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    • Recovered virus concentrations of the

    2020-07-28

    Recovered GSK2578215A receptor concentrations of the particular swab types were similar at different time points as illustrated by Fig. 1. Thus, CMV DNA recovery of the different swabbing materials seems to be independent of the duration of storage reflecting the transport time. However, the swab and transport medium seemed to affect recovery efficiency to a larger extent. Swab no. 1 revealed a significantly lower recovery than the same swab used with VTM (swab no. 3) at each time point (Fig. 1a, b). In contrast, there is obviously no such difference between the second dry swab (swab no. 2) and the same swab soaked with VTM (swab no. 4). The reproducibility within the same swabs was predominantly excellent. To quantitatively assess potential differences in CMV DNA recovery between different sampling procedures, the mean CMV DNA recovery for both virus suspensions at all time points in both laboratories was calculated for each swab type (Fig. 2). Swabs no. 3 and 6 showed the highest mean recovery of CMV DNA. Statistical analysis of CMV DNA recovery rates of the swabs analysed (no. 1-6) using one-way ANOVA showed significant differences (p < 0.0001). Results of the group to group comparisons with the Tukey´s Multiple Comparison Test are shown in Fig. 2. With swab no. 1 the lowest recovery rate of all swab types could be detected (40% of hypothetical value, Fig. 2) – this amount is significantly lower than in three other swab types (Fig. 2). Furthermore, flocked swab with eNat medium (swab no. 6) had the highest mean recovery rate (160% of hypothetical value, Fig. 2) that differed significantly from the recovery rates from swab 1, 4 and 5 (Fig. 2).
    Discussion Congenital CMV infection is the most common non-genetic cause of SNHL in childhood. The fact that most congenitally infected infants are asymptomatic at birth underlines the need for postnatal CMV screening, as early detection is essential for directed care [7]. The ideal screening method should be rapid, sensitive, cost effective and amenable to high-throughput testing. Saliva is easy to collect with swabs and PCR methods are widely used. Reports in the past indicate that this approach has good analytical performance and clinical sensitivity and may be able to fulfill the criteria for qualitative screening purposes [4]. Nevertheless the rate of false positive quantitative real time (qrt) PCR results from saliva compared to confirmation with PCR from urine was reported discordantly ranging from 1.2% [8] to 16% [9]. Importantly, contamination of saliva with CMV after breastfeeding leading to false positive PCR results seems to be less critical than previously thought [10]. Our study demonstrates that the duration of storage under the conditions analysed did not have a major effect on the recovery efficiency for any of the swabs (Fig. 1). However, up to 4-fold differences in the recovery efficiencies were observed between the different swabs tested (Fig. 2). Using the flocked swab with the eNAT-medium (swab no. 6) or VTM (swab no. 3), the highest amounts of CMV DNA could be recovered (Fig. 2). In contrast to the study done by [11], the traditional swab without transport medium (swab no. 2) performed well in our study. It recovered comparable amounts of virus DNA at different time points like the traditional swab with VTM (swab no. 4) and the flocked swab with UTM (swab no. 5). The difference could be due to the fact that in the study by Goshen et al. distilled water was used as a diluent of dry swabs prior to extraction. In an initial feasibility study we observed that distilled water proved not to be suitable as diluent for the dry swabs (data not shown). For some of the preparations the recovered virus concentration after 8 days was slightly higher in comparison to the recovery after two hours. Maybe this is due to the fact that after a longer storage period of the swab in the UTM, more viral particles got released from the matrix of the swab. In our study, dry swabs were pre-incubated in PBS for 10 min (and vortexing for 10 s) before extraction of nucleic acids. Preliminary tests showed that this pre-incubation leads only to a slight improvement in CMV DNA extraction (data not shown). Therefore this step can be dropped where necessary to make the laboratory screening procedure even faster and with reduced hands-on time.