Use of time-saving flow cytometry for rapid determination of resistance of human cytomegalovirus to ganciclovir.
There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL 97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL 54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC(50)) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC(50) values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M 460 V, and the GCV IC(50) results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.
Lee
Gyu-Cheol
GC
Clinical Research Institute, St. Mary's Hospital, The Catholic University of Korea, College of Medicine, 150-713 Seoul, Korea.
Lee
Dong-Gun
DG
Choi
Su-Mi
SM
Yoo
Jin-Hong
JH
Park
Sun-Hee
SH
Choi
Jung-Hyun
JH
Min
Woo-Sung
WS
Cho
Ok-Hee
OH
Lee
Chan-Hee
CH
Shin
Wan-Shik
WS
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D003587:Cytomegalovirus / Q000187:drug effects*; D003586:Cytomegalovirus Infections / Q000821:virology; D024882:Drug Resistance, Viral / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015774:Ganciclovir / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D013997:Time Factors; D010948:Viral Plaque Assay
D000998:Antiviral Agents; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir