Vol. 30, No. 7
JUIY 1992, p. 1874-1875 0095-1137/92/071874-02$02.00/0 Copyright © 1992, American Society for Microbiology
JOURNAL OF CLINICAL MICROBIOLOGY,
Comparative Evaluation of Four Commercially Available Monoclonal Antibodies for Culture Confirmation of Herpes Simplex Virus Infection SHERYL L. G. JOHNSTON'* AND KATHLEEN WELLENS2 Virology Laboratory, St. Vincent Hospital, Green Bay, Wisconsin 54301,1 and Colorado State University, Fort Collins, Colorado 805232 Received 23 December 1991/Accepted 13 April 1992
Four fluorescein isothiocyanate-labelled monoclonal antibody typing reagents were compared for intensity of fluorescence and sensitivity in confirming herpes simplex virus (HSV) in cell culture. A total of 125 (50 HSV type 1 [HSV-1] and 75 HSV-2) specimens positive for HSV were concurrently stained with type-specific monoclonal antibodies from Bartels (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), Kallestad (Pathfinder; Kallestad Diagnostics, Chaska, Minn.), Diagnostic Products Corp. (DPC) (PathoDx; DPC, Los Angeles, Calif.), and Syva (Microtrak; Syva Co., Palo Alto, Calif.). Fifty cultured specimens not displaying HSV-associated cytopathic effect were also stained. Each of the four reagents confirmed the 50 negative cultures and 125 positive cultures. One HSV-1-positive culture was missed by the Kallestad antibody. Ten HSV-1 isolates displayed dull fluorescence when stained with the Syva HSV-2 reagent. The other three reagents stained only the HSV-1 well with these 10 specimens. The Syva and Bartels stains were brighter, in general, than the Kallestad and DPC stains. However, the Kallestad and DPC stains were brilliant enough to allow interpretation without hesitation. The differences in intensity and background staining emphasize the need for laboratories to routinely perform quality checks on their reagents and to test new products which become commercially available. was scraped from the tube into approximately 0.5 ml of culture supernatant and the mixture was dropped onto the rings of an eight-well glass slide. The slides were air dried and acetone fixed. Each of the eight wells was then covered with type-specific antibodies from Bartels, Kallestad, DPC, and Syva. The slide was incubated in a humidity chamber for 30 min at 35°C, rinsed, and washed twice in fresh phosphatebuffered saline (PBS) (pH 7.6) for approximately 5 min. The slides were then blotted to remove excess PBS, mounted with a buffered glycerol-mounting medium, coverslipped, and evaluated under a fluorescent microscope (excitation wavelength = 490 nm) at 400x power. The intensity of the individual stains was rated on a scale of 1 to 4+, with 4+ representing the most brilliant. A total of 175 clinical samples-of which 50 were HSV-1 culture positive, 75 were HSV-2 culture positive, and 50 were negative-were inoculated into cell cultures and examined by using the four fluorescein isothiocyanate-conjugated monoclonal antibodies. A total of 125 HSV-positive isolates (50 type 1 and 75 type 2) and 50 negative cultures were stained. There was positive and negative agreement of the four reagents on all the specimens with the exception of one in which a single HSV-1 culture was stained by all but the Kallestad reagent. This isolate was documented at a 3+ intensity reading with each of the other three reagents. When this virus was reisolated and retested, similar results were obtained. The Syva, Bartels, and Kallestad stains displayed a more uniform cytoplasmic- and/or nuclear-staining pattern than did the DPC stain, which was more granular. All four stains demonstrated equal clarity. The intensities of the stains were graded on a scale of I to 4+, and the means are presented in Table 1. Bartels and Syva stains had similar overall means of 3.79 and 3.78, respectively, while the DPC overall mean was 3.45 and the Kallestad overall mean was 3.38.
Cell culture isolation remains the "gold standard" for herpes simplex virus (HSV) detection (6). Confirmation of cultures with fluorescein isothiocyanate-labelled monoclonal antibodies is the most cost-effective and least time-consuming method of verifying and typing isolates in the clinical laboratory (2, 8). The ever-growing commercial market for monoclonal antibody stains for HSV has placed a responsibility on clinical virology laboratories to continually monitor the performance of the stains which they use and also to investigate the use of new products (1, 5, 10). Typing of HSV isolates is generally not necessary for treatment of HSV infection, but it may affect patient management in terms of counseling and prognosis in that the rate of recurrence of genital HSV type 1 (HSV-1) infections is less than that of HSV-2 infections (2, 3, 7). Epidemiologically, the information can help identify patterns of correlation with other infections within specific populations such as immunocompromised patients (2). In this evaluation, four type-specific monoclonal antibody stains, one being a new commercial reagent, PathoDx (Diagnostic Products Corp. [DPC]), were compared for intensity of staining and sensitivity to HSV isolated from clinical specimens. (The work was done, in part, at Bellin Hospital, Green Bay, Wis.) Specimens were obtained from men and women primarily from genital sites, although oral and dermal sites were included. The specimens arrived in the laboratory on wet ice within 16 h of collection. The samples were vortexed, and 0.5 ml was inoculated into one tube each of locally produced rhabdomyosarcoma and mink lung cell cultures. These were incubated at 35°C for up to 7 days on a roller drum (30 rpm) and checked daily for cytopathic effect. Once cytopathic effect was observed in 50 to 75% of the cell culture monolayer, the rhabdomyosarcoma cell culture
*
Corresponding author. 1874
VOL. 30, 1992
NOTES
TABLE 1. Mean intensity ratings of four HSV strains HSV type
1 2 Total '
Intensity' of stain
No. of cultures
Bartels
DPC PathoDx
Kallestad Pathfinder
Syva Microtrak
50 75
3.56 3.95
3.44 3.45
3.10 3.56
3.52 3.96
125
3.79
3.45
3.38
3.78
Based on a rating scale of 1 to 4+, 4+ being the most brilliant.
1875
currently used products and investigate new products on the market. Individual laboratories must be aware of these differences among reagents and take into account their own needs when selecting a reagent. This study indicated that with the rhabdomyosarcoma cells prepared in our laboratory, the Bartels stain performed the best, being the most clear and intense stain without the crossover fluorescence observed with the Syva stain in a few HSV-1 isolates. The new DPC stain, although slightly less intense than the Bartels stain, was very clear and specific and came in an easy, ready-touse dropper bottle.
REFERENCES A Student's t test was used to determine the statistical significance of these values. There was no significant difference (P > 0.05) between the mean scores of the Bartels and Syva reagents for either type or between those of the Syva and DPC HSV-1 reagents. In all other combinations, mean scores were significantly different from each other. Using this rating system to identify the best overall stain demonstrated that the Bartels and Syva stains were nearly equal in intensity for both HSV-1 and HSV-2. While both the DPC and Kallestad stains were less intense than the other two, the DPC stain was significantly more intense than the Kallestad stain with both HSV types. Each isolate was stained with all four stains on the same slide and read by the same microscopist to ensure consistent intensity and clarity comparisons. Rhabdomyosarcoma cells, which in the past have been shown to have good staining abilities, were stained throughout the study (7-9). Because certain cells have been shown to have lower staining intensities with certain reagents, cell type should also be considered when stains are being evaluated (3). No isolates were determined to be from dual infections. However, 10 HSV-1 isolates displayed a dull fluorescence in the HSV-2 Syva-stained wells. There was no fluorescence in the HSV-2 wells with any of the other three reagents with these isolates. On the basis of the staining patterns of the other three stains, these 10 specimens were determined to be solely HSV-1. In all 10 of these specimens, all four HSV-1 stains were extremely brilliant (4+). The overlapping of HSV-1 isolate fluorescence with fluorescence in the HSV-2-stained wells creates the potential for misinterpretation by an inexperienced technologist. Unless these seemingly dual isolates are confirmed by staining with another reagent or by restriction endonuclease analysis, the Syva stain may lead to mistyping. The clinical virology reagent market continues to have an influx of monoclonal antibody stains from different manufacturers who are capitalizing on the growing number of clinical virology laboratories (1, 4, 5, 7, 10-12). With an increasing number of small community hospitals and laboratories delving into the area of HSV isolation, the demand for highquality low-cost HSV reagents has grown. Furthermore, as the technology and production of these reagents advance, commercially available reagents may undergo production changes that are not reported to the consumer. It is therefore up to the user of commercially marketed monoclonal antibody stains to routinely evaluate
1. Aarnaes, S. L., E. M. Peterson, and L. M. de la Maza. 1989. Evaluation of a new herpes simplex virus typing reagent for tissue culture confirmation. Diagn. Microbiol. Infect. Dis. 12: 269-270. 2. Arvin, A. M., and C. G. Prober. 1991. Herpes simplex viruses, p. 822-828. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 3. Gleaves, C. A., C. F. Lee, J. A. Dragavon, and J. D. Meyers. 1989. Detection of herpes simplex virus from clinical specimens by centrifugation enhanced cell culture in MRC-5, primary rabbit kidney and mink lung cells. Serodiagn. Immunother. Infect. Dis. 3:87-92. 4. Gleaves, C. A., D. H. Rice, R. Bindra, D. A. Hursch, S. E. Curtis, C. F. Lee, and S. F. Wendt. 1989. Evaluation of a HSV specific monoclonal antibody reagent for laboratory diagnosis of herpes simplex virus infection. Diagn. Microbiol. Infect. Dis. 12:315-318. 5. Gleaves, C. A., E. M. Swierkosz, C. F. Lee, and J. D. Meyers. 1988. Serologic confirmation and typing of clinical herpes simplex virus isolates: comparison of two different monoclonal antibody immunofluorescence kits. Serodiagn. Immunother. Infect. Dis. 2:151-156. 6. Hsiung, G. D. 1989. The impact of cell culture sensitivity on rapid viral diagnosis: a historical perspective. Yale J. Biol. Med. 62:79-88. 7. Johnston, S. L. G., and C. A. Gleaves. 1990. Culture confirmation and typing of herpes simplex virus isolates from clinical specimens with new monoclonal antibody typing reagents. Serodiagn. Immunother. Infect. Dis. 4:295-297. 8. Johnston, S. L. G., and C. S. Siegel. 1990. Comparison of enzyme immunoassay, shell vial culture, and conventional cell culture for the rapid detection of herpes simplex virus. Diagn. Microbiol. Infect. Dis. 13:241-244. 9. Johnston, S. L. G., K. Wellens, and C. S. Siegel. 1990. Rapid isolation of herpes simplex virus by using mink lung and rhabdomyosarcoma cell cultures. J. Clin. Microbiol. 28:28062807. 10. Lipson, S. M., R. J. Salo, and G. P. Leonardi. 1991. Evaluation of five monoclonal antibody-based kits or reagents for the identification and culture confirmation of herpes simplex virus. J. Clin. Microbiol. 29:466-469. 11. Nahmias, A., I. delBunono, J. Pipkin, R. Hutton, and C. Wickliffe. 1971. Rapid identification and typing of herpes simplex virus types 1 and 2 by a direct immunofluorescence technique. Appl. Microbiol. 22:455-458. 12. Swierkosz, E. M., M. Q. Arens, R. R. Schmidt, and T. Armstrong. 1985. Evaluation of two immunofluorescence assays with monoclonal antibodies for typing of herpes simplex virus. J. Clin. Microbiol. 21:643-644.
JUIY 1992, p. 1874-1875 0095-1137/92/071874-02$02.00/0 Copyright © 1992, American Society for Microbiology
JOURNAL OF CLINICAL MICROBIOLOGY,
Comparative Evaluation of Four Commercially Available Monoclonal Antibodies for Culture Confirmation of Herpes Simplex Virus Infection SHERYL L. G. JOHNSTON'* AND KATHLEEN WELLENS2 Virology Laboratory, St. Vincent Hospital, Green Bay, Wisconsin 54301,1 and Colorado State University, Fort Collins, Colorado 805232 Received 23 December 1991/Accepted 13 April 1992
Four fluorescein isothiocyanate-labelled monoclonal antibody typing reagents were compared for intensity of fluorescence and sensitivity in confirming herpes simplex virus (HSV) in cell culture. A total of 125 (50 HSV type 1 [HSV-1] and 75 HSV-2) specimens positive for HSV were concurrently stained with type-specific monoclonal antibodies from Bartels (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), Kallestad (Pathfinder; Kallestad Diagnostics, Chaska, Minn.), Diagnostic Products Corp. (DPC) (PathoDx; DPC, Los Angeles, Calif.), and Syva (Microtrak; Syva Co., Palo Alto, Calif.). Fifty cultured specimens not displaying HSV-associated cytopathic effect were also stained. Each of the four reagents confirmed the 50 negative cultures and 125 positive cultures. One HSV-1-positive culture was missed by the Kallestad antibody. Ten HSV-1 isolates displayed dull fluorescence when stained with the Syva HSV-2 reagent. The other three reagents stained only the HSV-1 well with these 10 specimens. The Syva and Bartels stains were brighter, in general, than the Kallestad and DPC stains. However, the Kallestad and DPC stains were brilliant enough to allow interpretation without hesitation. The differences in intensity and background staining emphasize the need for laboratories to routinely perform quality checks on their reagents and to test new products which become commercially available. was scraped from the tube into approximately 0.5 ml of culture supernatant and the mixture was dropped onto the rings of an eight-well glass slide. The slides were air dried and acetone fixed. Each of the eight wells was then covered with type-specific antibodies from Bartels, Kallestad, DPC, and Syva. The slide was incubated in a humidity chamber for 30 min at 35°C, rinsed, and washed twice in fresh phosphatebuffered saline (PBS) (pH 7.6) for approximately 5 min. The slides were then blotted to remove excess PBS, mounted with a buffered glycerol-mounting medium, coverslipped, and evaluated under a fluorescent microscope (excitation wavelength = 490 nm) at 400x power. The intensity of the individual stains was rated on a scale of 1 to 4+, with 4+ representing the most brilliant. A total of 175 clinical samples-of which 50 were HSV-1 culture positive, 75 were HSV-2 culture positive, and 50 were negative-were inoculated into cell cultures and examined by using the four fluorescein isothiocyanate-conjugated monoclonal antibodies. A total of 125 HSV-positive isolates (50 type 1 and 75 type 2) and 50 negative cultures were stained. There was positive and negative agreement of the four reagents on all the specimens with the exception of one in which a single HSV-1 culture was stained by all but the Kallestad reagent. This isolate was documented at a 3+ intensity reading with each of the other three reagents. When this virus was reisolated and retested, similar results were obtained. The Syva, Bartels, and Kallestad stains displayed a more uniform cytoplasmic- and/or nuclear-staining pattern than did the DPC stain, which was more granular. All four stains demonstrated equal clarity. The intensities of the stains were graded on a scale of I to 4+, and the means are presented in Table 1. Bartels and Syva stains had similar overall means of 3.79 and 3.78, respectively, while the DPC overall mean was 3.45 and the Kallestad overall mean was 3.38.
Cell culture isolation remains the "gold standard" for herpes simplex virus (HSV) detection (6). Confirmation of cultures with fluorescein isothiocyanate-labelled monoclonal antibodies is the most cost-effective and least time-consuming method of verifying and typing isolates in the clinical laboratory (2, 8). The ever-growing commercial market for monoclonal antibody stains for HSV has placed a responsibility on clinical virology laboratories to continually monitor the performance of the stains which they use and also to investigate the use of new products (1, 5, 10). Typing of HSV isolates is generally not necessary for treatment of HSV infection, but it may affect patient management in terms of counseling and prognosis in that the rate of recurrence of genital HSV type 1 (HSV-1) infections is less than that of HSV-2 infections (2, 3, 7). Epidemiologically, the information can help identify patterns of correlation with other infections within specific populations such as immunocompromised patients (2). In this evaluation, four type-specific monoclonal antibody stains, one being a new commercial reagent, PathoDx (Diagnostic Products Corp. [DPC]), were compared for intensity of staining and sensitivity to HSV isolated from clinical specimens. (The work was done, in part, at Bellin Hospital, Green Bay, Wis.) Specimens were obtained from men and women primarily from genital sites, although oral and dermal sites were included. The specimens arrived in the laboratory on wet ice within 16 h of collection. The samples were vortexed, and 0.5 ml was inoculated into one tube each of locally produced rhabdomyosarcoma and mink lung cell cultures. These were incubated at 35°C for up to 7 days on a roller drum (30 rpm) and checked daily for cytopathic effect. Once cytopathic effect was observed in 50 to 75% of the cell culture monolayer, the rhabdomyosarcoma cell culture
*
Corresponding author. 1874
VOL. 30, 1992
NOTES
TABLE 1. Mean intensity ratings of four HSV strains HSV type
1 2 Total '
Intensity' of stain
No. of cultures
Bartels
DPC PathoDx
Kallestad Pathfinder
Syva Microtrak
50 75
3.56 3.95
3.44 3.45
3.10 3.56
3.52 3.96
125
3.79
3.45
3.38
3.78
Based on a rating scale of 1 to 4+, 4+ being the most brilliant.
1875
currently used products and investigate new products on the market. Individual laboratories must be aware of these differences among reagents and take into account their own needs when selecting a reagent. This study indicated that with the rhabdomyosarcoma cells prepared in our laboratory, the Bartels stain performed the best, being the most clear and intense stain without the crossover fluorescence observed with the Syva stain in a few HSV-1 isolates. The new DPC stain, although slightly less intense than the Bartels stain, was very clear and specific and came in an easy, ready-touse dropper bottle.
REFERENCES A Student's t test was used to determine the statistical significance of these values. There was no significant difference (P > 0.05) between the mean scores of the Bartels and Syva reagents for either type or between those of the Syva and DPC HSV-1 reagents. In all other combinations, mean scores were significantly different from each other. Using this rating system to identify the best overall stain demonstrated that the Bartels and Syva stains were nearly equal in intensity for both HSV-1 and HSV-2. While both the DPC and Kallestad stains were less intense than the other two, the DPC stain was significantly more intense than the Kallestad stain with both HSV types. Each isolate was stained with all four stains on the same slide and read by the same microscopist to ensure consistent intensity and clarity comparisons. Rhabdomyosarcoma cells, which in the past have been shown to have good staining abilities, were stained throughout the study (7-9). Because certain cells have been shown to have lower staining intensities with certain reagents, cell type should also be considered when stains are being evaluated (3). No isolates were determined to be from dual infections. However, 10 HSV-1 isolates displayed a dull fluorescence in the HSV-2 Syva-stained wells. There was no fluorescence in the HSV-2 wells with any of the other three reagents with these isolates. On the basis of the staining patterns of the other three stains, these 10 specimens were determined to be solely HSV-1. In all 10 of these specimens, all four HSV-1 stains were extremely brilliant (4+). The overlapping of HSV-1 isolate fluorescence with fluorescence in the HSV-2-stained wells creates the potential for misinterpretation by an inexperienced technologist. Unless these seemingly dual isolates are confirmed by staining with another reagent or by restriction endonuclease analysis, the Syva stain may lead to mistyping. The clinical virology reagent market continues to have an influx of monoclonal antibody stains from different manufacturers who are capitalizing on the growing number of clinical virology laboratories (1, 4, 5, 7, 10-12). With an increasing number of small community hospitals and laboratories delving into the area of HSV isolation, the demand for highquality low-cost HSV reagents has grown. Furthermore, as the technology and production of these reagents advance, commercially available reagents may undergo production changes that are not reported to the consumer. It is therefore up to the user of commercially marketed monoclonal antibody stains to routinely evaluate
1. Aarnaes, S. L., E. M. Peterson, and L. M. de la Maza. 1989. Evaluation of a new herpes simplex virus typing reagent for tissue culture confirmation. Diagn. Microbiol. Infect. Dis. 12: 269-270. 2. Arvin, A. M., and C. G. Prober. 1991. Herpes simplex viruses, p. 822-828. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 3. Gleaves, C. A., C. F. Lee, J. A. Dragavon, and J. D. Meyers. 1989. Detection of herpes simplex virus from clinical specimens by centrifugation enhanced cell culture in MRC-5, primary rabbit kidney and mink lung cells. Serodiagn. Immunother. Infect. Dis. 3:87-92. 4. Gleaves, C. A., D. H. Rice, R. Bindra, D. A. Hursch, S. E. Curtis, C. F. Lee, and S. F. Wendt. 1989. Evaluation of a HSV specific monoclonal antibody reagent for laboratory diagnosis of herpes simplex virus infection. Diagn. Microbiol. Infect. Dis. 12:315-318. 5. Gleaves, C. A., E. M. Swierkosz, C. F. Lee, and J. D. Meyers. 1988. Serologic confirmation and typing of clinical herpes simplex virus isolates: comparison of two different monoclonal antibody immunofluorescence kits. Serodiagn. Immunother. Infect. Dis. 2:151-156. 6. Hsiung, G. D. 1989. The impact of cell culture sensitivity on rapid viral diagnosis: a historical perspective. Yale J. Biol. Med. 62:79-88. 7. Johnston, S. L. G., and C. A. Gleaves. 1990. Culture confirmation and typing of herpes simplex virus isolates from clinical specimens with new monoclonal antibody typing reagents. Serodiagn. Immunother. Infect. Dis. 4:295-297. 8. Johnston, S. L. G., and C. S. Siegel. 1990. Comparison of enzyme immunoassay, shell vial culture, and conventional cell culture for the rapid detection of herpes simplex virus. Diagn. Microbiol. Infect. Dis. 13:241-244. 9. Johnston, S. L. G., K. Wellens, and C. S. Siegel. 1990. Rapid isolation of herpes simplex virus by using mink lung and rhabdomyosarcoma cell cultures. J. Clin. Microbiol. 28:28062807. 10. Lipson, S. M., R. J. Salo, and G. P. Leonardi. 1991. Evaluation of five monoclonal antibody-based kits or reagents for the identification and culture confirmation of herpes simplex virus. J. Clin. Microbiol. 29:466-469. 11. Nahmias, A., I. delBunono, J. Pipkin, R. Hutton, and C. Wickliffe. 1971. Rapid identification and typing of herpes simplex virus types 1 and 2 by a direct immunofluorescence technique. Appl. Microbiol. 22:455-458. 12. Swierkosz, E. M., M. Q. Arens, R. R. Schmidt, and T. Armstrong. 1985. Evaluation of two immunofluorescence assays with monoclonal antibodies for typing of herpes simplex virus. J. Clin. Microbiol. 21:643-644.
