Stability of DBS over the
Stability of DBS over the 60-day time points was encouraging, with a maximum of 0.1 log10 IU/mL degradation from the day 1 value (Supplementary Fig. 1 and Supplementary Table 3).
Discussion This study evaluated the sensitivity and specificity of the Aptima HCV Dx assay for HCV RNA detection from spotted whole blood DBS samples compared with the CAP/CTM and Aptima HCV Dx assay in plasma using HCV antibody positive remnant diagnostic samples. A high degree of correlation and diagnostic accuracy was observed in HCV RNA detection in DBS compared to plasma (venepuncture), especially when considering the clinically relevant threshold proposed by EASL . This is one of the first studies to compare the Aptima HCV dx assay in DBS and plasma samples. Finger-stick DBS sample collection and HCV RNA testing using the Aptima assay provides an opportunity for simplified diagnostic strategies to increase testing uptake particularly in settings where venepuncture is a barrier to testing. These findings provide support for further evaluation of the ‘real-world’ performance of DBS collection to enhance HCV testing, 93 6 to care, and treatment by simplifying sample collection. The sensitivity and specificity of the Aptima for HCV RNA detection in DBS when compared to the CAP/CTM (‘gold standard’) was 96.4% and 95.8% respectively, consistent with similar studies evaluating automated PCR and TMA methods, where sensitivity ranged between 90–98% and specificity was reported as 100% [, , , ]. In this study, the false negatives (n = 3) and false positive (n = 1) occurred at the lower assay range (<33 IU/mL (false negatives) and below the CAP/CTM < LLOQ (false positive). The false negatives could be attributed to hematocrit correction as 33IU/mL in plasma is equivalent to 1.3 IU/mL in DBS (33/25.97) which falls below the limit of detection. Encouragingly, the sensitivity and specificity increased to 100% when a clinically relevant threshold of 1000 IU/mL was used based on guidance for the minimum detectable threshold for HCV RNA detection proposed in the 2018 EASL recommendations . Furthermore, excellent agreement and a strong correlation (R2 = 0.929, p < 0.0001) was observed between both plasma and DBS when analyzed using the Aptima assay with a small bias of 0.51 log10 towards plasma. Clinical use of DBS described in published literature is limited and regulatory requirements vary from country to country, adding to the need for validated, standardized manufacturers protocols and registration of HCV quantitative and qualitative molecular diagnostic assays with DBS as a sample type . Further work is urgently needed to have DBS product monograms for in-vitro medical diagnostic devices (outside of research only setting) recognised and approved by regulatory authorities to allow for clinical implementation . There are some limitations to this study, firstly the remnant DBS samples were manufactured in the laboratory by spotting standard of care HCV antibody positive venous whole blood from clinical samples onto filter paper rather than patient collected fingerstick. As such, limited demographic information was available and information on injecting drug use risks were unavailable (e.g. injecting drug use). The sample set was also small and heavily biased (anti−HCV positive), with high HCV RNA detection (80%) and concentration (average viral load 6.04 log10 IU/mL) and genotype was only determined for 45 of the 83 patients with detectable RNA due to sample insufficiency and was restricted to genotypes 1-3. Finally, to replicate ‘real-world’ testing scenarios, further evaluations with larger sample sizes and fingerstick capillary DBS are required to assess analytical sensitivity and specificity in relevant cohorts (including people who inject drugs), especially at lower HCV RNA concentrations and with genotypes 4-6.