Journal of Medical Virology 38:36-43 (1992)

Improved Detection of Cytomegalovirus Viremia in AIDS Patients Using Shell Vial and Indirect Immunoperoxidase Methodologies Steven M. Lipson, Mark H. Kaplan, Jacob K. Simon, Zarui Ciamician, and LingFang Tseng Jane and Dayton Brown and Dayton T . Brown, Jr., Virology Laboratory, Division of Infectious Diseases, Department of Medicine, North Shore University Hospital (S.M.L., M.H.K., Z.C.) and Department of Medicine, Cornell University Medical College (S.M.L., M.H.K., Z.C.), Manhasset and Department of Health Sciences, Long Island University, Brookville (S.M.L., J.K.S., L . P . T . ) , New York One hundred twelve peripheral blood specimens were tested for the presence of cytomegalovirus (CMV) by the tube culture indirect immunoperoxidase (TC-IPA) procedure, the shell vial assay [shell vials were pre- and postinoculation treated with medium containing 2 of 10% fetal bovine serum (FBS) or 100 p,g% cortisol] (SV-IFA), and conventional (MRC-5) tube cultures (TC-CPE). CMV was detected in 25 (22%) of the 112 specimens tested by at least one of these methods. The detection/isolation of CMV among the 25 positive specimens in shell vials maintained with 2% FBS, 100 p.g% cortisol + 2% FBS, and 10% FBS was 36,44, and 52%, respectively. Detection/ isolation of the virus from blood by TC-IPA and TC-CPE was 52% and 76%, respectively. A significantly greater CMV detection rate occurred using TC-CPE compared t o SV-IFA treated with medium supplemented with an FBS concentration of 2% (P = ,01321, but not medium containing the higher serum supplement or the glucocorticoid (P > .05).Differences in the identification of a CMV viremia were observed by IPA, SV-IFA, and TC-CPE methodologies o n a patient-to-patient basis, denoting the necessity of incorporating each methodology into the CMV screening panel. Demographic analysis of 82 AIDS patients showed a CMV viremia prevalence of 9% (2128) in intravenous drug users, 57% (27/47) in homosexual patients, and 22% (2/9) in heterosexual and transfusion patients, Overnight (24 hr) storage of whole blood at 4 or 24"C, respectively, reduced CMV recovery by 40% and 6570, when tested by TC-CPE. Improved culture-based detectioniisolation of CMV in peripheral blood is contingent upon prompt specimen processing, and the utilization of combined SV-IFA, TC-IPA, and 1992 WiIey-Liss, Inc. TC-CPE methods.

INTRODUCTION Cytomegalovirus (CMV)is a n ubiquitous agent producing infection in individuals of all age groups. Although the majority of infections are asymptomatic, the immunocompromised patient such as the organ transplant recipient and individuals suffering from the acquired immune deficiency syndrome (AIDS)may develop severe or fatal disease including CMV pneumonia, colitis, adrenalitis, and retinitis [Drew, 1992; Murray e t al., 19901. The development and licensing of the antiviral drugs ganciclovir 19-(1,3-dihydroxy-2propoxymethyl) guinine] and foscarnet ( tri-sodium phosphonoformate) support the need to develop sensitive, specific, rapid, and clinically relevant assays for the identification of this infectious agent [Guerin et al., 1989; Gudnason et al., 1989; Jacobson and Mills, 1988; Schaeffer et al., 1991; Schmidt et al., 19911. The rapid culture-based detection of CMV using shell vial (SV-IFA)and tube culture indirect immunoperoxidase (TC-IPA) technologies have been applied primarily to urine, respiratory, or tissue specimens IDeGirolami et al., 1987; Gleaves et al., 1989; Lipson et al., 1990; Swenson and Kaplan, 1987; Woods e t al., 19901. Smith and coworkers evaluated the efficacy of the shell vial assay for the rapid detection of CMV in blood (of renal transplant patients), and found the assay more sensitive and rapid than conventional tube culture [Paya et al., 1987, 19881. Attainment of maximum virus detection necessitated the use of shell vial assay in conjunction with the conventional tube culture, The clinical significance of CMV viremia is now only recently being understood. The isolation/detection of CMV in blood has been shown to have prognostic value for the development of severe organ involvement in AIDS [Fiala et al., 1986; Segundy et al., 1991; Salmon et al., 19901 and bone marrow transplant [Meyers et al., 19901patients.

KEY WORDS: CMV viremia, AIDS, rapid CMV

Accepted for publication February 18,1992. Address reprint requests to S.M. Lipson, Dept. of Medicine, North Shore University Hospital-Cornell University Medical College, Manhasset, NY 11030.

Ic_i

detection 1992 WILEY-LISS, INC.

Cytomegalovirus Viremia The purpose of this study was to identify, under routine screening conditions, the prevalence of CMV viremia in our AIDS patients and to compare conventional tube culture isolation (TC-CPE) to two culture-based rapid antigen detection systems which might be most appropriate for use in the clinical setting. Additionally, variable culture medium supplements were compared in SV-IFA to identify which medium component displays the greatest efficacy in this assay for the rapid detection of CMV in peripheral blood.

MATERIALS AND METHODS Clinical Specimens One-hundred sixty-two peripheral blood specimens were collected for routine testing from 82 stage IV AIDS [MMWR, 19861 and 16 non-HIV patients during the months of September through December 1990. A total of approximately 9 ml of peripheral blood, collected in two 5 ml EDTA-containing vacutainer’” tubes (Becton Dickinson Vacutainer Systems, Rutherford, NJ), were accepted for processing by the Virology Laboratory. Multiple (i.e., 2-3 specimens) were collected from most of the patients on different days throughout the study. 1. One hundred twelve of the 162 specimens which were received within 1-2 h r of collection were utilized for comparative testing. Specimens received late in the afternoon could not be incorporated into the evaluation phase of this study, but were used in prevalence testing. The 112 blood specimens were tested in parallel by SV-IFA (including each medium supplement), TC-IPA, and TC-CPE. 2. For the purpose of prevalence determination, the Virology Laboratory tested all 162 blood specimens obtained from 98 patients provided that specimens were recently drawn (1-2 hr) or had been stored no longer than 24 h r at refrigeration (4°C)temperature. A patient was reported a s CMV viremic if the virus was detected in a t least one of the detection methods. Processing of Blood Specimens One vacutainer tube (4.5 ml whole blood) was allowed to settle at room temperature for a period of 45 to 60 min. The second vacutainer tube was stored for 24 h r a t 4 or 24 2 1°C for subsequent infectivity testing by TC-CPE (see below). After the 45 to 60 min settling period, the plasma phase containing the leukocyte (buffy coat) population was decanted into a 10 ml sterile plastic tube and centrifuged at 500g for 10 min at room temperature (24 _t 1°C) using a Beckman Accuspin FR centrifuge (Beckman Instruments, Inc., Somerset, NJ). The supernatant was discarded, and the pelleted cells were washed twice in 5 ml of a 1 x Hanks’ balanced salt solution (HBSS)without calcium or magnesium (GIBCO Laboratories, Grand Island, NY). At the termination of the final wash, the pelleted cells were resuspended by vortexing in 3 ml of cell culture maintenance medium (minimal essential medium with Earle’s salts supplemented with 2% FBS, 50 pg/ml gen-

37

tamicin, 1.5 units amphotericin B, and 1%of 200 mM L-glutamine) [Lipson e t al., 19881.

Comparison of Shell Vial, Indirect Immunoperoxidase Assays and Conventional Tube Culture for the Detection/Isolation of CMV From Buffy Coat Shell vial-immumfluorescence assay. Commercially prepared human embryonic lung fibroblast (MRC-5) shell vial (1 dram; 3.9 g ) cultures were re ceived weekly from Whittaker Bioproducts (Walkersville, MD). Assays were performed with shell vials which were no older than 6-10 days post seeding (according to manufacturer’s seeding date on the shell vial labels). Twenty-four to 48 h r prior to inoculation of duplicate shell vials, the medium was removed and replaced with cell culture maintenance medium containing 2% FBS, maintenance medium containing 2% FBS supplemented with 100 pg% cortisol, or maintenance medium containing 10% FBS [Leonardi and Lipson, in press]. Pretreated shell vials (after removal of culture medium) were inoculated with 0.20 ml of the buffy coat preparation, followed by centrifugation a t 700g for 1h r a t 36 2 1°C according to the procedure of [Lipson et al., 19901. After the 1h r centrifugation period, 1ml each of the previously described medium was added to shell vials followed by a 16-24 h r incubation. An incubation period of 2 days (i.e., 40-48 hr) was not performed, as extended incubation time resulted in monolayer degeneration and sloughing. After the “overnight” incubation, the shell vials were washed in PBS (containing Ca2+ and M g + ) , fixed in acetone, and the coverslips were permanently mounted on glass slides to facilitate staining and reading (Permaslip, Alban Scientific, Inc., St. Louis, MO) [Lipson, 19921. After the mounting glue was totally dry (ca., 4.0-4.5 hr), the coverslips were reacted with anti-CMV monoclonal antibody directed against the 72 kDa early nuclear antigen (Dupont Specialty Diagnostics, Wilmington, DE). The lyophilized antibody was reconstituted with 1ml of sterile water and then diluted 1:5 in PBS. Slides were incubated at 36.5”C with 30 pl of the primary antibody. After a 10 min wash in PBS, binding of the monoclonal antibody was detected using 30 pl of fluorescein isothiocyanate labeled goat anti-mouse IgG (heavy and light chains specific) (Organon Teknika, Durham, NC) diluted 1:20 with PBS. After the additional 30 min incubation, the slides were washed for 10 min in PBS, air dried, and mounted (FA buffered glycerol, pH 8.0-8.4; [Baxterl).The number of infected cells identified by the appearance of the typical apple green ellipsoidal immunofluorescent nucleus, were quantitated and reported as fluorescent focus units (FFUs)per coverslip using a Leitz Laborlux 12 epifluorescent microscope equipped with a tungsten-halogen illumination system, and a n IF 4 6 0 4 9 5 excitation filter. The slides were read using the 40 x objective. Confirmatory readings, when necessary, were performed using a 50 x oil immersion objective. Positive control shell vials consisted of vials which were inoculated with the laboratory-

Lipson et al.

38

adapted CMV strain AD-169. The negative control shell vials consisted of vials which were inoculated with PBS. Tube culture-indirect immunoperoxidase assay. The detection of CMV in venous blood was performed by TC-IPA in commercially prepared MRC-5 tube cultures according to the methodology of Swenson and Kaplan [1985]. Briefly, 0.2 ml of the buffy coat preparation was inoculated into a n MRC-5 tube culture for incubation a t 36.5”C for a period of 48 hr. In order to eliminate toxicity, tubes were washed once with PBS 24 h r after inoculation and then “refed” with maintenance medium containing 2% FBS. At the termination of the 48 h r incubation, the medium was removed, the tubes were washed 2~ with PBS, and the monolayer was fixed in cold (4°C) acetone for a period of 10 min. The acetone was removed and the monolayer was washed once with PBS. The production of early nuclear antigen was detected by adding 0.2 ml of a 1:5 dilution of antiCMV monoclonal antibody (72 kDa early nuclear protein; Dupont) to the tubes, followed by a 30 min incubation a t 36.5”C.The monolayer was rinsed 2 x with PBS, followed by the addition of 0.2 ml of a 1:60 dilution of peroxidase-conjugated goat anti-mouse IgG (Organon Teknika). The tubes were again incubated for 30 min at 36.5”C, rinsed with PBS, followed by the addition of 5-6 ml of freshly prepared substrate solution (60 mg 3,3’diamino-benzidine and 300 p1 of a 3% H202solution in 100 ml PBS) for 5 min a t room temperature. The substrate solution was removed and the tubes were rinsed with sterile distilled H20, and then half filled with fresh distilled H20. Monolayers were screened using a 20 x objective (40x a s a confirmatory read, if necessary) for the presence of the characteristic dark brownstained ellipsoidal nuclei. The tubes were examined using a Leitz Diavert binocular universal inverted research microscope equipped for bright-field transmitted light. The stage was modified to house a removable track to facilitate reading of the tubes. Negative and positive controls for each lot of tubes were performed a s described above. CMV isolation by conventional tube culture. Buffy coat preparation, 0.2 ml, was inoculated into duplicate MRC-5 tube cultures and maintained at 36.5”C for a period of 24 hr. After the 24 h r incubation, the cell cultures were washed once in PBS and refed with maintenance medium (2% FBS). Incubation was continued for a period of 40 days or to the appearance of a (CMVinduced) cytopathic effect (CPE).Tubes were read twice weekly. Monolayers which appeared to be undergoing initial stages of degeneration or any changes in cell morphology which remotely suggested the existence of a CPE were subjected to a subpassage. Subpassage of a tube culture was accomplished by trypsinization of the monolayer, washing, and then resuspension of the pellet in 1ml PBS, followed by inoculation of 0.2 ml of the preparation into a new MRC-5 tube. Negative and positive control tubes were maintained for each MRC-5 tube lot, using inocula a s described above. The medium for both TC-CPE and TC-IPA systems were changed prior to specimen inoculation.

Effect of Storage on the Recovery of CMV From Blood The effect of overnight storage at refrigeration (4°C and room temperature (24 1°C) on the survival of CMV in freshly collected peripheral blood was investigated using the TC-CPE method. Blood from vacutainer tubes were pooled and then aliquoted for immediate (i.e., within 1-2 hr) processing (To)or stored for a period of 24 hr (T2J a t 4°C (40 blood specimens) in one experiment or 24°C (55 blood specimens) in a second experiment. The blood specimens were inoculated into duplicate MRC-5 tube cultures, and monitored for the appearance of a CPE, as described above. Inoculum titers at To were compared semiquantitatively by counting the number of shell vial FFUskoverslip and TCIPA infectious focal units (IFUVtube.

*

Statistics In order to determine whether the proportion of positives on the three shell vial assays were equal, a Cochran’s Q test was used [Zar, 19841. McNemar’s test was employed to determine if any pair of assays was significantly different. A binomial exact test was used to further examine the McNemar’s test results. P s .05 was used to determine statistical significance. Any toxic cell culture tubeskhell vials were eliminated from the analyses, because of a nonusable result. The effect of storage on the recovery of CMV from blood was analyzed by the two-tailed Fisher exact test. The association between the onset of CPE and the number of infectious units as determined by the SV-IFA and TC-IPA was analyzed by the Spearman rank correlation coefficient test. RESULTS Parallel testing by SV-IFA, TC-IPA, and TC-CPE was performed on 112 blood specimens. CMV was detectedhsolated from 25 (22%) of the 112 specimens tested by either one or a combination of the detection/ isolation methodologies described in this study (Table I). Detectionhsolation by TC-IPA, the combined SVIFA, or TC-CPE resulted in sensitivities of 52% (13 of 25). 68% (17 of 25), and 76% (19 of 251, respectively. Among the positive specimens, detection of CMV from shell vials pre- and postinoculation treated with medium supplemented with 2% FBS, 100 pg% cortisol plus 2% FBS, and 10% FBS was 36%, 44%, and 5296, respectively (Table I). These differences were not statistically different ( P > .05). Similarly, detection of CMV from venous blood by TC-IPA was not significantly different from that of the combined or any individual (variable) SV-IFA system. There was a greater tendency for a recovery of CMV by isolation compared with TC-IPA (76 vs. 52%), but these differences were not significant ( P = .07; exact binomial test). However, a n increased rate of CMV detection occurred by TC-CPE ( P = .0132) and the combined SV-IFAs ( P = .016) compared to shell

Cytomegalovirus Viremia

39

TABLE I. Comparison of Methodologies Utilized in the Detection/Isolation of Cytomegalovirus From Peripheral Blood Shell vial assay variable Shell vial Specimen 2% FBS assays 2% FBS cortisol 10%FBS (combined) TC-IPA Isolation number

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Totale Percent

1.5a tb

-c

t

4.5

-

0.5

-

t

t

t

t

-

-

1.0d

5.5 t 1.0

5.5 t

-

-

0.5 5.0 1.5

-

t

-

-

t

-

-

-

-

21.5 0.5 5.5

22.0

0.5 4.5 40.0d

3.4 9.0

-

-

4.5 9 36

t

5.0 -

t

-

0.5 -

-

11.5 1.5 -

26.0 1.o 11.5 -

2.5 12.0 67.0 4.0 3.0

-

-

t

77.0 13 52

11 44

+ t + t ++ t ++ + + + + ++ + ++ + + -

+-

+-

+ ++ + + + + -

+ + ++ + ++ + + + -

+ + + + + + + + + +-

-

-

-

-

-

-

-

+

+ +

17 68

13 52

19 76

aNo. of fluorescent focus units (FFU); mean of duplicate shell vials. bToxicity in both shell vials. CNegative. dToxicity in one of two shell vials. eTwenty-five of 112 specimens tested were identified positive by at least one of the assays used in this study.

vials which were pre- and postinoculation treated with medium supplemented with a n FBS concentration of 2%. Differences in the detectionlisolation of CMV from blood were observed on a patient-to-patient basis. For example, CMV was not detected by SV-IFA or TC-IPA methodologies in specimen No. 2 (unreadable due to toxicity), 6, 18 or 24, but the virus was detected from each specimen by TC-CPE. Conversely, six patient specimens (No. 3, 9, 10, 11, 22, 23) failed to reveal a cytopathic effect by TC-CPE but were identified by at least one of the rapid antigen detection methods utilized in this study (Table I). In these six patients in question, CMV was subsequently isolated from the blood of two, and one patient expired prior to retesting. In one patient (9) who was on ganciclovir, CMV was isolated 1week earlier from the blood. In specimens 3 and 11from two other patients, CMV retinitis was confirmed by ophthalmoscopic examination in one, and a febrile wasting disease was found in another. These latter individuals both responded well to ganciclovir therapy. Overnight refrigeration (4°C) or maintenance at room temperature of freshly collected whole blood re-

sulted in a n absolute loss of CMV recovery to 40% and 64% of the control, respectively. This loss of recovery of CMV at 4°C retains a 95% confidence between 9% and 71% and in the 24°C group, occurs between 34% and 94%. However, no significant difference in the recovery rates between the two storage temperatures could be demonstrated using a two-tailed Fisher exact test ( P = .395; Table 11). Semiquantitative measurements (i.e., the counting of infectious units) of CMV titer among the 21 positive blood specimens suggested a relationship between the number of infectious particles in the inoculum and the time of appearance of a cytopathic effect (Table 11).Within the 24°C blood storage experiment, we observed that the number IFUs decreased as the number of days to cytopathic effect increased (r = -0.56) but this trend could not be proven statistically significant (Spearman rank correlation coefficient). Similarly, moderate negative correlations were identified between days to cytopathic effect in To controls and IFUs in the 4°C experiment (r = - .47) and the number of FFUs (the To controls) in the 24°C (r = -.29) and in the 4°C (r = -.54) storage experiments, but these also were not statistically significant.

Lipson et al.

40

TABLE 11. Effect of 24 hr Storage on the Recovery of Cytomegalovirus From Freshly Collected Peripheral Blood 4°C 24°C Number of Number of days to days to cytopathic cytopathic effect Specimen effect Specimen number TO T24 number TOa T24 1,286 29 827 8 -b 7 41 1,288 918 9 88c 40 1,316 931 9 8gC 23 1,339 1,005 16 1,430 9 8 42 1,053 1,446 7 20 1,054 7 >14 1,451 6 1,963 7 >14 1,461 10 1.081 13 >14 11111 8 1;462 5 55 1,483 16 54 1,113 13 1,576 25 Virus recovered (W) 60 36 "Specimens processed within 1-2 hr of collection; CMV isolated by TC-CPC and either one or both SV-IFA and TC-IPA. h-, CMV not isolated (TC-CPE) after 24 hr storage. cCMC isolated (TC-CPE)in one of duplicate tubes, and only after a second passage.

TABLE 111. Risk Factors and Percentage of CMV Viremia Among 84 AIDS and 16 Non-AIDS Patients Number of patients Risk factor Tested Positive (%) AIDS Homosexuala IV drug abuser Male Female Heterosexual Femaleb Transfusion Non-HIV Transplant OtherC Totalsd

47

27 (57)

23 3

2 (8) 0

6 3

107) 1 (33)

2 14 98

0 0 32

"Includes 1 female patient in which virus was not isolated. bIncludes 1 viremic infant. cLeukemia,multiple myeloma, Hodgkin's granuloma, renal failure. dA patient was considered. positive following detection/isolation of CMV from the blood by IPA or conventional tube culture on one or more occasions during the study period.

Over the 4 month testing period, 162 blood samples (98 patients) were processed for SV-IFA, TC-IPA, and TC-CPE. Of 26 intravenous substance abusers and 47 homosexual AIDS patients, 8% and 5776, respectively, were identified a s CMV viremic at least once during the testing period (Table 111).CMV viremia was identified relatively infrequently among nine heterosexual and transfusion AIDS patients. I t was absent among 16 non-HIV patients. An initial detection/isolation of CMV by SV-IFA, TC-IPA, or TC-CPE was almost invariably followed by a positive (blood)culture upon collection of a second specimen (data not shown).

DISCUSSION The rapid and accurate laboratory diagnosis of CMV infection is important in the care and management of the immunocompromised (transplant) and the AIDS patient populations IMarsano et al., 1990; Meyers et al., 1990; Segundy et al., 19901. Isolation of CMV from peripheral blood of AIDS patients appears to be associated with clinically significant organ specific infections, and with a n unfavorable prognosis [Salmon et al., 1990; Segundy et al., 1990; Webster, 19911. Human CMV in peripheral blood may occur in the latent state in mononuclear (MN) cells or a s infectious particles in the polymorphonuclear (PMN)cell population [Danker et al., 1990; Howell et al., 19791. In this report, the detectionlisolation of the virus from buffy coat preparations containing both MN and PMN obtained from our AIDS homosexual patients (Table 111) either equalled or commonly surpassed those rates reported among transplant and in other AIDS homosexual patient populations [Gerna et al., 1990a; Paya et al., 1987; Quinnan e t al., 1984; Salmon et al., 1990; Schirm et al., 1987; Segundy e t al., 1990; Shibata et al., 1988; Jacobson and Mills, 19881. Interestingly, the detectioniisolation of CMV in our homosexual AIDS patients is comparable if not better than those detection rates achieved through dextran gradient separation of PMN and even the polymerase chain reaction [Gerna et al., 1990; Shibata et al., 19881. Earlier attempts by one of us (S.M.L.)to isolate CMV from blood of AIDS patients was thwarted by a delayed (i.e., overnight) transport of specimens to the laboratory. As shown in Table 11, and in agreement with the protocols of Jacobson and Mills [1988] and Paya et al. [19881, rapid transport to the laboratory following the collection of peripheral blood is necessary to optimize the detection of CMV in blood. It has been shown t h a t the addition of cortisol or the synthetic derivative dexamethasone to monolayer cultures increases the recovery or infectivity of CMV from frozen original specimens (i.e., urine, bronchoalveolar lavage), selected strains of CMV, a s well as the laboratory adapted CMV strain AD-169 [Forbes et al., 1990; Koment, 1985,1989; Leonardi and Lipson, 1992; Li and Fong, 1990; Tanaka e t al., 1984; West et al., 19881. In contrast, other investigators found no enhanced detection of CMV by dexarnethasone from large numbers of clinical specimens; Control studies were however, successful in improving infectivity (i.e., increase in the number of FFU/slide) but not absolute recovery of the AD-169 or Towne strains of CMV [Espy et al., 1988; Fedorko e t al., 1990; Hagerty et al., 1990; Thiele and Woods, 19881. In the current study, glucocorticoid treatment of fibroblast monolayers prior to inoculation of freshly collected peripheral blood failed to affect any enhanced viral recovery. The purported modifying effects by glucocorticoids on the recovery of CMV might possibly be ascribed to artifact obtained by the testing of stored, laboratory adapted, or selected CMV strains. Importantly, the utilization of varying concentrations

Cytomegalovirus Viremia of FBS in the assay system appears to have a significant effect on virus recovery. Some investigators for example [Arens et al., 1991, Espy et al., 1988; Fedorko et al., 1990; Thiele and Woods, 19881, choose to incorporate a 10% FBS supplement into their culture-based CMV detection systems. Other workers have chosen to use a less enriched (i.e., 2% or 5% FBS) cell culture maintenance medium [Forbes et al., 1990; Koment, 1989; Lipson et al., 1990; Tanaka et al., 1984; West et al., 19881. In the current study, the rate of CMV detection in shell vials maintained in a medium supplemented with a n FBS concentration of 2% was significantly less ( P = .0312) compared with that isolated in conventional tube culture. However, no significant difference in the rate of virus detection in shell vials maintained in a 10% FBS supplement occurred in comparison to that of isolation ( P > .05). Accordingly, and in agreement with the work of others [Arens et al., 1991; Fedorko e t al., 1990; Gleaves et al., 19841, it is our recommendation to supplement shell vial cell culture medium with a n FBS concentration of 10%. The maintenance of conventional tube cultures in a n enriched (i.e., 10% FBS supplement) medium was not investigated. In our experiences the maintenance of tube cultures in a n elevated serum-containing medium would necessitate a n increased number of medium changes resulting in a premature aging of the fibroblast monolayer. The staining of shell vials prepared by commercial vendors at 40-48 rather than 16-24 h r after inoculation has been reported by some [Arens et al., 1991; Jesperson et al., 19891 but not others [Gerna et al., 1990b, 19911 to affect improved sensitivity by SV-IFA. Preliminary experiments in our laboratory revealed t h a t the maintenance of shell vials inoculated with buffy coat for a n extended postinoculation period of 4 0 4 8 h r resulted in the development of fragile monolayers which could be easily removed from their coverslips during washing and fixation. Termination of the shell vial incubation period a t 16-24 h r after inoculation satisfactorily addressed this issue in our laboratory. It is difficult to propose a mechanism(s) for the comparable and the relatively rapid (viz., 24 vs. 48 hr) detection rates between the SV-IFA and the TC-IPA (viz., stationary tube) methodologies. Most workers suggest, for example, t h a t specimen centrifugation onto monolayer cultures enhances contact of the virus with its host cell. Accordingly, utilization of a n appropriate “g” force increases virus recovery in monolayer culture (e.g., early nuclear antigen) or, at the very least, shortens the time for the reporting of a positive test result. This advantage may be offset in the TC-IPA method which utilizes a much greater monolayer surface area. Our studies with blood, in contrast to earlier findings utilizing primarily nonblood specimens [Swensen and Kaplan, 19871, showed no significant differences in the overall recovery of CMV using either SV-IFA or TCIPA methodologies. The reduced sensitivity of SV-IFA compared with TC-IPA described earlier by one of us

41

may be ascribed to a n enshrouded detection efficiency by the SV-IFA assay due primarily to urine-induced cytotoxicity. Preinoculation treatment of urine by low speed centrifugation has markedly reduced cytotoxicity in our laboratory and in turn, increased the number of readable shell vial monolayers [Lipson et al., 19901. Most diagnostic virology laboratories have incorporated SV-IFA rather than TC-IPA technology for the routine detection of CMV in clinical (e.g., urine, BAL, autopsy, blood) specimens (State of New York, Department of Health, Round 9102, “Virology General” Proficiency Testing Report 1990). The decision is understandable as SV-IFA technology requires considerably smaller reagent volumes, necessitates no or minimal reagent preparation (reagents for SV-IFA are now commercially available in kit f0rm-e.g. Bartels Immunodiagnostic Supplies, Inc., Bellevue, WA), and SV-IFA testing of blood may be terminated 16-24 rather than 40-48 h r postinoculation. The manipulation of the shell vial coverslip (i.e., removal from the vial, gluing of the coverslip onto a slide) [Lipson, 19921necessitates minimal effort. TC-IPA methodology, however, does present some inherent advantages. Readings may be performed by light rather than fluorescent microscopy, and TCIPA cultures may be stored for 6 months or longer in sterile distilled water at room temperature without losing signal intensity (S.M. Lipson, personal observation). Isolation of a virus in cell culture or the observed morbidity/mortality of a susceptible animal have classically been considered “gold standards” [Lipson e t al., 19881. Antigen detection technologies, specifically those described in the present study, currently vie for the titular designation of a gold standard. Both SV-IFA and TC-IPA technologies may justifiably be considered isolation assays, as initiation of the CMV replicative cycle is a prerequisite for the detection of immediate early antigens. The rapid detection of CMV immediate early antigens in peripheral blood leukocytes (viz., antigenemia) using immunoperoxidase or immunofluorescence indicator systems has been shown promising for the diagnosis of CMV viremia [Gerna et al., 1990b; Jiwa et al., 1989; Wirgart et al., 19901. However, low numbers of positive leukocytes result in a dissociation between antigenemia and viremia [Gerna et al., 19911, suggesting a further need to evaluate the direct antigen detection system with one or more gold standard technologies such a s those described in the current study. CMV is a n extremely labile infectious agent, as shown by a significant recovery loss from peripheral blood after 24 hr storage at 4°C and 24°C of 40% and 64%, respectively (Table 11). The reduction of CMV recovery from whole blood after storage might be related to a loss of leukocyte viability and in turn, loss of virion infectivity within the blood cell itself. Protectants to retain virus infectivity in whole blood have been reported [Dworkin et al., 1990; Howell and Miller, 19831. Such protectants, however, are not currently available in routine blood collection systems. The rapid transport of blood to the laboratory can not be overemphasized.

Lipson et al.

42

Inoculation in triplicate or higher multiples in any one of the culture-based assays described in this study might result in a further improved detection rate of the virus. However, constraints within each system occur. One must accept, for example, the afforded time necessary to obtain a CPE by TC-CPE. Multiple TC-IPA cultures, each requiring 0.2 ml of monoclonal antibody, proves costly. The use of SV-IFA technology is indeed rapid, but toxicity, albeit minimal, occurs. Accordingly, inoculation of duplicate shell vials [Arens et al., 19911 is essential, as detection of CMV in only one of two shell vials is not uncommon (Tables I, 11). In summary, our data suggest t h a t a combination of SV-IFA, TC-IPA, and conventional tube cultures be utilized to affect a n improved detection rate of CMV in peripheral blood. An adequate system requires at least two MRC-5 tube cultures per clinical specimen. Duplicate shell vials maintained in a n elevated FBS (i.e., 10%)concentration or a cortisol-supplemented culture medium is suggested as well. However, the use of a 10% FBS supplement requires little preparative effort a s FBS is readily available in the virology laboratory. Last, the immediate processing, and then inoculation of freshly collected blood into the culture-based assay systems described in this study, is a further corequisite for improving the rate of CMV detectiontisolation from blood.

ACKNOWLEDGMENTS The authors appreciate the excellent technical assistance of Mark Bornfreund and the expertise and assistance of Drs. Martin Lesser and Francine Mandel (Division of Biostatistics) in the statistical analysis of the data, The authors also appreciate the excellent secretarial support of Carole Cristiano and Ellen Sweeney. This work was generously supported by The J a n e and Dayton Brown and Dayton T. Brown, J r . Virology Laboratory. REFERENCES Arens M, Owen J, Hagerty CM, Reed CA, Storch GA (1991):Optimizing recovery of cytomegalovirus in the shell vial culture technique. Diagnostic Microbiology Infectious Disease 14:125-130. Danker WM, McCutchan JA, Richman DD, Hirata K, Spector SA (1990):Localization of human cytomegalovirus in peripheral blood leukocytes by in uitro hybridization. Journal of Infectious Diseases 161:3136. DeGirolami PC, Dakos J , Eichelberger K, Mills LS, DeLuca M (1988): Rapid detection of cytomegalovirus in clinical specimens by immunofluorescent staining of shell vial cultures. American Journal of Clinical Pathology 89:528-532. Drew LW (1992): Cytomegalovirus infection in patients with AIDS. Clinical Infectious Disease 14:60%615. Dworkin RJ, Drew WL, Miner RC, Mehalko S, Evans C, Baxter R (1990):Survival of cytomegalovirus in viremic blood under blood bank storage conditions. The Journal of Infectious Diseases 161:1310-1311. Espy MJ, Wald AD, Ilstrup DM, Smith TF (1988):Effect of treatment of shell vial cultures with dimethyl sulfoxide and dexamethasone for the detection of cytomegalovirus. Journal of Clinical Microbiology 26:1091-1093. Fedorko DF, Ilstrup DM, Smith TF (1990):Effect of treatment of shell vial cultures with dimethyl sulfoxide and dexamethasone and age of MRC-5 monolayers for detection of cytomegalovirus. Diagnostic Microbiology of Infectious Diseases 13:4144.

Fiala M, Cone LA, Chang CM, Mocarski ES (1986):Cytomegalovirus viremia increases with progressive immune deficiency in patients infected with HTLV-111. AIDS Research Human Retroviruses 2:175-181. Forbes BA, Bonville CA, Dock NL (1990):The effect of a promoter of cell differentiation and selected hormones on human cytomegalovirus infection using an in vitro cell system. The Journal of Infectious Diseases 162:3945. Gerna G, Parea M, Percivalle E, Zipeto D, Silini E, Barbarini G, Milanesi G (1990a): Human cytomegalovirus viremia in HIV-1seropositive patients at various clinical stages of infection. AIDS 4:1027-1031. Gerna G, Revello MG, Porcivalle E, Zavattoni M, Parea M, and Battaglia M ( 1990b):Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins. Journal of Clinical Microbiology 28:2681-2688. Gerna G, Zipeto D, Parea M, Resell0 MG, Silini E, Percivalle E, Zavattoni M, Grossi P, Milanesi G (1991):Monitoring of human cytomegalovirus infections and ganciclovir treatment in heart transplant recipients by determination of viremia, antigenemia, and DNAemia. The Journal of Infectious Diseases 164:988-998. 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 antibodies t o a n early antigen. Journal of Clinical Microbiology 19:917919. Gleaves CA, Myerson D, Bowden RA, Hackman RC, Meyers J D (1989): Direct detection of cytomegalovirus from bronchoalveolar lavage samples by using a rapid in site DNA hybridization assay. Journal of Clinical Microbiology 27:2429-2432. Gudnason T, Belani KK, Balfour HH (19891: Ganciclovir treatment of cytomegalovirus disease in immunocompromised children. Pediatric Infectious Disease Journal 8:436-440. Guerin C, Pozzetto B, Broyet C, Gaudin C, Berthoux F (1989):Ganciclovir therapy of symptomatic cytomegalovirus infection in renal transplant recipients. Nephrology Dialysis Transplantation 4:900-906. Hagerty G, Arens M, Owen J , Keener M, Crespi V, Buller R, Reed CA, Storch GA (1990):Abstracts of the Annual Meeting of the American Society of Microbiology C-92, p. 359. Howell CL, Miller MJ (1983):Effect of sucrose phosphate and sorbitol on infectivity of enveloped viruses during storage. Journal of Clinical Microbiology 18:658-662. Howell CL, Miller MJ, Martin WJ (1979):Comparison of rates of virus isolation from leukocyte populations separated from blood by conventional and Ficoll-PaqueiMacrodex methods. Journal of Clinical Microbiology 10533-537. Jacobson MA, Mills J (1988): Serious cytomegalovirus disease in the acquired immunodeficiency syndrome (AIDS). Annals of Internal Medicine 108:585-594. Jesperson DJ, Drew WL, Gleaves CA, Meyers JD, Warford AL, Smith TF (1989):Multisite evaluation of a monoclonal antibody reagent (Syva) for rapid diagnosis of cytomegalovirus in the shell vial assay. Journal of Clinical Microbiology 27:1502-1505. Jiwa NM, van de Rijke FM, Mulder A, van der Bij W, The TH, Rothbarth PH, Velzing J, van der Ploeg M, Raap AK (1989): An improved immunocytochemical method for the detection of human cytomegalovirus antigens in peripheral blood leukocytes. Histochemistry 91:345-349. Koment RW (1985): Lytic cytomegalovirus replication and the hormones of human pregnancy. Journal of Medical Virology 15:149156. Koment RW ( 1989):Restriction to human cytomegalovirus replication in vitro removed by physiological levels of cortisol. Journal of Medical Virology 27:44-49. Leonardi GP, Lipson SM (1992):Enhanced detection of cytomegalovirus in shell vial culture following MRCd monolayer pretreatment with glucocorticoids. International Journal of Medical Microbiology (in press). Li SB, Fong CKY (1990): Detection of human cytomegalovirus early and late antigen and DNA production in cell culture and the effects of dimethyl sulfoxide, dexamethasone and DNA inhibitors on early antigen detection. Journal of Medical Virology 30:97-102. Lipson SM (1992): Growth of human rotavirus by trypsin activation and centrifugation enhanced shell vial culture. In Isenberg HD (edl: “Clinical Microbiology Procedures Handbook.” (CMPN). Washington, DC: American Society of Microbiology (in press).

Cytomegalovirus Viremia Lipson SM, Walderman R, Costello P, Szabo K (1988):Sensitivity or rhabdomyosarcoma and guinea pig embryo cell cultures to field isolates of difficult-to-cultivate groups A coxsackieviruses.Journal of Clinical Microbiology 26:129%1303. Lipson SM, Costello P, Forlenza S, Agins B, Szabo K (1990):Enhanced detection of cytomegalovirus in shell vial cell culture monolayers by preinoculation treatment of urine with low-speed centrifugation. Current Microbiology 20:39-42. Marsano L, Perrillo RP, Flye MW, Hanto DW, Spitzer ED, Thomas JR, Murray PR, Windus DM, Brunt EM, Storch GA (1990):Comparison of culture and serology for the diagnosis of cytomegalovirus in kidney and liver transplant patients. The Journal of Infectious Diseases 161:45@61. Meyers JD, Ljungman P, Fisher LD (1990): Cytomegalovirus excretion as a predictor of cytomegalovirus disease after marrow transplantation: Importance of cytomegalovirus viremia. The Journal of Infectious Diseases 162:373-380. MMWR (1986):HTLV-IIIILAV classification system. Centers for Disease Control. 35[140.201:335337. Murray PK, Drew WL, Kobayashi GS, Thompson J H (eds) (1990): “Medical Microbiology.” St. Louis, MO: The C.V. Mosby Co., pp 523-529. Paya CV, Wald AD, Smith TF (1987): Detection of cytomegalovirus infection in specimens other than urine by the shell vial assay and conventional tube cell culture. Journal of Clinical Microbiology 25:755-757. Paya CV, Wald AD, Smith TF (1988):Detection of cytomegalovirus from blood leukocytes separated by Sepracell-MN and FicollPaque/Macodex methods. Journal of Clinical Microbiology 26:2031-2033. Salmon D, Lacassin F, Harzic M, Leport C, Perronne C, Bricaire F, Brun-Vezinet F, Vilde J L (1990):Predictive value of cytomegalovirus viraemia for the occurrence of CMV organ involvement in AIDS. Journal of Medical Virology 32:160-163. Schaefer MD, Stratta RJ, Markin RS, Cushing KA, Woods GL, Reed EC, Wood RP, Langras AN, Shaw BW (1991):Ganciclovir therapy for cytomegalovirus disease in liver transplant recipients. Transplantation Proceedings 23:1515-15 16. Schirm J , Timmerije W, van der Bij W, The TH, Wilterdink JB, Tegzess AM, van Son WJ, Schroder FP (1987): Rapid detection of infectious cytomegalovirus in blood with the aid of monoclonal antibodies. Journal of Medical Virology 23:31-40. Schmidt GM, Horak DA, Niland JC, Duncan SR, Forman SJ, Zaia JA,

43 City of Hope-Stanford-Syntax CMV Study Group (1991):A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants. The New England Journal of Medicine 324:10051011. Segundy M, Atoui N, Reynes J , Vendrell J P , Ducos J , Blanc P, Mandin J (1990): Cytomegalovirus viremia in HIV-infected patients treated with zidovidine. Scandinavian Journal of Infectious Diseases 22:653-658. Shibata D, Martin WJ, Appleman MD, Causey DM, Leedom JM, Arnhelm N (1988): Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. The Journal of Infectious Diseases 158:1185-1192. Swenson PD, Kaplan MH (1985):Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen. Journal of Clinical Microbiology 21:669-673. Swenson PD, Kaplan MH (1987): Comparison of two rapid culture methods for detection of cytomegalovirus in clinical specimens. Journal of Clinical Microbiology 25:2445-2446. Tanaka J, Ogura T, Kamiya S, Sat0 H, Yoshie T, Ogura H, Hatano M (1984):Enhanced replication of human cytomegalovirus in human fibroblasts treated with dexamethasone. Journal of General Virology 675:1759-1769. Thiele GM, Woods GD (1988): The effect of dexamethasone on the detection of cytomegalovirus in tissue culture and by immunofluorescence. Journal of Virological Methods 22:319-328. Webster A (1991):Cvtomeealovirus as a nossible cofactor in HIV disease process. Journal ouf Acquired Immune Deficiency Syndromes 4[Suppl lI:S47-s52. West PG, Aldrich B, Hartwig R, Heller GJ (1988):Enhanceddetection of cytomegalovirus in confluent MRC-5 cells treated with dexamethasone and dimethyl sulfoxide. Journal of Clinical Microbiology 26:2510-2514. Wirgart BZ, Hokeber I, Lanqvust M, Griller L (1990):Detection of cytomegalovirus directly in blood leukocytes and evaluation of a new CMV monoclonal antibody, AAC10. Serodiagnosis and Immunotherapy in Infectious Disease 4:419426. Woods GL, Johnson AM, Thiele GM (1990):Clinical comparison of two assays for rapid detection of cytomegalovirus early nuclear antigen. American Journal of Clinical Pathology 93:373-377. Zar J H (1984):“Biostatistical Analysis.”Englewood Cliffs, NJ: Prentice Hall, Inc.