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

Isolation of cytomegalovirus (CMV) in tissue culture is presently the most reliable means of proving active CMV infection. To improve both the cost-effectiveness and clinical usefulness of procedures for the isolation of CMV from fresh clinical specimens, we analyzed results obtained with standard isolation procedures and compared them with results obtained under different conditions. Cell monolayers from commercial sources were inoculated with fresh specimens and then were observed for a cytopathic effect typical of CMV. Of 1,375 specimens submitted over a 12-month period, 6.4% were CMV positive in WI-38 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 +/- standard deviation), 9.9 +/- 3.3, and 7.7 +/- 3.3 days, respectively, and 92% were positive within 14 days. When 1,058 subsequent specimens were inoculated in parallel onto WI-38 and MRC-5 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, but 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. Based on these data, we have developed a practical 2-week protocol for CMV isolation from fresh clinical specimens that includes the use of MRC-5 cell monolayers incubated at 36 degrees C.