Laboratorio de Hepatites Virais, Instituto Oswaldo Cruz, Helio and Peggy Pereira Pavillion - Ground Floor - Room B09, Fundação Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro, Rio de Janeiro, 210360-040, Brazil.
Departamento de Educação, Instituto Federal de Educação, Ciência E Tecnologia Do Ceará, Fortaleza, Ceará, Brazil.
Sci Rep. 2022 Jan 31;12(1):1651. doi: 10.1038/s41598-022-05264-1.
Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.
乙型肝炎病毒 (HBV) 的诊断是在血清样本上进行的，但在低收入地区，这种诊断方法难以获得。使用干血斑 (DBS) 样本进行采集、储存和运输时，无需特殊结构。本研究评估了使用 DBS 检测、定量和测序 HBV DNA 的方法，采用的是内部技术。研究纳入了两个组：92 名 HBsAg +个体和 49 名阴性对照者。血清和 DBS 样本均提交进行 S/pol 基因的定量和定性内部 PCR、测序和系统发育分析。共成功扩增了 84 份血清样本。其中，63 对 DBS 定性 PCR 也呈阳性。DBS 定性 PCR 的灵敏度为 75%，特异性为 100%（Kappa = 0.689）。DBS 定量 PCR 的检测下限为 852.5 拷贝/mL（250 IU/mL），灵敏度为 77.63%，特异性为 100%（Kappa = 0.731）。共对 63 份血清样本和 36 份 DBS 样本进行了测序，发现基因型 A（65.08%）、D（4.8%）、E（3.2%）和 F（27%）在血清和 DBS 之间的循环率为 100%，完全一致。所有测序样本的 HBsAg 基因均显示出多态性。一名 HIV 合并感染患者的血清和 DBS 中均出现 rtM204V/I-rtL180M 双重耐药突变。总之，DBS 是一种替代方法，可用于检测、定量和表征 HBV DNA，是在低收入环境中增加诊断的一种可能性，有助于缩小乙型肝炎控制方面的差距。
