da Rocha Anna Caroline Avila, Rodrigues Grazielle Motta, da Silva Hellwig Alessandra Helena, Pereira Dariane Castro, Volpato Fabiana Caroline Zempulski, Barth Afonso Luís, de-Paris Fernanda
Faculdade de Biomedicina, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre 90050-170, Rio Grande do Sul, Brazil.
LABRESIS-Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre 90035-903, Rio Grande do Sul, Brazil.
Diagnostics (Basel). 2025 Jan 18;15(2):214. doi: 10.3390/diagnostics15020214.
Human cytomegalovirus (HCMV) DNAemia remains a significant concern for transplant recipients, largely due to mutations in the viral genome that may lead to antiviral-resistant strains. Mutations in the gene are frequently associated with resistance to ganciclovir (GCV), highlighting the importance of early mutation detection to effectively manage viremia. This study aimed to optimize a Sanger sequencing protocol for analyzing GCV resistance-linked mutations in the HCMV gene from plasma samples of transplant patients treated at Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. A nested-PCR approach combined with a touchdown PCR method was employed to enhance the sensitivity and specificity of the sequencing analysis. The study sample included various transplants, encompassing solid organ and bone marrow recipients. Among 16 sequenced samples, 8 exhibited nucleotide substitutions resulting in amino acid changes. Notably, the A594V and C603W mutations, associated with GCV resistance, were identified in four samples. Additionally, three mutations with unknown phenotypic impact (P509L, A628T, and H662Y) and two viral polymorphisms (N510S and D605E) were detected. Furthermore, double peaks in the Sanger electropherograms, indicative of mixed viral populations of HCMV were observed in seven samples. The optimized Sanger sequencing protocol provides a cost-effective solution for detecting GCV resistance mutations in HCMV among transplant recipients. This approach could improve the understanding of HCMV strain dynamics and serve as a valuable tool for long-term patient monitoring, particularly within resource-constrained settings such as the public health systems of middle-income countries.
人巨细胞病毒（HCMV）血症仍然是移植受者的一个重大问题，这主要是由于病毒基因组中的突变可能导致抗病毒耐药菌株。该基因的突变经常与更昔洛韦（GCV）耐药相关，这凸显了早期突变检测对于有效控制病毒血症的重要性。本研究旨在优化一种桑格测序方案，用于分析来自巴西南里奥格兰德州阿雷格里港临床医院接受治疗的移植患者血浆样本中HCMV基因中与GCV耐药相关的突变。采用巢式PCR方法结合降落PCR方法来提高测序分析的灵敏度和特异性。研究样本包括各种移植类型，涵盖实体器官和骨髓受者。在16个测序样本中，有8个出现了导致氨基酸变化的核苷酸替换。值得注意的是，在4个样本中发现了与GCV耐药相关的A594V和C603W突变。此外，还检测到3个表型影响未知的突变（P509L、A628T和H662Y）以及2个病毒多态性（N510S和D605E）。此外，在7个样本的桑格电泳图中观察到双峰，这表明存在HCMV混合病毒群体。优化后的桑格测序方案为检测移植受者中HCMV的GCV耐药突变提供了一种经济有效的解决方案。这种方法可以增进对HCMV毒株动态的了解，并作为长期患者监测的有价值工具，特别是在中等收入国家公共卫生系统等资源有限的环境中。
