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  • Our study demonstrates that the

    2019-07-02

    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 glutathione reductase 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. The amount of CMV DNA detected for the different swabs diverged approximately 3-fold (0.5 log10) between the two laboratories. This minor variation is most likely due to differences in the standards used. Substantial interlaboratory variability in quantitative CMV viral load values up to 2 log10 copies/ml for individual samples was already found in a multicenter study [12] and could be also observed in the national ring trial (organized by the German Society for Promotion of Quality Assurance in Medical Laboratories, INSTAND e.V.), where a deviation from the expected result by up to a factor of 6.3 are tolerated in order to pass the test. This indicates that the interlaboratory variation may be larger than the variations observed between the different swabs and storage conditions. However, since both variations may sum-up, both need to be controlled for establishment of quantitative assays of CMV viral load in saliva samples.
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    Introduction Cytomegalovirus (CMV) is one of the most common and important pathogens encountered following solid organ transplantation.1, 2, 3, 4, 5, 6, 7, 8, 9 This ubiquitous herpes virus may affect up to 90% of the general population by adulthood. Whilst CMV infection usually remains latent and asymptomatic in immunocompetent hosts, it is a significant cause of morbidity and occasional mortality post solid organ transplantation.3, 4, 5, 6 In addition to directly mediated viral syndromes and tissue-invasive disease, CMV reactivation also indirectly impacts on a number of graft and patient outcomes.2, 4, 5, 6, 7, 8, 9 CMV reactivation has been associated with transplant specific complications such as chronic renal allograft nephropathy, accelerated hepatitis C recurrence and hepatic artery thrombosis after liver transplantation, allograft vasculopathy after cardiac transplantation and bronchiolitis obliterans in lung allografts. Across multiple transplant types, CMV is also associated with acute rejection, bacterial, fungal and viral infections, post-transplant lymphoproliferative disorders and mortality.2, 3, 4 Current international consensus recommendations suggest that using either universal prophylaxis or a pre-emptive strategy are both effective preventative approaches. Such strategies have significantly decreased the burden of CMV disease following solid organ transplantation; however, late onset CMV disease after discontinuation of prophylaxis is a significant problem2, 10, 11 and is associated with higher rates of mortality and graft loss.2, 4 Monitoring plasma CMV viral loads after the cessation of prophylaxis is one approach used to prevent CMV disease but has been shown to have limitations. Viral monitoring increases pathology costs and the viral load thresholds for initiating treatment are unclear, as a significant proportion of patients will have spontaneous clearance without antiviral therapy.2, 7 A recent study in Western Australian renal transplant recipients found that CMV viral loads of >665 copies/mL only were associated with increased risk of mortality. Prolongation of prophylaxis is also not without problems including increased toxicities of antiviral medications, higher drug costs and increased risk of CMV drug resistance.4, 8, 10, 12 Furthermore, the use of prophylaxis has been shown to delay the development of CMV-specific cell mediated immunity in heart transplant recipients. While there are known risk factors for development of CMV disease, including donor and recipient CMV serostatus, type of transplant, number of treated acute rejection episodes and exposure to T-cell depleting therapies, the assessment of risk in individuals has been prevented by the lack of tools to directly assess CMV specific T-cell immunity. Measuring the competence of a CMV memory response would help determine whether viraemia is likely to progress to disease or be controlled without intervention.2, 5, 6, 7, 8, 14