Persistent Symptomless Human Metapneumovirus Infection in Hematopoietic Stem Cell Transplant Recipients

Viruses associated with acute respiratory diseases (ARDs) are increasingly recognized as important pathogens in patients with hematological malignancies [1]. A new paramyxovirus, designated human metapneumovirus (hMPV), has been observed to be associated with respiratory diseases [2] and to have worldwide distribution. hMPV infects children and adults and accounts for ∼5%–10% of respiratory infections in which previously characterized viruses, including human respiratory syncytial virus (HRSV), orthomyxoviruses, parainfluenza viruses, adenoviruses, and picornaviruses, could not be detected

Because the diseases associated with hMPV and HRSV have similar clinical features, ranging from mild respiratory symptoms to severe bronchiolitis and pneumonia [3], it could be hypothesized that hMPV may generate morbidity in hematopoietic stem cell transplantation (HSCT) recipients, especially in the cases in which other respiratory pathogens cannot be identified. To address this question, a prospective study in a group of allogeneic HSCT recipients was planned, together with a parallel control study on the role that hMPV plays in the etiology of pediatric ARDs during the same period. All viral strains were genotyped by use of phylogenetic analysis to evaluate the molecular epidemiological aspects of hMPV infection in the same geographical region as well as in the HSCT recipients’ setting

Patients and methodsThe study was approved by the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo Ethical Committee, and nasopharyngeal aspirate (NPA) samples were collected after informed consent was obtained from patients. HSCT recipients were recruited at the Division of Hematology, IRCCS Policlinico San Matteo, from 1 October 2004 through 30 October 2005. All adult patients with hematological malignancies undergoing allogeneic HSCT were enrolled. Hematological malignances in HSCT recipients included in the study were as follows: acute myeloid leukemia (12 patients), acute lymphoblastic leukemia (5 patients), myelodysplastic syndrome (2 patients), non-Hodgkin lymphoma (1 patient), and multiple myeloma (1 patient). The median age of patients was 40 years (range, 25–58 years); 11 patients received grafts from siblings, and 10 received grafts from matched unrelated donor (MUDs); 15 were conditioned by a standard myeloablative regimen, and 6 received a reduced-intensity conditioning regimen. All patients were treated with cyclosporine A and methotrexate as acute graft-versus-host-disease prophylaxis; 7 patients who received grafts from MUDs were treated with an additional dose of antithymocite globulin to effect in vivo T cell depletion. No patient showed signs of ARD before transplantation

Samples from pediatric patients with ARDs were collected from 1 October 2004 through 30 September 2005 from the microbiology laboratory of 2 hospitals in northern Italy, Azienda Ospedaliera in Melegnano and Ospedali Riuniti in Bergamo. Clinical specimens were obtained from children aged <5 years who were hospitalized for an ARD (mostly bronchiolitis and bronchopneumonitis). In all cases, NPAs were collected as part of the investigation of their illness, because collecting such samples is standard practice to assess the presence of HRSV

In HSCT recipients, NPA samples were collected at 8 different time points (before admission to the hospital, before HSCT and during the conditioning regimen, and 15, 30, 60, 90, and 180 days after HSCT). Clinical data were recorded and examined for type of HSCT, presence of fever, and signs or symptoms of respiratory-tract infection

Detection of hMPV in NPA samples was performed by use of reverse transcription (RT)–polymerase chain reaction (PCR) and isolation on cell culture. All samples were extracted by use of a Qiagen RNA minikit (Qiagen), in accordance with the manufacturer’s protocol. RNA was reverse transcribed by use of random hexamers and amplified by use of a primer mix including 1 forward and 2 reverse primers. To amplify 150-bp regions of the N genes of all known or possible hMPV subtypes, primers were designed on the basis of GenBank-published virus sequences. The forward primer (nucleotide position 934–962) was MPV001DtmF (GAA ATG GGC CCT GAA TCT GGR CTT CTA CA); the reverse primers (nucleotide position 1052–1084) were hMPV001DtmR (TTG GYA CTC TCC CTC GAT ACA TAC CGA TTA TGC) and hMPV9875tmR (TTG GTA CTC TTC CTC TGT ACA TTC CGA TTA TAC). The RT-PCR assay was optimized to detect at least 10 copies of both strains by cloning amplified products from reference strains into PCR2.1 plasmid vector (TA Cloning Kit; Invitrogen), serially diluted from 10⁶ copies to 1 copy. To reduce the impact of known or possible base mismatches with virus variants, annealing temperature was decreased to 53°C, allowing hybridization of primers even in the presence of minimal sequence variations. For all samples, assay specificity was demonstrated by sequencing

The amplified 150-bp PCR products were separated by use of agarose gel electrophoresis, visualized by use of ethidium bromide staining, and sequenced directly by use of BigDye Terminator v3.1 and an ABI 3100 sequencer (both manufactured by Applied Biosystems). Alignments of sequences were generated using ClustalW (available at: http://www.ebi.ac.uk/clustalw), and manual corrections were made by use of BioEdit (version 5.0.6; available at: http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic relationships were estimated using MEGA software (version 3.1; available at: http://www.megasoftware.net) (neighbor-joining method by using the Tamura-Nei model as estimated by using Modeltest [available at: http://darwin.uvigo.es/software/modeltest.html]; the α value used in MEGA was previously estimated directly from the data by using HYPHY [available at: http://www.hyphy.org])

Finally, the samples were stored at −80°C, and those that tested positive for hMPV RNA were inoculated onto monolayers of rhesus monkey kidney (LLC-MK2) cells for hMPV isolation. Virus isolation in vitro was obtained by use of a combination of conventional virus-isolation procedures and molecular techniques, as described elsewhere [4], with some modifications. In brief, cell-culture monolayers were incubated in serum-free Eagle MEM, supplemented with 2.0 μg/mL trypsin, and examined daily for cytopathic effect (CPE). After ∼14 days of incubation, cell-culture supernatants were tested by RT-PCR to assess the presence of hMPV RNA sequences. Regardless of CPE, RT-PCR–positive cell cultures were washed twice with PBS, trypsinized, and subjected to subcultivation after mixing cells with an equal number of uninfected LLC-MK2 cells. Subcultures were observed for 3 weeks for the development of CPE

ResultsDuring the study period, a total of 107 NPA samples from 21 HSCT recipients and 244 NPA samples from 244 pediatric patients with ARDs were examined. For the HSCT recipients, 31 samples were collected before HSCT (15 before admission to the hospital and 16 during the conditioning regimen), 40 were collected within 30 days after HSCT, and 36 were collected 60–180 days after HSCT

The presence of hMPV RNA sequences was found in 53 (49.5%) of the 107 NPA samples from 18 (85.7%) of the 21 patients examined. Of the 53 hMPV-positive samples, 6 had been collected before admission of the patient to the hospital, 12 during the conditioning regimen, 21 within 30 days after HSCT, and 14 within 60–90 days after HSCT. hMPV RNA was persistently detected in NPA from HSCT recipients for 26–94 days during every period of the year, without differences in seasonal distribution. Of 18 positive patients, 3 had a first positive sample during October to December, 2 during January to March, and 13 during April to September, without significant differences in infection persistence (figure 1)

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Figure 1

Human metapneumovirus (hMPV) infections in pediatric patients with acute respiratory diseases (ARDs) from October 2004 to September 2005. hMPV-positive pediatric patients with ARDs (white columns) and total no. of tested pediatric patients with ARDs (black columns) per month are shown. Each horizontal bar represents the time course of detection of samples positive for hMPV in hematopoietic stem cell transplant (HSCT) recipients. In pediatric patients with ARDs, 20 (54%) of 37 hMPV-positive specimens were collected from January to March 2005. During this period, 20 (19.6%) of 102 respiratory specimens tested positive for hMPV RNA sequences; in the remaining quarters, positive samples were 8.7% (8/91), 15% (6/40), and 27% (3/11) from October 2004 to December 2004, April 2005 to June 2005, and July 2005 to September 2005, respectively

In pediatric patients with ARDs, hMPV RNA sequences were detected in 37 (15.1%) of the 244 NPA samples. The median age of affected children was 4 months (range, 0.5–60 months). Most positive samples were recovered during the winter months, with the peak of virus detection in January and February (figure 1). Bronchiolitis was diagnosed in 28 (76%), bronchopneumonia in 3 (8%), bronchitis in 2 (5%), and rhinitis in 1 (3%) of the hMPV-positive children. The clinical symptoms observed for the remaining 3 children included cough and fever

Viral sequence analysis documented infection by hMPV genotype A in all the specimens from HSCT recipients. On the other hand, of the specimens from pediatric patients with ARDs, 31 (84%) tested positive for genotype A and 6 tested positive for genotype B (16%) (figure 2). Of the 6 genotype B viral sequences, 5 were recovered from the end of March to April, and 1 was recovered in January. All sequences from HSCT recipients were identical. However, some minor differences were present in both the genotype B and genotype A strains amplified from samples from pediatric patients with ARDs, although 84% of sequences (26 of 31) in genotype A were identical

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Figure 2

Neighbor-joining tree (Tamura-Nei method, with Γ distribution) showing phylogenetic relationships between hMPV sequences from pediatric patients with ARDs and those from HSCT recipients. Two clusters are evident: sequences from the pediatric patients with ARDs are present in either genotype A or genotype B, whereas sequences from the HSCT recipients are identical for the region analyzed and all are in genotype A. The consensus sequence from the HSTC recipents (HSR-EMA) (▵) is shown in the tree, together with the sequences obtained from the pediatric patients with ARDs (▴) and reference sequences (○). GenBank accession nos. of reference sequences used for strain genotyping are as follows: AF371337 (NED001), AY355323 (HNED0130), AY145286 (HCAN9981), AY256874 (HQ01726), AF443830 (HQ01680), AY256873 (HQ01725), AY297749 (HCAN9783), AY145272 (HCAN0012), AY530090 (JPS03176), AY355327 (HNED0123), AY145284 (HCAN9878), AY297748 (HCAN9875), AY145279 (HCAN9873), AY355332 (HBIR0110), AY355333 (HFIN0101), AY530089 (HJPS0276), AY355331 (HNED0121), AY355329 (HNED0105), AY355330 (HNED0109), and AY145277 (HCAN9782). Accession nos. of sequences from pediatric patients with ARD and HSCT recipients are DQ450606–DQ450656

After infection of cell cultures, 7 of 10 samples from HSCT recipients tested for viral isolation showed CPE on LLC-MK2 cell subcultures after a minimum of 12 days of incubation. In these cases, the presence of replication-competent hMPV was documented in cell-culture supernatant by RT-PCR. In 2 patients, hMPV was isolated in 2 consecutive samples collected 56 and 12 days apart, respectively

No specific respiratory symptoms or signs were documented in HSCT recipients in parallel with detection of hMPV RNA in NPA samples. Mild upper-respiratory syndromes were observed in 9 patients at different time points during follow-up (rhinorrhea in 3 patients, dry cough in 6 patients). Three patients died within the third month after HSCT of causes, other than hMPV infection

DiscussionIn the study described here, we analyzed prospectively for hMPV infection a group of 21 adult patients with hematological malignancies undergoing allogeneic HSCT. Previous studies addressing symptomatic patients have documented hMPV infection in ∼9% of adults with hematological malignancies [5, 6] and in 3.4% of immunocompetent adults with respiratory illnesses [7]. hMPV has also been associated with severe pneumonia and fatal lower-respiratory-tract disease in HSCT recipients [8, 9]. The results of the present study document a very high incidence of hMPV infection, and, notably, the data show symptomless persistence of the virus in HSCT recipients. Further attention to this evidence is necessary, because it may have implications for the understanding of hMPV-host relationships and of the pathogenic potential of hMPV. First, the data indicate that hMPV may infect throughout the year, which may explain the high prevalence of infection in HSCT recipients. Second, prolonged detection of hMPV RNA in sequential NPA samples from HSCT recipients suggests that the virus persistently infects these patients with an impaired immunological response, which is probably unable to determine rapid virus clearance after a symptomatic or asymptomatic primary infection. Recently, hMPV persistence has been documented in lung transplant recipients [10]; this further underlines the potential role that immunosuppression may play in hMPV persistence. Third, hMPV infection was subclinical in almost all the HSCT cases evaluated. Although further data are clearly necessary to clarify this point, this is first evidence, to our knowledge, of asymptomatic hMPV infection in immunocompromised hosts, and it raises the question of the role that the host’s immune response plays in disease pathogenesis. Fourth, although it has recently been observed that significant hMPV activity can occur throughout every period of the year [11], the analysis performed in the present study indicates that hMPV infection in HSCT recipients has had a different seasonal distribution than that in pediatric patients with ARDs [12]

The viral region amplified and sequenced in the present study is highly conserved among known hMPV isolates; although this ensured sensitivity and specificity of hMPV RNA detection in clinical samples (thus perfectly addressing the main objective of the study—the analysis of the virus-host relationships in 2 groups of patients), sequencing of more-diverse hMPV regions could have better defined the epidemiological aspects of the HSCT cases. However, the differences in viral genome similarity between the 2 groups of patients (viral genomes were more similar in HSCT recipients than in pediatric patients with ARDs) and in seasonal distribution of hMPV-positive cases, together with a negative assay at time of admission to the hospital for the majority of HSTC recipients, suggest a hospital origin of the infection in a large proportion of adult patients with hematological malignancies

Overall, although limited in number of patients, this study documents for the first time, to our knowledge, a high rate of asymptomatic hMPV persistence in immunocompromised patients with hematological malignancies. Further analysis of a larger number of cases is necessary to clarify exactly the rates of asymptomatic/symptomatic hMPV infection in this subgroup of patients, as well as the role that antiviral immune response plays in hMPV clearance and in respiratory disease pathogenesis

• Received February 24, 2006.
• Accepted April 6, 2006.

• © 2006 by the Infectious Diseases Society of America