Shell Vial Culture Research Articles

Rapid detection of polyomavirus BK by a shell vial cell culture assay

Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.

Comparison of enzyme immunoassay, shell vial culture, and conventional cell culture for the rapid detection of herpes simplex virus

Specimens submitted for the detection of herpes simplex virus (HSV) were inoculated into conventional cell-culture tubes and fresh MRC-5 shell vials. The shell vial centrifugation cultures (SVCs) were examined at 16 hr postinoculation for HSV by using type-specific monoclonal antibodies (SVC-FA); they were also analyzed for HSV antigen by using an enzyme-linked immunoassay (SVC-ELISA). Mink Lung (ML) and rhabdomyosarcoma (RD) cells were used in the cell-culture tubes. Of 182 specimens, 35 (19%) were positive in cell-culture tubes, 16 (9%) were positive by SVC-ELISA, and 22 (12%) were positive SVC-FA. All specimens that were positive by SVC-ELISA, SVC-FA, and in culture tubes displayed cytopathic effect (CPE) at 16 hr. Those specimens that had a negative ELISA and/or FA result and were positive in culture were evaluated for the time in which it took to detect CPE. At 16 hr, 48% of the positive tubes were detected; at 40 hr (second day), 83% of the positive tubes were detected; and by the third day, 94% were detected. The RD cell line displayed CPE at the same time or earlier than ML cells did in 92% of the positive cases. The sensitivity of the SVC-ELISA at 16 hr was 46% with 100% specificity. The sensitivity of the SVC-FA at 16 hr was 63% with 99% specificity. Given the increased sensitivity, rapid display of CPE, and reduced cost and handling time of cell cultures, our laboratory found that rapid SVC-ELISA and SVC-FA procedures for HSV detection have no clinical or laboratory advantage.

Comparison of conventional and shell vial cultures for detecting cytomegalovirus infection

We tested 606 specimens for cytomegalovirus infection. Fifty-two specimens were positive by conventional culture versus forty-three by pre-cytopathic effect detection. Twenty-nine specimens were positive by both methods. Because of toxicity, specimens should be set up on both conventional and shell vial culture systems to achieve maximum sensitivity.

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 evaluated. Preliminary 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.

Enhanced detection of cytomegalovirus in shell vial cell culture monolayers by preinoculation treatment of urine with low-speed centrifugation

In an effort to reduce the number of unreadable MRC-5 cell culture monolayers (i.e., monolayers that are obscured by an opaque film or are detached from their coverslips), urine specimens were subjected to low-speed (500 g for 10 min at 4°C) centrifugation prior to their inoculation into shell vials for the detection of cytomegalovirus (CMV). A total of 182 urine specimens obtained from patients suffering from ARC or AIDS were subjected to routine centrifugation culture and to preinoculation centrifugation. Cytomegalovirus recovery among the two specimen-treatment procedures was determined by counting the number of immunofluorescent MRC 5-positive cells. Eleven of 19 positive specimens were in accord in both systems. However, routinely treated (i.e., without preinoculation centrifugation) specimens were difficult to read specifically among those specimens that were cloudy or opaque. Cytome-galovirus was recovered in 18 of the 19 positive specimens that were subjected to the preinoculation centrifugation procedure. Only 12 of 19 positive specimens were identified following direct inoculation of untreated urine into shell vials. The additional preinoculation centrifugation step is appropriate for the removal of obfuscating amorphous substances from the assay system without compromising CMV detection rates.

Continuous high-speed rolling versus centrifugation for detection of herpes simplex virus

Specimens submitted for diagnosis of herpes simplex virus infections were inoculated into shell vials and conventional culture tubes. Inoculated culture tubes were incubated with rolling at 96 rpm. Immunoperoxidase (IP) staining and cytopathic effects (CPE) were used to detect positive cultures. At 24 h, 42 (53%) of the rolled cultures were positive for CPE, while only 16 (21%) of the shell vials were CPE positive (P less than 0.01). No difference in sensitivity was seen between rolled and shell vial cultures that were inoculated with high-titered viral preparations and IP stained at 16 h. However, when low-titered preparations were used, 39 of 41 (95%) were IP positive by the high-speed roller method at 64 h postinoculation, while only 24 of 41 (58%) were IP positive with shell vials (P less than 0.01). These results indicate that high-speed roller method at 64 h postinoculation, while only 24 of 41 (58%) were IP positive with shell vials (P less than 0.01). These results indicate that high-speed rolling is better than the shell vial technique for the detection of herpes simplex virus by IP staining.

Evaluation of the Du Pont HERPCHEK herpes simplex virus antigen test with clinical specimens

The HERPCHEK herpes simplex virus (HSV) antigen test (Du Pont, Billerica, Mass.) is a biotinstreptavidin-amplified enzyme-linked immunoassay for the detection of HSV antigen directly in clinical specimens. In a total of 200 mucocutaneous specimens, predominantly lesions from nongenital sites, HERPCHEK had a sensitivity of 90% and a specificity of 99% compared with the shell vial tissue culture method which uses primary rabbit kidney cells. Five false-negative results by HERPCHEK were found with specimens that contained a low percentage of positive cells by the direct immunoperoxidase stain used in a tissue culture method (1 to 9%). The absorbance values of the clinical specimens by the HERPCHEK test do not correlate well with the percentage of positive cells. Our results indicate that the HERPCHEK HSV antigen test, when performed with specimens from lesions, is a sensitive and specific test as compared with shell vial culture.

Multisite evaluation of a monoclonal antibody reagent (Syva) for rapid diagnosis of cytomegalovirus in the shell vial assay

A pre-cytopathic effect (CPE) monoclonal antibody reagent (Syva Co., Palo Alto, Calif.) was evaluated in four laboratories for the rapid detection of cytomegalovirus (CMV) in shell vial cell cultures at 16 to 24 h and 40 to 48 h postinoculation. Results were compared with those obtained by inoculation of the specimen into conventional tube cell cultures that were examined for the presence of typical CMV CPE and subsequently tested by reaction with the monoclonal antibody reagent in an indirect immunofluorescence test. Of 937 specimens, CMV was positive in 184 (20%). CMV was detected twice as frequently in shell vials only (n = 29) as in conventional tube cell cultures (n = 14). Pre-CPE shell vial assay was 91% sensitive (range, 84 to 98%) and 96% specific (range, 93 to 98%) compared with the detection of CPE in conventional tube cell cultures. Overall, 137 of 166 (83%) and 143 of 166 (86%) of the CMV strains were detected at 16 to 24 h and 40 to 48 h postinoculation, respectively. The Syva reagent produced sensitive and specific results for the rapid detection of CMV infection in shell vial cell cultures and reliably confirmed the presence of the virus as detected by CPE in conventional tube cell cultures.

Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye

The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

Rapid Shell Vial Culture and Tissue Histology Compared With Serology for the Rapid Diagnosis of Cytomegalovirus Infection in Liver Transplantation

Sixty-six patients who had undergone 78 liver transplantations, with no detectable cytomegalovirus (CMV) infection before transplantation, were studied to assess the value of CMV serology compared with the rapid detection of the virus in shell vial cell cultures or histology for the diagnosis of (1) the first evidence of infection, (2) symptomatic disease, and (3) asymptomatic infection. Of 28 evaluable patients, shell vial cell culture assay and histologic findings provided the first evidence of CMV infection earlier in 22 (79%) or at the same time as the serologic response in 5 (18%). Serologic results yielded the first indication of CMV infection in only 1 of these 28 patients (3%). Of 17 evaluable transplantations in 15 patients in whom symptomatic invasive disease developed, shell vial culture or histology provided a laboratory diagnosis of CMV infection earlier or at the same time as serologic responses in 16 (94%). Similarly, shell vial culture or histology provided evidence of CMV infection in 10 of 11 patients (91%) who had asymptomatic CMV infection and remained asymptomatic. Urine surveillance cultures yielded the first evidence of CMV infection in 14 of 19 patients who initially had an asymptomatic infection, of which 6 became symptomatic. Culturing of the blood with use of the rapid shell vial technique showed that viremia preceded CMV organ involvement in 7 of 10 patients. We recommend culture by the shell vial assay as the most rapid and sensitive method of determining CMV infection in liver transplant patients.

Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures

A total of 1,915 clinical samples was inoculated by low-speed centrifugation into shell vials (Bartels Immunodiagnostics, Bellvue, Wash.) containing cover slip monolayers of MRC-5 fibroblasts. At 1 and 2 days postinoculation, one cover slip was stained by an indirect immunofluorescence technique using a monoclonal antibody (Biotech Research Laboratories for Dupont, Billerica, Mass.) to cytomegalovirus (CMV) early antigen (EA). Clinical samples were also inoculated into three MRC-5 or MRHF cell cultures which were observed for 30 days for the appearance of a cytopathic effect (CPE). Of 157 CMV-positive samples, 92 (59%) were identified by centrifugation-enhanced EA (CE-EA) and 131 (83%) produced a CPE. CE-EA was less sensitive than CPE for all types of samples, although 17% of CMV-positive samples were detected by CE-EA alone. Evaluation of the CMV status of patients with CE-EA-positive-CPE-negative samples indicated that these samples likely represented true CMV-positive results. The average elapsed time between culture inoculation and identification of CMV decreased as follows when both CE-EA and CPE, rather than CPE alone, were used: urines, 15 to 7 days; buffy coats, 18 to 9 days; lung samples, 13 to 8 days; throat samples, 18 to 7 days. Although CE-EA was less sensitive than 30-day cell culture, both CE-EA and CPE were identified as valuable in CMV detection, and neither could be discontinued without a decrease in the CMV isolation rate or an increase in the turnaround time.

Rapid detection of varicella‐zoster virus in clinical specimens using monoclonal antibodies on shell vials and smears

Monoclonal antibodies were used for detecting varicella-zoster virus (VZV) by immunofluorescence (IF) in clinical specimens inoculated on shell vial cultures. Vesicles of 74 patients with varicella or herpes zoster-like eruptions were tested by this method and by conventional cell culture. Further diagnostic tests were direct IF on smears (42 patients), the cytological Tzanck test (28 patients), and serology (30 patients). Both IF assays were performed with the commercial VZV-specific monoclonal antibody 3B3 (Ortho). Using the shell vial technique, we found VZV in 37 (50%) of the patients, on average after 3 days. The conventional culture method yielded 26 positives (35%), on average after 7.5 days. Twenty-nine of the shell vial IF-positive patients were also positive by direct IF on smears (30 tested), while 15 gave positive Tzanck smears (18 tested). The sensitivities of the shell vial IF test, the direct IF test, the conventional culture method, and the Tzanck test were about 95%, 97%, 70%, and 79%, respectively. For the specificities, we found at least 97% for the shell vial IF test, at least 91% for the direct IF test, and about 78% for the Tzanck test. We conclude that both VZV IF tests are much more reliable than the conventional cell culture method and the Tzanck test for the laboratory diagnosis of VZV infections.

Detection of herpes simplex virus by 8 h in shell vial cultures with primary rabbit kidney cells

Shell vial (SV) cultures for herpes simplex virus using primary rabbit kidney cells stained at 8 h after inoculation were compared with 20-h SV cultures as well as conventional tissue culture. Of the 326 clinical specimens examined, conventional culture detected 67, and of these, 61 (91%) and 42 (63%) were detected by 20-h SV cultures and 8-h SV cultures, respectively.

Screening with a shell vial assay for antiviral activity against cytomegalovirus

An assay based on the shell vial cell culture (SVA) was devised to assess differences in fluorescence patterns of 8 cytomegalovirus (CMV) strains incubated in the presence or absence of three antiviral substances. The drug concentrations selected for in vitro testing were in the range of peak serum levels achievable in humans (ganciclovir 20 microM, acyclovir 80 microM, and phosphonoformic acid 300 microM). Four days after inoculation of the shell vials, the cell monolayer was fixed, stained with a mixture of monoclonal antibodies to early and late antigens of CMV, and reacted with antimouse fluorescein-labeled globulin. Twenty microM of ganciclovir in the media inhibited the formation of plaque-like foci and specific cytoplasmic fluorescence of laboratory strain AD169 and four clinical isolates. Two documented resistant strains exhibited partial inhibition at 20 microM, and a clinical isolate exhibited incomplete inhibition in three or four occasions. None of the strains was fully inhibited by 80 microM of acyclovir; however, different degrees of partial inhibition were observed. All isolates exhibited high-grade inhibition by 300 microM of phosphonoformic acid. The SVA can be used as a simple screening test for anti-CMV substances and as a screening of CMV resistance to ganciclovir prior to quantitative testing by the more complex plaque reduction assay.

Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and Ficoll-Paque/Macrodex methods.

Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.

Detection of IgM antibody to cytomegalovirus and rapid diagnosis of this virus infection by the shell vial assay

Multiple serum specimens from 10 patients with known cytomegalovirus (CMV) infections and 498 sera consecutively submitted to the laboratory for the diagnosis of CMV infection were tested for anti-CMV IgM after treatment with goat antihuman IgG and QAE-Sephadex A50 column chromatography. Specimens from all 10 patients were positive for IgM after treatment with anti-IgG, but only 8 after the column procedure. Anti-CMV IgM was detected in 23 of 498 (4.6%) specimens pretreated with anti-IgG but in only 12 (2.4%) of these samples after the sera was passed through QAE-Sephadex A50 columns. Anti-CMV IgM was detected exclusively in 2 sera after QAE-Sephadex A50 column treatment (sensitivity, 83%) and in 13 specimens pretreated with anti-IgG sera (specificity, 97%). Serology (IgG and IgM) was compared with the shell vial cell culture assay and histology for providing the first evidence of CMV infection in 28 liver transplant patients. CMV infection was detected initially by the rapid shell vial assay (24) or histology (3) in 27 (96%) of these patients. Detection of anti-CMV IgM in these patients had little value for rapid diagnosis of these infections, and suggests that serology should be recommended mainly for the diagnosis of primary CMV infections in localities in which the rapid shell vial assay is not available.

New and sensitive standard cell culture technique for the detection of cytomegalovirus in clinical specimens

Conventional tube cell culture has been recognised as the most sensitive technique available for human cytomegalovirus (HCMV) detection. Low-speed centrifugation of specimen inocula onto cell culture monolayers has been shown to increase the efficiency of infection with the AD 169 strain of HCMV. Therefore a centrifugal force of 900g for 1 hour at 37 degrees C was used to enhance the detection of HCMV cytopathic effect (CPE) in shell vials that contained circular coverslips with a monolayer of human embryonic lung (HEL) fibroblasts. Of 195 specimens, HCMV CPE was detected in 18 specimens (9.02%) on shell vial culture assay, whereas conventional tube cell culture was positive in only 13 specimens (6.6%). The shell vial culture assay was significantly more sensitive (P less than 0.05). Furthermore the development of the cytopathic effect on shell vial culture assay was significantly earlier (P less than 0.01) and more extensive. Urine samples were sonicated and the results obtained with immunofluorescence using human immune serum demonstrated that sonication increased both the intensity of fluorescence and number of fluorescent foci of HCMV-infected cells and also decreased the non-specific fluorescence of the background.

Effect of treatment of shell vial cell cultures with dimethyl sulfoxide and dexamethasone for detection of cytomegalovirus.

Urine specimens submitted for the diagnosis of cytomegalovirus infection were inoculated into shell vials that had been pretreated with a combination of dimethyl sulfoxide (DMSO) and dexamethasone (DEX). The results were compared with those for inoculated shell vials which had received no drug treatment. Of 664 specimens, 100 (15%) were positive for cytomegalovirus. Of the 100 strains of cytomegalovirus, 88 (88%) were detected in both DMSO-DEX-treated and untreated shell vials. Of the remaining 12 positive specimens, 6 were detected with untreated shell vials exclusively and 6 were detected with DMSO-DEX-treated shell vials alone (not significant by the sign test). The median number of fluorescent foci was not significantly higher in DMSO-DEX-treated shell vials compared with that in untreated cultures (Wilcoxon signed-rank test; P = 0.1). DMSO-DEX-treated monolayers did not enhance the sensitivity detection of cytomegalovirus in shell vial cell cultures.

Detection of cytomegalovirus in clinical specimens using shell vial centrifugation and conventional cell culture

Shell vial centrifugation and conventional cell culture methods for detecting cytomegalovirus (CMV) were compared in clinical specimens. Our data confirm that the shell vial centrifugation method is more sensitive and rapid than conventional cell culture; however, due to the shell vial method's problems with toxicity to the fibroblast monolayer, both methods must be performed if all specimens positive for CMV are to be detected.

Rapid detection of cytomegalovirus in clinical specimens by immunofluorescent staining of shell vial cultures.

A recently described rapid technique for detection of cytomegalovirus (CMV) was evaluated in clinical specimens utilizing indirect immunofluorescent staining (IFA) of shell vial cultures. A total of 266 clinical specimens received for viral isolation were inoculated to commercially available shell vials seeded with human lung fibroblasts (MRC-5), centrifuged at 700 X g for one hour, and stained after 18 hours incubation with monoclonal antibody to CMV early nuclear protein (Biotech Research Laboratories) and fluorescein conjugated goat antimouse IgG (Cappel Laboratories). All specimens were also inoculated to tubes of human lung fibroblasts and observed for cytopathic effect (CPE) for 28 days. Of 54 specimens positive for CMV, 36 were positive by both IFA and CPE, 3 were positive by CPE only, and 15 were positive by IFA only (P less than 0.01 by the chi-square test). Failure to detect CMV associated CPE in 10 of these 15 samples was probably due to concomitant infection with herpes simplex virus or heavy bacterial or fungal contamination. Nine of the 13 patients with IFA-positive CPE-negative specimens had CMV infection documented by other positive cultures. It was concluded that the shell vial IFA rapid technique for detection of CMV is highly specific, more sensitive than conventional isolation, and well suited for application in a clinical virology laboratory.