Identificazione rapida di mutazioni associate a farmaco-resistenza in ceppi di citomegalovirus umano mediante nPCR-RFLP

We developed a nested-PCR followed by restriction fragment length polymorphism (RFLP) for the detection of human cytomegalovirus (HCMV) UL97 M460V/I, H520Q, C592Q,A594V, L595S/F and C603W mutations associated to ganciclovir (GCV) resistance.The method uses five primer pairs and seven enzymes already published and newly combined.The detection limit of nPCR was assessed in a single serial dilution assay to be about 0.13 PFU/reaction. Expected restriction fragment patterns were obtained by nPCR-RFLP on either wild-type reference strains or strains and sequences of HCMV containing mutations. Then the nPCR-RFLP was used on 24 sera/plasma belonging to 22 transplant recipients (kidney, bone marrow, or kidney-pancreas): 13 subjects never treated with GCV (control group) and 9 subjects treated with GCV oral profilaxis (study group). All codons detected from the control group (six in 8 cases and four in 1 case) were identified as wild-type. All codons detected from the study group (six in 6 cases, three in 2 cases, and four in the second sample of 1 case whose first sample was negative by nPCR) were wild-type except one, which showed a restriction pattern referring to M460V and/or M460I ATA-codified, definitively proved to be M460V by sequence analysis.This was the case of a renal transplant recipient at the end of profilaxis. In conclusion, the procedure seems to be quite sensitive and specific as well as able to detect mixed population of mutants or mutants and wild-type. It could represent a good tool in monitoring the emergence of HCMV mutants in renal transplant recipients treated with GCV.