Abstract The development of a new fast, portable and reagent-free diagnostic technique for hepatitis B (HBV) and hepatitis C (HCV) viruses would be an enormous benefit to society. Here, we evalulate the ability of Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis to classify human serum samples based on the presence of HBV and HCV infection. Sera samples were prepared using three different methodologies: i) Sera depsoited onto glass cover slips, airdried and placed onto the ATR crystal. ii) Whole serum dried directly onto the ATR crystal. iii) Serum separated into high and low molecular weight compounds using a filtration approach and the high molecular weight fraction placed directly onto the ATR-FTIR diamond window and dried. For methodology i) the Partial Least Squares Discriminate Analysis (PLS-DA) calibration set included 313 (70 %) samples and the validation set 93 (30 %) samples. For HBV vs control the sensitivity and specificity was found to be 69.4 % and 73.7 % (10 latent variables (LV)), respectively. For HCV vs control the sensitivity and specificity was 51.3 % and 90.9 % (LV 11), respectively. In the second set of experiments the serum samples were dried directly onto the ATR diamond. PLS-DA models were constructed using 144 (70 %) samples for the calibration set and tested using an independent test set containing 62 (30 %) samples. For HBV versus control the sensitivity and the specificity was 84.4 % and 93.1 %, respectively (LV 8). For HCV versus control the sensitivity and specificity was 80.0 % and 97.2 %, respectively (LV 9). For HBV versus HCV the sensitivity and the specificity was 77.4 % and 83.3 %, respectively (LV 5). To increase the sensitivity and specificity serum sample was fractionated into high and low molecular weight components. In PLS-DA cross validated model (LV 8) the sensitivity and specificity was 87.5 % and 94.9 %, respectively for HBV vs control (high molecular concentrate). The PLS-DA cross-validated model (LV 8) for HCV vs control high molecular fraction produced a sensitivity and specificity of 81.6 % and 89.6 %, respectively. No linear correlation was observed for sera samples spiked with known viral loads using Partial Least Squares Regression (PLS-R) modelling. Spectra of positive serum (HBV and HCV) showed a strong band observed at 1631 cm−1, which was absent in the spectra of controls and assigned to the β-pleated sheet protein marker of immunoglobulin (Ig). A band at 1093 cm−1, observed in spectra of HBV infected sera, was assigned to C C and CO modes of polysaccharide N-glycan from hepatitis B surface antigen (HBsAg). The assignment was confirmed by atomic force microsocpy infrared (AFM-IR) spectroscopy of the isolated protein. This band represents a unique marker for HBV infection. In summary, ATR-FTIR spectroscopy is a powerful tool to study blood composition and identify potential disease markers but care must be taken to ensure that the modelling is not biased by inflammation markers, which may confound diagnosis.

中文翻译：

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光谱病毒化：使用 ATR-FTIR 光谱诊断人体血清中的乙型和丙型肝炎病毒感染

摘要 开发一种新的快速、便携和免试剂的乙型肝炎（HBV）和丙型肝炎（HCV）病毒诊断技术将给社会带来巨大的利益。在这里，我们评估了衰减全反射傅里叶变换红外 (ATR-FTIR) 光谱结合多变量数据分析根据 HBV 和 HCV 感染对人血清样本进行分类的能力。使用三种不同的方法制备血清样品： i) 将血清沉积在玻璃盖玻片上，风干并置于 ATR 晶体上。ii) 将全血清直接干燥到 ATR 晶体上。iii) 使用过滤方法将血清分离成高分子量和低分子量化合物，并将高分子量部分直接放在 ATR-FTIR 金刚石窗口上并干燥。对于方法 i)，偏最小二乘判别分析 (PLS-DA) 校准集包括 313 (70%) 个样本和验证集 93 (30%) 个样本。对于 HBV 与对照，发现敏感性和特异性分别为 69.4 % 和 73.7 %（10 个潜在变量 (LV)）。对于 HCV 与对照，敏感性和特异性分别为 51.3 % 和 90.9 % (LV 11)。在第二组实验中，将血清样品直接干燥到 ATR 金刚石上。PLS-DA 模型使用 144 个 (70%) 样本作为校准集构建，并使用包含 62 个 (30%) 样本的独立测试集进行测试。对于 HBV 与对照，敏感性和特异性分别为 84.4 % 和 93.1 % (LV 8)。对于 HCV 与对照，敏感性和特异性分别为 80.0 % 和 97.2 % (LV 9)。对于 HBV 与 HCV，敏感性和特异性分别为 77.4 % 和 83.3 % (LV 5)。为了提高灵敏度和特异性，血清样品被分成高分子量和低分子量成分。在 PLS-DA 交叉验证模型 (LV 8) 中，HBV 与对照（高分子浓缩物）的敏感性和特异性分别为 87.5 % 和 94.9 %。HCV 与对照高分子部分的 PLS-DA 交叉验证模型 (LV 8) 分别产生了 81.6 % 和 89.6 % 的敏感性和特异性。使用偏最小二乘回归 (PLS-R) 建模未观察到添加已知病毒载量的血清样本的线性相关性。阳性血清（HBV 和 HCV）的光谱显示在 1631 cm-1 处观察到强条带，它在对照的光谱中不存在，并分配给免疫球蛋白 (Ig) 的 β-折叠片蛋白标记。在 HBV 感染的血清光谱中观察到的 1093 cm-1 条带被指定为来自乙型肝炎表面抗原 (HBsAg) 的多糖 N-聚糖的 CC 和 CO 模式。通过分离蛋白质的原子力显微镜红外 (AFM-IR) 光谱证实了该分配。该条带代表 HBV 感染的独特标志物。总之，ATR-FTIR 光谱是研究血液成分和识别潜在疾病标志物的有力工具，但必须注意确保建模不受炎症标志物的影响，这可能会混淆诊断。被分配到来自乙型肝炎表面抗原 (HBsAg) 的多糖 N-聚糖的 CC 和 CO 模式。通过分离蛋白质的原子力显微镜红外 (AFM-IR) 光谱证实了该分配。该条带代表 HBV 感染的独特标志物。总之，ATR-FTIR 光谱是研究血液成分和识别潜在疾病标志物的有力工具，但必须注意确保建模不受炎症标志物的影响，这可能会混淆诊断。被分配到来自乙型肝炎表面抗原 (HBsAg) 的多糖 N-聚糖的 CC 和 CO 模式。通过分离蛋白质的原子力显微镜红外 (AFM-IR) 光谱证实了该分配。该条带代表 HBV 感染的独特标志物。总之，ATR-FTIR 光谱是研究血液成分和识别潜在疾病标志物的有力工具，但必须注意确保建模不受炎症标志物的影响，这可能会混淆诊断。