DIAG. MICROBIOL.INFECT. DIS. 1990;13:241-244
241
VIROLOGY
Comparison of Enzyme Immunoassay, Shell Vial Culture, and Conventional Cell Culture for the Rapid Detection of Herpes Simplex Virus Sheryl L. Ginnow Johnston and Charles S. Siegel
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 anaI~/zed for HSV antigen by using an enzymelinked 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 SVCELISA and SVC-FA procedures for HSV detection have no clinical or laboratory advantage.
INTRODUCTION
Tse et al., 1989), and all are eventually compared to the "gold standard" of viral detection, conventional cell culture. Recently, conventional cell culture has been modified by the use of shell vials and centrifugation (Gleaves et al., 1985; Oefinger et al., 1988; Woods and Mills, 1988). By using the shell vial culture (SVC) technique, theoretically, the conventional cell culture has been made rapid. To complement this procedure, enzyme-linked immunoassay (ELISA) has been used to detect HSV in SVC rather than the typical monoclonal antibody stain. Our experience in the laboratory has indicated that conventional cell culture with fresh rhabdomyosarcoma (RD) cells provides rapid, sensitive results in HSV detection. Therefore, in efforts to meet the needs of the physician in obtaining fast, accurate, and reliable culture results, we compared our conventional cell-culture isolation with SVCs stained
In the past several years, the diagnostic virology laboratory has been challenged with the task of detecting the presence of herpes simplex virus (HSV) from clinical specimens as rapidly as possible. Numerous methods have been employed for this purpose (Land et al., 1984; Gleaves et al., 1985; Halstead et al., 1987; Espy and Smith, 1988; MacDonald et al., 1988; Oefinger et al., 1988; Woods and Mills, 1988; From the Virology Laboratory, Department of Laboratory Medicine, Bellin Memorial Hospital, Green Bay, Wisconsin. Address reprint requests to: Sheryl L. Ginnow Johnston, MS, RM (AAM), Bellin Hospital, 744 South Webster, Green Bay, WI 54305. Received November 10, 1989; revised and accepted January 19, 1990. © 1990Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010. 0732-8893/90/$3.50
242
with a monoclonal antibody at 16 hr and SVCs examined with the ELISA system at 16 hr. The results indicated that the SVC systems offered no advantage over the conventional cell cultures.
MATERIALS A N D METHODS Specimens Clinical specimens were gathered from hospitals and clinics in northeastern Wisconsin. The specimens were primarily from female genital sources, although lesion specimens from other sites on both males and females were submitted. The specimen was collected on a sterile dacron swab and placed in 2 ml of viral transport medium. This medium was prepared inhouse and contained Eagle minimum essential medium (MEM) (GIBCO, Grand Island, NY), antibiotics (100 U penicillin/ml, 100 ~g streptomycin/ml, 2.5 ~g amphotericin/ml, and 10 ~g gentamycin/ml), and 1% gelatin. Specimens were received in the laboratory and processed within 24 hr of collection.
S.L. Ginnow Johnston and C.S. Siegel
monoclonal antibody (Syva Company) for HSV. Media were removed from the vials, and acetone was added for a period of 10-15 mins. The acetone was then removed, and phosphate buffered saline (PBS) was used to rinse the vial. Monoclonal antibody stain (0.1 ml undiluted) was then added to cover the coverslip in the bottom of the vial. The vial was covered and incubated at 35°C for 30 min. After incubation, the vial was rinsed again with PBS and the coverslip was removed and evaluated for fluorescence.
SVC-ELISA The third SVC was tested at 16-24 hr (overnight) by using the Ortho*HSV antigen ELISA test (Ortho Diagnostic Systems, Raritan, NJ). After incubation, two drops of cell lysis agent (provided in the test kit) was added to the vial, mixed, and allowed to stand for 5-10 min. This culture supernatant was then processed per assay instructions in the kit manual, and results were interpreted by spectrophotometric reading.
Conventional Cell Culture One RD tube and one ML tube (original cell culture from Viromed Laboratories, Inc., Minneapolis, MN) were inoculated with 0.30 ml of sample, placed at 35°C in a CO2 incubator, and rotated for 5 days. These specimens were inspected daily for cytopathic effect (CPE). All cell-culture tubes were no more than 14 days postseeding. Before inoculation, the media in the tubes were changed to media containing dimethyl sulfoxide (DMSO) and dexamethasone (Warford et al., 1989; West et al., 1989). All positive cultures were confirmed by typing with a type-specific monoclonal antibody stain (Syva Company, Palo Alto, CA).
Shell Vial Culture Three shell vials containing MRC-5 cells, no older than 14 days after seeding in our laboratory (original cells obtained from Viromed Laboratories) were inoculated with 0.30 ml of sample. The vials were then centrifuged for 60 min at 700 g at room temperature. After centrifugation, 1 ml of media containing DMSO and dexamethasone was added to each vial. The vials were incubated for 16-24 hr (overnight) at 35°C in a CO2 incubator. Evaluation of the SVCs was made by staining with a type-specific monoclonal antibody (SVC-FA) and by ELISA (SVC-ELISA), as described below. SVC-FA Evaluation of two of the 16-hr shell vials was made by staining with a fluorescein-labeled type-specific
RESULTS Conventional Cultures Of the 182 cultures tested, 35 (9 type 1 and 26 type 2) were positive, giving an overall isolation rate of 19%. In 46% of these positive cases, CPE was detected on the RD cell line before the ML cell line. In another 46% of the positive cases, there was simultaneous detection of HSV on the RD and ML cell lines. In only 8% of the positive cases did the ML cells display CPE before the RD cells. At 16 hr of incubation (overnight), 48% of the positive cultures were detected; by the second day of culture ( - 40 hr), 83% of the positive cultures were detected; and by the third day of culture, 94% of the positive cultures were detected. In all of the cases in which both the cell culture and corresponding SVC were positive, the positive cell culture was detected during the first day (16 hr) of observation. In cases of positive cell culture and negative SVC, 37.5% of the discrepancies were detected by CPE at 16 hr. A total of 66% of the discrepancies were detected by the second day of observation. By the third day of tissue-culture observation, 30 of the 32 discrepant results (94%) were detected. The average time needed to process the cell culture after initial incubation was 1 rain per tube per day to observe CPE or 10 rain total observation time for a negative patient. A positive culture required scraping, drying, staining, and reading for confirmation, with an average time requirement of 45 min.
243
Rapid HSV Testing
SVC-FA Of the 35 positive cell-culture cases, only 22 (8 type 1 and 14 type 2) were detected in 16-hr SVCs. The cell culture was positive and the SVC-FA was negative in 13 cases (1 type 1 and 12 type 2). There was a positive SVC and a negative cell culture in one case (type 2). The sensitivity of this method was 63%, and the specificity was 99%. The average amount of time to process the SVCFA from the end of the incubation period was 55 rain for one vial and controls. Additional vials added only 1-2 rain each.
SVC-ELISA Of the 35 positive cell-culture cases, 16 (8 type I and 8 type 2) were positive by the SVC-ELISA method. The cell culture was positive and the SVC-ELISA was negative in 19 cases (1 type 1 and 18 type 2). There was no positive SVC-ELISA/negative cell-culture cases. The sensitivity was 46%, and the specificity was 100%. The average amount of time to process one vial and controls from the end of the incubation period was 172 rain. Additional vials added
241
VIROLOGY
Comparison of Enzyme Immunoassay, Shell Vial Culture, and Conventional Cell Culture for the Rapid Detection of Herpes Simplex Virus Sheryl L. Ginnow Johnston and Charles S. Siegel
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 anaI~/zed for HSV antigen by using an enzymelinked 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 SVCELISA and SVC-FA procedures for HSV detection have no clinical or laboratory advantage.
INTRODUCTION
Tse et al., 1989), and all are eventually compared to the "gold standard" of viral detection, conventional cell culture. Recently, conventional cell culture has been modified by the use of shell vials and centrifugation (Gleaves et al., 1985; Oefinger et al., 1988; Woods and Mills, 1988). By using the shell vial culture (SVC) technique, theoretically, the conventional cell culture has been made rapid. To complement this procedure, enzyme-linked immunoassay (ELISA) has been used to detect HSV in SVC rather than the typical monoclonal antibody stain. Our experience in the laboratory has indicated that conventional cell culture with fresh rhabdomyosarcoma (RD) cells provides rapid, sensitive results in HSV detection. Therefore, in efforts to meet the needs of the physician in obtaining fast, accurate, and reliable culture results, we compared our conventional cell-culture isolation with SVCs stained
In the past several years, the diagnostic virology laboratory has been challenged with the task of detecting the presence of herpes simplex virus (HSV) from clinical specimens as rapidly as possible. Numerous methods have been employed for this purpose (Land et al., 1984; Gleaves et al., 1985; Halstead et al., 1987; Espy and Smith, 1988; MacDonald et al., 1988; Oefinger et al., 1988; Woods and Mills, 1988; From the Virology Laboratory, Department of Laboratory Medicine, Bellin Memorial Hospital, Green Bay, Wisconsin. Address reprint requests to: Sheryl L. Ginnow Johnston, MS, RM (AAM), Bellin Hospital, 744 South Webster, Green Bay, WI 54305. Received November 10, 1989; revised and accepted January 19, 1990. © 1990Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010. 0732-8893/90/$3.50
242
with a monoclonal antibody at 16 hr and SVCs examined with the ELISA system at 16 hr. The results indicated that the SVC systems offered no advantage over the conventional cell cultures.
MATERIALS A N D METHODS Specimens Clinical specimens were gathered from hospitals and clinics in northeastern Wisconsin. The specimens were primarily from female genital sources, although lesion specimens from other sites on both males and females were submitted. The specimen was collected on a sterile dacron swab and placed in 2 ml of viral transport medium. This medium was prepared inhouse and contained Eagle minimum essential medium (MEM) (GIBCO, Grand Island, NY), antibiotics (100 U penicillin/ml, 100 ~g streptomycin/ml, 2.5 ~g amphotericin/ml, and 10 ~g gentamycin/ml), and 1% gelatin. Specimens were received in the laboratory and processed within 24 hr of collection.
S.L. Ginnow Johnston and C.S. Siegel
monoclonal antibody (Syva Company) for HSV. Media were removed from the vials, and acetone was added for a period of 10-15 mins. The acetone was then removed, and phosphate buffered saline (PBS) was used to rinse the vial. Monoclonal antibody stain (0.1 ml undiluted) was then added to cover the coverslip in the bottom of the vial. The vial was covered and incubated at 35°C for 30 min. After incubation, the vial was rinsed again with PBS and the coverslip was removed and evaluated for fluorescence.
SVC-ELISA The third SVC was tested at 16-24 hr (overnight) by using the Ortho*HSV antigen ELISA test (Ortho Diagnostic Systems, Raritan, NJ). After incubation, two drops of cell lysis agent (provided in the test kit) was added to the vial, mixed, and allowed to stand for 5-10 min. This culture supernatant was then processed per assay instructions in the kit manual, and results were interpreted by spectrophotometric reading.
Conventional Cell Culture One RD tube and one ML tube (original cell culture from Viromed Laboratories, Inc., Minneapolis, MN) were inoculated with 0.30 ml of sample, placed at 35°C in a CO2 incubator, and rotated for 5 days. These specimens were inspected daily for cytopathic effect (CPE). All cell-culture tubes were no more than 14 days postseeding. Before inoculation, the media in the tubes were changed to media containing dimethyl sulfoxide (DMSO) and dexamethasone (Warford et al., 1989; West et al., 1989). All positive cultures were confirmed by typing with a type-specific monoclonal antibody stain (Syva Company, Palo Alto, CA).
Shell Vial Culture Three shell vials containing MRC-5 cells, no older than 14 days after seeding in our laboratory (original cells obtained from Viromed Laboratories) were inoculated with 0.30 ml of sample. The vials were then centrifuged for 60 min at 700 g at room temperature. After centrifugation, 1 ml of media containing DMSO and dexamethasone was added to each vial. The vials were incubated for 16-24 hr (overnight) at 35°C in a CO2 incubator. Evaluation of the SVCs was made by staining with a type-specific monoclonal antibody (SVC-FA) and by ELISA (SVC-ELISA), as described below. SVC-FA Evaluation of two of the 16-hr shell vials was made by staining with a fluorescein-labeled type-specific
RESULTS Conventional Cultures Of the 182 cultures tested, 35 (9 type 1 and 26 type 2) were positive, giving an overall isolation rate of 19%. In 46% of these positive cases, CPE was detected on the RD cell line before the ML cell line. In another 46% of the positive cases, there was simultaneous detection of HSV on the RD and ML cell lines. In only 8% of the positive cases did the ML cells display CPE before the RD cells. At 16 hr of incubation (overnight), 48% of the positive cultures were detected; by the second day of culture ( - 40 hr), 83% of the positive cultures were detected; and by the third day of culture, 94% of the positive cultures were detected. In all of the cases in which both the cell culture and corresponding SVC were positive, the positive cell culture was detected during the first day (16 hr) of observation. In cases of positive cell culture and negative SVC, 37.5% of the discrepancies were detected by CPE at 16 hr. A total of 66% of the discrepancies were detected by the second day of observation. By the third day of tissue-culture observation, 30 of the 32 discrepant results (94%) were detected. The average time needed to process the cell culture after initial incubation was 1 rain per tube per day to observe CPE or 10 rain total observation time for a negative patient. A positive culture required scraping, drying, staining, and reading for confirmation, with an average time requirement of 45 min.
243
Rapid HSV Testing
SVC-FA Of the 35 positive cell-culture cases, only 22 (8 type 1 and 14 type 2) were detected in 16-hr SVCs. The cell culture was positive and the SVC-FA was negative in 13 cases (1 type 1 and 12 type 2). There was a positive SVC and a negative cell culture in one case (type 2). The sensitivity of this method was 63%, and the specificity was 99%. The average amount of time to process the SVCFA from the end of the incubation period was 55 rain for one vial and controls. Additional vials added only 1-2 rain each.
SVC-ELISA Of the 35 positive cell-culture cases, 16 (8 type I and 8 type 2) were positive by the SVC-ELISA method. The cell culture was positive and the SVC-ELISA was negative in 19 cases (1 type 1 and 18 type 2). There was no positive SVC-ELISA/negative cell-culture cases. The sensitivity was 46%, and the specificity was 100%. The average amount of time to process one vial and controls from the end of the incubation period was 172 rain. Additional vials added
