Andreani Julien, Truffot Aurélie, Tilloy Valentin, Jardin Hugo, Lespinasse Matilda, Usal Marie, Larrat Sylvie, Morand Patrice, Lupo Julien, Hantz Sébastien, Alain Sophie, Germi Raphaële
Univ. Grenoble Alpes, CNRS, CEA, IRIG IBS, Grenoble, France.
Virology Laboratory, Institut de Biologie et de Pathologie, Grenoble, France.
Antimicrob Agents Chemother. 2025 Aug 6;69(8):e0014125. doi: 10.1128/aac.00141-25. Epub 2025 Jun 25.
Cytomegalovirus (CMV) infections occur in 10%-40% of organ or stem cell transplant patients. Despite the low prevalence, CMV antiviral resistance has an important impact on patient outcomes. Guidelines for transplant recipients recommend that resistance should be suspected in cases of unchanged or increasing CMV viral loads after a minimum of 2 weeks of antiviral therapy at an appropriate dose or >6 weeks of ganciclovir exposure. Next-generation amplicon sequencing (NGS) makes it possible to directly target the genes involved in this resistance. Currently, six drugs are available, and six CMV genes (UL54-UL97-UL89-UL56-UL51-UL27 genes) can harbor mutations affecting drug efficacy. Here, we developed different primers targeting these six genes with long-range polymerase chain reaction (PCR). Based on clinical requirements, all genes or a subset could be sequenced in a single run using Oxford Nanopore technology and combined with an automatic bioinformatics pipeline to detect and report mutations. We utilized 46 blood samples, five external quality controls, and 10 mixes of two bacmids provided by the national reference center (CNR) Herpesvirus Limoges, each carrying distinct mutations. Assay performance (sensitivity, specificity, and accuracy) was evaluated through an interlaboratory exchange with CNR Herpesvirus. Long-range PCR combined with next-generation sequencing analysis enables earlier and more comprehensive discrimination of the double population and determines whether the detected single-nucleotide polymorphisms are present on single or multiple CMV strains. We developed a next-generation sequencing assay combined with eight long-range PCRs to sequence all genes involved in CMV antiviral resistance and to detect early low-frequency mutations.
巨细胞病毒（CMV）感染发生在10%-40%的器官或干细胞移植患者中。尽管患病率较低，但CMV抗病毒耐药性对患者的预后有重要影响。移植受者指南建议，在以适当剂量进行至少2周的抗病毒治疗后，若CMV病毒载量未改变或增加，或在接受更昔洛韦治疗超过6周后，应怀疑存在耐药性。新一代扩增子测序（NGS）使得直接靶向参与这种耐药性的基因成为可能。目前有六种药物可用，六个CMV基因（UL54-UL97-UL89-UL56-UL51-UL27基因）可能存在影响药物疗效的突变。在此，我们使用长程聚合酶链反应（PCR）开发了针对这六个基因的不同引物。根据临床需求，所有基因或一个子集可以在一次运行中使用牛津纳米孔技术进行测序，并与自动生物信息学管道相结合，以检测和报告突变。我们使用了46份血液样本、5份外部质量控制样本以及法国利摩日国家疱疹病毒参考中心（CNR）提供的10种携带不同突变的两种杆粒混合物。通过与CNR疱疹病毒进行实验室间交换来评估检测性能（敏感性、特异性和准确性）。长程PCR结合新一代测序分析能够更早、更全面地区分双种群，并确定检测到的单核苷酸多态性是存在于单个还是多个CMV菌株上。我们开发了一种结合八个长程PCR的新一代测序检测方法，用于对所有参与CMV抗病毒耐药性的基因进行测序，并检测早期低频突变。
