Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

Abstract

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.

Adenovirus Infections in Immunocompetent and Immunocompromised Patients

“…Gastrointestinal manifestations are mainly associated with HAdV-F40 and -F41, but HAdV-G52 and different members of species D, including some of the most recently identified types (types 65 and 67), have also been observed (20,36,43,46,89). Respiratory tract involvement has been associated mainly with HAdV-B3, -B7, -B16, -B21, and -E4 and various members of species C (43,90). These examples indicate that certain adenoviruses have strong tropisms for specific tissues, but the same clinical manifestations can be caused by other HAdV types and species, thus requiring diagnostic screening methods with broad specificity.…”

Quantitative real-time PCR assays for detection and type-specific identification of the endemic species C human adenoviruses

“…However, these methods are often complex and time consuming, and are not optimally designed for viral load determinations, important for monitoring HAdV disease progression and therapeutic efficacy. Quantitative real-time PCR (qPCR) has been proposed as a rapid, sensitive and widely available technology for detection and identification of HAdVs more suited for the clinical diagnostic setting (Heim et al, 2003;Metzgar et al, 2010). We recently http://dx.doi.org/10.1016/j.jviromet.2016.05.020 0166-0934/Published by Elsevier B.V.…”

Computational and Serologic Analysis of Novel and Known Viruses in Species Human Adenovirus D in Which Serology and Genomics Do Not Correlate

“…The species groupings, in part, may reflect the cell tropism of the viruses, as well as the resulting symptoms and diseases caused by the individual HAdV types. For example, species HAdV-B1 viruses are known to cause respiratory infections of the lower lung [11] whereas viruses in species HAdV-D can cause ocular disease, including epidemic keratoconjunctivitis [12], and gastrointestinal disease [3], [13].…”