L Prix1, J Maierl, G Jahn, K Hamprecht. 1. Department of Medical Virology and Epidemiology of Viral Diseases, University of Tübingen, Germany.
Abstract
BACKGROUND: Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. OBJECTIVE: The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. STUDY DESIGN: Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. RESULTS: All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined 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. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.
BACKGROUND: Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. OBJECTIVE: The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. STUDY DESIGN: Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. RESULTS: All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined 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. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.