DIAGN MICROBIOL INFECT DIS 1990;13:41-44

41

VIROLOGY

Effect of Treatment of Shell Vial Cell Cultures with Dimethyl Sulfoxide and Dexamethasone and Age of MRC-5 Monolayers for Detection of Cytomegalovirus Daniel P. Fedorko, Duane M. Ilstrup, and Thomas F. Smith

Pretreatment of MRC-5 cell monolayers in commercially prepared shell vials with 1 ~ dimethyl sulfoxide (DMSO) and 10-~ mol/L dexamethasone (DEX) was evaluated. Prelimina~, experiments indicated enhanced infectivity of AD-169 for pretreated MRC-5 cells in shell vials of ages 9 and 16 days. Compared with untreated shell vials, DMSO-DEX increased positivity (day 9, 19 vs. 26 shell vials, p < 0.03; day 16, 13 vs. 29 shell vials, p < 0.001) and increased the mean number of fluorescent foci (days 9 and 16, p < 0.001). Pretreatment of 8-15-day-old monolayers was evaluated clinically using 146

urine specimens. Fifty specimens were positive for cytomegalovirus (CMV) in both treated and untreated shell vials with ten positive in untreated only and three positive in treated only (p = NS). The median number of fluorescent foci was not significantly higher in treated shell vials. Increased toxicity of MRC-5 cells was observed in treated monolayers (p < 0.0001). Pretreatment with DMSO-DEX did not enhance CMV isolation from clinical specimens and can be toxic to MRC-5 monolayers.

Infections with cytomegalovirus (CMV) are c o m m o n in all age groups and are especially severe in neonates and i m m u n o s u p p r e s s e d patients (Lamberson, 1985). The shell vial cell culture technique is a rapid detection m e t h o d that uses MRC-5 cell monolayers on cover slips in shell vials and monoclonal antibodies specific for an early antigen of CMV. This technique has reduced the time of detection of CMV in clinical specimens from 7-21 days to 16 hr (Gleaves et al., 1984, 1985). Several reports have described e n h a n c e d viral replication and an increased n u m b e r

of infected cells after treatment of cells with dimethylsulfoxide (DMSO), and d e x a m e t h a s o n e (DEX), or a combination of these two agents (Tanaka et al., 1984a, 1984b, 1985; West et al., 1988; Thiele and Woods, 1988). In a previous study, we found DMSODEX treatment did not enhance the sensitivity of detection of CMV in 3-7-day-old shell vial cell monolayers prepared in our laboratory (Espy et al., 1988). Due to production and shipping schedules, cell monolayers in shell vials from commercial v e n d o r s may be at least 5-7 days old w h e n received by a laboratory. Therefore, we c o m p a r e d the detection of CMV from 146 urine specimens inoculated into commercially p r e p a r e d shell vials with cell m o n o l a y e r s in the presence and absence of DMSO-DEX to assess the effect of these drugs on the sensitivity of 8-15day-old cell monolayers. Shell vials containing m o n o l a y e r s of MRC-5 cells were obtained from Viromed Laboratories, Minneapolis, Minnesota. The AD-169 strain (ATCC UR-

From the Sections of Clinical Microbiology (D.P.F., T.F.S.) and Biostatics (D.M.I.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Address reprint requests to: Dr. Thomas F. S:nith, Section of Clinical Microbiology, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905. Received July 6, 1989; revised and accepted October 19, 1989. © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010. 0732-8893/90/$3.50

42

538) of CMV was used as a positive control and for preliminary studies of the effect of DMSO-DEX on the infectivity of the virus in MRC-5 cells. DMSO was purchased from Sigma Chemical Co., St. Louis, Missouri (catalog no. D-5879), and DEX was obtained from Goldline Laboratories, Ft. Lauderdale, Florida (catalog no. 0182-1614-19). Both drugs were used at concentrations of 1% and 10 ~ tool/L, respectively, in Eagle Minimal Essential Medial (MEM) containing 10°/c fetal bovine serum, sodium bicarbonate, tris buffer, penicillin, streptomycin, and gentamicin (Espy et al., 1988). Shell vials were treated with I ml MEM containing DMSO-DEX 24 hr preinoculation. Stock virus was diluted with MEM to obtain approximately 100 focus forming units/ml. Both treated and untreated monolayers of ages 9 and 16 days (29 shell vials in each group), were inoculated with 0.2 ml of the viral suspension, centrifuged, incubated, and stained as previously described (Espy et al., 1988). After the shell vials were centrifuged for 40 min at 700 x g, 1 ml of MEM or MEM containing DMSO-DEX was added and the cultures were incubated at 36°C for 16 hr. The infected monolayers on the cover slips within the shell vials were washed twice with phosphate buffered saline and then fixed in cold acetone for 10 min. The fixed monolayers were stained by the indirect immunofluorescence technique (Smith, 1985) using a monoclonal antibody, commercially available from Dupont Specialty Diagnostics, Wilmington, Delaware, which was specific for the 72,000-dalton early nuclear protein of CMV (Shuster et al., 1985) and goat anti-mouse immunoglobulin G-fluorescein isothiocionate-labeled conjugate (Cappel Laboratories, Westchester, Pennsylvania). Stained cover slips were numerically coded and then examined for fluorescent foci in a blinded fashion. After completion of the experiment with the AD169 laboratory strain of CMV, a study using clinical specimens was undertaken. Urine specimens from 84 (57%) patients from a wide variety of clinical services at the Mayo Clinic and 62 (43%) specimens from patients submitted through Mayo Medical Reference Laboratories were processed in our study. The specimens from 73 of the Mayo Clinic patients came from a variety of clinical areas: gastroenterology (23), renal/liver/bone marrow/heart transplants (19), general medical oncology (9), respiratory intensive care unit (7), dialysis (5), ophthalmology (3), pediatrics (3), rheumatology (2), and hematology (2). The monolayers were 8-15 days old when used for inoculation of urine specimens. MEM was removed from the shell vial cultures before inoculation and 0.2 ml of urine was added to each of two treated and two untreated shell vials. The shell vial cultures were centrifuged, incubated, and the infected monolayers stained as previously described.

D.P. Fedorko et al.

40

ffl 0

"-I ,-r 10

Untreated T r e a t e d

Untreated Treated

9-day-old cells

16-day-oldcells

FIGURE 1. Mean number of ADI69 fluorescent foci in MRC-5 monolayers ages 9 and 16 days.

Both 9-day- and 16-day-old monolayers of MRC5 ceils within commercially prepared shell vials demonstrated enhanced infectivity of the AD-169 strain of CMV after DMSO-DEX treatment. Compared with untreated monolayers, DMSO-DEX pretreatment increased positivity; 9-day-old monolayers were positive in 19 vs. 26 shell vials (p < 0.03, Fisher's exact test) and 16-day-old monolayers positive in 13 vs. 29 shell vials (p ( 0.001, Fisher's exact test). An increase in the number of fluorescent foci was also observed in treated monolayers after 16 hours of incubation (9 and 16 day, p < 0.001, Rank-sum test) (Fig. 1). Cytomegalovirus was detected in 63 of 146 urine specimens. The results were evaluated by comparing the shell vials as groups of 8-11-day-old (68 specimens), 12-15-day-old (78 specimens), and 8-15-dayold (all 146 specimens) monolayers. Although a trend towards increased positivity in untreated monolayers was observed for all shell vial age groups, the results were not statistically significant (Table 1). The sum of counts of fluorescent foci from both cover slips inoculated, when both treatment groups were positive, was not significantly higher in treated vs. untreated monolayers for all age groups. Increased toxicity was observed in MRC-5 cells of all age groups when treated with DMSO-DEX. Toxicity was defined as either complete loss of the monolayer from the cover slip after fixation and staining or greater than one-half of a monolayer lost when there were no fluorescent loci present. In the 8-11-day and 12-15-day groups, toxicity was most pronounced in the older group of cells (p ( 0.01 vs. p ( 0.0003, Wilcoxon signed-rank test) (data not shown). For all shell vials, toxicity was significantly greater in treated monolayers were one or both monolayers were destroyed in 67 (46%) specimens compared to destruction in 39 (27%) specimens in

Cytomegalovirus Isolation in Treated Cells

TABLE 1.

43

Number of Urine Specimens Positive for CMV Number of Cultures

Age Group of Commercial Shell Vials (days)

Positive in B o t h Treatments

Positive Only in Untreated Cells

Positive Only in Treated Cells

Negative in Both Treatments

p values

8-11 12-15 8-15

19 31 50

5 5 10

2 1 3

42 41 83

>0.10 >0.10 0.5 >p > 0.10

untreated monolayers (p < 0.0001, Wilcoxon signedrank test). It is well established that mechanical assistance, i.e., centrifugation, greatly increases the sensitivitv of the rapid shell vial method for the detection of CMV (Gleaves et al., 1984; Thiele et al., 1987). The addition of chemical agents to the culture media in shell vials could further enhance this already sensitive technique. This idea was brought to the forefront by reports of enhanced detection of CMV in cell culture using DMSO and DEX (Tanaka et al., 1984a, 1984b, 1985). In a previous study, we pretreated MRC-5 monolayers in shell vials with 10 5 mol/L DEX and 1'7c DMSO and found neither an increase in the number of fluorescent foci nor increased detection of CMV (Espy et al., 1988). Using the same concentrations of DEX and DMSO, West et al. (1988) have reported both an increase in the number and size of fluorescent foci and enhanced detection of CMV in commercially prepared shell vials. Using 24 well plates, Thiele and Woods (1988) demonstrated an increase in the number of CMV fluorescent foci per coverslip after DEX treatment. However, they found no significant difference in the detection of CMV in treated and untreated MRC-5 cells. The optimal concentration of DEX was reported to be 10-5 mol/L. The results of our present study, using 10 5 mol/ L DEX and 1% DMSO pretreatment of commercially prepared shell vials for the detection of CMV from urine, are consistent with those of our previous study (Espy et al., 1988) and with those of Thiele and Woods (1988) in terms of the lack of enhancing the detection of CMV. However, we did observe an increase in the number of fluorescent foci and increased positivity using the AD169 laboratory strain of CMV. We feel there may be significant differences in replication of laboratory and clinical strains of CMV in relationship to cells treated with DMSO-DEX. We have noted inconsistent findings with DMSO

and DEX treatment not only among publications, but also within publications. For example, with a significant increase in the number of fluorescent foci per coverslip in treated cells an increase in positivity would also be expected. However, no significant increase in positivity of the rapid CMV detection system was reported by Thiele and Woods (1988). In two different experiments using identical concentrations of DMSO-DEX and pretreatment times, West et al. (1988) reported an 87% increase in the number of specimens with more inclusions in one experiment, but only a 4% increase in a second experiment. Thus, the interaction of these compounds with CMV infected cells in rapid detection systems is not a simple one. Optimal treatment times and concentrations may have yet to be determined. Although the concentrations of DMSO and DEX used in our study have been reported to be nontoxic (West et al., 1988), we observed increased toxicity of cells treated with these compounds. This increase in toxicity was not observed in the laboratory-prepared shell vials used in our previous study (Espy et al., 1988). Commercially prepared MRC-5 cell monolayers in shell vials are generally older than laboratory-prepared monolayers when used in the clinical virology laboratory. Toxicity in the 12-15day-old shell vials was greater than that observed in 8-11-day-old shell vials. We do not know if the DMSO-DEX was itself toxic to these older cells or the DMSO-DEX made these cells more sensitive to the toxic effects of urine. In summary, potentiation of CMV interaction with cells using chemicals is an important concept. The determination of appropriate regimens, to obtain optimal sensitivity without increased toxicity, is critical before DMSO-DEX can be routinely used in the clinical laboratory. Therefore, we do not recommend that shell vial MRC-5 cell monolayers be treated with DMSO-DEX in an attempt to enhance the detection of CMV in clinical specimens.

REFERENCES Espy MJ, Wold AD, Ilstrup DM, Smith TF (1988) Effect of treatment of shell vial cell cultures with dimethyl sulf-

oxide and dexamethasone for detection of cytomegalovirus. J Clin Microbiol 26:1091-1093.

44

Gleaves CA, Smith TF, Shuster EA, Pearson GR (1985) Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J Clin Microbiol 21:217-221. Gleaves CA, Smith TF, Shuster EA, Pearson GR (1984) Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J Clin Microbiol 19:917-919. Lamberson HV (1985) Cytomegalovirus (CMV): The agent, its pathogenesis, and its epidemiology. Prog Clin Biol Res 182:149-173. Shuster EA, Beneke JS, Tegtmeier GE, Pearson GR, Gleaves CA, Wold AD, Smith TF (1985) Monoclonal antibody for rapid laboratory detection of cytomegalovirus infections: characterization and diagnostic application. Mayo Clin Proc 60:577-585. Smith TF (1985) Viruses. In Laborato~ Procedures in Clinical Microbiology. Ed., JA Washington. New York: SpringerVerlag, pp 537-624. Tanaka J, Kamiya S, Ogura T, Sato H, Ogura H, Hatano M (1985) Effect of dimethyl sulfoxide on interaction of human cytomegalovirus with host cell: conversion of

D.P. Fedorko et al.

a nonproductive state of cell to a productive state for virus replication. Virology 146:165-176. Tanaka J, Ogura T, Kamiya S, Sato H, Yoshie T, Ogura H, Hatano M (1984a) Enhanced replication of human cytomegalovirus in human fibroblasts treated with dexamethasone. J Gen Virol 65:1759-1767. Tanaka J, Ogura T, Kamiya S, Yoshie T, Yabuki Y, Hatano M (1984b) Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures. Virology 136:448-452. Thiele GM and Woods GL (1988) The effect of dexamethasone on the detection of cytomegalovirus in tissue culture and by immunofluorescence. J Virol Methods 22:319-328. Thiele GM, Bicak MS, Young A, Kinsey J, White RJ, Purtilo DT (1987) Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen. ] Virol Methods 16:327-338. West PG, Aldrich B, Hartwig R, and Hailer GJ (1988) Enhanced detection of cytomegalovirus in confluent MRC5 cells treated with dexamethasone and dimethyl sulfoxide. J Clin Microbh~I 26:2510-2514.

Effect of treatment of shell vial cell cultures with dimethyl sulfoxide and dexamethasone and age of MRC-5 monolayers for detection of cytomegalovirus.

Pretreatment of MRC-5 cell monolayers in commercially prepared shell vials with 1% dimethyl sulfoxide (DMSO) and 10(-5) mol/L dexamethasone (DEX) was ...
311KB Sizes 0 Downloads 0 Views