Patients and stool samples.

A total of 125 clinical stool samples from 40 patients were included in the study, with 50 retrospective samples collected between December 2012 and August 2013 (kept frozen at −80°C) and 75 fresh prospective samples.

Initially, to assess the intra-assay reproducibility of the results after SPD processing, a preliminary retrospective study was conducted using 20 HAdV DNA-positive frozen stool samples from 12 HSCT recipients hospitalized at Robert-Debré Hospital (Paris, France). All samples were preprocessed in triplicate using the SPD. Each triplicate sample was extracted with two extraction systems, the easyMAG (bioMérieux, Marcy l'Etoile, France) and QIAsymphony (Qiagen, Courtaboeuf, France). Each extract was then quantified in triplicate for HAdV DNA using the quantitative real-time PCR Adenovirus R-gene assay (bioMérieux/Argene, Verniolle, France) on an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA).

To evaluate the differences in HAdV DNA quantification between the easyMAG and QIAsymphony NA extraction systems and between the ABI 7500 and LightCycler 480 (LC480) thermocycler (Roche Applied Science, Meylan, France) amplification systems, 30 additional frozen stool samples from the follow-ups of 5 HSCT recipients with active HAdV infection were tested in duplicate. This series included for each patient the last HAdV DNA-negative sample before the first HAdV DNA-positive sample and the first HAdV DNA-negative sample after the last HAdV DNA-positive sample, with the aim to evaluate if SPD preprocessing improved the sensitivity of HAdV detection compared to that of the routine preprocessing technique, thereby enabling the detection of HAdV reactivation earlier and for longer time periods.

Finally, 75 fresh stool samples from 23 pediatric HSCT recipients at risk for HAdV infections (cord blood graft or age <6 years) were prospectively tested with the routine preprocessing technique and the SPD. The same NA extraction system (QIAsymphony) and HAdV DNA quantification assay (Adenovirus R-gene assay on ABI 7500) were used for the two methods. All stool samples were qualified using the Bristol scale. The Bristol stool scale classifies the form of human feces into seven categories. Types 1 and 2 indicate constipation, types 3 and 4 are the ideal stools, and types 5, 6, and 7 tend toward diarrhea (19). HSCT patients usually do not have constipation in the weeks after transplantation, especially when an HAdV infection occurs. We thus did not have any type 1 or 2 stools to test.