Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir

Background of study: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variab le frequency under GCV therapy and is conferred by mutations (i) in the UL9 7 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which i s essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV. Objective: The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus is olation and nucleotide sequencing procedures. Study design: A nested PCR (nPCR) amplifying UL97 codons 450-672 was develo ped. Nested amplicons were subsequently sequenced directly. Oligonucleotide s for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD16 9 served as a wild-type control. Results: UL97-specific nPCR amplicons were obtained from Is EDTA blood samp les of ten transplant recipients receiving GCV for more than 30 days. In th ree consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-a dapted HCMV isolates known to exhibit GCV resistance in vitro revealed muta tions at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybri dization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleo tide sequence analysis for all 23 amplicons examined. Conclusions: Reverse hybridization appeared to be a rapid and convenient al ternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance. (C) 1999 Elsevier Science B.V. All rig hts reserved.