Effect of Age of Shell Vial Monolayers on Detection of ...

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of MRC-5 cells on 12-mm round cover slips and MRC-5 cells in cell culture flasks used for the preparation of shell vials in our laboratory were obtained from ...


Vol. 27, No. 9

0095-1137/89/092107-03$02.00/0 Copyright © 1989, American Society for Microbiology

Effect of Age of Shell Vial Monolayers on Detection of Cytomegalovirus from Urine Specimens DANIEL P.






Sections of Clinical Microbiology' and Biostatistics,2 Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 Received 31 January 1989/Accepted 15 May 1989

The effect of age of MRC-5 cell monolayers in shell vials on the detection of cytomegalovirus (CMV) from urine was evaluated. When the AD169 strain of CMV was used, 8-day-old monolayers had a higher mean count of fluorescent foci than 15-day-old monolayers did (5.78 versus 2.86) (P < 0.02) and were more frequently positive (21 of 23 shell vials versus 14 of 22 shell vials) (P < 0.04). Commercial shell vials used for clinical specimens were evaluated in groups of 8- to 11-, 12- to 15-, and 8- to 15-day-old monolayers. When compared with laboratory-prepared shell vials ranging in age from 3 to 9 days, commercial shell vials had a lower number of fluorescent foci in all groups (P < 0.03, P < 0.0001, and P < 0.0001, respectively), the 12- to 15- and 8- to 15-day-old groups were less frequently positive (P s 0.02 and P < 0.02, respectively), and ail three groups were more susceptible to the toxic effects of urine (P < 0.0001, P < 0.01, and P < 0.0001, respectively). For all 191 specimens cultured (8- to 15-day-old group), one or both monolayers were destroyed in 60 (31.4%) specimens compared with 9 (4.7%) specimens toxic to laboratory-prepared shell vials (P < 0.0001). Both the decreased sensitivity of older MRC-5 cells and the increased sensitivity to the toxic effects of urine made commercial shell vials less sensitive than laboratory-prepared shell vials for the detection of CMV.

Infections with cytomegalovirus (CMV) are a major cause of morbidity and mortality in immunosuppressed patients such as transplant recipients or patients with acquired immunodeficiency syndrome (5). The development of ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine], an effective therapeutic agent for CMV infections, has made rapid detection of the virus essential for patient management (1). Specific monoclonal antibodies against an early antigen of CMV (7) combined with the shell vial cell culture technique (3, 4) enable the laboratory to detect CMV within 16 h after inoculation of the specimen. Shell vials containing cell monolayers are routinely obtained from commercial sources because of considerations such as a low volume of specimens, cost, and technical demands relative to the preparation of these cell cultures. Shell vial cell culture monolayers of MRC-5 cells prepared by our laboratory are usually confluent and ready for inoculation 3 days after seeding; they are not used for culture if they are over 9 days old. Because of production and shipping schedules, MRC-5 cell monolayers in shell vials from commercial vendors may be at least 5 to 7 days old when received by a laboratory. With shipments of shell vials only on a weekly basis, the age of monolayers used for the isolation of CMV can range from 6 to 14 days. Thiele et al. demonstrated that monolayers older than 11 days may lose their susceptibility to infection by CMV (9). We examined the effect of age of commercially prepared shell vials with MRC-5 cell monolayers 8 and 15 days old on infection with the AD169 laboratory strain of CMV. In addition, the recovery of CMV from 191 urine specimens inoculated into shell vial cell cultures (8 to 15 days after seeding) was compared with recovery of the virus in shell vials prepared in our laboratory (3 to 9 days after seeding). Commercially prepared shell vials containing monolayers

of MRC-5 cells on 12-mm round cover slips and MRC-5 cells in cell culture flasks used for the preparation of shell vials in our laboratory were obtained from Viromed Laboratories, Minneapolis, Minn. Shell vials (1 dram; ca. 3.7 ml) with a 12-mm cover slip in each were seeded with 50,000 MRC-5 cells per ml to produce a monolayer within 3 days and were maintained for up to 9 days at 36°C with Eagle minimal essential medium containing 10% fetal bovine serum, sodium bicarbonate, Tris buffer, penicillin, streptomycin, and gentamicin (8). The AD169 strain (ATCC VR-538) was used as a positive control and for studies of the effect of age of MRC-5 monolayers on viral infectivity. Commercial shell vial monolayers were 6 to 8 days old when received by our laboratory. Medium was not changed in the commercial vials because the cell culture did not appear acidic, even after storage at 36°C for 7 to 9 days. Stock virus was diluted with minimal essential medium to obtain approximately 75 focus-forming units per ml. Monolayers 8 and 15 days old were inoculated with 0.2 ml of the viral suspension, centrifuged, incubated, and stained as previously described (3, 4). After the shell vials were centrifuged for 40 min at 700 x g, 1 ml of minimal essential medium was added, and the cultures were incubated at 36°C for 16 h. The infected monolayers on the cover slips within the shell vials were washed two times with phosphatebuffered saline and then fixed in cold acetone for 10 min. The fixed monolayers were stained by the indirect immunofluorescence technique (8) by using a monoclonal antibody specific for the 72,000-dalton early nuclear protein of CMV (Dupont Specialty Diagnostics, Wilmington, Del.) and goat anti-mouse immunoglobulin G fluorescein isothiocyanatelabeled conjugate (Cooper BioMedical, Inc., West Chester, Penn.). Stained cover slips were numerically coded and examined for fluorescent foci in a blinded fashion. Laboratory-prepared shell vial monolayers were between 3 and 9 days old, and commercial sheil vial monolayers were 8 to 15 days old when used for inoculation of urine specimens. Before inoculation, all shell vial monolayers were screened by using an inverted microscope to ensure the

* Corresponding author. t Present address: Department of Pathology, Hurley Medical Center, Flint, MI 48502.






TABLE 1. Number of urine specimens positive and negative for CMV No. of cultures

Age group of


commercial shell vials (days)

CL o'-,

2 0


a b


Pstv Positive

Both Laboratory systems prepared

Commercially prepared



in both systems







12-15 8-15

28 49

7 14

0 3

44 125

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