Enhanced detection of cytomegalovirus in MRC-5 shell vials
Journal of Clinical Microbiology
West and coworkers (6) reported differences in the sensitivities of MRC-5 monolayers to cytomegalovirus (CMV) among different suppliers (i.e., Whittaker Bioproducts, Bartels, and ViroMed). The authors further suggested that pretreatment of shell vials with the (glucocorticoid) dexamethasone plus dimethyl sulfoxide (DEX-DMSO) and DEX-DMSO plus Ca enhanced CMV recovery and that this effect was most pronounced in shell vials supplied by Whittaker. On the basis of recent studies by this worker and
... olleagues (4, 5), some important questions in experimental design should be brought to the attention of the readership of the Journal of Clinical Microbiology before the conclusions by the SmithKline investigators are drawn. Firstly, and perhaps most importantly, studies by Leonardi and Lipson (4) and Lipson et al. (5) using laboratoryadapted and wild CMV strains as well as freshly collected peripheral blood demonstrated that MRC-5 monolayer sensitivity may be enhanced following a medium enrichment effect. In our studies specifically, monolayer sensitivity was enhanced and CMV isolation was increased in shell vials pretreated with Eagle minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum (EMEM-10% FBS) compared with EMEM-2% FBS. Regarding the West et al. (6) study, I cannot help but question whether refeeding with an enriched culture medium, that is, EMEM-10% FBS (toxic cellular metabolites would be removed from the system by this step as well), might have increased the sensitivity and overall recovery rate of CMV in the untreated shell vials to the levels obtained by using monolayers pretreated with EMEM-2% FBS plus DEX-DMSO or DEX-DMSO-Ca. According to our data, for example, no significant differences were identified in the rates of CMV recovery from blood specimens which were inoculated into shell vials pretreated with either DMSO or EMEM-10% FBS (5). A second point arises with the fact that most diagnostic virology laboratories do not recommend that potentially CMV-positive clinical specimens remain in storage (at a refrigeration temperature [i.e., 4°C]) longer than from 24 to 48 h. Ideally, such specimens should be received and processed by the laboratory within hours of collection (1, 5). The testing of 1to 2-week-old preselected positive clinical specimens (the authors claim that "the number of [shell] vials available from each supplier was limited"), although extremely convenient for such comparative studies, may not necessarily reflect that which may take place when freshly collected clinical specimens are tested. Although the data of West et al. (6) imply differences in the sensitivities of MRC-5 cultures supplied by different manufacturers, the development of artifacts (possibly due to, e.g., the formation of particulates-precipitates or changes in pH) in the 1to 2-week-old clinical specimens cannot be ignored as possible confounding factors which might influence interpretation of the data. In summary, a valid protocol to investigate the issues raised by West and colleagues may be appropriately addressed by the performance of comparative testing utilizing different concentrations of FBS (i.e., at the very least, the routinely used EMEM-2% FBS and an enriched culture medium containing 10% FBS) and DEX, as well as the screening offieshly collected clinical specimens. We are in agreement with others (2, 3) that additional testing utilizing clinical specimens secured from varied sources is needed to substantiate the recommended use of dexamethasone as an enhancement agent of CMV infectivity. REFERENCES 1. Hodinka, R. L., and H. M. Friedman. 1991. Human cytomegalovirus, p. 829-837. (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 4. Leonardi, G. P., and S. M. Lipson. 1992. Enhanced detection of cytomegalovirus in shell vial culture following MRC-5 monolayer pretreatment with glucocorticoids. Zentralbl. Bakteriol. 277:90-99. 5. Lipson, S. M., MComparison of sensitivities of three commercial MRC-5 cell lines grown in shell vials to cytomegalovirus and responses to enhancing agents.