Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes

Abstract

The "gold standard" for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed.Today, 52 subtypes of adenoviruses (AV; family Adenoviridae, genus Mastadenovirus) are known to infect humans. They are transmitted by the fecal-oral route and the respiratory route and are associated with acute respiratory disease (accounting for 10% of febrile respiratory diseases in children), conjunctivitis, genitourinary infections, and infant gastroenteritis. AV have proved to be associated with the induction of malignant tumors in animals; however, this correlation has not been shown in humans. Human AV serotyping is based on resistance to neutralization by antisera to other AV serotypes. Of the 52 known serotypes, 51 have been sorted into six serogroups, based on their ability to agglutinate red blood cells from different species; serotype 52 has been found only recently. Most AV diseases are caused by a few serotypes (1 to 7) usually producing only mild infections in the immunocompetent host. The agent causing the infection can be isolated from stool samples during periods of no illness. This phenomenon hinders the establishment of a causal association of AV with disease and limits the significance of the diagnostic detection of these viruses. Based on virus culture studies, it is known that different serotypes of AV can cause different clinical syndromes; however, we may have underestimated the incidence of AV disease in some patient groups because some strains are difficult to cu...

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