Tan Susanna K, Shen Peidong, Lefterova Martina I, Sahoo Malaya K, Fung Eula, Odegaard Justin I, Davis Ronald W, Pinsky Benjamin A, Scharfe Curt
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA.
Stanford Genome Technology Center, Stanford University, Palo Alto, CA.
J Appl Lab Med. 2018 Mar;2(5):757-769. doi: 10.1373/jalm.2017.024521.
Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation.
We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients.
For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR.
Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.
病毒感染是实体器官和造血细胞移植中并发症和死亡的主要原因。
我们开发了一种多重病毒测序检测方法（mVseq），用于从小临床样本中同时检测20种与移植相关的DNA病毒。该检测方法使用单管多重PCR扩增高度保守的病毒基因组区域，无需事先进行病毒富集或宿主核酸扣除。使用Illumina MiSeq进行多重样本测序，并将读数与目标序列数据库进行比对。使用掺入人血浆的参考病毒以及患者血浆和非血浆样本（包括免疫功能低下的移植受者的支气管肺泡灌洗液、脑脊液、尿液和组织）评估分析性能和临床性能。
对于病毒掺入样本，mVseq的分析灵敏度和动态范围与定量PCR（qPCR）相似。在临床标本中，mVseq与单靶点qPCR显示出高度一致性（92%；k统计量，0.77；282次病毒检测中的259次）；然而，临床灵敏度降低（81%），特定病毒的灵敏度范围为62%至100%。在47名接受检测的患者中，有12名患者通过mVseq检测出先前未知的BK病毒、人类疱疹病毒7型和EB病毒感染，这些感染经qPCR证实。
我们的结果揭示了可能影响临床灵敏度的因素，例如宿主DNA背景水平高以及当一种病毒浓度远高于其他病毒时在合并感染中检测失败。mVseq检测方法具有灵活性和可扩展性，可纳入RNA病毒、感兴趣的新兴病毒以及对移植受者重要的其他病原体。
