title: Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools.
abstract: The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3-9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a 'hyper sensitive ELISA' for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant.
authors.affiliation: Centre de Recherche Public-Gabriel Lippmann, Department of Environment and Agro-biotechnologies (EVA), Belvaux, Luxembourg.
authors.forename: S
authors.initials: S
authors.lastname: Bonot
authors.forename: L
authors.initials: L
authors.lastname: Ogorzaly
authors.forename: B
authors.initials: B
authors.lastname: El Moualij
authors.forename: W
authors.initials: W
authors.lastname: Zorzi
authors.forename: H-M
authors.initials: HM
authors.lastname: Cauchie
mesh_terms: D000257:Adenoviridae Infections / Q000175:diagnosis*; D000260:Adenoviruses, Human / Q000302:isolation & purification*; D002675:Child, Preschool; D003955:Diagnostic Tests, Routine / Q000379:methods*; D005243:Feces / Q000821:virology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: human adenoviruses; immuno real-time PCR; quantitative real-time PCR; sandwich ELISA; stool samples