Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil
<b>Background/Objectives:</b> 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 <i>UL97</i> gene are frequently associated w...
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MDPI AG
2025-01-01
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| Online Access: | https://www.mdpi.com/2075-4418/15/2/214 |
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| author | Anna Caroline Avila da Rocha Grazielle Motta Rodrigues Alessandra Helena da Silva Hellwig Dariane Castro Pereira Fabiana Caroline Zempulski Volpato Afonso Luís Barth Fernanda de-Paris |
| author_facet | Anna Caroline Avila da Rocha Grazielle Motta Rodrigues Alessandra Helena da Silva Hellwig Dariane Castro Pereira Fabiana Caroline Zempulski Volpato Afonso Luís Barth Fernanda de-Paris |
| author_sort | Anna Caroline Avila da Rocha |
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| description | <b>Background/Objectives:</b> 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 <i>UL97</i> 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 <i>UL97</i> gene from plasma samples of transplant patients treated at Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. <b>Methods:</b> A nested-PCR approach combined with a touchdown PCR method was employed to enhance the sensitivity and specificity of the sequencing analysis. <b>Results:</b> 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. <b>Conclusions:</b> The optimized Sanger sequencing protocol provides a cost-effective solution for detecting GCV resistance mutations in HCMV <i>UL97</i> 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. |
| format | Article |
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| institution | Kabale University |
| issn | 2075-4418 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
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| series | Diagnostics |
| spelling | doaj-art-6a7abb76b6d947d0af589547e2e560e52025-01-24T13:29:08ZengMDPI AGDiagnostics2075-44182025-01-0115221410.3390/diagnostics15020214Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern BrazilAnna Caroline Avila da Rocha0Grazielle Motta Rodrigues1Alessandra Helena da Silva Hellwig2Dariane Castro Pereira3Fabiana Caroline Zempulski Volpato4Afonso Luís Barth5Fernanda de-Paris6Faculdade de Biomedicina, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre 90050-170, Rio Grande do Sul, BrazilPrograma de Pós-Graduação em Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre 90160-093, Rio Grande do Sul, BrazilPrograma de Pós-Graduação em Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre 90160-093, Rio Grande do Sul, BrazilLABRESIS–Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre 90035-903, Rio Grande do Sul, BrazilDepartamento de Biociências, Universidade Federal do Paraná, Setor Palotina, Palotina 85953-128, Paraná, BrazilLABRESIS–Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre 90035-903, Rio Grande do Sul, BrazilLABRESIS–Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre 90035-903, Rio Grande do Sul, Brazil<b>Background/Objectives:</b> 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 <i>UL97</i> 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 <i>UL97</i> gene from plasma samples of transplant patients treated at Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. <b>Methods:</b> A nested-PCR approach combined with a touchdown PCR method was employed to enhance the sensitivity and specificity of the sequencing analysis. <b>Results:</b> 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. <b>Conclusions:</b> The optimized Sanger sequencing protocol provides a cost-effective solution for detecting GCV resistance mutations in HCMV <i>UL97</i> 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.https://www.mdpi.com/2075-4418/15/2/214cytomegalovirusmutationganciclovir resistancetransplant recipients |
| spellingShingle | Anna Caroline Avila da Rocha Grazielle Motta Rodrigues Alessandra Helena da Silva Hellwig Dariane Castro Pereira Fabiana Caroline Zempulski Volpato Afonso Luís Barth Fernanda de-Paris Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil Diagnostics cytomegalovirus mutation ganciclovir resistance transplant recipients |
| title | Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil |
| title_full | Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil |
| title_fullStr | Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil |
| title_full_unstemmed | Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil |
| title_short | Ganciclovir Resistance-Linked Mutations in the HCMV <i>UL97</i> Gene: Sanger Sequencing Analysis in Samples from Transplant Recipients at a Tertiary Hospital in Southern Brazil |
| title_sort | ganciclovir resistance linked mutations in the hcmv i ul97 i gene sanger sequencing analysis in samples from transplant recipients at a tertiary hospital in southern brazil |
| topic | cytomegalovirus mutation ganciclovir resistance transplant recipients |
| url | https://www.mdpi.com/2075-4418/15/2/214 |
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