -negative patients post HSCT (allogeneic),55,56 our genoprevalence rate appears markedly higher. Medical records B19V-PCR B19V-PCR P-value 54 positivea (n ¼ 16) negative (n ¼ 37) Soliman et al. found B19V PCR-positive pediatric oncology patients to have a significantly higher frequency of unexplained Diagnosis ALL 62.5% ALL 70.3% NS anemia and red cell transfusions, and a longer hospital stay than (n ¼ 10) (n ¼ 26) PCR-negative patients. Lindblom et al.15 found children with ALL AML 25% (n ¼ 4) AML 16.2% NS and B19V viremia to develop cytopenia, leading to reduced (n ¼ 6) CML 12.5% CML 5.4% (n ¼ 2) NS treatment intensity and complicated course. However, in that (n ¼ 2) retrospective study the infection was clinically apparent in only Others 0% (n ¼ 0) Others 8.1% NS one patient out of 18. A recent review identified 98 solid organ and (n ¼ 3) hematopoietic transplanted patients (24 with an allogeneic HSCT) with symptomatic B19V infection after transplantation. The authors Age (years), median 7.7 (4.2–14.8) 8.3 (0.9–19.0) NS concluded the predominant manifestation to be refractory anemia, (range) a complication occurring commonly after transplantation along 57 Year of HSCT 0.008b with other cytopenias. It has been reported that B19V DNA may 2001 or before (n ¼ 28) 81% (13/16) 41% (15/37) be detectable by PCR for extended periods of up to years in bone 2002 or later (n ¼ 25) 19% (3/16) 59% (22/37) marrow and even serum.58 Donor-derived nosocomial infection has also been described (blood products; stem cells).1,59 The Respiratory symptoms 40% (6/15) 50% (16/32) NS infection has been seen in association with other post-transplant Gastro-intestinal 87% (13/15) 75% (27/36) NS complications, such as allograft dysfunction, hepatitis, myocarditis symptoms and glomerulonephritis, and post-transplant mortality directly Exanthema 93% (13/14) 91% (32/35) NS 10,57 Fever 93% (13/14) 97% (30/31) NS attributable to B19V has been estimated at 3%. B19V exanthema can also mimic cutaneous acute GVHD (aGVHD).12,60 Anemia (Hb o100) In our study none of the patients with B19V viremia had any Duration (mean, days) 24.8 25.2 NS specific signs or suspicion of B19V infection. Neither Need for red blood retrospectively did any clinical manifestation (including anemia) cells correlate significantly with the B19V viremia. However, many 43 months post HSCT 33% (5/15) 47% (17/36) NS symptoms common after HSCT (fever, exanthema and anemia) (%) resemble the symptoms of B19V infection, but as surveillance is Reticulocytesp0.5% not routine, the frequency of B19V infections might have been 41 month post HSCT 27% (3/11) 48% (16/33) NS underestimated. Thrombocytopenia B19V transmission occurs most commonly via aerosols or (o50) respiratory secretions, but virus-containing blood products can Duration (mean, days) 40.5 34.9 NS also be a source of transmission.61 In Finland, at the Red Cross Neutropenia 21.8 20.2 NS Blood Service all blood donations have been screened by PCR for ( 1.0 Â 109/L), duration B19V since April 2002. As cellular products with DNA levels below o 4 (mean, days) 10 IU/mL are rarely infectious, the European Pharmacopoeia stipulates that the B19V concentration in pooled blood should not GVHD 88% (14/16) 86% (32/37) NS exceed 104 IU/mL. However, it has been recommended to screen Grade 1, n (%) 3 (21%) 9 (28%) plasmas by minipool testing, to supply immunosuppressed Grade 2, n (%) 2 (14%) 5 (16%) 62 Grade 3, n (%) 8 (57%) 16 (50%) patients with B19V-negative blood components. Grade 4, n (%) 1 (7%) 2 (6%) In our study B19V viremia occurred mostly (81%, 13/16) among patients transplanted prior to 2002, that is, before blood product Corticosteroids 57% (8/14) 62% (21/34) NS screening. Yet, were B19V in this context transmitted via blood Mortality 25% (4/16) 51% (19/37) NS components, it should have been present also in the Finnish adult 55 Abbreviation: HSCT ¼ hematopoietic SCT. aB19PCR (max/patient) viral load allogeneic HSCT study. The clinical importance of transfusion- 9 2.8 Â 109 (mean), 1.2 Â 103 (median), 2.9 Â 101–4.4 Â 1010 (range). bStatis- transmitted infections is still controversial and reports scarce. tically significant difference in B19V PCR-positive patients when compared Yet, B19V infections among the immunocompromised might be HSCT performed before the year 2002 or later (odds ratio 6.4, P ¼ 0.008; underdiagnosed because of mildness or habituality of symptoms. Fisher’s exact test). Our results point to the importance of blood product screening for B19V DNA; after the onset of screening only three B19V viremias ensued in this cohort. durations of neutropenia, thrombocytopenia or anemia during the Susceptibility to an infection may depend on the presence or first 100 days post HSCT did not differ between those with or absence of neutralizing antibodies. Immunosuppression decreases without B19V viremia. Also, the proportions of patients requiring the ability of the transplant patients to produce neutralizing red cells beyond the first 100 days post HSCT were similar. antibodies.63 In a review by Eid et al.57 many of the transplant No PARV4 or HBoV1 DNA was detected in the serum of any of patients did not possess specific IgM at the onset of clinical the cases pre- or post-transplant. disease, while almost all had B19V viremia. PARV4 or HBoV1 viremias were not demonstrable in this group of immunocompromised patients. The absence of PARV4 in these DISCUSSION immunocompromised patients is in agreement with the recent The key finding in our study was that among pediatric patients study by Tolfvenstam et al. and with the previous studies with hematologic malignancy and an allogeneic HSCT, B19V reporting PARV4 appearing to be primarily blood-borne in viremia is frequently detectable. The prevalence of B19V PCR Northern Europe, but could possibly also reflect a lower positivity among our children was 30%. This is in agreement prevalence in Northern Europe.38,41–44,47 It has been found with the occurrences (22–27%) reported for children with among infants in West Africa with no evidence of parenteral malignancies.53,54 However, compared to two reports in which exposure.35,36

Bone Marrow Transplantation (2013) 1308 – 1312 & 2013 Macmillan Publishers Limited Pediatric patients with HSCT J Rahiala et al 1311 Children and infants are ubiquitously infected by HBoV1, 12 Muetherig A, Christopeit M, Muller LP, Grothe W, Weber T, Theurich S et al. Human leading to respiratory illnesses. The incidence of serologically parvovirus B19 infection with GvHD-like erythema in two allogeneic stem cell verified acute HBoV1 infections has been shown to be highest transplant recipients. Bone Marrow Transplant 2007; 39: 315–316. during the second year of life, and short-term viremia has also 13 Broliden K. Parvovirus B19 infection in pediatric solid-organ and bone marrow been found to be an excellent marker of an acute HBoV1 transplantation. Pediatr Transplant 2001; 5: 320–330. infection.21,27,28,64 Studies of nasopharyngeal samples have shown 14 Broliden K, Tolfvenstam T, Norbeck O. Clinical aspects of parvovirus B19 infection. persistence of HBoV1 DNA for several months.65–68 HBoV1 J Intern Med 2006; 260: 285–304. seroprevalence ranges from 25% in infants younger than 1 year 15 Lindblom A, Heyman M, Gustafsson I, Norbeck O, Kaldensjo¨ T, Vernby Å et al. (ref. 28) (ref. 27) Parvovirus B19 infection in children with acute lymphoblastic leukemia is asso- to 93% and 100% in children of 3 to 7 years of age, ciated with cytopenia resulting in prolonged interruptions of chemotherapy. Clin respectively. The causative role of HBoV1 in clinical manifestations Infect Dis 2008; 46: 528–536. 64 other than respiratory diseases remains to be assessed. Accurate 16 Calvo C, Garcia-Garcia ML, Pozo F, Carvajal O, Perez-Brena P, Casas I. Clinical diagnosis requires antibody analysis or PCR of serum; PCR of characteristics of human bocavirus infections compared with other respiratory nasopharyngeal samples alone is insufficient.27 In our study all viruses in Spanish children. Pediatr Infect Dis J 2008; 27: 677–680. serum samples investigated were HBoV1 PCR negative. With a 17 Pozo F, Garcıá-Garcıá ML, Calvo C, Cuesta I, Pe´rez-Brenã P, Casas I. High incidence median age of 6 years, most of our patients had probably been of human bocavirus infection in children in Spain. J Clin Virol 2007; 40: 224–228. infected by HBoV1 previously. Other reasons for PCR negativity 18 Franz A, Adams O, Willems R, Bonzel L, Neuhausen N, Schweizer-Krantz S et al. may be the short duration of HBoV1 viremia, as well as the fact Correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection. J Clin Virol that these children may not have had respiratory infections due to 2010; 48: 239–245. their separation from other children (especially day-care) during 19 Christensen A, Nordbø SA, Krokstad S, Rognlien AG, Døllner H. 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