Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Fort Collins, Colorado, United States of America.
PLoS Negl Trop Dis. 2022 Jun 15;16(6):e0010487. doi: 10.1371/journal.pntd.0010487. eCollection 2022 Jun.
Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.
黄热病病毒（YFV）是一种黄病毒，经常在非洲和南美洲引起出血热爆发，被认为是一种重新出现的公共卫生威胁。准确诊断黄热病（YF）疾病至关重要，因为一个确诊病例构成一次爆发，并可能引发大规模疫苗接种运动。已经开发出高度敏感和特异的分子诊断方法；然而，这些检测需要在标本运输过程中保持冷链，以防止病毒 RNA 在检测前降解。在一些地区，这种冷链要求很难满足。在这项研究中，我们研究了 Whatman FTA 卡作为用于分子诊断的 YFV RNA 替代稳定方法。使用模拟标本的线性回归分析表明，从单个 6mm FTA 卡打孔中检测到的 RNA 明显不如传统 RNA 提取敏感；然而，从两个 FTA 打孔中提取的 RNA 混合显著降低了检测限，使其与传统 RNA 提取金标准相等。在针对 FTA 卡方法在不同温度下稳定 YFV RNA 的能力的实验中，在 25°C 下储存超过两周后仍可检测到 RNA。更有希望的是，如果在接种前使卡片干燥，YFV RNA 可在 37°C 下储存的卡片上从两天到两周以上进行检测。FTA 卡还被证明可以在高湿度下稳定 YFV RNA，如果卡片在接种前干燥。这些结果表明，FTA 卡可以在 YF 的分子诊断中具有成本效益且易于使用，保存病毒 RNA 以在无法维持冷链的情况下进行阳性诊断。
