A SIMPLIFIED ASSAY FOR SCREENING OF DRUG-RESISTANCE OF CELL-ASSOCIATED CYTOMEGALOVIRUS STRAINS

Background: Conventional phenotypic drug resistance determination of c ell-free clinical human cytomegalovirus (HCMV) isolates is usually ver y laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a su fficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplifie d, less work-intensive methods are employed. Objective: The aim of thi s work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaqu e reduction assay should be simplified or omitted, but on the other ha nd, the assay should be reliable and reproducible. Study design: Twent y-six clinical HCMV isolates from 20 immunocompromised patients (ten p re-treatment and 16 post-treatment with ganciclovir) were tested for d rug susceptibility with the simplified plaque reduction assay. Most is olates were tested at least twice in independent assays. Virus titrati on could be avoided by using four different doses of cell-associated v irus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Pro bit analysis. Results: All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 mu M, (range 0.2-12.2; median 0.95) an d a mean foscarnet ID50 value of 92.4 mu M (range 35.7-181; median 81) . All except one isolate were classified ganciclovir sensitive when co mpared to ID50 values of two ganciclovir resistant control stains (53. 7 +/- 6.4 and 12.7 +/- 0.9 mu M) and the sensitive laboratory strain T owne (2.1 +/- 0.8 mu M). Repeated tests of individual isolates were re producible, although the infectivity of the inoculum has not been dete rmined prior of the assay. The mean time which elapsed between receipt of the clinical specimen and read-out of the assay was circa 4 weeks. Conclusions: Phenotypic resistance testing of HCMV isolates following to this protocol drastically reduces expenditure of time and work. Th e assay allows reliably the discrimination of HCMV isolates as drug re sistant or sensitive according to the recent classification criteria o f the AIDS Clinical Trials Group (ACTG). The simple handling and uncom plicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use. (C) 1998 Elsevier Science B.V. All rights reserved.