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Typing System for the detection and typing of herpes simplex virus from clinical specimens
Journal of Clinical Virology 12 (1999) 65 – 69
Short communication
Evaluation of ELVIS™ HSV ID/Typing System for the detection and typing of herpes simplex virus from clinical specimens Brian M. Turchek, Yung T. Huang * Department of Pathology, Uni6ersity Hospitals of Cle6eland, Case Western Reser6e Uni6ersity, 2085 Adelbert Road, Cle6eland, OH 44106, USA Received 29 July 1998; received in revised form 9 October 1998; accepted 13 October 1998
Abstract Background: Herpes simplex virus (HSV) is a common pathogen with two serotypes: HSV-1 and HSV-2. HSV infection does not pose much of a threat to an immunocompetent host but to an immunocompromised host or a neonate the infection can be fatal. The Enzyme-Linked Virus Inducible System™ (ELVIS™) employs a genetically altered baby hamster kidney (BHK) cell line that allows for the rapid overnight detection of HSV but also includes an immunofluorescent stain for the simultaneous detection and typing of HSV-1 and HSV-2. Objective: To evaluate the ELVIS™ HSV ID/Typing System in comparison with HSV identification and typing in primary rabbit kidney (PRK) cells grown in shell vials. Study design: Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK shell vials and one ELVIS™ BHK shell vial were inoculated with patient specimen. PRK shell vials were observed for cytopathic effect (CPE) for up to 4 days. When CPE was observed the PRK shell vials were fixed and one shell vial was stained with HSV-1 monoclonal antibody (Mab) and the other was stained with HSV-2 Mab. The coverslips were observed under the fluorescent microscope for specific apple-green fluorescence. The BHK shell vials were incubated overnight, fixed, and stained with galactopyranoside (X-Gal). If blue cells were present, the specimen was positive for HSV. The coverslip was then observed under the fluorescent microscope for the presence of specific apple-green fluorescence, indicating HSV-2. If no specific apple-green stain was observed, the coverslip was stained with a fluorescent conjugated goat anti-mouse IgG to determine the presence of HSV-1.
Abbre6iations: BHK, baby hamster kidney; CPE, cytopathic effect; ELVIS™, Enzyme-Linked Virus Inducible System™; HSV, herpes simplex virus; Mab, monoclonal antibody; PI, post-inoculation; PRK, primary rabbit kidney; X-Gal, galactopyranoside. * Corresponding author. Tel.: +1-216-844-8611; fax: +1-216-368-0495. 1386-6532/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 0 1 9 7 ( 9 8 ) 0 0 0 6 6 - X
66
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
Results: Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 were HSV-2 and 13 were HSV-1. The ELVIS™ BHK shell vials detected 42 positive HSV; 30 were HSV-2 and 12 were HSV-1. One low titer specimen was not identified as being HSV positive. Two specimens were not directly typed by the ELVIS™ system. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one fluorescent cell present that was difficult to type. As suggested by the manufacturer’s instructions, both specimens that were not directly typed were re-grown overnight from their supernatants and were correctly identified and typed. Conclusion: The ELVIS™ HSV ID/Typing System is a rapid, highly specific and sensitive method of overnight HSV detection and typing. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Herpes simplex virus; ELVIS™; X-Gal; Baby hamster kidney; Primary rabbit kidney; Monoclonal antibody
Herpes simplex virus (HSV) is a common human pathogen that can produce lesions at a variety of sites in the human body. There are two distinct serotypes of HSV, HSV-1 and HSV-2. HSV-2 is primarily associated with genital infections, while HSV-1 is associated with oral infection. Neither serotype, though, exclusively found in those locations (Mattison et al., 1991). With readily available and effective acyclovir to treat HSV infection, rapid detection will greatly assist patient management. There are reasons for the typing of HSV positive specimens. The type is important in following the epidemiology of HSV. In the case of genital herpes, the recurrence rate and asymptomatic shedding of the virus is significantly lower in HSV-1 infection than in HSV-2 (Corey et al., 1983). In terms of long-term suppressive therapy with acyclovir in cases of frequently recurring HSV infection, it is important to correlate the HSV type and site of infection (Kinghorn, 1988; Mostow et al., 1988; Rooney et al., 1993). In the case of pregnancy, mothers who are newly infected with HSV-2 are 33% more likely to pass the virus to their babies (Brown et al., 1991). Congenital HSV infection in neonates can cause conjunctivitis, severe neurodevelopmental problems, and death (Hirsch, 1990). Several methods have been used to identify HSV infection in an individual: the direct immunohistochemical staining of cells collected from an active lesion, enzyme immunoassay to detect HSV antigens (Wu et al., 1989; Dorian et al., 1990; Verano and Michalski, 1990), use of DNA probes on inoculated cell cultures (Espy and
Smith, 1988), in situ hybridization of inoculated cells in culture (Heggie and Huang, 1993), immunofluorescent staining of inoculated cells grown in shell vials (Gleaves et al., 1985), and inoculated cell culture for the formation of cytopathic effect (CPE) (Landry and Hsiung, 1994). The direct staining of cells is the fastest method of identifying HSV in a patient but a specimen with enough cells is not always achieved. Cell culture identification of HSV is the gold standard but it is more time consuming and usually requires a week of incubation. Once CPE is observed, the cells must be stained with type specific monoclonal antibody (Mab) in order to type the HSV. A rapid method is the centrifugation enhanced shell vial technique which requires an overnight incubation but two shell vials need to be inoculated and stained with HSV-1 and HSV-2 specific Mabs, respectively for typing. A recently introduced Enzyme-linked Virus Inducible System™ (ELVIS™) HSV Test System (Diagnostic Hybrids, Athens, OH) is a rapid, highly specific, and highly sensitive system for the detection of HSV (Stabell et al., 1993; Proffitt and Schindler, 1995). The ELVIS™ Test utilizes genetically altered BHK cells. The BHK cells have been transfected with an inducible HSV promoter gene linked to an Escherichia coli LacZ reporter gene (Stabell and Olivo, 1992). When the cell becomes infected with HSV, the virus induces the HSV promoter gene to express the LacZ gene, to produce b-Galactosidase. The infected cells, when stained with X-Gal, show blue, indicating a specimen positive for HSV. This system allows for the
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
67
Table 1 Type of specimens and type of virus isolated in PRK cultures
rapid detection of HSV, but does not offer typing. However, the new ELVIS™ HSV ID/Typing System (Diagnostic Hybrids, Athens, OH) utilizes the same modified BHK cells to detect HSV but also allows for direct typing at the same time. The X-Gal detection solution contains not only galactopyranoside but also fluorescein labeled Mab to HSV-2 and non-fluorescein labeled Mab to HSV1 for typing. Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK (BioWhittaker, Walkersville, MD) and one ELVIS™ BHK shell vial were inoculated with 200 ml of each specimen. Prior to inoculation the media in each vial was aspirated and replaced with 1 ml of Replacement Media (EMEM, 7% fetal bovine serum, 1.25 mg/ml fungizone, 100 U penicillin, 100 mg/ml streptomycin) provided by Diagnostic Hybrids. After inoculation, the shell vials were centrifuged at 700× g for 60 min at 24°C and incubated at 37°C with 5% CO2. The PRK shell vials were examined daily for CPE (up to 4 days). If CPE was present, one shell vial was stained for HSV-1 and the other was stained for HSV-2, using the Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit (Behring Diagnostics, Cupertino, CA). After an overnight incubation, the ELVIS™ BHK shell vial was fixed with the ELVIS™ HSV ID/Typing System solution c1 for 5 min and then stained with solution c2 (X-Gal, fluorescein conjugated HSV-2 Mab, and non-fluorescein labeled HSV-1 Mab). Each shell vial was examined for the presence of blue cells. If blue cells were not present, the specimen was called negative for HSV. If blue cells were present, the specimen was called positive for HSV, then examined under the fluorescent microscope
Specimen source
PRK cultures HSV-1
HSV-2
Genital Rectal Oral/facial Other
3 0 10 0
21 6 1 2
Totals
13
30
for the presence of apple-green staining cells. If specific staining was observed, it indicated HSV-2. If no specific staining was present, then the shell vial was stained with solution c3 (fluorescein conjugated anti-mouse IgG) to confirm the presence of HSV-1 and then re-examined under the fluorescent microscope. Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 (70%) were HSV-2 and 13 (30%) were HSV-1. As shown in Tables 1 and 2, of the 24 positive genital specimens: three were HSV-1 and 21 were HSV-2; all of the six rectal specimens were HSV-2; of the 11 oral-facial specimens, ten were HSV-1 and one was HSV-2. The two unidentified specimens were both HSV-2. Of the 43 HSV positives detected by PRK shell vials, 32 (74%) were detected on day 1 post-inoculation (PI), six (14%) were detected on day 2 PI, and five (12%) were detected on day 3 PI. No positives were detected after day 3 PI. The staining of PRK cells after observing CPE is done to save the cost of staining reagent. If the PRK cells were stained blindly after an overnight culture, the results may be similar to the ELVIS™ BHK cells. The ELVIS™ BHK shell vials detected 42 positives: 30 were HSV-2 and 12 were HSV-1. One specimen was not detected as being HSV positive by
Table 2 Time of virus detection in PRK cultures and ELVIS™ cultures Day 1
PRK cultures ELVIS™ cultures
Day 2
Day 3
HSV-1
HSV-2
HSV-1
HSV-2
HSV-1
HSV-2
7 12
25 30
3 –
3 –
3 –
2 –
68
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
ELVIS™. This was a nose specimen that was not detected by PRK shell vials until day 3 PI, indicating a low viral titer specimen. Two specimens were not directly typed by the ELVIS™ HSV ID/Typing System. One of them was an oral specimen that was not detected by PRK shell vials until day 3 PI. This specimen had only five blue cells present and weakly stained for HSV-2 but was not distinct enough to identify during the initial microscope reading and subsequently stained brighter after the conjugate was added making it difficult to identify type. The other specimen was a lip specimen that also was not detected by PRK shell vials until day 3 PI. This specimen had only one blue cell present and did not show specific fluorescent staining for either HSV-1 or HSV-2. The supernatants from both specimens were re-inoculated into ELVIS™ BHK shell vials and were detected and typed correctly after an overnight incubation. The sensitivity of the ELVIS™ detection of HSV was 98% (42/43) and the specificity for HSV was 100%. The sensitivity of the ELVIS™ HSV typing was 95% (40/ 43) and the specificity of the typing was 98% (42/43). The ELVIS™ system for HSV identification is proven to be highly sensitive and specific for the detection of HSV. Our results are comparable to previous reports (Stabell et al., 1993; Proffitt and Schindler, 1995) with a sensitivity of 98% and a specificity of 100% for the detection of HSV. The new ELVIS™ HSV ID/Typing System not only provides a rapid method for HSV identification but also allows for the simultaneous typing of HSV positive specimens. The results for the typing of HSV positive specimens using the ELVIS™ HSV ID/Typing System were comparable with the standard method. The sensitivity was 95% and the specificity was 98%. The specimens that were not able to be typed directly were specimens of low virus titer. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one cell was weakly stained fluorescent for HSV-2 but was not distinct enough to call HSV-2. After staining with the HSV-1 confirmatory stain, that one cell became more brightly stained making it difficulty to
give a definite type. In the package insert, the manufacturer suggests that specimens with ten or fewer blue cells may not show fluorescence and recommends re-testing of the specimen. Both specimens were re-inoculated with supernatant from their original ELVIS™ shell vials and were correctly identified and typed the following day. However, there were five other specimens that had ten or fewer blue cells that were correctly typed. If the manufacturer’s recommendations were followed in this study, the sensitivity and specificity for the typing would have been 100% and 100%, respectively. One other unique feature about the ELVIS™ HSV ID/Typing System is that the HSV-1 Mab primarily stains the nucleus (targeting an HSV nuclear antigen), while the HSV-2 Mab only stains the cytoplasm. This feature provides additional specificity in the identification of HSV type. Overall, the ELVIS™ HSV ID/Typing System is a rapid, highly specific, and sensitive method for overnight HSV detection and typing. A cost comparison was done between two shell vials with the Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit and one shell vial with the ELVIS™ HSV ID/Typing Test System. The Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit costs US$260 for 20 assays and the PRK shell vials cost US$2 each, which totals US$17 per specimen in order to blind stain and type. The ELVIS™ HSV ID/Typing Test System costs US$265 for 60 assays and the BHK shell vials cost US$5.52 each, which totals US$9.94 per specimen in order to blind stain and type. Therefore, the system requires less hands-on time than other methods for HSV detection and typing, requires less reagents and cultured cells, and costs less.
References Brown ZA, Benedetti J, Ashely R, Burchett S, Selke S, Berry S, Vontver LA, Corey L. Neonatal herpes simplex virus infection in relation to asymptomatic maternal infection at the time of labor. N Engl J Med 1991;324:1247 – 52. Corey L, Adams HG, Brown ZA, Holmes KK. Genital herpes simplex virus infections: clinical manifestations, course, and complications. Ann Intern Med 1983;98:958 – 72.
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69 Dorian KJ, Beatty E, Atterbury KE. Detection of herpes simplex virus by the Kodak SureCell Herpes Test. J Clin Microbiol 1990;28:2117–9. Espy MJ, Smith TH. Detection of herpes simplex virus in conventional tube cultures and in shell vials with a DNA probe kit and monoclonal antibodies. J Clin Microbiol 1988;26:22 – 4. Gleaves CA, Wilson DJ, Wold AD, Smith TF. Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation. J Clin Microbiol 1985;21:29–32. Heggie AD, Huang YT. Rapid detection of herpes simplex virus in culture by in situ hybridization. J Virol Methods 1993;41:1 – 8. Hirsch MS. Herpes simplex virus. In: Mandell GL, Douglas RG, Bennett JE, editors. Principles and Practice of Infectious Diseases, 3rd Edition. New York: Churchill Livingstone, 1990:1144 – 53. Kinghorn GR. Long-term suppression with oral acyclovir of recurrent herpes simplex virus infections in otherwise healthy patients. Am J Med 1988;85(2A):26–9. Landry ML, Hsiung GD. Herpesviridae. In: Hsiung GD, Fong CKY, Landry ML, editors. Hsiung’s Diagnostic Virology, 3rd Edition. New Haven, CT: Yale University Press, 1994:238 – 68. Mattison HR, Eisenberg RJ, Reichman RC. Herpes simplex virus. In: Belshe RB, editor. Textbook of Human Virology,
.
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Second Edition. St. Louis, MO: Mosby, 1991:822 – 41. Mostow SR, Mayfield JL, Marr JJ, Drucker JL. Suppression of recurrent genital herpes by single daily dosages of acyclovir. Am J Med 1988;85(2A):30– 3. Proffitt MR, Schindler SA. Rapid detection of HSV with an enzyme-linked virus inducible system™ (ELVIS™) employing a genetically modified cell line. Clin Diagn Virol 1995;4:175 – 82. Rooney JF, Straus SE, Mannix ML, Wohlenberg CR, Alling DW, Dumois JA, Notkins AL. Oral acyclovir to suppress frequently recurrent herpes labialis — double-blind, placebo-controlled trial. Ann Intern Med 1993;118:268 – 72. Stabell EC, Olivo PD. Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus. J Virol Methods 1992;38:195 – 204. Stabell EC, O’Rourke SR, Storch GA, Olivo PD. Evaluation of a genetically engineered cell line and a histochemical b-galactosidase assay to detect herpes simplex virus in clinical specimens. J Clin Microbiol 1993;31:1296 – 8. Verano L, Michalski F. Herpes simplex antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay. J Clin Microbiol 1990;28:2555 – 8. Wu TC, Zaza S, Callaway J. Evaluation of the Du Pont Herpchek herpes simplex virus antigen test with clinical specimens. J Clin Microbiol 1989;27:1903 – 5.
Short communication
Evaluation of ELVIS™ HSV ID/Typing System for the detection and typing of herpes simplex virus from clinical specimens Brian M. Turchek, Yung T. Huang * Department of Pathology, Uni6ersity Hospitals of Cle6eland, Case Western Reser6e Uni6ersity, 2085 Adelbert Road, Cle6eland, OH 44106, USA Received 29 July 1998; received in revised form 9 October 1998; accepted 13 October 1998
Abstract Background: Herpes simplex virus (HSV) is a common pathogen with two serotypes: HSV-1 and HSV-2. HSV infection does not pose much of a threat to an immunocompetent host but to an immunocompromised host or a neonate the infection can be fatal. The Enzyme-Linked Virus Inducible System™ (ELVIS™) employs a genetically altered baby hamster kidney (BHK) cell line that allows for the rapid overnight detection of HSV but also includes an immunofluorescent stain for the simultaneous detection and typing of HSV-1 and HSV-2. Objective: To evaluate the ELVIS™ HSV ID/Typing System in comparison with HSV identification and typing in primary rabbit kidney (PRK) cells grown in shell vials. Study design: Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK shell vials and one ELVIS™ BHK shell vial were inoculated with patient specimen. PRK shell vials were observed for cytopathic effect (CPE) for up to 4 days. When CPE was observed the PRK shell vials were fixed and one shell vial was stained with HSV-1 monoclonal antibody (Mab) and the other was stained with HSV-2 Mab. The coverslips were observed under the fluorescent microscope for specific apple-green fluorescence. The BHK shell vials were incubated overnight, fixed, and stained with galactopyranoside (X-Gal). If blue cells were present, the specimen was positive for HSV. The coverslip was then observed under the fluorescent microscope for the presence of specific apple-green fluorescence, indicating HSV-2. If no specific apple-green stain was observed, the coverslip was stained with a fluorescent conjugated goat anti-mouse IgG to determine the presence of HSV-1.
Abbre6iations: BHK, baby hamster kidney; CPE, cytopathic effect; ELVIS™, Enzyme-Linked Virus Inducible System™; HSV, herpes simplex virus; Mab, monoclonal antibody; PI, post-inoculation; PRK, primary rabbit kidney; X-Gal, galactopyranoside. * Corresponding author. Tel.: +1-216-844-8611; fax: +1-216-368-0495. 1386-6532/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 0 1 9 7 ( 9 8 ) 0 0 0 6 6 - X
66
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
Results: Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 were HSV-2 and 13 were HSV-1. The ELVIS™ BHK shell vials detected 42 positive HSV; 30 were HSV-2 and 12 were HSV-1. One low titer specimen was not identified as being HSV positive. Two specimens were not directly typed by the ELVIS™ system. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one fluorescent cell present that was difficult to type. As suggested by the manufacturer’s instructions, both specimens that were not directly typed were re-grown overnight from their supernatants and were correctly identified and typed. Conclusion: The ELVIS™ HSV ID/Typing System is a rapid, highly specific and sensitive method of overnight HSV detection and typing. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Herpes simplex virus; ELVIS™; X-Gal; Baby hamster kidney; Primary rabbit kidney; Monoclonal antibody
Herpes simplex virus (HSV) is a common human pathogen that can produce lesions at a variety of sites in the human body. There are two distinct serotypes of HSV, HSV-1 and HSV-2. HSV-2 is primarily associated with genital infections, while HSV-1 is associated with oral infection. Neither serotype, though, exclusively found in those locations (Mattison et al., 1991). With readily available and effective acyclovir to treat HSV infection, rapid detection will greatly assist patient management. There are reasons for the typing of HSV positive specimens. The type is important in following the epidemiology of HSV. In the case of genital herpes, the recurrence rate and asymptomatic shedding of the virus is significantly lower in HSV-1 infection than in HSV-2 (Corey et al., 1983). In terms of long-term suppressive therapy with acyclovir in cases of frequently recurring HSV infection, it is important to correlate the HSV type and site of infection (Kinghorn, 1988; Mostow et al., 1988; Rooney et al., 1993). In the case of pregnancy, mothers who are newly infected with HSV-2 are 33% more likely to pass the virus to their babies (Brown et al., 1991). Congenital HSV infection in neonates can cause conjunctivitis, severe neurodevelopmental problems, and death (Hirsch, 1990). Several methods have been used to identify HSV infection in an individual: the direct immunohistochemical staining of cells collected from an active lesion, enzyme immunoassay to detect HSV antigens (Wu et al., 1989; Dorian et al., 1990; Verano and Michalski, 1990), use of DNA probes on inoculated cell cultures (Espy and
Smith, 1988), in situ hybridization of inoculated cells in culture (Heggie and Huang, 1993), immunofluorescent staining of inoculated cells grown in shell vials (Gleaves et al., 1985), and inoculated cell culture for the formation of cytopathic effect (CPE) (Landry and Hsiung, 1994). The direct staining of cells is the fastest method of identifying HSV in a patient but a specimen with enough cells is not always achieved. Cell culture identification of HSV is the gold standard but it is more time consuming and usually requires a week of incubation. Once CPE is observed, the cells must be stained with type specific monoclonal antibody (Mab) in order to type the HSV. A rapid method is the centrifugation enhanced shell vial technique which requires an overnight incubation but two shell vials need to be inoculated and stained with HSV-1 and HSV-2 specific Mabs, respectively for typing. A recently introduced Enzyme-linked Virus Inducible System™ (ELVIS™) HSV Test System (Diagnostic Hybrids, Athens, OH) is a rapid, highly specific, and highly sensitive system for the detection of HSV (Stabell et al., 1993; Proffitt and Schindler, 1995). The ELVIS™ Test utilizes genetically altered BHK cells. The BHK cells have been transfected with an inducible HSV promoter gene linked to an Escherichia coli LacZ reporter gene (Stabell and Olivo, 1992). When the cell becomes infected with HSV, the virus induces the HSV promoter gene to express the LacZ gene, to produce b-Galactosidase. The infected cells, when stained with X-Gal, show blue, indicating a specimen positive for HSV. This system allows for the
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
67
Table 1 Type of specimens and type of virus isolated in PRK cultures
rapid detection of HSV, but does not offer typing. However, the new ELVIS™ HSV ID/Typing System (Diagnostic Hybrids, Athens, OH) utilizes the same modified BHK cells to detect HSV but also allows for direct typing at the same time. The X-Gal detection solution contains not only galactopyranoside but also fluorescein labeled Mab to HSV-2 and non-fluorescein labeled Mab to HSV1 for typing. Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK (BioWhittaker, Walkersville, MD) and one ELVIS™ BHK shell vial were inoculated with 200 ml of each specimen. Prior to inoculation the media in each vial was aspirated and replaced with 1 ml of Replacement Media (EMEM, 7% fetal bovine serum, 1.25 mg/ml fungizone, 100 U penicillin, 100 mg/ml streptomycin) provided by Diagnostic Hybrids. After inoculation, the shell vials were centrifuged at 700× g for 60 min at 24°C and incubated at 37°C with 5% CO2. The PRK shell vials were examined daily for CPE (up to 4 days). If CPE was present, one shell vial was stained for HSV-1 and the other was stained for HSV-2, using the Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit (Behring Diagnostics, Cupertino, CA). After an overnight incubation, the ELVIS™ BHK shell vial was fixed with the ELVIS™ HSV ID/Typing System solution c1 for 5 min and then stained with solution c2 (X-Gal, fluorescein conjugated HSV-2 Mab, and non-fluorescein labeled HSV-1 Mab). Each shell vial was examined for the presence of blue cells. If blue cells were not present, the specimen was called negative for HSV. If blue cells were present, the specimen was called positive for HSV, then examined under the fluorescent microscope
Specimen source
PRK cultures HSV-1
HSV-2
Genital Rectal Oral/facial Other
3 0 10 0
21 6 1 2
Totals
13
30
for the presence of apple-green staining cells. If specific staining was observed, it indicated HSV-2. If no specific staining was present, then the shell vial was stained with solution c3 (fluorescein conjugated anti-mouse IgG) to confirm the presence of HSV-1 and then re-examined under the fluorescent microscope. Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 (70%) were HSV-2 and 13 (30%) were HSV-1. As shown in Tables 1 and 2, of the 24 positive genital specimens: three were HSV-1 and 21 were HSV-2; all of the six rectal specimens were HSV-2; of the 11 oral-facial specimens, ten were HSV-1 and one was HSV-2. The two unidentified specimens were both HSV-2. Of the 43 HSV positives detected by PRK shell vials, 32 (74%) were detected on day 1 post-inoculation (PI), six (14%) were detected on day 2 PI, and five (12%) were detected on day 3 PI. No positives were detected after day 3 PI. The staining of PRK cells after observing CPE is done to save the cost of staining reagent. If the PRK cells were stained blindly after an overnight culture, the results may be similar to the ELVIS™ BHK cells. The ELVIS™ BHK shell vials detected 42 positives: 30 were HSV-2 and 12 were HSV-1. One specimen was not detected as being HSV positive by
Table 2 Time of virus detection in PRK cultures and ELVIS™ cultures Day 1
PRK cultures ELVIS™ cultures
Day 2
Day 3
HSV-1
HSV-2
HSV-1
HSV-2
HSV-1
HSV-2
7 12
25 30
3 –
3 –
3 –
2 –
68
B.M. Turchek, Y.T. Huang / Journal of Clinical Virology 12 (1999) 65–69
ELVIS™. This was a nose specimen that was not detected by PRK shell vials until day 3 PI, indicating a low viral titer specimen. Two specimens were not directly typed by the ELVIS™ HSV ID/Typing System. One of them was an oral specimen that was not detected by PRK shell vials until day 3 PI. This specimen had only five blue cells present and weakly stained for HSV-2 but was not distinct enough to identify during the initial microscope reading and subsequently stained brighter after the conjugate was added making it difficult to identify type. The other specimen was a lip specimen that also was not detected by PRK shell vials until day 3 PI. This specimen had only one blue cell present and did not show specific fluorescent staining for either HSV-1 or HSV-2. The supernatants from both specimens were re-inoculated into ELVIS™ BHK shell vials and were detected and typed correctly after an overnight incubation. The sensitivity of the ELVIS™ detection of HSV was 98% (42/43) and the specificity for HSV was 100%. The sensitivity of the ELVIS™ HSV typing was 95% (40/ 43) and the specificity of the typing was 98% (42/43). The ELVIS™ system for HSV identification is proven to be highly sensitive and specific for the detection of HSV. Our results are comparable to previous reports (Stabell et al., 1993; Proffitt and Schindler, 1995) with a sensitivity of 98% and a specificity of 100% for the detection of HSV. The new ELVIS™ HSV ID/Typing System not only provides a rapid method for HSV identification but also allows for the simultaneous typing of HSV positive specimens. The results for the typing of HSV positive specimens using the ELVIS™ HSV ID/Typing System were comparable with the standard method. The sensitivity was 95% and the specificity was 98%. The specimens that were not able to be typed directly were specimens of low virus titer. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one cell was weakly stained fluorescent for HSV-2 but was not distinct enough to call HSV-2. After staining with the HSV-1 confirmatory stain, that one cell became more brightly stained making it difficulty to
give a definite type. In the package insert, the manufacturer suggests that specimens with ten or fewer blue cells may not show fluorescence and recommends re-testing of the specimen. Both specimens were re-inoculated with supernatant from their original ELVIS™ shell vials and were correctly identified and typed the following day. However, there were five other specimens that had ten or fewer blue cells that were correctly typed. If the manufacturer’s recommendations were followed in this study, the sensitivity and specificity for the typing would have been 100% and 100%, respectively. One other unique feature about the ELVIS™ HSV ID/Typing System is that the HSV-1 Mab primarily stains the nucleus (targeting an HSV nuclear antigen), while the HSV-2 Mab only stains the cytoplasm. This feature provides additional specificity in the identification of HSV type. Overall, the ELVIS™ HSV ID/Typing System is a rapid, highly specific, and sensitive method for overnight HSV detection and typing. A cost comparison was done between two shell vials with the Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit and one shell vial with the ELVIS™ HSV ID/Typing Test System. The Syva Microtrak® HSV-1/HSV-2 Culture Confirmation/Typing Kit costs US$260 for 20 assays and the PRK shell vials cost US$2 each, which totals US$17 per specimen in order to blind stain and type. The ELVIS™ HSV ID/Typing Test System costs US$265 for 60 assays and the BHK shell vials cost US$5.52 each, which totals US$9.94 per specimen in order to blind stain and type. Therefore, the system requires less hands-on time than other methods for HSV detection and typing, requires less reagents and cultured cells, and costs less.
References Brown ZA, Benedetti J, Ashely R, Burchett S, Selke S, Berry S, Vontver LA, Corey L. Neonatal herpes simplex virus infection in relation to asymptomatic maternal infection at the time of labor. N Engl J Med 1991;324:1247 – 52. Corey L, Adams HG, Brown ZA, Holmes KK. Genital herpes simplex virus infections: clinical manifestations, course, and complications. Ann Intern Med 1983;98:958 – 72.
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