Abstract
Background/Objectives: 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 UL97 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 UL97 gene from plasma samples of transplant patients treated at Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. Methods: A nested-PCR approach combined with a touchdown PCR method was employed to enhance the sensitivity and specificity of the sequencing analysis. Results: 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. Conclusions: The optimized Sanger sequencing protocol provides a cost-effective solution for detecting GCV resistance mutations in HCMV UL97 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.