PROLONGED DETECTION OF HUMAN BOCAVIRUS DNA IN NASOPHARYNGEAL ASPIRATES OF CHILDREN WITH RESPIRATORY TRACT DISEASE

Abstract

Increasing evidence suggests that the recently identified human bocavirus (hBoV) is a cause of acute respiratory illness. However, the duration of hBoV shedding from the respiratory tract as demonstrated by positive hBoV polymerase chain reaction is unclear. We describe the virologic and clinical characteristics of 6 immunocompetent children with hBoV persistence in the respiratory tract for up to 4.5 months.

The human bocavirus (hBoV), a novel member of the Parvoviridae, was recently identified by molecular screening methods in respiratory tract samples of Swedish children.¹ HBoV is a virus of worldwide distribution and its DNA has been found by polymerase chain reaction in 1.5% to 19% of respiratory secretions from children with acute respiratory tract diseases.² Elucidation of the clinical relevance of hBoV has been difficult because of a high coinfection rate of hBoV DNA positive samples with other respiratory viruses.² Furthermore, there are no cell culture systems or animal models for hBoV. Based on the combination of a high hBoV load in nasopharyngeal samples (>10⁴ copies/mL) and concomitant hBoV DNA detection in sera, a model has been proposed in which hBoV is associated with acute respiratory tract diseases but persists in the respiratory tract for a longer period of time than other respiratory viruses after primary infection.³ Recently, serological evidence of primary hBoV infection has been demonstrated in children with acute respiratory tract diseases.^4,5 Although the combined evidence suggests that at least a proportion of positive hBoV DNA findings in respiratory tract samples is indicative of hBoV disease associated with primary infection, the clinical significance of low amounts of hBoV DNA in nasopharyngeal samples is unclear. In a child with acute lymphoblastic leukemia, hBoV DNA was detected over a period of 6 months during 5 consecutive febrile episodes.⁶ However, data on the duration of hBoV DNA shedding in immunocompetent children are largely unavailable.

To study this aspect, we identified children in whom hBoV DNA was repeatedly detected in nasopharyngeal aspirates (NPA) during successive episodes of acute respiratory tract diseases. Here, we describe the virologic and clinical characteristics of these patients. They were part of a cohort of children in whom detection of hBoV DNA by conventional and quantitative real-time polymerase chain reaction has been described previously.^7,8 The study was approved by the ethics committee of the medical faculty at the University of Würzburg.

CASE REPORTS

Case 1.

Case 1 describes a 1-year-old boy who was admitted to the hospital in February 2003 with high fever and signs of respiratory tract infection. Pneumonia was diagnosed by chest radiography. Antigen screening for respiratory viruses (influenza virus A/B, parainfluenza virus 1/2/3, respiratory syncytial virus [RSV], adenoviruses) by immunofluorescence assay was negative. Three weeks after discharge, he was rehospitalized with symptoms of high fever, cough, diarrhea, dehydration, and otitis media. Bronchiolitis was diagnosed and treated symptomatically. Influenza virus A antigen was identified in NPA. HBoV DNA was detected retrospectively in stored NPA obtained during both episodes of hospitalization (Table 1).

TABLE 1: Clinical and Diagnostic Findings of Patients With Prolonged Detection of hBoV DNA in Nasopharyngeal Aspirates

Case 2.

His twin brother (case 2) was hospitalized with clinical symptoms including fever, cough, tachypnea, and dehydration one week after the first admission of his sibling. Pneumonia was also diagnosed. Together with his twin brother, he was readmitted 2 weeks later with high fever, cough, rhinitis, and dehydration. Influenza virus A antigen was positive by immunofluorescence assay, and hBoV DNA was detected retrospectively in stored NPA from both episodes of hospitalization.

Case 3.

Case 3 describes a 2-month-old girl who was admitted with symptoms of cough, vomiting and fever. On the following day, she was transferred to the intensive care unit because of severe dyspnea, hypercapnia, and increasing oxygen demand. Her diagnosis was bronchiolitis. Oxygen was administered for 4 days. RSV antigen was detected in NPA. Five weeks later, she was again hospitalized because of bronchiolitis. Antigen screening for respiratory viruses was negative. Retrospective analysis of stored NPA obtained during the 2 episodes of hospitalization was positive for hBoV DNA.

Case 4.

Case 4 describes a 2-year-old girl with Sturge-Weber-syndrome, severe epilepsy, and recurrent respiratory tract infections. She was hospitalized in January, February, and March 2005 for repeated episodes of pneumonia of suspected viral origin. In February 2005, she developed severe respiratory failure and required mechanical ventilation for one week. Immunofluorescence screening for respiratory viruses was negative in January and March 2005, but positive for RSV antigen in February 2005. Five NPA samples collected during the 3 hospital stays were retrospectively tested for hBoV DNA. Two samples from January 2005 were negative, whereas hBoV DNA was detected in 3 samples in February and March.

Case 5.

Case 5 describes a 1.7-year-old boy who was hospitalized 7 times between October 2006 and May 2007 because of respiratory tract infections. Immunofluorescence screening for respiratory viruses was only positive for influenza A antigen in February 2007. When retrospectively tested for hBoV DNA, 5 samples obtained between December 2006 and May 2007 were positive.

Case 6.

Case 6 describes an 8-month-old girl, who was hospitalized with a clinical history of respiratory tract infection for 3 weeks with symptoms of cough, wheezing, and tachypnea. Pneumonia was diagnosed. Screening for viral antigens in NPA was negative. Two weeks after discharge from hospital, she was readmitted with fever, cough, and wheezing. Chest radiography showed infiltrates. NPA analysis was positive for RSV antigen. HBoV DNA was retrospectively detected in both NPA.

DISCUSSION

We describe prolonged hBoV DNA shedding in follow-up NPA samples from 6 infants and children. In one patient (case 5), hBoV DNA was slowly cleared during a period of 4.5 months. All patients were immunocompetent. Our findings are in agreement with a recent prospective study in Danish children.⁹ Based on the model suggested by Allander et al,³ primary hBoV infection at the disease episode of initial hBoV detection seems likely in cases 1, 2, 4, and 5, because the initial hBoV loads in NPA were high (>10,000 copies/mL). The clinical relevance of persistent detection of hBoV DNA in respiratory tract samples in the course of several weeks and months is unclear. The observations in our patients could be explained by several hypotheses. These include persistent hBoV infection as a predisposing or aggravating factor of acute respiratory tract infection with other viral agents, hBoV reactivation induced by other infections, or hBoV as an “innocent bystander” that is detected by chance.

Moreover, we cannot completely exclude reinfection as the cause of repetitive detection of hBoV DNA in NPA. To study this issue, we sequenced the hBoV VP2 region in 11 of the 17 hBoV DNA positive NPA⁸ and found 100% sequence identity within samples from individual patients. This finding is compatible with persistent infection or reactivation. However, because of the high sequence conservation (>98%) of all hBoV strains described to date, full sequence identity does not entirely rule out reinfection. The sequence identity between patients in this study was >98.9%.

Our results clearly show that qualitative hBoV DNA detection in single NPA samples is not sufficient to implicate hBoV as the causative agent of acute respiratory tract disease. This is in contrast to most other respiratory viruses, where positive results by direct detection methods in the context of acute respiratory tract disease are generally considered adequate proof of a causal relationship. Serologic assays to diagnose primary infection, detection of hBoV viremia, or high viral loads in respiratory tract samples may be more useful predictors of hBoV-associated clinical disease than only qualitative detection of hBoV DNA in respiratory tract samples.

Our data indicate that hBoV persistence for at least several weeks are not uncommon. Further studies are necessary to elucidate the duration, the target site, and the clinical relevance of hBoV persistence.