Optimizing recovery of cytomegalovirus in the shell vial culture procedure

Abstract

We have investigated three factors that may be related to the recovery of cytomegalovirus (CMV) using the shell vial culture procedure. First, we compared fluorescent-antibody staining of shell vial cultures using a monoclonal antibody to a CMV immediate early antigen at 16 vs 40 hr after inoculation. Of 332 routinely submitted specimens cultured in duplicate and stained at the different times, 25 (7.5%) were positive at 16 hr and 32 (9.6%) were positive at 40 hr. The increased yield was 28%. Second, we analyzed the effect of using duplicate shell vials (both stained at 40 hr) for all routinely submitted CMV cultures. During a 6-month period, 272 (12.5%) of the 2157 cultures processed with duplicate shell vials were positive, including 222 positive in both vials and 50 positive in only one. Assuming that a single-vial setup would have detected 50% of those positive in only one of the two vials, the increased yield attributable to the duplicate vial was estimated at 10% (25/(222 + 25)). Third, we investigated the effects of seeding density and culture age on the shell vial assay. Cell age of greater than 1 day was associated with a decrease in sensitivity both in cultures that were confluent and in those that were subconfluent at the time of inoculation. Incorporating these findings in the routine shell vial culture procedure used in our Clinical Virology Laboratory has resulted in a greater overall detection of CMV in shell vial cultures than in conventional 6-week tube cultures.