Vol. 29, No. 3
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 436-438 0095-1137/91/030436-03$02.00/0 Copyright © 1991, American Society for Microbiology
Comparison of Two Enzyme-Linked Immunosorbent Assays for Detection of Herpes Simplex Virus Antigen BERNARD GONIK,1* MARILYN SEIBEL,1 ALBERT BERKOWITZ,' MARY BETH WOODIN,2 AND KAREN MILLS3 Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Texas Medical School at Houston, 6431 Fannin,' and Department of Microbiology, Hermann Hospital,3 Houston, Texas 77030, and E. I. DuPont de Nemours and Company, North Billerica, Massachusetts 018622 Received 1 October 1990/Accepted 15 December 1990
Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.
viral transport media containing Hanks balanced salt solution, gelatin, antibiotic-antimycotic solution, and 5% NaHCO3 (for the Ortho ELISA analysis and fresh culture inoculation) or in specified ELISA collection media (for later analysis with the DuPont test kit). Laboratory analysis of the samples was carried out without knowledge of the clinical presentation or culture results. This protocol was approved by the Committee for the Protection of Human Subjects at the authors' institution. Each ELISA kit was run according to the manufacturer's directions. Each test run was validated with both positive and negative controls. Both test kits are sandwich-type ELISAs. Briefly, a sample of the clinical specimen is added to a microtiter plate which has been coated with capture antibody. For both ELISAs, the capture antibody is polyclonal rabbit anti-HSV types 1 and 2. After the antigen incubation step, both ELISAs are washed. At this point the two manufacturers' methods differ. The DuPont test utilizes a pool of biotin-labeled monoclonal antibodies directed to a variety of HSV type 1 and 2 glycoproteins as the detector antibody reagent. After incubation with this reagent, the ELISA is washed and horseradish peroxidase-labeled streptavidin is added. If the biotin-labeled antibody pool has reacted with HSV antigen, then the streptavidin-horseradish peroxidase reagent will bind to the antigen-antibody complex by the available biotin molecules. After streptavidinhorseradish peroxidase incubation, a final wash step is performed and peroxide substrate is added along with orthophenylenediamine chromogen as an indicator of peroxide catalysis. Oxidation of ortho-phenylenediamine by catalyzed peroxide causes a color change from light yellow to dark yellow or orange. The degree of color reaction is determined with a spectrophotometric ELISA plate reader at 490 nm with a 620-nm reference wavelength. A positive result is
The purpose of a herpes simplex virus (HSV) diagnostic test is to confirm, as rapidly as possible, a clinical impression that observed symptoms result from HSV infection. Although cell culture is considered the "gold standard" for HSV detection, it has several inherent limitations, including a lack of 100% sensitivity, a need for rapid transport to an appropriate laboratory facility, and delays in diagnosis (5, 8). Currently available rapid diagnostic tests, primarily of the enzyme-linked immunosorbent assay (ELISA) type, are not extensively or independently used for direct-site HSV diagnosis. This is due to their relative lack of sensitivity in identifying HSV antigen from direct preparations. As the ELISA technology continues to improve, better methods have been developed to capture viral antigen and therefore
improve sensitivity. In this prospective, blinded study we compared the results of two ELISA kits with the results of cultures from patients suspected of having recurrent oral or genital HSV infection. Sensitivity, specificity, and positive and negative predictive values were calculated for each of these two ELISA systems in relationship to culture results. MATERIALS AND METHODS One hundred fifty-three men and women who reported symptomatology consistent with a recurrent episode of HSV infection or who were being monitored for asymptomatic HSV shedding were identified. A subset of 15 of these subjects were identified early in the course of a single HSV-presumed recurrence and were sampled daily until symptom resolution. Direct-site specimens were collected with two cotton-tipped swabs and randomly placed in either *
Corresponding author. 436
VOL. 29, 1991
ELISAS FOR HSV DETECTION
TABLE 1. Viral culture versus Ortho and DuPont ELISA for the detection of HSV No. of samples with culture/ELISA results:
ELISA
Ortho DuPont
+/+
-/-
+/-
-/+
14 38
145 138
26 3
0 7
determined by comparison of the specimen optical density (OD) with a cutoff OD. This is derived by adding 0.07 to the mean OD of the three negative-control wells. The DuPont assay requires two wells per specimen, and both wells must have an OD value above the cutoff OD to be positive. If both wells are below the cutoff, the sample is negative. An indeterminate result is one well above and one well below the cutoff, requiring repeat testing of the sample. The Ortho assay utilizes polyclonal rabbit anti-HSV type 1 and 2 antibodies directly labeled with horseradish peroxidase as the detector antibody. After incubation with this reagent, the ELISA is washed and ortho-phenylenediamine substrate is added as described above. The reaction is read on an ELISA plate reader at 490 nm, and a cutoff is derived by adding 0.150 to the mean OD of the two negative-control wells. The Ortho assay uses one well per sample, and if that well has an OD above the cutoff, it is considered positive. If it is below the cutoff, it is negative. Total assay times are 3.75 and 2.5 h for the DuPont and Ortho assays, respectively. For viral culture, 0.2 ml of specimen is inoculated into primary rabbit kidney cell cultures. After a 1-h incubation, the sample is centrifuged onto the cells (spin amplification) and refed with nutrient media. Each of the viral cultures are examined daily for up to 4 days. A diagnosis of HSV infection is made when established criteria for viral cytopathic effect are observed. Comparisons were made between culture results and each ELISA result by McNemar's test for change (6). A P value of
4
-
FE|
2
_IVeslcle/0F-u. Crusting Prodrome n=8
Ulcer n=20
n=16
FIG. 1. Stage of lesion versus laboratory positivity (by Ortho ELISA, DuPont ELISA, and/or viral culture) in 15 patients sampled daily during a recurrent HSV infection. n, Number of samples collected for each stage of lesion.
infection. Although historical and physical examination findings are at times pathognomonic for HSV, appropriate medical management mandates confirmatory laboratory documentation. Until recently, viral culture has been the primary means to accomplish this goal. As previously stated, viral culture is not without its limitations, including a lack of 100% sensitivity. Aside from technical errors, one would empirically suspect that the size of the in situ viral inoculum could significantly affect culture results; that is, when the lesion is in its early stages or when healing has begun, the amount of viable virus is probably diminished and therefore positive culture results are less likely. Newer rapid diagnostic technologies such as those utilizing immunologic detection of viral antigen might therefore be preferable to culture for HSV diagnosis. As evidenced from our data, currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens and therefore are not interchangeable. Of note, the sensitivities calculated in this report are in keeping with other separately published data for both test kits and reflect clinical recommendations by the manufacturers for the use of these kits (3, 9). In particular, both the DuPont and Ortho ELISAs can be used on direct patient specimens, although backup culture is recommended for negative results with the latter test. Either ELISA is potentially useful in conjunction with culture to shorten the time interval from sampling to the identification of a positive result. The specimen transport systems utilized for these ELISAs reflect the above requirements. That is, the Ortho sample is placed in viral transport medium to allow for subsequent culturing if needed. The DuPont transport medium does not support viral growth, specifically inactivating the virus and making the sample noninfective. Of interest, data are available suggesting that sensitivity is minimally altered if the DuPont ELISA is performed on specimens collected in standard viral transport medium (4).
The differentiation between immunologic and biologic detection of HSV is important. As is clear from our blocking antibody and serial culture data, HSV antigen detection by the more sensitive ELISA methodology is not always synonymous with the state of viral infectivity as judged by cell culture cytopathic effect. In high-infectious-virus and highantigen states (blisters or ulcer lesions), the DuPont rapid diagnostic test is equally as efficacious as culture in diagnosing viral infection. However, during presumed high-antigen but low-infectious-virus states (crusting lesions), this same ELISA appears to be superior to culture in diagnosing the presence of HSV. These distinctions are critically important, depending on the indications for testing. For example, if diagnosis is the issue, the DuPont ELISA would be preferable over a broader range of clinical conditions (e.g., late presentation or previous therapy, etc.). Conversely, if risk of infectivity (i.e., neonatal transmission) is the issue, the immunologic assay will most likely overestimate these risks. One caveat, however, is that even though culture, by definition, better represents infectivity, this information is necessarily delayed by a minimum of 24 h and therefore can be utilized only in a retrospective fashion. Two other potential benefits to culture, viral typing and antiviral susceptibility testing, are rarely needed in the clinical setting. ACKNOWLEDGMENT The study was partially funded by a grant from E. I. DuPont de Nemours and Co. REFERENCES 1. Baker, D. A., B. Gonik, P. 0. Milch, A. Berkowitz, S. Lipson, and V. Verma. 1989. Clinical evaluation of a new herpes simplex virus ELISA: a rapid diagnostic test for herpes simplex virus. Obstet. Gynecol. 73:322-325. 2. Clayton, A. L., C. Roberts, M. Godley, J. M. Best, and S. M. Chantler. 1986. Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion samples and specimen treatment. J. Med. Virol. 20:89-97. 3. Dascal, A., J. Chan-Thim, M. Morahan, J. Portnoy, and J. Mendelson. 1989. Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassay kit. J. Clin. Microbiol. 27:700-704. 4. Dascal, A., J. Chan-Thim, M. Morahan, J. Portnoy, and J. Mendelson. 1989. Replacement of special enzyme immunoassay transport medium by a standard viral transport medium in the Herpcheck herpes simplex virus antigen detection test. Diagn. Microbiol. Infect. Dis. 12:473-475. 5. Drew, W. L., and W. E. Rawls. 1985. Herpes simplex viruses, p. 705-710. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 6. Glantz, S. A. 1981. Primer of biostatistics, p. 261-264. McGrawHill Book Co., New York. 7. Kohl, S., and B. Gonik. 1990. Herpes simplex virus, p. 16461651. In W. N. Kelley (ed.), Textbook of internal medicine, 2nd ed. J. P. Lippincott, Philadelphia. 8. Tada, A., N. Sekine, M. Toba, and K. Yoshino. 1977. Analysis of factors in influencing the isolation of herpes simplex virus. Microbiol. Immunol. 21:219-229. 9. Warford, A. L., R. A. Levy, and K. A. Rekrut. 1984. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of herpes simplex virus antigen. J. Clin. Microbiol. 20:490493.
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 436-438 0095-1137/91/030436-03$02.00/0 Copyright © 1991, American Society for Microbiology
Comparison of Two Enzyme-Linked Immunosorbent Assays for Detection of Herpes Simplex Virus Antigen BERNARD GONIK,1* MARILYN SEIBEL,1 ALBERT BERKOWITZ,' MARY BETH WOODIN,2 AND KAREN MILLS3 Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Texas Medical School at Houston, 6431 Fannin,' and Department of Microbiology, Hermann Hospital,3 Houston, Texas 77030, and E. I. DuPont de Nemours and Company, North Billerica, Massachusetts 018622 Received 1 October 1990/Accepted 15 December 1990
Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.
viral transport media containing Hanks balanced salt solution, gelatin, antibiotic-antimycotic solution, and 5% NaHCO3 (for the Ortho ELISA analysis and fresh culture inoculation) or in specified ELISA collection media (for later analysis with the DuPont test kit). Laboratory analysis of the samples was carried out without knowledge of the clinical presentation or culture results. This protocol was approved by the Committee for the Protection of Human Subjects at the authors' institution. Each ELISA kit was run according to the manufacturer's directions. Each test run was validated with both positive and negative controls. Both test kits are sandwich-type ELISAs. Briefly, a sample of the clinical specimen is added to a microtiter plate which has been coated with capture antibody. For both ELISAs, the capture antibody is polyclonal rabbit anti-HSV types 1 and 2. After the antigen incubation step, both ELISAs are washed. At this point the two manufacturers' methods differ. The DuPont test utilizes a pool of biotin-labeled monoclonal antibodies directed to a variety of HSV type 1 and 2 glycoproteins as the detector antibody reagent. After incubation with this reagent, the ELISA is washed and horseradish peroxidase-labeled streptavidin is added. If the biotin-labeled antibody pool has reacted with HSV antigen, then the streptavidin-horseradish peroxidase reagent will bind to the antigen-antibody complex by the available biotin molecules. After streptavidinhorseradish peroxidase incubation, a final wash step is performed and peroxide substrate is added along with orthophenylenediamine chromogen as an indicator of peroxide catalysis. Oxidation of ortho-phenylenediamine by catalyzed peroxide causes a color change from light yellow to dark yellow or orange. The degree of color reaction is determined with a spectrophotometric ELISA plate reader at 490 nm with a 620-nm reference wavelength. A positive result is
The purpose of a herpes simplex virus (HSV) diagnostic test is to confirm, as rapidly as possible, a clinical impression that observed symptoms result from HSV infection. Although cell culture is considered the "gold standard" for HSV detection, it has several inherent limitations, including a lack of 100% sensitivity, a need for rapid transport to an appropriate laboratory facility, and delays in diagnosis (5, 8). Currently available rapid diagnostic tests, primarily of the enzyme-linked immunosorbent assay (ELISA) type, are not extensively or independently used for direct-site HSV diagnosis. This is due to their relative lack of sensitivity in identifying HSV antigen from direct preparations. As the ELISA technology continues to improve, better methods have been developed to capture viral antigen and therefore
improve sensitivity. In this prospective, blinded study we compared the results of two ELISA kits with the results of cultures from patients suspected of having recurrent oral or genital HSV infection. Sensitivity, specificity, and positive and negative predictive values were calculated for each of these two ELISA systems in relationship to culture results. MATERIALS AND METHODS One hundred fifty-three men and women who reported symptomatology consistent with a recurrent episode of HSV infection or who were being monitored for asymptomatic HSV shedding were identified. A subset of 15 of these subjects were identified early in the course of a single HSV-presumed recurrence and were sampled daily until symptom resolution. Direct-site specimens were collected with two cotton-tipped swabs and randomly placed in either *
Corresponding author. 436
VOL. 29, 1991
ELISAS FOR HSV DETECTION
TABLE 1. Viral culture versus Ortho and DuPont ELISA for the detection of HSV No. of samples with culture/ELISA results:
ELISA
Ortho DuPont
+/+
-/-
+/-
-/+
14 38
145 138
26 3
0 7
determined by comparison of the specimen optical density (OD) with a cutoff OD. This is derived by adding 0.07 to the mean OD of the three negative-control wells. The DuPont assay requires two wells per specimen, and both wells must have an OD value above the cutoff OD to be positive. If both wells are below the cutoff, the sample is negative. An indeterminate result is one well above and one well below the cutoff, requiring repeat testing of the sample. The Ortho assay utilizes polyclonal rabbit anti-HSV type 1 and 2 antibodies directly labeled with horseradish peroxidase as the detector antibody. After incubation with this reagent, the ELISA is washed and ortho-phenylenediamine substrate is added as described above. The reaction is read on an ELISA plate reader at 490 nm, and a cutoff is derived by adding 0.150 to the mean OD of the two negative-control wells. The Ortho assay uses one well per sample, and if that well has an OD above the cutoff, it is considered positive. If it is below the cutoff, it is negative. Total assay times are 3.75 and 2.5 h for the DuPont and Ortho assays, respectively. For viral culture, 0.2 ml of specimen is inoculated into primary rabbit kidney cell cultures. After a 1-h incubation, the sample is centrifuged onto the cells (spin amplification) and refed with nutrient media. Each of the viral cultures are examined daily for up to 4 days. A diagnosis of HSV infection is made when established criteria for viral cytopathic effect are observed. Comparisons were made between culture results and each ELISA result by McNemar's test for change (6). A P value of
4
-
FE|
2
_IVeslcle/0F-u. Crusting Prodrome n=8
Ulcer n=20
n=16
FIG. 1. Stage of lesion versus laboratory positivity (by Ortho ELISA, DuPont ELISA, and/or viral culture) in 15 patients sampled daily during a recurrent HSV infection. n, Number of samples collected for each stage of lesion.
infection. Although historical and physical examination findings are at times pathognomonic for HSV, appropriate medical management mandates confirmatory laboratory documentation. Until recently, viral culture has been the primary means to accomplish this goal. As previously stated, viral culture is not without its limitations, including a lack of 100% sensitivity. Aside from technical errors, one would empirically suspect that the size of the in situ viral inoculum could significantly affect culture results; that is, when the lesion is in its early stages or when healing has begun, the amount of viable virus is probably diminished and therefore positive culture results are less likely. Newer rapid diagnostic technologies such as those utilizing immunologic detection of viral antigen might therefore be preferable to culture for HSV diagnosis. As evidenced from our data, currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens and therefore are not interchangeable. Of note, the sensitivities calculated in this report are in keeping with other separately published data for both test kits and reflect clinical recommendations by the manufacturers for the use of these kits (3, 9). In particular, both the DuPont and Ortho ELISAs can be used on direct patient specimens, although backup culture is recommended for negative results with the latter test. Either ELISA is potentially useful in conjunction with culture to shorten the time interval from sampling to the identification of a positive result. The specimen transport systems utilized for these ELISAs reflect the above requirements. That is, the Ortho sample is placed in viral transport medium to allow for subsequent culturing if needed. The DuPont transport medium does not support viral growth, specifically inactivating the virus and making the sample noninfective. Of interest, data are available suggesting that sensitivity is minimally altered if the DuPont ELISA is performed on specimens collected in standard viral transport medium (4).
The differentiation between immunologic and biologic detection of HSV is important. As is clear from our blocking antibody and serial culture data, HSV antigen detection by the more sensitive ELISA methodology is not always synonymous with the state of viral infectivity as judged by cell culture cytopathic effect. In high-infectious-virus and highantigen states (blisters or ulcer lesions), the DuPont rapid diagnostic test is equally as efficacious as culture in diagnosing viral infection. However, during presumed high-antigen but low-infectious-virus states (crusting lesions), this same ELISA appears to be superior to culture in diagnosing the presence of HSV. These distinctions are critically important, depending on the indications for testing. For example, if diagnosis is the issue, the DuPont ELISA would be preferable over a broader range of clinical conditions (e.g., late presentation or previous therapy, etc.). Conversely, if risk of infectivity (i.e., neonatal transmission) is the issue, the immunologic assay will most likely overestimate these risks. One caveat, however, is that even though culture, by definition, better represents infectivity, this information is necessarily delayed by a minimum of 24 h and therefore can be utilized only in a retrospective fashion. Two other potential benefits to culture, viral typing and antiviral susceptibility testing, are rarely needed in the clinical setting. ACKNOWLEDGMENT The study was partially funded by a grant from E. I. DuPont de Nemours and Co. REFERENCES 1. Baker, D. A., B. Gonik, P. 0. Milch, A. Berkowitz, S. Lipson, and V. Verma. 1989. Clinical evaluation of a new herpes simplex virus ELISA: a rapid diagnostic test for herpes simplex virus. Obstet. Gynecol. 73:322-325. 2. Clayton, A. L., C. Roberts, M. Godley, J. M. Best, and S. M. Chantler. 1986. Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion samples and specimen treatment. J. Med. Virol. 20:89-97. 3. Dascal, A., J. Chan-Thim, M. Morahan, J. Portnoy, and J. Mendelson. 1989. Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassay kit. J. Clin. Microbiol. 27:700-704. 4. Dascal, A., J. Chan-Thim, M. Morahan, J. Portnoy, and J. Mendelson. 1989. Replacement of special enzyme immunoassay transport medium by a standard viral transport medium in the Herpcheck herpes simplex virus antigen detection test. Diagn. Microbiol. Infect. Dis. 12:473-475. 5. Drew, W. L., and W. E. Rawls. 1985. Herpes simplex viruses, p. 705-710. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 6. Glantz, S. A. 1981. Primer of biostatistics, p. 261-264. McGrawHill Book Co., New York. 7. Kohl, S., and B. Gonik. 1990. Herpes simplex virus, p. 16461651. In W. N. Kelley (ed.), Textbook of internal medicine, 2nd ed. J. P. Lippincott, Philadelphia. 8. Tada, A., N. Sekine, M. Toba, and K. Yoshino. 1977. Analysis of factors in influencing the isolation of herpes simplex virus. Microbiol. Immunol. 21:219-229. 9. Warford, A. L., R. A. Levy, and K. A. Rekrut. 1984. Evaluation of a commercial enzyme-linked immunosorbent assay for detection of herpes simplex virus antigen. J. Clin. Microbiol. 20:490493.
