title: Comparison of in-house real-time quantitative PCR to the Adenovirus R-Gene kit for determination of adenovirus load in clinical samples.
abstract: In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the "gold standard" for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra- and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r = 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.
authors.affiliation: Laboratoire de Virologie, CHU de Nancy Brabois, rue de Neufchâteau, Vandoeuvre-lès-Nancy 54511, France. h.jeulin@chu-nancy.fr
authors.forename: Hélène
authors.initials: H
authors.lastname: Jeulin
authors.forename: Alexandra
authors.initials: A
authors.lastname: Salmon
authors.forename: Pierre
authors.initials: P
authors.lastname: Bordigoni
authors.forename: Véronique
authors.initials: V
authors.lastname: Venard
mesh_terms: D000257:Adenoviridae Infections / Q000821:virology*; D000260:Adenoviruses, Human / Q000302:isolation & purification*; D001331:Automation / Q000379:methods; D001769:Blood / Q000821:virology; D002555:Cerebrospinal Fluid / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral