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1.
Med Microbiol Immunol ; 200(2): 109-13, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21165648

RESUMEN

Prenatally acquired human cytomegalovirus (HCMV) infection is the most frequent viral infection of newborns in developed countries. Virostatic therapy is accompanied by side effects and stepwise emergence of resistant virus variants. Different genotypic approaches show limited sensitivity in detecting on-growing minor resistant virus populations. Here, we demonstrate the superiority of pyrosequencing for the monitoring of mutant emergence. In a preterm baby born after 28 weeks of gestation and suffering from disseminated congenital HCMV infection, long-term control could not be achieved under ganciclovir/valganciclovir therapy and the infant died on the 113th day of life. Resistance-associated mutations in the HCMV UL97 gene were not detected by conventional DNA sequencing but postmortem pyrosequencing. Four different CMV variants carrying resistance-associated mutations each representing 11-17% of the total CMV population were found.


Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/virología , Citomegalovirus/efectos de los fármacos , Farmacorresistencia Viral/genética , Ganciclovir/farmacología , Enfermedades del Prematuro/virología , Mutación , Análisis de Secuencia de ADN/métodos , Antivirales/uso terapéutico , Autopsia , Citomegalovirus/genética , Infecciones por Citomegalovirus/tratamiento farmacológico , ADN Viral/genética , Farmacorresistencia Viral/efectos de los fármacos , Femenino , Ganciclovir/uso terapéutico , Humanos , Recién Nacido , Recien Nacido Prematuro , Enfermedades del Prematuro/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa/métodos
2.
Antimicrob Agents Chemother ; 54(12): 5234-41, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20876379

RESUMEN

Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.


Asunto(s)
Citomegalovirus/genética , ADN Viral/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Citomegalovirus/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad
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