Detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real‐time PCR assay

Abstract Human adenoviruses (hAdVs) are associated with acute respiratory tract infections in pediatric populations and have been identified as a cause of outbreaks in institutional settings. Rapid diagnosis of hAdV infection is critical for appropriate and timely management. This study reports the design and validation of a sensitive and specific multiplex real‐time PCR for the detection of a broad range of hAdV serotypes in respiratory samples. The assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay was evaluated using retrospectively (n = 864) and prospectively (n = 11,451) collected samples from November 2005 to July 2006. Seasonality studies and analysis of outbreaks was conducted over a 2‐year period from January 2005 to December 2006 (n = 33,067 samples). The assay gave a hAdV positive rate of 7.1% (n = 811) for specimens tested prospectively and was able to detect a broad range of hAdV serotypes with good sensitivity and specificity. A high rate of co‐infection was noted (21.7%). Adenovirus infections were more prevalent in the young with a median age of 24 months for positive patients. Sequence analysis of hAdV positives showed that serotype 7 was the most prevalent followed by serotypes 2 and 3. Association of hAdVs with respiratory outbreaks was low at 2.3% (6 of 266 outbreaks tested) and no seasonal variation was observed for hAdV infections during the 2‐year study period. This assay can improve the detection of hAdVs in respiratory samples and can be used to provide valuable epidemiological information. J. Med. Virol. 80:856–865, 2008. © 2008 Wiley‐Liss, Inc.