Practical protocol for cytomegalovirus isolation: use of MRC-5 cell monolayers incubated for 2 weeks

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Abstract
Isolation of cytomegalovirus (CMV) in tissue culture is presently the most reliable means of proving active CMV infection. To improve the cost-effectiveness and clinical usefulness of procedures for the isolation of CMV from fresh clinical specimens, results obtained with standard isolation procedures were analyzed and compared with results obtained under different conditions. Cell monolayers from commercial sources were inoculated with fresh specimens and then observed for a cytopathic effect typical of CMV. Of 1375 specimens submitted over 12-mo., 6.4% were CMV positive in WI-38 [human fetal lung fibroblast] monolayers within 28 days after inoculation. The mean day of CMV detection for 45 urine, 13 cervical-vaginal and 5 saliva specimens was 6.7 .+-. 3.1 (mean .+-. SD), 9.9 .+-. 3.3 and 7.8 .+-. 3.3 days, respectively; 92% were positive within 14 days. When 1058 subsequent specimens were inoculated in parallel onto WI-38 and MRC-5 [human embryonic lung diploid] cell monolayers, 8.7% were positive for CMV. MRC-5 cells were significantly more sensitive than WI-38 cells: 98% of all positive specimens appropriate for comparison were detected in MRC-5 cultures; only 85% were detected in WI-38 cells. Although 1 specimen was positive in WI-38 cells only, 38% of all isolates were positive earlier (16 specimens) or only (10 specimens) in MRC-5 cultures. Thus, a practical 2 wk protocol for CMV isolation from fresh clinical specimens was developed that includes the use of MRC-5 cell monolayers incubated at 36.degree. C.