DEVELOPMENT, EVALUATION AND COMPARISON OF REAL-TIME PCR ASSAYS FOR DETECTION OF HUMAN BOCAVIRUS IN RESPIRATORY SAMPLES

Background: Human bocavirus (hBoV) is a newly discovered respiratory virus belonging to the Parvoviridae family, genus Bocavirus. The virus has been associated with acute respiratory tract infections in pediatric populations.

Study aims: 1) To design and validate a real-time PCR assay for sensitive and specific detection of hBoV. 2) To study the prevalence, seasonality and age distribution for hBoV infections. 

Methods: A hydrolysis probe-based real-time PCR assay was designed targeting the NP gene of hBoV (NP assay). The assay was used for retrospective detection and analysis of hBoV in respiratory specimens (n=2203) which included upper respiratory tract specimens (n=1592), lower respiratory tract specimens (n=562), and unspecified respiratory samples (n=49) collected between November 2005 and July 2007 from patients with acute respiratory symptoms of all age groups. Nucleic acid was extracted using the automated easyMAG® extractor (bioMérieux). Amplified DNA from a positive hBoV specimen was cloned and used as a plasmid control.  Amplification and detection of PCR products was performed on the ABI 7000/7500 SDS with universal master mix and thermocycling profile. An alternate real-time assay was designed in the 5’-region of the viral genome and used, along with a previously published NS1 gene assay, for comparison with and confirmation of hBoV NP assay positives.

Results: The limit of detection for the assay was 8 copies/ reaction of hBoV cloned plasmid.  Specificity of the assay was 100.0% as determined by testing RNA/DNA of other respiratory viral and bacterial pathogens. Human bocavirus was detected in 67/2203 (3.0%) of the specimens tested. The age of infected patients ranged from 5 weeks to 12 years with a median age of 17 months. A high rate of co-infection was noted for the hBoV positive samples (46.3%). The highest rate of hBoV infection was seen in patients between 1 to 4 years of age, at 14.7%. The virus was detected more commonly in upper than lower respiratory specimens 3.5% compared with 2.0% positive, respectively). Good correlation was observed between the two in-house designed assays and the published assay for the detection of positives. Association of hBoV with respiratory outbreaks was low (1/78 or 1.3% of outbreaks tested). No particular seasonality was noted for hBoV infections.

Conclusions: A sensitive and specific assay for detection of hBoV was developed and validated. This assay will be useful in resolving respiratory infections of previously unknown etiology and will enhance our understanding of the epidemiology and clinical impact of hBoV infections.