Objective Real-time PCR is a validated technique for the detection of RSV viral loads. However, test processing includes adequate laboratory performance (isolation of RNA, production of cDNA, and amplification) and clinical sampling. Whether laboratory processing and clinical sampling influences test precision needs to be determined.

Methods During the winter of 2007–2008 ventilated infants with an RSV bronchiolitis at the PICU were enrolled. Undiluted nasopharyngeal aspirates (NPAs) and tracheal aspirates (TAs) were taken at admission to determine viral loads with real-time PCR (t1). To investigate laboratory processing (I) aspirates were divided into three aliquots to detect viral load. Ct values (Cts) of the different aliquots were averaged and differences calculated. To investigate the influence of the time point of clinical sampling (II), sampling was repeated after 2–6 hours (t2) and viral loads were compared (t1–t2).

Results

I: On 10 NPA’s 3 PCR tests were performed; the mean (SD) viral load was 24.9 (3.0) Ct. The maximal difference from the mean was 1.07 Ct. On 9 TA’s the 3 PCR tests showed a mean of 23.6 (4.9) Ct. The maximal difference from the mean was 0.67 Ct.

II: Six NPAs and six TAs were tested at t1 and t2. The mean difference between t1 and t2 was 1.2 (3.9) and 3.7 (3.5) Ct, respectively.

Conclusions Laboratory processing does not lead to variability in viral load detection by real-time PCR. However, preliminary data show that clinical sampling within relatively short time intervals can lead to considerable variability in test results.