Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
Transfusion. 2014 Oct;54(10):2485-95. doi: 10.1111/trf.12653. Epub 2014 Apr 17.
Sensitive triplex nucleic acid tests (NATs) are implemented for blood donation screening worldwide. Assays have variable ability to detect low-level hepatitis B virus (HBV) DNA. At borderline DNA detection levels, where Poisson distribution impacts results, distinguishing true-positive from false-positive results is challenging. Algorithms are needed to confirm such low-level HBV DNA-positive samples.
A total of 135 blood donor samples reactive by one or more HBV markers that provided discrepant results were tested undiluted with four commercial NATs: Ultrio, Ultrio Plus, MPX, and a quantitative assay (SuperQuant). To further explore discrepancies, three additional in-house NATs including real-time polymerase chain reaction (PCR) and nested PCR and sequencing were performed.
The numbers reactive of these 135 "difficult" samples by four commercial NATs were as follows: 39 of 107 (36%) with SuperQuant, 40 (30%) with Ultrio, 100 (74%) with Ultrio Plus, and 102 (76%) with MPX. Of the seven NATs, 109 (81%) samples were reactive by at least two assays and thus considered confirmed positive of which 67 (50%) generated a sequence. Ultrio Plus and MPX performed similarly as above (80%-85% detected of 109 and 81%-90% of 67, respectively). Older (median, 49 years), HBV core antibody-reactive donors carried predominantly Genotype A (58%) with high-frequency amino acid substitutions in the major hydrophilic region of the S-protein. Younger (median, 24 years) hepatitis B surface antigen-positive donors carried wild-type strains predominantly Genotype B (32%) and E (24%), the latter in an apparent cluster.
Highly sensitive NATs require new confirmatory algorithms as presented optimally using different genomic regions or sequence generation. The introduction of immigration-related HBV genotypes may impact HBV epidemiology in the United States.
敏感三重复合核酸检测（NAT）已在全球范围内用于献血筛查。检测方法对低水平乙型肝炎病毒（HBV）DNA 的检测能力各不相同。在泊松分布影响结果的边界 DNA 检测水平，区分真正的阳性和假阳性结果具有挑战性。需要算法来确认此类低水平 HBV DNA 阳性样本。
共有 135 份经一种或多种 HBV 标志物反应的献血者样本，这些样本提供了不一致的结果，这些样本未经稀释，使用四种商业 NAT 进行了检测：Ultrio、Ultrio Plus、MPX 和定量检测（SuperQuant）。为了进一步探讨差异，还进行了另外三种内部 NAT，包括实时聚合酶链反应（PCR）、巢式 PCR 和测序。
四种商业 NAT 检测的 135 份“困难”样本的反应数量如下：SuperQuant 检测 39 份（36%），Ultrio 检测 40 份（30%），Ultrio Plus 检测 100 份（74%），MPX 检测 102 份（76%）。在七种 NAT 中，有 109 份（81%）样本至少有两种检测呈阳性，因此被认为是阳性确认，其中 67 份（50%）产生了序列。Ultrio Plus 和 MPX 的表现与上述相似（80%-85%检测到 109 份，81%-90%检测到 67 份）。较年长（中位数，49 岁）的 HBV 核心抗体反应供体主要携带基因型 A（58%），其 S 蛋白主要亲水区域的氨基酸替换高频。较年轻（中位数，24 岁）的乙型肝炎表面抗原阳性供体主要携带野生型株，主要为基因型 B（32%）和 E（24%），后者呈明显聚集状态。
高度敏感的 NAT 需要新的确认算法，最好使用不同的基因组区域或序列生成来优化。移民相关 HBV 基因型的引入可能会影响美国的 HBV 流行病学。
