In this study, we evaluated the stability of HCV RNA stored as DBS from whole blood or plasma samples using two separate card devices under different storage conditions. In our hands, we observed lower RT-PCR sensitivity during the first times tested, although it increased after 120 days, possibly due to optimizations made in the FTA card purification protocol. Additionally, it should be noted that the boards were exposed to thaw/freeze cycles throughout the study which may have influenced its stability.
Despite these facts, we were able to determine that the 903 Protein Saver cards provide stability and integrity of HCV RNA for longer periods of at least 9 months. However, slight differences were observed on the stability of HCV RNA in maps stored at -20°C, since in the 30% of samples analyzed so far, it lasted up to 10 months. Additionally, although longer RNA stability was originally expected for samples stored at −20 °C21.25we could not determine any temperature-dependent effect on HCV RNA stability using the 903 Protein Saver cards.
Although FTA Classic cards are believed to retain nucleic acids longer than untreated cards like 903 Protein Saver, our analysis showed no significant difference between them. Additionally, we determined that viral RNA can be stored at room temperature for 9 months without undergoing significant degradation using 903 Protein Saver cards. Our results agree with other studies also showing that these cards are suitable for longer storage of nucleic acids.26. In this line, our results are in agreement with the studies carried out by Keeler et al.27.
Regarding sample type, our results showed higher detection rates in whole blood than in plasma samples. During separation from plasma, a fraction of HCV RNA may be lost due to virus adherence to red blood cells or even through the formation of virus-immunoglobulin complexes. Intracellular RNA from these granule complexes will be lost in the plasma fraction and then reduce viral load in plasma relative to whole blood, as described by Schmidt et al.28.
We have determined that whole blood can be an adequate source of HCV RNA and that no viral load is lost compared to the most often preferred plasma sample. Next, the 903 Protein Saver Cards are an ideal sampling device, less expensive than similar developed sampling methods such as the Cobas Plasma Separation Card (PSC), developed for HIV-1 quantitation29.30. To determine if 903 Protein Saver cards could replace PSC cards, further studies analyzing the stability of 903 Protein Saver cards under high humidity conditions are needed.
Considering the above results, we suggest 903 Protein Saver Cards as the best and most cost-effective storage system for DBS, as blood samples remained stable longer than FTA Classic Cards, even under harsh conditions. ambient temperature. This feature is essential for low-resource environments because no additional sample processing is required for collecting, storing, or shipping whole blood samples. In addition, the long-term stability of RNA is crucial for patient follow-up.
Long-term storage of reference samples remains essential to assess relapse of viremia after treatment by RT-PCR and sequencing of reference and post-treatment paired samples. Sequencing and phylogenetic analysis allow us to distinguish between reinfection (different virus than reference) and treatment failure (same virus). This is particularly important in settings where reinfection is common, such as among people who inject drugs.
However, 903 Protein Saver cards have an untreated matrix which does not guarantee virus inactivation. In fact, a recent study showed that purified Newcastle disease virus remained infectious in DBS after 24 h, unlike FTA cards.31. Further studies analyzing viral stability in patient samples would be needed to verify the safety levels of 903 Protein Saver Cards for short- and long-term storage of blood samples.
Previous studies using other viruses have been able to detect African swine fever virus (ASFV) viral DNA or peste des petits ruminants (PPR) viral RNA in whole animal blood-soaked FTA cards. infected stored as DBS for 9 months at 22, 32 or 37°C32. In contrast, other studies establish a maximum storage of the FTA card at room temperature in 30 days for the detection of avian influenza virus RNA.27. Different detection methods used in these studies, such as real-time RT-PCR versus conventional RT-PCR, as well as other procedural differences such as sample volume or extraction method yields of RNA, could explain the reported discrepancies. Because of our primary objective throughout this study, which was to evaluate the FTA Classic and 903 Protein Saver cards as an alternative sampling method for HCV-infected patients, our primary objective was to determine whether RNA whether or not HCV was detected after storage in DBS, and for how long it remained detectable by RT-PCR, the methodology used in low- and middle-income countries. Conventional RT-PCR is a convenient method for this purpose as no RNA quantification was required. However, to confidently conclude that DBS maps are suitable for current HCV diagnostic methods, FTA maps must be validated with established routine qRT-PCR methods.
Due to the presumed weakness of RNA, it is best stored at -80°C. However, our primary focus was to analyze the integrity of RNA as DBS for implementation in LMICs, with virtually no access to -80°C storage or shipping conditions. Thus, our results show that 903 Protein Saver cards, or even FTA Classic cards, could be a suitable method for long-term storage of HCV RNA at room temperature.
In summary, this study shows that unpurified HCV RNA from blood samples can be stored at room temperature in 903 Protein Saver Cards for at least 9 months without affecting RNA integrity, making 903 Protein Saver cards a recommended sampling method for HCV diagnosis in PRITI countries.